Laboratory Models for

Foodborne Infections
 Series Editor
Dongyou Liu

Laboratory Models for Foodborne Infections, edited by Dongyou Liu (2017)

Food Spoilage Microorganisms: Ecology and Control, edited by Yanbo Wang (2017)
Foodborne Viral Pathogens, edited by Peter A. White, Natalie E. Netzler, and Grant S.
Hansman (2016)
Molecular Biology of Food and Water Borne Mycotoxigenic and Mycotic Fungi,
edited by R. Russell M. Paterson, and Nelson Lima (2015)
Biology of Foodborne Parasites, edited by Lihua Xiao, Una Ryan, and Yaoyu Feng (2015)
 Laboratory Models for
Foodborne Infections

Edited by
Dongyou Liu
Royal College of Pathologists of Australasia Quality Assurance Programs
New South Wales, Australia
CRC Press
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Library of Congress Cataloging‑in‑Publication Data

Names: Names: Liu, Dongyou, editor.

Title: Laboratory models for foodborne infections / [edited by] Dongyou Liu.
Other titles: Food microbiology series.
Description: Boca Raton : CRC Press/Taylor & Francis, 2017. | Series: Food
microbiology series | Includes bibliographical references and index.
Identifiers: LCCN 2016040619 | ISBN 9781498721677 (hardback : alk. paper)
Subjects: | MESH: Foodborne Diseases | Models, Animal | Models, Biological |
Food Microbiology
Classification: LCC QR201.F62 | NLM WC 268 | DDC 615.9/5293--dc23
LC record available at https://lccn.loc.gov/2016040619

Visit the Taylor & Francis Web site at

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Contents

Preface for Food Microbiology Series....................................................................................................... ix

Preface....................................................................................................................................................... xi
Editor.......................................................................................................................................................xiii
Contributors.............................................................................................................................................. xv

1. Introductory Remarks...................................................................................................................... 1
Dongyou Liu

Section I  Foodborne Infections due to Viruses

2. Adenoviruses.................................................................................................................................... 13
Anthony P. Malanoski and Baochuan Lin

3. Astrovirus......................................................................................................................................... 29
Matthew D. Koci and Stacey L. Schultz-Cherry

4. Hepatitis E Virus..............................................................................................................................41
Kavita Lole, Prudhvi Lal Bhukya, and Subhashis Chatterjee

5. Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation.................. 75

Doris H. D’Souza and Snehal S. Joshi

6. Rotavirus.......................................................................................................................................... 95
Lijuan Yuan and Ke Wen

7. Prions...............................................................................................................................................117
Akikazu Sakudo and Takashi Onodera

Section II  Foodborne Infections due to Gram-Positive Bacteria

8. Bacillus............................................................................................................................................131
Jessica Minnaard, Ivanna S. Rolny, and Pablo F. Pérez

9. Clostridium......................................................................................................................................155
Emilio Aranda, María G. Córdoba, María J. Benito, and Juan José Córdoba

10. Enterococcus...................................................................................................................................175
Dongyou Liu

11. Listeria monocytogenes..................................................................................................................185

Sarah E.F. D’Orazio

v
vi Contents

12. Mycobacterium............................................................................................................................... 197

Flábio R. de Araújo and Nalvo F. Almeida

13. Staphylococcus............................................................................................................................... 209

Mar Rodríguez, Alicia Rodríguez, María Jesús Andrade, Elena Bermúdez, and
Juan José Córdoba

14. Streptococcus.................................................................................................................................. 223

Dongyou Liu

Section III  Foodborne Infections due to Gram-Negative Bacteria

15. Aeromonas...................................................................................................................................... 237
Dongyou Liu

16. Bacteroides..................................................................................................................................... 247

Mario Julio Avila-Campos

17. Brucella.......................................................................................................................................... 259

S.C. Olsen and B. Bricker

18. Burkholderia.................................................................................................................................. 271

Danielle L. Peters, Fatima Kamal, and Jonathan J. Dennis

19. Campylobacter............................................................................................................................... 289

Martin Stahl and Bruce A. Vallance

20. Cronobacter: Virulence and Pathogenesis.................................................................................. 305

Nemani V. Prasadarao

21. Escherichia......................................................................................................................................317
Dongyou Liu

22. Helicobacter....................................................................................................................................331
Tetsuya Tsukamoto, Yuka Kiriyama, and Masae Tatematsu

23. Klebsiella: Caenorhabditis elegans as a Laboratory Model for

Klebsiella pneumoniae Infection.................................................................................................. 343
Arumugam Kamaladevi and Krishnaswamy Balamurugan

24. Proteus.............................................................................................................................................355
Paola Scavone, Victoria Iribarnegaray, and Pablo Zunino

25. Pseudomonas aeruginosa.............................................................................................................. 373

Stavria Panayidou and Yiorgos Apidianakis

26. Salmonella.......................................................................................................................................391
Dongyou Liu

27. Shigella........................................................................................................................................... 401

Soumik Barman and Yoshifumi Takeda
Contents vii

28. Vibrio: Caenorhabditis elegans as a Laboratory Model for Vibrio Infections.........................413

Sellegounder Durai and Krishnaswamy Balamurugan

29. Yersinia........................................................................................................................................... 427

Xin Wang, Ran Duan, Junrong Liang, Wenpeng Gu, and Huaiqi Jing

Section IV  Foodborne Infections due to Fungi

30. Alternaria....................................................................................................................................... 441
Alicia Rodríguez, Andrea Patriarca, Mar Rodríguez, María Jesús Andrade, and
Juan José Córdoba

31. Aspergillus.......................................................................................................................................455
László Kredics, János Varga, Rajagopalaboopathi Jayasudha, Sándor Kocsubé,
Nikolett Baranyi, Coimbatore Subramanian Shobana, Muthusamy Chandrasekaran,
Shine Kadaikunnan, Venkatapathy Narendran, Csaba Vágvölgyi, and
Palanisamy Manikandan

32. Candida.......................................................................................................................................... 497

María Jesús Andrade, Mar Rodríguez, Alicia Rodríguez, and Juan José Córdoba

33. Enterocytozoon bieneusi.................................................................................................................511

Hirotake Mori and Aongart Mahittikorn

34. Fusarium........................................................................................................................................ 523

Palanisamy Manikandan, Coimbatore Subramanian Shobana, Mónika Homa,
Sándor Kocsubé, János Varga, Muthusamy Chandrasekaran, Naiyf S. Alharbi,
Venkatapathy Narendran, Csaba Vágvölgyi, and László Kredics

35. Penicillium and Talaromyces.........................................................................................................555

Elena Bermúdez, Félix Núñez, Josué Delgado, and Miguel A. Asensio

Section V  Foodborne Infections due to Protozoa

36. Acanthamoeba................................................................................................................................ 579
Dongyou Liu

37. Cryptosporidium............................................................................................................................ 589

Dongyou Liu

38. Cystoisospora belli......................................................................................................................... 599

Chaturong Putaporntip and Somchai Jongwutiwes

39. Entamoeba histolytica....................................................................................................................617

Mineko Shibayama, Nidia León-Sicairos, Jesús Serrano-Luna, and Mireya de la Garza

40. Giardia lamblia.............................................................................................................................. 635

Steven M. Singer, Jenny G. Maloney, and Camila H. Coelho

41. Toxoplasma: Animal and In Vitro Models on Toxoplasmosis................................................... 655

Renato Augusto DaMatta, Andrea Cristina Vetö Arnholdt, and Farlen José Bebber Miranda
viii Contents

Section VI  Foodborne Infections due to Helminths

42. Anisakis.......................................................................................................................................... 679
Mauricio Afonso Vericimo, Gerlinde Teixeira, Israel Figueiredo Jr., Janaina Ribeiro,
Maria Augusta Moulin Fantezia, and Sergio Carmona São Clemente

43. Clonorchis sinensis........................................................................................................................ 703

Bayissa Chala Legissa and Sung-Tae Hong

44. Fasciola and Fasciolosis.................................................................................................................717

Antonio Muro and Jose Rojas-Caraballo

45. Haplorchis...................................................................................................................................... 735

Dongyou Liu

46. Metagonimus...................................................................................................................................743
Jong-Yil Chai

47. Opisthorchis viverrini.................................................................................................................... 765

Thidarut Boonmars

48. Paragonimus.................................................................................................................................. 773

Dongyou Liu

49. Taenia............................................................................................................................................. 783

Dongyou Liu

50. Trichinella...................................................................................................................................... 793

Ljiljana Sofronic-Milosavljevic, Natasa Ilic, and Alisa Gruden-Movsesijan

Index....................................................................................................................................................... 809
Preface for Food Microbiology Series

Microorganisms (including viruses, bacteria, molds, yeasts, protozoa, and helminths) represent abundant
and diverse forms of life that occupy various ecological niches of earth. Those utilizing food and food
products for growth and maintenance are important to human society due not only to their positive and
negative impacts on food supply, but also to their potential pathogenicity to human and animal hosts.
On one hand, foodborne microorganisms are known to play a critical role in fermentation and modi-
fication of foods, leading to a variety of nutritious food products (e.g., bread, beverage, yogurt, cheese,
etc.) that have helped sustain the human civilization from time immemorial. On the other hand, food-
borne microorganisms may be responsible for food spoilage, which, albeit a necessary step in keeping
up ecological balance, reduces the quality and quantity of foods for human and animal consumption.
Furthermore, some foodborne microorganisms are pathogenic to humans and animals, which, besides
creating havoc on human health and animal welfare, decrease the availability of meat and other animal-
related products.
Food microbiology is a continuously evolving field of biological sciences that addresses issues arising
from the interactions between food-/waterborne microorganisms and foods. Topics of relevance to food
microbiology include, but are not limited to, adoption of innovative fermentation and other techniques
to improve food production; optimization of effective preservation procedures to reduce food spoilage;
development of rapid, sensitive, and specific methods to identify and monitor foodborne microbes and
toxins, helping alleviate food safety concerns among consumers; use of -omic approaches to unravel
the pathogenicity of foodborne microbes and toxins; selection of nonpathogenic foodborne microbes as
probiotics to inhibit and eliminate pathogenic viruses, bacteria, fungi, and parasites; design and imple-
mentation of novel control and prevention strategies against foodborne diseases in human and animal
populations.
The Food Microbiology Series aims to present a state-of-art coverage on topics central to the under-
standing of the interactions between food-/waterborne microorganisms and foods. The series consists
of individual volumes, each of which focuses on a particular aspect/group of foodborne microbes and
toxins, in relation to their biology, ecology, epidemiology, immunology, clinical features, pathogenesis,
diagnosis, antibiotic resistance, stress responses, treatment and prevention, etc. The volume editors and
the authors are professionals with expertise in their respective fields of food microbiology, and the chap-
ter contributors are scientists directly involved in foodborne microbe and toxin research.
Extending the contents of classical textbooks on food microbiology, this series serves as an indispens-
able tool for food microbiology researchers, industry food microbiologists, and food regulation authori-
ties wishing to keep abreast with latest developments in food microbiology. In addition, the series offers
a reliable reference for undergraduate and graduate students in their pursuit to becoming competent and
consummate future food microbiologists. Moreover, the series provides a trustworthy source of informa-
tion to the general public interested in food safety and other related issues.

ix
Preface

Foodborne infections result from the ingestion of foods and beverages (including drinking water) that are
contaminated by pathogenic microorganisms (including viruses, bacteria, fungi, and parasites). While
some microbial pathogens stay in the gastrointestinal system and produce toxins (e.g., enterotoxins, exo-
toxins, and mycotoxins) that are absorbed into the bloodstream, others may directly invade deeper body
tissues. Although foodborne infections generally tend to induce mild clinical symptoms (e.g., nausea,
vomiting, fever, abdominal cramps, and diarrhea) in immunocompetent individuals, they may have seri-
ous consequences in young children and people with suppressed immune functions.
With the increasing consumption of manufactured foods and beverages, foodborne infections are
becoming a common and expensive public health problem worldwide. The World Health Organization
(WHO) estimates that food-/waterborne diarrheal diseases kill about 2.2 million people (mostly chil-
dren) annually. Based on FoodNet data collected between 2000 and 2007 by the Centers for Disease
Control and Prevention (CDC), 48 million foodborne illness cases (16,000 cases for 100,000 inhabitants)
occur in the United States every year, including 128,000 hospitalizations and 3,000 deaths. Interestingly,
31 foodborne pathogens have been implicated in 9.4 of the 48 million foodborne illness cases, with
7 (Salmonella, norovirus, Campylobacter, Toxoplasma, Escherichia coli O157:H7, Listeria, and
Clostridium perfringens) accounting for 90% of these illnesses alone. Similarly, an estimated 4.1 mil-
lion cases of foodborne gastroenteritis were documented in Australia in 2010, with norovirus, pathogenic
E. coli, Campylobacter spp., and nontyphoidal Salmonella spp. being the main culprits.
Although proper storage and refrigeration of food play a vital role in the prevention of foodborne infec-
tions, other good food safety practices (handwashing, preventing cross-contamination, and maintaining
cooking temperatures in the kitchen) are also valuable. In addition, accurate diagnosis and prompt medi-
cal intervention are crucial in reducing the mortality due to foodborne infections. However, thorough
understanding of host–pathogen interactions and elucidation of molecular mechanisms of pathogenesis
are critical for the development of effective vaccines that will lead to ultimate elimination of foodborne
infections in human population. Toward this goal, application of laboratory models (including both in
vivo and in vitro models) is essential.
As a part of the Food Microbiology Series, this book focuses on the value and utility of various
animal and cellular systems (ranging from mice, rats, hamsters, guinea pigs, rabbits, nonhuman pri-
mates, birds, zebrafish, frogs, chicken embryo, fruit fly, nematode, and waxworm to established and
nonestablished cell lines) in the study of foodborne infections. Written by experts involved in foodborne
pathogen research, each chapter presents a state-of-the-art review of laboratory models in the study of a
particular foodborne pathogen (of viral, bacterial, fungal, or parasitic origin) in relation to its life cycle,
host–pathogen interaction, pathogenesis, immunity, and other related aspects. Besides providing a reli-
able reference for undergraduates and postgraduates of food microbiology, this book is a valuable guide
for scientists using laboratory models in their investigation of foodborne infections.
Given the diversity of foodborne pathogens, a comprehensive book such as this is clearly beyond an
individual’s capacity. I am fortunate and honored to have a large group of scientists as chapter contribu-
tors, whose in-depth knowledge and technical insights on foodborne pathogens have greatly enriched
this book. Additionally, the professionalism and dedication of the senior editor, Stephen Zollo, have
enhanced its presentation. Finally, the understanding and support from my family—Liling Ma, Brenda,
and Cathy—have helped me keep focused during the compilation of this all-inclusive volume.

Dongyou Liu

xi
Editor

Dongyou Liu, PhD,studied veterinary science at Hunan Agricultural University, China, and completed
his postgraduate training at the University of Melbourne, Victoria, Australia. Over the past two decades,
he has worked at several research and clinical laboratories in Australia and the United States of America,
focusing on molecular characterization and virulence determination of microbial pathogens such as
ovine footrot bacterium (Dichelobacter nodosus), dermatophyte fungi (Trichophyton, Microsporum,
and Epidermophyton), and listeriae (Listeria spp.), as well as development of nucleic-acid-based quality
assurance models for security-sensitive and emerging viral pathogens. He is the author of over 50 origi-
nal research and review articles in various international journals, a contributor of 165 book chapters,
and the editor of Handbook of Listeria monocytogenes (2008), Handbook of Nucleic Acid Purification
(2009), Molecular Detection of Foodborne Pathogens (2009), Molecular Detection of Human Viral
Pathogens (2010), Molecular Detection of Human Bacterial Pathogens (2011), Molecular Detection
of Human Fungal Pathogens (2011), Molecular Detection of Human Parasitic Pathogens (2012),
Manual of Security Sensitive Microbes and Toxins (2014), and Molecular Detection of Animal Viral
Pathogens (2016), all of which are published by CRC Press. He is also a coeditor of Molecular Medical
Microbiology, 2nd edition (2014), which was published by Elsevier.

xiii
Contributors

Naiyf S. Alharbi Krishnaswamy Balamurugan

Botany and Microbiology Department Department of Biotechnology
King Saud University Alagappa University
Riyadh, Saudi Arabia Karaikudi, Tamil Nadu, India

Nalvo F. Almeida Nikolett Baranyi

School of Computing Department of Microbiology
Federal University of Mato Grosso do Sul University of Szeged
Campo Grande, Mato Grosso do Sul, Brazil Szeged, Hungary

María Jesús Andrade Soumik Barman

Food Hygiene and Safety National Institute of Cholera and Enteric
Research Institute of Meat and Meat Products Diseases
University of Extremadura Beleghata, Kolkata, India
Cáceres, Spain
María J. Benito
Yiorgos Apidianakis Food Science and Technology
Department of Biological Sciences Agricultural Resources Research Institute
University of Cyprus University of Extremadura
Aglatzia, Cyprus Badajoz, Spain

Emilio Aranda Elena Bermúdez

Food Science and Technology Food Hygiene and Safety
Agricultural Resources Research Institute Research Institute of Meat and Meat
University of Extremadura Products
Badajoz, Spain University of Extremadura
Cáceres, Spain
Andrea Cristina Vetö Arnholdt
Laboratório de Biologia do Reconhecer Prudhvi Lal Bhukya
Centro de Biociências e Biotecnologia Hepatitis Group
Universidade Estadual do Norte Fluminense National Institute of Virology
Darcy Ribeiro Pashan, Pune, India
Campos dos Goytacazes, Rio de Janeiro, Brazil
Thidarut Boonmars
Miguel A. Asensio Department of Parasitology
Food Hygiene and Safety Khon Kaen University
Research Institute of Meat and Meat Products Khon Kaen, Thailand
University of Extremadura
Cáceres, Spain B. Bricker
Bacterial Diseases of Livestock Research
Mario Julio Avila-Campos Unit
Department of Microbiology National Animal Disease Center Agricultural
Institute of Biomedical Science Research Service
University of São Paulo United States Department of Agriculture
São Paulo, Brazil Ames, Iowa

xv
xvi Contributors

Jong-Yil Chai Mireya de la Garza

Department of Parasitology and Tropical Departamento de Biología Celular
Medicine Centro de Investigación y de Estudios Avanzados
Seoul National University College of del Instituto Politécnico Nacional
Medicine Mexico City, Mexico
Seoul, South Korea
Josué Delgado
Muthusamy Chandrasekaran Food Hygiene and Safety
Botany and Microbiology Department Research Institute of Meat and Meat
King Saud University Products
Riyadh, Saudi Arabia University of Extremadura
Cáceres, Spain
Subhashis Chatterjee
Hepatitis Group Jonathan J. Dennis
National Institute of Virology Department of Biological Sciences
Pashan, Pune, India University of Alberta
Edmonton, Alberta, Canada
Sergio Carmona São Clemente
Departamento Tecnologia de Produtos de Sarah E.F. D’Orazio
Origem Animal Department of Microbiology, Immunology, &
Universidade Federal Fluminense Molecular Genetics
Niterói, Rio de Janeiro, Brazil University of Kentucky
Lexington, Kentucky
Camila H. Coelho
Department of Biology Doris H. D’Souza
Georgetown University Department of Food Science and
Washington, DC Technology
University of Tennessee, Knoxville
Juan José Córdoba Knoxville, Tennessee
Food Hygiene and Safety
Research Institute of Meat and Meat Products Ran Duan
University of Extremadura National Institute for Communicable Disease
Cáceres, Spain Control and Prevention
Chinese Centre for Disease Control and
María G. Córdoba Prevention
Food Science and Technology Beijing, People’s Republic of China
Agricultural Resources Research Institute
University of Extremadura Sellegounder Durai
Badajoz, Spain Department of Biotechnology
Alagappa University
Renato Augusto DaMatta Karaikudi, Tamil Nadu, India
Laboratório de Biologia Celular e Tecidual
Centro de Biociências e Biotecnologia Maria Augusta Moulin Fantezia
Universidade Estadual do Norte Fluminense Departamento de Imunobiologia
Darcy Ribeiro Universidade Federal Fluminense
Campos dos Goytacazes, Rio de Janeiro, Niterói, Rio de Janeiro, Brazil
Brazil
Israel Figueiredo Jr.
Flábio R. de Araújo Departamento Materno-Infantil
Embrapa Beef Cattle Universidade Federal Fluminense
Campo Grande, Mato Grosso do Sul, Brazil Niterói, Rio de Janeiro, Brazil
Contributors xvii

Alisa Gruden-Movsesijan Somchai Jongwutiwes

Department for Immunology and Department of Parasitology
Immunoparasitology Chulalongkorn University
Institute for the Application of Nuclear Bangkok, Thailand
Energy
University of Belgrade Snehal S. Joshi
Belgrade, Serbia Department of Food Science and
Technology
Wenpeng Gu University of Tennessee, Knoxville
National Institute for Communicable Disease Knoxville, Tennessee
Control and Prevention
Chinese Centre for Disease Control and Shine Kadaikunnan
Prevention Botany and Microbiology Department
Beijing, People’s Republic of China King Saud University
Riyadh, Saudi Arabia
Mónika Homa
Department of Microbiology Fatima Kamal
University of Szeged Department of Biological Sciences
Szeged, Hungary University of Alberta
Edmonton, Alberta, Canada
Sung-Tae Hong
Department of Parasitology and Tropical Arumugam Kamaladevi
Medicine Department of Biotechnology
Seoul National University College of Alagappa University
Medicine Karaikudi, Tamil Nadu, India
Seoul, South Korea
Yuka Kiriyama
Natasa Ilic Department of Diagnostic Pathology
Department for Immunology and Fujita Health University
Immunoparasitology Toyoake, Aichi, Japan
Institute for the Application of Nuclear Energy
University of Belgrade Matthew D. Koci
Belgrade, Serbia Prestage Department of Poultry Science
North Carolina State University
Victoria Iribarnegaray Raleigh, North Carolina
Department of Microbiology
Instituto de Investigaciones Biológicas Clemente Sándor Kocsubé
Estable Department of Microbiology
Montevideo, Uruguay University of Szeged
Szeged, Hungary
Rajagopalaboopathi Jayasudha
Department of Biotechnology László Kredics
Bharathiar University Department of Microbiology
Coimbatore, Tamil Nadu, India University of Szeged
Szeged, Hungary
Huaiqi Jing
National Institute for Communicable Disease Bayissa Chala Legissa
Control and Prevention Department of Parasitology and Tropical
Chinese Centre for Disease Control and Medicine
Prevention Seoul National University College of Medicine
Beijing, People’s Republic of China Seoul, South Korea
xviii Contributors

Nidia León-Sicairos Jessica Minnaard

Facultad de Medicina Centro de Investigación y Desarrollo en
Universidad Autónoma de Sinaloa Criotecnología de Alimentos
Culiacán Sin, Mexico Consejo Nacional de Investigaciones Científicas
y Técnicas
Junrong Liang La Plata, Argentina
National Institute for Communicable Disease
Control and Prevention Farlen José Bebber Miranda
Chinese Centre for Disease Control and Laboratório de Imunopatologia
Prevention Instituto René Rachou
Beijing, People’s Republic of China Belo Horizonte, Minas Gerais, Brazil

Baochuan Lin Hirotake Mori

Center for Bio/Molecular Science & Department of Protozoology
Engineering Mahidol University
US Naval Research Laboratory Ratchathewi, Bangkok, Thailand
Washington, DC
Antonio Muro
Dongyou Liu Parasite and Molecular Immunology
Royal College of Pathologists of Australasia Laboratory
Quality Assurance Programs Universidad de Salamanca
St Leonards, New South Wales, Australia Salamanca, Castilla y León, Spain
Kavita Lole Venkatapathy Narendran
Hepatitis Group Aravind Eye Hospital and Postgraduate Institute
National Institute of Virology of Ophthalmology
Pashan, Pune, India Coimbatore, Tamil Nadu, India
Aongart Mahittikorn
Félix Núñez
Department of Protozoology
Food Hygiene and Safety
Mahidol University
Research Institute of Meat and Meat Products
Ratchathewi, Bangkok, Thailand
University of Extremadura
Cáceres, Spain
Anthony P. Malanoski
Center for Bio/Molecular Science &
S.C. Olsen
Engineering
Bacterial Diseases of Livestock Research
US Naval Research Laboratory
Unit
Washington, DC
National Animal Disease Center Agricultural
Jenny G. Maloney Research Service
Department of Biology United States Department of
Georgetown University Agriculture
Washington, DC Ames, Iowa

Palanisamy Manikandan Takashi Onodera

Aravind Eye Hospital and Postgraduate Research Center for Food Safety
Institute of Ophthalmology University of Tokyo
Coimbatore, Tamil Nadu, India Tokyo, Japan
and
Department of Medical Laboratory Stavria Panayidou
Sciences Department of Biological Sciences
Majmaah University University of Cyprus
Kingdom of Saudi Arabia Aglatzia, Cyprus
Contributors xix

Andrea Patriarca Jose Rojas-Caraballo

Departamento de Química Orgánica Centro de Investigación en Salud para el
Universidad de Buenos Aires Trópico
Buenos Aires, Argentina Universidad Cooperativa de Colombia
Santa Marta, Magdalena, Colombia
Pablo F. Pérez
Centro de Investigación y Desarrollo en Ivanna S. Rolny
Criotecnología de Alimentos Cátedra de Microbiología
Consejo Nacional de Investigaciones Científicas Universidad Nacional de La Plata
y Técnicas La Plata, Argentina
La Plata, Argentina
and Akikazu Sakudo
Cátedra de Microbiología Laboratory of Biometabolic Chemistry
Universidad Nacional de La Plata University of the Ryukyus
La Plata, Argentina Nishihara, Okinawa, Japan

Danielle L. Peters Paola Scavone

Department of Biological Sciences Department of Microbiology
University of Alberta Instituto de Investigaciones Biológicas Clemente
Edmonton, Alberta, Canada Estable
Montevideo, Uruguay
Nemani V. Prasadarao
Departments of Pediatrics and Surgery Stacey L. Schultz-Cherry
Children’s Hospital Los Angeles Infectious Diseases
and St. Jude Children’s Research Hospital
Molecular Microbiology and Memphis, Tennessee
Immunology
University of Southern California
Jesús Serrano-Luna
Los Angeles, California
Departamento de Biología Celular
Centro de Investigación y de Estudios
Chaturong Putaporntip
Avanzados del Instituto Politécnico
Department of Parasitology
Nacional
Chulalongkorn University
Mexico City, Mexico
Bangkok, Thailand
Mineko Shibayama
Janaina Ribeiro
Departamento de Infectómica y Patogénesis
Departamento Tecnologia de Produtos de
Molecular
Origem Animal
Centro de Investigación y de Estudios
Universidade Federal Fluminense
Avanzados del Instituto Politécnico
Niterói, Rio de Janeiro, Brazil
Nacional
Mexico City, Mexico
Alicia Rodríguez
Food Hygiene and Safety
Research Institute of Meat and Meat Products Coimbatore Subramanian Shobana
University of Extremadura Department of Microbiology
Cáceres, Spain PSG College of Arts & Science
Coimbatore, Tamil Nadu, India
Mar Rodríguez
Food Hygiene and Safety Steven M. Singer
Research Institute of Meat and Meat Products Department of Biology
University of Extremadura Georgetown University
Cáceres, Spain Washington, DC
xx Contributors

Ljiljana Sofronic-Milosavljevic János Varga

Department for Immunology and Department of Microbiology
Immunoparasitology University of Szeged
Institute for the Application of Nuclear Energy Szeged, Hungary
University of Belgrade
Belgrade, Serbia Mauricio Afonso Vericimo
Departamento de Imunobiologia
Martin Stahl Universidade Federal Fluminense
Division of Gastroenterology Niterói, Rio de Janeiro, Brazil
Child and Family Research Institute and
University of British Columbia Xin Wang
Vancouver, British Columbia, Canada National Institute for Communicable Disease
Control and Prevention
Yoshifumi Takeda Chinese Centre for Disease Control and
Hideyo Noguchi Memorial Foundation Prevention
Inawashiro, Fukushima, Japan Beijing, People’s Republic of China

Masae Tatematsu Ke Wen

Department of Diagnostic Pathology Department of Biomedical Sciences and
Fujita Health University Pathobiology
Toyoake, Aichi, Japan Virginia-Maryland College of Veterinary
Medicine
Gerlinde Teixeira Virginia Polytechnic Institute and State
Departamento de Imunobiologia University
Universidade Federal Fluminense Blacksburg, Virginia
Niterói, Rio de Janeiro, Brazil
Lijuan Yuan
Tetsuya Tsukamoto Department of Biomedical Sciences and
Department of Diagnostic Pathology Pathobiology
Fujita Health University Virginia-Maryland College of Veterinary
Toyoake, Aichi, Japan Medicine
Virginia Polytechnic Institute and State
Csaba Vágvölgyi University
Department of Microbiology Blacksburg, Virginia
University of Szeged
Szeged, Hungary Pablo Zunino
and Department of Microbiology
Botany and Microbiology Department Instituto de Investigaciones Biológicas Clemente
King Saud University Estable
Riyadh, Saudi Arabia Montevideo, Uruguay

Bruce A. Vallance
Division of Gastroenterology
Child and Family Research Institute and
University of British Columbia
Vancouver, British Columbia, Canada
1
Introductory Remarks

Dongyou Liu

CONTENTS
1.1 Preamble............................................................................................................................................ 1
1.2 Foodborne Pathogens and Diseases.................................................................................................. 1
1.2.1 Foodborne Pathogens........................................................................................................... 1
1.2.2 Foodborne Diseases.............................................................................................................. 3
1.3 Laboratory Models............................................................................................................................ 3
1.3.1 Rationales for Using Laboratory Models............................................................................. 3
1.3.2 Milestones in the Use of Laboratory Models....................................................................... 4
1.3.3 Characteristics of Laboratory Models.................................................................................. 4
1.4 Future Perspective............................................................................................................................. 9
References................................................................................................................................................... 9

1.1  P reamble
Foodborne disease (also known as foodborne illness or colloquially as foodborne poisoning) is largely attrib-
utable to microbial pathogens and their toxins contained in food and food products that are inappropriately
prepared or stored before consumption. Once inside the host, these pathogens establish in their predilection
sites and cause damages to the host either through direct physical/mechanical destruction or through secre-
tion of toxins and antigens that provoke host innate and acquired immune responses, leading to a range of
clinical symptoms (e.g., diarrhea, abdominal cramps, nausea, fever, joint/back aches, and fatigue).
Although foodborne disease is a current buzzword that appears in various popular media outlets with
alarming frequency, it has a long and tortuous history. Our awareness of as well as our struggle against
foodborne disease goes hand in hand with our attempts to survive and prosper in a constantly chang-
ing, and challenging, world, with significant milestones marked by the use of fire, the development of
crop cultivation, the luxury of food storage, the evolution of culinary art, the sophistication of sewage
system, the observation of disease-causing microbes, the application of refrigeration, and the discovery
of antibiotics [1,2].
From scavengers who searched for the scraps left by other predators for survival, humans have
made enormous technological advances that overcome the barrier of seasonality for food supply, that
reduce the proliferation of foodborne disease, and that enable rapid identification and tracking of food-
borne pathogens implicated in any food-related disease outbreaks. Nonetheless, it is still a long way
before we can call foodborne pathogens the genie in the bottle, foodborne disease a memory of the past,
and foodborne outbreak an absolute nonevent.

1.2  Foodborne Pathogens and Diseases

1.2.1  Foodborne Pathogens
In a narrow sense, foodborne pathogens refer to microbes that contaminate the foods and related products
(e.g., pasteurized carrot juice, peanut butter, broccoli powder on a children’s snack food, frozen pot pies,

1
2 Laboratory Models for Foodborne Infections

canned chili sauce, hot peppers, white and black pepper, raw cookie dough, hazelnuts, fenugreek sprouts,
papayas, pine nuts, raw frozen scraped ground tuna, etc.), the consumption of which by humans leads to
infections and diseases. In a broader sense, foodborne pathogens include microbes that occur in animals
(in farm/zoo animals and pets), the environment (soil, water, and air), and foods, the ingestion, inhalation,
and contact of which by humans result in discomfort and illness. Based on the latter premise, this book
covers not only microbial pathogens that come along with foods and food products (foodborne), but also
those that may occasionally enter into human host via water (water-borne), air (airborne), or direct contact
(skin wound), as well as those that cause diseases not through infection, but through production of toxins
and antigens that disturb/upset/confuse the gut, neurological, and immune systems of the human host.
As steadfast survivors, microbes (e.g., viruses, bacteria, fungi, and parasites) utilize animals (includ-
ing humans), foods, beverages, and water as growth or maintenance media. Some microbes remain in
humans (e.g., Salmonella Typhi and norovirus) or animal reservoirs and contaminate the food supply
via excreta, meat, milk, or eggs. Others persist in the environment and contaminate the ecosystems that
are fundamental to food production. Some microbes demonstrate the unusual ability to endure extreme
temperature, pH, and osmolarity, to sustain for long periods on dry surfaces, food processing plants,
and to exploit any temporary weakness in human innate and acquired immune defense networks (as
seen in pregnant women, infants, the elderly, and individuals under immune suppressing therapies).
Although a large number of foodborne microbes are known to infect humans and cause diseases
of varying severity, those having the most significant impact on human health in terms of prevalence,
morbidity, and mortality include Escherichia coli O157:H7, Campylobacter jejuni, Salmonella enteriti-
dis (e.g., serotypes Typhi and Typhimurium), Staphylococcus aureus, Listeria monocytogenes, Bacillus
cereus, Clostridium botulinum, Clostridium perfringens, Streptococcus pyogenes, Vibrio cholerae,
Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, hepatitis A virus, hepatitis E virus, norovirus,
rotavirus, Cryptosporidium, Cyclospora cayetanensis, and Toxoplasma gondii [3]. It is notable that a
majority of these high-impact foodborne pathogens are bacteria, and the remainder are viruses and
parasites. Interestingly, some of these pathogens have emerged only in the past 30 years presumably due
to the increased consumption of processed food products, globalization of food trade, and population
ageing. For instance, E. coli O157:H7 is a shiga-toxin-producing bacterial strain that was first recognized
as a human pathogen in foodborne outbreaks associated with ground beef in 1982, producing symptoms
ranging from simple diarrhea and hemorrhagic colitis to hemolytic-uremic syndrome (which is charac-
terized by hemolytic anemia, thrombocytopenia, and renal injury) [4,5]. Subsequently, lettuce grown
in close proximity to a dairy farm from which wastewater contaminated with animal feces was used to
irrigate the plant was linked to a 2006 outbreak of E. coli O157:H7 infection in Iowa and Minnesota (see
Chapter 21 in this book). Another recently emerged foodborne pathogen of note is Aeromonas (mainly
A. hydrophila, A. caviae, and A. veronii), which is responsible for both intestinal and extraintestinal
diseases in humans (see Chapter 15 in this book). There is no doubt that new foodborne pathogens will
likely emerge or reemerge in the future.
Apart from infections with foodborne viruses, bacteria, fungi, and parasites, another important cause
of foodborne diseases is toxins or toxic chemicals produced by foodborne bacteria and fungi as well as
those associated with shellfish and plants [6]. Toxins originated from foodborne bacteria can be sepa-
rated into exotoxins (which remain part of the bacteria, and are secreted, or, similar to endotoxins,
released during bacterial lysis) and endotoxins (which form part of the bacterial outer membrane, and are
released during bacterial lysis). Some well-known foodborne bacterial exotoxins include superantigens
from S. aureus and Streptococcus  pyogenes; pore-forming toxins (PFTs) from E. coli, L. monocyto-
genes, and Streptococcus pneumoniae; heat-stable enterotoxins (ST, exotoxins targeting the intestine)
from pathogenic strains of E.  coli; and botulinum neurotoxin (BoNT) from C. botulinum. A notable
foodborne bacterial endotoxin is lipopolysaccharide (LPS, which is made up of O antigen, core oligosac-
charide, and lipid A) from Gram-negative bacteria. As water-soluble proteins, PFTs induce host mem-
brane damages as amphiphilic surfactants and phospholipases. On the other hand, endotoxins (e.g., LPS)
cause severe inflammation, endotoxemia (septic shock), and autoimmune disease. Being the by-products
of foodborne fungi, mycotoxins are responsible for alimentary mycotoxicoses in humans through food
consumption. The most common foodborne mycotoxins consist of aflatoxins (from Aspergillus para-
siticus and Aspergillus flavus), altertoxins (from Alternaria), fumonisins (from Fusarium moniliforme),
Introductory Remarks 3

ochratoxins (from Aspergillus ochraceus, Aspergillus carbonarius, Penicillium verrucosum), patulin

(from Aspergillus, Penicillium), and trichothecenes (from Fusarium).

1.2.2  Foodborne Diseases

Foodborne diseases usually arise from consumption of improperly handled, prepared, or stored foods
that are contaminated with foodborne pathogens and/or toxins. With incubation period of several hours
to 1 week, the initial symptoms of foodborne disease consist of diarrhea, vomiting, abdominal cramps,
nausea, fever, joint/back aches, and fatigue, which may last for a week or so. However, some foodborne
pathogens (e.g., Streptococcus pyogenes) may cause a spectrum of clinical diseases, including (1) local-
ized inflammatory lesions; (2) both local and systemic diseases; and (3) immune dysfunction.
In localized inflammatory lesions, inflammation linked to foodborne pathogens is responsible for
lesions in various locations, accompanied by other symptoms. As in the case of Streptococcus pyogenes
(group A Streptococcus or GAS) infection of the pharynx (i.e., pharyngitis, or strep sore throat), inflam-
mation in the pharynx and tonsils leads to sore throat, along with sudden-onset fever, headache, nausea,
abdominal pain, vomiting, and patchy exudates. Similarly, GAS infection of the skin (i.e., impetigo)
results in the formation of pustules that enlarge and rupture to become thick, honey-colored scabs
(see Chapter 14 in this book) [7].
In local and systemic diseases, toxins (e.g., streptococcal pyogenic exotoxins) produced by foodborne
pathogens (e.g., GAS) induce a local disease with a deep red, finely papular, erythematous rash (straw-
berry tongue) and exudates in the pharynx (scarlet fever), or cause soft tissue infection at a surgical site
(surgical scarlet fever). Additionally, following minor nonpenetrating trauma, suction lipectomy, hyster-
ectomy, vaginal delivery, bunionectomy, and bone pinning, GAS invades and produces streptococcal
toxins that contribute to streptococcal toxic shock syndrome (STSS) (see Chapter 14 in this book) [7].
Regarding immune dysfunction, some foodborne pathogens produce antigens that confuse host
immune systems, leading to autoimmune diseases. For example, as a sequela of untreated GAS pharyn-
geal infection, acute rheumatic fever (ARF) results from the activity of antigens produced by GAS that
cause inflammation in the joints (arthritis) and the heart (carditis, also known as rheumatic heart disease
or RHD). Another sequela of GAS infection is acute poststreptococcal glomerulonephritis (APSGN),
which is a disorder of the kidneys mediated by the immune complex, with symptoms ranging from
edema, hypertension, and urinary sediment abnormalities to reduced serum complement components
(see Chapter 14 in this book) [7].
While all people are at risk for foodborne illness and most recover without any lasting effects, some may
show serious long-term consequences such as kidney failure, chronic arthritis, brain and nerve damage,
and even death, especially infants and toddlers, the elderly, pregnant women, transplant recipients, and
individuals with chronic illnesses (e.g., cancer, diabetes, or HIV/AIDS) and compromised immune systems.

1.3  Laboratory Models

1.3.1  R ationales for Using Laboratory Models
In spite of our unrelenting past efforts, including the implementation of procedures to reduce pre- and
postharvest contamination, the introduction of best-practice in food products processing, package and stor-
age, the education of the general public about the danger of and effective prevention measures against
foodborne diseases, the application of antibiotic and antimicrobial therapies, and the redirection of public
resources into research on the mechanisms of foodborne infections and diseases, the war against foodborne
pathogens and diseases is far from being over [3]. Among many other things, we are still uncertain how
foodborne pathogens sabotage host immune defense and manipulate host cell machinery for their own gain.
Since the best way to observe a battle is to get close to the battlefield, use of laboratory models (i.e., in
vivo animal models and in vitro culture models) provides a unique opportunity to determine the infectiv-
ity, host specificity, and life cycle of foodborne pathogens; to compare the virulence potential of various
microbial strains and serotypes; to generate large quantities of pathogenic microbes for detailed analysis;
4 Laboratory Models for Foodborne Infections

to examine host immune responses to foodborne pathogens; to uncover the pathological and histological
details resulting from foodborne infections; to validate the accuracy of clinical diagnostic techniques;
and to evaluate the efficacy of newly developed antimicrobial and vaccine preparations against food-
borne pathogens without harming human hosts. This is made possible by the common ancestry of all
living organisms, the similarity of anatomical structures and functions (e.g., breathing, digestion, move-
ment, sight, hearing, reproduction, immunity, etc.), the homology of genetic materials, the sharing of
hundreds of illnesses, and the conservation of cell biological and developmental pathways among verte-
brates as well as between vertebrates and invertebrates [8].

1.3.2  M ilestones in the Use of Laboratory Models

Animals have long been employed as laboratory models for investigation of the anatomy, physiology, epi-
demiology, and disease mechanisms of vertebrates. The available records suggest that in the 6th century
BCE, Alcmaeon of Croton examined dogs to demonstrate the brain as the seat of intelligence and sen-
sory integration; Aristotle (384–322 BCE) studied embryogenesis and ontogeny in chickens; after analy-
sis of the cardiovascular system in live animals, Erasistratus (304–258 BCE) postulated that the heart
functions as a pump; in the 2nd century, Galen of Pergamum employed live animals for extensive studies
of cardiovascular and neuroanatomy; in the 12th century, Avenzoar polished his tracheotomy skill on
animals before applying to humans; in the mid-16th century, Servetus and Lusitano identified pulmonary
and systemic circulation as two connected but distinct blood circuits in the body through animal experi-
ments; in the 17th century, through comparison of the anatomic and functional properties of the heart
and vasculature in eels, chicks, and pigeons, William Harvey provided accurate and detailed descrip-
tions of the function of the cardiovascular and other systems; in the 18th century, Antoine Lavoisier used
guinea pigs in a calorimeter to prove respiration as a form of combustion; in the 19th century, Louis
Pasteur demonstrated the germ theory of disease using a sheep model of anthrax; in the late 19th century,
Emil von Behring observed the effect of diphtheria toxin in guinea pigs that led to the development of an
antitoxin against diphtheria in animals and humans.
Another significant milestone in the use of laboratory animals for microbial research was achieved
in 1902, after William Castle and Abbie Lathrop generated the DBA (“dilute, brown, and non-agouti”)
inbred mouse strain and other inbred mice for genetic studies. Between 1910 and 1927, working with
the fruit fly Drosophila melanogaster, Thomas Hunt Morgan pinpointed chromosomes as the vector of
inheritance for genes. In the 1920s, Frederick Banting utilized the isolates of pancreatic secretion to
treat dogs with diabetes; in the 1930s, Little and MacDowell produced the first fully inbred mouse (20
brother × sister matings); in the 1940s, John Cade discovered the anticonvulsant properties of lithium
using guinea pigs, which helped replace lobotomy or electroconvulsive therapy for the treatment of
bipolar disorder (manic depression); also in the 1940s, Jonas Salk isolated the most virulent forms of the
polio virus from the rhesus monkey and created a highly effective polio vaccine; in the 1960s, Albert
Sabin passed the polio virus through animal hosts (including monkeys) to improve the effectiveness
of the Sabin vaccine for mass application; in 1976, Rudolf Jaenisch and colleagues developed the first
transgenic mouse; in 1987, Capecchi, Evans, and Smithies developed the first knockout mouse; in 1997,
Wilmut and Campbell obtained the first cloned animal (Dolly the sheep) from an adult somatic cell; in
2009, Aron Geurts and colleagues developed the first knockout rat [9].

1.3.3  Characteristics of Laboratory Models

Laboratory models used for the study of foodborne infections are of two main types: in vivo animal models,
and in vitro culture models. The in vivo animal models involve vertebrates [nonhuman primates (e.g., rhesus
monkey, cynomolgus monkey, chimpanzee, baboon), rodents (e.g., mice, rats, gerbils, hamsters, chinchil-
las, guinea pigs), rabbits, cats, dogs, pigs, sheep, cattle, chicken, zebrafish (Danio rerio), etc.] and inverte-
brates [fruit fly (D. melanogaster), silkworm (Bombayx mori), waxworm (Galleria mellonella), roundworm
(Caenorhabditis elegans), protozoa (Tetrahymena thermophila or Tetrahymena pyriformis), etc.]; the listing
order reflects the evolutionary relationship between these animals and humans, with nonhuman primates being
most close and roundworm being least close to humans (Table 1.1) [10–14]. The in vitro culture models rely
Introductory Remarks 5

on the use various established and non-established cell lines (derived from epithelia, endothelia, macro-
phage, etc.), embryonated eggs, and organs and tissues from hosts (Table 1.1).
Among various in vivo animal models, nonhuman primates (NHPs, with genomes of 2.85–3.09 Gb dis-
persed in 21–24 chromosome pairs) are the closest relatives to humans (with a genome of 3.23 Gb dispersed
in 23 chromosome pairs), and represent ideal models for investigation of foodborne infections and other
human diseases, on the basis of biological, physiological, immunological, and genetic similarities. However,
because of limited availability, prohibitive cost, and ethical concerns, NHPs are rarely used nowadays [15].

TABLE 1.1
Characteristics of Laboratory Models for Foodborne Infections
Common Species/
Modela Cell Type Characteristics Exemplary Application
In Vivo
Nonhuman primates Chimpanzee (Pan Chimpanzee has a genome of 3.02 Gb, Helicobacter pylori,
(family Hominidae, troglodytes), rhesus rhesus monkey 3.09 Gb, cynomolgus L. monocytogenes,
order Primates) monkey (Macaca monkey (crab-eating macaque, Mycobacterium,
mulatta), cynomolgus long-tailed macaque, or Java macaque) hepatitis E virus
monkey (Macaca 2.85 Gb, olive baboon 2.94 Gb. Ideal
fascicularis), olive models for foodborne infections and
baboon (Papio anubis) other human diseases, but limited by
availability, cost, and ethical concerns
Mice (family Muridae, House mouse (Mus Mice (house mice) possess a genome L. monocytogenes,
order Rodentia) musculus) strains: of 2.67 Gb, are small, readily S. aureus, Salmonella
BALB/c/(inbred, available, easy to handle, amenable
albino), C57BL/6/ to genetic manipulation, and
(inbred, dark brown), reproduce quickly, representing an
athymic nude mice efficient, cost-effective, and widely
(outbred) applicable animal model for
experimentation on foodborne
infections and other human diseases
Rats (family Muridae, Norway rat (Rattus Norway rat (brown rat) has a genome Salmonella, S. aureus,
order Rodentia) norvegicus) (inbred), of 2.61 Gb. Developed in 1906, Yersinia,
Wistar rat (outbred, Wistar rat (outbred albino) is the Acanthamoeba
albino), Lewis rat ancestor of most laboratory rats used
(inbred) today, including the Lewis rat. Wistar
rat shows albino coloring, a docile
behavior, and low fertility, and
tolerates crowding
Gerbils (family Mongolian gerbil Mongolian gerbil (Mongolian jird) is H. pylori,
Muridae, order (Meriones easy to keep as it adapts to a new L. monocytogenes,
Rodentia) unguiculatus) (outbred) setting well Giardia
Hamsters (family Syrian hamster Syrian hamster (golden hamster) Mycobacterium,
Cricetidae, order (Mesocricetus auratus) possesses a genome of 2.50 Gb, Acanthamoeba
Rodentia) (outbred), Chinese Chinese hamster 2.36 Gb. Hamsters
hamster (Cricetulus have a short life cycle and breed well
griseus) in captivity; being relatively free from
natural diseases, hamsters are
susceptible to experimental pathogens
Chinchillas (family Long-tailed chinchilla Chinchilla has a genome of 2.39 Gb. L. monocytogenes,
Chinchillidae, order (Chinchilla lanigera) Being crepuscular rodents, chinchilla Yesinia
Rodentia) is a robust host for experimental study
Guinea pigs (family Hartley Guinea pig Guinea pig has a genome of 2.72 Gb, and L. monocytogenes,
Caviidae, order (Cavia porcellus) shows similarity to humans in disease S. aureus
Rodentia) (outbred, albino) symptoms, immune response, and
pathogenesis
(Continued)
6 Laboratory Models for Foodborne Infections

TABLE 1.1 (Continued)

Characteristics of Laboratory Models for Foodborne Infections
Common Species/
Modela Cell Type Characteristics Exemplary Application
Rabbits (family New Zealand white New Zealand white rabbit possesses a L. monocytogenes,
Leporidae, order rabbit (Oryctolagus genome of 2.73 Gb, and represents a Salmonella
Lagomorpha) cuniculus) (outbred) nonaggressive host for experimental work
Cats (family Felidae, Domestic cat (Felis Domestic cat has a genome of 2.9 Gb, S. aureus
order Carnivora) catus) and is useful for modeling some
foodborne infections
Dogs (family Canidae, Domestic dog (Canis Domestic dog possesses a genome of H. pylori
order Carnivora) familiaris) 2.25 Gb, and may be used
experimentally for a number of
foodborne infections
Pigs (family Suidae, Domestic pig (Sus scrofa Domestic pig has a genome of 2.5 Gb. L. monocytogenes,
order Artiodactyla) domesticus) Being truly omnivorous, pigs (piglets) S. aureus, Taenia
show strikingly similar nutritional solium
requirements to those of humans. Pigs
practice coprophagy, and represent a
useful model for a number of foodborne
infections
Sheep (family Bovidae, Sheep (Ovis aries) Sheep harbors a genome of 2.61 Gb, and L. monocytogenes,
order Artiodactyla) is useful for modeling some foodborne bovine spongiform
infections encephalopathy
(BSE)
Cattle (family Bovidae, Cattle (Bos taurus) Cattle possess a genome of 2.69 Gb, and E. coli, Taenia
order Artiodactyla) may be used for a number of foodborne saginata
infections
Chicken (family Domestic chicken Domestic chickens have a genome of E. coli, Aspergillus
Phasianidae, order (Gallus gallus 1.23 Gb, are noted for their rapid growth fumigatus
Galliformes) domesticus) rate, distinct anatomy, relatively small
size, and low cost
Zebrafish (family Zebrafish (D. rerio) Zebrafish possess a genome of 1.4 Gb. Mycobacterium
Cyprinidae, order Due to small size, zebrafish are easy to
Cypriniformes) house and care for, easy to introduce
genetic changes, and easy to observe the
impact of any genetic mutation (with
transparent early life stages)
Fruit fly (family Common fruit fly Common fruit fly has a genome of L. monocytogenes,
Drosophilidae, order (D. melanogaster) 139 Mb, and shares 75% of known S. aureus
Diptera) human disease genes. Due to its small
size, simple anatomy, high fecundity,
and short life cycle (about 30 days at
29°C), the fruit fly is easy and
inexpensive to maintain. However, the
fruit fly does not have an adaptive
immune system and is not an
appropriate model for the study of
antibody- and lymphocyte-dependent
adaptive immune defenses
Silkworm (family Domestic silkworm Domestic silkworm possesses a genome Pseudomonas
Bombycidae, order (B. mori) of 397 Mb, and represents a low-cost aeruginosa, S. aureus
Lepidoptera) model for some foodborne infection. It
has a body size large enough for easy
handling (e.g., injecting sample solution
into the hemolymph)
(Continued)
Introductory Remarks 7

TABLE 1.1 (Continued)

Characteristics of Laboratory Models for Foodborne Infections
Common Species/
Modela Cell Type Characteristics Exemplary Application
Waxworm (family Greater wax moth or Despite lacking an adaptive immune Streptococcus pyogenes
Pyralidae, order honeycomb moth response, greater wax moth (waxworm)
Lepidoptera) (G. mellonella) shows an innate immune response
functionally similar to that of mammals,
and provides a rapid, inexpensive, and
reliable model for certain foodborne
infections
Roundworm (family Soil nematode Soil nematode possesses a genome of Enterococcus faecalis,
Rhabditidae, order (C. elegans) 100 Mb, and lacks an adaptive immune E. coli, S. aureus
Rhabditida) system. It has a short life cycle, simple
anatomy, is easy to handle, and has low
cost maintenance. C. elegans intestine is
composed of cells that share striking
similarities to human intestinal epithelial
cells
Protozoa (family Ciliated protozoan Ciliated protozoan T. thermophila has a Aeromonas, E. coli,
Tetrahymenidae, order (T. thermophila or genome of 104 Mb. Being able to switch Listeria, Vibrio,
Hymenostomatida) T. pyriformis) from commensalistic to pathogenic Yesinia
modes of survival, Tetrahymena offers a
low-cost and easy to handle alternative
for modeling foodborne infections
In Vitro
Epithelial cell lines Human colorectal cells Easy and low-cost maintenance, high E. coli O157:H7,
Caco-2 and HT29, sensitivity, and broad spectrum L. monocytogenes
human colonic cell
T84, human cervical
cell HeLa, African
green monkey kidney
cell Vero, Madin-Darby
canine kidney cell
(MDCK)
Endothelial cell lines Human umbilical vein Easy and low-cost maintenance, high E. coli O157:H7,
endothelial cell sensitivity, and broad spectrum L. monocytogenes
(HUVEC), human
glomerular microvascular
endothelial cell
(GMVEC)
Macrophage cell lines Mouse macrophage cell Easy and low-cost maintenance, high E. coli O157:H7,
J774, human sensitivity, and broad spectrum L. monocytogenes
macrophage cell U937
Embryonated eggs Chicken eggs Cost-effective and easy maintenance, C. perfringens,
ready availability, sterile, and wide Aspergillus fumigatus
ranging fluids and tissues
In vivo grown organ Various organs or tissues Close to native live tissue; IVOC usage E. coli O157:H7,
cultures (IVOC) is limited to several hours (when the Salmonella, norovirus
tissue dies). Further, it is technically
challenging and shows sample
variability
Ussing chamber Epithelial tissues Ussing chamber detects and quantifies E. coli O157:H7,
transport and barrier functions of Salmonella
living tissue
a Animal models are listed (in descending order) according to their evolutionary closeness to humans, with nonhuman
primates being the closest and roundworm being the most distant.
8 Laboratory Models for Foodborne Infections

On the other hand, as the next in the order of closeness to humans, rodents (especially mice) are
increasingly employed as preferred animal models for foodborne infections and other human diseases.
Mice possess a genome of 2.67 Gb dispersed in 20 chromosome pairs with ∼25,000 genes, 99% of
which have human counterparts. Having a relatively small body size, 18-day gestation, 10–15 pups per
litter, 7 weeks to sexual maturity, and a 2–3-year lifespan (1 mouse year equals about 30 human years),
mice provide an efficient and cost-effective model for human disease research including foodborne infec-
tions. It should be noted that mice practice coprophagy, an aspect that may be considered in experimental
design for certain disease types.
Mice are highly amenable to manipulation, and can be inbred to yield genetically identical strains,
which allows for more accurate and repeatable experiments. Through practice of cesarian birth, flexible-
film isolator cages, and irradiated food, mice (and other animal species) can be maintained in completely
germ-free conditions or colonized with one or more defined bacterial species (gnotobiotics). In addition,
use of genetic selection and manipulation technologies enables insertion of extra genetic materials into
genome, creating a variety of transgenic mice (including knockout, knock-in, and humanized mice as
well as mice with conditional gene modifications or chromosomal rearrangement) [8].
For example, athymic nude mice are selected for the nude spontaneous mutation (Foxn1nu, formerly
Hfh11nu) (which results in abnormal hair growth) and in the defective development of the thymic epithe-
lium (which abrogates a cell-mediated immunity, despite the presence of T-cell precursors). Homozygous
nude mice show partial defect in B cell development probably due to the absence of functional T cells,
and their responses to thymus-dependent antigens are primarily limited to IgM due to a defect in helper
T-cell activity.
Knockout mice are created by inserting a specific mutation into the endogenous gene. This leads to
inactivation/silencing of the gene of interest, suppressing its expression and function. Knock-in mice are
created by inserting a transgene into an exact location for overexpression. Both knockout and knock-in
animals rely on the use of embryonic stem (ES) cells containing null or point mutations and complex
chromosomal rearrangements (e.g., large deletions, translocations, or inversions), which are injected into
the host mouse embryo, and subsequently implanted into a foster mother.
Humanized mice are created by inserting human genes (more recently entire human systems) into
mice for subsequent expression. For instance, mice with human “immune systems” were generated by
implanting either fetal lymphoid tissue or peripheral blood leukocytes into mice with spontaneous severe
combined immunodeficiency. Humanized mice are capable of accepting a variety of human cells (blood,
immune, cancer, etc.) without rejection.
Mice with conditional gene modifications are created with two different types of genetic alterations:
one contains a conditional vector [through inserting recognition sequences for the bacterial Cre recom-
binase (loxP sites) using homologous recombination in ES cells], which functions as an “on switch”
for the mutation, and the other contains specific sites (called loxP) inserted on either side of a whole
gene, or part of a gene, that encodes a certain component of a protein that will be deleted. Similarly,
mice with chromosomal rearrangement are created using the Cre/loxP recombination system to induce
site-specific mutations that display defects resembling those caused by human chromosomal rearrange-
ments (e.g., chromosome deletions, duplications, inversions, translocations, and nested chromosome
deletions) [8].
Depending on the levels of simulation to human disease, animal models may be separated into three
types: homologous, isomorphic, and predictive. Homologous animals demonstrate identical causes,
symptoms, and treatments relative to human diseases; isomorphic animals have identical symptoms
and treatments; predictive models share only a couple of aspects of human disease with humans, but
nevertheless provide useful predictions about mechanisms of particular disease features. Similarly,
depending on the way in which animal disease is induced, animal models may be divided into four
categories: experimental, spontaneous, negative, and orphan. Experimental disease models resemble
human disease conditions in phenotype or response to treatment but are induced artificially in the labo-
ratory. Spontaneous disease models are analogous to human disease conditions that occur naturally.
Negative disease models are essentially control animals, and are used to validate an experimental result.
Orphan disease models have no human analog and occur exclusively in the species studied. Furthermore,
to examine a particular disease, various approaches may be used. For example, inflammation may be
Introductory Remarks 9

studied via Carrageenan footpad edema (CFE) model, collagen-induced arthritis (CIA) model, pristane-
induced arthritis (PIA) model, adjuvant-induced arthritis (AIA) model, ovalbumin-induced arthritis (OIA)
model, air pouch model, and delayed-type hypersensitivity (DTH) model [8].
The in vitro culture models provide an alternative to the in vivo animals for mechanistic studies, by
preserving the physiology of the living cell, without the need to sacrifice an animal. The advantages
of the in vitro culture models include low cost, easy maintenance, relatively high efficiency, and little
ethical concern. For instance, Caco-2 cells (of human colonic origin) can differentiate in culture, form
brush border membranes, demonstrate transport properties (similar to intestinal epithelia), and express
abundant intestinal microvilli, enzymes, and differentiation markers (typical of human small intestinal
enterocytes), offering a valuable model for investigation of vectorial epithelial passage by para- and
transcellular routes. Apart from the established cell lines (of epithelial, endothelial, and macrophage
origins), other cells, organs, and tissues may be obtained from animal and human hosts for in vitro mod-
eling. These include enterocyte suspensions, brush border membranes and vesicles, perfused duodenal
segment, everted gut sacs, lymphocytes, etc.
When selecting an animal model for research, considerations should include: (1) appropriateness as
an analog, (2) transferability of information, (3) genetic uniformity of organisms, (4) background knowl-
edge of biological properties, (5) cost and availability, (6) generalizability of the results, (7) ease of and
adaptability to experimental manipulation, (8) ecological consequences, and (9) ethical implications. If
possible, three basic principles should be applied: replacement, reduction, and refinement. Replacement
aims to use alternatives [e.g., computer models, tissues and cells, “lower-order” animals (cold-blooded
animals, invertebrates, bacteria) instead of “higher-order” animals (primates and mammals) for experi-
mentation]. Reduction employs mathematical calculations of statistical power to minimize the number of
animals used. For example, by using an alternative way to LD50 for result interpretation, the number of
experimental mouse groups for assessing L. monocytogenes virulence may be reduced from four to two,
with further advantage of obviating the necessity to perform colony forming unit (CFU) estimation [16].
Refinement aims to minimize the suffering of each animal subject through experimental design that is
as painless and efficient as possible [8].

1.4  F uture Perspective

Despite our nonstopping efforts in the past, foodborne disease continues to savage human society at
random and cause particular misery to vulnerable population groups. Naturally, we can point our fingers
to the fact that foodborne pathogens have uncanny ability to constantly evolve and develop phenotypic
and genetic traits that enable their evasion of host innate and acquired immune defense mechanisms,
and their sabotage of our every intervention attempt. However, this does not hide the reality that some
obvious gaps exist in our knowledge about the molecular basis of pathogenicity of foodborne organisms.
Use of laboratory models including animal and cell culture models has contributed greatly to our past
understanding of foodborne pathogens and diseases, and more will have to be learned via this approach
in combination with other emerging technologies. The documentation and summation of the existing
findings in this area provide a platform from which new insights will be uncovered and innovative miti-
gation measures will be launched.

REFERENCES
1.
Bell A. Foodborne illness: the history of an invisible enemy. Available at https://antiquitynow.
org/2014/06/05/foodborne-illness-the-history-of-an-invisible-enemy/.
2.
Satin M. History of foodborne disease—Part 1—ancient history. Encyclopedia of Food Safety
(Internet). 2014. Available at http://tinyurl.com/kmzt56u.
3.
Behravesh CB, Williams IT, Tauxe RV. Emerging foodborne pathogens and problems: expanding pre-
vention efforts before slaughter and harvest. In: Institute of Medicine (US). Improving Food Safety
through a One Health Approach: Workshop Summary. Washington, DC: National Academies Press
(US); 2012. A14. Available at http://www.ncbi.nlm.nih.gov/books/NBK114501/.
10 Laboratory Models for Foodborne Infections

4. Riley LW, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. New Engl J Med.
1983;308(12):681, 684–685.
5. Rangel JM, et al. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002.
Emerg Infect Dis. 2005;11(4):603.
6. Henkel JS, Baldwin MR, Barbieri JT. Toxins from bacteria. Experienta Suppl. 2010;100:1–29.
7. Walker MJ, et al. Disease manifestations and pathogenic mechanisms of group A Streptococcus. Clin
Microbiol Rev. 2014;27(2):264–301.
8. McGonigle P, Ruggeri B. Animal models of human disease: challenges in enabling translation. Biochem
Pharmacol. 2014;87(1):162–171.
9. Ericsson AC, Crim MJ, Franklin CL. A brief history of animal modeling. Mo Med. 2013;110(3):201–205.
10. Wolfgang MJ, Golos TG. Nonhuman primate transgenesis: progress and prospects. Trends Biotechnol.
2002;20:479–484.
11. Padilla-Carlin DJ, McMurray DN, Hickey AJ. The guinea pig as a model of infectious diseases. Comp
Med. 2008;58(4):324–340.
12. Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. The pig: a model for human infectious
diseases. Trends Microbiol. 2012;20(1):50–57.
13. Bou Ghanem EN, Myers-Morales T, D’Orazio SE. A mouse model of foodborne Listeria monocyto-
genes infection. Curr Protoc Microbiol. 2013;31:9B.3.1–9B.3.16.
14. Disson O, Lecuit M. In vitro and in vivo models to study human listeriosis: mind the gap. Microbes
Infect. 2013;15(14–15):971–980.
15. García Y, Díaz-Castro J. Advantages and disadvantages of the animal models v. in vitro studies in iron
metabolism: a review. Animal. 2013;7(10):1651–1658.
16. Liu D. Listeria monocytogenes: comparative interpretation of mouse virulence assay. FEMS Microbiol
Lett. 2004;233(1):159–164.
 Section I

Foodborne Infections due to Viruses

2
Adenoviruses

Anthony P. Malanoski and Baochuan Lin

CONTENTS
2.1 Introduction..................................................................................................................................... 13
2.2 Classification and Morphology........................................................................................................14
2.3 Genome Organization and Conserved Features............................................................................. 15
2.4 Protein Function...............................................................................................................................16
2.5 Life Cycle........................................................................................................................................ 20
2.5.1  Cell Entry and Replication.................................................................................................... 20
2.5.2  DNA Packaging and Virion Assembly.................................................................................. 21
2.6 Pathogenesis.................................................................................................................................... 21
2.7 Host–Pathogen Interaction.............................................................................................................. 22
2.8 Immunity......................................................................................................................................... 22
2.9 Conclusions and Future Perspectives.............................................................................................. 23
References................................................................................................................................................. 23

2.1  I ntroduction
Adenovirus (AdV) is an important human pathogen and is estimated to account for 8% of clini-
cally relevant viral diseases globally.1 First identified in 1953 as the cause for acute febrile respira-
tory disease, AdVs are endemic in the pediatric population worldwide, affecting children younger
than 5 years old with mild symptoms and generally self-limiting illnesses.2–4 Self-limiting infections
may also occur in adults, but some serotypes have been associated with severe respiratory illness
and potentially fatal outbreaks of pneumonia in residential facilities and military bases. The main
etiologic agents for these outbreaks are serotype 4 and occasionally serotypes 3, 7, 14, and 21. It is
possible that stress and crowding may contribute to AdV transmission and susceptibility.5 AdVs have
serious complications, impacting morbidity and mortality in immunocompromised individuals of
any age.6–8
With more AdV serotypes being identified, it becomes clear that AdVs cause an array of clinical
diseases, including epidemic keratoconjunctivitis (EKC), acute hemorrhagic cystitis, hepatitis, gastroen-
teritis, myocarditis, and pneumonia. Being one of the most prevalent enteropathogens causing infantile
gastroenteritis, enteric AdVs are implicated in sporadic cases as well as in outbreaks of food-borne
illness in kindergartens, schools, and hospitals.9 Gastroenteritis due to AdVs often occurs in children
younger than 5 years of age, accounting for ∼12% of all enteropathogenic viruses identified, and is
most commonly associated with serotypes 40 and 41; however, other types including 1, 2, 7, 9, 10, 18,
19, 22, 31, 42, 52, 58, 65, and 67 have also been reported as etiologic agents of viral gastroenteritis.9–20
Serotypes 40 and 41 account for 5%–20% of hospital-admitted diarrhea cases in children under 2 years
old. As children age, the incidence of AdV gastroenteritis decreases due to increasing levels of popula-
tion immunity to AdV infection.
AdVs can be easily propagated in cell culture, and there are several cell lines that can be used as
laboratory models for AdVs. The primary human embryonic kidney (HEK) cells are the best host for

13
14 Laboratory Models for Foodborne Infections

replicating various serotypes of AdVs. The lung epithelial cell line A549 and other epithelial cell lines,
such as HEP-2, HeLa, and KB, are also good hosts for AdVs. For enteric AdVs, such as AdV40 and
AdV41, the HEK 293 cell offers a convenient laboratory model.2 In addition, Sigmodon hispidus cotton
rats and mice, such as C57BL/6N, C57BL/lOScN, CBA/N, and C3H/N strains, were used as animal
models to investigate the molecular pathogenesis of pneumonia caused by AdV infection.21 AdVs have
been used as models of virus–cell interaction. Decades of studies have contributed to the extensive
understanding of the molecular biology, including life cycles, the host–pathogen interaction, genet-
ics, epidemiology, and pathogenesis of AdVs, which are discussed in this chapter. AdVs continue to be
studied as delivery vehicles for gene therapy, vaccination, and cancer treatment, which underscores the
importance of understanding these viruses.

2.2  C
 lassification and Morphology
AdVs constitute the Adenoviridae family, which is divided into the genera Mastadenovirus and
Aviadenovirus. The genus Mastadenovirus covers viruses of several different animals, including
bat, bovine, canine, equine, human, murine, ovine, porcine, simian, and so on, whereas the genus
Aviadenovirus is limited to viruses of birds.2 Currently, there are 68 reported human AdVs according
to the National Center for Biotechnology Information Taxonomy Browser, representing seven different
species or subgroups (A–G). The classification of AdVs was originally based on their hemagglutina-
tion patterns and serologic profiles. Recent advancements in sequencing capability have allowed the
discovery and classification of new AdVs (types 52–68), where the differentiation of strains is based
on bioinformatics analysis of their genomic sequencing (Table 2.1).2,4,11–15,22–37 The majority of these
newly discovered AdVs are products of homologous recombination, a common evolutionary adaptation
of AdVs. Among the seven different species, species B can be further divided into B1 and B2 based on
their organ tropisms.22 There is a correlation between the species and their tissue tropisms, which deter-
mines the clinical manifestation of AdV infection. Species A, F, and G show tissue tropisms toward the
gastrointestinal tract and induce gastroenteritis. Species B1, C, and E mainly cause respiratory illness;
species B1, B2, D, and E produce ocular infection, whereas B2 AdVs cause kidney and urinary tract
infection.8,37
AdVs are nonenveloped double-stranded DNA (ds DNA) viruses with icosahedral shells and nucleo-
protein cores, ranging in size from 65 to 100 nm in diameter. The capsid of the viral particle is composed
of seven proteins: hexon, three hexon-associated proteins, penton, a penton-associated protein, and fiber.
The proteins form 252 capsomeres, which consist of 240 trimers of the major capsid protein hexon and
12 pentons. The fiber protein, which has a length that varies among the different serotypes, embeds in
the penton base and projects out from the capsid. These 12 extensions out of the particle serve crucial

TABLE 2.1
Classification of Human Adenoviruses
Species Hemagglutination Serotype References
A IV 12, 18, 31, 61 2,12
B1 I 3, 7, 16, 21, 50, 66, 68 22–25
B2 I 11, 14, 34, 35, 55 22,26,27
C III 1, 2, 5, 6, 57 2,28
D II 8–10, 13, 15, 17, 19, 20, 22–30, 32, 33, 36–39, 2,13–15,25,29–36,120
42–49, 51, 53, 54, 56, 58, 59, 60a, 62–65, 67
E III 4 2
F III 40, 41 2
G 52 11
I, complete agglutination of monkey erythrocytes; II, complete agglutination of rat erythrocytes; III, partial
agglutination of rat erythrocytes; IV, little or no agglutination.
Adenoviruses 15

(A) (B)

Core-V
Terminal protein
Hexon
Penton base
Fiber

FIGURE 2.1  Image and schematic depiction of the structure of adenovirus. (A) Transmission electron micrograph (TEM)
of adenovirus virions. Visible at this high magnification are the capsomeres, which are hexagonally shaped, also called
hexons. Hexons comprised the outer shell of the adenovirus known as a capsid. These adenoviruses displayed an icosahe-
dral symmetry, characterized by 12 vertices and 20 facets. Each virion was 70–80 nm and exhibited no spikes. Image by
Dr. G. William Gary, Jr. was downloaded from the public health image library (ID #237) of Centers for Disease Control
and Prevention, USA. (B) Schematic depiction of the icosahedral capsid of human adenovirus (HAdV) showing the major
structural proteins, hexon, penton based, and fiber in the virion. The ds DNA genome is shown as a dark line inside the
capsid. (Modified from Russell, W.C., J. Gen. Virol., 90, 1–20, 2009.)

functions in cell attachment and entry (Figure 2.1).2,6 All the reported AdVs encode a single fiber protein
except AdV40, AdV41, and AdV52, which encode two fiber proteins. Fiber proteins mediate viral entry
by interacting with receptors on susceptible cells; the expression of two fiber proteins might expand the
range of susceptible cells for these AdVs.2,11 The nucleoprotein core of AdVs contains the viral genome
and five structural proteins, V, VII, Mu (μ), IVa2, and a terminal protein (TP).2,6

2.3  G
 enome Organization and Conserved Features
The AdV genome consists of a linear ds DNA of ∼35 kb encoding more than 30 structural and nonstruc-
tural proteins in most serotypes. All AdV serotypes have the same general organization; that is, the genes
encoding specific functions are located at the same position on the viral chromosome. However, DNA
sequence homology among the strains within a human AdV species ranges from 48% to 99%, with the
highest homology among strains of species C. The homology between species is <20%.2,4 The 5′ and
3′ ends of the human AdV genome consist of inverted terminal repeats (ITRs) ranging from 40 to 212
nucleotides, which contain the origin of DNA replication and are hallmark motifs of the HAdV genome.
All HAdV genomes contain five early (E) transcribed genes (E1A, E1B, E2–E4), two intermediate genes
(IX, IVa2), and five late (L) genes (L1–L5) (Figure 2.2). The early genes are mainly involved in the
control of DNA replication, transcription, transportation of Ad mRNAs (messenger ribonucleic acids) to
the cytoplasm, and immunoregulation; the intermediate genes control DNA packing and also function as
transcription factors, whereas the late genes are coded for viral structural proteins.38
The E1A gene is the first to be transcribed and expressed after infection, and multiple E1A proteins
are generated through alternative splicing, which regulate the transcription of viral and cellular genes.
The E1B gene encodes proteins that block apoptosis and potentiate viral replication. The E2 region is
composed of two transcripts, E2A and E2B, and produces proteins required for viral replications, which
include DNA-binding protein (DBP), terminal protein precursor (pTP), and DNA polymerase (pol).29,39–41
Proteins encoded by E3 gene are not essential for viral growth, but function as modulators of the host
immune response.38,42 The E4 gene encodes proteins with various functions, including promoting viral
gene expression and replication, regulating cellular transcription factor E2F, and controlling protein
phosphorylation.43 The two intermediate genes produce proteins, IX and IVa2, which control DNA
16 Laboratory Models for Foodborne Infections

1 2K 4K 6K 8K 10 K 12 K 14 K 16 K 18 K 20 K 22 K 24 K 26 K 28 K 30 K 32 K 32, 214

ITR ITR
L3 L4A L5A
E2B E2A Fiber-2
E1A
ssDBP E4
IVa2 L1 E2A-L U
L2 L5
E1B VAI Fiber
E3B
IX
E3A

FIGURE 2.2  Genome structure of human AdV-type 40. The genome structure was constructed based on the sequence
deposited in GenBank (NC_001454). The half-filled rectangle indicates a gene, while the fully filled rectangle indicates
CDS. The arrows indicate the direction of the gene that is transcribed. VAI, virus-associated RNA I; ssDBP, single-
stranded DNA-binding protein.

packing and also function as transcription factors. The five late genes encode viral proteins involved in
capsid production for mature virions, assembly, and viral maturation, such as 52-kDa protein, IIIa, pen-
ton base (III), V, VII, X, minor capsid protein (VI), hexon, 23-kDa protease, 100-kDa protein, 33-kDa
protein, VIII, and fiber.2,39,40
There are 16 genus-common proteins; that is, there is homology in all genera in AdV genomes, which
are probably rooted in a common ancestral AdV. These proteins primarily function in DNA replication,
DNA encapsidation, and viral particle structural formation. The genus-common proteins involved in
DNA replication are DBP, pTP, and pol, and two proteins are involved in DNA encapsidation, 52-kDa
protein and IVa2. Viral proteins, such as IIIa, III, VII, X, VI, hexon, protease, 100-kDa protein, 33-kDa
protein, pVIII, and fiber, necessary for virion formation are also genus common.41 Additionally, U exon
is also considered as a genus-common feature, even though it has been lost in certain members of the
Mastadenovirus genus (PAdV-5 and MAdV-1). U exon locates between the E3 region and the fiber gene,
encodes the U exon protein (UXP, ∼24 kDa), which initiates in U exon, continues in frame in the DBP
leader, and extends throughout the DBP coding region but with a different reading frame from DBP that
has functions relevant to DNA replication or RNA transcription.41,44 For HAdVs, most genus-specific
genes are located within the E1 and E4 regions.

2.4  P rotein Function

There are seven structural proteins that form the capsid of the viral particle: hexon (polypeptide II),
hexon-associated proteins (pVI, pVIII, pIX), L1 peripentonal hexon-associated protein (pIIIa), fiber
(polypeptide IV), and penton (polypeptide III). Inside the capsid, the nucleoprotein core contains a viral
genome and five additional proteins (pV, pVII, μ, IVa2, and TP) as well as a viral protease.37
Hexon, the main building block of AdV capsid, forms homotrimers, which in general are referred to
as hexon capsomeres and arrange in a pseudohexagonal symmetry. The size of the hexon monomer var-
ies with the serotype ranging from 917 to 978 amino acids, with the largest one belonging to HAdV6.28
Each viral particle consists of 240 capsomeres, which form the facets of the icosahedral virus shell. The
N- and C-terminals of hexon can adopt different conformations and provide interactions between hexons
or between hexons and hexon-associated proteins pIIIa and pVIII. In the base of each hexon molecule, an
eight-stranded β-barrel arranges in a “jellyroll” topology, which provides the means for interacting with
neighboring capsomeres.37,45,46 Hexon capsomeres are further stabilized by minor coat proteins pIIIa,
pVI, pVIII, and pIX, also called hexon-associated proteins. In addition, these minor coat proteins also
serve as extensions of major capsid proteins (i.e., hexon, penton, and fiber), which mediate the protein–
protein interactions and stabilization of all capsomeres.2,47
Minor coat protein pIIIa is a highly helical protein and serves to stabilize the vertex region by tether-
ing peripentonal hexons and penton bases via its N-terminal domain. In addition, pIIIa also interacts
with pVIII, which tethers the peripenton hexons to the hexons in the central plate of the facet. Studies
also indicated that pIIIa promotes correct genome packaging during viral assembly by interacting with
the putative scaffold protein L1 52/55 kDa via its N-terminal domain as well as signaling for vertex and
genome release during uncoating.45,48 Similar to pIIIa, pVI also plays multiple roles during AdV infection
Adenoviruses 17

in addition to stabilizing hexon capsomeres and bridging the core to the icosahedral shell. In the early
phase of viral infection, the N-terminal amphipathic α-helix is responsible for inducing curvature of the
endosome membrane, which leads to membrane disruption, thereby allowing the virus to escape into
the cell’s cytosol. pVI also contains a ubiquitin ligase-interacting motif (PPxY), which not only facili-
tates microtubule-dependent trafficking toward the nucleus, but also activates E1A promoter and leads
to initiation of adenoviral gene expression. In later phases of AdV infection, the nuclear export signals
of pVI interact with importin α/β, which mediates the nuclear import of hexon and the subsequent pro-
teolytic removal of C-terminal nuclear export signal transition pVI to support infectious viral particle
assembly.45,49–51 Minor coat protein pVIII is not as well characterized, and its only known function is
stabilizing capsomeres. pIX, located on the outer part of the capsid, also has multiple functions in the life
cycle of AdV infection. Upon viral entry and escaping from endosome, pIX is responsible for interacting
with kinesin-1, which results in capsid dismantling and promotion of transport of the viral genome into
nucleus. Subsequently, the C-terminal leucine repeat is important for enhancing gene expression from
the E1A promoter in the early stage of infection, and the N-terminal region is required for incorporation
into viral capsid at the later stage of infection. pIX also plays a role in virus-induced nuclear reorganiza-
tion, that is, the formation of specific nuclear structures, which appear as dispersed nuclear globules on
immunofluorescence staining and as clear amorphous spherical inclusions on the electron microscopy
of cells that are in the later stages of infection. It is suggested that the reorganization of nuclear proteins
provides a favorable environment for viral replication. Evidence also suggests that pIX may play a role in
modulating the viral tropism and/or interfering with the immune response.45,52–54
In addition to hexon capsomeres, the penton base and fiber proteins are also the main building blocks
of AdV capsid and form penton capsomeres, which locate at 12 vertices of the icosahedral facets. The
penton capsomere consists of a homopentameric penton base and homotrimeric fibers, with the fiber
molecules projecting from the penton base at each vertex. Penton and fiber molecules are the key play-
ers in AdV attachment and cell entry.45,55 The C-terminal knob domain of fiber molecules initiates the
interaction with cell receptors. Once the cell binding is initiated, the integrins (coreceptor) αvβ3 and αvβ5
bind to conserved RGD domain at penton base, which in turn starts the receptor-mediated endocytosis
with the exception of species F enteric AdVs, which neither have the RGD motif nor use integrin for
cell infection.56 The size of the penton protein varies with the serotype, ranging from 493 to 594 amino
acids. The most significant size variation of the major capsid proteins of AdVs is observed in fiber pro-
tein, which ranges from 319 to 587 amino acids (aa). Serotypes within species A and C have long fibers
(>500 aa), serotypes within B and D species have short fibers (<400 aa), and species E has intermediate
length of fibers (425 aa). Interestingly, serotypes belonging to species F and G have two fibers (long and
short fibers) and all cause gastroenteritis. For species F, the long fiber has been shown to bind to the AdV
receptor, while the function of the short fiber remains unknown, but it likely plays a role in enteric AdV
tropism and cell infection.57
Capsid core proteins include pV, pVII, Mu, TP, pIVa2, and the protease, and all but the protease are
associated with the viral genome. The minor core protein pV, containing a basic arginine-rich domain,
interacts with viral DNA. Functional studies indicated that pV is not essential for viral replication and
infectious viral particle assembly in cancerous cells, but it mediates the translocation of nucleophosmin
1/NPM1/B23.1 from the nucleoli to the nucleoplasm that is essential for productive infection in normal
cells.37,58,59 The major core protein pVII, with four basic lysine- and arginine-rich domains, serves as a
histone-like structure that viral DNA wraps around. pVII is synthesized as a precursor protein (preVII)
and is matured by the removal of the N-terminal 24 amino acids via the viral protease. Interestingly,
preVII is involved in condensing the viral DNA during particle assembly, but only mature pVII is pres-
ent in the viral nucleoprotein core. In addition, pVII also mediates virus DNA import into the nucleus
through the transportin pathway during infection, potentially through its nuclear and nucleolar local-
ization signals. Furthermore, pVII interacts with pIVa2 and L1 52/55 kDa during viral particle assem-
bly.37,60,61 pIVa2 performs multiple tasks during the life cycle of an AdV. Encapsidation of viral DNA
requires the binding of pIVa2 to the packaging domain as a multimeric complex with the L4 22-kDa
protein and the L1 52/55-kDa protein, as well as pVII. The binding of pIVa2 to the packaging domain
initiates genome recognition and recruits the L4 22-kDa protein to the packaging domain. In addi-
tion, pIVa2 forms a dimer with the L4 33-kDa protein and functions as a transcription activator of the
18 Laboratory Models for Foodborne Infections

viral major late promoter and regulates the synthesis of the majority of late structural proteins of the
virus.37,62–64 Core protein Mu (μ), generated from a precursor protein (79 aa) by the removal of the N- and
C-terminal amino acids via the viral protease, is a function partner of pV and pVII in condensing the
viral genome and DNA encapsidation.65,66 TP is a 55-kDa protein, covalently linked to the 5′ end of ITR,
which allows the circularization of viral DNA and enhances in vitro DNA replication.67–69 The TP is
synthesized as an 80-kDa pTP, which forms a heterodimer with pol that plays a key role in DNA replica-
tion by serving as a primer for initiating viral DNA replication. Furthermore, pTP is a key participant
in the AdV replication complex consisting of pTP, pol, and DBP as well as cellular factors NFI, Oct-1,
and type I topoisomerase NFII. This replication complex is tightly bound to the nuclear matrix through
interaction with the pTP, thereby anchoring the viral genome to the nuclear matrix.68,70–72 Protease plays
a key role in several steps of the viral life cycle, including uncoating the viral particles upon viral entry
into cells and enabling the cleavage of core precursor proteins that facilitates the formation of infec-
tious viral particles and the cleavage of cytokeratins leading to host cell lysis. It is synthesized in an
inactive form and becomes partially activated by binding to the viral DNA, which then cleaves out an
11-amino-acid viral peptide (pVIc, GVQSLKRRRCF) from the C-terminal of the precursor of pVI. pVIc
binds to the protease and fully activates its function.73–78
The majority of nonstructural proteins of AdVs are encoded by the early-transcribed genes. E1A is
the first gene expressed upon viral infection and plays an essential role in regulating the viral transcrip-
tion of genes necessary for replication and reprograming the cellular transcription to facilitate viral rep-
lication. Two major E1A proteins, 12S and 13S E1A, are synthesized via alternative splicing of mRNA
transcripts, which results in a 46 aa conserved region (CR3) that is unique in 13S E1A. Despite their
similarity, 13S E1A is primarily responsible for activating the viral gene expression via CR3, while
12S E1A represses transcription, which is achieved via temporal regulation.79–82 E1A proteins activate
transcription through a TATA motif, YY1 recognition sites, by interacting with a variety of cellular
transcription factors and relieving transcription repression.2 In addition to modulating transcription,
E1A proteins also regulate signaling pathways and 26S proteasome, interfere with pathways involved
in immune regulation, as well as involve the evasion of T-cell immunity to ensure viral survival and
infectivity.82
E1B gene products are required for efficient viral replication during AdV infection. At least five E1B
gene products were identified in Ad5, which were generated through alternative splicing. One of these
proteins, E1B-55-kDa protein, in conjunction with E4 open reading frame (ORF) 6 protein (E4ORF6)
forms an E3 ubiquitin ligase complex consisting of cellular proteins cullin 5 (Cul5), RING-box 1 (Rbx1),
and elongins B and C. This viral ubiquitin ligase regulates the degradation of cellular proteins, such as
p53, the MRE11-Rad50-NBS1 (MRN) DNA damage recognition/repair complex, and DNA ligase IV,
that have a detrimental effect on viral replication during the early stage of viral infection. In addi-
tion, the E1B-55-kDa/E4ORF6 ubiquitin ligase complex also prevents DNA damage repair mechanisms
by interfering with the MRN complex. During AdV infection, promyelocytic leukemia (PML) bodies,
consisting of several critical DNA response proteins including components of the MRN complex, are
disrupted and relocated to nuclear track-like structures. The MRN complex is initially relocalized into
PML-containing nuclear tracks by another E4 protein, E4ORF3, where it subsequently binds to the E1B-
55-kDa protein. The interaction of E1B-55 kDa and E4ORF3 enables the localization of E1B-55 kDa to
the PML-containing nuclear track, and forms complexes containing MRN, E1B-55 kDa, E4ORF3, and
E4ORF6 proteins, which are then transported out of the nucleus into cytoplasmic aggresomes to further
MRN proteasomal degradation by the E1B-55K/E4orf6 ubiquitin ligase complex. E1B-55K protein can
also contribute to the deregulation of the cell cycle during lytic infection. During the later stages of
viral infection, the E1B-55-kDa/E4orf6 ubiquitin ligase complex inhibits the export of host cell mRNA
while promoting the export of late viral mRNA from the nucleus and its translation.83,84 Another E1B
gene product, E1B-19-kDa protein (small T antigen), is the first viral BCL-2 homolog discovered, and it
inhibits apoptosis triggered by AdV infection by blocking the BIK/NBK pathway to ensure successful
virus production.85,86
E2 encodes three replication proteins, DBP, pol, and pTP. These proteins along with three cellu-
lar transcription factors, NFI, Oct-1, and type I topoisomerase, constitute the AdV DNA replication
system.2 DBP, a product of the E2A gene, binds dsDNA, ssDNA, and RNA without apparent sequence
Adenoviruses 19

specificity. It is involved in initiating DNA replication and is also essential for elongation by enhancing
the activity of pol, unwinding dsDNA, removing secondary structures, and protecting ssDNA. DBP is
a multifunctional protein involved in DNA replication, transcription regulation, mRNA stability, trans-
formation, virion assembly, and host range determination. During DNA replication, DBP first stimulates
the initiation by increasing the binding of NFI and recruiting the pTP–pol complex to the replication
origin. Then, DBP unwinds the parental strand and enhances the progress of the polymerase by binding
to the displaced strand and protecting it from nuclease digestion, which enhances strand displacement
activity during the elongation phase. Finally, DBP enhances the rewinding of complementary displaced
strands.87–89 AdV pol is a DNA-dependent DNA-pol encoded by E2B transcript with a molecular weight
of 140 kDa and belongs to a distinct family of protein priming pol, eukaryotic pol α family, that uses a
protein primer for the initiation of replication. It also shows 3′→5′ exonuclease activity for proofread-
ing. AdV pol forms a stable heterodimer with pTP, protein primer, which initiates the viral DNA rep-
lication by binding to NFI and Oct-1 and results in a pre-initiation complex which then binds the viral
core origin.90–92
The E3 region is distinct for each species varying in length and number of ORFs representing areas
of major sequence divergence.93 Common to all species, E3 encodes three proteins, gp19K, receptor
internalization and degradation (RIDα/β, 10.4 kDa/14.5 kDa), and 14.7-kDa protein, which function in
modulating host immune response and AdV death protein (ADP, 11.6 kDa), which is expressed in the
later stages of infection.38,94 The main function of gp19K is to suppress the cell surface expression of
class I major histocompatibility complex (MHC) by inhibiting the transport of class I MHC from the
endoplasmic reticulum (ER) to the plasma membrane and the processing of peptides by tapasin, thus
decreasing the amount of peptide presented by class I MHC in infected cells. The E3 14.7-kDa protein
inhibits apoptosis induced by TNF-α via reducing the secretion of arachidonic acid, while RIDα/β inhib-
its apoptosis induced by Fas-L and TRAIL. RIDα/β not only destroys the receptors for Fas-L and TRAIL
to prevent cell death caused by these factors but also inhibits apoptosis induced by TNF-α through the
same mechanism as the E3 14.7-kDa protein.38 In contrast, ADP is proapoptotic and mediates the release
of AdV by lysing the infected cells at very late stages of infection.94
E4 is predicted to encode seven different polypeptides that are required for lytic growth, and all but
one are expressed. Both ORF3 and ORF6 are involved in viral DNA replication by interacting with
E1B-55-kDa protein, as described earlier (see section above).83 Both ORF3 and ORF6 promote viral
gene expression by facilitating the accumulation of relevant mRNAs at a posttranscriptional level in
the cytoplasm and stabilizing the unprocessed late RNA in the nucleus. Evidence suggests that the
E1B-55-kDa–E4ORF6 complex causes the redistribution of cellular factors necessary for RNA biogen-
esis and that ORF3 directly affects the distribution of essential transcription/replication factors in the
nucleus. In addition, ORF6 interacts with p53 to inactivate its function, which is crucial for successful
infection by AdV.43 Besides ORF3 and ORF6, studies suggested that ORF1 is a transforming protein and
may stimulate quiescent cells to promote lytic infection and oncogenesis. E4ORF6/7 forms dimers to
interact and modulate the activity of the cellular transcription factor E2F and subsequently activate cel-
lular factors that are important for the S phase of cell cycle.43,95 The function of E4ORF4 is to negatively
regulate E1A and E4 transcription via protein phosphorylation by interacting with protein phosphatase
(PP)2A, which creates a regulatory loop limiting cytotoxicity in the early stages of infection. The func-
tional information for E4ORF2 and E4ORF3/4 still awaits discovery.43
AdVs possess two intermediate (also called delay early) genes IVa2 and IX, which encode pIVa2 and
pIX, respectively. pIVa2 interacts with L1 52/55-kDa protein to activate a major late promoter which is
required for viral DNA packaging.96 pIX is a minor coat protein and its function has been described in
the previous section. In addition, pIX also stimulates an AdV major late promoter containing TATA box
elements and contributes to the efficient induction of the late transcription unit.97
One of the nonstructural proteins is encoded by L4 gene. The L4-100-kDa protein inhibits the transla-
tion of cellular mRNA by eliminating the cap-dependent translation pathway via binding to eukaryotic
initiation factor 4G (eIF4G) and displacing Mnk1 from eIF4G. It also promotes translation of late viral
mRNAs through ribosome shunting, which leads to nuclear accumulation of viral products for capsid
assembly. The underlying mechanism for these functions is the interaction of L4-100 kDa with the tri-
partite leader (TL) sequence present in all the late viral transcripts and eIF4G, which is the scaffolding
20 Laboratory Models for Foodborne Infections

element of the cap-dependent translation initiation complex. In addition, L4-100-kDa protein not only
acts as a chaperone facilitating hexon trimerization but also assists in nuclear accumulation of hexon
trimers and in the scaffolding process of AdV capsid.98–101 Another function of L4-100 kDa is to prevent
apoptosis induced by granzyme B in infected cells. Granzyme B, one of the lymphocyte granule serine
proteases, catalyzes the cleavage and activation of several caspases which induce apoptotic events in
infected cells. By interacting with granzyme B, the L4-100-kDa protein prevents access of substrates
to the proteinase catalytic site through steric hindrance and inhibits its activity.102 A unique feature of
L4-100 kDa is its posttranslational modification by arginine methyltransferase 1 (RPMT1). The methyla-
tion of L4-100 kDa has regulatory effects on modulating its interaction with hexon and TL mRNA as
well as on promoting the capsid assembly.101
A conserved ORF in Mastadenovirus genus, U exon, located between E3 and L5, was first identified in
Ad40 genome sequence, and later identified to be a 24-kDa protein, UXP. UXP is a late protein encoded
in an overlapping reading frame with DBP and part of the 100-kDa protein and has been found in all
species C serotypes so far. The characterization of UXP indicated that it localizes within the nucleoli,
nucleus, and the periphery of the replication center, suggesting its involvement in DNA replication or
RNA transcription.44,103

2.5  Life Cycle

2.5.1  Cell Entry and Replication
As nonenveloped viruses, AdVs require engaging host cell factors, that is, protein or carbohydrate
receptors, to gain cell entry via interactions between the viral fiber protein and host cell receptor.
Host cell factors for species A, C, E, and F have been identified as coxsackievirus and AdV receptor
(CAR), and CD46 or desmoglein-2 (DSG-2) are used by species B. The majority of species D uses
CAR except subtypes 8, 19, and 37, which use sialic acid and GD1a glycan for attachment.104 After
initial attachment, the interaction of the penton base (via RGD motif) and αvβ3 or αvβ5 integrins is the
required second step for cell entry, which triggers clathrin-mediated endocytosis of virions. Upon
entry into cells, capsids are partially disassembled and escape from the endosome, which are then
transported along microtubules to the perinuclear envelope, where further disintegration of the capsid
allows import of the viral genome into the nucleus (Figure 2.3).55 It should be noted that the relative
importance of the fiber–CAR interactions and penton base attachment mechanism in the determina-
tion of successful infection depends on the length of the fiber protein. For example, fibers in group C
species that are consistently on the longer side required less of the penton base attachment mechanism
for successful infection.38
DNA replication of HAdVs employs a unique mechanism similar to the protein-primed DNA replica-
tion of several bacteriophages and requires three viral proteins—DBP, pTP, and pol—as well as three
cellular factors—NFI, Oct-1, and type I topoisomerase. Located within the ITRs of HAdV genomes
are DNA replication sites, which consist of core and auxiliary origins (the terminal nucleotides form
the minimal origin and the remainder of ITR). For initiating DNA replication, the formation of a pre-
initiation complex consisting of DBP, pTP, pol, NFI, and Oct-1 is needed. The interactions of NFI with
pol and Oct-1 with pTP enable the pTP/pol dimer to be recruited to the replication origin. NFI and Oct-1
also bind to the auxiliary origin, which enhances the initiation and changes the origin structure of the
DBP. The covalent addition of dCMP to pTP was followed by the formation of a pTP–trinucleotide
intermediate, pTP–CAT, which uses nucleotides 4–6 as a template. This pTP–CAT intermediate then
jumps back three bases and becomes paired with template residues 1–3. This jumping-back mechanism
explains the presence of a short 3-bp (or sometimes 2- or 4-bp) repeat sequence in the first 10 bp of all
HAdV origins. Either during or shortly after jumping back, pol dissociates from pTP and in conjunction
with the action of DBP elongates the protein primer, leading to the formation of a new duplex genome
and the displacement of the nontemplate strand. Full-length replication of the genome requires the pres-
ence of NFII/topoisomerase I. In the final stages of replication, pTP is cleaved by a viral protease to TP,
resulting in the progeny DNA, which is subsequently packaged in virions.71,89,92
Adenoviruses 21

Endocytosis

Endosomal lysis
Structural L1–L5 mRNAs
proteins

Vesicle escape

Assembly

DNA replication
E1–E4 mRNAs

Nonstructural
proteins

FIGURE 2.3  The life cycle of HAdV. First, the virus attaches to a primary receptor on the cell surface via fiber. This is
followed by penton base interaction with αvβ3 and αvβ5 integrins, which facilitates clathrin-mediated endocytosis. Inside
the endosome, the virion begins to dissociate in the low pH environment and releases the vertex proteins including pVI,
which has been implicated in the disruption of the endosomal membrane, allowing the virion to escape from the endo-
some. The partially disassembled virion is transported to the nuclear pore complex. At the nuclear pore, the viral DNA is
imported into the nucleus allowing the transcription and replication of the virion. At the final stage, the viral proteinase
cleaves its multiple substrates and an icosahedral structure is formed, which contains the full-length DNA. (Modified from
Nemerow, G.R. et al., Virology 384, 380–388, 2009; Shenk, T.E., Adenoviridae. in Fields Virology, Vol. 2. eds. Knipe,
D.M., Howley, P.M., Lippincott Williams & Wilkins, Philadelphia, 2265–2300, 2001.)

2.5.2  DNA Packaging and Virion Assembly

The assembly of infectious AdV particles requires cis-acting packaging sequence at the left end of the
AdV genome, and genetic analysis has identified seven functionally redundant repeated A/T-rich motifs,
A repeats 1–7, from left to right that direct the packaging of the DNA. Among these repeats, A1, A2, A5,
and A6 are functionally dominant with a bipartite consensus: the TTTGN8CG sequence. Three proteins,
IVa2, L1 52/55 kDa, and L4 22 kDa, have been identified to bind to the packaging domain and trigger
the interaction with the structural proteins, hexon, IIIa, penton, fiber, pVI, and VIII, as well as nonstruc-
tural proteins (scaffolding proteins), L4 100 and 33 kDa, L1 52/55 kDa, and IVa2, forming a “procapsid”
structure with the viral DNA. Encapsidation of the complete viral genome into the procapsid is driven by
a putative packaging motor through the portal complex, presumably an ATP-dependent incorporation of
the remaining viral DNA. The mechanism is still unclear, but pV and pVII are incorporated into interme-
diate viral particles containing full-length DNA. At the final stage of virion maturation, viral proteinase
cleaves its multiple substrates and forms an icosahedral structure, which contains full-length DNA and
has a density of 1.34 g/cc in cesium chloride, but does not contain proteins L4 100 and 33 kDa.64,105

2.6  Pathogenesis
While AdVs generally infect the mucosal epithelium, individual serotypes differ in their tissue specific-
ity and cause a number of distinct clinical syndromes, including gastroenteritis, respiratory disease, con-
junctivitis, hemorrhagic cystitis, and exanthema. Thus, the pathogenicity of AdVs varies depending on
22 Laboratory Models for Foodborne Infections

the species, serotype, and organ specificity. In general, AdV-infected cells degenerate, and the infected
respiratory epithelial cells have enlarged nuclei containing inclusion bodies.3 Most of our understanding
on AdV pathogenesis comes from studies using S. hispidus cotton rats and mice for respiratory illness.
For gastroenteritis, it is likely that AdV infection causes destruction of functional mature cells, which in
turn disrupts the water readsorption from the gut and consequently diarrhea ensues. In response to the
damage, the villi retract and the crypt cells undergo rapid division to repopulate the villi with young and
undifferentiated cells which are resistant to virus infection. However, the malabsorption continues until
these undifferentiated cells mature and develop the necessary functional capabilities.106,107

2.7  Host–Pathogen Interaction

In addition to the interaction of structural proteins and host cell receptors to gain entry, AdVs also
employ various strategies interacting with cytokines to modulate the innate and the adaptive host
immune defenses.108 Interferons (IFNs) provide the first line of defense against viral infection by binding
to their receptors, which enhances transcription of cellular factors that restrict viral growth and signal
to the adaptive immune system the presence of a viral pathogen. For AdVs, E1A is responsible for the
anti-IFN effect by blocking the IFN signal transduction pathway mediated by IFN-stimulated gene fac-
tor 3 (ISGF3) via binding to p300, one of the transcriptional adaptor proteins that is required for the
activation of IFN-regulated genes.109,110 Similarly, AdE1A also interferes with the expression and signal
transduction of interleukin (IL)-6, an inflammatory cytokine, via signal transduction and activation of
transcription (STAT) family members.111
Tumor necrosis factor (TNF), produced by activated macrophages in response to inflammatory stimuli,
can directly inhibit virus replication and lyse virus-infected cells in vitro. There are several viral proteins
that interact with TNF during its life cycle to provide an optimal viral replication environment. The
various functions of E1A proteins are associated with their binding with various cellular factors. Among
these, the binding of E1A with cellular factors p300 and p105 Rb (retinoblastoma gene product) resulted
in cell cycle dysregulation, that is, the activation of quiescent cells for optimal viral replication; however,
these interactions also induce TNF sensitivity and potential apoptosis. The E1A-induced TNF sensitiv-
ity and apoptosis are countered by E1B 19-kDa protein, which inhibits the cytolysis activity of TNF
by blocking TNF-mediated signal transduction or by counteracting cytotoxicity through activating the
transcription of protective gene products, such as Mn superoxide dismutase. Furthermore, E3 10.4, 14.5,
and 14.7-kDa proteins provide TNF resistance via a 10.4-kDa/14.5-kDa heterodimer or 14.7-kDa protein.
The 10.4-kDa/14.5-kDa heterodimer also downregulates the epidermal growth factor receptor (EGF-R)
by stimulating endosome-mediated internalization of the receptor, which induces cell proliferation.42,108,112
Cytotoxic CD8+ T cells are critical in controlling primary viral infections via major histocompat-
ibility complex class I proteins (MHC-I). MHC-I binds to proteolytic cleaved viral peptides and is trans-
ported to the cell surface, termed as antigen processing and presentation, which will be recognized by
receptors on CD8+ T cells, and as a result, the T cells are triggered to kill the virus-infected cells.108,113
To evade this reaction, species B–E AdVs encode the E3 19-kDa protein, which binds to MHC-I and
anchors it to ER, preventing its transport to cell surface, thereby inhibiting the lysis of virus-infected
cells by CD8+ T cells. Alternatively, species A and F AdVs do not code for E3 19 kDa, but use 13S E1A
instead to downregulate MHC-I. 13S E1A binds to cellular factor p105-NFκB1, the precursor of p50
NFκB1, resulting in reduced amounts of NFκB and KBF1, which are needed to activate MHC-I expres-
sion, and consequently, a lesser amount of MHC-I is expressed.108,114

2.8  I mmunity
Murine pneumonia models (nonpermissive infection with HAdVs) have been used to study the cytokine
response induced by AdV infections. During early-phase infiltration, the infected mouse lungs showed
high titers of TNF-α, IL-1, and IL-6, but only IL-6 was found in the peripheral blood. IL-6 reached max-
imum titers 6–24 h after infection, whereas maximum levels of TNF-α and IL-1 were attained 2–3 days
Adenoviruses 23

after infection.21 IL-8, a proinflammatory cytokine, secreted by airway macrophages and the epithelial
cells within the lung epithelium, was also induced by AdV infections. The higher titer of IL-8 increases
the protein synthesis and apical localization of CAR in polarized cells, which in turn tethers the infiltrat-
ing neutrophils on the apical surface of polarized epithelia. The adherence of neutrophils on the apical
surface provides for amplification of AdV infection.115 IL-12 is also induced by AdV infections, which
may be important in innate immune activation and the subsequent adaptive immune response to AdV
infection; however, the exact mechanism remains unclear.116 AdV infections also induce the produc-
tion of high levels of type I IFN. The increased levels of type I IFN enhance the transcription of IFN-
stimulated genes; however, the E1A gene products specifically suppressed this transcription.70,109 Recent
studies also indicated that antimicrobial peptides, such as defensins and cathelicidins, play a role during
AdV infection. Defensins inhibit the infectivity of wild-type AdV via blocking the uncoating events of
AdVs, thus preventing genome exposure and nuclear entry by α-defensin HD5 and HNP1, while catheli-
cidins are upregulated by cytokines induced by AdV, which are chemotactic for neutrophils, monocytes,
and T cells, thus causing the inflammatory infiltrate to be seen during AdV infection.117

2.9  Conclusions and Future Perspectives

AdV infections mainly cause childhood illness with mild symptoms and are generally self-limiting with
the exception of AdV caused acute respiratory distress in residential facilities and military bases. Over
six decades of studies have (1) established that AdVs are useful model systems for uncovering fundamen-
tal aspects of cell and molecular biology; (2) provided important insights into AdV life cycle as well as
its interactions with components of the host immune system; and (3) shown that AdVs are useful vectors
in gene transfer for gene therapy, vaccination, and cancer treatment. However, most of the studies are
focused on core virus functions and respiratory disease-related aspects of AdV infection. Little is known
about the characteristics specific to AdV gastroenteric infections. So much is known about the virus
because it is easily grown and serves the useful role of a model system, but less research has been focused
toward clinical questions as infections most often produce mild effects.
Without the rising population of immunosuppressed potential hosts, there would be little urgency
to further study the remaining unresolved aspects of AdVs; however, it has been recognized that AdV
infections are responsible for significant morbidity and mortality among immunocompromised patients.
Severe gastroenteritis caused by AdVs has been reported in immunocompromised individuals.118,119
Without any further understanding on AdV-caused gastroenteritis, only symptomatic treatment is pos-
sible. New research to better characterize the molecular features that are specific to the gastroenteric
infectious AdVs may allow effective antiviral agents to be developed or may help to determine effective
preventive measures for the infections.

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3
Astrovirus

Matthew D. Koci and Stacey L. Schultz-Cherry

CONTENTS
3.1 Introduction..................................................................................................................................... 29
3.2 Life Cycle.........................................................................................................................................31
3.3 Pathogenesis.................................................................................................................................... 32
3.4 Immunity......................................................................................................................................... 34
3.5 Future Perspectives......................................................................................................................... 35
References................................................................................................................................................. 35

3.1  I ntroduction
Astroviruses are small, round, nonenveloped, positive-sense single-stranded RNA viruses. They were
named for their “star-like” appearance when visualized by negative staining electron microscopy
in the feces of infants suffering from gastroenteritis.1,2 Since their original discovery, astroviruses
have been identified in almost all animal species examined.3 In the vast majority of these species,
astrovirus infection is transmitted through the fecal–oral route and causes acute gastroenteritis. In
humans, the classical astroviruses are associated with mild to moderate diarrhea and are recognized
as one of the leading causes of enteritis in children and the immunocompromised.4,5 More recently,
astroviruses have been associated with encephalitis.6,7 In other species, especially poultry, diarrhea
can be severe and can involve other organ systems including the liver and kidneys.8,9 Historically,
astroviruses have been thought to be species-specific, with only limited evidence of interspecies
transmission.10 However, there is increasing evidence that astroviruses can infect multiple species.
Convincing evidence for this was first found for avian astroviruses,11–17 but recent reports suggest the
same may be true for mammalian astroviruses,18–20 and that there may even be transmission between
birds and mammals.21 More recently, astrovirus genotypes associated with human infections were
detected in nonhuman primates, strongly supporting the suggestion that astroviruses can cross spe-
cies barriers.21a
Since the Astroviridae family was established by the ICTV in 1995, there have been considerable
changes in our understanding of the diversity, molecular evolution, and host range of the family.
In just the last decade, the numbers of host species detected with astroviruses have increased more
than fourfold.22 Additionally our appreciation of the diversity of astrovirus genotypes capable of
infecting a given host has led to a reevaluation of their classification system. Currently, Astroviridae
is divided into two genera (Table 3.1), Mamastrovirus (MAstV) and Avastrovirus (AAstV), repre-
senting viruses that affect mammals and avian species, respectively. Within each of these two gen-
era, viruses are classified by distinct genotypic differences within their polymerase and/or capsid
genomic sequences.23
Based on this nomenclature system, there are 19 species of MAstV recognized to infect humans
(MAstV 1–19). Of these species, MAstV 1 encompasses the human astroviruses (HAstVs) first rec-
ognized and most extensively studied.22 This group of classical HAstVs contains eight distinct serotypes
(HAstV-1 to HAstV-8), which are known to have a worldwide distribution and are recognized as one of

29
30 Laboratory Models for Foodborne Infections

TABLE 3.1
Astroviridae
Genus Species Host(s)
Mamastrovirus (MAstV) MAstV 1 Human
MAstV 2 Cat, cheetah
MAstV 3 Pig
MAstV 4 California sea lion
MAstV 5 Dog
MAstV 6 Human
MAstV 7 Bottlenose dolphin
MAstV 8 Human
MAstV 9 Human
MAstV 10 Mink
MAstV 11 California sea lion
MAstV 12 Bat
MAstV 13 Sheep
MAstV 14 Bat
MAstV 15 Bat
MAstV 16 Bat
MAstV 17 Bat
MAstV 18 Bat
MAstV 19 Bat
Unassigned Pig, sheep, mouse, rat, cattle, deer
Avastrovirus (AAstV) AAvstV 1 Turkey, chicken
AAvstV 2 Turkey, guinea fowl, duck
AAvstV 3 Chicken, turkey
Unassigned Dove, pigeon, heron, pintail

Environmental
Swimming/bathing
Fresh or saltwater

Fomites

Direct fecal–oral

Contaminated
Drinking water
Fruits and vegetables
Bivalves
Food Water systems
Freshwater
Groundwater
Water Saltwater

FIGURE 3.1  Direct and indirect routes of astrovirus transmission. As the cause of gastroenteritis, the direct fecal–oral
transmission of astroviruses is well established, as is its ability to be transmitted by fomites; however, astroviruses are
environmentally very stable, allowing them to be transmitted indirectly to new hosts via water and the environment.
Astrovirus 31

the top three causes of diarrhea in children under 2 years of age.24–26 The other HAstVs found in MAstV
were identified through pathogen discovery surveys of fecal material.27–32
Astroviruses are transmitted through the fecal–oral route (Figure 3.1), as demonstrated by several
volunteer studies.33,34 This direct transmission of fecal material from an infected person to a new host, or
through fomites is thought to be the major mechanism for the spread of astroviruses through a popula-
tion; however, astroviruses are highly stable in the environment.35,36 This stability allows them to spread
through wastewater, groundwater, saltwater, and even drinking water.37–45 These astrovirus-contaminated
waters can then infect a new patient following drinking, eating fruits and vegetables treated with these
waters,46 consuming bivalves harvested from these waters,38,39,47 or even bathing, swimming, or surfing
in astrovirus-contaminated waters.48,49 Given this link between astrovirus stability and water, it is espe-
cially important to recognize that most municipal water treatment systems do not filter out, or inactivate
astroviruses as part of their process, as studies have demonstrated by isolating astroviruses from house-
hold tap water.4,50,51

3.2  Life Cycle

After ingestion, the virus passes through the gastrointestinal tract where it is likely activated by host
trypsin-like proteases (Figure 3.2).52–56 The virus then binds to an as yet unidentified receptor(s) on host
cells and enters intestinal epithelial cells of the small intestine (host cells for most of the known astrovi-
ruses) through clathrin-mediated endocytosis.57 Following its entry, the virus releases its positive-sense
single-stranded RNA genome into the cytoplasm, which is immediately recognized by the host cell as
mRNA, leading to the expression of a polyprotein containing the nonstructural proteins (nsPs) and the
viral RNA-dependent RNA polymerase (RdRp).58 Once these proteins have been produced, they quickly

(A)

Immature virion Trypsin “activated” virion Cross-section of mature

virion

(B)
ORF1a ORF1b ORF2

FIGURE 3.2  Model of astrovirus virion (A) and diagram of astrovirus genome (B). The astrovirus virion is a nonenvel-
oped capsid ranging in size from 28 to 41 nm depending on the species and propagation conditions. Studies of HAstV-8 by
Dryden et al. suggest that astroviruses are released as immature virions, which become highly infectious mature virions
following digestion by trypsin.112 The mature virion surface is comprised of multiple spike-like projections (dark gray,
VP27 and VP25), extending from the shell (light gray, VP34), giving the virus its distinctive five- or six-pointed star-like
appearance when visualized under negative staining electron microscopy. Inside the capsid, the astrovirus genome is com-
prised of single-stranded, positive-sense RNA ranging from 6.4 to 7.7 kb in size. The genome contains three open reading
frames: ORF1a encodes the nonstructural proteins; ORF1b encodes the RNA-dependent RNA polymerase; and ORF2
encodes the capsid protein. The retrovirus-like frameshift structure formed between ORF1a and ORF1b allows these two
ORFs to be translated as one polyprotein and is one of the molecular hallmarks that distinguishes the astrovirus replication
strategy from other small, round RNA viruses.
32 Laboratory Models for Foodborne Infections

establish replication complex vesicles.59 As part of this process, the virus also produces a subgenomic
RNA encoding the viral capsid protein (ORF2) (Figure 3.2).60 The subgenomic message is then used
to produce ample copies of the virus structural protein, while the genomic RNA is used to produce
more nsPs and RdRp, as well as a template for the progeny genome.58 Once enough capsid and progeny
genomes have been produced, the daughter viruses are assembled. This initial assembly step involves the
full 90-kDa capsid protein, which is then digested by host caspases to yield progeny viruses made up of
70-kDa capsid proteins.55,61–63 These immature progeny viruses are then released from the infected cell,
where they will be further digested by host trypsin-like proteases, activating them and allowing them to
infect new cells.52–56

3.3  Pathogenesis
Classical HAstVs cause gastrointestinal disease in young children, aged adults, and the immunocom-
promised,5,64–68 but are only rarely associated with the disease in normal healthy adults.33,69,70 Molecular
epidemiology and serology studies suggest that humans are exposed to HAstVs regularly and antibodies
against HAstVs develop early in life.22 In fact, preexisting anti-HAstV antibody levels seem to explain the
biphasic age distribution of HAstV infections, as well as the sporadic development of HAstV-mediated
diarrhea during adult volunteer studies.54,71,72
In susceptible people, following an incubation period of 4–5 days,73 HAstV infection leads to mild,
watery diarrhea, lasting 48–72 h.5,22 Clinical symptoms can also include vomiting, fever, and abdominal
pain. Overall, astrovirus-induced disease is generally less severe than that caused by rotavirus or calici-
virus infections, and rarely requires hospitalization in otherwise healthy people.5,22
While HAstVs are regarded as a disease of the intestinal tract, recent evidence suggests that they
can induce viremia, potentially causing more severe infections in immunocompromised patients.74–77
This concept of extraintestinal astrovirus was first reported in studies examining the pathology of avian
astroviruses,12,74,78–80 and was more recently demonstrated in a newly established immunodeficient mouse
model of astrovirus infection81 as well as in immunocompetent mice orally inoculated with murine astro-
virus (unpublished observations).
Despite the well-established disease burden caused by HAstVs, the mechanisms involved in astrovirus-
mediated disease in humans are still poorly understood. Much of what has been reported regarding the
pathophysiological changes induced by astrovirus infection has come from animal models; however, the
changes noted and the cell types involved appear to vary considerably between models.
Studies using experimentally infected lambs reported atrophy of the villi of the jejunum and ileum,
with astroviruses found in the mature enterocytes.82 In astrovirus-infected calves, the virus was found
in the M cells and enterocytes associated with Peyer’s patches in the jejunum and ileum.83 In ducks and
chickens, astroviruses are more commonly associated with other tissues, causing fetal hepatitis in ducks
and mild nephritis in chickens.12,78,84,85 The young turkey model, however, has been the most extensively
used to understand astrovirus pathogenesis.74,79,86–92 These studies demonstrate that astrovirus infection
leads to very few, if any, remarkable changes in the intestinal histology. No significant inflammation, flux
of immune cells, or changes in the villus height, width, or surface area were observed.74,79,88,91 While not
wholly unique to astroviruses, the absence of histological changes correlating with the onset of diarrhea
is uncommon among diarrhea-inducing agents. These observations in turkeys have been supported by
histological analysis of small intestine biopsy samples from an immunosuppressed child suffering from
persistent diarrhea.93
The lack of histological changes consistent with the severity of diarrhea observed has led to a more
specific examination of the changes in intestinal function to determine how astroviruses cause diseases.
These studies have demonstrated decreased expression of digestive enzymes, specifically maltase and
sucrose, in the brush border of the small intestine, suggesting the development of malabsorption of
carbohydrates, leading to osmotic diarrhea (Figures 3.3 and 3.4).91,92 More recent studies suggest that
astrovirus infection leads to a rearrangement of actin in intestinal epithelial cells.88,94 These changes
were associated with decreases in the electrical resistance (decreased barrier function) in the jejunum
that allowed for increased movement of ions but not larger macromolecules (Figure 3.3).88 In addition,
Astrovirus 33

+ +
- + -
+- +
- + -
-
+
-
+
-
+

+
-
+
-
-
+
+
+
-
-
+
-
+
-

Decreasing barrier function

FIGURE 3.3  Astrovirus infection and exposure to astrovirus capsid protein lead to changes in intestinal barrier function.
Under normal conditions, the tight junctions (gray boxes) between neighboring cells form water-tight barriers, are held
in place and regulated by actin (red lines). Following astrovirus replication, or even just on exposure to astrovirus capsid
protein, the actin at the apical ends of epithelial cells is rearranged. The more significant this rearrangement is, the greater
the change in barrier function. Smaller changes can allow for the diffusion of solutes and ions across the epithelium, while
still larger disruptions allow diffusion of macromolecules.

Increased disruption of enzymes and exchangers recycling in the brush border

FIGURE 3.4  Astrovirus-induced changes in the apical expression of digestive enzymes and exchangers. Following infec-
tion or exposure to astrovirus capsid protein, the apical expression of digestive enzymes such as maltase and the NHE-3
have been reported to be significantly decreased, resulting in malabsorption and osmotic diarrhea. While the specific
mechanism(s) involved is not known, it is likely that the astrovirus-induced disruption to actin (red lines) prevents the
normal recycling of vesicles and proteins whose function is regulated by a near-constant shuttling in and out of the brush
border, and they are more dramatically affected by astrovirus-induced actin rearrangement.
34 Laboratory Models for Foodborne Infections

there was a decrease in the absorption of sodium ions that correlated with decreased apical expression
of the sodium/hydrogen exchanger-3 (NHE-3) molecule in the jejunum.88 Collectively, these findings
suggested an infection established by sodium malabsorption, leading to osmotic diarrhea. The changes
in the turkey jejunum were similar, but not completely consistent with studies of HAstV in vitro where
viral infection as well as exposure to inactivated HAstV capsid was sufficient to induce actin rearrange-
ment leading to tight-junction disruption, allowing macromolecules to diffuse across the barrier.88,94 The
observation that the capsid protein alone is capable of inducing barrier changes in vitro suggests that it
possesses enterotoxin-like activity and could be capable of inducing even diarrhea. Current experiments
using turkey,94a as well as other animal models (unpublished observation), have confirmed this hypoth-
esis, but they also suggest that the potential impact a nonhuman astrovirus may have on a human disease
could be greatly underappreciated if replication is not required to induce clinical signs.95

3.4  I mmunity
Epidemiological evidence suggests that immunity to HAstV is largely mediated through antibodies.
Clinical signs of astrovirus infection are most pronounced, if evident at all, in those with no preexisting
antibodies or those who are immunocompromised due to age, HIV status, or on immunosuppressive
therapy.67,71,72 The levels of anti-HAstV antibodies observed in normal healthy adults, in some studies,
suggest that we may be under frequent exposure to these pathogens.33,34,96 In addition to antibodies,
anti-HAstV-specific T cells have been recovered from intestinal biopsy samples from a normal healthy
adult97,98 suggesting that both the cellular and humoral arms of the adaptive immune system respond to
HAstV infections. The relative importance of each in clearing the pathogen and preventing subsequent
infection is still unknown.
Given that astrovirus infection is most severe when the adaptive immune system is least active, many
researchers have focused on the role of the innate immune response to understand how the body fights
the infection under these conditions. Most interestingly, recent studies by Guix et al. have demonstrated
that HAstVs can inhibit the production of type I interferon in the early phase of replication allowing for
greater viral production. This effect seems to correlate with mutations in one of the mature nsP proteins
(nsP1a/4), suggesting that some strains and some genotypes may have different abilities to modulate the
interferon response.99
This is not the only evidence that astroviruses are able to circumvent the innate immune response. The
astrovirus capsid protein can inhibit activation of the lectin and classical complement pathways.100–103
Amino acids in the N-terminal region of the capsid interact with the C1 and MBL molecules, prevent-
ing the formation of C3 convertase and subsequent cleavage of C3 into C3a and C3b. The blockage of
these complement activation pathways prevents the formation of a very powerful pro-inflammatory and
immune-activating signal,71 one that host cells use to detect the presence of numerous RNA, DNA, and
bacterial infections.104 The specific role the inhibition of the complement pathway serves in astrovirus
pathology is unclear, but it is easy to speculate that it helps to mute the inflammatory signal and is at least
partially responsible for the astrovirus’s immune evasion in vivo.71
As with astrovirus pathogenesis, most of our understanding of the immune response to astroviruses
in vivo comes from the turkey model. In this model, however, the adaptive immune response seems to play
little to no role in controlling the infection,79,86 and in fact some researchers have suggested that it induces
an immune dysfunction that can persist long after the virus has cleared.105–107 Macrophages isolated from
astrovirus-infected turkeys demonstrate reduced phagocytosis and the ability to kill engulfed bacteria
in ex vivo assays, along with the reduced production of pro-inflammatory cytokines.108–111 Lymphocytes
from astrovirus-infected turkeys also demonstrated reduced proliferation in response to mitogenic stim-
ulation. The apparent inhibition of these immune cells could be a response to increased levels of active
TGFβ in the serum of infected turkeys.79 The mechanism leading to the activation of TGFβ in these
animals is unclear but given its role as a potent anti-inflammatory cytokine, it is likely key to the limited
role the adaptive system plays in response to astrovirus infection.
In spite of numerous immune factors being downregulated following astrovirus infection, the treat-
ment of avian macrophages with astrovirus capsid was seen to stimulate the expression of inducible
Astrovirus 35

nitric oxide synthase (iNOS) and its antimicrobial mediator NO.86 The modulation of this response in an
in ovo culture system demonstrated that the iNOS response could modulate virus replication; however,
there was no evidence that macrophages responded to the sites of infection.79,86,88 Subsequent studies
demonstrated that the intestinal epithelial cells can and do express iNOS following astrovirus infection
suggesting that in the absence of professional immune cells the intestinal cells are capable of mounting
their own response.87

3.5  F uture Perspectives

Over the past decade, we have learned about the diversity of astroviruses, the diseases they can cause,
and that their ability to move between species is far more complicated than what was originally thought.
These revelations underscore the long-held idea that humans are exposed to astroviruses on a frequent
basis, and highlight the need for greater attention to fully appreciate their role in gastroenteritis. Given
their ubiquity and environmental stability, astroviruses may serve as an ideal indicator virus to assess
the sterility and safety of water and food preparation systems. Any treatment system capable of inac-
tivating and removing astroviruses would likely be devoid of other, more episodic, foodborne patho-
gens. Studies are underway to develop desperately needed small-animal models of infection as well
as improved in vitro culture systems. This is especially important given that the majority of astrovirus
strains are currently unculturable. Given the rapid increase in the number of host species in which astro-
viruses have been detected, evidence of possible zoonotic/reverse zoonotic infections, and the possibility
that the astrovirus capsid protein may be a unique enterotoxin, astrovirus-associated disease is likely to
increase in the future.

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4
Hepatitis E Virus

Kavita Lole, Prudhvi Lal Bhukya, and Subhashis Chatterjee

CONTENTS
4.1 Introduction......................................................................................................................................41
4.1.1 HEV Transmission............................................................................................................. 43
4.1.2 HEV Infection in Humans.................................................................................................. 44
4.1.3 Clinical Course/Manifestations of HEV Infection............................................................ 46
4.1.4 Immune Response.............................................................................................................. 46
4.1.4.1 Innate Immune Response................................................................................... 46
4.1.4.2 HEV-Induced Adaptive Immune Response........................................................ 47
4.1.5 Diagnosis............................................................................................................................ 47
4.1.6 HEV Vaccine...................................................................................................................... 48
4.1.7 Antiviral Therapy against HEV......................................................................................... 48
4.1.8 HEV Biology...................................................................................................................... 49
4.1.8.1 Proteins Encoded by the HEV Genome............................................................. 50
4.1.8.2  cis-Regulatory Genomic Regions....................................................................... 52
4.1.9 HEV Life Cycle.................................................................................................................. 52
4.2 Tools That Can Be Used for Studying HEV................................................................................... 53
4.2.1 Use of Infectious cDNA Clones of HEV............................................................................ 53
4.2.2 HEV Cell-Culture Models.................................................................................................. 54
4.2.2.1 Genotype 3 Virus Culture Systems.................................................................... 55
4.2.2.2 Kernow C1 Virus Strain..................................................................................... 55
4.2.2.3 3D-Cell-Culture System..................................................................................... 55
4.2.2.4 Primary Hepatocytes as the Culture System...................................................... 56
4.2.2.5 Genotype 4 Virus Culture System...................................................................... 56
4.2.2.6 Propagation of HEV Strains Circulating in Blood............................................. 56
4.2.3 HEV Animal Models.......................................................................................................... 58
4.2.3.1 Nonhuman Primates........................................................................................... 58
4.2.3.2 Pigs...................................................................................................................... 59
4.2.3.3 Rodents............................................................................................................... 59
4.2.3.4 Rabbits................................................................................................................ 60
4.2.3.5 Ferrets................................................................................................................. 60
4.2.3.6 Chickens.............................................................................................................. 60
4.3 Conclusions......................................................................................................................................61
References..................................................................................................................................................61

4.1 Introduction
Hepatitis E virus (HEV) is a major causative agent of acute viral hepatitis in developing countries.
HEV is primarily transmitted via the fecal–oral route through contaminated water and is endemic in
several countries in Asia, Africa, and Central America1–3 due to poor sanitary conditions. It is also

41
42 Laboratory Models for Foodborne Infections

highly endemic in India, Bangladesh, Egypt, Mexico, and China, where hepatitis E can occur either
in the form of sporadic cases or occasional large outbreaks. Hepatitis E represents the major cause of
waterborne outbreaks in these countries, and contamination of water with sewage has been a common
feature preceding such outbreaks.4–10 HEV is believed to be the most common cause of sporadic acute
hepatitis (>50%–60% cases) in adults in regions where the virus is endemic.11–13 There have also been
large outbreaks among displaced persons in Sudan, Chad, and Uganda.14–17
Although, previously, sporadic cases of hepatitis E in developed countries were attributed to travel to
hyperendemic areas, in recent years, a surprisingly higher number of cases of autochthonous transmis-
sion of hepatitis E with high seroprevalence have been documented from the United States, Canada,
Europe (including UK, France, the Netherlands, Austria, Denmark, Spain, Italy, Greece, and Germany),
and Asia–Pacific countries (Japan, Taiwan, Korea, Hong Kong, Australia, and New Zealand).18,19 It is
estimated that about 2 billion people live in HEV-endemic areas. A global disease burden study esti-
mated that HEV genotypes 1 and 2 account for approximately 20.1 million incidents of HEV infections,
3.4 million cases of symptomatic disease, 70,000 deaths, and 3,000 stillbirths. Clinical HEV infection is
most common in adolescents and young adults, but also occurs to a lesser extent in children.20–22 During
epidemics of HEV, the ratio of clinical to subclinical or anicteric infection was shown to be 1:26 in
pregnant women, 1:4 in children, and a­ pproximately 1:10 in adults.20,23 In developing countries, although
HEV is endemic, seroprevalence usually remains below 40% as against hepatitis A virus, which shows
almost 100% seropositivity in adults.1
Nucleotide sequence analysis of HEV genomes from different geographical isolates has revealed wide
genetic diversity among HEV strains forming four distinct phylogenetic clusters. Of these, genotype 1
and 2 strains are restricted to humans. Genotype 1 strains are associated with large outbreaks of acute
hepatitis E in humans in Asia. Genotype 2 includes one Mexican strain and several African strains.
Genotype 3 is distributed worldwide. Distribution of genotype 2 virus is restricted to few countries and is
not a major problem in comparison with the more widely distributed genotype 1 virus. Genotype 3 is also
associated with sporadic, cluster, and chronic cases of hepatitis E in humans, mostly in industrialized
countries. Genotype 3 strains have also been isolated from domestic pigs, wild pigs, deer, mongoose, and
rabbits.24–26 Wang et al.27 identified an additional HEV strain in the sera of Chinese patients with acute
hepatitis, classified as genotype 4. Genotype 4 virus can also infect pigs.28,29 Genotype 3 and 4 strains
have been found to infect a broader range of hosts including deer and boars.26,30–32 Intra- and intergeno-
type recombination has been recently documented.33
HEV is enzootic in domestic pigs even though the virus may not be endemic in human population or
belongs to a different genotype than that is circulating in humans in endemic settings. Detection of HEV
RNA and antibodies in wild boar populations has been reported from Japan, Germany, Italy, Spain, the
Netherlands, and Australia.34–39 In addition, several divergent strains of HEV have been isolated from
animals such as bats,40 rats,31 wild boar,41 ferrets,42 mongoose,25 fish, and cutthroat trout.43
HEV strains within genotypes 1 and 2 are less divergent as compared with HEV strains from
genotypes 3 and 4. These HEV genotypes have been classified further into 24 subgenotypes.44,45
Currently, four putative genera have been proposed in the family Hepeviridae, one comprising human
HEV genotypes and closely related animal viruses and the other three including viruses of rodent (rat),
chiropteran (bat), and avian (chicken) origins.40 HEV isolates from rats share approximately 59.9% and
49.9% sequence identities with human and avian HEVs, respectively, while the ferret HEV sequences
share 72.3% identity with the rat HEV and have been proposed to be grouped in the genus Ortho
hepevirus.42,46 Bat HEV strains from Africa, Central America, and Europe have a very high sequence
divergence (53% at amino acid level) from known HEV isolates and are suggested to be classified in
a separate genus Chiropteranhepevirus.47,48 Avian HEV strains share 50%–60% nucleotide sequence
identity with swine or human HEV strains, and recently it was proposed that all three known genotypes
of avian HEV in chickens (genotype 1 in Australia and Korea, genotype 2 in the United States, and
genotype 3 in Europe and China) should be grouped into a new genus Avihepevirus.47,49,50 The cutthroat
trout HEV from the United States exhibits only 13%–27% sequence homology leading to a proposal
of another genus Piscihepevirus.43,47 Interestingly, the range of different animal species that appear
to harbor HEV/HEV-like agents is continuously extending, indicating the ubiquity of HEV/HEV-like
agents in different species from many regions. We expect many more additions in this family in the
Hepatitis E Virus 43

e2
Genotype 4

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FIGURE 4.1  HEV phylogenetic tree: Full genome/capsid region nucleotide sequence analysis of global hepatitis E virus
isolates. Genotypes 1 and 2 (HEV-1 and HEV-2) circulate among humans, primarily in Africa and Asia, while genotypes 3
and 4 (HEV-3 and HEV-4) have animal reservoirs. Recently discovered rabbit strains appear to form a closely related clade.
Alignment was done using MEGA 7 and tree by using software available on www.itol.embl.de.

coming years. A phylogenetic tree based on a portion of the nucleotide sequence encoding the capsid
protein is shown in Figure 4.1.

4.1.1 HEV Transmission
The virus is relatively stable in the environment51 and is sensitive to heat, chlorination, and ultraviolet
light.52,53 In endemic regions, HEV remains in circulation due to the excretion of the virus by humans and
other animals in the acute phase of infection and environment acts as the major reservoir of the virus.54
Contamination of drinking and irrigation water mainly occurs due to improper sewage disposal, leading to
epidemics in developing countries. Water scarcity in summers or floods during rainy season can increase
the possibility of the virus spread. Surface water bodies can easily get contaminated with human and ani-
mal wastes. Inadequate treatment and release of sewage in rivers has been documented, with 4.9% HEV
RNA positivity in 403 water samples from different locations of a river in one Indian city (Mutha River,
Pune).55 Waterborne route could also be important for genotype 3 and 4 HEVs. Genotype 3 virus has
been detected in waste/runoff waters from pig slaughterhouses, swine manure, and swine slurry storage
facilities.56,57 An increased risk of hepatitis E in sewage treatment plant workers has been documented.58,59
Person-to-person transmission of HEV is rare in both epidemic60 and sporadic settings.61 Occasionally,
nosocomial spread has also been reported.62 Vertical transmission from mother to infant is also known
to occur.63 In endemic as well as nonendemic settings, transfusion-associated hepatitis E infection is
possible64–69; however, this route of transmission is not so common.70,71 Plasma products have been
44 Laboratory Models for Foodborne Infections

documented as the source of HEV infections.72,73 Easy and rapid HEV infectivity laboratory assays to
evaluate virus inactivation processes would be useful for monitoring plasma-derived products.
Close phylogenetic relationships between human and animal viruses prove that HEV is transmitted
from animals to humans. Domestic pigs are the major animal reservoirs of HEV. Hepatitis E is now
a recognized zoonotic disease, with swine and other likely animals serving as the reservoir for these
viruses.74,75 Naturally acquired anti-HEV antibodies have been shown in many animal species including
swine, sheep, cattle, goats, horses, macaques, cats, dogs, rats, and mice, raising the possibility of these
animals being the reservoirs and causing zoonotic infections.10,76,77 Occupational exposure to infected
pigs poses the risk of acquiring HEV infection.56,78–80
Consumption of undercooked or raw pig liver, pork, and game meat (wild boar, deer, or rabbit) plays
a significant role in HEV transmission in industrialized countries. Studies from Japan have documented
foodborne HEV infections that occurred due to the consumption of undercooked pork81,82 and wild boar
or deer meat.83–86 HEV infections due to wild boar meat have also been reported from Germany86 and
South Korea.32 Consumption of pig liver figatellu sausages has been documented to cause human HEV
infections in France, as seen from the identical HEV sequences from the infected individuals and sau-
sages from the local grocery stores.87,88 Infectious nature of HEV particles in the sausages was demon-
strated by Berto et al.89 HEV presence in pig liver or meat has also been reported from countries such
as India,90 United States,91 Germany,92 Canada,93 Democratic Republic of Congo,94 Japan,95 Italy,96 and
the Netherlands.97
HEV has been detected in varied shellfish collected from European and Asian countries. Genotype 3
HEV has been detected in mussels and oysters.98–102 Human HEV infections due to the consumption of
shellfish are documented.103–105 Bivalves are known to concentrate viral particles during their filter feed-
ing process, and as they are mostly consumed raw or cooked for a very short period, they pose a serious
risk of HEV infection. Viral RNA sequences have been detected in soft fruits like strawberries and green
leafy vegetables such as lettuce, with irrigation water being the suspected contamination source.106,107
The concentration of the viable virus in an environment or food is an important factor in the outcome of
clinical hepatitis E infection. HEV is known to remain stable in frozen conditions even after 10 years.
HEV remains viable after heating to 56°C for 1 h,108 and a cooking temperature of 71°C for 20 min is
required for the complete inactivation of the virus.109 Use of contaminated water for drinking, cooking,
cleaning, and irrigation107 can spread the virus.

4.1.2 HEV Infection in Humans

Hepatitis E is generally a self-limiting disease which lasts for 4–6 weeks. Entry of the virus into the host
primarily occurs by the oral route. Infection is presumed to be initiated via cells lining the alimentary
tract. The virus then enters the liver, presumably via the portal vein, where it replicates in the cytoplasm
of hepatocytes without causing any direct catalytic effect, and is released into the bile and blood. The
incubation period for hepatitis E infection ranges from 15 to 60 days with a mean of 40 days as seen from
the two studies wherein the clinical course of HEV infection was monitored in two human volunteers
who ingested the virus from fecal suspensions.110,111 HEV was detected in feces by immuno electron
microscopy (IEM) on day 28, and the liver enzyme level peaked on day 42.110 Viremia was detected from
day 22 onward up to the alanine aminotransferase (ALT) peak. Anti-HEV antibody was first detected on
day 41 and was detectable until 2 years later.111
HEV infection can lead to liver injury of variable severity. Clinically, HEV infection manifests in
different ways from asymptomatic infection, acute hepatitis to fulminant hepatitis. The disease occurs
in two stages: the prodromal (pre-icteric) stage and the icteric stage. The illness is often clinically and
biochemically similar to other hepatotropic viruses, such as hepatitis A virus or hepatitis B virus. Typical
symptoms of HEV infection include fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain,
jaundice, dark urine, clay-colored stool, and enlargement of liver. Few patients with hepatitis E infection
develop prolonged cholestasis, characterized by persistence of jaundice, marked itching, and elevated
alkaline phosphates.112 The prodromal symptoms usually subside at the onset of clinical jaundice; how-
ever, some patients may not show visible signs of jaundice despite experiencing severe symptoms. The
ALT level peaks at the onset of symptoms followed by conjugated hyperbilirubinemia. The level of
Hepatitis E Virus 45

ALT, however, does not correlate with the degree of liver cell damage (peak levels vary from 1000 to
2000 U/L at the onset). Peak serum total bilirubin levels range from 5 to 25 mg/dL; both conjugated and
unconjugated fractions are increased. ALT progressively diminishes during the recovery phase. Patients
with anicteric or subclinical acute hepatitis E infection only exhibit elevated ALT levels, which is use-
ful in the early diagnosis of clinically suspected cases, along with the presence of anti-HEV IgM in the
serum. Liver damage results, presumably mediated by host cellular immune responses. The course of
hepatitis E in human subjects strongly suggests that HEV, like other hepatitis viruses, is noncytopathic in
most circumstances, and liver disease is probably caused by immunological and hormonal factors.113–116
The severity of hepatitis E is considered to be dose-dependent, and associated host factors such as
chronic liver disease or alcohol overuse may enhance it further.3 Patients with HEV infection superim-
posed with other viral or nonviral chronic liver disease (acute on chronic liver disease) are at the higher
risk of having a poor outcome.117,118 Case fatality rates in hepatitis E epidemics range from 0.1% to 4%,
but pregnant women, especially during the third trimester, are at a higher risk of severe disease, with
case fatality rates of 10%–25%.119–121 Hepatitis E is also associated with premature delivery, with low
birth weight and an increased risk of perinatal mortality.122 HEV transmission from mother to fetus was
reported in 33% of cases; the virus has been detected in human colostrum as well.117,123
The outcome of hepatitis E infection during pregnancy is probably influenced by nutritional status,
host immune response, and hormonal factors.113,115,116 It is not yet understood how HEV infections dur-
ing pregnancy are less severe in Egypt124 while they are more severe with higher mortality rates in
sub-Saharan Africa and South and South-East Asia,115 although genotype 1 HEV predominates in these
regions. Thus, severity during pregnancy is not the unique feature of genotype 1 or 2 viral infections, but
there seem to be additional contributing factors that influence the disease severity. Only occasional cases
of hepatitis E during pregnancy with less severity have been reported from low-endemic, developed
countries.125–127 Thus, understanding HEV pathogenesis during pregnancy still remains a big challenge.
There are certain observations to suggest that HEV strains may vary in their virulence. Genotype 3
strain (3c) circulating in Europe is known to cause mostly subclinical infections.128 A comparative study
of genotype 3 and 4 HEV-infected individuals in Japan revealed that genotype 4 HEV is associated
with significantly higher ALT and total bilirubin levels, higher viral loads, and an aggressive disease
course.129 Similar observations have been reported by Jeblaoui et al.130 in a study from France that the
clinical presentation is more severe in patients with HEV4 infections than in patients with HEV3 infec-
tions. Purcell et al.131 examined the relative virulence of human genotypes 1 and 2 and swine genotype 3
in rhesus monkeys and found that genotype 3 is significantly less virulent as compared with human
genotypes 1 and 2. There are studies indicating the association of point mutations in the HEV genome
with disease severity; however, the underlying mechanism still remains unknown. Silent substitutions of
U at the nucleotide 3148 (in the helicase domain) and C at the nucleotide 5907 (in the capsid gene) in the
genomes of HEV strains of genotypes 3 and 4 have been documented to have an association with fulmi-
nant hepatitis and disease severity in patients. C5907 mutation was also associated with high viral loads
in the infected individuals.132,133 Takahashi et al.134 have reported the association of V239A mutation (in
the helicase domain) with increased virulence of genotype 3 virus. Similarly, L1110F and V1120I muta-
tions in the helicase domain of genotype 1 sequences were found to be associated with fulminant hepa-
titis cases.135 Shukla et al.136 have recently reported the integration of human S17 rRNA sequence into
the genotype 3 HEV genome in a patient with chronic HEV infection. This patient had both neurologic
and hepatic symptoms. Nguyen et al.137 have also reported the integration of S19 in an isolate analyzed
from a chronic hepatitis E patient with organ transplant in the United States. These integrations in HEV
genomes probably altered tissue specificity and pathogenicity of the virus.
HEV normally does not lead to chronic infection in immunocompetent individuals. However, chronic
HEV infections have been observed in immunocompromised organ transplant recipients138,139 and in
patients with other conditions of immunosuppression such as HIV infection140 and hematological malig-
nancies.141,142 HEV infection results in persistent viral shedding and may rapidly progress to liver cir-
rhosis and may also cause extrahepatic manifestations such as neurological disorders and impaired renal
functions in these patients.143 Chronic HEV infections were thought to exclusively involve genotype 3
viral strains, however, recently a report from China documented the chronic course of HEV infection
with genotype 4 virus in a boy who was receiving chemotherapy for an acute lymphoblastic leukemia.144
46 Laboratory Models for Foodborne Infections

A study from India showed no evidence of chronic hepatitis E in a cohort of kidney transplant recipients,
suggesting that HEV genotype 1 may not cause chronic hepatitis E.145 However, there is a need for more
data from other endemic regions to confirm these findings.

4.1.3 Clinical Course/Manifestations of HEV Infection

In macaques, the incubation period is generally 3–8 weeks, but shorter and longer incubation periods
have been observed.146–149 In intravenously inoculated macaques, the expression of HEV antigens can
be seen in hepatocytes 7 days postinfection with a parallel appearance in bile and feces, suggesting the
release of the virus from hepatocytes into bile and then into feces.150 Peak viremia and peak shedding
of HEV into the feces occurs during the incubation period and early acute phase of the disease.146,151–153
HEV RNA could be detected in serum, bile, and feces several days before the elevation of serum ALT
levels.146–148 Histopathological changes in the liver were observed in parallel with ALT elevation. High
mortality (∼20% particularly in the third trimester) of hepatitis E during human pregnancy was not
reproducible in pregnant rhesus macaques.152,153 HEV RNA detection in experimentally infected chim-
panzees revealed viral genomic sequences in serum and stools from the very beginning of the infection
and a sudden drop of the viral titer with the development of an antibody response.154 All known mamma-
lian HEV genotypes (1–4) and swine HEV genotypes 3 (United States)155,156 and 4 (India)157,158 have been
transmitted to nonhuman primates. The course of hepatitis E in human subjects and in experimentally
infected primates strongly suggests that HEV, like other hepatitis viruses, is noncytopathic in most cir-
cumstances and liver disease is probably caused by immunological factors.113–116
Swine is both a reservoir and a host of HEV. Both natural and experimental HEV (genotypes 3 and
4) infections in swine are asymptomatic with only mild microscopic lesions in the liver and associated
lymph.35,157–160 There is a transient viremia while fecal shedding continues from 3 to 7 weeks and anti-
HEV antibodies are seen in 4–6 weeks.28,156,159 The acquisition of anti-HEV is age dependent in pigs, the
majority acquiring antibodies by 4–5 months of age.159,161
Avian HEV also shows mostly subclinical infections with mortality rates up to 0.3%–1.0%.162–164
Clinical signs may include egg drop in some flocks up to 20%, enlargement of the liver and spleen, and
acute death of affected birds. Since avian HEV does not infect humans, it is not a concern for food and
environmental safety.

4.1.4 Immune Response
4.1.4.1 Innate Immune Response
After entry into the host, HEV needs to overcome the host innate immune response and establish infec-
tion in the host. In the past decade, remarkable progress has been made in understanding strategies of
HEV in combating host innate immune responses. HEV interferes with type 1 interferon (IFN) induction
and IFN-activated signaling. HEV inhibits IFN-α signaling and manages to replicate in the presence of
IFN-α. There is a study demonstrating the reduced ubiquitination and degradation of IkBα in hepatoma
cells expressing the open reading frame (ORF) 2 protein, affecting the nuclear translocation of NF-κB.
Recently, the macro domain and papain-like cysteine protease (PCP) domain from HEV ORF1 were
identified as the putative IFN antagonists by Nan et al.165 In contrast, HEV ORF3 was shown to enhance
RIG-I-mediated signaling leading to enhanced IFN-α synthesis, indicating complex strategies of the
virus during its establishment in the host cells.166
A microarray-based gene expression analysis of serial liver biopsy samples from HEV and HCV in
chimpanzees has shown attenuated adaptive immune response in HEV-infected animals as compared
with HCV.167 However, there was a robust innate immune response correlating well with viremia in both
HCV- and HEV-infected chimpanzees. A major component of the host response to HEV infection was
type I interferon-induced genes (ISGs). HEV infection was completely resolved without any recurrence
in the infected chimpanzees, indicating that HEV is highly susceptible to ISGs. This indicated that HEV
infection is mostly taken care of by the robust innate immune response. Altered peripheral frequencies
and activation status of NK and NKT cells in HEV-infected individuals suggest that innate immunity
Hepatitis E Virus 47

plays a role in the pathogenesis of hepatitis E.168 It was speculated that the reduced NK cell levels during
pregnancy could contribute toward the increased severity of hepatitis E during pregnancy. Robust non-
specific IFN-γ-producing T-cell response in acute hepatitis E patients suggesting innate immune mecha-
nisms involving NK/NKT/T reg cells has been documented.169 Higher frequencies of CD4+ CD25+
Foxp3+ regulatory T cells, elevated levels of IL-10, and suppressive functionality of the regulatory T
cells in acute hepatitis E patients have been documented.170 Overall, innate immune responses seem to
play a major role during HEV infection.

4.1.4.2 HEV-Induced Adaptive Immune Response

HEV usually causes mild disease or subclinical infections. Icteric symptoms in hepatitis E coincide with
the rise in anti-HEV antibodies. Experimental animal infections and human studies suggest that liver
damage mainly occurs due to immune response rather than by the virus itself. The immunohistochem-
istry of liver biopsies from patients with HEV-induced acute liver failure revealed significant infiltration
of CD8+ T cells containing granzyme suggesting their role in liver damage.171 In comparison to patients
with acute hepatitis E and healthy controls, patients with fulminant hepatitis E have a less marked expan-
sion of HEV-specific IFN-γ or TNF-α-secreting CD4+ T cells. Husain et al.172 provided evidence for the
activation of effector T cells during acute hepatitis E. Srivastava et al.173 observed less-marked cellular
immune responses and a heightened antiviral humoral response in severe HEV infection. In contrast,
significantly higher levels of both Th1 (IFN-γ, IL-2, and TNF-α) and Th2 (IL-10) cytokines and higher
anti-HEV IgM and IgG titers were recorded in fulminant hepatic failure (FHF) patients as compared with
acute hepatitis E patients by Saravanabalaji et al.174 The existence of a Th2 bias in pregnant women with
acute hepatitis E infection is reported.18 Impairment of HEV-specific T-cell responses was shown to be
associated with chronic HEV infection in immunocompromised organ transplant patients.175 Association
of TNF-α and IFN-γ cytokine gene polymorphism with the susceptibility and clinical outcome of hepati-
tis E has been shown.176 Authors noted an overrepresentation of the T (vs. A) allele at IFN-γ+874, which
is linked to higher IFN-γ production, among symptomatic cases. These observations suggest a probable
role of higher acquired anti-HEV response in serious complications in FHF with HEV.

4.1.5 Diagnosis
Hepatitis E diagnosis mainly depends on the clinical features and exclusion of other causes of acute
hepatitis, especially hepatitis A, in the endemic regions. The presence of anti-HEV IgM is a marker
of acute infection, while anti-HEV IgG alone is a marker of past infection. HEV was first identified by
immunoelectron microscopy.110 Serological as well as molecular assays were developed eventually after
successful cloning of the HEV genome.177,178 Anti-HEV IgM appears during the early acute phase of ill-
ness and may be detected as early as 4 days after the onset of jaundice and lasts for up to 2–5 months.179
Anti-HEV IgG has been shown to persist for long periods of time (>14 years) and provides protection
against subsequent infections. Anti-HEV IgA has also been detected in the serum of naturally infected
individuals.180 For practical purposes, HEV IgM can be detected from blood to confirm the clinical diag-
nosis. Detection of viral RNA in HEV IgM-positive patients provides additional confirmation and further
information about the virus strain, enabling molecular epidemiological studies. In immunocompromised
patients, HEV RNA testing is essential for diagnosis since patients have impaired immune response.
There are several HEV RNA detection assays developed during the past decade.181–185 However, the assay
developed by Jothikumar et al.185 seems to be very commonly used. For optimal diagnosis, a combination
of serological and nucleic acid-based assays has been recommended.186,187
HEV ORF2 protein expressed in SF9 cells has been shown to have glycosylation and performs
well in anti-HEV IgM and IgG diagnostic assays. All the four genotypes of HEV belong to a single
serotype,188 thus suggesting that diagnostic antigens from a single HEV genotype should detect
antibodies against HEV strains of different genotypes. Broad cross immunoreactivity among HEV
genotypes has been documented.189–192 Commercially available assays for hepatitis E diagnosis have
problems of sensitivity and specificity, and many studies have shown poor concordance of these
48 Laboratory Models for Foodborne Infections

tests.193,194 There is a need to have a validated assay that would have reasonable sensitivity and
specificity in detecting infections with different HEV genotypes. Such an assay would be extremely
useful in generating worldwide seroprevalence data to understand the realistic disease burden of
hepatitis E.

4.1.6 HEV Vaccine
Being refractory to cell culture, the conventional approach of developing killed or attenuated virus-
based vaccines for HEV was not possible and hence recombinant DNA technology has been employed.
Four mammalian HEV genotypes are believed to represent a single serotype. Cross-challenge studies
in rhesus monkeys using various strains of genotype 1 HEV have shown that animals are protected
from the disease up to 5 years after primary infection, indicating the possible utility of the vaccine to
prevent hepatitis E.1,12,64,195 HEV capsid (ORF2) protein is highly immunogenic and has been used as
the target antigen for vaccine development. Several approaches based on recombinant DNA technol-
ogy have been attempted to develop candidate subunit vaccines, either using a complete or partial
ORF2 protein. A 56-kDa (112–607 amino acids) recombinant protein-based candidate vaccine
phase III clinical trial, with 95% efficacy, was successfully completed in 2007 by the NIH group.196
This subunit vaccine protected against genotype 1 (homologous) as well as genotype 2 and 3 HEV
(heterologous) challenges in the monkey model.197 However, since then, there has been no progress in
the commercialization of this vaccine. The second candidate vaccine that was found to be safe and
immunogenic was based on the bacterially expressed genotype 1 HEV ORF2 protein (p239, 368–606
amino acids). Phase III clinical trial of this vaccine showed >95% efficacy and cross protection against
the heterologous genotype 4 virus.198 The vaccine (Hecolin) is currently licensed and commercially
available only in China.

4.1.7 Antiviral Therapy against HEV

There has been no established therapy for treating acute HEV infection and associated disease.
Infections with genotype 1 and 2 HEVs are generally self-limiting and therefore do not require spe-
cific therapy in the majority of infections. Similarly, the disease course of genotype 3 infections is
generally self-limiting illness that lasts for 4–6 weeks in immunocompetent individuals and hence gen-
erally does not require specific treatment. Therapy is essential only when patients are at the risk of
having severe manifestations of the disease. Recent evidences of the chronic course of HEV genotype 3
infections in immunocompromised patients with rapid progression into cirrhosis showed the need to
control the virus replication. This has increased the awareness for this ignored disease, and several stud-
ies have been initiated to understand the mechanisms leading to persistent infection and for the testing of
different antiviral therapies in such patients. Pegylated IFN-α-2a has been successfully used for treating
chronic hepatitis E in transplant recipients.199 However, IFN-α-2a treatment is known to have side effects
and may also increase the risk of graft rejection. Ribavirin is emerging as the treatment of choice for
chronic HEV infection as it results in sustained virological response; however, there are treatment fail-
ures.200–202 Ribavirin needs to be administered for at least 3 months in chronically infected individuals.
Ribavirin monotherapy has also been used in immunocompetent patients suffering from severe acute
hepatitis E.203,204
In HEV-endemic regions, genotype 1 HEV infections in patients with chronic liver disease have
been known to worsen rapidly to a condition known as acute-on-chronic liver failure (ACLF), leading
to very high mortality. Several studies have reported HEV as one of the leading causes of ACLF in
Asia and Africa, where HEV is endemic.205–208 Goyal et al.209 have recently demonstrated the effective-
ness of ribavirin therapy in treating genotype 1 HEV infections. However, the evaluation of ribavirin
therapy needs to be carried out in a well-designed study, with a large number of patients in the active
viremic phase. Overall, considering the limitations of the existing therapies, alternative antivirals are
needed. Table 4.1 describes the overall characteristics of different HEV genotypes.
Hepatitis E Virus 49

TABLE 4.1
Characteristics of HEV Genotypes 1–4
Characteristics Genotype 1 Genotype 2 Genotype 3 Genotype 4

Distribution of Asia, Africa, and the Mexico, West Africa North America, China, Taiwan, and
virus in humans Middle East Europe, Latin South-East Asia
America, and Japan
Distribution of Not reported and Not reported and Widespread and China, Taiwan, and
virus in animals identified identified reported in all India, with a few
continents recent reports from
Europe and North
America
Interspecies Only human to Only human to Animal to human Animal to human
transmission human and no human and no (pigs, wild boar, and (pigs, wild boar)
interspecies interspecies deer)
transmission transmission
observed observed
Waterborne Yes, frequent (source Yes, frequent (source Not reported Not reported
transmission is from human is from human
feces) feces)
Food-borne Not reported and Not reported and Reported from Reported from
transmission recognized recognized contaminated animal contaminated
meat animal meat
Zoonotic Not reported Not reported Yes Yes
transmission
Mortality among High Not reported Not reported Not reported
pregnant women
Infection and attack Most common in Most common in Middle age and older Middle age and older
rate with respect young adults young adults above 55 years, above 55 years
to age (15–44 years) (15–44 years) males, and
immunocompromised
persons
Outbreaks Common Smaller scale Uncommon Uncommon
outbreaks
Symptoms Fever, fatigue, loss of Fever, fatigue, loss of Fever, fatigue, loss of Fever, fatigue, loss of
appetite, nausea, appetite, nausea, appetite, nausea, appetite, nausea,
vomiting, vomiting, vomiting, abdominal vomiting, abdominal
abdominal pain, abdominal pain, pain, jaundice, dark pain, jaundice, dark
jaundice, dark jaundice, dark urine, clay-colored urine, clay-colored
urine, clay-colored urine, clay-colored stool, and joint pain stool, and joint pain
stool, and joint pain stool, and joint pain
Diagnosis Anti-HEV test Anti-HEV test Anti-HEV test Anti-HEV test
Prevention Good sanitation and Good sanitation and Avoiding raw pork and Avoiding raw pork
the availability of the availability of other contaminated and other
clean drinking clean drinking animal meat can contaminated
water water reduce the risk animal meat can
reduce the risk

4.1.8 HEV Biology
HEV is a nonenveloped, spherical particle of approximately 30–34 nm in diameter.110 The HEV genome
is a positive-sense single-stranded RNA of approximately 7.2 kb with a 5ʹ-methylguanine cap and 3ʹ-poly
(A) stretch. Viral RNA contains short 5ʹ (27–35 nt) and 3ʹ (65–74 nt) untranslated regions (UTRs) and
three ORFs, ORF1, ORF2, and ORF3.178 ORF1 encodes the nonstructural polyprotein, ORF2 encodes
50 Laboratory Models for Foodborne Infections

(nt 26–5107) (nt 5108–5130) (nt 5145–7127)

5´ NCR JR NCR 3´

me7G ORF1 ORF2 (Poly A)

ORF3

(nt 5131–5475)

Met Y Protease P Macro Helicase RdRp

FIGURE 4.2  Schematic diagram of the HEV genome: The three ORFs are labeled and shown as boxes with the putative
ORF1 domains indicated outside the box. JR, junction region; me7G, 5ʹ Cap; NCR, noncoding region; ORF, open reading
frame; P, Proline-rich hinge region; RdRp, RNA-dependent RNA-polymerase; Y, Y domain (nucleotide numbering done
as per prototype genotype 1 sequence SAR55).

the capsid protein, and ORF3 encodes a multifunctional protein. ORF2 and ORF3 are proposed to be
translated from a single bicistronic messenger ribonucleic acid (mRNA) and overlap with each other.210,211
Figure 4.2 shows a schematic diagram of the HEV genome.

4.1.8.1 Proteins Encoded by the HEV Genome

4.1.8.1.1 Nonstructural Proteins
The ORF1 encodes for a 1693 amino acid polypeptide (180 kDa) (for the genotype 1 SAR-55 strain),
predicted to encode different domains required for virus replication such as methyltransferase (MeT),
a “Y” domain, PCP, a proline-rich region that contains a hypervariable region (HVR), an “X” domain,
RNA helicase (Hel), and RNA-dependent RNA-polymerase (RdRp).212

4.1.8.1.2 Methyltransferase
It is the first functional domain of ORF1.213 Recombinant protein-encompassing amino acids 1–979 from
HEV ORF1 were shown to have guanine-7-methyltransferase as well as guanyltransferase activities. The
presence of an m7G cap at the 5ʹ end of the HEV genomic RNA confirmed the functional role of HEV
methyltransferase.214 The source of RNA triphosphatase activity, that is required for 5ʹ cap formation,
was later confirmed to be a HEV helicase protein.215

4.1.8.1.3 PCP
The most intriguing question of HEV biology that has not yet been answered is whether the ORF1 pro-
tein (pORF1) is processed into functional biochemical subunits or whether it remains as a single protein.
The postulated role of PCP is the polyprotein processing of ORF1 polyprotein. The above hypothesis
was put to test by many researchers, but none could show the evidence of ORF1 processing and the role
of PCP in polyprotein processing.216–221 A recent study from our group showed that HEV MeT-PCP pro-
tein recognizes the LXGG motif and shows in vitro deubiquitination (DUB) activity. The protein also
showed deISGylation of cellular proteins, suggesting its probable role in combating cellular antiviral
innate immunity during the HEV life cycle.222 Paliwal et al.223 recently demonstrated the role of PCP in
ORF1 polyprotein processing.

4.1.8.1.4 Helicase
The putative RNA helicase of HEV was shown to contain all the seven conserved motifs found in SF-1
helicases and was proposed to have the NTPase as well as RNA-binding domains.212 Functionality of
HEV helicase was recently demonstrated by our group. HEV Hel exhibited NTPase and RNA unwinding
activities. Enzyme hydrolyzed all rNTPs efficiently; dATP and dCTP were hydrolyzed more efficiently
as compared with dGTP and dTTP. Enzyme showed the unwinding of only RNA duplexes with 5ʹ over-
hangs showing a 5ʹ-to-3ʹ polarity.224
Hepatitis E Virus 51

4.1.8.1.5  RdRp
HEV RdRp contains eight conserved motifs (motif I–VIII) that are similar to the RdRps from other
­positive-sense RNA viruses. RdRp containing polypeptide, encoded by 3546–5106 nt in ORF1, was
shown to interact with the 3ʹNCR of HEV genomic RNA and synthesizes the complementary strand
in vitro.225 Rehman et al.226 demonstrated the localization of RdRp to the endoplasmic reticulum (ER),
suggesting that HEV replicates probably in the ER in the cytosolic compartment of the cells.

4.1.8.1.6 Macro Domain (X-Domain)

It is proposed that viral macro domains bind viral poly(A) tails and recruit poly(ADP-ribose)-modified
cellular factors to the site of replication. Polypeptide encompassing amino acids 775–960 (Burmese
strain, genotype 1) of HEV ORF1 was expressed in Escherichia coli. The purified protein showed
ADPR-1″ phosphatase activity and in vitro binding to poly(ADP-ribose) and ADP ribose.227,228 The affin-
ity for poly(ADP-ribose) was high as compared with ADP ribose. However, the significance of these
functions is not yet clear.

4.1.8.1.7 HVR
The hypervariable region overlaps with the proline-rich sequence located between the N-terminus of the
X-domain and the C-terminal portion of the putative PCP domain. It varies both in length and sequence
among different HEV strains. HEV can tolerate small deletions in the HVR229; however, the replica-
tion efficiency of HVR deletion mutants was found to be reduced when tested in Huh7 cells.230 HVR
sequences were found to be interchangeable between different HEV genotypes, resulting in differential
replication efficiencies. From these observations, it was suggested that the HVR probably could influence
the efficiency of HEV replication by interacting with viral and/or host factors.

4.1.8.1.8 The Capsid Protein

ORF2 codes for the structural (capsid) protein (pORF2) of the predicted mass of approximately 72 kDa
(660 amino acids) and has three putative glycosylation sites at the asparagine residues, Asn137, Asn310,
and Asn562. The pORF2 amino-terminal signal sequence and all three N-linked glycosylation sites are
universally conserved in all isolates of human HEV, suggesting the functional significance of glycosyl-
ation.231 The N-terminal part of pORF2 contains a putative ER-directing signal peptide and is rich in
codons for arginine and highly basic in charge.178 It is synthesized as a precursor and co-translationally
translocated into the ER, where it is processed into the mature protein through signal peptide cleavage.235
ORF2 was reported to occur in glycosylated and nonglycosylated forms (88 and 72 kDa, respectively)
when expressed in animal cells in culture.232 The proposed encapsidation of the viral genome by pORF2
was experimentally demonstrated by Surjit et al.233 by the specific binding of N-terminal 111 amino
acids. Many constructs corresponding to the short and long regions of ORF2 have been expressed in
E. coli. These include the p239 (amino acids 268–607), E2 (amino acids 394–606), E2a (amino acids
459–660), and E2s (amino acids 455–602). All of these constructs fold to form higher-order structures.
By carrying out truncations at the N-terminus, C-terminus, or both, it was proved that amino acids
126–601 are essential elements required for the initiation of the VLP assembly. X-ray studies on ORF2
crystals obtained recently showed that capsid protein monomers are composed of three linear domains:
the shell domain (S) (amino acids 129–319), the middle domain (M) (amino acids 320–455), and the
protruding domain (P) (amino acids 456–606), harboring the key viral neutralizing domain.234,235

4.1.8.1.9 ORF3 Protein
HEV ORF3 is translated from the bicistronic subgenomic RNA.211 It overlaps with ORF2 at its 3ʹ end
and is most variable among the different HEV strains. It is a small 114-amino acid phosphoprotein that
remains associated with the cytoskeleton.236 ORF3 contains two large hydrophobic domains—D1 (amino
acids 7–23) and D2 (amino acids 28–53) at the N-terminus—and two proline-rich domains—P1 (amino
acids 66–77) and P2 (amino acids 95–111) toward the end.237,238 Of these, the cysteine-rich D1 domain
was shown to be required for the association of ORF3 to the cytoskeleton244 to bind microtubules239 and
52 Laboratory Models for Foodborne Infections

mitogen-activated protein kinase (MAPK)-phosphates,240 while the D2 domain was shown to bind to the
hemopexin and proposed to aid in viral infection by affecting cellular iron homeostasis.241 The C-terminal
region of the ORF3 protein is multifunctional and appears to be involved in virion morphogenesis and
pathogenesis. Using an in vitro replication system, it was shown that the ORF3 protein is not essential for
replication in cell lines;242,243 however, intrahepatic inoculations of these constructs in rhesus macaques
revealed that ORF3 is essential for the establishment of infection.211 ORF3 protein was detected on the
surface of HEV particles produced in cell culture, and these ORF3-coated viral particles were seen to be
lighter than uncoated particles, indicating a probable interaction with lipids.244 ORF3 coat was present on
the virus particles present in the serum, but not in the feces of HEV-infected individuals, suggesting that
the lipid-ORF3 “coat” was shed as the virus passed through the enteric system due to exposure to bile salts.
An intact PSAP motif was shown to be required for the formation of membrane-associated HEV particles
with the ORF3 protein on their surface.244 Taken together, these results suggest that the ORF3 protein is a
multifunctional protein that appears to play an important role in both HEV replication and pathogenesis.

4.1.8.2  c is-Regulatory Genomic Regions

ʹNCR
4.1.8.2.1 5
The 5ʹNCR of the HEV genome is only 25–27 nt in length, has a methylated cap, and plays a role in the
initiation of HEV replication. Mapping experiments have revealed that ORF2 binds to the 76 nt region at
the 5ʹ end of the HEV genome. This region includes the 51 nt sequence, conserved across alphaviruses.
Secondary structure predictions and the location of the ORF2 binding region within the HEV genome
indicate that this interaction may play a role in viral encapsidation.233

4.1.8.2.2 3ʹNCR
The interaction of viral RdRp with 3ʹNCR and the adjacent region containing two stem-loop (SL)
structures (SL1 and SL2) and its importance in virus replication were documented by Agrawal et al.225
Using gel shift assays, they could demonstrate that RdRp bound specifically to 3ʹNCR, while 3ʹNCR
without a poly A tail failed to bind with RdRp. Similarly, a single nucleotide change in the SL2 of the
genotype 1 viral genome significantly affected virus replication, indicating the important role of both
poly A tail and SL structures in HEV replication.244,245

4.1.8.2.3 Putative Subgenomic Promoter

A junction region (JR) between ORF1 and ORF2 has a highly conserved SL structure found to be important
for HEV replication.210,246 The putative subgenomic promoter required for the synthesis of the subgenomic
mRNA is located in this region of the HEV genome. Graff et al.245 reported that silent mutations in this
region abolish the synthesis of ORF2 and ORF3 proteins, thus giving the first evidence of this region being
the regulatory region. An in-depth study involving genotype 3 swine HEV and mutants with changes in the
junction region identified a double SL structure of approximately 50 nucleotides in this region.246

4.1.8.2.4 Subgenomic RNA
Graff et al.211 detected two positive-sense RNA species, approximately 7.3 and 2.2 kb in size, in HEV
replicating cells. The 2.2-kb subgenomic RNA was capped, and its 5ʹ end matched the nucleotide number
5122 in genotype 1, SAR55 HEV genome. With these results, this group proposed the ORF2 start at 5145
and 5131 as the ORF3 start, and the translation of ORF3 and ORF3 proteins from the same bicistronic
subgenomic mRNA. This model was confirmed by using the intrahepatic inoculation of wild-type and
mutant genotype 3 swine HEV replicons into pigs by Huang et al.210

4.1.9 HEV Life Cycle

The proposed model of life cycle is mainly based on the existing knowledge of well-characterized simi-
lar positive-sense RNA viruses. The target cells are hepatocytes; however, several extra hepatic tissues
also support replication. Virus capsid protein is believed to be involved in binding to a cell receptor for
Hepatitis E Virus 53

entry. It is suggested that the C-terminal portion of pORF2 may bind to heat shock cognate protein 70
(HSPC70) on the cell surface to initiate cell entry.247 Heparan sulfate proteoglycans (HSPGs) appear to
be required as attachment factors.248 The intracellular trafficking following entry is also poorly under-
stood; HSP90 and tubulin appear to be involved in this process.249 A recent study has documented that
HEV enters through clathrin-mediated endocytosis.250 However, it is still not clear whether these are
specific receptors or just attachment factors. Holla et al.251 have recently reported that following inter-
nalization, the HEV-LP initially traffics into Rab5-positive compartments en route to acidic lysosomal
compartments. HEV entry requires dynamin-2, clathrin, membrane cholesterol, and actin, but is inde-
pendent of factors associated with macropinocytosis.
Once viral RNA is released in the cytosol, ORF1 translation is initiated by the cap-dependent recruitment
of ribosomes. It is not clear whether the ORF1 encoded nonstructural polyprotein functions as a single pro-
tein or whether it is processed into individual functional units. However, the regions predicted to encode viral
methyltransferase, helicase, and RdRp produce functionally active proteins when expressed in heterologous
systems. The genomic RNA is copied into a negative-sense RNA intermediate by viral RdRp. These RNA
intermediates then serve as templates for the synthesis of genomic as well as subgenomic p­ ositive-sense
RNA species. Genomic intermediates have been detected in replicon-transfected cells220 in the livers of
experimentally infected macaques252 and pigs.155 It was demonstrated using a replicon system that there is
an alternate cycle of positive- and negative-sense RNA synthesis.253 Single subgenomic RNA is translated
to synthesize ORF2 and ORF3 proteins.210,211,245 The ORF2 protein packages the genomic ­positive-sense
RNA into progeny virions. Immunocapture PCR analysis showed the association of ORF3 on the surface
of the cell-culture-generated HEV, which also showed a lower density than the ORF3 deficient virus. These
observations suggested that the ORF3 protein is present on the virion surface in association with cellular
lipids and probably plays some role during viral egress.254 Emerson et al.243 and Nagashima et al.255 have
documented the importance of the PSAP motif within the P2 domain of the ORF3 protein in the virus
egress and the probable role of SRC homology 3 signaling pathways in HEV maturation and egress. PSAP
motif is required for the formation of membrane-associated HEV particles with ORF3 on their surface,
which is mediated by cellular Tsg101 protein.256,257 These findings suggest that HEV follows the vacuolar
protein sorting pathway and uses cellular proteins such as Tsg101 for its release from infected cells.

4.2 Tools That Can Be Used for Studying HEV

Molecular mechanisms of HEV replication, cell surface receptors, tissue/species specificity of different
viral strains, and immunopathogenesis of HEV are still not understood. Researchers have used tools
such as established cell lines, primary cells lines, and animal models including nonhuman primates,
pigs, rabbits, rats, and chickens to understand the different aspects of HEV biology. Although not very
efficient, these models have helped in shedding some light on the various aspects of virus pathogenesis,
immune response, virus–host interactions, and virus replication.

4.2.1 Use of Infectious cDNA Clones of HEV

HEV has been a difficult virus to grow in cell culture. Reverse genetics approach is a tool that can be
very useful in such situations. Direct genetic manipulation using infectious cDNA clones has been an
indispensable tool for studying HEV replication and pathogenesis. The development of infectious cDNA
clones has allowed the exploration of the structural and functional relationship of HEV genes. The suc-
cessful development of a genotype 1 full-genome cDNA clone encompassing the complete HEV genome
from an Indian isolate (Accession Number AF076239) was performed by Panda et al.217 Transfection
studies were carried out in HepG2 cells using in vitro transcribed uncapped RNA transcripts. The pres-
ence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells.
The culture supernatant from the transfected cells was able to produce HEV infection in rhesus monkeys
following intravenous injection, indicating the successful generation of viable HEV particles following
the transfection of cells with in vitro synthesized HEV genomic RNA.
54 Laboratory Models for Foodborne Infections

The second report of an infectious clone from the Sar-55 strain of genotype 1 HEV (Pakistan) was
reported by Emerson et al.244 This recombinant genome could establish infection in rhesus macaques and
chimpanzees, and developed symptoms like hepatitis. They used capped transcripts for these studies. It
was controversial whether capping of RNA transcripts was essential for the infectivity since a previous
study by Panda et al. showed the successful generation of infectious particles using uncapped RNA tran-
scripts. Subsequently, Emerson et al.258 reported that HEV capped full-genome transcripts are 32–38 times
more efficiently translated than their uncapped counterparts. Several primate cell lines such as PLC/PRF/5,
Huh-7, Caco-2, HepG2/3CA, FKRHK, Vero, and AGMK were shown to support HEV replication; how-
ever, none of the nonprimate cells were able to support replication, indicating the requirement of species-
specific factors for HEV replication. Further studies from the same group using an infectious cDNA tool
demonstrated that the highly conserved regions of HEV ORF3 harbor cis-reactive regulatory elements
and that mutations in this region eliminate ORF2 and ORF3 synthesis. A single unique nucleotide change
within the stem structure at the 3ʹ end of the HEV genome similarly reduced the efficiency of replication
in both Huh-7 cells and rhesus macaques.245,257 Quantitative measurement of HEV replication became pos-
sible due to the development of fused ORF2-GFP, ORF2-luciferase subgenomic HEV replicons.246,258,259
Successful replication of a virus in a host is a complex phenomenon. With the wider range of host spe-
cies for genotype 3 and 4 HEVs, it was evident that these viruses are comparatively flexible in their host
specificity than genotype 1 and 2 viruses which have a very narrow host range. It is still not understood
how HEV determines its host specificity at the molecular level. A recent report proposes that the host
restriction for the genotype 1 virus could be due to efficiency of the virus to synthesize the ORF2 protein
in the given host.260 Lack of compatibility between cell surface receptor and receptor-binding region
(456–605 amino acids) in the capsid protein of the virus was suggested as the deciding factor. This group
used genotype 3 virus replicon (P6), developed from the Kernow C-1 virus,261 for developing chimeric
genotype 1/3 viral genomes. P6 is known to have a 171-nucleotide insertion (a sequence from the human
S17 ribosomal protein encoding gene) in the HVR region of the viral genome. It was previously reported
by this group that the P6 virus, that was isolated from a chronic HEV case, can cross the species bar-
rier.136 Ability of the S17 sequence to alter species specificity was confirmed by inserting this sequence in
SAR55 (genotype 1). This insertion enabled the replication of the chimeric virus in LLC-PK (pig) cells,
which otherwise was refractory to SAR55 1 virus infection.
Pigs being the most promising model for HEV studies, full-length infectious cDNA clones were also
developed from genotype 3 swine HEV.262 Capped transcripts from these clones were demonstrated to be
competent for forming infectious virions in Huh-7 cells, and these virions were able to establish infection
in pigs. Rescue of a genotype 4 human HEV from cloned cDNA in Huh7 cells and the infectivity of this
virus in HepG2/C3A cells were reported by Cordoba et al.263 Capped in vitro transcripts from this clone
could also establish infection in pigs. Zhu et al.264 recently reported cloning of the genotype 4 virus and
successful infection in rats.
Considering chickens as the practical animal model for HEV pathogenesis and replication studies,
cDNA clones from avian viruses were developed.265 Direct intrahepatic inoculation of RNA transcripts
from these infectious cDNA clones could establish infections in chickens and showed fecal virus shed-
ding, viremia, seroconversion, and histopathological lesions characteristic of avian HEV infection.

4.2.2 HEV Cell-Culture Models

HEV has proven to be very refractory to cell culture. Establishment of an efficient cell-culture system that
will facilitate HEV propagation is very critical for understanding HEV biology. HEV propagation has
been attempted by many researchers in primary hepatocytes from nonhuman primates (chimpanzees,
cynomolgus macaques, tamarins, and African green monkeys)266,267 in cell lines, such as human normal
embryonic liver cells (WRL68), human hepatoma cell lines (PLC/PRF/5, HepG2, and Huh-7 cells),
human colon carcinoma cells (Caco-2), human embryo lung diploid cells (2BS), human lung embry-
onic fibroblast cells (MRC-5), human lung cancer cells (A549), human chorio carcinoma cells (HCCM),
African green monkey kidney cells (Vero), and Rhesus monkey kidney cells (LLC-MK2).268–273 However,
these cell-culture systems either were nonpermissive or failed to support the generation of an infectious
progeny virus or were not very efficient.
Hepatitis E Virus 55

4.2.2.1 Genotype 3 Virus Culture Systems

Recently, an efficient cell-culture system for HEV replication has been reported. A fecal sample
taken from a sporadic acute hepatitis E case from Japan with a high HEV load (the JE03–1760F
strain of genotype 3, 2 × 107 copies/ml per 10% fecal suspension) was used as an inoculum to evalu-
ate the replication in 21 different established cell lines derived from humans, monkeys, cows, dogs,
rats, and mice, including three human hepatocellular carcinoma cell lines (Huh7, HepG2, and PLC/
PRF/5). The virus efficiently replicated in two cell lines, PLC/PRF/5 and A549 from human lung
cancer.274 PLC/PRF/5 and A549 cells showed the presence of HEV on days 12 and 14 after inocula-
tion. HEV progeny virus released in cell-culture supernatants could be passaged >50 times in both
cell lines to achieve cell titers as high as 10 −9 –1010 copies/mL. The successful culturing of the virus
was attributed to the high viral load of the fecal sample, which possibly helped selecting viral vari-
ant from the pool which efficiently adapted to the cell culture as it had favorable mutations. The
JE03–1760F strain harbored the 29 unique point mutations, which were not possessed by any of the
reported genotype 3 HEV strains. Yamada et al.254 constructed an infectious cDNA clone of the virus
successfully.
This cell-culture system was utilized to assess the thermal stability of HEV, and it was observed that
HEV incubated at 56°C for 30 min remained infectious, while the virus incubated at higher than 70°C
was completely inactivated. Further, it was used to show that convalescent serum samples obtained from
patients infected with HEV genotypes 1, 3, or 4 can neutralize genotype 3 virus, indicating the broad
cross-reactivity of anti-HEV antibodies. Serum samples obtained from patients 8.7 to 24.0 years after
the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the
presence of long-lasting anti-HEV antibodies with a neutralizing activity.274 However, the use of this sys-
tem for various applications is yet to be established. It is known that HEV does not cause any cytopathic
changes in the cell culture irrespective of the cells used.

4.2.2.2 Kernow C1 Virus Strain

A genotype 3 virus strain, Kernow C-1, isolated from a chronically infected patient was shown to grow
efficiently in HepG2/C3A cells by Shukla et al.136 This virus contained a 58-amino acid human S17
ribosomal protein sequence insert in the HVR1 region of the viral genome. An infectious cDNA clone
of the virus was developed to analyze the significance of insertion.261 Their mutagenesis studies showed
that the S17 insertion was mainly responsible for the cell-culture adaptation. Introduction of 54 syn-
onymous mutations into the insert had no detectable effect, thus implicating the protein, rather than
the RNA, as the important component responsible for altered abilities of the virus replication. Both the
sequence length and the amino acid composition of the insert were important in increasing the replica-
tion efficiency. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-
activating protein sequence resulted in the partial enhancement of virus replication. On insertion of this
S17 sequence in genotype 1 cDNA clone, the chimeric virus acquired the ability to cross the species
barrier and replicated in hamster cells (BHK-21), though with a low efficiency.

4.2.2.3  3D -Cell-Culture System

Berto et al.89 used a rotating wall vessel for the culture of PLC/PRF/5 cells in a three-dimensional
(3D) configuration and infected them with HEV obtained from the liver of an experimentally infected
pig containing 1.38 × 106 genome equivalents/mL of the virus. This study demonstrated that HEV can
replicate efficiently in these cells for up to 5 months as seen by the presence of a virus in the super-
natants. The virions generated from the culture were able to infect fresh 3D cultures, showing that
this method is able to produce infectious hepatitis E virions. Since these researchers were not able to
cultivate genotype 3 HEV on the monolayer cultures of HepG2/C3A and PLC/PRF5 cells as described
by Tanaka et al.,274 it suggested the possibility of these cells being permissive for only selective viral
strains and not for all. Overall, existing HEV cell-culture systems need to be evaluated with a wide
range of HEV strains.
56 Laboratory Models for Foodborne Infections

4.2.2.4 Primary Hepatocytes as the Culture System

The propagation and production of HEV in vitro have been attempted by many researchers in primary hepa-
tocytes from nonhuman primates as well as in cells from other tissues (chimpanzees, cynomolgus macaques,
tamarins, and African green monkeys), and in Rhesus monkey kidney cells (LLC-MK2).20,159,243,249,266–273
Overall, the results concluded that HEV is refractory to the cell culture. However, recent results from Rogee
et al.275 are encouraging. Their group used a human hepatoma-derived cell line, HepaRG, and a porcine
embryonic stem-cell-derived cell line, PICM-19, for HEV replication studies. These cells have morphologi-
cal and functional properties similar to primary hepatocytes. These researchers used matrigel-embedded
cell cultures to retain a hepatocyte-like morphology and liver-specific gene expression patterns of the cells.
These cells supported the HEV replication and release of virions. However, the virus release in the superna-
tants of infected cell cultures was not as efficient as reported by Tanaka et al. (103 vs. 107). Using these two
cell lines, HEV suspensions heated at 56°C for 60 min were shown to be noninfectious. This result is dif-
ferent from previous studies, where HEV genotype 1 or HEV genotype 3 present in the pork liver or stool
sample were found to be infectious after the 56°C treatment.108,273,274 Since this culture system was unable
to generate high titers of infectious progeny virions, its usefulness as a model system would be limited.

4.2.2.5 Genotype 4 Virus Culture System

After the successful development of a culture system for genotype 3 virus by Okamoto’s laboratory in
Japan, their group opted for a similar strategy to test the genotype 4 virus culture in PLC/PRF/5 and A549
cell lines. A HEV RNA-positive fecal sample from a fulminant hepatitis E patient was used as a virus
source for culturing experiments (the HE-JF5/15F strain of genotype 4, 1.3 × 107 copies/mL).276 The genotype
4 HE-JF5/15F virus strain was found to be even better in its replication efficiency as compared with the
JE03–1760F strain. This culture system will be very useful in delineating the role of viral factors with
respect to the recently observed association of genotype 4 virus with fulminant hepatitis E cases in Japan.
Zhang et al.277 have recently reported a swine cell-culture system for genotype 4 virus. They used
rectal swabs/liver as the virus source and infected swine cells (IBRS-2, derived from swine kidney,
ATCCCRL-1835) and human cells, A549. HEV RNA was detectable for up to 12 passages inIBRS-2
cells and 24 passages in A549 cells. Visible cytopathic effects could be seen from passages 8–12 in
infected IBRS-2 cells, and passages 22–24 in infected A549 cells. However, the authors did not check
for the release of infectious virions in the culture supernatants.

4.2.2.6 Propagation of HEV Strains Circulating in Blood

The cell-culture systems developed for genotype 3 and 4 viruses used primarily fecal samples as the
virus source. In order to check whether the virus present in the serum would grow as efficiently as the
virus that is shed in feces, Takahashi and his group 278 studied the replication of HEV strains from serum
samples in PLC/PRF/5 and A549 cells and found that HEV strains of genotypes 1, 3, or 4 can replicate
efficiently in PLC/PRF/5 and A549 cells. HEV strains in all serum samples tested, with or without con-
current HEV antibodies, were successfully propagated in cultured cells when inoculated at an HEV load
of ≥105 ­copies/well of a 6-well plate. Progeny viruses in the culture supernatant could be successfully
­passaged in the same cells, indicating their infectious nature. Viral concentrations of <105 particles were
not ­efficient, which could be a major hurdle in using this culture system. Moreover, it is not easy to get
viremic serum samples due to transient viremia during HEV infection.
Overall, it is observed that HEV does not cause any cytopathic changes in the cell culture irrespective
of the cells used. Use of current cell-culture systems for various applications is yet to be established.
Further, the reproducibility of these culture systems in different laboratories is apparently not uniform.
Robust cell-culture systems for genotype 1 and 2 viruses are still lacking. Virus infectivity assays are
still dependent on either PCR-based assays, IFA or FACS, which would mean that there is a requirement
for a well-equipped laboratory for studying these viruses. Considering the abundance of these viruses in
developing countries and the major need of such tools there, we still need to develop practical HEV study
tools. Table 4.2 gives an overview of different cell-culture models used in HEV studies.
Hepatitis E Virus 57

TABLE 4.2
Occurrence of HEV in Animal Species
Serological Evidence Experimental Applications as Animal
Animal Species HEV Genotype (Anti HEV) Susceptibility Model for HEV Studies

Cynomolgus Human 1,2 Positive Yes Pathogenesis,

monkey Rabbit HEV molecular biology,
and cross-species
infection
Rhesus monkeys Human 1,2,3,4 Positive Yes Vaccine, pathogenesis,
Swine 3,4 molecular biology,
Avian HEV and cross-species
infection
Chimpanzee Human 1,2,3,4 Positive Yes Pathogenesis,
Swine 3,4 molecular biology,
and cross-species
infection
Tamarins Human 1,2 Positive Yes Not reported
Owl monkey Human 1,2 Positive Yes Cross-species
infection
Vervets Human 1,2 Positive Infection studies
Squirrel monkeys Human 1,2 Positive Yes Not reported
Domestic swine Genotype 3,4 Positive Yes Vaccine, pathogenesis,
molecular biology,
and cross-species
infection
Wild boar Genotype 3,4 Positive Yes Vaccine, pathogenesis,
molecular biology,
cross-species infection
Rabbit Genotype 3 Positive Yes Vaccine, pathogenesis,
and cross-species
infection
Chicken Avian HEV Positive Yes Vaccine, pathogenesis,
molecular biology,
and cross-species
infection
Turkey Avian HEV Positive Yes No reports
Rodents Unknown Positive Yes Vaccine, cross-species
infection
Rat Human 1,2,3,4 Positive Yes Molecular biology and
Swine 3 pathogenesis studies
Avian HEV
Balb/c mice Swine 4 Positive Yes Molecular biology and
pathogenesis studies
C57BL/6 Human 1 Positive Yes
Swine 3,4
Mongolian gerbil Genotype 4 Positive Yes Pathogenesis,
molecular biology,
and cross-species
infection
Deer Genotype 3,4 Positive Not reported Not reported
Goat Genetically Positive Not reported Not reported
divergent from the
known HEV
strains
(Continued)
58 Laboratory Models for Foodborne Infections

TABLE 4.2 (Continued)
Occurrence of HEV in Animal Species
Serological Evidence Experimental Applications as Animal
Animal Species HEV Genotype (Anti HEV) Susceptibility Model for HEV Studies

Dog Genetically Positive Not reported Not reported

divergent from the
known HEV
strains
Cats Genetically Positive Not reported Not reported
divergent from the
known HEV
strains
Bats Bat HEV Positive Not reported Not reported
Ferrets Genetically Positive Not reported Not reported
divergent from the
known HEV
strains
Fish Genotype 3 Positive Not reported Not reported
Camel Unknown new Positive Not reported Not reported
HEV genotype
Sheep Genotype 4 Positive Not reported Not reported
Foxes Divergent from Positive Not reported Not reported
known HEV
genotypes
Horses Genotype 3 Positive Not reported Not reported

4.2.3 HEV Animal Models

4.2.3.1 Nonhuman Primates
Rhesus/cynomolgus macaques and chimpanzees have been used to understand HEV infectivity and
pathogenesis. Animal species differ in virus excretion, liver enzyme elevation, and histopathological
changes in the liver.131,148,224 There are diverse outcomes of experimental infections done in different
nonhuman primate species. Tamarins were found to be inconsistent in developing HEV infection, while
chimpanzees,20 pig-tailed macaques,148 vervets,148 owl monkeys,147 squirrel monkeys,148 and patas mon-
keys were found to be susceptible. All known mammalian HEV genotypes (1–4) and swine HEV geno-
types 3 (United States)155,156 and 4 (India)157,158 have been transmitted to nonhuman primates. Among
primates, chimpanzees, cynomolgus, and rhesus macaques were found to be the best animal models110,148
since experimental HEV infections result in elevated liver enzymes and histopathologic changes in the
liver of these animals similar to hepatitis E patients. Although chimpanzees have been used to study the
clinical course, and host gene responses to HEV infection,167 the use of this model system is not practi-
cal.110,148,279 Although HEV is transmitted naturally via the feco–oral route, this route is not preferred for
experimental infections in primates as this requires a much higher virus dose. Most studies in nonhuman
primates have utilized the intravenous route for virus inoculation. Direct intrahepatic inoculation of
chimpanzees and rhesus monkeys with RNA transcripts from full-length HEV cDNA clones has helped
in establishing a direct link between the disease and the genetic material of HEV by inducing virologic,
pathologic, and serologic characteristics typical of hepatitis E infection.
The disease course of HEV infection in nonhuman primates was comparable to that in humans,
without leading to chronicity. The incubation period to peak serum levels of liver enzymes is gener-
ally 3–8  weeks, but shorter and longer incubation periods have been observed.110,149 Enzyme eleva-
tions are usually unimodal; however bimodal curves have also been observed.146–149 Peak viremia and
peak shedding of HEV into the feces occurs during the incubation period and early acute phase of
the disease.110,148,151 Detection of HEV antigens in the liver generally precedes or parallels viremia and
Hepatitis E Virus 59

fecal shedding.150,151 The clinical presentation of hepatitis E infection in nonhuman primates was dose
dependent. Severity of infection was directly related to the infectivity titer of the challenge virus. For
demonstration of consistent hepatitis E infection in nonhuman primates, challenge virus doses at least
1000 times greater than the minimum dose needed for infection were required. Also, the oral route of
inoculation was not efficient and required a 10,000-fold higher challenge virus than that was used for
the intravenous inoculation of nonhuman primates.195 The observed high mortality (approximately 20%
particularly in the third trimester) of hepatitis E during human pregnancy was not reproducible in preg-
nant rhesus macaques.153,280
Rhesus monkeys infected with one Indian HEV isolate were found to be immune 1.5–2.75 years after
the primary infection when challenged with closely related Indian strains.1 Further, a long-term serologi-
cal follow-up of HEV immunity in experimentally infected rhesus monkeys indicated that anti-HEV IgG
titers declined to undetectable levels, but still protected the animals from homologous and heterologous
HEV challenge 5 years after the initial infection.64 It was previously documented that naturally acquired
anti-HEV in nonhuman primates renders them resistant to experimental infection.153
In general, rhesus monkeys and cynomolgus macaques were found to be most suitable model systems
for hepatitis E vaccine efficacy studies.195,281–286 Macaques immunized with an HEV candidate vaccine
developed using one genotype showed protection against homologous and heterologous HEV challenges.
The noteworthy vaccine studies that were taken further for phase 2 and phase 3 trials in populations of
hepatitis E endemic countries were from Nepal and China.
Cumbersome procurement procedures, limited animal resources, and ethical concerns have limited
the use of nonhuman primate models in HEV research today.

4.2.3.2 Pigs
HEV research and animal model development got a boost after Meng et al.159 discovered a novel strain
of the virus in pigs. As discussed earlier, swine serve as a major reservoir for zoonotic genotype 3 and 4
HEVs. Wild boars are assumed to be a natural reservoir of HEV. Although attempts to infect pigs with
genotype 1 or 2 of human HEVs were not successful,155 they were susceptible to genotype 3 and 4 human
HEV strains under experimental conditions.156,287 The pig model system has been successfully used to
study the structural and functional relationship of HEV genes and in understanding the mechanism of
HEV replication, pathogenesis, and species specificity.
Pathogenesis study with the specific-pathogen-free (SPF) pig model using a genotype 3 human HEV
strain revealed more severe and persistent hepatic lesions (multifocal lymphoplasmacytic hepatitis and
focal hepatocellular necrosis) than those infected with the swine HEV strain.156 Extra hepatic sites of
HEV replication observed were the small intestine, lymph nodes, and colon.288, 289 Pigs also shed virus in
feces, became viremic, and seroconverted to IgG anti-HEV when infected with human HEV genotype 4
(Taiwanese strain).287 The HEV pig model was used to determine the infectivity of the first infectious
clone of HEV genotype 3.262 This model also helped in the identification of the actual initiation site of the
HEV ORF3. Recently, HEV genotype 3 mutants with deletions in the HVR were shown to be infectious
in pigs, indicating dispensability of the HVR during HEV replication and infection. Thus, pigs seem to
be a promising model of HEV infection, as they are relatively inexpensive to house, feed, maintain, and
breed than nonhuman primates. However, since they do not reproduce hepatic disease with overt clinical
symptoms, their usefulness in understanding HEV pathogenesis will be limited.

4.2.3.3 Rodents
The rat strain of HEV was first identified in Hamburg, Germany, in 2009.41 Huang et al.290 have reported
the successful infection of Balb/c nude mice with a strain of genotype 4 HEV recovered from a pig; how-
ever, Li et al.291 failed to infect C57BL/6 mice with genotype 1, 3, and 4 HEV strains. Wistar rats could
be infected with the human strain of HEV,292 but subsequent studies aimed to confirm that the transmis-
sion of genotypes 1, 2, and 4 of human HEV and an avian HEV was unsuccessful. Mongolian gerbils
(desert rats) were recently shown to be susceptible to experimental infection with a genotype 4 strain
of HEV.293 Viremia and fecal virus shedding were detected in inoculated gerbils, and HEV antigen was
60 Laboratory Models for Foodborne Infections

detected in the liver, kidneys, spleen, and small intestine. Naturally infected rats do not show any HEV-
related illness. In experimental infections, laboratory rats infected with rat HEV showed seroconversion
and fecal shedding of the virus without any apparent clinical signs. Histopathological analysis of tissues
revealed mild portal inflammation, foci of parenchymal necrosis, and aggregates of lymphocytes, and
Kupffer cells within the lobules, indicating evidence of mild hepatitis consistent with human acute HEV
infection. Availability of a small animal model would give a boost to HEV research. Although labora-
tory mice and rats have been found to be susceptible to HEV, the lack of reproducibility is the major
drawback, and more work is needed in optimizing this model.

4.2.3.4 Rabbits
The potential of rabbits as an animal model was tested after the discovery of genotype 3 virus in farmed
rabbits from the Gansu province in China.26 Subsequent to these findings, HEV strains related to gen-
otype 3 were detected in farms as well as in wild rabbits in the United States and France. Rabbits
inoculated with rabbit HEV shed virus in the feces, become viremic, and have elevated levels of serum
ALT with irregular multifocal lymphohistiocytic infiltrates and local hepatocellular necrosis.294 Rabbits
experimentally inoculated with human HEV genotype 4 showed viremia and fecal virus shedding in 2/9
rabbits but in none inoculated with genotype 1 human HEV.294 This indicated that the rabbit model of
HEV infection may be useful for studying genotype 3 and 4 HEVs; however, its use for studying geno-
types 1 and 2 would be limited.
Han et al.295 utilized the rabbit model to see whether they can establish an animal model for chronic
HEV infections. On infecting SPF rabbits with a homologous rabbit HEV isolate and a heterologous
genotype 4 swine HEV, they observed the development of chronic hepatitis and an associated liver fibro-
sis in some rabbits infected with homologous HEV. Persistent fecal shedding of the virus and elevated
liver enzymes were evident for more than 6 months after infection in the chronic infections. The detec-
tion of both the positive-/negative-sense RNA and HEV antigen in the extra hepatic tissues such as
brain, stomach, duodenum, and kidney further confirmed similarity with human chronic HEV infection.
However, the underlying mechanism was not clear. In humans, HEV can establish chronic infections
only in immunocompromised individuals, this chronic disease pathology was not observed in the rabbits
infected with the heterologous genotype 4 swine HEV. Rabbit HEV has 31 amino acid insertions in the
X-domain, which probably broadened the tissue specificity of the virus as previously documented by
Shukla et al.261 in the case of chronic HEV infection in human patients.

4.2.3.5 Ferrets
HEV was first detected in ferrets (Mustela putorius) in Netherlands43 followed by detection in the United
States. Phylogenetic analysis has shown that ferret HEV strains are closer to rat strains. Nucleotide
sequence analyses indicated that the ferret HEV genome shares the highest nucleotide sequence identity
(72.3%) with rat HEV. A putative ORF4 was observed in the ferret HEV genome similar to the rat HEV
genome. Information about ferret HEV epidemiology, distribution, transmission, and pathogenesis is still
not available. Usefulness of ferret HEV as an animal model needs to be evaluated considering its small
size and extensive use as an animal model for studying the pathogenesis of respiratory viruses.

4.2.3.6 Chickens
Haqshenas et al.296 discovered avian HEV and facilitated the use of chickens as a model system for avian
HEV infection. Avian HEV has been shown to cross species barriers and infect turkeys.163 Although chick-
ens are not susceptible to human strains, upon inoculation with avian HEV, infection results in subcapsular
hemorrhages, focal lymphocytic periphlebitis, and phlebitis in the liver; extra hepatic sites of HEV repli-
cation are also evident.297 A recent study by Kwon et al.265 showed that an infectious cDNA clone of the
variant avian HEV (isolated from healthy chicken) could still induce histological liver lesions. This study
also proved that RNA transcripts may serve as an alternative for live virus in animal pathogenesis studies.
Hepatitis E Virus 61

Avian ORF2 capsid protein expressed in E. coli having a similar antigenic structure as that of
human HEV containing major neutralizing epitopes was assessed for immunogenicity in chickens.
All the tested mock-immunized control chickens developed typical avian HEV infection character-
ized by viremia, fecal virus shedding, and seroconversion to avian HEV antibodies, while none of the
tested chickens immunized with avian HEV capsid protein had detectable viremia, fecal virus shed-
ding, or observable gross hepatitis lesions. The results from this study suggested that immunization
of chickens with the avian HEV recombinant ORF2 capsid protein with aluminum as the adjuvant can
induce protective immunity against avian HEV infection.298 Overall, avian HEV seem to be useful
for HEV studies.
Animal models such as chimpanzees, rhesus/cynomolgus macaques, owl monkeys, rodents, pigs,
chickens, and rabbits have been used for studying HEV biology. Although nonhuman primates and pigs
seem to be useful animal models, ethical issues, high prices, and difficulties in handling, manipulating,
and housing these animals are major hurdles. Rats seem to be a promising laboratory model due to their
small size and easy handling. Table 4.2 shows different animal models being used for HEV studies.

4.3 Conclusions
HEV transmission occurs primarily due to contaminated water in developing countries while transmis-
sion via uncooked or partially cooked pig meat and meat products is mainly responsible for hepatitis E
infection in developed countries. For waterborne transmission, effective prevention and control depends
on ensuring a safe drinking water supply, adequate sanitation, and proper personal and environmental
hygiene. For the prevention of food-borne infections, a stringent screening of meat products is needed.
HEV infections in pigs are mostly silent and difficult to detect with traditional meat-screening proce-
dures. In such situations, a quick on-site diagnostic test is needed. PCR-based detection is comparatively
time consuming and needs well-equipped laboratories.
The proper and timely diagnosis of human HEV infections in nonendemic regions is technically chal-
lenging. Lack of consistency of serological tests and viral load quantification in terms of sensitivity and
specificity are the limiting factors. Future research should focus on developing broadly applicable sero-
logical and molecular assays for hepatitis E diagnosis.
The unsolved issues such as the relative importance of HEV transmission pathways, inactivation prop-
erties of the virus, and dose–response relationship of HEV infection need to be addressed. It is essential
to know the factors that lead to clinical hepatitis E infection in humans. For this, there is a need for a
practical animal model that would mimic the natural course of HEV infection in humans and its out-
come. With the ever expanding host range of HEV, we hope to find new animal strains that can be used
as model systems. Development of a reliable cell-culture system(s) that would support the complete life
cycle of HEV will help in knowing the virus better. Most therapeutic approaches and discovery efforts
to suppress HEV propagation will not progress unless we have these basic tools.

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5
Noroviruses: Laboratory Surrogates for
Determining Survival and Inactivation

Doris H. D’Souza and Snehal S. Joshi

CONTENTS
5.1 Introduction..................................................................................................................................... 75
5.2 HNoVs and the Use of Surrogates................................................................................................... 76
5.3 Human Feeding Studies and Associated Drawbacks..................................................................... 77
5.4 Animal Viruses as HNoV Surrogates and Model Systems to Study HNoVs................................. 77
5.4.1 Feline Calicivirus............................................................................................................... 77
5.4.2 Murine Norovirus............................................................................................................... 80
5.4.3 Tulane Virus....................................................................................................................... 84
5.4.4 Porcine Sapovirus............................................................................................................... 85
5.4.5 Bovine Noroviruses............................................................................................................ 86
5.4.6 Rabbit Caliciviruses........................................................................................................... 86
5.4.7 Norovirus-Like Particles ................................................................................................... 86
5.5 Bacteriophage MS2......................................................................................................................... 87
5.6 Conclusions..................................................................................................................................... 88
Acknowledgments..................................................................................................................................... 88
References................................................................................................................................................. 88

5.1 Introduction
Noroviruses (NoVs) belong to the Caliciviridae family that currently comprises of five genera: Norovirus,
Sapovirus, Lagovirus, Vesivirus, and Nebovirus, with potentially additional genera as new sequence
information becomes available on unclassified viruses [1,2]. The name “Norovirus” was shortened from
the initial “Norwalk-like” virus term that was associated with an epidemic outbreak that occurred in
Norwalk, Ohio, in 1968 in an elementary school [3]. Infected school children upon returning home trans-
mitted the secondary infection to family members resulting in 50% of students and teachers developing
nonbacterial gastroenteritis.
NoVs are small viruses about 27–32 nm in size and round in structure with an icosahedral sym-
metry. The human norovirus (HNoV) genome contains a single-stranded positive-sense RNA about
7.6 kb in length that is enclosed in a capsid without an envelope [3]. The capsid is made of 90 cap-
somers protruding from the shell that has 90 dimers of capsid protein. The genome has three open
reading frames (ORFs). ORF1 (nucleotides 146–5359) is about 5 kb in size and encodes a ∼200 kDa
nonstructural polyprotein. This nonstructural protein is cleaved to produce the N-terminal protein, the
enzyme nucleoside triphosphatase, a 3A-like protein, a genome-linked viral protein (VpG), a 3C-like
protease, and RNA-dependent RNA-polymerase (RdRp) [4]. ORF2 (nucleotides 5346–6935) is ∼1.8 kb
in size and encodes the 57 kDa major structural capsid viral protein VP1; ORF3 (nucleotides 6938–
7573) is ∼0.6 kb in size and encodes a small 22 kDa minor viral structural protein, VP2, reported to
package the genome into virions [5,6]. The NoV genus at the time of this submission, is composed of

75
76 Laboratory Models for Foodborne Infections

five genogroups based on sequence analysis: genogroup I (GI) (prototype Norwalk virus); GII (pro-
totype Snow Mountain virus); GIII (prototype bovine enteric calicivirus); GIV (prototype Alphatron
and Ft. Lauderdale viruses); and GV (prototype Murine NoV) [7,8].
From the five genogroups of NoV, genogroups GI, GII, and GIV specifically infect humans (and are
commonly referred to as human noroviruses or HNoV) and as indicated above, GIII includes bovine
enteric calicivirus that infects cattle, and GV is associated with the infection of mice. There have been
at least 32 genetic clusters identified based on the amino acid-sequence similarity between these geno-
groups [1,9]. Eight genotypes are recognized in GI and 19 genotypes are recognized in GII, and of these,
HNoV genotype GII.4 has been the most prevalent during the past few decades. Indeed, the majority of
HNoV outbreaks are reported to be caused by the GII.4 genocluster and its variants, where in 2002, a
GII.4 variant, called the Farmington Hill strain, was responsible for 80% of acute HNoV outbreaks in the
United States [10]. In addition, the Hunter strain GII.4 variant was found to be circulating in Australia,
Europe, and Asia in 2004, and then the Sakai strain (reported in Southeast Asia) and Minerva strain
(found in the United States and the Netherlands) were found to be circulating in 2006, replacing the
Hunter GII.4 variant [11]. Another HNoV GII.6 strain (previously not reported in California) classified
as Seacroft was reported to be responsible for causing illness in 30 individuals at a local university in
Los Angeles, California, in October 2008 [12]. It is also noteworthy to report that outbreaks of the HNoV
genogroup GII.4 in health-care settings in the United States, Europe, and Oceania were responsible for
70%–80% of all HNoV outbreaks [13]. The HNoV strains continue to remain a worldwide concern and
are continually evolving, with these emerging HNoV GII.4 variants also becoming virulent and known
to cause death in the elderly and immunocompromised [14].
HNoVs have a very short incubation period of 18–48 h, with typically self-limiting infection that lasts
for up to 72 h, characterized by mild gastroenteritis symptoms that include nausea, vomiting, abdomi-
nal cramps, fever, and malaise. “Winter vomiting disease” is the common seasonal syndrome associ-
ated with HNoV infection, with peak outbreaks in winter, even though HNoV transmission reportedly
occurs all year round [15]. It is also important to keep in mind that from a public health aspect, infected
individuals can shed viruses for 72 h (and even longer, greater than a week) after signs of the first symp-
toms appear [16]. Outbreaks have been reported in restaurants, cruise ships, health-care settings, closed
environments, and nursing homes, either due to the consumption of at-risk ready-to-eat or undercooked
contaminated foods, such as seafood, shellfish, produce, deli items, and bakery products, and also due to
environmental transmission and person-to-person transmission [17]. Therefore, adequate control strate-
gies, proper sanitation, food processing, storage, and hygienic practices of workers are important to
prevent further transmission especially in food environments and health-care settings.

5.2 HNoVs and the Use of Surrogates

HNoVs are epidemiologically significant as they are considered the leading cause of nonbacterial
gastroenterititis to date worldwide. Given the prevalence of HNoV outbreaks worldwide and the
spread of emerging viruses that cause severe symptoms even leading to death, and the fact that
HNoVs cannot be cultivated in cell culture in the laboratory to date, cultivable viral surrogates are
used to determine the infectivity and inactivation of HNoVs. In fact, with regard to the cultivation
and propagation of HNoVs, even 3D cell-culture models using Int-407 or Caco-2 cells did not sup-
port HNoV replication [18]. However, more recently, it was reported that Histoblood group antigen
(HBGA)-expressing enteric bacteria were required for the HNoV infection of B cells, though vali-
dation of HNoV replication in these cells is still pending, and the replication of HNoV needs to be
demonstrated and validated by researchers from other laboratories [19].
Due to the numerous issues and challenges facing the cultivation of HNoVs, cultivable animal viral
surrogates such as feline calicivirus (FCV-F9), followed by murine norovirus (MNV-1) and then Tulane
virus (TV) and porcine enteric sapovirus have been considered as alternate HNoV surrogates to enable
progress in research toward prophylactic therapies and control measures against HNoVs. Initially, during
environmental studies, bacteriophage MS2 (viruses that infect bacteria) was also used as a surrogate to
determine survival and persistence.
Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation 77

5.3 Human Feeding Studies and Associated Drawbacks

Indeed, it is apparent that humans are the most relevant models to study HNoV biology and replication.
However, for these feeding or challenge studies, volunteers are needed along with privacy protection,
requiring appropriate institutional and board approvals, labor, cost, and time. Also, health-care facilities
should be easily accessible with a team of skilled technicians, scientists, researchers, and health-care
professionals. In addition, as noted by other researchers [20], sampling difficulties and expenses, biocon-
tainment due to the presence of live and infectious viruses, fecal and sera sample collection and storage,
invasive biopsies, and monitoring of the health of eligible volunteers need to be also considered as some
limitations. It must be noted that feces and sera provide valuable data to understand HNoV survival,
persistence, prevalence, disease symptoms, mechanisms of associated diarrhea, immune responses,
and diagnostics and also have potential to develop vaccines and control strategies [20]. In fact, human
volunteer studies played a key role in resistance marker identification (related to HBGA blood groups)
and understanding the immune selection mechanism responsible for the persistence of HNoV GII.4 in
human populations [20].
Thus, the only source of live infectious HNoVs remains the human population during outbreaks
and/or human volunteer feeding or challenge studies. A randomized, double-blinded clinical trial was
carried out to determine the inactivation of Norwalk virus (HNoV genogroup I.1) by high hydrostatic
pressure processing (HPP) in virus-seeded oysters that were ingested by adults [21]. Healthy, positive-
secretor adults who consumed oysters artificially seeded with Norwalk virus (8FIIb, 1.0 × 104 genomic
equivalent copies) that were treated with 600 MPa HPP at 6°C for 5 min (but not 400 MPa at 6°C
or 25°C) did not show any HNoV infection, as determined using reverse transcription-PCR of their
stool or vomitus samples, indicating the complete inactivation of HNoV in seeded oysters [21]. Thus,
these researchers showed the use of HPP as a potential intervention to inactivate infectious HNoV in
oysters for the commercial shellfish industry. In another earlier clinical trial carried out in Australia,
shellfish testing positive for HNoV were depurated and subsequently fed to volunteers who became ill,
which demonstrated that commercial depuration was not effective in eliminating HNoV in contami-
nated oysters [22]. Human feeding studies do provide reliable results on the effectiveness of inactivation
methods, and represent the best model for determination of the inactivation, pathogenesis, and immu-
nity of HNoVs for research in the absence of cell-culture infectivity assays and inability to discriminate
infectious from noninfectious HNoVs. Richards has suggested and recommended a movement from
surrogate research to human volunteer studies to identify practical and valid processing methods for
improving food safety [23]. However, due to research-funding limitations, extensive labor, and the time
associated with human feeding studies, other models and surrogates have to be relied on to determine
the best approaches for HNoV inactivation.

5.4 Animal Viruses as HNoV Surrogates and Model Systems to Study HNoVs

5.4.1 Feline Calicivirus
FCV-F9 belongs to the genus Vesivirus of the Caliciviridae family and is responsible for acute oral and
upper respiratory tract disease in cats characterized by oral ulcerations, limping syndrome, and ocular
and nasal discharge [24]. It is typically transmitted via the nasal, oral, or conjunctival routes [25]. This
virus has been extensively utilized as a model surrogate for HNoVs and was the first virus surrogate
to be used to conduct research on HNoV inactivation, since HNoVs are considered unculturable in the
laboratory to date as mentioned above [26]. FCV-F9 was considered a suitable model system as it is a
nonenveloped single-stranded RNA virus with an approximate diameter of 35–39 nm and icosahedral
symmetry, being similar to HNoVs in these aspects. Also, the RNA genome is 7.5 kb in size and has
three ORFs [27]. However, there are many issues with this surrogate that question its suitability as a
surrogate and model system. FCV-F9 is transmitted through the respiratory route unlike the fecal–
oral route of HNoVs. Additionally, given its respiratory route of transmission and sensitivity to the low
78 Laboratory Models for Foodborne Infections

pH of 2.0–4.0 encountered in the gastrointestinal tract [6], unlike HNoVs that are known to be resistant
to low pH conditions, alternate surrogates and model systems are being researched.
FCV-F9 has been utilized as a surrogate in determining improved inactivation strategies against
HNoVs in foods and on contact surfaces, such as chemical, nonthermal, and thermal treatments (refer
to Tables 5.1 through 5.3). Broad-spectrum contact surface disinfectants have been evaluated using

TABLE 5.1
Effect of Chemicals on the Feline Calicivirus (FCV-F9) Used as a Cultivable HNoV Surrogate to Develop
Disinfection and Control Strategies
Virus Treatment Reduction (log PFU/mL) References
Feline calicivirus Hydrogen peroxide vapors; 25 mL volume: [70,109]
70% ethanol, 5 min 2.6
Formaldehyde; 0.7%, 60 min at 20°C 4 [110]
Glutaraldehyde; 0.5%, 1 min at room temperature 5 [26]
Bacoban WB (quaternary ammonium compound); 4 [110]
2%; 240 min at 20°C
Benzalkonium chloride; 0.25 mg/mL, 2 h at room 3.1 [28]
temperature
β-Propiolactone; 0.1%, 60 min at 22°C 5.2 [111]
Free chlorine; 500 ppm, 10 min at room temperature 1.9 [112]

TABLE 5.2
Effects of Surface Inactivation Methods Using Feline Calicivirus (FCV-F9) as a Cultivable HNoV Surrogate
Virus Medium Treatment Reduction References
Feline calicivirus Plastic surfaces Steam (130°C)–ultrasound 30–50 kHz for 3 s 4.8 log [36]
Low protein virus stock Gamma radiation 5.9 log PFU [113]
1 µM methylene blue; human plasma, 5 min 3.9 log [114]
illumination
Virus + cell-culture media 200 MPa—0°C, 4 min 4.4 log [115]
250 MPa—0°C, 4 min 4.8 log
300 MPa—10°C, 3 min 5 log
Glass dishes ClO2 gas (1.7 mg/h at 25°C for 5 h) 3 log [116]

TABLE 5.3
Evaluation of Heat Treatments on Feline Calicivirus (FCV-F9) as a HNoV Surrogate
Virus Medium Inactivation Parameters References
Feline calicivirus Buffer 56°C; D-value 6.36 [73]
65°C; D-value 0.32
72°C; D-value 0.11
Seafood 56°C; D-value 3.33 [75]
65°C; D-value 0.33
72°C; D-value 0.07
Spinach 56°C; D-value 5.83 [40]
65°C; D-value 0.27
72°C; D-value 0.15
Virus stock 56°C; D-value 3.473 [6]
63°C; D-value 0.435
72°C; D-value 0.166
Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation 79

FCV-F9 for their antiviral properties. Potassium peroxymonosulfate (KPMS) at concentrations of 5, 10,
and 20 mg/mL was reported to reduce FCV-F9 titers to undetectable levels from an initial 5 and 7 log
PFU/mL within 2 h at room temperature [28]. Similarly, benzalkonium chloride (BAC) at 0.1, 0.25, and
0.5 mg/mL was reported to reduce FCV-F9 by 2.87, 3.08, and 3.25 log PFU/mL from an initial 7 log PFU/mL
after 2 h at room temperature [28]. D’Souza and Su reported that FCV-F9 could be inactivated to non-
detectable levels (6.84 log PFU/mL reduction) after a contact time of 30 s with 10% bleach (0.6% sodium
hypochlorite, 5000 ppm available chlorine) and 2% trisodium phosphate (TSP) [29]. A neutral solution
of electrochemically activated anolyte Ecasol was reported to reduce FCV-F9 titers by more than 5 log
within 1 min of treatment at room temperature [30].
FCV-F9 was also used as a model virus in a study to determine the effect of chlorine dioxide (ClO2) gas
against the virus inoculated on glass surfaces in a test room (39 m3), where a constant low-concentration
ClO2 gas was produced [31]. These researchers found that a low-concentration ClO2 gas (mean 0.05 ppmv,
0.14 mg/m3) inactivated FCV-F9 (by >2 logs) in the wet state on glass dishes within 5 h. Other research-
ers used FCV-F9 as a cultivable HNoV surrogate and model to determine the effect of aqueous ClO2
at concentrations ranging from 0.2 to 0.8 mg/L at pH 7 and 20°C, and found that at a concentration of
0.8 mg/L ClO2, a complete elimination of FCV-F9 was obtained in 2 min, while lower concentrations
of 0.2 mg/L required 30 min for inactivation [32]. FCV-F9 was also used as a model to determine the
inactivation of enteric viruses by ozone treatment, where for a 4-log (99.99%) inactivation of FCV at 5°C
and pH 7 about <0.01–0.03 mg/L min of ozone was required [the Ct value which is described as the con-
centration of ozone multiplied by the virus contact time, using application of the efficiency factor Hom
(EFH) model] [33]. In another study using FCV-F9 as an HNoV surrogate, ozone treatment at 6.25 ppm
for 4 min showed >6 log TCID50/mL reduction of FCV-F9 in water and ∼2-log TCID50/mL on lettuce and
green onions [34].
High-pressure processing and ultraviolet (UV) light treatments are commonly used processing methods
in the food industry. A study reported a decrease of 3.48, 3.82, and 4.62 log TCID50 in FCV-F9 titers on let-
tuce after exposure to UV light at doses of 16, 40, and 75 mW s/cm2, respectively [35]. High HPP treatments
of 500 and 600 MPa were reported to reduce FCV-F9 titers to undetectable levels after 1, 3, 5, and 7 min,
while lower pressures of 300 and 400 MPa caused 1.13 and 0.55 log decreases after 1 min, respectively [32].
A combination of pressurized steam (130°C) and high-power ultrasound (30–50 kHz) was reported to
reduce FCV-F9 titers on plastic surfaces by >99.99% corresponding to >4.8-log after 3 s of treatment [36].
FCV-F9 in milk showed reductions of ≥4 log PFU/mL after high-pressure homogenization at 300 MPa,
∼1.3 log PFU/mL at 250 MPa, and insignificant reduction at ≤200 MPa [37]. These researchers showed that
in orange juice, FCV-F9 was reduced by ≥4 and ∼1 log PFU/mL only after high-pressure homogenization at
300 and 250 MPa, respectively [37]. FCV-F9 in PBS at titers of ∼4 log PFU/mL after a treatment at 20 kHz
of high-intensity ultrasound (HIU) for 5 min could be reduced to undetectable levels [38].
FCV-F9 has also been used as a model/surrogate for small, round-structured viruses on lettuce, where
the lettuce was inoculated with FCV-F9 by immersion to simulate contamination from irrigation or wash
water. Then the lettuce was subjected to electron beam irradiation at various dose levels [39]. These
researchers reported the D10-value of FCV-F9 on lettuce to be 2.95 kGy. When FCV-F9 was used as a
model to determine heat inactivation of HNoVs in spinach, the D-values calculated from the first-order
model (50°C–72°C) ranged from 0.15 to 17.39 min [40].
Significant research has been conducted on natural oils and extracts for their antiviral properties,
including 0.25% natural mulberry (Morus alba) juice that caused a 50% reduction in FCV-F9 titers [41].
Essential oil thymol at 0.5% and 1% was reported to reduce FCV-F9 titers to undetectable levels from an
initial 6 log TCID50 after 2 h at 37°C [42]. Black raspberry juice showed antiviral activity when Crandell
Reese feline kidney cells were pretreated with the juice or directly or FCV-F9 itself [43]. After treatment
with cranberry juice and cranberry proanthocyanidins at 0.30, 0.60, and 1.20 mg/mL for 1 h at room
temperature, FCV-F9 from initial 5 log PFU/mL was reported to be reduced to undetectable levels [44].
When FCV-F9 was used as a surrogate to determine the effect of grape-seed extract (GSE) as a wash to
disinfect lettuce and jalapeno peppers, FCV-F9 was reported to be reduced by 2.33, 2.58, and 2.71 log
PFU on lettuce, and 2.20, 2.74, and 3.05 log PFU on peppers after 1 min using 0.25, 0.50, and 1 mg/mL
GSE, respectively [45]. Thus, FCV-F9 has also been extensively used as a model to understand antiviral
options.
80 Laboratory Models for Foodborne Infections

The survivability/persistence of FCV is surprisingly high on surfaces and was detectable for up to
28 days at room temperature [26]. FCV-F9 was detected on three surfaces (formica, stainless steel,
and ceramic) for up to 7 days postinoculation at room temperature [46]. A persistence/survival study of
FCV-F9 at storage (7°C) and room temperatures over 70 days showed that the infectivity of FCV-F9 at
room temperature decreased after 6 h, 1 day, and 7 days by 1 log, 3 log, and to below the detection limit
(∼4.5 log), respectively [47]. However, FCV-F9 was shown to survive longer at 7°C, with only a reduction
of 2  log after 7 days and a titer decrease of 3 log after 70 days being reported [47]. Similarly, FCV-F9
at 6 log PFU/mL was reported to be reduced to undetectable levels after 14 days in orange as well as
pomegranate juice and by ∼3 logs after 21 days in milk at 4°C [48]. However, FCV-F9 was reported to
be completely reduced to undetectable levels after 1 day in the blend of orange and pomegranate juice
at 4°C [48]. In another study, FCV-F9 (at initial titers of ∼5 log PFU/mL) was reported to be reduced to
undetectable levels after 1 day in blueberry juice at 4°C [49].
Thus, FCV-F9 has been widely used as a surrogate for HNoVs due to its ease of cultivation in vitro,
its relatively straightforward infectivity assays to develop and understand antiviral agents and for envi-
ronmental persistence and stability studies, and because it is readily manipulated genetically for use
as a model for replication and translation [20]. The development of the FCV reverse genetics system
made FCV a desirable model to study the molecular mechanism of calicivirus translation and genome
replication [50]. Infection with FCV was reported to lead to the inhibition of cellular protein synthesis
associated with the cleavage of host translation initiation factors [51] and also to the first report and
identification of a functional protein receptor molecule for a calicivirus [52]. Subsequent studies with
FCV showed conformational changes in the viral capsid protein upon receptor binding and FCV was
used to identify the first functional host cell factor–viral genome interaction required for FCV replica-
tion. Similar interactions are reported to occur for HNoVs [20]. However, when compared to the other
surrogates including MNV-1 and the newer cultivable TV, FCV does not appear to be the most robust
surrogate model for the determination of HNoV survival and inactivation, specifically because it causes
a respiratory or systemic disease in some cases, unlike the gastrointestinal illness caused by HNoVs.

5.4.2 Murine Norovirus
MNV-1 is a member of the Caliciviridae family and since it falls under the Norovirus genus, it shares
closer biological and molecular properties with HNoVs as compared to FCV-F9 [53]. Besides, MNV-1
can replicate to high titers in tissue culture having a dendritic cell and macrophage tropism and the infec-
tious assays are relatively straightforward as well. MNV-1 is clearly an intestinal pathogen spread via
the fecal–oral route, and MNV infection exacerbates the onset of inflammatory bowel disease similar to
HNoVs [20]. These properties make MNV-1 a suitable model or surrogate to study HNoVs. MNV-1 was
isolated from mice brain tissue with clinical symptoms including diarrhea, fever, nausea, and abdom-
inal pain [53,54]. Similar to HNoVs, with transmission via the fecal–oral route, MNV-1 is shed for
up to 8 weeks after infection and/or inoculation [2]. It is the only NoV that currently replicates in cell
culture in a reproducible manner, which has made HNoV surrogate studies adaptable and somewhat eco-
nomical [54]. On an average the size of this nonenveloped virus lies between 28 and 35 nm in diameter,
with an icosahedral symmetry. MNV-1 contains a single-stranded positive-sense RNA with three ORFs,
where ORF1 encodes a predicted 187.5-kDa polyprotein that comprises of the 2C helicase, 3C protease,
and 3D polymerase motifs typically observed in other caliciviruses and picornaviruses, while ORF2
encodes a 58.9-kDa capsid protein which self-assembles into virus-like particles (VLPs) in baculovirus
expression systems, and ORF3 encodes a putative 22.1-kDa basic protein [54].
As a laboratory model to understand HNoV survival, MNV-1 has been shown to be more stable and
survive longer under various environmental conditions than FCV-F9. Upon exposure to a low pH of 2
at 37°C for 30 min, MNV-1 titers were reported to be reduced by <1 log [6]. MNV-1 titers were found
to decrease by a mere 0.09 log PFU/mL/day in environmental waters at 25°C over 3–5 weeks [55]. In a
study conducted for its survival in the hospital environment, MNV-1 infectivity was reduced by <2 log
over 40 days at both −20°C and 4°C on the surface of gauze and diaper materials; however, at a higher
temperature of 30°C, a higher reduction of 5 log was reported after 24 days [56]. On stainless steel, under
wet and dry conditions, MNV-1 was shown to survive for 7 days with <2 log PFU/mL reduction at 4°C,
Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation 81

with similar reductions at room temperature [6]. MNV-1 is also known to survive freezing temperatures
of −21°C, and no change in infectivity on frozen onions and frozen spinach was reported after 6 months
[57]. MNV-1 was found to survive in commercial blueberry juice (BJ) even after 21 days at 4°C [37]. In
orange juice and milk, MNV-1 titers were not reported to be reduced after 21 days; however, in pome-
granate juice a reduction of 1.4 log PFU/mL was reported. MNV-1 was reduced to undetectable levels
after 7 days in the orange and pomegranate juice blend at 4°C [48]. Escudero et al. also showed that infec-
tious MNV-1 could be detected until 21 days on inoculated stainless steel, formica, or ceramic surfaces,
and did not persist on lettuce stored at room temperature or 4°C [58]. Taken together, these survival stud-
ies provide an indication of the ease of transmission of HNoVs in the environment.
MNV-1 has also been studied as a laboratory model to understand the effect of various inactivation
methods against HNoV (Tables 5.4 through 5.6). With free chlorine concentrations of 0.193 mg/L, MNV
infective titers were found to be reduced to undetectable levels within 2 min of contact time at 5°C and
1 min of contact time at 20°C [59]. Upon exposure to 0.255 mg/L ClO2, MNV-1 was reduced to unde-
tectable levels (<3.5 log) within 1 min of contact time at 5°C and 30 s of contact time at 20°C [59]. It

TABLE 5.4
Inactivation of Murine Norovirus (MNV-1) as an HNoV Surrogate by Physical and Chemical Approaches
Virus Treatment Reduction References
Murine norovirus 15 ppm active chlorine 1.4 log [60]
0.193 mg/L free chlorine; 2 min at 5°C 5 log PFU/mL [59]
0.255 mg/L ClO2; 1 min at 5°C 3.5-log PFU/mL [59]
100 ppm peroxyacetic-biocide; 2 min, room temperature 2.3 log RT-PCR units [60]
2.5% liquid H2O2; 5 min, room temperature 1 log PFU/mL [61]
6.25 ppm ozone; 10 min, room temperature 2.5 log PFU/plant [86]
2% trisodium phosphate; 30 s, room temperature 3 log PFU/mL [86]
0.2 mg/mL benzalkonium chloride; 2 h, room temperature 5 log PFU/mL [28]
5 mg/mL potassium peroxymonosulfate; 2 h, room temperature 5 log PFU/mL [28]
5% levulinic acid and 2% sodium dodecyl sulfate; 1 min 1.50 log PFU/mL [65]
Tween 20 (50 ppm) and chlorine (200 ppm); 1 h 3 log PFU/mL [66]
450-MPa; high hydrostatic pressure in cell-culture media 6.85 log PFU [117]
400-MPa; high hydrostatic pressure; oysters 4.05 log PFU [69]
300 MPa; high-pressure homogenization; blueberry juice 0.71 log PFU/mL [37]
UV at 25 mJ/cm2; 200 s 3.6 log [56]
Sonication; 20 kHz 4 log PFU/mL [38]

TABLE 5.5
Thermal Inactivation of Murine Norovirus (MNV-1) as a Cultivable Surrogate for HNoVs
Murine norovirus Buffered cell-culture media 56°C; D-value 3.74 [73]
65°C; D-value 0.77
72°C; D-value 0.25
Seafood 56°C; D-value 6.12 [75]
65°C; D-value 0.14
72°C; D-value 0.18
Spinach 56°C; D-value 3.29 [40]
65°C; D-value 0.40
72°C; D-value 0.16
Virus stock in cell-culture media, 72°C for 25 s 1 log PFU/mL [6]
Soft shelled clams; 90 s at 90°C 3.33 log cycles [76]
60°C for 5 min; buffer 3.03 log TCID50/mL [77]
60°C for 5 min 0.78 log TCID50/mL
82 Laboratory Models for Foodborne Infections

TABLE 5.6
Inactivation of Murine Norovirus (MNV-1) as a Cultivable HNoV Surrogate Using Natural Plant Extracts
Murine norovirus Grape seed extract (0.35 mg/mL); 37°C for 2 h 0.82 log PFU/mL [78]
Cranberry proanthocyanidins (0.6 mg/mL); 2 h, room temperature 2.9 log PFU/mL [44]
Pomegranate juice; 1 h, room temperature 1.32 log PFU/mL [79]
Pomegranate polyphenols (4 mg/mL); 1 h, room temperature 1.30 log PFU/mL [79]
Black raspberry juice (6%); 1 h at 37°C 99% reduction in plaque titers [43]
Aqueous hibiscus extracts (40 mg/mL); 6 h, 37°C 1.78 log PFU/mL [80]
Oregano oil (4%); 24 h, room temperature 1.07 log TCID50 [82]

was  reported that 15 ppm of active chlorine could reduce MNV-1 by 1.4 log units (detected by PCR)
and the application of 100 ppm peroxyacetic-based biocide on lettuce as a produce wash could decrease
MNV-1 titers by 2.3 log RT-PCR units in 2 min [60]. Liquid hydrogen peroxide (H2O2) at 2.5% when used
as an antiviral produce wash was reported to reduce MNV-1 by ∼1 log after 5 min [61]. Ozone is known to
decompose in the water phase of foods, though ozone might be able to diffuse and act on viruses as shown
in a study where the internalized MNV-1 in green onions was reduced by 2.5 log PFU/plant after an ozone
treatment of 6.25 ppm for 10 min [62]. When the green onion plants that were inoculated with MNV-1
were sprayed with calcium hypochlorite (150 ppm, 4°C) or ozone (6.25 ppm for 10 min), a reduction of 2
and 2.5 log PFU/plant, respectively, from an initial titer of 4.92 log PFU/plant was reported [63]. TSP at
2% and 5% after 30 s as a produce wash for lettuce and peppers was reported to reduce MNV-1 by 3 log
PFU/mL and to undetectable levels, respectively, from an initial titer of 5 log PFU/mL [64]. BAC, KPMS,
tannic acid (TA), and gallic acid (GA) were also evaluated for their antiviral effect using MNV-1 as a labo-
ratory model, and MNV-1 (5 log PFU/mL) was reported to be reduced to undetectable levels with BAC
at 0.2, 0.5, and 1 mg/mL after 2 h at room temperature [28]. KPMS at 2.5 and 5 mg/mL was reported to
reduce low-titer MNV-1 (5 log PFU/mL) to undetectable levels and high-titer MNV-1 (7 log PFU/mL) by
0.92 and 3.44 log PFU/mL, respectively, after 2 h at room temperature. TA at 0.1 mg/mL and GA at 0.2 and
0.4 mg/mL were reported to have caused no reduction in MNV-1 titers after 2 h at room temperature [28].
Another novel sanitizer consisting of 0.5% levulinic acid in combination with an anionic detergent
sodium dodecyl sulfate (SDS) at 0.5% was shown to decrease MNV-1 titers by 3 log PFU/mL after
1 min, while a solution of 5% levulinic acid and 2% SDS was reported to reduce MNV-1 titer by 1.50 log
PFU/mL after 1 min and by 3.3 log PFU/mL after 5 min on stainless steel surfaces [65]. Another study
explored the effects of combinations of surfactants (SDS) and polysorbates (Tween 20, Tween 65, and
Tween 80) along with the traditional chlorine (200 ppm) washes against MNV-1 from fresh produce [66].
The study reported that the combination of Tween 20 (50 ppm) and chlorine (200 ppm) after 1 h at room
temperature was most effective, with MNV-1 being reduced by 3 log PFU/mL from an initial titer of
∼6.5 log PFU/mL.
MNV-1 was also used as a laboratory model and surrogate for HNoV to determine inactivation by
physical and chemical inactivation methods that include high HPP, high-pressure homogenization, UV
light, and ozone. The high hydrostatic pressure inactivation of viruses is shown to be due to changes in the
capsid proteins or by disruption of capsid proteins and thereby their inability to bind to host cells [67,68].
MNV-1 titers after a high hydrostatic pressure treatment of 450-MPa were shown to decrease by 6.85 log
PFU in cell-culture media [69]. A reduction of 4.05 log was reported in MNV-1-contaminated oysters
after 5 min at 5°C at 400-MPa high hydrostatic pressure [69]. The high hydrostatic pressure treatment
of 300 MPa was shown to inactivate MNV-1 completely from initial titers of ∼7–8 log PFU/mL after
1 min [70], while MNV-1 in inoculated clams was reported to be reduced by a mere 1 log after high
hydrostatic pressure treatment of 400 MPa for 5 min [71]. When the effect of UV treatment on MNV-1
infectivity was evaluated, 3.6 log reductions after 200 s with UV at 25 mJ/cm2 were obtained [56]. When
green onions were inoculated with MNV-1 and treated with UV (240 mJ s/cm2), 1.2 log PFU reduction
per plant was obtained from an initial titer of 4.92 log PFU/plant [62]. These researchers also showed
that high hydrostatic pressures (500 MPa for 5 min at 20°C) could decrease MNV-1 to undetectable lev-
els. When MNV-1 was used as a surrogate to understand the effect of electron beam irradiation against
Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation 83

enteric viruses, the electron-beam dose required to reduce MNV-1 titers by 90% (D10-value) in whole
oysters was reported to be 4.05 ± 0.63 kGy [72].
When sonication was studied as another physical inactivation method for HNoV, MNV-1 titers were
found to be decreased by ∼4 log PFU/mL after a 30-min treatment at 20 kHz [44]. When exposed to high-
pressure homogenization, MNV-1 in blueberry juice was reported to be reduced by 0.33 log PFU/mL at
250 MPa and by 0.71 log PFU/mL at 300 MPa [37]. Studies carried out by Bozkurt et al. aimed to charac-
terize the thermal inactivation kinetics of MNV-1 as a laboratory surrogate to understand the inactivation
of HNoV at 50°C, 56°C, 60°C, 65°C, and 72°C in buffer as well as different food samples including spin-
ach and blue mussels [40,73,74]. The D-values for MNV-1 using the capillary tube method at 50°C, 56°C,
60°C, 65°C, and 72°C were reported to be 34.49, 3.65, 0.57, 0.30, and 0.15 min, respectively [73]. In buff-
ered cell-culture media, D-values of 36.28, 3.74, 1.09, 0.77, and 0.25 at 50°C, 56°C, 60°C, 65°C, and 72°C,
respectively, were reported [74]. In blue mussels, the reported D-values were 20.19, 6.12, 2.64, 0.41, and
0.18 at temperatures of 50°C, 56°C, 60°C, 65°C, and 72°C, respectively [75]. In spinach, the D-values were
comparatively lower than in blue mussels, where values of 14.57, 3.29, 0.98, 0.40, and 0.16 were obtained
at 50°C, 56°C, 60°C, 65°C, and 72°C, respectively [40]. The effect of heat treatment on MNV-1-infected
soft shell clams was studied using RT-PCR as a method of detection that showed a reduction of 3.33 log
cycles after 90 s at 90°C and 5.47 log cycles after 180 s at 90°C [76]. MNV-1 was reported to be reduced by
3.03, 3.69, 4.35, 5.05, and 5.88 log TCID50/mL in suspensions at temperature–time combinations of 60°C
for 5 min, 60°C for 15 min, 60°C for 30 min, 85°C for 3 min, and 85°C for 6 min, respectively, and in dried
mussels, titer reductions of 0.78, 2.00, 3.35, 1.95, and 3.20 log, respectively, were reported [77].
A variety of natural plant and fruit extracts have been examined for their antiviral properties against
enteric viruses using MNV-1 as a cultivable laboratory surrogate for HNoVs. GSE at 0.25, 0.5, and 1.0 mg/
mL was shown to reduce MNV-1 by 0.82, 1.35, and 1.73 log PFU/mL, respectively, at 37°C for 2 h [78].
Cranberry proanthocyanidins were also reported to decrease MNV-1 titers by 1.6, 2.4, and 2.9 log PFU/
mL after 0, 10, and 60 min, respectively [44]. Su et al. showed that commercial pomegranate juice could
reduce MNV-1 by 1.32 log PFU/mL, while pomegranate polyphenols at 4 mg/mL reduced MNV-1 by
1.30 log PFU/mL after 1 h at room temperature [79]. Black raspberry juice (6%) was also shown to have
antiviral effects on MNV-1 causing reduction in plaque titers by 99% after 1 h [43]. Cotreatment (simul-
taneous addition of treatment and virus to the host cells) was demonstrated to exert maximal antiviral
activity on MNV-1, suggesting that the juice exerted its antiviral effect by inhibiting viral attachment to
host cells, though gallic acid and quercetin were not shown to have any significant effect [43]. Aqueous
hibiscus extracts were shown to reduce MNV-1 by 1.89 and 1.78 log PFU/mL after 6 h with 100 and
40 mg/mL of the extract, respectively, and to undetectable levels after 24 h with both 100 and 40 mg/mL
of the extract at 37°C, respectively [80]. When host cells RAW 264.7 were pretreated with Korean Red
Ginseng at 5, 6.7, and 10 μg/mL, a reduction in the infectivity of MNV-1 by 0.38 ± 0.41, 0.73 ± 0.19,
and 1.48 ± 0.27 log TCID50/mL from an initial titer of 6.7 ± 0.2 log TCID50/mL after 24 h at 37°C was
reported [81]. Oregano oil at 4% was reported to cause a reduction of 1.07 log TCID50 in MNV-1 titers after
24 h, while carvacrol caused a 3.87 log TCID50 decrease after 1 h at room temperature [82].
As immune knockout mice are readily available, MNV-1 is often used as a model to study the immune
responses to HNoV infection and to examine the host responses required for norovirus pathogenesis,
clearance and establishment of immunity, and insights for the future development of vaccines [21].
MNV-1 has also gained popularity as the surrogate or model system of choice to study HNoVs due to the
development of two reverse genetics systems, one based on the polymerase II-driven expression of viral
RNA and the second based on the T7 RNA polymerase-driven expression of viral RNA with the recov-
ery of fully infectious virions by both systems that are used to determine the fundamentals of norovirus
genome translation and replication [21]. The cleavage of the MNV protease–polymerase precursor form
of RdRp into separate units and RNA secondary structures was shown to be essential for replication,
while the correct sequence of the last nucleotide of the MNV genome, immediately upstream of the poly
A tail was found to be essential for the recovery of infectious virus. Terminal sialic acid moieties on
gangliosides function as an attachment receptor for MNV on murine macrophages [20,83].
However, the limitations of using MNV-1 as a model and surrogate need to be addressed. MNV-1
causes a persistent disease in its host that is different from HNoV as mice do not routinely develop diar-
rhea and cannot vomit, while HNoV causes acute rapid infection and subsequent shedding and clearance
84 Laboratory Models for Foodborne Infections

with sometimes longer shedding periods that can last for more than a week. MNV-1 is less genetically
variable than the variable HNoV genome that is associated with repeated infection and short-term immu-
nity [20]. The relevance of the apparent immune-cell-tropism of MNV-1 is currently unknown as HNoV
tropism in vivo remains unclear to date [20]. Therefore, research on suitable cultivable surrogates and
model systems for understanding HNoV infections and inactivation is ongoing.

5.4.3 Tulane Virus
Tulane virus (TV) is a cultivable enteric calicivirus and is also known to recognize the same receptors
(human histoblood group antigens) as HNoVs [84]. TV is 36 nm in size with a genome size of about
6.7 kb single-stranded RNA with a poly(A) tail comprising of one of the shortest known genomes in
the Caliciviridae family and belongs to the Recovirus genus. The viral genome that is enclosed in a
nonenveloped capsid is divided into three ORFs, where ORF1 encodes for one nonstructural polyprot-
ein, ORF2 encodes for the capsid protein, and ORF3 encodes for a minor structural protein [85]. TV is
closely genetically related to HNoVs and pairwise homology has revealed the highest amino acid identity
with HNoVs among other members of the Caliciviridae family [85]. TV was isolated from stool samples
of monkeys without typical symptoms of gastrointestinal disease, questioning the ability of monkeys to
be robust animal models. TV is known to bind to all B antigens (types 1–4) and type 3 of the A antigen.
Since TV can be easily cultured and grown in vitro, it remains an important cultivable surrogate for
HNoVs and a strong candidate as a laboratory model to study HNoVs.
TV has been used as a cultivable HNoV surrogate to determine inactivation and survival under
different environmental stresses and conditions that include different pH, temperature, and chlorine
levels [86]. Hirneisen and Kniel reported that TV could be reduced by 1.59, 0.96, and 0.82 log PFU/mL
at pH values 2.0, 3.0, and 4.0, after 30 min at room temperature, respectively [62]. At temperatures of
50°C, 55°C, 60°C, and 65°C for 2 min, TV was reported to be reduced by 1.79, 1.83, 2.90, and 3.07 log
PFU/mL, respectively [62]. Upon chlorine treatments of 0.2, 2, 20, and 200 ppm, TV was reported to be
reduced by 1.33, 2.11, 1.53, and 2.93 log PFU/mL after 5 min. TV was also examined for its survival on
spinach plants with and without exposure to sunlight [62]. It was found to persist on whole spinach plants
and on both semisavoy and smooth spinach plants maintained in a greenhouse chamber for 7 days [62].
To assess the role of sunlight, these researchers exposed the spinach plants to UVA/UVB using a lamp to
mimic sunlight for 10 h/day for 7 days; however, they did not find any significant difference in TV titers
from the initial titer values.
The survival and dissemination of TV was also studied by inoculating the roots of romaine lettuce
with TV and growing them for 2 weeks in hydroponic feed water [87]. These researchers detected about
3 log PFU/g of TV infectious particles in leaves and shoots after 2 days postinoculation; however, the
levels of detected infectious particles in leaves and shoots reached up to 6 log PFU/g after 7 days postin-
oculation. Thus, TV was found to internalize in the roots of plants of lettuce and can be disseminated to
the leaves and root shoots. It was also found to persist on alfalfa seeds for over 50 days at 22°C, where
TV was reported to be reduced from an initial titer of 3.87–0.85 log PFU/g [88].
TV was also used to determine the effects of high hydrostatic pressure on its inactivation in cell-
culture media, blueberries, and oysters [89]. These researchers showed that in cell-culture media, a
treatment of 350 MPa caused a reduction of 3.8 and 2.4 log PFU/mL of TV at pH 7 and 4, respectively,
after 2 min at 21°C, while 600 MPa at temperatures of 4°C, 21°C, and 35°C was shown to not cause any
reduction of TV on dry blueberries after 2 min. However, it was shown that complete inactivation of TV
occurred when blueberries were immersed in phosphate-buffered saline after 300 MPa treatment for
2 min at 4°C and also at 400 MPa for 2 min at 4°C. In oysters, TV reductions of 2.9 and 2 log PFU/mL
were reported after a treatment of 250 MPa at 4°C and 21°C, respectively.
TV was also used as a surrogate to understand the effects of ionizing radiation by an electron beam,
as a nonthermal processing approach on the viral infectivity of spiked produce, and to determine the
mechanism of inactivation [90]. TV was reported to be reduced from 7 log PFU/mL to undetectable
levels with 16 kGy or higher doses in strawberries and lettuce, while with lower target doses of 4 kGy,
reductions of 1.5 and 1.8 log PFU/mL were reported in PBS and cell-culture media (Opti-MEM), respec-
tively (Table 5.7). Transmission electron microscopy (TEM) did not reveal any significant damage to
Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation 85

TABLE 5.7
Thermal and Nonthermal Approaches for the Inactivation of Tulane Virus (TV) as a Cultivable HNoV Surrogate
Virus Treatment Reduction References
Tulane virus 50°C, 2 min 1.79 log PFU/mL [86]
55°C, 2 min 1.83
60°C, 2 min 2.90
65°C, 2 min 3.07
High hydrostatic pressure; 350 MPa; cell-culture media 3.8 log PFU/mL [89]
High hydrostatic pressure; 600 MPa; dry blueberries No reduction [89]
High hydrostatic pressure; 600 MPa; oysters 2.9 log PFU/mL [89]
16 kGy electron beam; strawberries 7 log PFU/mL [90]
70% ethanol; 20 s 5 log TCID50 [91]
UVC; 60 mJ/cm2; 4 min 4 log TCID50 [91]
300 ppm free chlorine; 10 min 4 log TCID50 [91]

the treated viral particles, though the numbers of detectable virions were reported to be reduced in the
treated samples. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the treated
viral particles showed degraded viral proteins compared to the controls posttreatment [90]. TV was also
used as an HNoV surrogate to determine inactivation by 50%–70% ethanol [91], where TV was reduced
by 5 log TCID50 after 20 s treatment, and heating at 63°C for 5 min and 56°C for 30 min, UVC exposure
of 60 mJ/cm2 for 4 min, and 300 ppm of free chlorine for 10 min were also reported to cause a reduction
of 4 log TCID50 [91].
TV infectivity was also determined in the presence of bacterial cell-free supernatants (CFS), show-
ing reduction by 0.44, 0.37, 0.38, and 0.81 logs with CFS of Bacillus coagulans, Escherichia coli,
Enterococcus faecalis, Staphylococcus epidermidis grown in tryptic soy broth (TSB), when host cells
were overlaid with CFS at 37°C for 44 h [92]. These results should help toward understanding the factors
affecting viral infections and how colonization of bacteria in the gut or the native microbiome can inhibit
HNoV or other viral infections through competitive attachment [92].
Replication and biological studies have demonstrated that an authentic 5′ end of TV RNA or the
N-terminal protein appears to be essential for infectivity, indicating that while nonstructural proteins
appear to be sufficient for TV genomic RNA replication and translation, structural proteins are still
needed to generate progeny viruses [20].
However, TV causes an uncharacterized disease in its rhesus macaque host and represents a separate
genus than HNoV. Nonhuman primates such as common marmosets, cotton-top tamarins, and rhesus
macaques serve as a good alternate model to study HNoV biology, as they seem to be susceptible to
the HNoV GI Norwalk virus and shed virus after infection, though without exhibiting any disease symp-
toms [20]. Thus, based on the information provided above, TV may be another appropriate alternate
model to study HNoV pathogenesis, survival, and transmission.

5.4.4 Porcine Sapovirus
Porcine sapovirus belongs to the genus Sapovirus within the Caliciviridae family, and the porcine sapovi-
rus Cowden strain has been successfully propagated in the continuous porcine kidney cell line (LLC-PK)
[93]. The presence of bile acid or intestinal content fluid filtrate obtained from uninfected gnotobiotic
pigs was found to be necessary during preinfection for viral growth in cell-culture media [94]. Human
sapoviruses are known to cause gastroenteritis in humans with similar symptoms to HNoVs. However,
similar to HNoVs, they currently remain unculturable in the laboratory. From an epidemiological per-
spective, the HNoVs are considered to be more significant due to their prevalence/number of outbreaks as
compared to Sapoviruses. The properties of porcine sapovirus that make it a suitable surrogate are that
it is an enteric virus, is genetically related to HNoVs, is cultivable in the lab, replicates in intestinal cells
of pigs, and causes gastroenteritis in piglets [95–97].
86 Laboratory Models for Foodborne Infections

TABLE 5.8
Evaluation of Various Inactivation Methods on Porcine Enteric Virus (PEV) as a Cultivable Surrogate
Virus Treatment Reduction References
Porcine enteric virus 56°C, 20 min 2 log TCID50 [70]
70% and 90% ethanol, 1 min 0.38 × 106 TCID50 [70]
200 ppm chlorine, 5 min 0.4 log TCID50 [70]
1000 ppm Chlorine, 5 min 1.3 log TCID50
High hydrostatic pressure; 700 MPa 7 log TCID50 [70]
High hydrostatic pressure; 400 MPa 4 log TCID50

Porcine enteric caliciviruses (PEC) are reported to infect pigs of all ages, but cause diarrhea only
in young piglets. Gnotobiotic pigs can become infected with HNoVs and therefore have been reported
to be used to study and model antibody and cytokine responses [20]. PEC is subclinical in pigs, yet
may play an important role in evolutionary modeling if they are a natural reservoir or are involved in
zoonotic disease transfer and have been used as a model for tissue culture–derived enteric calicivirus
attenuation [20]. To gain insight into PEC replication, a reverse genetics system that was developed for
PEC showed that capped RNA transcripts derived from a cDNA clone were fully infectious [20].
Porcine sapovirus appears to be suitable as a cultivable HNoV for determining inactivation by various
methods (Table 5.8) as PEC infectious (using TCID50 assays) titers were reported to have no significant
changes after exposure to pH 4–8 at room temperature for 1 h and showed <l-log reduction at pH 3 [88].
Porcine sapovirus was also reported to attach to lettuce leaves at its capsid isoelectric point (pH 5.0), and
even remained infectious on lettuce after 1 week of storage at 4°C [88]. These researchers also showed
that porcine sapovirus and HNoV shared similar resistance to heat and chlorine treatment. However, the
stability of porcine sapovirus to food-processing technologies is yet to be determined. Hence, further
studies using PEC as a model or surrogate for HNoVs and human sapoviruses are ongoing.

5.4.5 Bovine Noroviruses
Bovine NoVs are reported to be less closely related to HNoVs than porcine viruses, and the prototype
bovine NoV strain Newbury agent 2 was identified in calves suffering from diarrhea. The overall simi-
larity of bovine NoVs and their potential zoonotic transfer make them an alternative model for in vivo
studies, although recently NoVs have also been identified in sheep [20].

5.4.6 Rabbit Caliciviruses
Rabbit hemorrhagic disease virus (RHDV), rabbit calicivirus (RCV) and, European brown hare syn-
drome virus (EBHSV) are caliciviruses of the Lagovirus genus, mainly of concern in America and
China, that most often cause fatal disease with symptoms such as hepatitis and hemorrhages, and are
virulent viruses of rabbits [20]. Their suitability as model systems for HNoVs is unclear as they cause
different disease symptoms than HNoVs, while RCV is nonpathogenic and has not been completely
characterized.
However, for RHDV, an animal model, a tissue culture system, and a reverse genetics system are
reportedly available, and it has been demonstrated that the minor capsid protein VP2 of RHDV does not
play a role in virus infectivity and also that the 3′ poly A tail has no effect on virus replication in vitro
[20]. Thus, RHDV may be considered for use in an alternate animal model system to study HNoV
biology.

5.4.7 Norovirus-Like Particles
Norovirus-like particles have also been used as a model and alternate surrogate in the laboratory for
inactivation studies on HNoVs in order to investigate the structural and biochemical characteristics of
Noroviruses: Laboratory Surrogates for Determining Survival and Inactivation 87

HNoVs after inactivation treatments. The recombinant expression of the VP1 major capsid protein of
HNoV has been shown to result in self-assembly to form empty, noninfectious VLPs that have similar
morphology and antigenic properties to the native infectious virus particle [114]. The protruding (P)
domain of the major structural capsid protein that forms the outermost surface of the capsid contains the
elements required for viral capsid binding to host carbohydrate receptors [89,95,98]. A variety of expres-
sion systems have been reported to be used for the expression of VLPs, including baculovirus insect cell
and transgenic plant expression systems [99–102]. When the P protein is expressed in E. coli, subviral
particles, namely P particles, are formed that may be used as a surrogate to study the binding ability of
HNoVs, and it is the VLP receptor binding ability that has been proposed as a unique indicator for virus
survival and infectivity [95], as well as for the determination of the damage of VLPs by using electron
microscopy and SDS–PAGE techniques. These VLPs have been used in research to demonstrate the
ability of gamma irradiation to disrupt the VLP structure causing degradation of the VP1 protein [61].
VLPs have also been used as a surrogate for determining the interaction/binding of HNoV with fresh
produce, seafood, and other at-risk foods [95]. HNoV P particles, using saliva binding enzyme-linked
immunosorbent assays, were used as an HNoV surrogate, and the presence of cranberry and pomegran-
ate juices, showed reduced HNoV binding [103]. However, some fresh produce extracts that included
strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce, or raspberry did not demon-
strate any effect on the binding ability of these particles [103].

5.5 Bacteriophage MS2
Bacteriophage MS2 is a bacterial virus, commonly found in sewage and wastewaters, that infects the
bacteria E. coli [in particular, American Type Culture Collection (ATCC) 15597B]. Bacteriophage MS2
is a single-stranded icosahedral RNA virus, between 27 and 34 nm in diameter, that belongs to the
Leviviridae family in group 1 of the RNA coliphages and is adapted to the intestinal tract [104].
Bacteriophage MS2 has been used by various researchers as a gastrointestinal virus surrogate to deter-
mine disinfection and inactivation efficacy as well as survival and transmission in the environment,
including the use of electrochemical oxidants (ECOs) for surface disinfection, due to its ease of propaga-
tion and relatively simplistic assays [105]. ECOs were generated using battery power to electrolyze brine
(NaCl) solutions, which contain both chlorine [HOCl, OCl(−)] and reactive oxygen species (e.g., ·OH,
O3, H2O2, and O2−), and at free available chlorine concentrations of 2500 ppm for a 30-s contact time,
infective MS2 bacteriophage was reported to be inactivated by >7 log compared to the MNV-1 inactiva-
tion of ∼2 log [105]. When these researchers compared genomic RNA inactivation, MS2 RNA inactiva-
tion was reported to be around 5 log compared to the MNV-1 RNA inactivation of around 1.5 log, with
comparable inactivation efficacy to household bleach at similar free available chlorine concentrations.
MS2 was reported to be reduced by ≥6 log PFU/mL at 5% TSP, and a 4.5 log PFU/mL reduction was
reported to be achieved with 1% TSP after a contact time of 30 s [29]. Lee and Ko reported that MS2 was
highly resistant to disinfection by UVA, but the addition of TiO2 enhanced the efficacy of UVA, where a
UVA dose of 1379 mJ/cm2 resulted in a 4 log reduction, while UVB alone inactivated both MS2 by 4 log
with a dose of 367 mJ/cm2 [59]. A study of the virucidal inactivation efficacy of an in-house-designed
atmospheric pressure, nonthermal plasma jet operated at varying helium–oxygen feed gas concentrations
used MS2 bacteriophage as a surrogate and model for HNoV, showed that the inactivation rate constant
increased with increasing oxygen concentrations up to 0.75% [106]. These researchers reported that
3 log (99.9%) reductions in infectious MS2 were obtained after 3 min of exposure in a helium–oxygen
(99.25%:0.75%) gas mixture, with increased reduction after 9 min exposure by >7 log. Black et al. found
that MS2 was inactivated by <1 log PFU/mL at hydrostatic pressures of 500 MPa for 5 min at 20°C,
showing its resistance as compared to other surrogates and making it a suitable surrogate and model
virus for hydrostatic pressure studies [107]. Dawson et al. studied the survivability/persistence of MS2
bacteriophage on fresh iceberg lettuce, baton carrot, cabbage, spring onion, curly leaf parsley, capsicum
pepper, tomato, cucumber, raspberries, and strawberries and showed that MS2 survived for a very long
time and even extended the shelf life of the produce, where <1 log PFU/mL reduction was observed after
50 days at 4°C and 8°C [104]. In a study determining the survival of HNoV in juices, bacteriophage MS2
88 Laboratory Models for Foodborne Infections

(∼6 log PFU/mL) was used as a surrogate and model that showed significant reduction (1.93 log PFU/mL)
after 2 days and was undetectable after 7 days in blueberry juice at 4°C [48].

5.6 Conclusions
HNoVs are a worldwide public health and food safety concern. However, in the absence of reproducible
cell-culture-based infectivity assays and due to the lack of available vaccines to date, cultivable surro-
gates and model systems are used in order to determine survival, transmission, and inactivation methods
to prevent outbreaks and minimize the risk of HNoV contamination. Typically, cultivable surrogates that
mimic the characteristics of HNoVs are genetically related and are used in laboratory settings. These
surrogates include cultivable animal viruses from the Caliciviridae family as well as bacteriophages and
noroviral-like particles. The application of these surrogates in environmental studies and to enhance
food safety remains ongoing. However, the ideal situation of having reproducible infectivity assays for
HNoVs appears to be a challenge and research efforts continue to be made toward attaining this goal.
It is evident that a reproducible and validated cell-culture system for the propagation of HNoVs is
needed to understand HNoV biology, replication, and molecular aspects. Toward this end, researchers
have still to validate a B-cell replication system for HNoVs [19]. Virus entry and genome uncoating are
considered the “vital missing link” for HNoV propagation [20]. Despite the availability of a variety of
animal models and surrogates for understanding HNoVs, there does not appear to be one single overall
“ideal” model system, whose suitability varies and depends on the topic being researched. As indicated
and described in detail by Vashist et al., for water-related and environmental studies, bacteriophages
such as MS2 and phiX-174 have been used as indicators and surrogates in the past to determine survival
and transmission routes. For studying pathogenesis, animal caliciviruses appear to be most suitable, yet
none of them reproduce all the HNoV disease aspects. Most animal caliciviruses appear to be suitable
models for genome translation and replication studies, and MNV-1 appears to be most closely related
or suitable for HNoVs [20]. With regards to vaccines and antivirals, progress seems to be limited and
slow, and small molecule inhibitors, natural plant polyphenols, and antisense RNA technologies show
potential for future application (further suggested reading on NoVs in reference [20,108]). Thus, in
conclusion, research emphasis should continue to be placed on the propagation of HNoV in cell-culture
systems in vitro to obtain reproducible HNoV infectivity assays.

Acknowledgments
The authors gratefully acknowledge the support provided by the University of Tennessee-Institute of
Agriculture. The authors also wish to thank Carrie Yard for her assistance with the formatting some of
the references during the final stage of this chapter submission.

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6
Rotavirus

Lijuan Yuan and Ke Wen

CONTENTS
6.1 Introduction..................................................................................................................................... 95
6.2 Overview of Animal Models for Human Rotavirus (HRV) Research........................................... 97
6.2.1 History of Mouse Models for Rotavirus Research............................................................. 98
6.2.2 History of the Gnotobiotic (Gn) Pig Model for HRV Research......................................... 99
6.2.3 History of Nonhuman Primate Models for HRV Research............................................... 99
6.3 Rotavirus Replication and Pathogenesis........................................................................................101
6.3.1 Recent Major Advancements Made Using Mouse Models...............................................101
6.3.2 Major Advancements Made Using the Gn Pig Model..................................................... 102
6.4 Rotavirus Immunity and Vaccine Development........................................................................... 102
6.4.1 Mouse Models in the Study of Rotavirus Protective Immunity and Vaccines................ 102
6.4.2 Gnotobiotic Pig Model in the Study of Rotavirus Immunity and Vaccines.................... 103
6.4.2.1 Unique Features of the Gn Pig Model.............................................................. 103
6.4.2.2 HRV Vaccines and Therapeutics Evaluated in the Gn Pig Model of Wa
HRV Infection and Diarrhea���������������������������������������������������������������������������� 104
6.4.2.3 Gnotobiotic Pig Model Colonized with Probiotic Bacteria or Human Gut
Microbiota�������������������������������������������������������������������������������������������������������� 104
6.5 Cell Lines and Organoid/Enteroid Models for Rotavirus Infection............................................. 106
6.6 Concluding Remarks..................................................................................................................... 107
References............................................................................................................................................... 107

6.1 Introduction
Rotaviruses are recognized as the leading cause of severe gastroenteritis in infants and young chil-
dren worldwide since the discovery of human rotavirus (HRV) in the early 1970s.1–3 Rotaviruses are
transmitted via the fecal oral route, are highly contagious and stable in the environment,4 and post a
high risk of foodborne infections. Rotavirus is a nonenveloped, 70-nm icosahedral virus in the family
of Reoviridae. It has a genome consisting of 11 double-stranded RNA segments surrounded by a dis-
tinctive three-layered protein capsid.5 These 11 segments encode six structural proteins, VP1–4, VP6,
and VP7, and six nonstructural proteins, NSP1–6.6 Among the 11 viral genes, the properties of the
proteins encoded by genes 3, 4, 5, 9, and 10 are known to be related to rotavirus virulence in the host.7
Gene 3 encodes the capping enzyme that affects the level of viral RNA replication.8,9 Genes 4 and 9
produce the outer capsid proteins VP4 and VP7 required to initiate infection, and they induce virus
neutralizing antibodies independently from each other.10,11 Gene 5 codes NSP1 that functions as an
interferon antagonist.12–14 Gene 10 codes for the nonstructural protein NSP4, which regulates calcium
homeostasis and virus replication and acts as an enterotoxin.7 NSP2 (encoded by gene segment 8) is
also involved in virulence, especially in mice.15
Seven rotavirus groups (A to G) are known based on the antigenicity of VP6 and sequences of
genomic RNA.16 Among them, Groups A, B, and C are associated with illness in humans and animals,

95
96 Laboratory Models for Foodborne Infections

and Groups  D through G with illness in animals only. Group A rotaviruses are the most common
cause of illness in infants and young children worldwide. The antigenicity of Group A rotaviruses
is highly diverse, and about 19 G (based on VP7) and 28 [P] (based on VP4) sero/genotypes have
been ­identified.17 Serotypes G1, G3, G4, and G9 are responsible for 90% of all HRV infections in
North America and Europe but for less than 70% infections in Africa.18 Genotypes P[8] and P[4]
account for over 90% of circulating P types worldwide, whereas in Africa, the relative frequency of
these two P types is lower and P[6] accounts for around a third of all detected P types.18 There are
potentially “211” different combinations of G and P proteins that can be generated; however, the actual
number of G and P combinations is less than the possible number because most combinations do not
survive subsequent rounds of replication in the host due to lack of fitness. The different rotaviruses
circulating in humans are categorized as (1) common human genotypes (G1P[8], G2P[4], G3P[8],
G4P[8]); (2) reassortants among human genotypes (G1P[4], G2P[8], G4P[4]); (3) reassortants between
animal and human genotypes (G1P[9], G4P[6], G9P[8], G12P[8]); and (4) likely zoonotic introductions
(G9P[6], G9P[11], G10P[11], G12P[6]).17,19 Among these, G1, G2, G3, and G4 in combination with P[4]
and P[8] represented over 88% of strains worldwide based on studies published between 1998 and
2004.18 The data from the surveillance networks of the WHO also indicate that G1P[8], G9P[8], and
G2P[4] account for 75% of samples genotyped in North America, Europe, southeast Asia, and western
Pacific regions.
Rotavirus replicates in the small intestine and causes gastroenteritis. The symptoms of rotavirus
infection are severe watery diarrhea, fever, and vomiting, leading to fluid and electrolyte disequi-
librium. Severe diarrhea without fluid and electrolyte replacement may result in other secondary
complications (e.g., renal failure and death).20 Before the implementation of rotavirus vaccines in
many developed and some developing countries starting from 2006, it was estimated that rotavirus
infections annually accounted for more than 125 million cases of diarrhea (20%–50% of hospitaliza-
tions for gastroenteritis) and 600,000–870,000 deaths (around 20% of all diarrhea-associated deaths)
in children under 5 years of age worldwide.21 In the United States, rotavirus infections caused an
estimated 410,000 physician visits and 50,000–70,000 hospitalizations each year before rotavirus
vaccines were implemented in the vaccination program.22 Following implementation of rotavirus vac-
cination in 2006, all-cause acute gastroenteritis hospitalization rates among US children younger than
5 years declined by 31%–55% in each of the postvaccine years from 2008 to 201223 and the laboratory
detection rate of rotavirus declined by 57.8%–89.9% in each of the seven postvaccine years from 2007
to 2014.24
Because rotaviruses are highly contagious and stable in the environment, exposure in closed envi-
ronments usually results in rapid, widespread infections among susceptible individuals. Outbreaks of
rotavirus infection are common in childcare centers, and rotavirus is one of the most common causes of
nosocomial infection, especially in pediatric hospitals.25,26 In spite of improved sanitation and water sup-
ply, rotavirus infections remain an important cause of pediatric hospitalization in many countries where
rotavirus vaccines have not been implemented.
Given that rotaviruses are the single most important cause of severe acute gastroenteritis in infants and
young children, the worldwide impact of rotaviruses on public health has led to major research efforts in
the past 40 years in understanding rotavirus replication, host–pathogen interactions, pathogenesis, and
immunity, and in vaccine development to control rotavirus diarrhea. Animal models including mice,
rats, rabbits, pigs, lambs, calves, and nonhuman primates have been used extensively in these stud-
ies. Among these animal models, gnotobiotic (Gn) piglets were among the first animal models used to
study HRV right after the virus was associated with infantile gastroenteritis. Since then, the model has
continuously contributed to our understanding of rotavirus pathogenesis and immunity. Various mouse
models with different genetic backgrounds and immune competence are used extensively for studies
of rotavirus–host interactions, mechanisms of host range restriction, and protective immunity. In this
chapter, animal models pertaining to the establishment and applications in studies of rotavirus–host
interactions, pathogenesis, immunity, and vaccine development are reviewed with emphasis on mouse
and Gn pig models. The advantages, disadvantages, and limitations of the animal models are discussed.
Cell lines and tissue culture models that support rotavirus replication and have been used in rotavirus
research are summarized.
Rotavirus 97

6.2 Overview of Animal Models for Human Rotavirus (HRV) Research

Mice are resistant to HRV infection due to the species barrier. However, several murine rotaviruses iso-
lated from mouse colonies are used for studies of rotavirus biology and immunity as homologous model
systems. The diversity of murine rotaviruses is more limited as compared with HRV. All the known
murine rotaviruses belong to one single G type, G3, and two P types, P[16] or P[20]. The epidemic diar-
rhea of infant mice (EDIM) strain and all other murine rotavirus strains (EW, EB, EC, EL, EMcN, YR-1,
YR-2) are G3P[16], except for EHP which is G3P[20].27
In comparison to murine rotaviruses, porcine rotaviruses are more closely related to HRV in genetic
composition (gene constellation). There is a common origin between Wa-like HRV strains and porcine
rotavirus strains.28 The interspecies reassortment between porcine rotaviruses and HRV and zoonotic
transmissions of reassortant porcine rotaviruses to human patients are frequently reported in the litera-
ture.29–33 This close relationship forms the basis for the establishment of the Gn pig model of HRV infec-
tion and disease. The mortality of rotavirus infection in pigs is usually low, and the severity of disease
is dependent on the rotavirus strain and the age of inoculation, with more severe disease and mortality
in pigs inoculated with porcine rotavirus strains than with HRV.34 However, colostrum-deprived neo-
natal Gn pigs can be readily infected by a non-cell-culture-adapted virulent HRV Wa strain (G1P[8])
and M strain G3P[8]) and develop gastroenteritis similar to human infants and young children.35–37 It is
important to note that only Gn pigs develop diarrhea for up to 8 weeks of age from HRV infection. No
conventional laboratory animals such as mice, rats, and rabbits develop rotavirus diarrhea for nearly as
long. This makes the Gn pig model the only animal model of HRV disease that allows for the assessment
of active immunity against diarrhea over an extended period.
Newborn Gn calves and lambs are also susceptible to HRV infection.38,39 After oral inoculation of
calves with a “reovirus-like agent” of human infantile gastroenteritis, the same rotavirus inoculum used
in Gn pigs,39 fecal virus shedding was detected for 2–7 days using electron microscopy. Diarrhea (for
less than 24 h) was observed in seven calves on the second to fourth serial passage of HRV in calves but
in none of the four animals inoculated with the first passage.40 Rotavirus infection in newborn calves
leads to a change in the villus epithelium from columnar to cuboidal, causing villi to become stunted and
shortened.41 The Gn calf model was then used to test the feasibility of heterotypic protective immunity
against HRV. Gn calves were infected in utero with the bovine rotavirus NCDV strain and challenged
with the HRV D strain or NCDV strain shortly after birth.42 Infection in utero with bovine rotavirus
induced resistance to diarrhea caused by HRV as well as the homologous bovine virus. This finding led
to the attempts of using bovine rotavirus as a candidate vaccine for human infants.43,44 Gn lambs are
limited to studies of immunity to lamb and bovine rotavirus infection and vaccines.45–47
Nonhuman primates are animals genetically most closely related to humans. They are naturally
infected with simian rotaviruses; these viruses isolated so far include SA11 (G3P5B[2] or G3P6[1]), RRV
MMU18006 (G3P5B[3]), YK-1 (G3P[3]), TUCH (G3P23[24]), and PTRV (G8P6[1]).48–51 However, the
genetic and antigenic relationships between HRV and simian rotaviruses are more distant than those
between HRV and porcine rotaviruses.28 Various simian rotaviruses isolated from different nonhuman
primates and several HRV isolates have been tested or suggested for use in animal challenge models for
studies of rotavirus pathogenesis and immunity, but all with limited success.48,49,51–57
Other small laboratory models for rotavirus research are rabbits and rats.58,59 In rabbits, like in
mice, rotavirus disease is age restricted.60 In rabbits infected with lapine rotavirus between 1 week
and 2 months of age, diarrhea was observed only within the first 2 weeks of age, but histopathological
changes including villous shortening and fusion, increased vacuolation of epithelial cells, and mono-
nuclear cell infiltration of the lamina propria were observed throughout the small intestine. Five-day-old
rats are susceptible to various group A animal rotaviruses and HRV infections, and most of the viruses
resulted in diarrhea in rats that lasted from 1 to 10 days.59 The severity of disease and spread of infection
to naive rat littermates differed depending on the rotavirus strain. The rabbit model was used for assess-
ing host range restriction, active immunity, and protection after infection or vaccination with virus or
virus-like particles (VLPs),61–64 and the neonatal rat model of rotavirus infection was used to determine
the kinetics of viremia, spread, and pathology of rotavirus in extraintestinal organs.59,65
98 Laboratory Models for Foodborne Infections

6.2.1 History of Mouse Models for Rotavirus Research

In the landmark study by Eydelloth et al.,66 CD-1 mice from Charles River Breeding Laboratories
(Wilmington, MA) were used for the investigation of rotavirus replication and immune responses. Mice
orally inoculated with the murine EDIM virus, a murine rotavirus first reported in 1957 as causing diar-
rhea in 3- to 11-day-old mice,67,68 were used to examine the kinetics of rotavirus replication, the effect of
age on rotavirus infection and diseases, and the relationship of viral replication to the immune response
and to the development and resolution of disease. The study demonstrated the following: (1) Younger
mice (1, 7, and 14 days old) were more susceptible to EDIM infection than older mice; when the mice
were inoculated at 28 days of age, only minimal viral replication was detected; (2) mice older than 7 days
can still be infected with EDIM, but they do not develop diarrhea as seen in the younger mice beyond 14
days of age; (3) the increase in intestinal antibody levels at 7 days postinoculation coincided with a rapid
decline in the intestinal EDIM virus antigen in animals inoculated at 7 days of age; (4) mice inoculated
at 7 or 14 days of age developed the highest titers of serum antibodies; these titers reached peak levels at
days 10 and 7 postinoculation, respectively.66
Thus, in mice under 15 days of age, homologous rotavirus infection causes diarrhea and lethargy,
but only mild intestinal lesions consisting of vacuolization of villous tip epithelial cells with little or
no villous atrophy. An adult mouse model of rotavirus infection was later established with the EDIM
virus using infection as the endpoint in the BALB/c strain of mice.69 This adult mouse model has been
extensively used to study the determinants of immunity against rotavirus infection. Overall, the majority
of mechanistic studies in history have been conducted with the adult mouse model of murine rotavirus
infection.70
However, caution needs to be taken when using the murine rotavirus for challenge studies in mice
with different genetic backgrounds and using virus shedding as the readout. Rotavirus fecal shedding
detected after the EDIM challenge of mouse strains other than BALB/c (H2-d), for example, C57BL/6
(H2-b), and genetically modified mice on a C57BL/6 background, has been found to be less reliable
and sometimes undetectable.71 The reasons for the different infectivities of murine rotaviruses could
be the difference in available rotavirus receptors on intestinal epithelial cells or the difference in the
innate immunity of the mouse strains.72 Such factors can affect the extrapolation of mechanisms of
adaptive immunity induced by rotavirus vaccines using mouse models.73 Conclusions drawn with a
specific mouse and/or rotavirus vaccine set cannot be generalized to other sets.72 Another murine rota-
virus strain, EMcN, was later reported to shed in much greater quantities in adult BALB/c mice than
in EDIM. In addition, in contrast to the EDIM strain, EMcN was shown to consistently shed in large
quantities in adult C57BL/6 mice and in genetically modified mice of this background.71 Therefore,
rotavirus studies on adult mice with the C57BL/6 background should use the EMcN strain as the chal-
lenge virus.
Neonatal mice at 5–7 days of age can develop diarrhea after inoculation with high doses of a heterolo-
gous rotavirus, including human, simian, or bovine rotaviruses,74 but virus shedding is not consistently
detected75 and virus replication is not required for the development of diarrhea. Neonatal mice inocu-
lated with recombinant NSP4 develop diarrhea comparable to that after challenge with live virus, sug-
gesting the enterotoxigenic properties of rotavirus NSP4.76,77
In addition to wild-type mice, various gene knockout adult mice that have defective adaptive
immune systems (i.e., Rag-2 mice devoid of both T and B cells, β2m mice that lack cytotoxic T cell
responses, JHD mice that lack B cell responses, and IgA-knockout mice that have no detectable
IgA in the serum or in any secretions) have been extensively used in studying determinants of adap-
tive protective immunity. These studies have produced important knowledge regarding the role of
humoral and cellular immunity in protection against rotavirus reinfection.78–85 Other gene knockout
mice that have defective innate immune pathways (i.e., TLR3, MAVS, STAT1, IFN-α/βR, IFN-γR,
and IFN-λR knockout) have provided critical tools for determining the role of innate immune
responses in controlling homologous versus heterologous rotavirus, intestinal versus extraintestinal
rotavirus replication, virus systemic dissemination, and age-dependent susceptibility.86–89 For com-
prehensive reviews on rotavirus studies using mouse models prior to 2007, see Feng et al.,90 Franco
et al.,91,92 and Ward et al.27,93
Rotavirus 99

6.2.2 History of the Gnotobiotic (Gn) Pig Model for HRV Research

Although mouse models for rotavirus research are relatively easy to set up and do not require spe-
cially designed laboratory facilities for husbandry, mice older than 15 days do not develop diarrhea
after rotavirus infection and the pathogenesis of rotavirus in mice is different from that in humans.27,94
Animal models of HRV diseases, not subclinical infection, are more desirable, especially for preclini-
cal studies on the safety and efficacy of rotavirus vaccines. Gn pigs have successfully fulfilled this
need. Germfree isolators provide a sterile environment free of bacteria, virus, fungus, and so on. Once
an agent, such as rotavirus, is introduced into the isolator, it is no longer truly germfree and is referred
to as being gnotobiotic. In the literature, however, the words germfree pig, Gn pig, and isolator pig are
used interchangeably.
The first study showing that Gn pigs were productively infected with HRV and developed diarrhea
and virus shedding was reported by Dr. Albert Kapikian’s group in 1976.39,95 Nineteen of twenty-one
piglets developed diarrhea after being inoculated at 1–4 days of age with the rotavirus isolate acquired
from human infants with acute gastroenteritis in 1974, which is the original Wa strain HRV that belongs
to G1P1A[8] serotypes with the genome constellation of G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1.96 Virus
particles were observed in pig intestinal contents and/or fecal samples by electron microscopy. The
total duration of rotavirus shedding was 2–6 days after the onset of diarrhea. The onset of diarrhea was
2–7 days after virus inoculation. The same infectivity was reproduced in Gn pigs for up to five serial
passages.39 Two years later, another group from Australia reported that three HRV isolates induced only
subclinical infection in newborn Gn pigs and failed to infect Gn calves and lambs.97 The different out-
come is likely due to the different HRV strains used in the studies. Gn pigs can be infected with G1 (Wa
strain) and G3 (M strain) HRV and develop diarrhea, but not G2 (DS-1 strain) HRV36,98; however, the
genotypes of the viruses used in the Australian study were not reported.
The Gn pig model of HRV infection and diarrhea was formally established in 1996 using the same
virus isolate, the HRV Wa strain, from Wyatt and Kapikian’s group.37,99,100 The virulence of Wa HRV in
Gn pigs was maintained by passaging the virus solely through the intestinal contents of infected neonatal
Gn pigs. Over the past two decades, the Gn pig model of Wa HRV infection and diarrhea has been the
key element for many studies published in over 50 peer-reviewed journal articles, testifying to the useful-
ness of this unique animal model in rotavirus research.37,101–150
Recently, the human gut microbiota (HGM) transplanted Gn pig model for HRV infection and diar-
rhea has been established.105 The gut microbiota of the recipient pigs was similar to that of the human
donor.102 The HGM Gn pigs more closely mimic the intestine environment of human infants and provide
a more robust model for virus–host interaction studies and for evaluating intervention approaches to
reduce rotavirus diarrhea.151

6.2.3 History of Nonhuman Primate Models for HRV Research

An early study showed that diarrhea developed in five colostrum-deprived newborn rhesus monkeys
(Macaca mulatta) inoculated orally on the first day of their life with a “reovirus-like agent” of infantile
gastroenteritis (later named as Wa HRV).54 As reported, the onset of diarrhea ranged from 2 to 5 days;
virus particles were detected in stools by electron microscopy in association with illness, and virus shed-
ding lasted 1–3 days. The virus derived from monkeys that developed illness following inoculation was
infectious for other monkeys but did not induce diarrhea.54
Similarly, after oral administration of HRV (strain or genotype unknown) to six newborn cynomol-
gus monkeys (Macaca fascicularis) naturally delivered and normally nursed, five developed diarrhea.55
Virus excretion was observed in the stool of four animals. This virus was transmitted to four out of six
other monkeys, but only caused diarrhea in one animal. Another attempt was made to infect nonhuman
primates with a HRV G8P[6] isolate.56 Rotavirus was detected in the stool of all the rotavirus-inoculated
vervets and in one out of the two rotavirus-inoculated baboons; however, none showed any clinical dis-
ease. Therefore, nonhuman primates have not become a principal animal model for HRV research. The
advantages, disadvantages, and limitations of the three most commonly used animal models (mice, Gn
pigs, and nonhuman primates) are listed in Table 6.1.
100 Laboratory Models for Foodborne Infections

TABLE 6.1
Advantages and Disadvantages of the Major Animal Models Used in Rotavirus Research
Animal Species and Age Advantages Disadvantages
Neonatal mice (between • Small size • Resistant to infection by HRV
1 and 14 days of age) • Inbred; homogeneous response; allow immune • Different from humans in
cell transfer experiments gastrointestinal physiology and
• Availability of many genetically modified immune system
strains (gene knockout mice) • Different histopathology from
• Availability of molecular biology and humans after rotavirus infection
immunology reagents • Cannot be used to study infection-
• Produce clinical signs of disease (diarrhea) for or vaccine-induced active immunity
a limited time after infection by murine against rotavirus diarrhea due to
rotavirus and high-dose heterologous rotavirus age-dependent disease
strains • Maternal antibodies can interfere
• Can be used to study passive protective with rotavirus infectivity and
immunity against rotavirus diarrhea by development of immune responses
immunizing dams
Adult mice (older than • Small size • Resistant to infection by HRV
15 days of age) • Inbred; homogeneous response; allow immune • Different from humans in
cell transfer experiments gastrointestinal physiology and
• Availability of many genetically modified immune system
strains (gene knockout mice) • Mouse strains with different genetic
• Availability of molecular biology and backgrounds display different
immunology reagents susceptibility to infection for the
• Can be used to study rotavirus infection or same rotavirus strain, and shed
vaccine-induced protective immunity using different titers of rotavirus
virus shedding as the readout • Cannot be used to study infection- or
vaccine-induced active immunity
against rotavirus diarrhea
Gnotobiotic pigs • Susceptible to infection by HRV (Wa and M • Outbred; variability in host response
strains) without prior adaptation and develop • Although increasing, the availability
diarrhea up to 8 week of age of reagents is still less than that for
• Display clinical signs of disease (diarrhea) and mice and humans
histopathological changes similar to human • Expensive
infants • Special facilities and trainings are
• Similarity to human infants in physiology, required
immune system, body size, milk diet, and
metabolism
• Large amount of cells can be isolated from
lymphoid tissues for comprehensive
immunological analysis
• Devoid of maternal antibodies and other
maternal and environmental confounding
factors
• Can be colonized with human gut microbiota
to more closely mimic humans
Nonhuman primates • High similarity with humans in genomics, • Fail to induce clinical signs of
gastrointestinal physiology, and immune disease by second passage HRV
system (after the HRV isolate is passaged in
• Neonates are susceptible to HRV infection and nonhuman primates)
diarrhea • Outbred; high variability in host
• Broad availability of immunology and response
molecular biology reagents • Varying levels of maternal
antibodies
• Ethical concerns
• Very expensive
• Special facilities and trainings are
required
Rotavirus 101

6.3 Rotavirus Replication and Pathogenesis

6.3.1 Recent Major Advancements Made Using Mouse Models
Rotavirus primarily infects and replicates in villous mature epithelial cells of the small intestine. An innate
immune response triggered by rotavirus infection is the first line of defense. One of the most distinct advan-
tages of using the mouse model for research is the availability of a wide variety of genetically modified
(gene knockout) mouse strains that allow the identification of molecular mechanisms in the studies. Using
knockout mouse models of murine rotavirus infection, studies have clearly identified the role of innate
immune effectors induced by homologous rotavirus that control the virus replication after oral inocula-
tion.87,152 In the study by Angel et al.,152 type I IFN (IFN-α/β) receptor or type II IFN (IFN-γ) knockout
suckling mice developed diarrhea with similar characteristics and duration and had comparable quantities
of viral antigen in the intestines as wild-type mice. Furthermore, type I IFN receptor knockout adult mice
infected with murine rotavirus also shed equivalent quantities of viral antigen and with similar kinetics as
the wild-type mice. Thus, type I and II IFNs are not major inhibitors of rotavirus diarrhea or replication in
mice. Further, in another study,87 both suckling and adult IFN-λ receptor knockout mice had increased sus-
ceptibility to EDIM rotavirus infection, whereas IFN-α/β receptor knockout mice were similar to wild-type
mice. In addition, the systemic treatment (injection) of suckling mice with IFN-λ repressed rotavirus repli-
cation in the gut, whereas treatment with type I IFN was not effective. These studies clearly demonstrated
that type III IFN (IFN-λ), not type I or II IFN, plays a critical role in the intestinal epithelial antiviral host
defense in mice and that this function is nonredundant in the mucosal antiviral innate immune system.87
Homologous and heterologous rotaviruses have different replication efficiencies in the intestine. In
general, heterologous rotaviruses are less virulent, or totally attenuated, compared to homologous rota-
viruses. The host species-specific restriction of rotavirus replication and virulence forms the basis for
heterologous attenuated oral rotavirus vaccines. These include RotaShield, RotaTaq, and LLR, in which
an animal rotavirus that is naturally restricted for replication and virulence in humans is used either as
the vaccine (LLR)153 or as the genetic backbone to produce the reassortant rotaviruses included in the
multivalent vaccine (RotaShield and RotaTaq).154 To identify the genetic basis and mechanism for the
host range restriction, intestinal replication of a series of EW × RRV reassortants in suckling mice was
used to identify rotavirus genes that influence rotavirus replication in the intestine.155 It was found that
permissive replication of a homologous murine rotavirus is primarily regulated by VP4 and NSP1, and
high-efficiency replication in the mouse intestine requires a constellation of murine genes encoding VP3,
NSP2, and NSP3 along with NSP1. VP4 acts as a determinant of host range restriction from having mod-
est to very strong effects, depending on the species origin of the heterologous VP4.155
The most dominant host factor in rotavirus pathogenesis is age: Only newborn mice, rats, and rabbits
less than 2 weeks of age develop diarrhea after rotavirus infection. On the other hand, newborn humans
infected with rotavirus rarely have symptomatic disease. This protection is thought to be mediated pri-
marily by the transplacental transfer of maternal antibodies,156 but recent studies have indicated that the
age-dependent pathogenesis of rotavirus may also derive from the age-dependent expression of TLR389
and N-acetyllactosamine (LacNAc), a precursor of the human histo-blood group antigen (HBGA) on
intestinal epithelial cells.157 A study by Pott et al.89 investigated the age-dependent mechanisms of the
intestinal epithelial innate immune response to rotavirus infection. TLR3 expression is low in the epithe-
lium of wild-type suckling mice but is strongly increased during the postnatal period. TLR3 expression
is inversely correlated with rotavirus susceptibility, viral shedding, and histological damage. After oral
infection with EDIM rotavirus, TLR3 or the adaptor molecule TRIF knockout adult mice, but not neona-
tal knockout mice, had significantly higher viral shedding and decreased epithelial expression of proin-
flammatory and antiviral genes as compared to wild-type mice. This study identified the age-dependent
expression of TLR3 as a factor of rotavirus susceptibility.89
For most HRV strains, the spike protein VP8* binds to the human HBGAs as a potential receptor or
attachment factor similar to that of human noroviruses158; strains in the P[4] and P[8] genotypes share
the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bind the H type
1 antigen only. Notably, only rotavirus strains in the P[11] genotype have a preference for neonates
102 Laboratory Models for Foodborne Infections

(human 116E and bovine B223 strains). In vitro studies have suggested that the P[11] rotavirus specifi-
cally recognizes LacNAc that are likely expressed on the intestinal epithelial cells of only neonates and
infants, not in older children or adults, as potential receptors for P[11] rotaviruses.157 Rotavirus can also
infect adults, but severe symptomatic disease is relatively uncommon and can result from immunosup-
pression,159 infection with an unusual virus strain, or extremely high doses of rotavirus.7 Further studies
are needed to better understand the age-dependent susceptibility and pathogenesis of rotaviruses, and
to fully identify the host genetic factors in susceptibility to rotavirus illness and the mechanisms behind
asymptomatic rotavirus shedding in adults.

6.3.2 Major Advancements Made Using the Gn Pig Model

Pathological changes induced by HRV infections in the intestines of humans have been examined in
only a limited numbers of studies due to the difficulty of collecting specimens. In pigs, the macroscopic
changes induced by porcine rotavirus infection demonstrate the thinning of the intestinal wall, and the
microscopic changes include villus atrophy, villus blunting, and conversion to a cuboidal epithelium.15
Based on duodenal biopsies from children with acute rotavirus infection, the histopathologic changes
are similar to those found in piglets.37,160,161 The general progression of events in rotavirus infections in
humans and pigs consists of virus replication in and destruction of the mature differentiated villous epi-
thelial cells of the small intestine, leading to necrosis and desquamation of these cells.162,163 As a conse-
quence, crypt cells invade the villus surface to cause a decrease in the digestive and absorptive capacities
of the intestine and generate malabsorptive and maldigestive diarrhea.164 In neonatal Gn pigs infected
with virulent Wa HRV, diarrhea developed at 13 h postinoculation and correlated with the presence of
viral antigens within villous epithelial cells.37 Villous atrophy was observed at 24–48 h postinoculation,
coincident with the peak of virus replication. Diarrhea and virus shedding persisted for 4–7 postinocula-
tion days. Recovery from disease correlated with the return of morphologically normal villi.37
Rotavirus pathogenesis is multifactorial. Both viral and host factors affect the outcome of rotavirus
infection. Using porcine SB-1A × HRV DS-1 reassortants in neonatal Gn pigs and using diarrhea as the
readout (replication was not measured), VP3, VP4, VP7, and NSP4 together were associated with the capac-
ity of the porcine strain to induce diarrhea in neonatal Gn pigs. Reassortants that possessed only one, two,
or three of these porcine rotavirus genes on a background of HRV genes failed to induce diarrhea.98
Rotavirus primarily infects intestinal villus enterocytes. However, all infected individuals and ani-
mals undergo at least a short period of viremia, and rotavirus can be detected in several other tissues in
addition to the intestine.165–167 A study using Gn pigs provided clear evidence that virulent HRV causes
transient viremia and upper respiratory tract infection in addition to gastrointestinal infection.128 In Gn
pigs, after oral, intranasal, or gavage inoculation with virulent Wa HRV, all pigs had viremia along with
nasal and rectal virus shedding and diarrhea.128 The HRV in serum was confirmed to be infectious by
orally inoculating native Gn pigs. Serum-inoculated pigs developed diarrhea and fecal and nasal virus
shedding. It is important to note that pigs inoculated intravenously with serum or intestinal contents from
the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to
the orally inoculated pigs, suggesting that intestinal infection might be initiated from the basolateral side
of the epithelial cells via viremia.128
The Gn pig model can be used to address many unaddressed questions in rotavirus research, such as
the influence of interactions between gut microbiota and rotavirus in virus replication and pathogenesis.
In addition, genetically modified pigs have become available.168,169 This opens up a new toolbox for using
the Gn pig model in mechanistic studies of rotavirus pathogenesis and immunity.

6.4 Rotavirus Immunity and Vaccine Development

6.4.1 Mouse Models in the Study of Rotavirus Protective Immunity and Vaccines
In the history of rotavirus immunity research, mouse models have contributed to a number of mile-
stone studies that have greatly improved our understanding of the mechanisms of rotavirus protective
Rotavirus 103

immunity. In sucking mice orally inoculated with a heterologous simian or bovine rotavirus (RRV or
NCDV) strain or a homologous murine rotavirus (wild-type or tissue culture-adapted EHP) strain and
challenged after 6 weeks with a virulent murine rotavirus (wild-type strain ECW), a fecal IgA antibody
induced by homologous infection was identified as the correlate of protection.74 A murine model with
“backpack tumor” transplantation was used to determine the protective effects of antibodies against VP4
(the outer capsid viral protein) and VP6 (the major inner capsid viral protein). The study indicated that
in vivo intracellular viral inactivation by anti-VP6 secretory IgA during transcytosis is a mechanism of
host defense against rotavirus infection.170
Various gene knockout mice have been used to identify the determinants of rotavirus protective immu-
nity,78–85 including Rag-2 (devoid of both T and B cells), β2m (lack cytotoxic T cell responses), JHD (lack
B cell responses), and IgA knockout mice (no detectable IgA in the serum or in any secretions). These
studies have examined the roles of various components of humoral and cellular immunity in the resolu-
tion of primary infection or protection against rotavirus reinfection. The findings indicate that the IgA
antibody, CD4, and CD8 T cells are all important in mediating protective immunity; however, none of
the cellular effectors is absolutely indispensable in mediating protective immunity against rotavirus infec-
tion, suggesting the redundant nature of the host immune defense system of mice. The B-cell-­dependent
humoral immunity appears to be the main mechanism of protection from rotavirus infection.171
The effectiveness of various novel rotavirus vaccines including DNA plasmids,172 VLP,173 and sub-
unit174 vaccines was first evaluated in mouse models of rotavirus infection for protection against virus
shedding before it was further evaluated in the Gn pig model of rotavirus diarrhea.104,127,136

6.4.2 Gnotobiotic Pig Model in the Study of Rotavirus Immunity and Vaccines

6.4.2.1 Unique Features of the Gn Pig Model
Hysterectomy-derived colostrum-deprived Gn pigs have been used extensively in rotavirus research to
understand protective immunity and for the evaluation of rotavirus vaccine efficacy.130 Gn pigs are kept
in sterile isolator units and are raised on sterilized milk supplements.175 They are susceptible to HRV
­disease for at least 8 weeks.138 The minimal infectious dose of virulent Wa HRV in Gn pigs is one plaque
or fluorescent forming unit (PFU or FFU) or less,37 similar to that of porcine rotaviruses.176 However,
after HRV infection of Gn pigs, clinical disease is usually milder compared to that produced with por-
cine rotaviruses and mortality is uncommon. Gn pigs from birth to 8 weeks of age develop diarrhea in
1–3 days, and diarrhea usually continues for 3–5 days after oral inoculation with HRV.37,110 In compari-
son to other animal models such as mice, rats, and rabbits, Gn pigs present a number of important advan-
tages (Table 6.1). Although the rodents serve as useful models for the evaluation of immune responses
to rotaviruses, older mice, rats, and rabbits are refractory to disease, and therefore, the readout is only
for protection against viral infection; it is not useful for the evaluation of active immunity to clinical
disease. In comparison, Gn pigs are susceptible for up to at least 8 weeks of age to infection and diarrhea
when inoculated with several HRV strains.99 Although the evaluation of relatively short-term protection
(3–8 weeks) is a limitation of this model, no other animal model is susceptible to HRV infection and
clinical disease, even for this limited period. Another important feature regarding Gn pigs is that expo-
sure to extraneous enteric pathogens, especially the wild-type rotaviruses as a confounding variable, is
excluded; therefore, true primary immune responses to a single rotavirus infection or vaccination can
be evaluated. Gn pigs are also valuable in the identification of virulence factors that are often masked in
studies using conventional animals.177
Pigs are monogastric and closely resemble humans physiologically and immunologically.178,179 The
ontogeny of the porcine immune system, especially the development of immunoglobulins, antibody rep-
ertoire, and B cells, has been extensively reviewed by Butler and colleagues.179–181 The development of
lymphoid organs from birth to the adult stage in pigs and T- and B-cell immune responses have been doc-
umented and show great similarity to humans.182 Because of the physiological and immunological simi-
larities to humans, the pig is an important large animal model for human biomedical research.177,178,183
The ability to isolate large numbers of lymphocytes following the disruption of solid organs offers con-
siderable advantages for the study of the intestinal and systemic immune systems.112,114,134
104 Laboratory Models for Foodborne Infections

Although there are many similarities between porcine and human physiology and immune systems,
there are also differences that need to be considered. One significant difference between humans and
pigs is that humans acquire maternal antibodies transplacentally, whereas the placenta of pigs acts
as a barrier to the transfer of macromolecules, including maternal antibodies and cytokines. Unlike
mice and humans, no maternal antibodies are transferred from the mother to the porcine fetus during
intrauterine development due to the special structure of the placenta wall in swine; hence, Gn pigs
are agammaglobulinemic but are immunocompetent at birth.184 Piglets acquire immunoglobulins (Ig)
solely by intestinal absorption of colostral Ig for about 36 h after birth before gut closure occurs.185
This feature permits basic studies of the ontogeny of neonatal immune responses and a true primary
antibody response to rotavirus to be evaluated. This is also an advantage for using this model to study
the effects of maternal antibodies and other maternal immune regulators on rotavirus vaccine efficacy
because the levels and titers of those introduced into the circulation or intestines of piglets can be
manipulated experimentally.125,144,145,150

6.4.2.2 HRV Vaccines and Therapeutics Evaluated in the Gn Pig Model of

Wa HRV Infection and Diarrhea
The Gn pig model has been widely accepted to be a valuable model for studying HRV pathogenesis and
as an ideal model for preclinical testing of the safety and efficacy of rotavirus candidate vaccines.99,130
The Gn pig model of Wa HRV infection and diarrhea has been used in evaluating numerous rotavirus
candidate vaccines including live attenuated (Wa), reassortants (RotaTaq, LLR), inactivated (Wa, CDC-9),
recombinant protein (P2-VP8*), DNA plasmid (VP6), and VLP (2/6 and 2/6/7) vaccines with different
adjuvants (LT-R192G, ISCOM, aluminum phosphate), immunostimulating supplements (probiotics and
rice bran) and immunization routes (oral, intramuscular, intranasal, intradermal by gene gun), and with
or without maternal antibodies.103,104,118,124,125,127,133,136,138,141,144,186,187 The main criteria used in these stud-
ies to establish the efficacy of HRV vaccines include the protection of immunized animals from fecal
virus shedding and rotavirus-induced diarrhea (onset, duration, and fecal consistence scores). Immune
responses associated with protection against rotavirus diarrhea identified in these studies include intes-
tinal IgA antibody secreting cells141 and memory B cells,134 intestinal IFN-γ-producing T cells,188 and
serum IgA, intestinal IgA, and intestinal IgG antibodies.139 Through these studies, the Gn pig model has
established its role as the most reliable animal model for the preclinical evaluation of rotavirus vaccines
and therapeutics.
The Gn pig model has been used to evaluate the therapeutic effect of passive antibodies and dietary rice
bran supplementation on protection against rotavirus diarrhea.103,109 Oral administration of VP6-specific
llama-derived single domain nanoantibodies was shown to be an effective treatment against virulent
Wa HRV-induced diarrhea.109 Rice bran demonstrated strong effects on the stimulation of nonspecific
and HRV-specific immune responses and protection against HRV diarrhea.103 In addition, rice bran sig-
nificantly enhanced the growth and colonization of probiotic Lactobacillus rhamnosus GG (LGG) and
Escherichia coli Nissle 1917 (EcN) in the intestine of Gn pigs, protected against damage to intestinal
epithelium while maintaining intestinal homeostasis, maintained intestine permeability, enhanced the
IFN-γ and IgA protective immune responses during HRV infection, and provided complete protection
against HRV diarrhea in LGG- and EcN-colonized pigs.189

6.4.2.3 Gnotobiotic Pig Model Colonized with Probiotic Bacteria or

Human Gut Microbiota
Gn animal models are an important tool for studying the role of probiotics and commensal microbiota in
the development of the mucosal immune system.190 Despite the differences from conventional animals,
Gn pigs offer distinct advantages for studies on the effects and immunomodulating mechanisms of pro-
biotics and gut microbiota on enteric virus infections and vaccines. The most distinctive advantage of
using the Gn pig model is that neonatal pigs provide an immunologically and microbiologically naive
background that allows the identification of host responses to a single pathogen or a single vaccine in
hosts colonized with a clearly defined probiotic strain or gut microbiota.183
Rotavirus 105

The mechanisms of probiotic LGG on reducing HRV pathogenesis were examined in Gn pigs fed with
high-dose LGG and challenged with virulent Wa HRV.108,110 LGG treatment before and during HRV
gastroenteritis partially prevented virus-induced intestinal tissue damage, reduced autophagy marker
expression to normal levels, induced apoptosis,108 and partially prevented HRV-induced compensatory
increases of the adherent junction proteins, α-catenin, and β-catenin; tight junction proteins, occludin,
claudin-3, and claudin-4; and leak protein claudin-2 in the ileal epithelium.110
The immune-modulating effects of probiotic Lactobacillus strains L. acidophilus NCFM, L. reuteri
and LGG on intestinal and systemic B cells, CD4 and CD8 T cells, γ/δ T cell, macrophage/dendritic
cell, toll-like receptor, and cytokine responses to virulent HRV infection or the live attenuated Wa HRV
vaccine were comprehensively examined in a series of studies on Gn pigs fed with different doses of
L. acidophilus NCFM or LGG101,106,113,191 or the mixture of L. acidophilus NCFM and L. reuteri.116,117,120–123
L. acidophilus NCFM and LGG are shown to have therapeutic as well as immune-stimulating effects,
and have dual functions in partial protection against HRV diarrhea and as adjuvants for the HRV vac-
cine when used in the proper dosage.101,106 These studies also demonstrated that both the dose (CFU/day)
and dosing regimen (total number of days’ intake) of probiotics have significant effects on the immune
modulatory functions of probiotics. Different LGG dosages differentially modulated immune responses
to favor either the mucosal IgA response (five doses) or the T-cell response (nine doses).101 High-dosage
L. acidophilus NCFM intake induced regulatory immune responses, similar to the effect of microbiota
colonization in infants, which had a negative impact on the immunostimulatory effect of probiotics and
abolished its effectiveness as a vaccine adjuvant.106
The lack of gut microbiota is a unique feature of the Gn pig model that provides an indispensable tool
for the study of the consequences of bacterial colonization, but it can also be a drawback when results
from preclinical vaccine studies need to be extrapolated to humans. In normal humans and conven-
tional animals, commensal microbiota helps the host to defend against pathogenic microorganisms.190
These bacteria provide the colonization resistance against attachment, multiplication, and invasion of
pathogenic microorganisms into epithelial cells and their potential circulation in the host. Furthermore,
they exert a major influence on the development of the mucosal immune system and regulation of host
immune responses. In the very early stage of the postnatal period, the immune responses are biased
toward Th2, and the Th1-mediated immune response is lacking.192,193 Thereafter, intestinal commensal
microbiota stimulates the development of both local and systemic immune systems, preferentially Th1
immune responses, but later induces regulatory mechanisms to maintain homeostasis.194 The intesti-
nal colonization of germfree animals with commensal microbes not only significantly stimulates the
development of mucosal and systemic immune systems, but may also promote integrity of the epithe-
lial barrier by regulating tight junctions and protecting it from injury with enhanced proliferation and
cytoprotective protein production.195–199 In addition, commensal bacteria regulate cell migration to rein-
force the epithelial barrier.200 Intestinal colonization also stimulates mucin secretion from goblet cells
to limit intestinal infections through binding between mucin and pathogens.201,202 Intestinal microbiota
also drive the development of Th17 cells203 and Treg cells.204 Therefore, the lack of gut microbiota in Gn
pigs can cause deviations in the pathogenesis of HRV infection and in the patterns of immune responses
induced by HRV infection or on vaccination, as compared to those observed in human infants.
In order to identify the influence of microbiota on the Gn pig’s response to HRV and to more closely
mimic human infants, Gn pigs transplanted with newborn HGM and infected with HRV have been estab-
lished. This model has been used to test the effects of probiotics on the gut microbiome structure during
an HRV infection.102,105 The development of HRV vaccine-induced immune responses has been compared
between the HGM and non-HGM-transplanted Gn pigs. HGM successfully colonized the Gn pig intestine
after three oral inoculations. Sequencing of the V4 region of 16S rRNA genes demonstrated that the pigs
carried a microbiome similar to that of a C-section delivered human infant.102 The attenuated Wa HRV
vaccine conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and
HGM-transplanted Gn pigs. HGM promoted the development of the neonatal immune system, significantly
enhanced IFN-γ-producing T-cell responses, and reduced Treg cell responses in the AttHRV-vaccinated
pigs.105 Given the many advantages of HGM pigs (reviewed by Wang et al.151), the HGM pig model will
have wide applications for the studies of viral pathogenesis; interactions between host-microbiota, host-
pathogens, and microbiota-pathogens; and for the evaluation of vaccines and therapeutics.143
106 Laboratory Models for Foodborne Infections

6.5 Cell Lines and Organoid/Enteroid Models for Rotavirus Infection

Unlike the notoriously difficult human noroviruses,205 HRV is very easy to adapt into cell cultures where
primary cells can support virus growth directly from fecal specimens.206 When stool samples from 454
diarrheic episodes were attempted, rotavirus was cultured from 381 of the 423 specimens that do not
contain other interfering agents (a 90% success rate).207 In the study, two initial cell-culture passages
were made in primary African green monkey kidney (AGMK) cells, followed by additional passages
in MA104 cells. Another study showed that the Wa HRV strain was adapted to cell culture through 14
passages in primary AGMK cells.100 This passage series was successfully initiated only with a virus
that had been serially passaged 11 times in newborn Gn pigs. The virus present in the stool of patient
Wa and the virus from the first three passages in Gn pigs could not be propagated in AGMK cells. This
cell-culture adaptation process simultaneously started the attenuation process of the virulence of the Wa
HRV strain.37 The virus became fully attenuated for pigs after an additional 27 passages in MA104 cells,
and its complete genome sequence was determined.96 Because it has the same G and P types as the cur-
rently licensed Rotarix vaccine, it has been serving as the prototype of the rotavirus vaccine in studies
of live oral rotavirus vaccines in Gn pigs.141,142
For studies of rotavirus replication, innate immune responses, and mechanisms of immune evasion,
various mammalian cell lines have been utilized. Some recent examples are listed in Table 6.2; however,
the findings from these studies are beyond the scope of this chapter.
A new, emerging research area in laboratory models for rotavirus is the development of stem-
cell-derived human intestinal organoid208 and human enteroid209 models for rotavirus infection and as
ex vivo models of host–pathogen interactions in the gastrointestinal tract. Organoids are generated from
induced-pluripotent adult stem cells, whereas enteroids are from intestinal crypts isolated from human
surgical specimens or endoscopic biopsies.209 Human stem cell technology has allowed the in vitro
re-creation of 3D organoids or enteroids that better mimic human intestinal tissues than cell lines.
These models may complement and expand upon the limitations of cell and animal models currently
used to study rotavirus infection and diseases.209

TABLE 6.2
Cell Lines Used in Rotavirus Research
Cell Line Rotavirus Strain References
MA104 cells Bovine rotavirus RF and UK 141,210–218
Simian rotavirus RRV and SA-11
HRV Wa, RV4, M4, Wi, M69 strains, and from clinical
stool samples
Porcine rotavirus OSU, CRW-8, and YM
Murine rotavirus EB
Caco-2 RRV and SA-11 217,219,220
HRV Wa, Wi, and M69
Porcine small intestinal epithelial cell Porcine rotavirus OSU 221,222
line (IPEC-J2) HRV Wa
Vero cells HRV Wa and CDC-9 223,224
Primary AGMK cells HRV from clinical stool samples, murine rotavirus EB 100,207,218
Cos-7 cells Bovine rotavirus RF 216,225
HRV M4
HT-29 SA11–4F, Wa, Bovine rotavirus A5–13 226
Stem-cell-derived human intestinal SA11 and rotavirus from clinical stool samples 208
organoids
Human enteroids Laboratory strains and rotavirus from clinical stool 209
samples
Rotavirus 107

6.6 Concluding Remarks
Animal models have been critical in our understanding of rotavirus pathogenesis and immunity and in
the preclinical assessment of the safety and efficacy of rotavirus vaccines and therapeutics. The most
widely used animal models, namely mice and Gn pigs, present different sets of advantages and limita-
tions, and will continue to be indispensable tools for rotavirus research. Further optimizations of the
models, including humanization of the immune system through stem cell transfer, transplantation with
HGM from donors representing different health and immune statuses, and genetic modification using
CRISPR/Cas9 technology, will further improve the usefulness and reliability of the models for mimick-
ing rotavirus infection in humans.

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Rotavirus 113

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7
Prions

Akikazu Sakudo and Takashi Onodera

CONTENTS
7.1 Introduction....................................................................................................................................117
7.2 In Vitro Models..............................................................................................................................119
7.3 Cell-Culture Models..................................................................................................................... 120
7.4 Animal Models............................................................................................................................. 122
7.5 Conclusions................................................................................................................................... 123
Acknowledgments................................................................................................................................... 123
References............................................................................................................................................... 124

7.1 Introduction
Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are fatal neurological
disorders that include Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine
spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, transmissible
mink encephalopathy (TME) in mink, feline spongiform encephalopathy (FSE) in cats, and exotic ungu-
late encephalopathy (EUE) in zoo animals such as kudu, nyala, gemsbok, eland, and oryx [1]. The prion
agent associated with each disease is named after the disease itself (e.g., CJD agent in CJD, scrapie
agent in scrapie, etc.). A key event in the development of prion diseases is the conversion of the cellular,
host-encoded prion protein (PrPC) to its abnormal isoform (PrPSc) predominantly in the central nervous
system (CNS) of the infected host [1]. Numerous compelling observations support the notion that PrPSc
is the main component of prion agents [2,3]. PrPSc, in contrast to PrPC, is resistant to complete digestion
with proteinase K (PK). Consequently, the detection of PK-resistant prion protein (PrPres) is generally
used for identifying the presence of a prion agent, because PrPres contains PrPSc. Therefore, most diag-
nostic methods for prion diseases are based on the index of PrPSc.
Several diagnostic methods have been used for prion diseases. These include enzyme-linked immu-
nosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) [4]. As an index of prion
infection, representative changes in the brain are generally assayed. Prion protein (PrP) accumulates in
the brain to form deposits. PrPres is biochemically detected after treatment with PK using western blot-
ting (Figure 7.1) and ELISA (Figure 7.2). In the case of ELISA for BSE, a homogenate is usually prepared
from the obex region of the brain, which is subsequently treated with PK. The PK-treated sample is then
applied to a microtiter plate for absorption and reacted with an anti-PrP antibody. This method is com-
mercially exploited in the Bio-Rad TeSE BSE kit (Bio-Rad, France), Enfer-TSE kit (Abbott Laboratories,
USA), and FRELISA BSE Kit (Fujirebio Inc., Japan). Although the extensively employed ELISA is a
sensitive and high-throughput method, the large number of false positives remains a significant problem.
Therefore, if a result is positive, ELISA must be repeated to validate the finding. If a positive result is
obtained again, western blotting and IHC are performed.
Western blotting initially involves separation of the PK-treated proteins by sodium dodecyl sulfate
(SDS)–polyacrylamide gel electrophoresis (PAGE) followed by electroblotting onto a suitable membrane
support. After blotting, PrPres present as a membrane-bound protein can be detected using an anti-PrP

117
118 Laboratory Models for Foodborne Infections

Uninfected mice tissues Infected mice tissues

PrPC PrPSc
PrPC

PK PK

(+) (–) Western blotting (+) (–)

Band
pattern

FIGURE 7.1  Western blotting for detection of PrPSc. Most methods for diagnosing prion diseases are based on the char-
acteristics of the PrPSc, which is resistant to PK. PK completely degrades the PrPC but only partially digests PrPSc because
the latter forms protease-resistant aggregates. After protease treatment, PrPres is detected by western blotting with an
anti-PrP antibody. Western blotting reveals that PK-resistant PrP shifts to a lower molecular weight compared to untreated
PrP (−) because the N-terminal region of PrPSc is susceptible to PK (+). A schematic representation of a Western blot for
PrP derived from representative tissues of prion-infected and uninfected mice before (−) and after (+) PK treatment is
shown. The tissue of choice for PrPSc analysis is the brain.

Uninfected mice tissues Infected mice tissues

PrPC PrPC PrPSc

PK PK

ELISA
Well
color

FIGURE 7.2  ELISA for detection of PrPSc. ELISA diagnosis of prion diseases is based on the characteristics of PrPSc,
which is resistant to PK. PK completely degrades PrP C but only partially digests PrPSc, because PrPSc forms protease-
resistant aggregates. After PK treatment, ELISA is performed using an anti-PrP antibody to detect PK-resistant PrP
(when PrPSc is present in the sample). Because the N-terminal portion of PrPSc is digested with PK, an antibody should
be chosen that recognizes an alternative region of PrP. In the case of a sandwich ELISA, which is becoming increas-
ingly popular, two discrete anti-PrP antibodies recognizing different epitopes of PrP are used. Specifically, one acts as
a capture antibody, which is coated onto the well of the microplate, while the other is a detection antibody conjugated
with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). After reaction with the detection
antibody, the corresponding substrate for the conjugated enzyme is added. The resulting color change caused by the
enzyme reaction is then measured using a microplate reader on the basis of absorbance.
Prions 119

antibody. Importantly, western blotting provides information on both the prion infection and the mobil-
ity of peptides, which is influenced by the host genotype and strain of prion [5]. In the case of IHC, the
indexes for prion infection are neuronal cell loss, astrocytosis, and vacuolation in addition to PrP accu-
mulation (amyloid plaques) [6]. In IHC-based analyses of brain sections, these changes are examined by
light microscopy.
A number of novel, alternative diagnostic methods for prion diseases have also been developed, and
recently reviewed [4,7,8]. Readers may refer to these articles for further technical details.
In this chapter, we describe laboratory models used for analyzing prion infections. The laboratory
models for prion infections are divided into in vitro models, cellular models, and animal models. In all
three cases, prion infection is diagnosed by detection of PrPSc as described earlier.

7.2  In Vitro Models

The representative in vitro model for prion infection is protein misfolding cyclic amplification (PMCA)
(Figure 7.3). PMCA is a PrPres amplification method that is conceptually analogous to DNA amplification
by PCR. This in vitro model could be used as a surrogate for animal bioassay.
PMCA [9] exploits the fact that PrPSc can convert PrPC to PrPSc. Specifically, PMCA enables the in vitro
amplification of PrPres in an accelerated manner from a small quantity of PrPSc, which acts as the seed,
via sequential cycles of incubation and sonication [10]. In the first phase, small quantities of PrPres are
incubated with a large quantity of PrPC, leading to the growth of PrPres aggregates. In the second phase,
the PrPres aggregates are broken up by sonication, causing a proliferation in the number of nuclei for fur-
ther PrPres amplification. By repeated cycles of incubation followed by sonication, the number of seeds
increases in an exponential fashion resulting in the amplification of PrPres. Healthy brain homogenate is
usually used as the source of PrPC. Optimization of the buffer conditions used for the amplification step
may be required. Successful PrPres amplification after PMCA is generally verified by western blotting
using an anti-PrP antibody following PK treatment. The levels of PrPres amplified by this method are then
correlated with the prion infectivity titer [11]. The results from this analysis indicate that amplified PrPres
includes PrPSc, suggesting that PrPSc can be amplified by PMCA. Furthermore, PMCA is able to detect

PrPSc
PrPC Incubation

Conformational
change

Sonication/
incubation

FIGURE 7.3  PMCA for amplification of PrPSc. Recently, a powerful diagnostic method for prion diseases, known as
PMCA, has been developed for the amplification of PrPSc prions from body fluids such as blood, cerebral spinal fluid,
semen, milk, urine, and saliva during the preclinical and clinical phases of prion diseases [61]. In this method, a PrPSc seed
is incubated with the PrPC source. PrPSc in the presence of excess PrPC induces the formation of large aggregates that are
subsequently broken up by sonication. Repeated cycles of this procedure efficiently amplify PrPSc.
120 Laboratory Models for Foodborne Infections

prions in blood [12]. PMCA has been successfully used to diagnose both terminally diseased hamsters
and prion-infected presymptomatic hamsters [13] and deer [14]. In summary, this method can be used to
amplify PK-resistant PrP derived from various species including cervid, ferret, hamster, mouse, sheep,
bovine, and human [12–17].
Although conventional PMCA utilizes normal brain homogenate as a source of PrP C, a novel
adaptation of the PMCA procedure is to amplify PrP res and generate highly infectious prions using
recombinant PrP derived from Escherichia coli [18] or baculovirus [19] in the absence of brain
homogenate. This novel procedure requires RNA and lipids, such as 1-palmitoyl-2-oleolyl-sn-
glycero-3-phospho(1′-rac-glycerol) (POPG) or phosphatidylethanolamine (PE), to act as conversion
cofactors [18,20,21].
Recently, the real-time quaking-induced conversion (RT-QuIC) test [22], which is a modified version
of PMCA, has been developed. This method is based on the prion-seeded fibrillization of recombi-
nant PrP. In RT-QuIC reactions, prion-associated seeds induce the amyloid fibril formation of bacte-
rially expressed recombinant PrP in multiwell plates. The resulting amyloid fibrils are then detected
by the enhanced fluorescence of an amyloid sensitive dye, thioflavin T, present in the reaction mix.
RT-QuIC is known to be highly specific and sensitive for the detection of multiple human and animal
prion diseases [23]. Epidemiological surveillance of CJD, which currently relies heavily on autopsy-
based diagnosis, could be more efficient, cost-effective, and broadly applicable with RT-QuIC testing for
cerebrospinal fluid (CSF) samples that can be obtained without autopsies [24]. The second-generation
RT-QuIC assay markedly improves the speed and sensitivity of detecting prion seeds in CSF specimens
from CJD patients [24].
In addition, the RT-QuIC assay has been used successfully to detect multiple human, bovine, cer-
vid, ovine hamster, and mouse strains in a variety of biological tissues such as saliva, blood, and nasal
fluids [25]. Indeed, numerous CJD diagnostic laboratories around the world are currently implement-
ing and validating RT-QuIC testing for human sCJD CSF using standard conditions [26]. RT-QuIC
enables an estimation of the quantity of CWD prion infectivity in tissues, such as the obex, left ventricle,
pancreas, jejunum, and spleen, as well as body fluids, such as saliva and urine [27]. Such an assay will
contribute to the risk assessment of deer tissues, biological fluids, excreta, or environmental samples
obtained from CWD-endemic areas.

7.3  Cell-Culture Models

Several prion-susceptible cell lines have been exploited to generate cell-culture models for prion infec-
tion [4,28]. The general cell-culture procedures for prion infection have been described previously
[29,30]. Brain homogenates of prion-infected animals are added to the culture medium for 1–2 days
before culture passage. Pretreatment by heating at 80°C for 20 min, sonication for 3 min, and filtration
through a 0.22 µm filter unit reduce toxicity of the brain homogenate, which displays an efficient rate of
infection. As an alternative approach, it is known that immortalized cell groups are observed in prion-
infected animals. Thus, primary cultures from a prion-infected brain can act as a source of cells by
continuous passage (e.g., ScHB and SMB produced from prion-infected animals) [31]. Consequently,
established cell lines derived from prion-infected animals sometimes result in the establishment of
persistent prion-infected cells after multiple passages. Indeed, such cell lines of neuronal and nonneu-
ronal origin have been established. After persistent infection, these cells proliferate without any overt
cytopathic effect.
Infectivity or PrPSc production per cell can be assayed to determine the percentage of infection of
prions. The infectivity of harvested cells is measured after the brain sample has been injected into
experimental animals via intracerebral inoculation. After performing behavioral observations for
1 year, the effective lethal dose (LD50) can be derived from the survival curve of lysate-injected ani-
mals. PrPSc detection in cells and animals is usually performed by western blotting. After confirming
persistent infection of prions using these methods, the persistently infected cells are established and used
for analysis.
Prions 121

To date, numerous cell lines have been established for prion infection (Table 7.1). The representa-
tive neuronal cell, rat pheochromocytoma PC12, can be differentiated by nerve growth factor (NGF)
and infected with prions [32], probably due to increased levels of PrP through NGF stimulation. Rabbit
kidney–derived RK13 cells overexpressed with ovine PrP are susceptible to sheep scrapie infection [33].
Furthermore, mouse fibroblast cells NIH/3T3 and L929 are also susceptible to mouse-adapted scrapie
infection [34]. In other cell types, microglial cells as well as epithelial cells and myoblasts can be suc-
cessfully infected with prions. Thus, under certain conditions, nonneuronal cells can also be infected
with prions. Indeed, this is consistent with the observation that PrPSc accumulates not only in the CNS
but also in other tissues such as the placenta, lymphoreticular system, and muscle tissue [35,36].
The use of the prion-susceptible cell line N2a is attractive because it is easily cultured. The N2a cell
line has been extensively used since 1970 to study prion infection [37], although the observed low postin-
fection titers and rapid attenuation in titers remain problematic [38,39]. The hypothalamic cell line GT1
displays higher PrPC expression levels and is more susceptible to prion infection than other cell lines
[40,41]. Although the human neuroblastoma cell line SH-SYS5 was reported to be infected with CJD pri-
ons, reproducibility of the result has yet to be confirmed by other laboratories [42]. Therefore, cell-culture
systems for CJD prion-infection have not been established. For BSE prion, only two cell lines, the mouse
microglial cell line MG20 overexpressing mouse PrP and the rabbit epithelial cell line RK13 overexpress-
ing bank vole PrP, can be infected with mouse-adapted BSE and bank vole-proliferated BSE, respectively.

TABLE 7.1
Representative Cell Lines Used for Prion Infection
Cell Lines Origins Prion Strains References
PC12 Rat pheochromocytoma 139A, ME7 [32,62,63]
C2C12 Mouse skeletal myoblast cell 22L [64]
CAD Mouse catecholaminergic cell RML, 22L, 22F, 79A, 139A, ME7 [44,65–67]
CF10 Mouse brain–derived PrP- 22L [68]
deficient cell
GT1 Mouse hippocampal neuron Chandler, RML, 139A, 22L, kCJD, FU [40,41,69–72]
CJD, M1000
HaB Hamster brain–derived cell Sc237 [73]
HpL3–4 Mouse hippocampal neuronal cell 22L, Chandler [74,75]
L. fibroblasts Mouse fibroblast ME7, Chandler [76]
L929 Mouse fibroblast 22L, ME7, RML [34]
MDB Deer immortalized brain cell CWD [77]
MG20 Mouse microglial cell Chandler, ME7, Obihiro, mouse-adapted [78]
BSE
MNB Mouse neuroblastoma Chandler [39]
MovS Mouse Schwann cell-like dorsal PG127, SSBP/1, scrapie field isolates [79,80]
root ganglia
MSC-80 Mouse Schwann cell Chandler [81]
N2a Mouse neuroblastoma Chandler, RML, 139A, 22L, C506, [38–40,71,82–86]
Fukuoka-1, FU CJD
NIH/3T3 Mouse fibroblast 22L [34]
RK13 Rabbit kidney epithelial cell Fukuoka-1, 22L, Chandler, M1000, [33,69,80,87–91]
mo sCJD, voBSE, PG127, LA404,
SSBP/1, scrapie field isolates, CWD
ScHB Hamster brain cell Scrapie (Chandler) [31]
SH-SY5Y Human neuroblastoma CJD [42]
SMB Mouse brain cell Chandler, 139A, 22F, 79A [31,92,93]
SN56 Mouse cholinergic septal cell Chandler, ME7, 22L [94]
BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt–Jakob disease; kCJD, Kuru CJD; sCJD, Sporadic CJD; CWD,
chronic wasting disease; RML, Rocky Mountain Laboratory isolate.
122 Laboratory Models for Foodborne Infections

It should be noted that cell lines susceptible to some prion strains demonstrate a remarkable resistance
to other strains [43,44]. For example, mouse fibroblast 3T3 cells are only susceptible to prion strain 22L,
while mouse fibroblast L929 cells can be infected with prion strain 22L, RML, and ME7 [34,44]. In addi-
tion, although most of the cell lines and primary cells have been shown to express PrPC [45,46], only a lim-
ited number of these cells are susceptible to prion infection. Moreover, it is intriguing that both neuronal
and nonneuronal cell lines are susceptible to prion infection [47]. However, the reason for this difference
in susceptibility to prion infection among cell lines and prion strains remains unclear. Such differences
may be related to the cellular properties of the receptor expression or possibly cofactors contributing to
PrPSc internalization, such as laminin receptor precursor (LRP/LR) [48], low-density lipoprotein receptor-
related protein 1 (LPR1) [49], proteoglycans, and glycosaminoglycans (GAGS) [50]. Indeed, some cells
infected with prions secrete exosomes associated with the prion agent, suggesting that exosome release
from cells into the cell-culture medium might be related to susceptibility to infection [51,52].

7.4  Animal Models

The most common experimental animal models for studying prion infection are mouse and hamster [53].
For example, infection of a hamster with 263K prion leads to the onset of prion disease within a short
incubation period (∼100 days). The experimental route of infection is normally intracerebral and intra-
peritoneal inoculation (Figure 7.4). In the case of intracerebral inoculation, a sample is injected into the
cerebral ventricular system of mice using a microsyringe [54]. Intraperitoneal inoculation is the injection
of the sample into the peritoneum (body cavity). In some cases, such as during an inactivation or removal
assay for prions, stainless steel wires contaminated with prions are often used [55].
To estimate the titer of infectious prion, samples used to inoculate animals should be serially diluted
and assayed. The length of the incubation period following inoculation with the serially diluted prions
before the onset of the disease is shortened in a dose-dependent manner. Clinical symptoms such as
tremors and ataxia are used for the calculation of incubation time. Thus, using this protocol, it is possible
to estimate the infectious titer of prions by monitoring clinical TSE signs and comparing mice inoculated
with serially diluted prions. In addition to the incubation time, the survival rate after inoculation is also
important. By monitoring the survival curve, quantitative bioassays can be performed using biostatistics.
Kaplan–Meier estimators have been used to generate survival curves and the results were analyzed by
using other tests such as the log-rank test or generalized Wilcoxon test.
TSE infected mice have a prolonged asymptomatic incubation period, lasting between 4 months and the
full lifespan of the mice (over 2 years), depending on the particular mouse and prion strains under inves-
tigation. However, the incubation time after intracerebral inoculation is uniform for any given mouse and

Intracerebral inoculation

Intraperitoneal inoculation

FIGURE 7.4  Animal infection models of prion diseases. Animal infection models of prion diseases generally involve
intracerebral or intraperitoneal inoculation. In both cases, the survival time and incubation period are measured as an
index of prion infection and infectious titer. Both the survival time and incubation period after intracerebral infection are
shorter than after intraperitoneal infection. The survival curve of the mice is then examined by statistical analysis (e.g.,
the log-rank test).
Prions 123

prion strain combination. Nonetheless, markedly different incubation times are observed when different
prion strains are tested against a single inbred mouse strain [56]. Likewise, strikingly different incubation
times are observed when a single prion strain is tested against different mouse strains, especially those
having different genotypes of the PrP gene (Prnp). There are two alleles of mouse Prnp, namely Prnpa and
Prnpb, encoding PrP that differ by two amino acid residues at codons 108 and 189 [57]. Mice with differ-
ent PrP genotypes may have an incubation period that differs by hundreds of days after infection with the
same prion strain. Subtle changes in the behavior of infected mice are often seen for a few weeks before
the onset of definite neurological signs during the last 2–3 weeks of the incubation period.
Behavioral changes are closely related to neuropathology. Images of histological sections of the brains
of infected animals are dramatically different among prion strains [58]. The areas of vacuolation and
PrP deposits depend on the prion strain, but also to some extent on PrP and other genetic factors. The
so-called “lesion profile” is an objective analysis that involves scoring the area of vacuolation and/or PrP
deposits in nine gray matter and three white matter brain areas. Interestingly, each combination of prion
strain and mouse strain gives a characteristic lesion profile [56,59,60].
Due to the extended incubation period, animal bioassays take a very long time to complete (e.g., up to
2.5 years for primary transmission in mice, then at least 6 months for each serial mouse passage). In addi-
tion, a special biosafety level facility is required, which makes the whole procedure very costly. Therefore,
an appropriate sample size (number of animals, type of animals) for each assay should be carefully con-
sidered. Given the difficulties and expense of performing such experiments, researchers should consider
whether an alternative approach, such as in vitro testing or cell-culture assays, can be used.

7.5 Conclusions
Studies of prion biology have until recently used conventional laboratory models for prion infection,
cell-culture assays, and bioassays. The development of novel tube-based methods involving PrPres
­amplification, such as PMCA and RT-QuIC, has revolutionized the analysis of prion diseases [61].
In p­ articular, PMCA and RT-QuIC have dramatically improved the diagnosis of prion diseases by
­facilitating the sensitive detection of prions. Furthermore, RT-QuIC provides quantitative data on PrP res,
while PMCA enables amplification of PrPSc in vitro. Novel future applications of these methods in prion
biology are anticipated.
Studies using cell lines suggest that PrPC is not the sole determinant for prion susceptibility. Indeed,
other cellular factors may be required for efficient infection. Under certain conditions, loss of host factors
for susceptibility to prion infection may occur. Furthermore, most cell lines have restricted prion-strain
specificity, that is, matching pairs between the host cell and prion strain. Similarly, constituents of the
buffer used for PMCA vary depending on the particular prion strain under investigation. Research into
anti-prion drugs using cell lines and PMCA systems must take these factors into consideration, because
some of the anti-prion activity might be attributed to a special property of the prion strain or cell line. For
example, an anti-prion activity may be caused by an indirect effect on cellular metabolism. Thus, studies
using different strains and cell lines are necessary in order to identify an anti-prion compound effec-
tive against a broad range of prion strains. Unfortunately, animal bioassays are often time ­consuming,
laborious, and expensive to perform. Nonetheless, animal bioassays are still valuable in determining the
biological activity of prions and remain the gold standard laboratory model for prion infections.

Acknowledgments
This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education,
Science, Culture and Technology of Japan (25450447, 24110717, 23780299, 22110514, 20780219,
17780228), Grants-in-Aid from the Research Committee of Prion Disease and Slow Virus Infection,
the Ministry of Health, Labour and Welfare of Japan, and Japan Agency for Medical Research and
Development (AMED).
124 Laboratory Models for Foodborne Infections

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like growth factor-1 receptor in scrapie-infected neuroblastoma cells. J Biol Chem 276, 36110–5 (2001).
85. Race, R. The scrapie agent in vitro. Curr Top Microbiol Immunol 172, 181–93 (1991).
86. Scott, M.R., Kohler, R., Foster, D. & Prusiner, S.B. Chimeric prion protein expression in cultured cells
and transgenic mice. Protein Sci 1, 986–97 (1992).
87. Courageot, M.P. et al. A cell line infectible by prion strains from different species. J Gen Virol 89,
341–7 (2008).
88. Lawson, V.A. et al. Mouse-adapted sporadic human Creutzfeldt–Jakob disease prions propagate in cell
culture. Int J Biochem Cell Biol 40, 2793–801 (2008).
89. Sabuncu, E. et al. PrP polymorphisms tightly control sheep prion replication in cultured cells. J Virol 77,
2696–700 (2003).
90. Bian, J. et al. Cell-based quantification of chronic wasting disease prions. J Virol 84, 8322–6 (2010).
91. Kim, H.J. et al. Establishment of a cell line persistently infected with chronic wasting disease prions.
J Vet Med Sci 74, 1377–80 (2012).
92. Birkett, C.R. et al. Scrapie strains maintain biological phenotypes on propagation in a cell line in cul-
ture. EMBO J 20, 3351–8 (2001).
93. Kanu, N. et al. Transfer of scrapie prion infectivity by cell contact in culture. Curr Biol 12, 523–30 (2002).
94. Baron, T.G., Biacabe, A.G., Bencsik, A. & Langeveld, J.P. Transmission of new bovine prion to mice.
Emerg Infect Dis 12, 1125–8 (2006).
 Section II

Foodborne Infections due to

Gram-Positive Bacteria
8
Bacillus

Jessica Minnaard, Ivanna S. Rolny, and Pablo F. Pérez

CONTENTS
8.1 Introduction....................................................................................................................................131
8.2 Virulence Factors: An Overview...................................................................................................133
8.3 In Vitro Models............................................................................................................................. 134
8.3.1 Cultured Eukaryotic Cells................................................................................................ 134
8.3.2 Regulation of Toxin Production....................................................................................... 138
8.3.3 Direct Interactions with Eukaryotic Cells: Other Virulence Factors............................... 139
8.3.4 Virulence Factors in Other Bacillus spp.......................................................................... 140
8.4 Ex Vivo and In Vivo Models.......................................................................................................... 140
8.4.1 Ex Vivo Models..................................................................................................................141
8.4.2 In Vivo Models...................................................................................................................141
8.5 Invertebrates...................................................................................................................................141
8.5.1 Nematodes.........................................................................................................................141
8.5.2 Insects................................................................................................................................142
8.5.2.1 Galleria mellonella.............................................................................................142
8.5.2.2 Bombyx mori.....................................................................................................143
8.6 Vertebrates.....................................................................................................................................143
8.6.1 Rabbit.................................................................................................................................143
8.6.2 Zebrafish........................................................................................................................... 144
8.6.3 Monkeys........................................................................................................................... 144
8.6.4 Asian House Shrew.......................................................................................................... 144
8.6.5 Mice.................................................................................................................................. 144
8.6.6 Rats................................................................................................................................... 146
8.7 Concluding Remarks..................................................................................................................... 146
Acknowledgments....................................................................................................................................147
References................................................................................................................................................147

8.1 Introduction
The ability of prokaryotes to sporulate is one of the evolutionary strategies to cope with stress. Spore-
forming microorganisms invest considerable amounts of energy in a refined differentiation process
(sporogenesis) that involves the expression of hundreds of genes in order to give a morphologically
distinct cell that presents several barriers related to the resistance to physical, chemical, and biological
challenges.1
Among spore-forming microorganisms, those belonging to the genus Bacillus play an important role
in several aspects of human activities. Indeed, the ability of fast growth and the diversity of metabolic
capabilities along with the capacity to survive in adverse conditions lead to a versatile genus that actively

131
132 Laboratory Models for Foodborne Infections

participates in shaping the environment. However, some members of the genus Bacillus have evolved
pathogenic potential and are thus associated with distinct pathological processes.
The genus Bacillus includes spore-forming Gram-positive, low G + C content, rod-shaped bacteria.
Members of the genus are ubiquitous and capable of surviving and growing in very different, even
extreme, conditions such as high temperatures, high saline concentrations, and low pH.2
The early works of Pollender, Brauell, Delafond, and Davaine on anthrax or carbuncle prompted
Robert Koch to enunciate the paramount postulates of the germ theory of disease and Louis Pasteur
to establish effective immunization for anthrax prevention in the seminal experiment of massive live-
stock vaccination at Pouilly-le-Fort.3–5 It is known that some Bacillus species constitute a very definite
cluster sharing many characteristics. This so-called “cereus group” includes microorganisms that can
be identified as Bacillus cereus (B. cereus sensu lato) on the basis of conventional microbiological pro-
cedures, but the use of more refined techniques lead to a more precise identification. The B. cereus
group encompasses seven species, that is, Bacillus anthracis—the etiological agent of anthrax, B. cereus
(sensu stricto), Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihen-
stephanensis, and Bacillus cytotoxicus.6–8 The division between species of the B. cereus group is based
on phenotypic characteristics related to differences in ecology and pathogenesis. However, adoption of
molecular approaches strongly suggests that members of the B. cereus group belong to one single spe-
cies.9–12 In this chapter, we will include bacterial species stated in the original publications irrespective
of the exact taxonomic nomenclature.
It has been demonstrated that the B. cereus group diversified along evolution. This evolutionary path-
way includes thermotolerant lineages as the older microorganisms evolved into mesophilic members,
with a more recent shift to psychrotrophic groups.13 Interestingly, the food-poisoning risk is not neces-
sarily related to species but to the phylogenetic group.7,14
Although the exact cause of many historical epidemics may never be known, there are relevant refer-
ences compatible with epidemic anthrax. Even though no diagnostic methods were available, descriptions
from historians and physicians revealed an illness; their symptoms probably implicated anthrax, that is,
the fifth and sixth plagues of Egypt described in the Book of Exodus, the Plague of Athens (430–427
BCE), and the epizootics described in Virgil’s Aeneid (70–19 BCE).15 However, there are other reports
that postulated that anthrax is unlikely the basis of the Plague of Athens16–18 or other biblical plagues.19
Beyond controversial historical and epidemiological aspects, it is evident that B. anthracis is an impor-
tant zoonotic pathogen and that several outbreaks are well documented. In the 18th century, there was
an outbreak of intestinal anthrax due to consumption of smoked or salted meat from ill animals after
an earthquake occurred in Saint Domingue (modern Haiti). In this episode, 15,000 people were killed.3
Human gastrointestinal anthrax outbreaks are described in different studies, and they are often associ-
ated with consumption of foods originating from diseased animals.20–24 However, unusual sources of
contamination such as animal-hide drums have been reported.24 Intestinal anthrax constitutes the main
presentation for livestock that normally ingests spores in foods.25,26
The B. cereus group represents an example of how natural plasmid transfer within a genus leads to
different microorganisms that are allocated to different species.
Indeed, in the same chromosomal genetic background, the presence of PXO1 and PXO2 character-
izes B. anthracis, a microorganism with high pathogenic potential, and the presence of large transfer-
able plasmids in B. thuringiensis enables its production of insecticidal proteins.27 It is worth noting that
B. cereus harboring B. anthracis toxin genes leads to life-threatening pathology.28–30 Because of these
particularities, members of the group other than B. anthracis were not clearly differentiated until the
late 1960s. Indeed, aerobic endospore-forming rods were not identified at all or assigned to the Bacillus
subtilis species.31 Earlier reports that univocally identified B. cereus as the causative agent of foodborne
diseases were published between 1950 and 1955.31
Foods are suitable culture media for Bacillus spp., and thus, growth in high numbers can occur in
many steps of food preparation and preservation. In addition, some strains are psychrotrophic.32 To
a lesser extent, other Bacillus species have been associated with gastrointestinal illness, for example,
B. subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus brevis, and Bacillus mojavensis, although
these microorganisms are more often involved in food spoilage.31,33–35
Bacillus 133

8.2 Virulence Factors: An Overview

Virulence traits differ between members of the B. cereus group. Certainly, B. anthracis possesses the
highest pathogenic potential because of its ability to produce protein toxins with high biological activ-
ity36 and its potential for phagosomal escape.37 The virulence of this microorganism is related to the
presence of two plasmids, namely, pXO1 and pXO2 encoding for the protective antigen (PA) (pXO1),
edema factor (EF) (pXO1), lethal factor (LF) (pXO1), and capsule (pXO2).25 The expression of toxin
genes is regulated by AtxA, a global regulator encoded by atxA located in the pathogenicity island
within pXO1.38
Virulence factors produced by members of the genus Bacillus differ between species. There is a well-
defined difference between virulence factors of B. anthracis and the more diffuse traits of other mem-
bers of the B. cereus group and other Bacillus spp. Concerning B. anthracis, there are three main routes
of infection: inhalational, cutaneous, and digestive. Even though severity differs according to the infec-
tion route, the same virulence factors are involved.
The pathogenesis of B. anthracis is related to the production of an A2B toxin that includes a B subunit,
that is, PA, and two catalytic subunits, the EF and LF. In addition, it has the ability to synthesize a poly-
δ-d-glutamic capsule, an anti phagocytic factor.
On a mechanistic basis, the mode of action of the AB toxin involves binding of PA that, after proteo-
lytic cleavage, binds either EF or LF that are now able to be internalized to trigger the biological effect.
EF activates adenylate cyclase, thus leading to the secretion of water and electrolytes. On the other hand,
LF cleaves MAPKK, thus interfering with signal transduction.
After secretion and dissemination, PA binds to cell surface receptors, that is, anthrax toxin receptor 1
(TEM8) and anthrax toxin receptor 2 (CMG2). The latter has the higher binding affinity in vivo.36 The
binding of LF or EF to PA requires the proteolytic activation of PA. Indeed, PA83 (83 kDa) splits into
PA63 and PA20 by furin or furin-like proteases, with PA63 being the receptor for LF and EF. After the
proteolytic cleavage of PA83, PA63 oligomerizes on the plasma membrane of the target cell, binds LF or
EF, and is further internalized by receptor-mediated endocytosis.
LF inhibits signaling events of the MAPK pathway, that is, those mediated by ERK, p38, and JNK.
This inhibition occurs after the proteolytic cleavage of MAPKK.36 Since this signaling pathway is para-
mount for cellular homeostasis, LF leads to major adverse effects on the eukaryotic cells. Particularly,
macrophages are very susceptible to LF that triggers apoptosis in these cells. However, this effect is
species dependent and seems to be inversely correlated with the host’s susceptibility to B. anthracis
infection.39,40 EF is a calmodulin-dependent adenylate cyclase that converts ATP to AMPc. It has been
suggested that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved as it is for
Vibrio cholerae.36 The phosphorylation of CFTR leads to the efflux of Cl− followed by Na+ and subse-
quent water movement to the extracellular milieu.
The other main player of B. anthracis virulence is a unique poly-δ-d-glutamic capsule that endows
the microorganism the ability to disseminate.41 Even though the mechanisms behind this are not fully
understood, the role of the capsule in shielding bacterial surface structures and inhibiting the activity of
antimicrobial peptides such as α and β defensins has been emphasized, thus allowing the microorgan-
isms to survive in intracellular compartments.37
Concerning other members of the Bacillus genus, virulence factors are not well identified and they are
still under debate.
The virulence of B. cereus sensu lato has been ascribed to several factors, for example, hemolysin
BL (HBL),42,43 nonhemolytic enterotoxin (NHE),44 enterotoxin FM, cytolysin K, phosphatidylinositol-
specific phospholipase C, enterotoxin S, sphingomyelinase, cereolysin O,45,46 hemolysin II (HLyII),47
and camelysin.48 Emetic strains produce cereulide, a thermostable cyclic dodecadepsipeptide.49,50
The mechanisms behind this plethora of extracellular factors are not completely understood, and the
relevance of these factors in the intestinal pathology is still under discussion. Besides these tradi-
tional factors, the biological effect of direct bacteria–enterocyte interaction has been demonstrated
in vitro.51–53
134 Laboratory Models for Foodborne Infections

Other virulence factors have been described in different species of the Bacillus genus. Interestingly,
B. subtilis and B. mojavensis involved in food poisoning have been demonstrated to produce amylosin,
a heat-stable ion channel forming toxin, which also reported for B. amyloliquefaciens.54,55 Some strains
are able to produce this toxin during growth at refrigeration temperatures.33 In addition, different factors
with biological activity on mammalian cells have been associated to B. licheniformis (lichenysin)56 and
B. pumilus (pumilacidin).57
In the next sections, models that contributed to the understanding of the pathogenesis of Bacillus spp.
will be discussed in the context of the biological activities they are able to detect.

8.3  I n Vitro Models

8.3.1 Cultured Eukaryotic Cells
Studies with cultured eukaryotic cells have contributed to the knowledge of the virulence factors of
many pathogens. These models mimic specific steps of the interaction between microorganisms and
the host, and some cell lines have been demonstrated to generate responses similar to those of the more
sophisticated models.
Several eukaryotic cell lines have been employed to assess the virulence factors of Bacillus spp.
(Table  8.1). Although the modeling of gastrointestinal pathologies can be achieved with cultured
­enterocytes, other cell lines have successfully been employed (Table 8.1).46,47,51,58–78
The experiments with cell-free culture supernatants of B. cereus have provided general information
about the effects of extracellular factors. One of the first reports showed that filtered-dialyzed spent cul-
ture supernatants of strain B-48 grown in a fresh beef infusion were cytotoxic for two cell lines (guinea
pig spleen and buccal carcinoma cells) and the primary explants of mouse-embryo tissue.79 This effect
was observed after 1 h of incubation, and this activity was partially conserved after heating filtrates at
60°C for 2 h.79 In contrast, B. anthracis culture filtrates did not detach cells from monolayers but are
lethal to rats.79 These findings demonstrate that the biological activity on cultured cells does not neces-
sarily correlate with the presence of known factors in the spent culture supernatants.
Supernatants from cultures of B. cereus induce a decrease in the metabolic activity, rounding-up,
cell detachment, and/or death in Caco-2, CHO, Hep-2, HT1080, Raw264.7, McCoy, and human poly-
morphonuclear (neutrophils) cells.51,58,61,63,68,69 Heat or trypsin treatment of the supernatants reduced or
eliminated the cytotoxic effects in Hep-2 and Caco-2 cells.63
Susceptibility to extracellular factors varies between cell lines; that is, the Ped-2E9 cell line was 25- to
58-fold more susceptible than CHO cells when the cytotoxicity of two diarrheal strains was evaluated by
assessing the release of alkaline phosphatase and impairment of mitochondrial dehydrogenase activity.
Fractions with molecular sizes higher than 30 kDa yielded the highest cytotoxicity values.59 Only one
isolate, identified as B. cereus, was active on Ped-2E9 cells as assessed by alkaline phosphatase release.
Other microorganisms identified as B. subtilis or Bacillus spp. were inactive.59
Dramatic changes on the surface of Caco-2 cells were observed in the presence of sterile filtrate super-
natants from B. cereus.58 These changes include microvilli effacement and correlate with cytoskeleton
disorganization (Figure 8.1).
Assays on cultured human enterocyte-like cells (Caco-2) allowed refining studies on the extracel-
lular factors present in spent culture supernatants of B. cereus. This cell line has been considered as a
reference model for the study of the interaction between intestinal microorganisms and the host since
Caco-2 cells spontaneously differentiate in culture and show morphological and functional polariza-
tion.80 In this context, three levels of biological activity were defined on the basis of dose–response
curves in detachment vs. supernatant concentration plots. These different behaviors were high, low, and
non-detaching strains. Culture filtrate supernatants from high detaching strains are able to completely
detach cells from monolayers at least in two serial dilutions. On the other hand, low detaching strains
show a dose–response curve in the entire range of concentrations studied, with the highest detachment
only at the highest dose. Non-detaching strains do not detach cells at any of the concentrations assayed.51
Bacillus 135

TABLE 8.1
In Vitro, In Vivo, and Ex Vivo Models Used for the Study of the Biological Activity of Bacillus spp.
Model

In vitro Epithelial Caco-2 (human colorectal HT1080 (human Vero (kidney epithelial cells from
cells adenocarcinoma) fibrosarcoma cells) an African green monkey)
CHO (Chinese hamster Hep-2 (human laryngeal Hep-G2 (human hepatocellular
ovary) carcinoma) carcinoma)
A549 (human lung HeLa (cervical cancer from Calu-3 (human lung
carcinoma) human) adenocarcinoma)
Paju (human neural Ptk6 null colonic epithelial A204 (human
crest-derived cell line) cells rhabdomyosarcoma)
McCoy (murine Huvet (human umbilical
fibroblasts) vein endothelial cells)
Immune Raw264.7 (monocytes J774 (monocyte/ Neutrophils (hPMNs)
cells from murine origin) macrophage from murine
origin)
Jurkat (human T Ped-2E9 (lymphocyte from U937 (monocytes from human
lymphoblastoid cell line) murine origin) origin)
Ex vivo Spermatozoon test
In vivo Invertebrates Caenorhabditis elegans
Galleria mellonella
Bombyx mori
Vertebrates Ligated rabbit or mouse ileal loop
Vascular permeability reaction
Zebrafish
Suncus murinus
Mice
Rats
Monkeys

This simple experimental approach allowed for the differentiation of the biological activity of B. cereus
strains, even though this activity is related to several extracellular factors present in spent culture super-
natants. In addition, a multivariate analysis demonstrated that high detaching strains were positive for
the sequences of the virulence genes entS, nheC, and sph.51
Cytotoxicity is strain-dependent51,58,68 with species-to-species variations.63 Since nutritional factors
influence the expression of virulence factors, the culture medium modifies the values of cytotoxicity, cell
adhesion, and invasion.63
Fractioning and purification of factors present in the culture supernatants allowed for the identifica-
tion and further study of several toxins of B. cereus. Hemolysin BL (HBL) and nonhemolytic enterotoxin
(NHE) are consistently described as key virulence factors. They are tripartite protein toxins expressed
in different proportions by members of the B. cereus group.61 HBL components are HBLB (37.5 kDa),
HBLL1 (38.2 kDa), and HBLL2 (45 kDa),81 and NHE components are NHA (41 kDa), NHB (39.8 kDa),
and NHC (36.5 kDa).67,82 HBL is expressed in 42%–73% of food-poisoning-associated strains, whereas
NHE is expressed in 97%–99% of strains.61,83,84 Both toxins are responsible for the cytotoxic effect on
several cell lines such as Vero, Hep-2, Hep-G2, Caco-2, Jurkat, RPMI 8226, A204, A549, HUVET, and
U937 cells.62 For NHE, the specificity of the observed biological effects was demonstrated in neutraliza-
tion experiments with monoclonal antibodies.85
The presence of the three components for maximum cytotoxic effect was necessary for the activity
of both HBL and NHE.60,66,81,82 Furthermore, the relative concentrations of the components are relevant
136 Laboratory Models for Foodborne Infections

FIGURE 8.1  Disorganization of the F-actin network following incubation of cultured human enterocytes (Caco-2 cells)
with spent culture supernatants of B. cereus. References: control cells (top), strain 2 (middle), and strain M2 (bottom).
Bacillus 137

for the observed effects (e.g., inhibition of the protein synthesis of Vero cells by NHE44,82). NHEB and
HBLL2 are crucial for the cytotoxic effect since when these components were not present significantly,
lower cytotoxic effects were observed in some cell lines.62
Studies on the structures involved in the binding of toxins to target cells by using CHO and red blood
cells demonstrated that for HBL, the binding component is B (Hbl-B) and L1 and L2 bind to Hbl-B.61,86
On the other hand, the NHE component responsible for the binding to eukaryotic cells is Nhe-C.61,87
Then, there is a sequential binding of Nhe-B and Nhe-A.87 It is worth noting that despite its name (non-
hemolytic enterotoxin), the NHE toxin also has hemolytic activity.88
Even though several extracellular factors have been described in the B. cereus group, NHE was pro-
posed as the main virulence factor84,89 showing the highest cytotoxic effect on Vero (fibroblasts) and
HUVEC (endothelial) cells.62 Albeit to a lesser extent, NHE is also active on U937 cells62 and is respon-
sible for the impairment of the plasma membrane of Caco-2 cells.72 Vero cells treated with spent culture
supernatants showed blebs, which subsequently burst, and these effects were not observed when super-
natants from nheBC mutants were used.72 By using a nheBC mutant strain and Ptk6 cells, it has been
demonstrated that NHE and sphingomyelinase have a concerted effect.71
In relation to the hemolytic action of toxins, B. cereus isolates with the ability to produce trimeric
toxin HBL lead to a characteristic ring-shaped zone of hemolysis in blood agar plates.86,90,91 Hemolysis
depends on the source of erythrocytes: Sheep or calf erythrocytes are the most susceptible, and human
or horse erythrocytes are the least susceptible.86
There is a high similarity in the structure and sequence between HBL and NHE toxins,72 but it is not
possible to complement or substitute components to give an active toxin.61 So far, little is known about
the mechanisms involved in the biological effects of these toxins. Studies performed with osmotic pro-
tectants such as carbohydrates or polyethylene glycol of increasing size allowed researchers to determine
that the mechanism involved in HBL and NHE lysis cells is colloid-osmotic72,92 leading to pore forma-
tion in planar lipid bilayers.72 Spent culture supernatants lead to the release of intracellular ATP and
LDH from Caco-2 and Vero cells; these effects were not detected when supernatants from cultures of
nheBC mutants were employed.72 Because of structural similarities between NHE and HBL components
with hemolysin E from E. coli, it is proposed that the biological effects of both toxins share common
mechanisms.61,93,94
Cytotoxin K (CytK) is another relevant extracellular factor of B. cereus associated with necrotic
enteritis. It is a single-component protein toxin of 34 kDa that is hemolytic to rabbit red blood cells (it
is a β-barrel pore-forming toxin) and cytotoxic to several cells lines.8,62,70 However, CytK has lower
biological activity as compared with other virulence factors such as NHE and HBL.62 Caco-2 and
Vero cells have been seen to be particularly susceptible to CytK.62 Toxicity to Caco-2 cells is related
to the ability to form nonselective pores in the plasma membrane.8,70 This effect is similar to that of C.
perfringens C toxin, although this toxin is cytotoxic and noncytolytic due to the formation of cation
selective pores.70
CytK, also known as hemolysin IV,95 has two variants: CytK1 (firstly reported by Lund et al.8) and
CytK2 which shares 89% identity with CytK1 and has a similar size.96 CytK2 is hemolytic to bovine
erythrocytes and cytotoxic to Caco-2 although to a lesser extent than the original variant, and it did not
show toxic effects on Vero-2 cells.96 Both variants lead to different distributions of pore sizes on lipid
bilayers.96
HlyII is another single-component protein toxin (42.3 kDa), which is a member of the family of
β-barrel pore-forming toxins.8,97–99 HlyII leads to a decrease of the mitochondrial membrane potential,
and it is lytic to Paju and Caco-2 cells.99 Besides, it has hemolytic activity on erythrocytes of different
species, with the highest effects on human and rabbit red blood cells.99 However, participation of HlyII
in diarrhea caused by B. cereus has not been demonstrated.95
The apoptosis of macrophages (J774 cell line) was induced by purified HlyII, and the involvement
of this factor in the observed effect was supported by studies with mutants with deletions in the hlyII
gene.47 This effect is related to the ability of HlyII to form pores because macrophages incubated with
purified HlyII in the presence of polyethylene glycol abolished apoptosis and preserved the perme-
ability of the cell membrane. There is also a contribution of caspase activation on the observed pro-
apoptotic effect.47
138 Laboratory Models for Foodborne Infections

Members of the B. cereus group are able to produce other pore-forming toxins such as cereolysin O,
hemolysin III, and three phospholipases C.95,100–102 Cereolysin O (or Hemolysin I), a heat-labile protein
toxin, is a member of the cholesterol-dependent cytolysin family; its best known members are listerio-
lysin O and perfringolysin O. They are able to form large pores in the cell membrane.103 Concerning
hemolysin III, it was first reported by Baida and Kuzmin in 1995.100 This is a heat-labile factor that is
not inhibited by cholesterol, but no further reports were found. Sphingomyelinase, phosphatidylcholine–
phospholipase C, and phosphatidylinositol–phospholipase C (PI-PLC) have cytolytic activity.42,46,104–107
Interestingly, PI-PLC from B. anthracis inhibits the activation of murine dendritic cells.108
The B. cereus emetic toxin is cereulide, a heat-stable dodecadepsipeptide preformed in food.109,110 It
is a toxin that causes mitochondria dysfunction due to its activity on K+ -channels.111,112 Since cereulide
is structurally closely related to valinomycin, several studies used this antibiotic as a surrogate mol-
ecule.110,113,114 However, the emetic toxin showed higher affinity for potassium than valinomycin thus
leading to a higher biological effect.113,114
Cereulide is produced by a non ribosomal peptide synthetase (NRPS) encoded in an operon situated
in a virulence plasmid related to the B. anthracis toxin plasmid pXO1.115,116 The gene sequence (ces) that
encodes the enzymatic machinery required for the synthesis of cereulide also includes genes encoding
a putative hydrolase (cesH), a phosphopantetheinyl transferase (cesP), a type II thioesterase (cesT), and
a putative ABC transporter (cesC7D).115,116 Because of its small size and low antigenicity, cereulide is
difficult to detect using immunological methods. Instead, assays with cultured cells (Hep-2) allow for the
determination of its biological activity leading to vacuole formation and impairment of mitochondrial
dehydrogenase activity.109,117–119
It has been demonstrated that there are different cereulide variants (isocereulides) that differ in toxic-
ity to Hep-2 and lead to different effects on the conductance of lipid bilayers.75,120 These in vitro effects
could correlate with the severity of the gastrointestinal disease.75
Although it has been established that cereulide doses of around 8 μg/kg body weight can be associ-
ated to severe emetic outbreaks,102 in vitro assays in Caco-2 cells showed that there is an accumulative
effect with successive subemetic doses of cereulide ranging from 0.125 to 4 ng/mL. Indeed, the effects on
viability, proliferation, and release of lactate dehydrogenase were detected at cereulide concentrations of
0.5–5 μg/mL.74 Interestingly, cereulide treatments also change the expression of proteins such as PCSK9,
cathepsin D, apolipoprotein A-I, proapolipoprotein, and apolipoprotein preprotein.74
Although Hep-2 cells are most widely used to evaluate the toxicity of cereulide, other cell lines such
as HepG2 (inhibition of RNA-synthesis),76 Hepa-1 (cytotoxicity),76 fetal porcine Langerhans islets (necro-
sis),121 natural killer (NK) cells (apoptosis and swelling of mitochondria)122 and HeLa, Calu-3, and Paju
(dissipation of the mitochondrial membrane potential)77 have also been used. These assays of cereulide
activity correlate with the gold standard assay for this toxin, that is, the boar sperm motility assay (see
ex vivo assays).

8.3.2 Regulation of Toxin Production

B. cereus exocellular factors HBL, NHE, CytK, proteases, phospholipases, and surface proteins are
controlled by PlcR, a pleiotropic regulator activated in the stationary phase by quorum sensing.103,123–128
This activity depends on the presence of a signal peptide, PAPR, which is exported and reimported by
a permease.129,130
Other cytotoxic factors such as HLyII are not controlled by PlcR. Instead, this extracellular factor
is negatively controlled by the global regulator Ferric Uptake Regulator (Fur) and the transcriptional
regulator HlyIIR.102,131,132 Glucose 6P and iron promote repression by binding to HlyIIR and Fur, respec-
tively.95 When glucose and iron diminish, the expression of HlyII is activated and induces macrophage
apoptosis and red blood cell lysis.95
Cereulide synthesis is partially controlled by Spo0A-AbrB regulon involved in the onset of sporula-
tion,133 and it is negatively regulated by CodY, a regulator involved in response to nutrient limitation in
Gram-positive bacteria.134 Besides, CodY activates enterotoxin synthesis and up-regulates the expression
of Plc-R.134
Bacillus 139

Also, environmental factors control the production of extracellular factors in B. cereus.102 Indeed,
growth in anaerobic conditions and low oxidation–reduction potential favor HBL and NHE production
through the participation of the two-component system, ResDE, and the transcriptional redox regulator,
Fnr.102 B. cereus has the ability to adapt to low pH conditions and survive within the gastrointestinal
tract,135,136 but the adaptation to sublethal low pH values inhibits HBL production.136

8.3.3 Direct Interactions with Eukaryotic Cells: Other Virulence Factors

Cultured eukaryotic cells were also useful models to gain insight into the effect of the direct interac-
tion of Bacillus spp. with the host’s cells. Even though the virulence of the members of this genus was
traditionally allocated to the production of extracellular factors, it has been demonstrated that adhesion/
invasion of the host’s cells is also relevant for pathogenesis.14,51–53,137,138
An early report from Andersson et al.137 suggests that the adhesion of B. cereus to Caco-2 cells and
subsequent spore germination could contribute to virulence. By using microscopy techniques, they found
that the adhesion of spores to enterocytes was strain-dependent and that high surface hydrophobicity
favored adhesion.137 Surface properties were also relevant for the interaction of spores with abiotic sur-
faces.139 Spore adhesion to Hep-2 cells was also observed.135 Interestingly, differentiated Caco-2 but not
Hep-2 cells induced spore germination.135
Since ingested spores can germinate after contact with epithelial cells, a series of studies were con-
ducted in order to gain insight into the relevance of the interaction of vegetative (i.e., harvested at
the logarithmic phase of growth) cells of Bacillus spp. B. cereus vegetative cells have the ability to
adhere to (Figure 8.2) and invade Caco-2 and HeLa cells.51–53,63,138 Calcium depletion experiments dem-
onstrate that invasion is favored by loosening tight junctions but no effects on the adhesion values
were observed.53 Also, the differentiation status of the cells is also relevant since when infection was
carried out with undifferentiated Caco-2 cells (4 days in culture), adhesion and invasion values were
significantly higher than those obtained with differentiated cells.53 By conducting experiments in the
presence of specific inhibitors of signaling pathways, it was demonstrated that the disassembly of the
F-actin cytoskeleton that follows the infection of epithelial cells involves signaling through phosphory-
lated lipids.53
Extracellular factors contribute to prokaryote–eukaryote interactions since infection carried out in the
presence of supernatants from 3-h-old cultures increases association (adhesion + invasion) of bacteria
with cultured enterocytes.53
A multivariate analysis revealed that the different effects observed in B. cereus-infected Caco-2 cells
can be correlated with the presence of virulence genes, that is, entS, entFM, nhe (A, B, and C), sph, hbl
(A, B, C, and D), piplC, and bceT.51

FIGURE 8.2  Adhesion of vegetative cells of B. cereus onto cultured human enterocytes (Caco-2 cells).
140 Laboratory Models for Foodborne Infections

Adhesion onto the epithelial cells of bacteria of the B. cereus group is related to the presence of flagella
components (i.e., FLhA) whose expression is not controlled by the PlcR regulator.138
Concerning models of professional phagocytic cells, studies with J774 macrophages demonstrate
that the metalloprotease InhA1 (main component of the exosporium) is involved in the escape of
Bacillus from macrophages.140 These findings are related to the ability of InhA1 to increase membrane
permeability.140
Spores of B. anthracis adhered to a diversity of epithelial cells like A549, CHO, and Caco-2.141
Interestingly, a model of M-like cells was developed by using cocultures of Caco-2 (epithelial) and Raji
B cells (human Burkitt’s lymphoma). In this model, there was an efficient translocation of B. anthracis
as compared with single cultures of Caco-2 cells.142 These findings encourage further studies on the
mechanisms involved in the pathogenesis of intestinal anthrax.

8.3.4 Virulence Factors in Other Bacillus spp.

Microorganisms of the genus Bacillus often contaminate raw and cooked foods. Even though
B. cereus is the most studied group, there are other Bacillus species having pathogenic potential.143–145
Models with cultured eukaryotic cells also contributed to the knowledge of this less virulent group of
Bacillus spp.
Some toxins associated with food borne illness are lichenysin A (B. licheniformis), amylopsin
(B. subtilis and B. mojavensis), and pumilacidin (B. pumilus).33,57,145 It is noteworthy that toxins in culture
supernatants from species other than those of the B. cereus group are less cytotoxic to HT-29 cells145 or
they were not toxic to Vero and Hep-2 cells.143
It has been demonstrated that cell-free alcohol-soluble extracts from B. subtilis strain depolarized
mitochondria of Caco-2 cells. This activity was related to the presence of amylopsin that is also produced
by B. mojavensis33 where three surfactin-like compounds were detected.57 These factors had toxic effects
on Vero cells with the inhibition of protein synthesis.57
Extracellular factors of Bacillus megaterium, a microorganism commonly found in honey and sel-
dom associated with severe pathologies, are cytotoxic to Caco-2 and Hep-2 cells in a strain-dependent
manner.63,146 However, a study performed by Alippi and coworkers146 demonstrated that most strains
are not cytotoxic, since from a total of 53 strains studied, only 7 strains detached Caco-2 cells.
Interestingly, some strains of B. megaterium have the ability to adhere to and invade both Caco-2 and
Hep-2 cells.63,146
The heat-stable toxin most well-known from the genus Bacillus is cereulide. Nevertheless, Bacillus
firmus, Bacillus simplex, B. megaterium, and B. licheniformis also produce heat-stable factors.147 Other
members of the Bacillus genus (i.e., B. firmus and B. simplex) lead to vacuolation of Hep-2 cells.147
Diagnostic kits were developed on the basis of the knowledge of secreted virulence factors. Bacillus
Diarrhoeal Enterotoxin Visual Immunoassay (BDE-VIA TM) (3M Tecra, St Paul, MN) detects NheA
and NheB components through polyclonal antibodies with detection limits90 of 2–5 ng mL −1. The
Duopath Cereus Enterotoxins assay (EMD Millipore, Darmtadt, Germany) is a lateral flow device that
detects the NheB and L2 components of Hbl through monoclonal antibodies with a detection limit of
6 and 20 ng mL −1, respectively.148 The BCET-RPLA (Oxoid Ltd., Basingstoke Hampshire, UK) detects
mainly the L2 component of Hbl with polyclonal antisera by reverse passive latex agglutination.68,149,150
Because of the multifactorial character of the virulence of Bacillus spp. and some divergences
with the results obtained with cultured cells, some authors recommended testing by more than one
method.59,68,69,151–153

8.4  E
 x Vivo and In Vivo Models
Different in vivo and ex vivo models of infection have been used to study the pathogenesis of Bacillus
spp. These models can determine both the importance of the route of infection and the virulence factors
involved. Table 8.1 summarizes different models reported in the scientific literature.
Bacillus 141

8.4.1  E x Vivo Models

Boar sperm motility inhibition bioassay detects the ability of toxins to inhibit sperm motility, which is
affected when mitochondria become damaged or the protonmotive force is reduced by dissipation of
the electrochemical potential across mitochondrial compartments.111 The loss of motility of boar sper-
matozoa after exposure to emetic B. cereus toxin (cereulide) or cereulide-containing samples resulted
in a sensitive, fast, and inexpensive bioassay for the detection of this toxin that acts as a potassium iono-
phore.77,111,154 Indeed, the spermatozoa bioassay has been used to detect the presence of cereulide77,154 in
bacterial cultures and contaminated foods.
Besides cereulide, other toxins showed similar effects on sperm motility. By this method, amy-
lopsin, produced by B. subtilis and B. mojavensis, that is involved in food-poisoning outbreaks has
been detected.33 Lichenysin, produced by B. licheniformis, was also detected through the inhibition of
sperm motility. The effect on motility is due to interference in the energy metabolism of cells,56 and the
strong correlation between lichenysin concentration and toxicity to boar sperm cells has been shown.155
B. pumilus, isolated from food-poisoning outbreaks, produced a toxin that induced damage of the cell
membrane and collapse of mitochondria, thus provoking the inhibition of motility in boar sperm cells.156
Jääskeläinen et al. found that commercial boar sperm was more sensitive to cereulide than commercial
bull semen.77 Additionally, boar sperm cells were as sensitive to cereulide as human cell lines (i.e., HeLa,
Caco-2, Calu-3, and Paju), indicating that this bioassay was appropriate for the in vitro assessment of the
cereulide effect on human cells.77
Since it is conducted by microscopic examination, the test of the inhibition of the motility of sperm
cells is prone to operator bias. Computer-assisted sperm analysis systems seem to be more objective and
offer the advantage of a numerical interpretation of the observed motility changes. Quantification is
based on software utilization and permits a real-time observation of the sperm motility.157
Quantitative analysis conducted by liquid chromatography connected to ion trap mass spectrometry
showed a high correlation with the biological activity of cereulide.112,114

8.4.2  In Vivo Models

Both invertebrate and vertebrate animal models were used to assess virulence mechanisms of Bacillus
spp. These experimental approaches add further insight in to the information obtained in the framework
of in vitro systems. In addition, vertebrate and invertebrate models give complementary data concerning
innate and adaptive immune responses.

8.5 Invertebrates
Despite evolutionary distances between mammals and invertebrates, they share common mechanisms
concerning detection, signaling, and response to pathogens. These characteristics have encouraged mod-
eling mammals’ host–microbe interactions by using different invertebrates.158

8.5.1 Nematodes
Caenorhabditis elegans is a bacteriovorous soil nematode that has gained attention as a model for the
study of host–microbe interactions.159 It is a natural host for Bacillus spp. and can be involved in the
spreading of these microorganisms.160 In addition, it is considered as a potential vector for human patho-
gens related to food borne diseases.161 It is worth noting that the defenses of C. elegans against pathogens
rely on epithelial cells as key players of its innate immune system.159 Interestingly, there are similarities
between structures of the intestinal epithelia of C. elegans and those of mammals.159
Since C. elegans feeds on a variety of microbes, it is usually maintained on agar plates seeded with
E. coli. Worms are motile and curvilinear, displaying typical internal structures when in healthy condi-
tions.162 The effects of pathogens on C. elegans are evidenced by assessing the survival ratio, ratio of
142 Laboratory Models for Foodborne Infections

infected worms, fecundity, development, microbial load, and more refined readouts including transcrip-
tional responses.159,162
C. elegans and other nematode species (Pristionchus pacificus) have been used to study virulence fac-
tors of Bacillus spp. isolated from different sources (soil, horse dung, and beetles).160 There was a strain-
dependent effect on the survival ratio of infected nematodes, and there were different responses for the
fecundity of the two nematodes studied. Interestingly, virulence patterns depended on both the genetic
background of worms and bacteria.160
By using this model, Franks et al.162 determined the correlation between the presence of genes related
to tellurite resistance and the virulence of B. anthracis. In this study, they demonstrated a correlation
between results obtained in this model and resistance to reactive oxygen species and antimicrobial pep-
tides as well as survival in the blood of humans and mice.162

8.5.2 Insects
8.5.2.1  Galleria mellonella
Caterpillars of the greater wax moth, G. mellonella, have been widely used to mimic gastrointestinal
infection caused by bacteria of the genus Bacillus.
G. mellonella eggs are incubated at 30°C, and the larvae are reared on beeswax and pollen at 25°C.
There are three infection routes: free ingestion, force feeding, or intrahemocoelic injection.163
The use of this model allowed for great strides in the knowledge of the factors involved in the viru-
lence of Bacillus spp. In this context, studies conducted with G. mellonella permitted the identification
of virulence factors that both B. cereus and B. thuringiensis have in common. These factors include the
pleiotropic regulator PlcR, the internaline/siderophore-like protein IlsA, the PlcR-regulated metallopro-
tease InhA2, the flagellar protein FlhA, and other adaptation and pathogenic genes.101,124,164,166
As described previously in this chapter, production of secreted virulence factors by B. thuringiensis
and B. cereus is regulated by the PlcR regulon, which is the major virulence regulator in these species.
This regulation has been demonstrated in G. mellonella where disruption of the plcR gene mitigates
the virulence of B. thuringiensis and B. cereus.124 These results were also seen in mice, suggesting that
extracellular factors that depend on the plcR regulon are required for the pathogenesis in both hosts.124
In a series of experiments, spores of strains from B. cereus of a clinical origin were administered by two
methods: free ingestion and force feeding. The survival ratio was evaluated and compared with those of
other microorganisms including some human pathogens such as Listeria monocytogenes. The lowest sur-
vival ratios were obtained with the entomopathogen Pseudomonas entomophila. Some B. cereus strains lead
to low ratios of mortality that were significantly increased in the presence of Cry toxin (entomotoxin from
B. thuringiensis). In contrast, one attenuated strain of B. anthracis and L. monocytogenes did not lead to lar-
vae mortality even in the presence of Cry toxin. These results demonstrate that the suitability of this model to
assess the biological activity of human pathogens by the oral route is both strain- and species-dependent.165
B. weihenstephanensis virulence was assayed for the first time in an in vivo model using G. mellonella.
B. weihenstephanensis is a psychrotolerant species of the B. cereus group that is potentially patho-
genic for humans. In this model, it has been demonstrated that the virulence is temperature-dependent.
A comparison between the virulence of B. cereus and B. weihenstephanensis showed that the virulence
of the latter is low at 37°C and maximal at 15°C. Interestingly, these findings correlated with those found
in vitro with cultured Vero cells.167
B. cereus sphingomyelinase (SMase) acquired relevance using this infection model. Doll et al. sug-
gested that SMase is a relevant virulence factor involved in the mortality rate of insect larvae infected with
B. cereus. In addition, their results have shown that Nhe and SMase complement each other for full virulence.71
Furthermore, this infection model, along with assays with cultured eukaryotic cells, has been used to
test the virulence of B. cereus strains isolated in a severe intestinal infection in neonates.168 Also, the rel-
evance of InhA1, NprA, and HlyII to differentiate pathogenic from nonpathogenic B. cereus strains was
assessed in G. mellonella.169 This model was also appropriate to gain insight into the expression of viru-
lent genes in vivo by using in vivo expression technology (IVET). Through this experimental approach,
the relevance of the expression of internalin under iron deprivation was demonstrated.164
Bacillus 143

8.5.2.2  Bombyx mori

The larvae of Bombyx mori (silkworm) constitute an alternative model to study host–pathogen interac-
tions. Their relatively big size is appropriate for manipulation although accumulated knowledge on this
lepidopteran is not comparable to that of C. elegans.158
This animal model was used to gain insight into the role of zinc metalloproteases (Inh) in B. thuringi-
ensis virulence. These proteases can specifically cleave insect antimicrobial peptides, thus overcoming
one of the main defense mechanisms. For the experimental procedures, B. mori eggs were incubated at
25°C and the resulting larvae were reared on a commercially available artificial diet.101 In this study,
Fedhila et al.101 used intrahemocelic inoculation for B. mori larvae and force feeding for G. mellonella.
They also conducted immunization experiments with E. coli in order to trigger the production of antimi-
crobial peptides in larvae. The role of Inh metalloproteases was assessed by using mutants in genes inhA
and inh2. Interestingly, the immunization of larvae did not protect B. mori inoculated with B. thuringi-
ensis by the intrahemocelic route, and deletions in the genes inhA and inhA2 did not modify the killing
activity. In contrast, these deletions significantly decreased the virulence for G. mellonella inoculated
by the oral route.101

8.6 Vertebrates
8.6.1 Rabbit
Early studies used this model to analyze crude cell-free spent culture supernatants of B. cereus cul-
tures.170 Biological activity was detected through two main experimental approaches: accumulation
of fluid in ligated rabbit ileal loops (RILs) and vascular permeability reaction (VPR). In the RIL test,
samples are injected into ileal loops and fluid accumulation is evaluated after euthanasia. Results
obtained with this technique correlated with those obtained in the VPR. This latter assay involves the
intradermal injection of samples in the shaved back of rabbits. Next, a Blue Evans solution is admin-
istered intravenously (ear vein), and the diameter of blue zones in the inoculation zone is evaluated. In
some cases, edema and hemorrhage are also present.170 It has been suggested that capillaries of blood
were affected by B. cereus enterotoxin, giving a local and also a systemic response since histologi-
cal changes were observed in the liver.171 Comparing with ligated ileal loop assays, the VPR is faster,
cheaper, and easier to perform.172
Both vegetative cells and cell-free culture filtrates from B. cereus cultures induce fluid accumulation.
However, ileal loop response did not differentiate strains isolated from food-poisoning outbreaks from
those isolated from other sources.173 Fluid accumulation was also positive for other Bacillus species, for
example, some B. thuringiensis strains. It is important to highlight that several strains that were positive
for the ileal loop assays failed to elicit diarrhea neither when injected intraluminally into normal ileum
rabbits nor when administered by a stomach catheter.173 This experimental approach (conducted in rab-
bits or mice) demonstrated histological changes and, in some instances, lethality in mice.64,171,173,174
Ligated RIL has been used to analyze purified toxins or their components. For example, Beecher
et al. studied hemolysin BL activity, a three-component toxin associated with diarrheal food poisoning.
The authors showed that all three components were necessary for maximal activity.42 In addition, Agata
et al. cloned the bceT gene, encoding one of the enterotoxic proteins of B. cereus. Supernatants of E. coli
cultures bearing this gene were positive in the VPR, resulting in fluid accumulation in murine ileal loops
(mouse), and had mouse lethal activity.64
Several enteropathogenic bacteria that induced fluid accumulation in ligated intestinal loop can also
produce a characteristic reaction when injected into the rabbit skin.172 However, other authors have found
no relationship between permeability reaction size and fluid accumulation in ligated RIL.175
Rabbit vascular permeability reaction has been used for the study of enterotoxins, either for the analy-
sis of toxins that could be related to outbreaks176 or for the study of isolated toxins.
Sometimes toxins caused vascular permeability, fluid accumulation, and lethal ­activity in mice— all
requested features for a diarrheal enterotoxin.64
144 Laboratory Models for Foodborne Infections

8.6.2 Zebrafish
Zebrafish (Danio rerio) is a vertebrate organism that shares some level of the functional conservation of genes
and signaling pathways with mammals. In addition, developmental particularities of the immune system of
zebrafish allow for the study of different levels of complexity of the immune response, that is, innate immune
response in embryos and larvae or fully developed adaptive response in adult fishes. It is noteworthy that
there are many similarities between components of zebrafish and human immune systems. Other advantages
are that each single experiment could include hundreds of embryos for examination of several conditions
and that the transparency of some stages of the fish cycle allows for the localization of infecting bacteria. In
the framework of intestinal infections, bacteria can be administered by the oral route or static immersion.177
There are few reports on the use of this model to study the virulence factors of Bacillus spp., and to
our knowledge, there is only one study conducted by using the oral route of infection with these micro-
organisms.178 In contrast, Bolcome et al. developed a zebrafish embryonic model to assess the effect
of B. anthracis toxins. They demonstrated an increased endothelial permeability by extravasation of
fluorescent microspheres in toxin-injected embryos.179 Further studies demonstrated the involvement of
the MAPK pathway in the biological effects of the lethal toxin. Interestingly, in this study, genetically
modified zebrafishes were developed.180

8.6.3 Monkeys
The concept of diarrheal and emetic syndromes in foodborne B. cereus pathologies started in the 1970s,
when most of the in vivo studies included the analysis of fluid accumulation in ligated rabbit or mice ileal
loop and alteration of vascular permeability.
Nausea and vomiting are pathognomonic signs of gastrointestinal infections and intoxications in
humans. However, emetic reflex is restricted to some mammals (e.g., pig, goat, dog, cat, seal, sperm
whale, and nonhuman primates) and nonmammals (e.g., some fish, reptiles, amphibians, and birds).
Although, overall, some of the above-mentioned animals could be suitable to study emesis following
food poisoning, there are intrinsic differences between humans and other animals.181 Consequently, stud-
ies in nonhuman primates fulfil most of the expected clinic outcomes although their implementation is
restricted due to ethical considerations.
Early studies in rhesus monkeys were used to analyze the effect (diarrhea and vomiting) of B. cereus
ingested with foods.182,183 Along with studies in rabbits (ligated ileal loop), the monkey feeding test
permitted the demonstration of factors involved in vomiting, and diarrheal syndromes were distinct enti-
ties.184 Some examples of cats and dogs suffering from diarrhea were described, but these models were
not practical for the study of B. cereus infection.173

8.6.4 Asian House Shrew

As stated before in this chapter, in vivo studies on cereulide have been limited because of the paucity of
animal models for the emetic syndrome. This prompted researchers to develop an easy-to-handle mam-
mal model of emesis. In this context, the Asian house shrew (Suncus murinus) was an attractive alter-
native due to its susceptibility to various emetic drugs administered orally.185 This model was used by
Agata et al. to induce emesis by the oral and intraperitoneal administration of purified cereulide. Animal
response was dose-dependent, and no animal death was observed. As a surrogate molecule, the antibi-
otic valinomycin was also tested.109 The authors suggested that the emetic effect of cereulide was caused
through the 5-HT receptor and stimulation of the vagus afferent. The same model was used by Isobe
et al. to analyze the emetic effect of a synthetic cereulide186 and by Ueda et al.187 to validate quantitative
analytical methods for cereulide.187

8.6.5 Mice
Mice (Mus musculus) have long been used for modeling host–pathogen interactions. They constitute an
easy-to-handle mammalian animal model that presents a fully developed immune system and shares
Bacillus 145

with humans many mechanisms of detection, signaling, and response to microorganisms. Furthermore,
a wide range of tools have been developed for this model and genetic engineering resulted in obtaining
strains of mice deficient in key steps of the host response against microorganisms.
Since Koch’s early reports on B. anthracis, mice were used in studies on this microorganism.5 Models
became more and more refined and mouse strains with specific deficiencies in key steps of the immune
response allowed for gaining further insight into the interactions between B. anthracis and the host.
As indicated in Section 8.1, the capsule of B. anthracis endows bacteria with the ability to evade
phagocytosis. It has been shown that A/J mice are susceptible to the noncapsulated Sterne strain of
B. anthracis. This infection model resembles the situation of conventional mice infected with wild-type
strains but, concomitantly, has reduced safety concerns. The susceptibility of A/J mice to noncapsulated
B. anthracis is due to deficiency in C5, a key component of the complement system. With this strain,
Duong et al. developed a murine model of systemic anthrax. Using both spores and vegetative cells, and
a subcutaneous infection, all infected animals became moribund or died.188 Mice showed severe dam-
age at the thoracic nodes, thymus, and spleen. However, there was a remarkable lack of hemorrhage or
inflammation in Peyer’s patches, mesenteric lymph nodes, and bronchial-associated lymphoid tissue.
Interestingly, in this model of infection, pathologies in the gastrointestinal tract were absent in contrast
with human systemic anthrax.188
A/J mice were also used for the development of a murine model of gastrointestinal anthrax.189 In this
study, infection was done by gavage with suspensions of Sterne strain (vegetative form). Results showed
animal death in a dose-dependent manner. It is worth mentioning that bacilli penetrated and grew within the
intestinal villi. As a consequence, bacteremia and systemic dissemination were observed with pathological
changes similar to those of systemic anthrax. The animals showed a decrease in weight, body temperature,
and activity along with evidence of intestinal hemorrhage. However, no immune activation was detected.189
A remarkable result obtained in this model was the early compromise of the intestinal epithelium.
Disruption of the normal villus structures, villus blunting, and ulceration at the ileum and jejunum showed
that gastrointestinal anthrax mainly affects the small intestine instead of the colon. Gastrointestinal tract
epithelium was the first site of infection. Xie et al. hypothesized that extracellular factors produced by
vegetative bacteria (e.g., LF) could be involved in the early steps of pathogenesis as well as in the immu-
nosuppressive effect of anthrax.189 An antibiotic treatment (amoxicillin + gentamicin) assayed 24 h after
infection resulted in complete recuperation with no deaths of the infected animals. These findings encour-
age the use of this in vivo model of gastrointestinal anthrax for the evaluation of potential therapeutic
approaches. Interestingly, A/J mice orally infected with spores of the Sterne strain showed a significant
breakdown of the intestinal barrier function. This leads to the systemic dissemination of B. anthracis and
commensal microorganisms.190 This model of gastrointestinal anthrax allowed for the demonstration of
the role of the MAPK pathway in the impairment of the immune response triggered by B. anthracis.
In Balb/cJ mice, a bioluminescent B. anthracis strain, unable to produce PA, was used for the real-time
analysis of gastrointestinal infection.26 In this study, the importance of the selection of the inoculation
technique was demonstrated. Rigid feeding needles cause abrasions in the laryngopharynx, which in
turn leads to systemic dissemination, whereas by using a flexible plastic tube, the infection initiated in
Peyer’s patches resembles gastrointestinal infection.26
The DBA/2 strain is a complement-deficient mouse susceptible to the Sterne strain. Tonry et al. ana-
lyzed gastrointestinal infection using this model. To maximize spore survival, neutralization of the stom-
ach acidity was performed before infection. In addition, a thiabendazole paste containing spores was
formulated to protect spores from gastric acidity. Intragastric inoculation of animals was performed by a
catheter syringe. The infected animals showed signs similar to those of human gastrointestinal anthrax,
that is, edema and hemorrhage.142
Also, the effects of B. anthracis toxins were studied in murine models. The effect of purified EF was
assayed by intravenous or intraperitoneal injection in Balb/cJ mice. The toxin caused rapid death of mice,
even at low doses, and these findings are related to the expression of toxin receptors in various tissues,
which in turn correlates with histological damage. The animals showed a marked intestinal dilatation due
to the extensive intestinal fluid accumulation in the gastrointestinal tract. Necrotic lesions were observed
in the mucosa. This experimental model demonstrated the toxic effects induced by purified EF, suggesting
that the death of animals could be related to multiorgan failure.191 On the other hand, anthrax LF caused
146 Laboratory Models for Foodborne Infections

intestinal damage in both C57BL/6J and Balb/c mice after being injected intravenously. Likewise, when A/J
mice were orally infected with spores of the Sterne strain,190 a systemic infection with commensal enteric
organisms was seen. An early breakdown of the gastrointestinal barrier, villus blunting, hemorrhage, and
ulceration, along with the known immunosuppressive effect of LF, provided a potential mechanism for
invasion via the enteric route.192 This toxin causes a dose-dependent disruption of the intestinal epithelial
integrity. Interestingly, the effects were partially attenuated by the co administration of antibiotics.
To investigate the toxic activity of B. cereus-secreted proteins, DBA/2J mice were inoculated intra-
peritoneally with filtered spent culture supernatants.61 Shortly after inoculation (10 min), a high ratio of
cells was observed in the peritoneal cavity. When viable bacteria were inoculated, the recruitment of
high numbers of neutrophils masked the effect on other cells. The authors concluded that Hbl and Nhe
cannot complement each other in spite of the coexpression of these factors.61
A murine model of B. cereus gastrointestinal infection was developed by Rolny et al.193 The vegeta-
tive B. cereus strain B10502 isolated from a food-borne outbreak was used to infect C57BL/6J mice by
gavage. To our knowledge, this was the first published data of an in vivo model of B. cereus gastrointes-
tinal infection. In contrast to murine gastrointestinal anthrax, which produced intestinal hemorrhage,
edema, systemic dissemination, and death, mouse infection with B. cereus led to a transient passage of
the bacteria through the digestive tract. Although self-limiting, B. cereus infection modifies the balance
of relevant immune cells in different regions of the intestinal mucosa and immune-associated tissues.
Ratios and activation statuses of different cell populations were studied in the spleen, Peyer’s patches,
and mesenteric lymph nodes. These results correlated with an increase in the size of Peyer’s patches in
infected mice. Additionally, the infected mice showed a higher ratio of intestinal goblet cells and the
presence of mononuclear cell infiltrates in the spleen. Cytokine mRNA expression showed a significant
increase in IFN-γ in mesenteric lymph nodes. At the same time, a slight increase in IL-12 mRNA and
TNF-α mRNA was observed. This study established relevant immune readouts to assess gastrointestinal
infection with B. cereus.193

8.6.6 Rats
The rat model is seldom used for the study of Bacillus spp. infections. Human-flora-associated rats
(Germfree Sprague–Dawley rats) were used to analyze the effect of B. cereus in the gastrointestinal
tract. Evaluation of the composition of the indigenous gut flora and examination of whether spores and
vegetative cells were able to persist in the gut were carried out. Experiments included administration
of irradiated spores, untreated spores, heat-activated spores, and vegetative cells of a ­k nown-to-cause
food-poisoning B. cereus strain.194 By DGGE analysis, it was demonstrated that when animals were
administered with spores, significant changes in the intestinal microbiota were evident. However,
­culture-dependent approaches did not reveal changes during the passage of B. cereus. Using in vitro
assays and commercial kits for detection of toxins, no enterotoxins were detected in the intestinal tract.
In addition, in those infectious conditions, only spores were able to resist the gastrointestinal passage.194

8.7 Concluding Remarks
The interaction of the genus Bacillus with different hosts leads to various outcomes: from a beneficial
relationship195 to life-threatening pathologies such as anthrax. The use of in vitro and in vivo mod-
els allowed for establishing key steps and mechanisms involved in infection and foodborne outbreaks.
Experimental models with cultured eukaryotic cells such as enterocyte-like Caco-2 cells helped unravel
extracellular factors involved in the gastrointestinal pathologies as well as to demonstrate the partici-
pation of direct bacteria–enterocyte interactions in the virulence. Further insights were obtained with
in vivo invertebrate and vertebrate models that elucidated many aspects of the pathogenesis in a more
complex cellular context. The recent advances in the field of in vivo expression technologies herald fur-
ther gains in our understanding of the control and treatment of foodborne pathologies associated with
Bacillus spp.
Bacillus 147

Acknowledgments
Jessica Minnaard is a Research Scientist at the Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET, Argentina), Ivanna S. Rolny is a Research Scientist at the Biological Sciences Department
(Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina), and Pablo F. Pérez is
a Research Scientist at the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET,
Argentina) and a Microbiology Professor at the Biological Sciences Department (Facultad de Ciencias
Exactas, Universidad Nacional de La Plata, Argentina).

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148. Krause, N. et al. Performance characteristics of the Duopath(R) cereus enterotoxins assay for rapid
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9
Clostridium

Emilio Aranda, María G. Córdoba, María J. Benito, and Juan José Córdoba

CONTENTS
9.1 Introduction: Clostridium Neurotoxins and Enterotoxins.............................................................155
9.2 Characteristics and Incidence of Foodborne Outbreaks due to Clostridium Neurotoxins
and Enterotoxins........................................................................................................................... 156
9.3 Investigation of Foodborne Intoxication by Clostridium Neurotoxins......................................... 157
9.3.1 Investigation of C. botulinum and BoNTs in Food Samples............................................ 157
9.3.2 Investigation of C. perfringens in Food Samples............................................................. 159
9.4 Laboratory Models for Foodborne Clostridium Intoxication....................................................... 160
9.4.1 Animal Models for Foodborne BoNT.............................................................................. 160
9.4.2 Animal Models for Foodborne C. perfringens Enterotoxins............................................162
9.4.3 Animal Models for Foodborne C. difficile....................................................................... 164
9.5 Cell Culture Models.......................................................................................................................165
9.6 Conclusions................................................................................................................................... 166
Acknowledgments....................................................................................................................................167
References................................................................................................................................................167

9.1 Introduction: Clostridium Neurotoxins and Enterotoxins

Species of the Clostridium genus are widely distributed in the environment (e.g., soil, sewage, and
marine sediments) and in the gastrointestinal tract of humans and domestic animals. Although most
Clostridium species are saprophytes, 34 species have been considered pathogenic to man and ani-
mals. Among the main pathogenic species are Clostridium botulinum and Clostridium perfringens
that usually are involved in foodborne outbreaks. C. botulinum produces the highly potent botuli-
num neurotoxin (BoNT), which is responsible for botulism, a severe foodborne disease with a high
fatality rate.1
C. botulinum belonging to groups I and II pose different types of risks in food processing. Group I
strains, the spores of which are highly heat-resistant, are frequently related to insufficiently processed
home-preserved foods such as canned vegetables and cured meats. The failure to effectively apply the
botulinum cook (121°C/3 min) to canned or bottled foods has led to many outbreaks of foodborne botu-
lism associated with C. botulinum group I.2
C. botulinum produces seven different neurotoxins, designated A to G, and a new type H was recently
reported.3,4 Dover et al.5 have described it as a B2 subtype toxin, with a minor toxin that could be
described as a new serotype (H) or as a chimeric A/F toxin. BoNT A, B, E, and F cause human botulism,
while C and D cause animal botulism. C. botulinum group I produces A, B, F, and H BoNT, and genes
are variable, with strains possessing from one to three neurotoxin genes, and forming one active, strains
form active type A neurotoxin, but not active type B neurotoxin or even two active toxins.2 C. botulinum
group II produces type B, E, and F BoNT; it was recently discovered that strains of C. botulinum group
II type F also contain a fragment of a type B and type E neurotoxin genes.6

155
156 Laboratory Models for Foodborne Infections

Other members of the genus Clostridium (Clostridium baratii and Clostridium butyricum) have occa-
sionally been reported to be involved in foodborne botulism and thus should also be considered as
potentially foodborne pathogens.
Furthermore, C. perfringens, which produces enterotoxins and is responsible for foodborne intoxica-
tion,7 and Clostridium difficile, which is involved in nosocomial, or institution-acquired, diarrhea,8 must
be considered as potentially pathogenic clostridia associated to foodborne outbreaks.
C. perfringens produces clostridial toxins activated in the gastrointestinal tract. Among 17 toxins
produced by C. perfringens, alpha (CPA), beta (CPB), epsilon (ETX), and iota (ITX) toxins are the four
major toxins present in five different toxinotypes (A, B, C, D, and E) of the bacterium.9–11 In addition,
C. perfringens may produce other toxins such as C. perfringens enterotoxin (CPE), C. perfringens beta2
toxin (CPB2), C. perfringens NetB toxin, and TpeL.9,12–14 CPE causes C. perfringens type A food poison-
ing as well as antibiotic-associated diarrhea (AAD) and sporadic diarrheas in humans. This toxin is also
responsible for enteric diseases and enterotoxemias in animals.15,16
Accurate methods for isolating and detecting these pathogenic clostridia in raw materials and pro-
cessed foods are necessary to take corrective measures throughout processing, avoiding clostridial food-
borne cases. In addition, laboratory (animal or cell culture) should be available to investigate these types
of foodborne cases.

9.2 Characteristics and Incidence of Foodborne Outbreaks due to

Clostridium Neurotoxins and Enterotoxins
The Centers for Disease Control and Prevention (CDC) categorizes human botulism cases into four
transmission categories: foodborne, infant, wound, and other.17
Foodborne botulism results from the ingestion of preformed botulinum toxin in foods. The toxin can
be found in food that has not been properly cooked, processed, handled, or canned and is often present
in canned food such as vegetables, meat, and seafood products.18 Foodborne botulism typically presents
18–36 h after ingestion of contaminated food; symptoms of botulism are often preceded by abdomi-
nal symptoms such as nausea, vomiting, and diarrhea, which are absent in botulism of the wound.19
Most foodborne botulism in Europe and North America has been associated with the use of canning
and bottling processes to extend food product shelf-life. The failure to effectively apply the botulinum
cook (121°C/3 min) to canned or bottled foods has led to many outbreaks of foodborne botulism associ-
ated with proteolytic C. botulinum or C. botulinum group I. Strains of nonproteolytic C. botulinum or
C. botulinum group II forming type B neurotoxin are often associated with foodborne botulism and meat
products in Europe, and sometimes with fish products in North America. Botulism outbreaks involving
C. botulinum group II type E have been most frequently associated with fish and home-prepared foods
in the north of Canada and Alaska.2
It should be noted that the true incidence of foodborne botulism is likely to be much higher, with
underreporting an issue. Foodborne botulism is not reportable in all countries and the efficiency of
investigating potential outbreaks also varies from country to country.20
Infant botulism (IB) is an infectious intestinal toxemia affecting only infants younger than 1 year.
During a transient period in infancy, gut flora may not inhibit intestinal outgrowth of ingested clostridial
spores. It occurs when infants (persons less than 1 year of age) ingest C. botulinum spores that then ger-
minate and produce the botulinum toxin in the intestines.21 Swallowed spores of C. botulinum (or, rarely,
toxigenic C. butyricum or C. baratii) germinate and temporarily colonize the lumen of the large intes-
tine, whereas vegetative cells produce BoNT.22 Because honey is a common dietary source of C. botuli-
num spores, infants should never be fed honey.18 However, it is recognized that C. botulinum spores are
present in soils and dust, and it is presumed that these may also be sources of infection. Environmental
factors and differences in the ability of physicians to recognize this disease may explain the wide varia-
tions in the report of IB between regions.23
Although IB has been reported in almost all continents, its epidemiological distribution varies widely
between continents and between countries. The United States (with approximately 70–110 cases of IB
Clostridium 157

each year), Argentina, Australia, Canada, Italy, and Japan have reported most of the cases.23 IB is a rare
disorder in Europe. From 1976, the year in which IB was first recognized,24 through 2006, 65 cases were
identified in 13 European countries, most of them in Italy and Spain, followed by the UK.25 In Europe,
IB is less common than foodborne botulism; however, IB is the most common form of human botulism
recognized in the United States.25
Foodborne illness caused by CPEs commonly occurs in industrialized countries.7 This food
poisoning is the second most common bacterial illness in the USA, where about 1 million cases/
year are reported.26 Outbreaks typically involve a large number of victims and are associated with
­temperature-abused meat or poultry dishes. The high incidence of C. perfringens type A food poison-
ing is well documented. From 1998 to 2010, 289 confirmed outbreaks of C. perfringens illness were
reported in the United States with 15,208 illnesses, 83 hospitalizations, and eight deaths.27 Sudden
infant death syndrome (SIDS) accounts for unexpected deaths in infants under the age of 1 year.
Strains of C. perfringens type A have been discovered to be commonly present in the intestines of
babies dying with SIDS.28
C. difficile belongs to the normal microbiota of the mammalian gastrointestinal tract. C. difficile prolif-
eration and infection in the human colon often occur after use of broad-spectrum antibiotics. C. difficile
destroys the intestinal lining and causes AAD, colitis, and pseudomembranous colitis. The pathogenicity
depends on the production of enterotoxin A and cytotoxin B. C. difficile toxin A is an enterotoxin that
induces fluid accumulation in the bowel, and toxin B is a cytopathic toxin that is extremely lethal. Both
toxins are highly unstable and tend to degrade at room temperature.10

9.3 Investigation of Foodborne Intoxication by Clostridium Neurotoxins

C. botulinum produces well-defined and very distinctive symptoms compared to C. perfringens and
C. difficile, and its foodborne investigation and differentiation relies on the use of laboratory procedures.

9.3.1 Investigation of C. botulinum and BoNTs in Food Samples

Isolation, detection, and characterization of C. botulinum from food samples by traditional microbio-
logical culture techniques require media deoxygenated by heating in a boiling water bath or by a con-
tinuous flow of an anaerobic gas mixture, and the use of anaerobic jars and anaerobic workstations is
necessary for successful diagnostics.29 Reddy et al.30 studied the effect of media, additives, and incuba-
tion conditions on the recovery of C. botulinum spores, and Mato Rodriguez and Alatossava31 studied the
effects of copper on germination, growth, and sporulation of Clostridium spp. Christian et al.32 evaluated
the effect of calcium, magnesium, and manganese spore resistance in different media. Calcium cations
give resistance to spores, while high amounts of magnesium cations appear to have a negative effect.
Manganese cations in low concentrations are important for the development resistance to heat and pres-
sure treatments, but not heat alone.
To enhance the isolation of C. botulinum from clinical samples (e.g., serum and feces), various
sample pre-preparation steps may be used including: (1) ethanol pretreatment to recover spores and
eliminate vegetative bacteria, (2) heating to discard nonspore-forming bacteria (i.e., 80°C for 10 min for
group I spores or 60°C for 10–20 min for group II spores), and (3) treatment with lysozyme (5 μg/mL)
or other heat-resistant lytic enzymes to facilitate germination of heat-stressed spores.29,33 In germina-
tion of spores, the effect of spore density system or proximity on the time of germination could be of
great utility.33
Cultivation of C. botulinum in liquid media could be carried out in chopped-meat-glucose-starch
medium; cooked-meat medium; broths containing various combinations of tryptone, peptone, glucose,
yeast extract, and trypsin; reinforced clostridial medium; and fastidious anaerobe broth.30,34 Pretreatment
with some of the above liquid culture media is of great importance to assure viability of spores, as it has
been reported by Stringer et al.35 However, all of these media are nonselective and thus allow the growth
of a range of other bacteria.
158 Laboratory Models for Foodborne Infections

To identify C. botulinum, unselective plating media such as blood agar and egg yolk agar (EYA)36 are
commonly used, since it could enable typical lipase production by this microorganism. However, other
clostridia species have lipase and may therefore confuse the identification.37 EYA medium alone does
not contain any inhibitory compounds, but it has been reported that medium supplemented with cyclo-
serine, sulfamethoxazole, and trimethoprim can be used for identifying select group I C. botulinum.38,39
Selection of the correct incubation temperature is essential to differentiate strains from the different
physiologies of groups I and II of Clostridium. Group I strains grow optimally at 35°C–37°C, whereas
temperatures of 25°C–30°C or even lower favor growth of group II strains.40
Quantification of C. botulinum by use of plate count procedures in samples containing other bac-
teria is difficult, since prevalence of C. botulinum in naturally contaminated samples is generally low
(10–1000 spores/kg) and proper selective media are not available.29
Polymerase chain reaction (PCR) represents an alternative to the traditional microbiological culture
technique to detect C. botulinum in foods. Fakruddin et al.41 described improvements and alternatives to
PCR such as loop-mediated isothermal amplification (LAMP), nucleic acid sequence based amplification
(NASBA), self-sustained sequence replication (3SR), and rolling circle amplification (RCA).
PCR detection of C. botulinum often targets the BoNT genes although other types of toxin genes are
also of diagnostic value.29 Use of multiplex PCR assays enables the simultaneous detection of two or more
types of BoNT genes.42–44 One such multiplex method was able to discriminate among BoNT serotypes
A, B, E, and F, corroborating mouse bioassay results.45 Furthermore, Peck and colleagues1 developed cul-
ture enrichment methods that when coupled with multiplex PCR can identify strains of C. botulinum that
are nonproteolytic (BoNT serotypes B, E, and F).1 However, PCR detection of neurotoxin genes does not
provide details on the physiological group and epidemiology of C. botulinum isolates. For ­differentiation
between group I (proteolytic) and group II (nonproteolytic) strains of C. botulinum, several molecular
typing methods are useful.46–48 Furthermore, Janda and Whitehouse49 have developed a multilocus PCR
followed by electrospray ionization mass spectrometry to identify C. botulinum, and Fach et al.50 have
described an innovative molecular detection tool based on the GeneDisc cycler for tracking all types
A, B, E, and F. Detection of C. botulinum in foods by the above mentioned traditional culture techniques
are highly time consuming and require good microbiological expertise. PCR offers the possibility to
detect any of the toxic strains regardless of the type of C. botulinum and BoNT produced. This strategy
is possible due to the use of universal primers that recognize all BoNT genes, but the nucleotide diversity
among the BoNT genes may present a potential problem.51 Nonetheless, BoNTs are generated as part of a
progenitor toxin complex, and a conserved component among serotypes is the nontoxic nonhemaglutinin
(NTNH). East and Collins52 demonstrated that the gene encoding NTNH shows a high level of similarity
and is present in all strains that produce BoNTs and absent from strains that are nontoxic. Aranda et al.53
developed a PCR protocol to detect all BoNTs-producing strains by using a single set of degenerate
primers. This protocol yields a single PCR product of 1.1 kb in agarose gel, providing a more specific
detection than hybridization with a BoNT probe.
Real-time PCR, biosensors, and DNA microarray offer the possibility of continuous and real-time
monitoring of the environment for the presence of infectious agents. Therefore, several real-time PCR
protocols54 along with biosensor technology55 and DNA microarrays56 have been reported to detect
Clostridium. More recently, Fenicia et al.57 proposed a real-time PCR method for detecting and typing
BoNT-producing C. botulinum types A, B, E, and F in clinical, food, and environmental samples and
thus support use of it as an international standard method. Microarray technology for toxin identifi-
cation of contaminated food has not been widely used. This may be due to the challenge in isolating
high-quality RNA samples from clostridia in food matrices. A recent oligonucleotide microarray with
62 different sequences based on known strain variable regions in the genome of C. botulinum strain
ATCC 3502 was constructed and used to differentiate different C. botulinum type A strains.58 Regions
corresponding to BoNT genes of various serotypes, and other markers were observed. Further develop-
ment of microarray-based assay approaches may provide a means to rapidly identify toxin-producing
strains.
In most of the cases of foodborne botulism investigation, detection of C. botulinum should be comple-
mented by detection of BoNT toxins. For BoNT detection, several procedures such as enzyme-linked
immunosorbent assay (ELISA)59,60 or mass spectrometric-based endopeptidase methods61 have been
Clostridium 159

reported. This approach has been successful in identifying BoNT serotypes A, B, E, and F in a variety of
food and clinical sample matrices with submouse bioassay sensitivities. To advance this technique even
further, a single, high-affinity mAb (4E17.1) that can simultaneously identify BoNT serotypes A, B, E,
and F has been developed.62 Recently, surface plasmon resonance (SPR) sensors have been also applied
for detecting BoNTs serotypes A, B, and F.63,64 This method uses a label-free biosensor assay for BoNT
B detection in food and human serum based on protein chip assay. Lastly, Ching et al.65 have reported
the use of an immunochromatographic test strip for the detection of BoNT serotypes A and B and Liu
et al.66 have developed an immunobiochemical assay to detect BoNT. In addition, ELISA and Endopep
MS analyses using fluorogenic reporters to detect BoNT in drinking water have been reported.67,68
Several protocols such as ELISA and immuno-PCR and ELISA based on protein antibody microarray
have been recently reported to detect BoNT neurotoxins A, B, C, D, E, and F in complex clinical, food,
and environmental samples at higher sensitivity than mouse bioassay.69,70 A number of rapid affinity
immunochromatography column (AICC) assays for the detection of BoNT serotypes A, B, E, and F in
food matrices have been developed. Brunt et al.71 reported a detection limit for BoNT/A of 0.5 ng, two-
fold more sensitive than earlier reported lateral flow methods. For serotypes B, E, and F, the minimum
detection limit ranged from 5 to 50 ng. Although not as sensitive as ELISA or mouse bioassays, immuno-
chromatographic methods generally are rapid assays, requiring only 15–30 min to complete, and do not
require enrichment steps, making them highly amenable to use in the field.

9.3.2 Investigation of C. perfringens in Food Samples

Detection of genes encoding C. perfringens toxins or the corresponding toxins is the most accepted
criterion in establishing a definitive diagnosis of this microorganism.72,73 Several procedures including
conventional and genomic molecular methods have been described.
Isolation, characterization, and detection of C. perfringens from food samples by traditional culture
techniques require culture media such as blood agar plates. In this medium, the microorganism produces
flat, rough, and translucent colonies, with regular or irregular margins, an inner zone of hemolysis, and
an outer zone of less complete clearing. Occasionally strains without inner zone hemolysis are seen.74 On
Nagler agar containing 5%–10% egg yolk, C. perfringens generates a characteristic white precipitate as
a result of its alpha toxin interacting with the lipids in egg yolk.10,75
Furthermore, several solid media have been reported to be suitable for detecting and isolating C. per-
fringens from different environmental samples. These media include neomycin blood agar, Shahidi
Ferguson perfringens (SFP) agar, sulfite polymyxin sulfadiazine (SPS) agar, oleandomycin polymyxin
sulfadiazine perfringens (OPSP), agar, tryptone sulfite neomycin (TSN) agar, and tryptose sulfite cyclo-
serine (TSC) agar with or without egg yolk.36,76 Isolation of typical colonies from media is followed
by confirmation of C. perfringens colonies with different tests to reveal phenotypic characteristics.
Observation of nitrate reduction, gelatine liquefaction, lactose fermentation, or lack of motility in sus-
pected C. perfringens isolates is usually enough to differentiate C. perfringens from other microor-
ganims.37,74 Identification of C. perfringens may be also carried out by using biochemical test kits such
as API.76 Several standard international methods are available to differentiate C. perfringens, includ-
ing methods published by the Association of Official Analytical Chemists (AOAC), the International
Standardization Organization (ISO), and the Nordic Committee on Food Analysis (NCFA).
Many PCR protocols focus on individual C. perfringens toxin genes,77 in particular the cpe gene78,79
and cpa gene.80 In addition, multiplex PCR protocols targeting several genes have also been reported.81–84
Baums et al.85 developed a reliable species-specific multiplex PCR for the detection of the cpa, cpb, cpb2,
cpe, etx, and iap genes of C. perfringens isolates without DNA purification. Furthermore, Joshy et al.86
reported a multiplex PCR for simultaneous detection of the enterotoxin gene of C. perfringens and BoNT
genes of C. botulinum. In addition to PCR, other approaches such as DNA microarray have also been
reported for detecting toxin genes of C. perfringens.72
Real-time PCR allows for quantifying the copy numbers of plasmid-borne toxin genes87,88 designed a
dual-labeled fluorescence hybridization probe (TaqMan)-based real-time multiplex PCR assay for detec-
tion of toxin genes α (cpa), β (cpb), ι (iap), ε (etx), β2 (cpb2), and enterotoxin (cpe) of C. perfringens
directly from cattle feces. Similarity, three real-time fluorogenic (TaqMan) multiplex PCRs have been
160 Laboratory Models for Foodborne Infections

reported for the detection of six toxic genes of C. perfringens.89,90 Recently, Chon et al.91 developed an
accurate real-time PCR for detection and quantification of C. perfringens in foods.
Given that not all C. perfringens produce toxins and consequently not all strains are related to dis-
eases or cause food poisonings, the confirmation of toxin productions is the most accepted criterion in
conventional diagnosis.92 Among the techniques for detection of major toxins of C. perfringens are the
mouse neutralization test (MNT),92 ELISAs,93–95 counter-immunoelectrophoresis,96 and latex agglutina-
tion test.97,98 Lastly, Seyer et al.99 reported a method to detect C. perfringens toxins in complex foods and
biological matrixes by immunopurification and ultra performance liquid chromatography-tandem mass
spectrometry.
The above methods give information about the presence of pathogenic Clostridium species and/
or the corresponding toxins in foods. However, no information is supplied about the effect of these
microorganisms in live cell that it is required in most of the cases during clostridial foodborne investi-
gation. Bioassays using laboratory animal or cells culture methods report data on effects of neurotoxin
or enterotoxins in live organisms. They are based on the ability of suspected food extracts to induce
symptoms in laboratory animal models and/or superantigenic action in cell culture models. These
methods offer a very valuable alternative for foodborne clostridia investigation and will be explored in
a separate section.

9.4 Laboratory Models for Foodborne Clostridium Intoxication

9.4.1 Animal Models for Foodborne BoNT
Nowadays, the diagnosis of foodborne diseases caused by the Clostridium-produced neurotoxin is car-
ried out by molecular, PCR, or immunological analysis but should be confirmed in all cases by the analy-
sis of stool samples with the mouse neutralization bioassay, using either monovalent, toxin-type-specific
botulinum antitoxin or polyvalent antibotulinum antitoxin. Despite many attempts and much research to
replace the use of animals, it is still the best assay to model all aspects of BoNT intoxication—binding,
translocation, and enzymatic activity60 (Table 9.1).
The samples used could be the serum of intoxicated people, their feces, or food directly involved. It
is desirable to obtain 10–15 mL serum; this allows specific identification of botulinum toxin involved
from inoculation serum neutralized all antitoxins, and repetition trial, if necessary. Since it is some-
times impossible to obtain this volume of serum in some patients, especially children, a lower quan-
tity is sufficient to confirm botulism, although in most cases the lower volume of 3 mL cannot provide
conclusive results. Approximately 25–50 g of feces sample is required, preferably collected before
treatment with antitoxin. However, botulism confirmation has been obtained with lower amounts,
and C. botulinum has also been isolated from stool after treatment with antitoxin. When performing
a rectal lavage due to constipation, it is necessary to use a minimum amount of fluid (preferably ster-
ile water without bacteriostatic) to obtain a sample where the toxin is not diluted. If the patient has
received a medication that may interfere with the assay for toxin or cultivation of stool, the laboratory
should be notified. For example, it has been shown that anticholinesterase drugs administered orally
to patients with myasthenia gravis can interfere with the detection assay used to identify toxin in stool
samples. When food samples are directly used, it is important to specify whether it is a commercial
or a homemade or handmade product. In all cases, refrigerated samples must be submitted to check
whether biosafety standards are met.
There are many studies using mouse bioassays to confirm the presence or type of botulinum toxin100,101
(Table 9.1), although additional techniques have been used. In this sense, López-Laso et al.,25 in the diag-
nosis of some of the cases of infant botulism, used PCR to detect C. botulinum neurotoxin genes, and
all cases were confirmed by analyses of stool samples with mouse neutralization bioassay. On the other
hand, Chun et al.102 described a steam treatment to reduce or eliminate C. botulinum type E viable cells
in Cladophora mats, thereby breaking the potential transmission route of the toxin up the food chain to
animals. They used mouse bioassay and antibody neutralization analysis can help in the identification of
C. botulinum neurotoxin serotypes.
Clostridium 161

TABLE 9.1
Animal Models Used for the Detection of Clostridium Neurotoxins and Enterotoxins
Animal Studies Toxins Tested References
Mouse Mouse neutralization bioassay— BoNT A, B, C, D, E, and F 25,60
binding, translocation, and enzymatic
activity of neurotoxins
Inactivation/neutralization effect of BoNT A, B, C, D, E, and F 102–109,111,115–117
botulinum toxin
Neuroimmunological changes BoNT A 114
Changes of ECG pattern of CPE 121
hyperpotassemia, fall of blood
pressure, and transient hyperpnea and
respiratory depression of
C. perfringens enterotoxins and death
Mucosal necrosis of small intestinal CPE 122
loops, without accumulation of fluid
in the lumen
Absorption of CPE from the intestine CPE 122,123
and formation of CH-1-like
complexes in the liver and kidney
Hemorrhagic cecitis C. difficile C. difficile Enterotoxin A and B 148–152
enterotoxins
Rat Developing, maintaining, and BoNT A 110
recovering from neuropathic pain
Neuroimmunological changes BoNT A 108,112,113
Changes of ECG pattern of CPE C. perfringens Enterotoxin 121
hyperpotassemia, fall of blood Type A
pressure, and transient hyperpnea and
respiratory depression caused by
C. perfringens enterotoxins and death
121 Rabbit Fluid and electrolyte secretion and CPE C. perfringens Enterotoxin 119,125,128
changes to the mucosa consist mainly Type A
of mucosal necrosis and hemorrhage
in small intestinal and colonic mucosa
Toxin binds to liver and kidney CPE C. perfringens Enterotoxin 122,123
Type A
Hemorrhagic lesions and luminal fluid C. perfringens Enterotoxin CPB 130
accumulation in these intestinal loops and CPE
Hamster Hemorrhagic cecitis C. difficile C. difficile Enterotoxin A and B 146,147
enterotoxins
Piglets, goats Endothelial damage of intestinal loops CPB in C. perfringens type C 134,135
Primates Developed vomiting and diarrhea CPE C. perfringens Enterotoxin 140
C. perfringens enterotoxins type A

Wohlfarth et al.103 reported that oral poisoning with BoNT serotype B was capable of activating the
immune system. These authors studied two cases of foodborne botulism following a meal consisting of
homemade canned beans. Four months later, one person developed a moderate and the other a borderline
titer of serum antibodies to BoNT serotype B, detected using an ex vivo assay (mouse phrenic-nerve
hemidiaphragm assay).
Moreover, bioassays in mice are used not only to confirm the type of C. botulinum, but also to ­investigate of
substances that neutralize the effect of Botulinum toxin. Examples in recent years are studies that use animal
models to find a small molecular weight (SMW) inhibitor of BoNTs that will neutralize the toxin. There have
been numerous publications reporting finding SMW inhibitors to BoNTA,104–106 but few reports on SMW
162 Laboratory Models for Foodborne Infections

inhibitors to BoNTB, one report on BoNTE,107 and none on BoNTF. Recently, Pirazzini et al.108 reported on
several inhibitors that were effective against BoNTA, BoNTC, and BoNTE, but Montgomery et al.109 found
an SMW inhibitor that might be useful against multiple serotypes of BoNT (Table 9.1). They tested two
promising SMW inhibitors to BoNTA against BoNTs B, C, E, and F in mice phrenic nerve-hemidiaphragm.
Although most studies are performed in mice, to have a confirmation in different species, additional
experiments must be carried out in other animal models, where it is also easier to verify the toxin effects.
Thus, Marinelli et al.110 studied in rats the effects of BoNT/A in developing, maintaining, and recover-
ing from neuropathic pain induced by the ligature of the sciatic nerve. They saw this effect was evident
starting 24 h after the administration of BoNT/A, and it was long-lasting—present 81 or 25 days after
intraplantar injection of the higher dose in mice (15 pg/paw) and 35 days after injection in rats (75 pg/rat).
Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing
compared to control groups.
Botulinum toxin type A used in the treatment of strabismus and other human diseases characterized
by hyperactivity of peripheral cholinergic nerve terminals, it could get some patients are or become
resistant to it. This can be overcome by using other botulinum toxins. Eleopra et al.111 investigated the
action of BoNT type D in mouse and human muscles and showed that botulinum toxin type D is poorly
effective in inducing human skeletal muscle paralysis but is very potent in mice. Mika et al.112 studied the
effect of BoNT A on sciatic-nerve-injury-induced neuroimmunological changes in rats and found evi-
dence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection
site using doses based on an approximate equivalence of 100 U of botox (4.8 ng tox) as reported by Cui
et al.,113 the dose injected in this experiment was 75 pg per paw, which corresponded to about 4–5 U/kg
in rats weighing 300–350 g. Also, Kato et al.,114 using 8 weeks old male animals, showed that BoNT A2
reduces incidence of seizures in mouse models of temporal lobe epilepsy.
Pirazzini et al.108 studied a Thioredoxin Reductase-thioredoxin Redox System cleaves the interchain
disulfide bond of BoNTs on the cytosolic surface of synaptic rat vesicles which blocked the differ-
ent BoNTs tested within a very similar concentration range, suggesting that the interchain disulfide
reduction is closely similar for different BoNTs serotypes. These in vitro results were validated in vivo
using digit abduction score (DAS) assay, a well-established model to compare potency and duration
of BoNTs.115 All tested molecules were very effective in reducing the degree and duration of paralysis
induced by the local injection of BoNT/A and BoNT/C. For the first time, it was shown that small mol-
ecules effectively prevent the paralytic activity of BoNTs. As a proof of concept, they also tested one
of these inhibitors, Ebselen, a compound reported to target both TrxR and Trx,116 in the lethality assay.
Ebselen, preventively administered via intraperitoneal injection, was very effective in protecting animals
from a lethal amount of BoNT/A, both by prolongation of the time to death and by reduction of the num-
ber of deaths108 (Table 9.1).
In another report, Pellet et al.117 analyzed in vivo the onset and duration of action of BoNT/A1–5 in female
ICR mice (Harlan) that were injected with sublethal amounts of BoNT/A1,/A2,/A3,/A4, or/A5 in 10 μL of
GelPhos buffer [30 mM sodium phosphate (pH 6.3) and 0.2% gelatin] into the right gastrocnemius muscle.
The amount of toxin injected IM per dilution was confirmed by mouse bioassay. Analyses of several of these
studied subtypes revealed distinct characteristics, ranging from differences in cell entry and enzyme kinetics
to differences in potency in mice and cell-model-specific potency. In a long-term activity study in cultured
primary neurons, it was indicated that BoNT/A1, 2, 4, and 5 have a similar duration of action, whereas
BoNT/A3 has a significantly shorter duration of action. This report describes an in vivo mouse study, show-
ing that local injection of BoNT/A2 resulted in faster onset of local paralysis than BoNT/A1, 3, 4, and 5,
whereas BoNT/A3 resulted in significantly faster recovery of motor-neuron deficiency.

9.4.2 Animal Models for Foodborne C. perfringens Enterotoxins

Several animal models have been used to study the role of the different toxins of C. perfringens in the
pathogenesis of infections produced by this microorganism9,118–120 (Table 9.1).
The pathodynamics of lethal intoxication in rats and mice by administration of enterotoxin of
C. perfringens type A was studied using whole animals and isolated organs. Rapid changes of the
­electrocardiogram (ECG) pattern suggestive of hyperpotassemia, rapid fall of blood pressure, and
Clostridium 163

transient hyperpnea followed by respiratory depression were observed. Analysis of plasma levels of
rations revealed hyperpotassemia in both animal species. On the other hand, enterotoxin (up to 100 µg)
showed little direct cardiotoxicity on the isolated heart.121 The in vivo effects of CPE have also been stud-
ied in rats,121 although much less extensively than in mice and rabbits. In rats, as in mice, this toxin pro-
duces lethality when injected i.v., and death in rats is preceded by respiratory difficulty, ECG alterations,
and hyperkalemia, because these effects were accompanied by an increase in liver enzymes (GPT, GOT,
and LDH). Sugimoto et al.121 suggested that CPE-induced hyperkalemia was a consequence of cytotoxic
action of CPE on hepatocytes. Mice have also been used to study the intestinal and systemic effects of
CPE.122 In these animals, lethality was associated with a rapid fall in blood pressure, respiratory dif-
ficulty, and changes in ECG. In the small intestinal loops of mice, as in rabbits, CPE causes dose- and
time-dependent mucosal necrosis. However, toxin administered in this manner does not cause fluid accu-
mulation in mice.122 Results of experiments inoculating CPE into intestinal loops of mice suggest that
death observed in constipated human patients with CPE-positive C. perfringens type A infection123 could
have been a consequence of absorption of CPE from the intestine. Mice that were inoculated with CPE in
the intestinal loops showed that CPE bound to and formed CH-1-like complexes in the liver and kidney.
A mouse intravenous lethality model was used to demonstrate that CPB is the main factor responsible for
systemic lethality in type C culture supernatants.124 In that study, lethality was abolished when culture
supernatants were preincubated with a CPB monoclonal antibody, but not when the cultures were incu-
bated with a CPA monoclonal antibody, which confirmed the role of CPB in mouse lethality.124
Furthermore, for many years, rabbit intestinal loops have been used, and are still used today, to study
the effects of CPE in vivo.125 Most of these rabbit models have been followed to evaluate the effect of CPE
in the small intestine, where it causes fluid and electrolyte secretion and produces significant damage to
the mucosa of the jejunum and ileum, but less damage in the duodenum.125–127
García et al.119 have also demonstrated that rabbit colon is sensitive to the action of purified CPE, with
both fluid secretion and mucosal damage observed. The histological changes caused by CPE in both
small intestinal and colonic loops of rabbits consist mainly of mucosal necrosis and hemorrhage. These
changes are both time- and dose-dependent.119,125
Rabbits have also been used to study the binding of CPE to extraintestinal tissues, which led to the
demonstration that this toxin binds to liver and kidney.122,123 This finding suggests that CPE absorbed
from the intestine can be responsible for systemic alterations, which may help explain the lethality
observed in some cases of experimental animals and human patients with the disease.122,123
A rabbit intestinal loop model was also used to determine the spatial distribution of the effects of CPB
along the alimentary canal.128 In that study, fluid accumulation and necrotizing enteritis were observed
only in the small intestine, with the jejunum and ileum being most severely affected. This result is in
agreement with natural type C disease in animals and humans, in which the jejunum and ileum are pri-
marily affected.73,129
Synergism between CPB and CPE for the virulence of CPE-positive type C strains of C. perfringens
was demonstrated by using a rabbit-ligated intestinal loop model,130 inducing significant hemorrhagic
lesions and luminal fluid accumulation in these intestinal loops. However, when lysate supernatants of
the cpb or cpe knockout mutants of these strains were inoculated into rabbit-ligated intestinal loops, no
significant damage or fluid accumulation were observed. Complementing the cpe mutant, or reversing
the cpb mutation, restored the virulent effects of culture lysates. Purified CPB and CPE, inoculated
together at concentrations similar to those found in wild-type CN3758 culture lysates, also produced
lesions and fluid accumulation in rabbit intestines. However, when either of these toxins was inoculated
independently, only higher doses caused damage to the intestine, suggesting that at low concentration,
both toxins act synergistically in the intestine.130 These experiments provided the first evidence of syner-
gistic toxin activity during intestinal C. perfringens infections.
Large animal models have also been used to study the pathogenesis of C. perfringens type C dis-
ease. Initially, piglets were experimentally used for this purpose.131–133 However, those experiments
were performed using whole cultures or crude culture supernatants, and although the results confirmed
that C. perfringens type C is a pathogen for piglets, they did not identify the main virulence factor(s)
involved in the pathogenesis of those infections. The mechanism of action of CPB in type C infection
was recently studied in intestinal loops of piglets.134 This study indicated that there is a tropism of CPB
164 Laboratory Models for Foodborne Infections

toward endothelial cells, suggesting that endothelial damage induced by CPB plays a role in the early
stages of C. perfringens type C enteritis in pigs. Koch’s postulates for type C disease were fulfilled in
goats,135 using the same set of C. perfringens type C mutants previously used to fulfill those postulates
in rabbits and mice.118,136 The results of the experiments in goats confirmed, this time in a natural host of
the disease, the key role of CPB in the pathogenesis of natural C. perfringens type C disease.
The effects of i.v. administration of extracts of sporulating cultures of CPE-positive C. perfringens
type A, and of CPE into ligated loops, also have been studied in calves and lambs, respectively.137–139 The
results of these experiments were variable and included diarrhea in calves and mild mucosal changes in
the intestinal loops of lambs.
Nonhuman primates have rarely been used to study the pathogenesis of CPE intoxication and/or
enterotoxigenic C. perfringens type A-associated disease.140 In the only published study, cynomolgus
monkeys fed purified CPE developed vomiting and diarrhea, while monkeys given CPE-positive C. per-
fringens type A orally developed only diarrhea. These effects were only observed when either CPE or
CPE-positive C. perfringens type A were given together with sodium bicarbonate to neutralize the low
gastric pH. Lethality was not observed in these nonhuman primates.140

9.4.3 Animal Models for Foodborne C. difficile

Various animal models have been used extensively for C. difficile research to study disease pathogen-
esis. Until recently, the most commonly used C. difficile disease model has utilized hamsters. However,
mouse and pig models have now been developed that unravel different aspects of C. difficile pathology141
(Table 9.1).
Furthermore, animal models have been developed to study various aspects of C. difficile infection,
including colonization, disease pathophysiology, intoxication, transmission, recurrence, efficacy testing of
potential therapeutics, and the impact of strain variability on all of these factors. Small animals that have
been utilized in these studies include mice, hamsters, rats, rabbits, hares, guinea pigs, prairie dogs, and
quails.142,143 More recently, zebrafish embryos have been used to study and compare the effects of C. difficile
variant toxin B derivatives isolated from a number of C. difficile strains.144,145 Larger animals such as foals,
Rhesus monkeys, and gnotobiotic piglets have been used in other studies to study C. difficile.142
Importantly, many of the animal models used for C. difficile research require pretreatment with antibiotics
to induce C. difficile infection, such as metronidazole, vancomycin, kanamycin, gentamicin, clindamycin,
and cefoperazone, by drinking a single antibiotic or cocktail with water or by intraperitoneal injection.141
C. difficile pathogenesis understanding has steadily increased since the development of hamster
and mouse models of C. difficile infection. The hamster model provides the foundation for C. difficile
research and is a useful and important model for studying C. difficile. After pretreatment with clindamy-
cin and challenge with toxigenic strains of C. difficile, hamsters develop hemorrhagic cecitis, which
presents as diarrhea or “wet tail” as well as other fulminant disease symptoms including ruffled fur,
hunching, and lethargy, leading to death.146,147 Histopathological analysis of cecal tissue isolated from
diseased hamsters shows mucosal ulceration associated with polymorphonuclear leucocyte influx and
tissue hemorrhage. However, the site of C. difficile infection in hamsters differs from that in humans, as
infection occurs in the cecum of hamsters but in the colon of humans.147
The development of new mouse models, combined with wide access to mouse-specific reagents and
tools, is offering new opportunities to study subtle features of disease. Three different mouse C. difficile
infection models have been described. The first employs gnotobiotic/germ-free mice,148,149 the second
uses a cocktail of antibiotics to disrupt the normal gut microbial communities and predispose the mice
to infection,150 and the third uses a single antibiotic to induce susceptibility to C. difficile infection.151,152
However, due to the notable interspecies differences in susceptibility to C. difficile infection and the
severity of disease outcomes, caution should be exercised when comparing results obtained using differ-
ent animal models or when extrapolating those results to humans.
Thus, the choice of appropriated animal model is critical when endeavoring to study C. difficile infec-
tion and it is likely that in coming years the use of various animal models will be reevaluated, particu-
larly as more become known about the complex relationship between C. difficile, host cell receptors, the
gut microbiota, and the host immune system.
Clostridium 165

9.5 Cell Culture Models

Cell-based assays measure BoNT receptor biding, translocation, and enzymatic activity and can be
alternatives to animal model and particularly to the mouse bioassay. A number of different neuronal and
nonneuronal derived cell lines have been generated for use in BoNT assays (Table 9.2). These include
rat spinal cord cells,153 chick embryo neuronal cells,154 neuroblastoma cells N2A,155 and BE(2)-M17
cells.156 The read out for most of the cell-based assays for detection of BoNT/A is the cleavage of
SNAP-25. Antibodies for SNAP-25 allow immunoblot detection of cleavage products, specifically
detecting a decrease in size of endogenous SNAP-25 protein.
Some studies in recent years use these types of cell culture, modifications of these, or new cell cul-
tures. Lyman et al.157 said no cell-based platform recapitulates the sensitivity of primary spinal cord
neurons to botulinum toxins (BoNTs), suggesting that neurogenic cell lines do not accurately model the
mechanisms of toxin internalization and activity. They developed a biologically relevant cell-culture
model for BoNT intoxication, in which glutamatergic neurons (ESNs), derived from murine embryonic
stem cells (ESCs), are highly sensitive to multiple BoNT serotypes. This study showed that ESNs were
susceptible to BoNT/A, /B, and /E with EC50’s of 0.81, 12.3, and 67.4 pM, respectively.
On the other hand, several continuous cell lines (Neuro-2a, SK-N-SH, M17, SHSY5Y, PC12 NT2) have
been tested for sensitivity to BoNT/A and are being used as research models158,159 (Table 9.2). However,
most of them lack the sensitivity necessary to compete with the commonly used mouse bioassay.160
Regina et al.161 demonstrated that mouse neuroblastoma rat glioma hybrid cell line NG108–15 was the
most sensitive cell line for detecting BoNT/A1. Even nowadays, human-derived cell systems have been
reported as a useful alternative. In this sense, Fonfria et al.162 compared results from two rodent-derived
[dorsal root ganglia (DRG) and cortical neurons (CTX)] and two human-derived neuronal cellular mod-
els (SiMa cells and iCell neurons) with data obtained from rat spinal cord neurons (SCN). The rank order
of BoNT/A1 sensitivity was: SCN = CTX = iCells > DRG > SiMa cells. Thus the iCell neurons had high

TABLE 9.2
Cell Culture Models Used for the Studies of Clostridium Neurotoxins and Enterotoxins
Cell Culture Studies Toxins Tested References
Rat spinal cord cells 153,163
Chick embryo neuronal cell Receptor binding, BoNT A, B, C, D, E, and F 154
translocation, and enzymatic
activity of neurotoxins
Neuroblastoma cells N2A 155
BE(2)-M17 cells 156
Murine embryonic stem cells BoNT A, B, and E 157
Rat spinal cord cells 153
Neuro-2a, SK-N-SH, M17, Sensitivity to BoNT BoNT A 158,159
SHSY5Y, PC12 NT2
Neuroblastoma cells NG108–15 161
Rodent ganglia cell 162
Cortical neurons
Human neuronal cell
Human intestinal epithelial Cytotoxicity of C. perfringens C. perfringens Enterotoxin 165–167
Caco-2 cells enterotoxins type A
Human colonic epithelial cell Cytotoxicity of C. difficile C. difficile Enterotoxin A and B 169,170
lines Caco-2, T84, HT-29 enterotoxins
Human monocytic THP-1 cells Inflammatory cytokine release C. difficile Enterotoxin A and B 168,171–173
stimulation of C. difficile
enterotoxins
166 Laboratory Models for Foodborne Infections

BoNT/A1 sensitivity comparable with rat SCN, but another human cell model, the SiMa cells, had the
lowest BoNT/A1 sensitivity of all the models tested.
Delaflotte et al.163 developed a procedure to isolate, enrich, and culture spinal motor neurons from rat
embryonic spinal cords and to assess sensitivity to BoNT/A1 (Table 9.2). This model obtained a highly
enriched culture derived from neonatal rat ventral spinal cord and provides a physiologically relevant
model system to understand the biology and characteristics of BoNTs acting at cholinergic terminals.
These cultures’ exhibited high sensitivity an EC50 (median effective concentration) below 0.5 pM to
BoNT/A1, which is more than five times the sensitivity measured in the standard rat spinal cord neuron
cultures and could be an ideal model to assess differences between BoNT serotypes and subtypes.
Regarding C. perfringens foodborne investigation, cell culture assays are possible alternatives to
replace in vivo neutralization tests. Animal experiments still play a central role in this kind of quality
control test; however, potency tests such as clostridial toxoids require a very high number of animals for
quality control testing.164 In this sense, Allaart et al.165 demonstrated that CPE expression is necessary
and sufficient for C. perfringens strains SM101 and F4969 to cause fluid accumulation and GI damage
in a rabbit ileal loop model; however, it remains unclear whether CPE is indispensable for bacterial cyto-
toxicity in vitro (Table 9.2).
The significance of toxins in the induction of in vitro cytotoxicity has been investigated using human
intestinal epithelial Caco-2 cells infected with toxin-gene-harboring C. perfringens strains and their
mutants or anti-toxin antibody.165,166 These authors revealed that β2 toxin is not involved in Caco-2 cell
cytotoxicity during infection with a cpb2-harboring C. perfringens strain. Yasugi et al.167 examined the
cytotoxicity of cpe-harboring C. perfringens isolates cocultured with human intestinal epithelial Caco-2
cells. The food poisoning strains showed severe cytotoxicity during sporulation and CPE production,
but not during vegetative cell growth. While Caco-2 cells were intact during coculturing with cpe-null
mutant derivative of strain SM101 (a food poisoning strain carrying a chromosomal cpe gene), the wild-
type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and
cpe-null mutant derivative of the non-foodborne strain F4969 induced Caco-2 cell death during both
vegetative and sporulation growth. The Caco-2 cell cytotoxicity caused by C. perfringens strain SM101
is considered to be exclusively dependent on CPE production.
Regarding C. difficile investigation, the human colonic epithelial cell lines Caco-2, T84, HT-29, and
human monocytic THP-1 cells have been used to study direct effects on intestinal cells168–170 (Table 9.2).
In human intestinal epithelial cell line, C. difficile enterotoxins initiate colonic inflammation in humans
by injuring epithelial cells and inducing production of IL8.169 Detachment of these cells from the base-
ment membrane leads to cell death by apoptosis. IL8 produced by the injured epithelial cells, as well as
responses by the exposed lamina propria cells (especially macrophages that lie just below the basement
membrane 43), to toxins A and B and other luminal contents would initiate a cascade of events character-
ized by migration of circulating polymorphonuclear cells into the mucosa to mediate tissue damage and
induce diarrhea. Most interestingly, colonic epithelial cells from some adults may be resistant to the effects
of even high concentrations of toxin A, possibly explaining why some people are resistant to disease.
In cells of the monocyte lineage, C. difficile toxins A and B stimulate inflammatory cytokine release,
including TNF-α, IL-1β, IL-6, and IL-8,171–173 and induce necrosis in human monocytes and in THP-1
human monocytic cells168 (Table 9.2).
Toxins A and B share 63% amino acid homology174 and possess similar domains. These toxins were
shown to block small GTP-binding proteins. In monocytes, toxins induce IL-8 production and necrosis
by unknown mechanisms. When these methods are used in foodborne investigation, confirmation of
toxin production is necessary.

9.6 Conclusions
Being the most widely distributed pathogenic species within the Clostridium genus, C. botulinum,
C. perfringens, and C. difficile are frequently involved in foodborne outbreaks. Accurate methods for
isolating and detecting these pathogenic clostridia in raw material and processed foods are necessary to
take corrective measures throughout food processing that reduce potential clostridial cases. For isolation,
Clostridium 167

detection, and characterization of C. botulinum, C. perfringens, and C. difficile from food samples, tradi-
tional microbiological culture techniques and molecular methods (PCR and qPCR) are available. When
these methods are used in foodborne investigation, confirmation of toxin production by rapid methods
such as ELISA, immuno-PCR, or ELISA based on protein antibody microarray is important. While these
methods reveal the presence of pathogenic Clostridium species and/or the corresponding toxins in food,
no information is available about the effect of these microorganisms in live cells, which is required in
most of the cases during clostridial foodborne investigation. Bioassays using laboratory animal or cells
culture methods help determine the effects of neurotoxin or enterotoxins in live organisms. Based on
the ability of a suspected food extracts to induce symptoms in laboratory animal models and/or supe-
rantigenic action in cell culture models, these methods offer a very valuable alternative for foodborne
clostridia investigation. The mouse neutralization bioassay, using either monovalent, toxin-type-specific
botulinum antitoxin or polyvalent antibotulinum antitoxin is the most valuable method for foodborne
botulism investigation. Rabbit intestinal loops, rat, and mice laboratory models are available for C. per-
fringens foodborne investigation. For C. difficile investigation, hamster, mouse, and pigs may be utilized.
In addition, cell lines provide an alternative to laboratory animal models for foodborne clostridial cases
investigation. For example, rat spinal cord, chick embryo neuronal, and neuroblastoma cell lines are avail-
able for accurate investigation of foodborne botulism. For the study of pathogenic C. perfringens and C.
difficile, human intestinal epithelial lines such as Caco-2 cells have been reported as sensitive method.

Acknowledgments
This work has been funded by the Spanish Instituto Nacional de Investigación Agraria y Agroalimentaria
(INIA) and the Spanish Comisión Interministerial de Ciencia y Tecnología with the projects RTA-2013-
00070-C03-03 and Carnisenusa CSD2007-00016, Consolider Ingenio 2010, and GRU09162 of the Junta
de Extremadura and FEDER.

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10
Enterococcus

Dongyou Liu

CONTENTS
10.1 Introduction....................................................................................................................................175
10.1.1 Classification, Morphology, and Genomics......................................................................175
10.1.1.1 Classification......................................................................................................175
10.1.1.2 Morphological and Biochemical Characteristics...............................................176
10.1.1.3 Genomics...........................................................................................................177
10.1.2 Biology and Epidemiology................................................................................................177
10.1.3 Clinical Features and Pathogenesis...................................................................................178
10.1.4 Identification......................................................................................................................178
10.1.5 Treatment and Prevention..................................................................................................179
10.2 Laboratory Models........................................................................................................................ 180
10.2.1 Animal Models................................................................................................................. 180
10.2.2 In Vitro Models..................................................................................................................181
10.3 Conclusion......................................................................................................................................181
References................................................................................................................................................181

10.1 Introduction
First observed by Thiercelin in 1899 as saprophytic Gram-positive diplococcus in the gastrointestinal
(GI) tract of humans, Enterococcus (initially named “Enterocoque” to emphasize its morphology and
its intestinal origin) was found to be associated with diarrhea, septicemia, and acute endocarditis.
In the early 1900s, similar organisms (then known as Streptococcus faecalis and Streptococcus fae-
cium) were described. In 1937, a classification scheme that separated streptococci into four groups—
pyogenic, viridans, lactic, and enterococcus—was proposed, with the “enterococcal group” covering
streptococci that grow between 10°C and 45°C, at pH 9.6, in 6.5% NaCl, and survive at 60°C for
30 min. This classification scheme correlated with a serological scheme developed by Lancefield in
the 1930s, in which the “enterococcal group” react with group D antisera (thus the so-called group
D streptococci), while nonenterococcal streptococci react with antiserum groups A, B, C, E, F, or G.
Following the description of additional members in the enterococcal group, a proposal to create the
genus Enterococcus was made in 1970 and formally accepted in 1984 upon DNA–DNA and DNA–
rRNA hybridization analyses [1].

10.1.1 Classification, Morphology, and Genomics

10.1.1.1 Classification
Taxonomically, the genus Enterococcus (from Greek éntero, “intestine” and coccos, “granule”) is
classified in the family Enterococcaceae, order Lactobacillales, class Bacilli, phylum Firmicutes, and
domain Bacteria. As one of the four genera (i.e., Enterococcus, Melissococcus, Tetragenococcus, and
Vagococcus) within the family Enterococcaceae, the genus Enterococcus encompasses over 50 recognized

175
176 Laboratory Models for Foodborne Infections

species (Enterococcus alcedinis, Enterococcus aquimarinus, Enterococcus asini, Enterococcus avium,

Enterococcus caccae, Enterococcus camelliae, Enterococcus canintestini, Enterococcus canis,
Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus devri-
esei, Enterococcus diestrammenae, Enterococcus dispar, Enterococcus durans, Enterococcus eurek-
ensis, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus,
Enterococcus haemoperoxidus, Enterococcus hermanniensis, Enterococcus hirae, Enterococcus itali-
cus, Enterococcus lactis, Enterococcus lemanii, Enterococcus malodoratus, Enterococcus moravien-
sis, Enterococcus mundtii, Enterococcus olivae, Enterococcus pallens, Enterococcus phoeniculicola,
Enterococcus plantarum, Enterococcus pseudoavium, Enterococcus quebecensis, Enterococcus raf-
finosus, Enterococcus ratti, Enterococcus rivorum, Enterococcus rotai, Enterococcus saccharolyti-
cus, Enterococcus silesiacus, Enterococcus solitarius, Enterococcus sulfureus, Enterococcus termitis,
Enterococcus thailandicus, Enterococcus ureasiticus, Enterococcus ureilyticus, Enterococcus viikkien-
sis, Enterococcus villorum, and Enterococcus xiangfangensis). On the bases of their varied tropisms,
habitats, metabolic, and phenotypic characteristics, members of the genus Enterococcus may be sepa-
rated into at least five groups, namely, E. faecalis group (E. faecalis, E. haemoperoxidus, E. moraviensis,
E. silesiacus, E. termitis, and E. caccae), E. faecium group (E. faecium, E. durans, E. hirae, E. mundtii,
E. villorum, E. canis, E. ratti, E. asini, E. phoeniculicola, E. canintestini, and E. thailandicus), E. avium
group (E. avium, E. pseudoavium, E. malodoratus, E. raffinosus, E. gilvus, E. pallens, E. hermanni-
ensis, E. devriesei, and E. viikkiensis), E. gallinarum group (E. gallinarum and E. casseliflavus), and
E. cecorum group (E. cecorum and E. columbae) in addition to ungrouped E. saccharolyticus, E. aqui-
marinus, E. sulfurous, E. dispar, E. italicus, and E. camelliae [2]. Of these, E. faecalis, E. faecium,
E. durans, E. mundtii, E. avium, E. raffinosus, E. gallinarum, and E. casseliflavus have been linked to
human diseases.
It is notable that being abundantly present as commensal organisms in the intestines of humans,
E. faecalis and E. faecium are implicated in various human nosocomial infections [e.g., urinary tract
infections (UTIs), endocarditis, bacteremia, neonatal infections, central nervous system (CNS) infec-
tions, and abdominal and pelvic infections] and have accounted for 90%–95% and 5%–10% of clinical
isolations, respectively, until the mid-1990s [3]. With the emergence and spread of antibiotic resistance
(especially vancomycin and ampicillin) in recent decades, the proportion of E. faecium-related infections
has shown a marked increase [1]. Other Enterococcus species (e.g., E. avium, E. gallinarum, E. casseli-
flavus, E. hirae, E. mundtii, and E. raffinosus, along with E. sanguinicola, E. gilvus, E. pallens, and
E. canintestini) are occasionally implicated in human infections, of which E. gallinarum and E. cas-
seliflavus are noted for their intrinsic resistance to vancomycin, an antibiotic used from the mid-1980s
to treat the aminoglycoside-resistant enterococcal infections. In addition, E. caccae has been detected in
human feces, while the remaining Enterococcus species commonly occur in animals, insects, traditional
fermented food and dairy products, plants, soil, and water [2].

10.1.1.2 Morphological and Biochemical Characteristics

Enterococci are spherical or ovoid in shape and appear in pairs (diplococci) or short chains. As Gram-
positive, non-spore-forming facultative anaerobes (with the capability of cellular respiration in both
­oxygen-rich and oxygen-poor environments) and obligately fermentative chemoorganotrophs, entero-
cocci grow optimally at 35°C, with a growth range from 10°C to 45°C. In addition, they grow in broth
containing 6.5% NaCl, hydrolyze esculin in the presence of 40% bile salts, and withstand heating at 60°C
for 30 min [1]. Although enterococci are considered catalase-negative, some species may appear catalase-
positive with weak effervescence (pseudocatalase). Being homofermentative, enterococci produce lactic
acid as the end product of glucose fermentation without gas. On sheep’s blood agar, enterococci typically
exhibit gamma-hemolysis. Apart from E. cecorum, E. columbae, E. pallens, and E.  saccharolyticus,
E. devriesei (variable among strains), E. canintestini, E. termitis, and E. viikiensis, most enterococci
are capable of hydrolyzing pyrrolidonyl-β-naphthylamide and producing leucine aminopeptidase. While
some species (e.g., E. gallinarum and E. casseliflavus) are motile, others (e.g., E. asini and E. phoenicu-
licola) are not. Some species, especially those found among plants (e.g., E. sulfureus, E. casseliflavus,
and E. mundtii), produce yellow pigments [1].
Enterococcus 177

10.1.1.3 Genomics
Phylogentic comparison of ∼1.4 kb of the 16S rRNA gene demonstrates a closer genetic relatedness
of Enterococcus to Vagococcus, Tetragenococcus, and Carnobacterium than to Streptococcus and
Lactococcus.
Multilocus sequence typing (MLST) targeting the internal regions of seven housekeeping genes
(gdh, gyd, pstS, gki, aroE, xpt, and yqiL) permits assignment of E. faecalis strains into sequence types
(ST). MLST analysis of E. faecalis strains isolated worldwide from the early 1900s to 2006 suggested
that acquired antibiotic resistance is enriched in the clonal complex 2 (CC2), CC8, and CC9 lineages,
whereas examination of more recently isolated strains from Europe (between 2006 and 2009) indicated
that multidrug resistance is enriched in the CC2, CC16, and CC87 lineages. Similarly, MLST analysis
of internal gene fragments (between 395 and 583 nucleotides [nt] in size) of seven housekeeping genes
(atpA, ddl, gdh, purK, gyd, pstS, and adk) in E. faecium resulted in the identification of a large cluster of
clinical E. faecium isolates that was first termed lineage C1 and later renamed clonal complex 17 (CC17),
after ST17, which are resistant to ampicillin and ciprofloxacin and are enriched for several genes with
putative roles in virulence (e.g., the large surface protein esp, and carbohydrate metabolism), with the
presence of IS16 being the most prominent marker. In addition, Bayesian analysis of genetic population
structure (BAPS) analysis of 491 distinct STs found among 1720 E. faecium isolates identified 13 groups
of related E. faecium strains. Phylogenetic analysis based on concatenated MLST gene sequences of
isolates contained in the two largest BAPS groups (BAPS 2–1 and BAPS 3–3) revealed that currently
circulating clinical isolates belong to three different lineages (lineage-17, lineage-18, and lineage-78),
which stem from at least three different ancestral strains and have independently acquired genes that
characterize clinical isolates through convergent evolution [4].
The genomes of enterococci are found to range from 2.7 to 3.6 Mb across species, with the G + C content
of DNA between 37 and 45 mol% [5]. Examination of E. faecalis V583 genome (3.36 Mb)—a bloodstream
infection (BSI)-derived, vancomycin-resistant (vanB phenotype) ST6/CC2 lineage strain—revealed the
presence of a large amount of mobile DNA (accounting for >25% of the genomic content), including
seven predicted prophages, multiple integrated plasmids, IS elements, and genomic islands (including
the pathogenicity-associated island or PAI), a vanB-type transposon that confers vancomycin resistance,
and three extrachromosomal plasmids [pTEF1 (66.3 kb), pTEF2 (57.7 kb), and pTEF3 (18.0 kb)] [6,7].
This highlights the propensity of enterococci to acquire and disseminate mobile elements, such as those
that encode antibiotic resistance genes. Other notable traits identified include a capsule, a novel adhesin
termed enterococcal surface protein (Esp), a bile acid hydrolase, and an operon for the enterococcal
cytolysin [8].

10.1.2 Biology and Epidemiology

Enterococci are common colonizers of the GI tract of humans and other mammals, birds, reptiles, and
insects, and are routinely isolated from soil and sediments, beach sand, aquatic and terrestrial vegetation,
and ambient waters (rivers, streams, and creeks) [9,10].
Although first implicated in infective endocarditis (IE) in 1899, Enterococcus emerged in the 1970s
as a leading cause of multidrug-resistant (MDR), hospital-acquired infections. Currently, Enterococcus
[dominated by E. faecalis (80%–90%) and E. faecium (10%–15%)] represents the third most common
nosocomial pathogen (12% of all hospital infections), causing IE, catheter-related BSIs, UTIs, wound
infections, endophthalmitis, and peritonitis.
Patient’s own endogenous flora was once thought as the main source of enterococcal infection; how-
ever, detailed studies pointed to the transmission of pathogenic enterococci among patients in hospital
settings through the hands of health-care workers (e.g., direct inoculation onto intravenous or urinary
catheters). Enterococci can persist on hands for as long as 60 min after inoculation onto hands, and as
long as 4 months on inanimate surfaces.
Risk factors for MDR enterococcal infections include patients with neutropenia, undergoing transplan-
tation, chronic renal failure, ICU stay, prior antibiotic use (including cephalosporins), bladder catheter-
ization, expression of cytolysin as an enterococcal virulence determinant, and prolonged hospitalization.
178 Laboratory Models for Foodborne Infections

The emergence and dissemination of MDR enterococci (especially E. faecium) exhibiting resistance to
ampicillin, vancomycin, and aminoglycosides have reduced the number of therapeutic options. There is
evidence that resistance is also emerging to newer agents used to treat vancomycin-resistant enterococci
(VRE) infections (e.g., linezolid, quinupristin/dalfopristin, and daptomycin) [1,11–13].

10.1.3 Clinical Features and Pathogenesis

As commensals of human GI tract, enterococci do not usually cause disease inside the intestine. However,
once outside of the gut, they may become pathogenic, inducing a variety of clinical syndromes, ranging
from UTIs, intra-abdominal, pelvic, and soft tissue infections, bacteremia, endocarditis, to other uncom-
mon infections [3].
UTIs are the most common type of enterococcal infections that are likely acquired in hospital or long-
term care settings (e.g., ICU). Lower UTIs (e.g., cystitis, prostatitis, and epididymitis) frequently occur
in older men, and upper UTIs in older men may lead to bacteremia [3].
Intra-abdominal, pelvic, and soft tissue infections are the next common enterococcal infections.
Peritonitis (an infection of the abdominal lining) may occur in conjunction with liver cirrhosis or in
patients who receive chronic peritoneal dialysis. In addition, enterococci may be detected in cultures
from decubiti and foot ulcers [3].
Bacteremia and endocarditis are the more common and serious manifestations of enterococcal infec-
tions, with between 5% and 15% of cases of infectious endocarditis (heart valve infections) attributing
to enterococci (particularly E. faecalis). Enterococcal endocarditis typically occurs in older persons
and usually evolves from bacteremia, which in turn originates from the genitourinary or GI tract
infections [3].
Other uncommon infections due to enterococci include meningitis, hematogenous osteomyelitis, sep-
tic arthritis, pneumonia, and chronic bacterial prostatitis. Enterococcal meningitis usually occurs as a
rare complication of neurosurgery [3].
Enterococci are remarkably thermotolerant (growing at 10°C–45°C), pH-tolerant (pH 4.8–9.6), salt-
resistant (up to 28% NaCl), and capable of surviving at 60°C for 30 min and extended desiccation. These
properties enable the bacterium to flourish in a multitude of environments, including the human gut, and
abiotic surfaces in hospitals (e.g., bedrails and medical station keyboards). The ability of Enterococcus to
enter into a viable but noncultivable state and form a biofilm also facilitates its environmental persistence
and provides protection against the host immune response and antibiotic intervention.
Enterococci are known to encode a number of virulence-associated proteins that are essential for their
pathogenicity. These include Clp ATPase (encoded by the clpB gene, with critical role in protein fold-
ing, assembly, and degradation of proteins), methionine sulfoxide reductase A (involved in antioxidant
repair), elatinase (GelE, a secreted zinc metalloproteinase), hyaluronidase, cytotoxin (a secreted two-
peptide lytic toxin encoded on a pheromone-responsive plasmid on pathogenicity island), enterococ-
cal surface protein (Esp, a surface adhesin that controls adherence to host tissues), the protein Ace (a
collagen- and laminin-binding microbial surface component recognizing adhesive matrix molecules or
MSCRAMM), and enterococcal aggregation substance (AS, a pheromone-responsive, surface-bound,
plasmid-encoded protein), etc. [14–20]. In particular, proteases and enzymes secreted by enterococci not
only degrade host tissues into peptide nutrients for the bacteria, but also cause direct destruction of both
cells and the extracellular matrix in the host tissue, activate host proteolytic cascades, and interfere with
host inflammatory processes [21,22].

10.1.4 Identification
Enterococci grow slowly in culture media and require further conventional biochemical tests or com-
mercial test systems [e.g., the BD GeneOhm VanR (BD Diagnostics, Spark, MD) and Xpert vanA/vanB
(Cepheid, Sunnydale, CA)] for identification. As many enterococcal species vary by only one pheno-
typic trait, the biochemical approach is far from being straightforward. For VRE, chromogenic media
(e.g., CHROM-agar, chromID, and Spectra VRE media) reduce turnaround times through early visual
Enterococcus 179

identification of colonies, although use of standard broth macrodilution or disk diffusion method for
vancomycin-susceptibility testing requires additional time [23].
While 16S rRNA sequence analysis is valuable for identification of many Enterococcus species
(using 97% identity as the threshold), it does not provide adequate resolution between E. casselifla-
vus and E. gallinarum (which share 99.9% identity). Therefore, other molecular methods are required
for routine identification of enterococcal isolates. These include amplification and sequencing of the
domain V of the 23S rRNA gene, rRNA or tRNA intergenic spacers, the D-ala:D-ala ligase genes
(ddl), the ­manganese-dependent superoxide dismutase (sodA) genes, the chaperonin 60 (cpn60) gene,
the Enterococcus protein A (efaA) gene, genes encoding the RNA polymerase α-subunit (rpoA), the
­phenylalanyl-tRNA synthase (pheS), and the elongation factor Tu (tufA), in addition to the application
of ribotyping, repetitive extragenic palindromic PCR (REP-PCR) or BOX-PCR, pulsed-field gel electro-
phoresis (PFGE), and multilocus sequence typing (MLST) [3].
PFGE represents the current standard in the clinical identification of Enterococcus spp. and strain
typing, while ribotyping techniques allow accurate discrimination among species with reduced cost.
Interestingly, MLST demonstrates an accuracy equivalent to that of PFGE for the identification of organ-
isms to the subspecies level. de Been et al. [24] developed a core genome MLST (cgMLST) scheme for
E. faecium that is capable of distinguishing between epidemiologically related and unrelated isolates,
even between those of identical ST. Hullahalli et al. [25] showed that clustered, regularly interspaced
short palindromic repeats (CRISPR) 2 analysis is an inexpensive alternative to MLST for assessing clon-
ality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination
events occurring between STs. Other novel approaches for enterococcal identification include quantita-
tive PCR (qPCR) targeting the esp gene of E. faecium (espfm), DNA microarrays, and use of bacterio-
phages specific to certain Enterococcus strains.

10.1.5 Treatment and Prevention

For susceptible Enterococcus isolates, β-lactam agents (ampicillin and penicillin/aminopenicillin) are
the drugs of choice for treating monomicrobial enterococcal infections (e.g., UTIs and nonendocarditis
bacteremia). Treatment of polymicrobial infections (e.g., skin and subcutaneous infections and intraab-
dominal or pelvic infections) relies on a combination of ampicillin and other broad-spectrum antibiotics
or a β-lactamase inhibitor (e.g., ampicillin-clavulanic acid or piperacillin-tazobactam). A glycopeptide
(vancomycin or teicoplanin) may be employed as a single agent to treat simple enterococcal infections
in patients with a serious allergy to penicillins. A combination of a glycopeptide and an aminoglycoside
(gentamicin or streptomycin) may be considered for patients with serious allergies. Additionally, nitro-
furantoin may be used to treat lower-tract urinary infections [3].
As Enterococcus species are intrinsically resistant to many antimicrobial agents (e.g., cephalospo-
rins, clindamycin, semisynthetic penicillinase-stable penicillins, and aminoglycosides) and have the
capacity to acquire resistance genes and mutations, treatment of severe infections with VRE or MDR
enterococci is challenging [1,11,12,26,27]. Interestingly, most E. faecalis isolates expressing vancomy-
cin resistance remain susceptible to ampicillin, whereas many E. faecium isolates are resistant to both.
Quinupristin/dalfopristin [a combination agent consisting of streptogramin A (70% dalfopristin) and
B (30% quinupristin) effective against E. faecium] and linezolid are FDA-approved agents for VRE.
With intrinsic activity against enterococci, nitrofurantoin, fosfomycin, and doxycycline are potential
oral options for treating simple VRE infections. Other antibiotic alternatives include daptomycin (DAP,
a lipopeptide antibiotic), tigecycline, lipoglycopeptides (oritavancin, telavancin, and dalvabancin), and
rifampicin [28,29].
The intervention measures for controlling the spread of VRE in health-care settings range from (1)
active periodic surveillance (cultures or molecular tests) of highest risk carriage patients, (2) decontami-
nation of the hands of health-care workers before and after all patients contact, (3) adherence to barrier
precautions (gloves and gowns) and hand antisepsis after glove removal, to (4) thorough terminal clean-
ing of rooms occupied by patient with VRE and daily cleaning of high-touch items such as bedside rails,
tables, toilets, and handles [3].
180 Laboratory Models for Foodborne Infections

10.2 Laboratory Models
10.2.1 Animal Models
Rabbits, rats, and mice have proven to be useful for the investigation in the molecular mechanisms of an
antibiotic therapy against enterococcal infections [30].
Utilizing a rabbit subdermal abscess model, Frank et al. [31] identified three in vivo-activated genes
in the E. faecalis OG1RF chromosome that encode glutamate 5-kinase (proB [EF0038]), the transcrip-
tional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]). They
showed that the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep
strain was severely attenuated in a rabbit model of endocarditis. In a separate study, Frank et al. [32]
employed a rabbit model of endocarditis to test E. faecalis strains with transposon insertions or in-frame
deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. They noted that
while only the ahrC mutant was significantly attenuated in endocarditis, the transcriptional regulator
AhrC and the protease Eep were also required for full virulence in murine catheter-associated urinary
tract infection (CAUTI), confirming AhrC and Eep as enterococcal biofilm-associated virulence factors.
More recently, Frank et al. [33] used Wistar rat model of acute foreign body osteomyelitis to examine
the relationship between biofilm formation and development of antimicrobial resistance, and found that
surface colonization alone is sufficient for E. faecalis cells to acquire the biofilm antimicrobial resistance
phenotype.
Eguchi et al. [34] assessed the pharmacodynamics of SMP-601 (also known as PTZ601, PZ-601,
or SM-216601, a novel parenteral carbapenem with potent activity against MDR Gram-positive
pathogens) against vancomycin-resistant E. faecium (VREF) in neutropenic murine thigh infec-
tion model, and found that SMP-601 had a sufficient therapeutic effect against VREF infections
at relatively low exposure conditions. In a rat model of plastic-catheter-induced left-sided entero-
coccal endocarditis, DAP monotherapy (20 mg/kg twice daily) demonstrated superior efficacy
for the treatment of penicillin-resistant E. faecalis in comparison with vancomycin. Similarly,
DAP (12 mg/kg every 8 h) was effective against vancomycin-resistant and gentamicin-susceptible
E. faecium [35,36].
Given the evolutionary conservation of both ancient innate host defenses and bacterial virulence
mechanisms, invertebrate hosts (e.g., the greater wax moth Galleria mellonella, the free-living bacte-
riovorus nematode Caenorhabditis elegans, and the common fruit fly Drosophila melanogaster) offer
valuable models to study innate immunity and host–pathogen interactions and help unravel the pathobio-
logic details in enterococcal infections [37–41].
Infection of the greater wax moth G. mellonella larvae with E. hirae led to the identification of sev-
eral novel enterococcal virulence factors in the insect hemolymph (e.g., GelE, Clp ATPase). Because of
its capacity to escape from the intestinal lumen to the body cavity during the larval to pupal transition,
E. hirae activates the host defense responses, yielding important clues in the pathogenic mechanisms
of enterococcal infection. As G. mellonella tolerates relatively high temperatures (37°C or higher), this
simple nonmammalian model is clearly more relevant to the study of human entercoccal infections than
C. elegans and D. melanogaster (maximum 25°C) [39].
The small roundworm C. elegans possesses 20 nonrenewing intestinal epithelial cells (IECs) that play
a key role in the mediation of intestinal immunity. As C. elegans IECs are morphologically similar to
mammalian IECs, including a “brush border” of microvilli, this worm represents an excellent model
for the study of gut immunity during enterococcal infections. Additionally, the relatively small size and
the ability to survive in liquid culture make C. elegans a cost-effective model for disease investigation
including enterococcal infections [42,43].
The common fruit fly D. melanogaster allows natural colonization of E. faecalis in the intes-
tine and thus offers a useful model for investigations related to its biology, immunity, and pathogen-
esis [44]. Indeed, examination of E. faecalis quorum regulatory system genes LrgAB and SprE, and
bacteriocin EF1097 in D. melanogaster confirmed their role in enhancing E. faecalis infectivity
and toxicity [45].
Enterococcus 181

10.2.2  In Vitro Models

Simulated endocardial vegetations (SEVs) prepared from enterococcal bacteria offer a useful model for
assessing the efficacy of antibiotic regimens. For example, DAP at dose equivalent to 6–8 mg/kg/day was
shown to exert rapid bactericidal activity at 8 h against a VREF isolate using a SEV model. Similarly,
DAP at dose of 10–12 mg/kg was sufficient to kill both of vancomycin-resistant E. faecalis and E. fae-
cium at 96 h [46].

10.3 Conclusion
The genus Enterococcus comprises over 50 species of Gram-positive, non-spore-forming bacteria that
mostly occur as commensal organisms in the GI tract of mammals and birds. However, when entero-
cocci escape from the gut and establish in other body sites, they may cause a diverse range of clinical
diseases, including UTIs, intra-abdominal, pelvic, and soft tissue infections, bacteremia, endocarditis, and
other uncommon infections. Further, with the ability to acquire and develop multidrug resistance traits,
enterococci have become a serious problem in health-care settings in recent decades, not only putting mil-
lions of people at risk, but also exerting a significant economic burden on the society. In order to devise
improved treatment and control strategies against enterococcal infections, it is important to clearly define
the molecular basis of disease progression and host–microbe interactions. Toward this end, application of
various laboratory models (both vertebrate and invertebrate; in vivo and in vitro) is invaluable.

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11
Listeria monocytogenes

Sarah E.F. D’Orazio

CONTENTS
11.1 Listeriosis Is a Significant Public Health Concern........................................................................185
11.2 L . monocytogenes Is a Facultative Intracellular Pathogen............................................................186
11.3 Cell Culture Models.......................................................................................................................186
11.4 Animal Models..............................................................................................................................187
11.4.1 Rabbits, Sheep, and Goats.................................................................................................187
11.4.2 Mice and Rats....................................................................................................................187
11.4.3 Guinea Pigs........................................................................................................................188
11.4.4 Gerbils...............................................................................................................................188
11.4.5 Nonhuman Primates..........................................................................................................189
11.5 L . monocytogenes Isolates Vary in Virulence...............................................................................189
11.6 M  ethods of Oral Transmission...................................................................................................... 190
11.7 O  ther Variables to Consider...........................................................................................................191
11.8 F  uture Directions...........................................................................................................................191
Acknowledgments................................................................................................................................... 192
References............................................................................................................................................... 192

11.1 Listeriosis Is a Significant Public Health Concern

Listeria monocytogenes typically causes infection in humans when ready-to-eat food products are
contaminated during processing1; the bacteria increase in number during refrigerated storage,2 and then
the food is consumed without adequate heating.3 Ingestion of L. monocytogenes leads to a wide spectrum
of clinical outcomes, ranging from self-limiting gastroenteritis to life-threatening systemic infections
of the blood, brain, and placenta that have a high mortality rate (25%–30%) even in patients receiving
antibiotic treatment.4 The true incidence of intestinal infection is not known, as most people do not seek
medical treatment for mild gastroenteritis, and L. monocytogenes is not commonly isolated from stool
without the use of a specific enrichment broth.5 However, it is thought that the infection rate for heav-
ily contaminated foods is likely to be high.6,7 After a short incubation period that can range from a few
days up to several weeks, L. monocytogenes disseminates and then crosses the blood–brain barrier or
the placenta in pregnant women. A retrospective analysis of recent outbreaks found that gastrointestinal
symptoms were noted within 24 h of ingestion and bacteremia occurred within 2 days.8 However, it took
an average of 9 days for central nervous system manifestations to develop, and much longer (17–67 days)
for pregnancy-associated cases to be reported.
The high fatality rate associated with listeriosis (20%–30%)4,9 makes it a significant public health
concern for high-risk groups including neonates, pregnant women, and people in other categories that
are steadily increasing due to medical advances (the elderly, transplant recipients, and patients with
chronic disease). L. monocytogenes infections can also occur in young, otherwise healthy individuals; in
fact, cases in people over 50 with no known underlying medical issues have increased recently.10–12 The
most deadly outbreak of foodborne disease in the United States in more than a decade occurred in 2011

185
186 Laboratory Models for Foodborne Infections

when cantaloupes from Jensen Farms were contaminated during a washing procedure, and were then
widely distributed to at least 28 states.13 Since then, increased awareness of the risk of L. monocytogenes
contamination in processed forms of fresh produce has resulted in a significant increase in the number
of food recalls by the FDA, with an average of 50–60 due to L. monocytogenes per year (www.fda.gov/
Safety/Recalls/).

11.2 
L . monocytogenes Is a Facultative Intracellular Pathogen
L. monocytogenes is a nonspore-forming, Gram-positive bacillus that is readily found in both soil and
water samples. A large portion of the L. monocytogenes genome is devoted to regulatory proteins, and
not surprisingly, the bacterium is able to adapt to changes in environmental temperature, pH, salt con-
centrations, and nutrient availability.14 Following transmission to a mammalian host, a large proportion
of L. monocytogenes continue to replicate extracellularly; however, a vital subset of the bacterium can
invade host cells and survive and replicate within this intracellular niche.15
L. monocytogenes mediates its own uptake into nonphagocytic cells via a family of surface-exposed
proteins called internalins. Internalin A (InlA) binds to E-cadherin and internalin B (InlB) binds to
c-Met, and engagement of these receptors results in bacterial internalization via clathrin-mediated endo-
cytosis.16 L. monocytogenes contained within host cell phagocytic or endocytic vacuoles produces lis-
teriolysin O (LLO), a cholesterol-dependent pore-forming toxin that lyses the vacuole, releasing the
bacterium into the cytosol.17–19 L. monocytogenes multiplies rapidly in the cytosol and begins to nucleate
actin filaments via the surface-exposed protein ActA. L. monocytogenes can move through the cytoplasm
by actin-based motility towards the cell membrane and can be enveloped in pseudopod-like structures
and then phagocytosed by neighboring cells.20,21 This results in a cell-to-cell spread of L. monocytogenes
without exposure to the extracellular environment.

11.3 Cell Culture Models

To cause infection, foodborne L. monocytogenes must survive passage through the stomach and then
colonize some portion of the intestines. If the bacterium does not attach firmly or invade the gut muco-
sal barrier, it will most likely be expelled in feces within just a few hours. Tissue culture studies have
been widely used to investigate these initial steps in the infection process. For example, in vitro studies
showed that several different proteins expressed on the surface of L. monocytogenes can bind mucin.22,23
This could serve to anchor the bacterium in the mucus layer overlying intestinal epithelial cells. The most
common model for studying invasion of enterocytes is Caco-2, a human colorectal adenocarcinoma cell
line that differentiates into polarized epithelial-like cells at confluence. Caco-2 cells were employed to
demonstrate that the bacterial surface protein InlA can bind to E-cadherin on enterocytes, triggering a
signaling cascade that leads to cytoskeletal rearrangements and internalization of the bacteria.16,24
The gut epithelium actually consists of several different cell types, and interspersed within the
enterocytes are goblet cells, Paneth cells, and M cells. M cells, in particular, are known to be a route of
invasion for many different bacteria, including L. monocytogenes.25 Corr et al. differentiated C2Bbe1
enterocytes into cells that resembled M cells by coculturing the epithelial cells with lymphocytes har-
vested from Peyer’s patches in the small intestine.26 Although cell culture models such as these have
been very useful for studying the dynamics of specific invasion pathways, a significant disadvantage
of this approach is that a single cell line cannot replicate the complexity of cell types that are actually
present in the gut. An innovative approach that shows great promise in this regard is the use of intes-
tinal organoids. Intestinal stem cells are cultured in vitro with the appropriate growth factors and this
leads to the formation of “miniguts” that contain fully differentiated cell types.27,28 Microinjection of
bacteria directly into the lumen of intestinal organoids may provide an excellent in vitro model system
for future studies to better understand how L. monocytogenes optimally uses the multiple invasion
pathways available in the gut.
Listeria monocytogenes 187

11.4 Animal Models
An ideal small animal model of L. monocytogenes infection should closely mimic all phases of human
listeriosis. Oral challenge is the preferred route of transmission to allow investigation of environmental
factors that promote bacterial infectivity and the host innate resistance mechanisms that limit coloniza-
tion of the gut. Only a small subset of humans exposed to L. monocytogenes develops severe systemic
infections; thus, dissemination from the gut to peripheral tissues should be a rate-limiting step. Ideally,
resistant animals should be able to inhibit bacterial replication rapidly, and there should be a lag period
before L. monocytogenes cross the blood–brain barrier or the maternal–fetal interface in susceptible
animals.
Most of the existing models of L. monocytogenes infection have caveats that either present significant
technical hurdles or limit the physiologic relevance of the model. One such limitation is the large inocu-
lum size required to establish infection in most laboratory animals. The infectious dose in humans has
been estimated at 106 –107 CFU.29,30 At least 100-fold larger doses (108–1010 CFU) are typically needed
for experimental infections in small animal models. Differences in the stomach pH, bile content, or
gastric enzyme composition may account for some of the relative resistance of experimental animals.
However, it is also possible that in otherwise healthy individuals, systemic listeriosis only occurs when
very large doses of bacteria are ingested. In that case, the range of inocula used in immune competent
laboratory animals may, in fact, closely model human transmission of the disease.
Another issue is the species specificity of the interaction between the L. monocytogenes surface
proteins InlA and InlB and the receptors found on mammalian cells.31,32 Early studies indicated that
InlA was important for invasion of epithelial cells,24,33 and InlB was needed for uptake in endothelial
cells.34,35 However, more recent work suggests that these two proteins may work in conjunction to allow
for efficient internalization of L. monocytogenes in nonphagocytic cells.36,37 InlA has a high affinity for
human E-cadherin, and the human Met protein serves as a receptor for InlB, so that both ligand/receptor
interactions are functional during human infections.38 As described below, most small animal models
of listeriosis are hampered by a low affinity interaction for either InlA or InlB. Importantly, intestinal
infection and fetoplacental transfer do still occur in some of these animals, which suggest that other
uptake mechanisms may be able to compensate, but it is not yet clear how accurately these systems
mimic human infections.

11.4.1 Rabbits, Sheep, and Goats

L. monocytogenes was first identified in 1926 as the causative agent of a mononuclear leukocytosis in
rabbits.39 Until the 1980s, the bacterium was thought of as mainly a veterinary pathogen, with naturally
occurring listeriosis found primarily in sheep and other ruminants.40 Thus, most early research studies
focused on feeding trials to model the disease observed in rabbits and sheep.41,42 Although some inves-
tigators still do use rabbit43 or goat44 models to study oral transmission, most effort is now focused on
rodent and nonhuman primate models. For a comprehensive review of infection studies in ruminants and
other veterinary models, see Hoelzer et al.45

11.4.2 Mice and Rats

Rodents are the preferred animal for most infectious disease studies because their small size allows
large-scale experiments to be performed affordably in a relatively small space. Mice, in particular, are
commonly used because many tools and reagents (antibodies, ELISA kits, transgenic, and knockout
strains) exist that facilitate experiments, and most of these are commercially available. Early studies
indicated that 108–1010 CFU were needed to establish gastrointestinal infection in mice, and that intra-
venous inoculation was more reproducibly lethal in mice46,47; however, it is clear that infectious dose is
dependent on the mouse strain used. For example, C57BL/6 mice are significantly more resistant than
either A/J or BALB/c/By/J mice48–50 that can be colonized by as few as 106 CFU administered orally.
188 Laboratory Models for Foodborne Infections

The differential susceptibility of these mouse strains is not likely due to the intestinal microbiota, since
fecal transplantation between C57BL/6 and BALB/c/By mice did not change the infection outcomes.51
Two different “humanized” mouse strains have been developed in an effort to enhance the effi-
ciency of oral infection in mice. In both cases, modifications were designed to promote interaction
between InlA expressed on the bacterial surface and E-cadherin expressed on the intestinal epithe-
lium. In the first mouse strain, human E-cadherin was ectopically expressed under the control of
the iFABP promoter, resulting in dual expression of both mouse and human E-cadherin in the small
intestine.52 The second mouse strain is a “knock-in” strain that has a single amino acid substitution
(E16P) in murine E-cadherin that allows the protein to serve as a high affinity receptor for InlA.53
Orally infected E16P mice showed enhanced colonization in the gut compared to wild-type mice;
therefore, this mouse strain represents the best option for mimicking the InlA/InlB-mediated inva-
sion events that can occur in the human gastrointestinal tract. However, as with most transgenic mice,
the knock-in was generated on a C57BL/6 background, and these mice are innately more resistant to
L. monocytogenes infection compared to other mouse strains. Although it would be time consum-
ing and expensive, it would be very useful to cross the E16P mutation onto more susceptible strain
backgrounds such as BALB/c mice.
Intravenous or intraperitoneal injection of L. monocytogenes in rats was frequently used in the 1980s
as an infection model to study immune responses against an intracellular pathogen. However, very few
studies have examined the oral transmission of listeriosis in rats. Czuprynski and Balish first showed that
germfree rats were more readily colonized than conventionally housed rats offered L. monocytogenes
in their drinking water.54 Later, Schlech et al. used a feeding tube to deliver the bacteria and found that
doses of at least 109 CFU were needed to consistently establish intestinal infection in young Sprague–
Dawley rats.55 Rats have the same species barrier that limits InlA-mediated invasion of the gut mucosa
as is found in mice,38 and thus, offer no particular advantage for use as a model organism.

11.4.3 Guinea Pigs
Young guinea pigs have also been used to study L. monocytogenes infection, and like mice or rats, doses
of 108–1010 CFU are typically needed to facilitate intestinal colonization.56,57 Melton-Witt et al. infected
guinea pigs with a mixture of 20 signature-tagged strains and showed that the spread from the MLN to
the spleen was a rate-limiting step for systemic dissemination, with only one in every 100–1000 bacteria
getting beyond this bottleneck.56 Using this model, the total number of L. monocytogenes that reached
the spleen or liver was very low, suggesting that the guinea pig is not the ideal model to study the sys-
temic phases of listeriosis.
As with most other rodents, the use of guinea pigs does present a species barrier for internaliza-
tion of L. monocytogenes. Guinea pig E-cadherin does bind efficiently to InlA, but the Met protein
in guinea pigs has an amino acid substitution that prevents optimal interaction with InlB.58 In spite of
this limitation, guinea pigs are considered the model of choice to study maternal–fetal transmission of
L. monocytogenes, largely due to the architecture of their placenta. Both humans and guinea pigs have
a hemochorial placenta, with only a single layer of cells separating the fetal and maternal blood sup-
plies. Particularly during the last stages of pregnancy, this means that bloodborne L. monocytogenes
need to traverse just a single trophoblast to invade the placenta.59 Williams et al. showed that infection
of pregnant guinea pigs with oral doses ranging from 104 to 108 CFU led to invasion in approximately
half of the fetuses.60,61

11.4.4 Gerbils
The E-cadherin and Met proteins in gerbils efficiently bind InlA and InlB, respectively.53 The lack of a
species barrier for interaction with these key bacterial surface proteins makes gerbils perhaps the most
physiologically relevant rodent model to study listeriosis. Disson et al. orally inoculated gerbils and
found significant colonization of both the small and large intestines.53 Furthermore, they demonstrated
100% lethality in fetuses when pregnant females were infected orally.53 The inocula used in those studies
were 109–1010 CFU, but lower doses were not tested, so it is possible that the infectious dose in gerbils
Listeria monocytogenes 189

could be closer to that observed in humans. Blanot et al. also demonstrated that gerbils could develop
rhombencephalitis, a localized brainstem infection, that closely mimicked the type of brain infections
seen in about 20% of human cases.62 However, in those studies, the gerbils were infected via the middle
ear, so it is not yet clear whether brainstem infections can naturally occur following the oral ingestion of
L. monocytogenes in gerbils. Unfortunately, a lack of available tools and reagents specific for gerbils has
limited the enthusiasm for studying host responses in this model.

11.4.5 Nonhuman Primates
Nonhuman primate models have the distinct disadvantage of being expensive to use, and so only small
numbers of animals are usually available for any given study. The clinical course of sporadic listeriosis
appears to mimic human disease closely, as evidenced by occasional outbreaks of L. monocytogenes
infection in nonhuman primate colonies that resulted in either meningoencephalitis or spontaneous abor-
tion in pregnant females.63,64 The few primate infection studies performed to date have been most useful
for approximating the infectious dose of L. monocytogenes in humans. For example, Farber et al. found
that cynomolgus monkeys fed at least 107 CFU of strain Scott A shed L. monocytogenes in feces for
3 weeks, but only the animals given 109 CFU displayed signs of disease (septicemia and occasional diar-
rhea).65 More recently, Smith et al. used a monkey clinical isolate of L. monocytogenes to infect pregnant
rhesus macaques.30 In that study, the overall LD50 was estimated to be 107 CFU, but it was noted that
monkeys with a stillborn fetus needed a significantly lower dose to establish an intestinal infection than
monkeys that had normal deliveries. This suggests that host susceptibility factors are also likely to play
an important role in determining the clinical outcomes following ingestion of L. monocytogenes, and
the use of an inbred animal model (such as laboratory mice) may be the easiest way to identify these
genetic loci.

11.5 
L . monocytogenes Isolates Vary in Virulence
Based on serological reactions between listerial antigens and specific antibodies, L. monocytogenes is
separated into at least 12 serovars (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7), which tend to
show varied virulence potential in human and animal hosts. It is notable that serovars 1/2a, 1/2b, 1/2c,
and 4b are responsible for >95% of human clinical listeriosis cases, and are also highly pathogenic to
animal hosts such as rodents via intragastric (i.g.) route. Most published studies of oral infection models
have utilized one of the two most common strains of L. monocytogenes: EGDe and 10403s. An advan-
tage of using one of these strains is that there are many isogenic derivatives (specific gene knockouts
and reporter strains) available, and it is easier to compare directly the results of unrelated experiments.
However, the choice of which L. monocytogenes isolate to use for oral challenge studies in small animal
models could greatly alter the course of the ensuing infection. Barbour et al. infected BALB/c mice
intragastrically with 66 different isolates of Listeria and found that the serovar 4b and 1/2a strains were
the most virulent, a pattern that has also been observed in humans.66 Both L. monocytogenes EGDe and
10403s are serovar 1/2a strains, but they have been extensively passaged in the lab. Each is probably a
good representative of pathogenic L. monocytogenes, but it is important to passage the bacteria through
an animal periodically to maintain virulence traits.
Alternatively, the use of strains that have been freshly isolated from particular sources may yield
results that more closely mimic specific types of human disease. For example, a recent microarray analy-
sis suggested there may be identifiable differences between strains that primarily cause febrile gas-
troenteritis and those that lead to more invasive disease.67 In support of this idea, Jensen et al. tested a
strain (La111) that persistently colonized a fish processing plant and demonstrated that the bacteria were
not more virulent in vitro and they did not persist well in the intestines of orally infected guinea pigs.
However, oral infection with strain La111 did result in a significantly greater rate of infected fetuses than
similar challenges with other clinical isolates.68,69 In another example, pregnant mice were significantly
more susceptible to oral challenge with L. monocytogenes 2203, a strain that was isolated during an
outbreak of listeriosis that affected primarily pregnant women.70,71
190 Laboratory Models for Foodborne Infections

Mouse-adapted strains of L. monocytogenes have also been developed to overcome the species bar-
rier to receptor-mediated invasion of epithelial cells in the gut mucosa. The modified InlA expressed
by these strains (InlAm) has a similar affinity for mouse E-cadherin as wild-type InlA has for human
E-cadherin.72,73 Foodborne transmission of InlAm-expressing L. monocytogenes can be achieved with
lower doses (106 –108 CFU) than wild-type strains and results in an infection course that closely mimics
all phases of human disease.49 However, the modified InlAm protein also acquired the ability to bind
N-cadherin in addition to E-cadherin, resulting in an enhanced uptake by villous M cells.74 Thus, the use
of an InlAm-expressing strain may result in altered tropism of bacterial invasion compared with human
infections.

11.6 Methods of Oral Transmission

There are multiple ways to inoculate experimental animals orally. One of the first methods employed
was to contaminate the drinking water of Swiss mice and then allow the animals to drink from the water
bottle for 24 h.75 This approach was not technically challenging, but had a major disadvantage because
it was difficult to control the exact dose of bacteria each mouse swallowed. Due to this limitation, most
laboratories began using oral gavage as a method of choice to place a defined dose directly into the stom-
ach of each experimental animal. Depending on the size of the animal, this can be performed by placing
a blunted feeding needle or flexible tubing attached to a syringe into the esophagus, or for a more invasive
approach, the delivery device can be pushed all the way down to the stomach.
Although i.g. injections allow for more control over the delivery of the inoculum, they may have
unintended consequences. First, when L. monocytogenes is suspended in a liquid, most of the bacteria
transit rapidly through the intestines, and the majority of the inoculum is shed in feces within the first
4 h after infection.56,76 Rapid passage through the gut may not provide enough exposure to the acidic pH
and bile present in the upper gastrointestinal tract to promote induction of transcriptional changes that
are known to make L. monocytogenes more virulent.77 I.g. inoculation also has the potential to cause
minor physical trauma to the lining of the esophagus, especially when a feeding needle is used, and
this may promote direct invasion of the bloodstream in a manner that does not efficiently occur when
Listeria-contaminated food is ingested. A comparative analysis of multiple previously published studies
that utilized i.g. inoculation indicates that some investigators observed rapid dissemination to the spleen
and liver in as few as 4 h,48,52,73,78 while studies found no systemic spread of L. monocytogenes until 48 h
postinfection.72,79,80 This suggests that i.g. injections can have variable outcomes that are highly depen-
dent on the technique used by the investigator, and therefore, this method of oral inoculation does not
yield reproducible results. In addition, the use of certain types of anesthesia for more invasive routes of
inoculation may also alter host susceptibility to infection. In this regard, Czuprynski et al. showed that
sodium pentobarbital transiently enhanced the severity of infection in mice that were inoculated by the
i.g. route, but isoflurane had no effect on infection rates.81,82
Another solution to the problem of controlled dosage is to place the bacterial solution directly into the
mouth of the animal. For larger animals such as guinea pigs, it is relatively easy to slowly drip a solution
into the oral cavity using a syringe.56 For smaller animals such as mice, Manohar et al. used a sterile
bacterial inoculating loop to place a bacterial solution into the mouth.83 This approach avoids the trauma
of an invasive injection, but still involves a bacterial inoculum suspended in a liquid.
Bou Ghanem et al. recently described a novel model of foodborne listeriosis in which mice are fed a
L. monocytogenes-contaminated piece of bread.84,85 This natural feeding model has several advantages.
First, the infection method is not invasive, does not cause physical trauma, and does not require special-
ized skills to perform. This greatly decreases the chance of investigator-dependent variability, making
it a much more reproducible model, and one that needs fewer animals to achieve statistical significance.
Furthermore, this model allows one to test the effect of different types of food and/or variable food stor-
age conditions on the transmissibility of disease.
It should be noted that there is some confusion in the literature concerning the methods used to
achieve oral transmission in experimental animal models. Not all published reports describe the inocu-
lation procedure in detail; some simply state that the animals were “orally infected.” Since the choice
Listeria monocytogenes 191

of inoculation procedure may affect the route used by L. monocytogenes to invade the gut barrier and
spread systemically, caution should be used when interpreting the results of a study that does not specify
the exact technique used.

11.7 Other Variables to Consider

As outlined above, each small animal model of listeriosis has both advantages and disadvantages.
Ultimately, the choice of which model to use may depend heavily on the availability of the laboratory
animals, or the exact phase of L. monocytogenes infection that is being studied. Regardless of which
in vivo model is chosen, there are other factors that should be taken into account to best mimic human
disease.
For example, it has been suggested that patients who take antacids or cimetidine to block acid reflux
are more susceptible to developing systemic listeriosis.86 Thus, neutralization of stomach acid might
enhance oral transmission of L. monocytogenes. Saklani-Jusforgues et al. showed that buffering an
i.g. inoculum with sodium bicarbonate or providing only buffered drinking water to mice significantly
increased the number of L. monocytogenes found in the stomach 15 min after inoculation.87 However,
longer-term studies using a variety of infection models are less conclusive. Sodium bicarbonate pretreat-
ment did increase the severity of infection in neutropenic mice, but not in mice with normal levels of
circulating neutrophils.88,89 Likewise, other studies found no difference in infectivity in mice pretreated
with cimetidine47 or in monkeys given a calcium carbonate solution 1 h prior to infection.65 However,
Schlech et al. demonstrated that pretreatment with cimetidine did significantly decrease the dose needed
to establish infection in rats.55
One explanation for the different outcomes in these studies may be the endpoints that each investi-
gator used to define infectivity. It is possible that buffering the stomach pH may increase the number
of L.  monocytogenes that survive and pass through to the lower intestines, and this would be most
apparent when examining tissues at very early time points following infection. However, efficient inva-
sion of the gut mucosal barrier in the ileum, cecum, or colon may be dependent on gene expression
changes that occur following exposure to an acidic environment.90,91 Thus, the relatively smaller number
of L. monocytogenes that might survive passage through the stomach could be “gut adapted” and better
able to invade the gut mucosa. If this were the case, then the acid-exposed bacteria would have a growth
advantage that would not be apparent until later in the infection.
The vehicle used to administer L. monocytogenes may also influence the infectivity rate during oral infec-
tion. The processed food products most commonly linked to outbreaks of listeriosis (deli meats, cheeses,
and raw milk) all share a relatively high-fat content, and it is possible that gastric secretions induced when
ingesting a fatty meal help promote the survival or infectivity of L. monocytogenes. Several studies have
examined whether a high-fat content can improve the efficiency of establishing infection in animal models.
Bou Ghanem et al. found that foodborne infection of mice was most consistent when the bacteria were
suspended in melted butter, rather than saline, prior to contamination of food particles.49 Likewise, Smith
et al. demonstrated that stillbirths in pregnant monkeys were more likely to happen when whipping cream,
rather than whole or skim milk, was used as the delivery vehicle.64 These studies would appear to suggest
that the consumption of fatty foods could be a risk factor for developing listeriosis. However, the deadly
2011 listeriosis outbreak associated with contaminated cantaloupes indicates that produce can be a vehicle
to transmit the disease,13 and suggests that a high-fat food product is not required to promote infectivity.
Further studies examining the growth rate of L. monocytogenes in various food products, as well as studies
of transmission rates in a small animal model are needed to address this question.

11.8 Future Directions
Orally transmitted models of L. monocytogenes infection have not been as widely used as intravenous or
intraperitoneal models due to a high degree of innate resistance in many species and significant pheno-
typic variability among infected animals. It is often difficult to directly compare the results obtained in
192 Laboratory Models for Foodborne Infections

previously published studies because of significant differences in bacterial strain choice, route of inocu-
lation, and various manipulations investigators have used to enhance the infectivity. Recent improve-
ments, particularly for the mouse model, have greatly improved the efficiency of intestinal infection
following oral transmission, and the use of a standardized model would help to greatly advance the field.
Future studies should focus on the natural pathways used by L. monocytogenes to disseminate from
the gut to peripheral tissues. Of particular relevance will be mechanisms used to cross the blood–brain
barrier and the placental barrier in pregnant females. Finally, the vast majority of human L. monocyto-
genes infections take place in the elderly.92 Studies that directly compare infections in young versus aged
animals may shed light on potential therapeutic strategies that could prevent these deadly infections.

Acknowledgments
S.E.F.D. was supported by a grant from the National Institutes of Health (R56 AI091918).

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12
Mycobacterium

Flábio R. de Araújo and Nalvo F. Almeida

CONTENTS
12.1 Introduction................................................................................................................................... 197
12.2 Etiology......................................................................................................................................... 198
12.3 Life Cycle...................................................................................................................................... 198
12.4 Virulence....................................................................................................................................... 199
12.4.1 Two M. bovis Strains and Their Virulence Genes........................................................... 199
12.4.2 PE and PPE Genes............................................................................................................ 199
12.4.3 Mycobactin....................................................................................................................... 199
12.4.4 Mce Genes........................................................................................................................ 200
12.5 Immunity....................................................................................................................................... 200
12.6 Foodborne Illnesses Associated with Mycobacterium Species.................................................... 200
12.7 Genomics....................................................................................................................................... 201
12.7.1 Genome Rearrangements................................................................................................. 203
12.7.2 Repeat Sequences............................................................................................................. 203
12.8 Conclusion..................................................................................................................................... 203
Acknowledgments................................................................................................................................... 204
References............................................................................................................................................... 204

12.1 Introduction
Mycobacterium spp. are intimately linked to human society. Leprosy, caused by Mycobacterium
leprae, has been described in historical texts since ancient times; archaeological and osteological findings
indicate its existence in human populations for thousands of years [1]. Evidences of Mycobacterium
tuberculosis complex (MTC) infection date from the Neolithic C period, more than 8000 years ago [2].
Evidences of human infection by Mycobacterium bovis (a bovine tuberculosis agent) have been described
in skeletons from South Siberia, with dates ranging from approximately 1761 to 2199 years before pres-
ent, placing the remains within the Iron Age period [3].
The zoonotic transmission of Mycobacterium spp. has been related, among other means, to the inges-
tion of contaminated food. Historically, M. bovis has been associated with extrapulmonary tuberculosis
in infants and children, usually occurring due to the consumption of milk, which had not been pasteur-
ized or boiled, from infected cattle [4].
This chapter presents an overview of foodborne Mycobacterium spp., including the background infor-
mation and recent findings relating to the etiology, life cycle, virulence, immunity, and clinical diseases,
as well as genomic aspects such as annotation, genome comparison of closely related organisms, and
virulence-related genes.

197
198 Laboratory Models for Foodborne Infections

12.2 Etiology
The genus Mycobacterium includes a group of high GC Gram-positive microorganisms, including
saprophytic species, and important human and animal pathogens. Pathogenic members are usually char-
acterized by their slow growth in culture, with generation times of 12–24 h, whereas nonpathogenic
members grow considerably faster [5]. In this section, we focus on mycobacteria species involved in
foodborne diseases.
Mycobacterial species causing tuberculosis in humans and animals belong to the Mycobacterium
tuberculosis complex (MTBC). The following organisms are considered members of the MTBC:
M. tuberculosis [6]; Mycobacterium africanum [7] and Mycobacterium canettii [8], which are mainly
human pathogens; M. bovis [9] and Mycobacterium caprae [10], which are mainly ruminant pathogens;
Mycobacterium microti, a pathogen of small rodents [11]; Mycobacterium pinnipedii [12] from marine
mammals; Mycobacterium mungi from mongooses [13]; and Mycobacterium orygis from oryx [14].
Members of the MTBC have been implicated in foodborne transmission to man. The consumption
of contaminated raw dairy products has been recognized as a major cause of transmission of M. bovis
to humans [15], generally associated with the development of extrapulmonary TB [16]. Another species
of the MTBC that infects man is M. caprae. Although the transmission to man by raw dairy products
has not been proven formally for M. caprae, the relatedness of the pathogens and the epidemiological
settings suggest that this is probably the case [17].
Interestingly, there are pre-Columbian evidences of zoonotic infection in Peruvian human skeletons,
revealing that a member of the M. tuberculosis complex caused human disease before contact. The
ancient strains are distinct from known human-adapted forms and are most closely related to those
adapted to seals and sea lions (M. pinnipedii) [18], raising the possibility of foodborne transmission by
ingestion of sea lions’ meat, hides, and bones, which was common practice in this region [19].
Nontuberculous mycobacteria (NTM) are ubiquitous organisms and were believed to represent envi-
ronmental contamination. These organisms are a significant cause of infection in both immunocompe-
tent [20] and immunocompromised humans [21].
M. avium subsp. paratuberculosis (MAP), which belongs to the Mycobacterium avium complex
(MAC), is a well-known NTM pathogen causing Johne’s disease (or paratuberculosis), a chronic
progressive, infectious granulomatous enteritis that principally affects ruminants [21]. There is a pos-
sible association between MAP and Crohn’s disease, a human inflammatory bowel disease. MAP is also
a potential human foodborne pathogen because it survives pasteurization treatments [22].
A series of other NTM have been found to be associated with food: M. nonchromogenicum,
M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii,
M. scrofulaceum, M. genavense, M. simiae, and M. szulgai, which were detected in milk [23,24],
and M. chelonae and M. kansasii, which were detected in water [25,26].

12.3 Life Cycle
As reviewed by Scherr and Nguyen [27], the developmental stages of mycobacterial life cycle are better
defined in M. tuberculosis. This pathogen infects human macrophages within granulomatous structures
of the lung, where they may undergo morphological alterations into filamentous cells within the infected
host cells. Later, host immune responses suppress the vegetative growth, inducing the formation of
persisters that are able to survive in a dormant state for years (the latent TB stage). These dormant cells
of M. tuberculosis may also become shorter, and some transform into a spherical, ovoid morphology
characterized by a thickened cell wall with altered chemical characteristics. Probably after a weakness in
the host immune defense, M. tuberculosis reactivates vegetative growth from the dormant state to cause
active TB. In this state of the disease, many M. tuberculosis cells may escape the host cells to replicate
extracellularly in the TB cavity within the lung. These actively growing cells also become transmissible
through aerosolized droplets expelled from the lung of TB patients, and continue their life cycle in
the newly infected patients who inhale the droplets.
Mycobacterium 199

12.4 Virulence
Despite the high overall phylogenetic relationship among species of the MTBC, they show wide
variability in their phenotypes, especially in their virulence characteristics. The same occurs with MAP
members. This section points out some important, recent works on MTBC and MAP virulence issues
present in the literature.

12.4.1 Two M. bovis Strains and Their Virulence Genes

Using next-generation whole genome sequencing (WGS), two M. bovis strains (04-303 and 534) were
recently sequenced. The first one was isolated from a wild boar and the other from cattle, both in
Argentina. These strains have been used in the experimental BALB/c model of progressive pulmonary
tuberculosis [28]. Mice were infected with 04-303, resulting in a mild inflammatory response, followed by
sudden pneumonia with extensive necrosis and high mortality. On the other hand, mice infected with 534
presented limited tissue damage and higher survival. Based on these results, 04-303 was designated to be a
virulent strain and 534 an attenuated strain.
In a transcriptome analysis of both strains [29], a total of 49 differentially expressed genes were
detected, 35 of which were at a higher level in 04-303 and 14 in strain 534. Their expression levels
dramatically changed during in vitro culture, with the same behavior inside bovine macrophages. The
analyses at the nucleotide level revealed that these genes were conserved in both strains, suggesting that
the differential expression could be related to mutations in regulatory regions.
In a genome analysis, a comparison of a set of virulence-related genes present in these genomes has been
made by using Blastn and Blastx [30] and having M. bovis AF2122/97 as a reference genome (Castelão
A.B. et al., unpublished data). A  better understanding of the virulence factors of these strains may be
essential to help researchers in developing new vaccines and diagnostics. In this analysis, 345 virulence
genes, as reviewed by Forrellad et al. [31] and/or listed in the TubercuList database (http://genolist.pastteur.
fr/TubercuList), were inspected in both strains 04-303 and 534, by looking for SNPs. The SNPs were then
classified as synonymous or nonsynonymous. Strain 04-303 has three synonymous mutations and eight non-
synonymous ones. On the other side, strain 534 presented six and nine mutations, respectively. Mutations
found in virulence genes, like hrcA, hpx, and esat6 (on the 04-303 side) and irtA, pks10, clgR, and tetR (on
the 534 side), agree with the attenuated phenotype of strain 534 while only being in partial consonance with
the hyper-virulent phenotype of the 04-303 strain. Special attention should be given to the mutation found
in hrcA from 04-303, a heat shock protein transcriptional repressor that could lead to an overexpression of
heat shock proteins that can be related with virulence (Castelão A.B. et al., unpublished data).

12.4.2 PE and PPE Genes

By doing genome comparisons (showed in Section 12.7), we could find the presence of the PE (Pro-Glu)
and PPE (Pro-Pro-Glu) protein families, involved in immune stimulation and virulence in all seven
compared genomes. Besides cell wall proteins, these polymorphic gene families have been indicated as
a major factor for differences among these species [32,33].
The total numbers of PE/PPE proteins found in MtH37Rv and MbAF2122/97 were 97 and 99, respec-
tively. On the other hand, in MAP strains, the numbers were 42, 45, 43, 49, and 45 in MapMAP4, MapK10,
Ma104, MapS397, and MapS5, respectively. Because these gene families used to be more frequent in
pathogenic members of the Mycobacterium genus, this small number of PE/PPE proteins in MAP strains
could suggest differences in the immunity response, compared to the MTBC species. A total of 18 fami-
lies containing at least one gene of each one of the MAP strains and not containing genes of the MTBC
strains have been found, showing a distinguishable group in MAP strains of this important protein class.

12.4.3 Mycobactin
Because some Mycobacterium species secrete iron-chelating siderophores as virulence factors in the
iron-limiting environments of their hosts to compete for ferric iron, they need to produce mycobactin,
200 Laboratory Models for Foodborne Infections

a siderophore responsible for transporting iron into cells. One important phenotypic of MAP when
compared with the MTBC and other MAC species is its inability to produce mycobactin [34,35]. Iron
transport into cells is associated with a 10-gene cluster mbtA-J. In MAP species, the first gene of this
cluster is shorter than the corresponding one in the MTBC and MAC. Because MbtA is responsible for
initiating mycobactin production, this truncation reported suggests that the cascade leading to mycobac-
tin production may be attenuated or disrupted in MAP [35].

12.4.4 Mce Genes
Virulence genes that are important for the entry and persistence of bacteria in the host deserve our
attention. The mammalian cell entry (Mce) gene has this property and has been identified in the MTBC
species. Four copies of the Mce gene are present in M. tuberculosis, and there are eight homologs of the
Mce gene in M. avium subsp. paratuberculosis K-10 [35]. Because there are Mce operons in both patho-
genic and nonpathogenic mycobacteria, it implies that the mere presence of these genes does not give to
the species the ability to be virulent. However, the role of this operon in virulence may be determined by
its expression under specific conditions.

12.5 Immunity
In this section, we briefly review the model of immune response against M. bovis. Following infec-
tion, innate immune responses are important for recruiting immune cells and establishing early lesion
formation; however, these responses do little to limit infection [36]. On the other hand, the adaptive
cell-mediated immune response by Th1 CD4 T cells and their soluble mediators are essential for control-
ling the disease [37]. Additional cells and soluble mediators that contribute to immunity include CD8 T
cells [38], IL-17 [39], nitric oxide [40], and IL-2 [41].

12.6 Foodborne Illnesses Associated with Mycobacterium Species

Foodborne diseases are a public health problem worldwide. With regards to Mycobacterium spp., prior
to mandatory milk pasteurization in many countries, M. bovis accounted for about 25% of tuberculosis
cases in children [42]. In Great Britain, human consumption of infected cows’ milk led to an estimated
2500 deaths and more than 50,000 new cases of TB per year in the early 1900s [43]. In Latin America,
M. bovis is involved in 2% pulmonary and 8% extrapulmonary human tuberculosis cases [44].
The significance of the public health threats from zoonotic tuberculosis resulted in the adoption of a
resolution by the World Organization for Animal Health (Office International des Epizooties; OIE) in
1983, calling for the eradication of M. bovis for public health and economic reasons, adoption of stringent
meat inspection and pasteurization or boiling of milk for human consumption, and continued research
into bovine tuberculosis, particularly in the improvement of diagnostic tests [45].
Experiments to determine the efficacy of high temperature, short time (HTST) pasteurization of milk
in terms of inactivation of pathogenic microorganisms were mainly performed between 1930 and 1960.
Among the target organisms were M. bovis and M. tuberculosis. As a result, the Codex Alimentarius
prescribes that the HTST treatment of milk should lead to a significant reduction of pathogenic microor-
ganisms during milk pasteurization [46].
While standard HTST pasteurization treatment results in the efficient inactivation of M. bovis and
M. caprae [46], NTB (including MAP) are more resistant to heat treatment and pasteurized milk may be
considered one source for NTM human infection [23,46].
Both M. bovis and MAP have already been detected in many types of cheese in different countries. In
Brazil, MAP was detected in Coalho cheese by PCR and culture [47]. In Mexico, MTBC DNA was found
in fresh cheeses that were obtained from municipal markets in the state of Hidalgo [48]. In Switzerland,
MAP DNA was detected in 4.2% of the raw milk cheese samples collected at the retail level, although
no viable MAP cells could be cultured [49].
Mycobacterium 201

Zoonotic TB has reemerged in some places of the world such as the Mexico–EUA border among
immigrants from regions where bovine TB is endemic, associated with the consumption of soft fresh
cheeses [50–53]. An epidemiological investigation conducted by the Centers for Disease Control and
Prevention (CDC) with 35 human cases (1.12% of all analyzed TB cases) of M. bovis infection in
New York City (NYC) also suggested that the fresh cheese brought to NYC from Mexico was a likely
source of infection [54]. In Brazil, this association has also been found. In a recent study, three of 189
patients (1.6%) diagnosed with TB exhibited a coinfection of M. bovis–M. tuberculosis associated with
the consumption of homemade cheeses, processed from raw milk [55].
Some artisanal cheeses are made with raw milk, followed by a ripening period, to ensure safety, since
the process of ripening can contribute significantly to the reduction of pathogens in such products [56].
Nevertheless, MAP could be cultured from Cheddar cheeses prepared from pasteurized milk artificially
contaminated with MAP strains after a 27-week ripening period [57]. The survival of M. bovis during
the ripening of cheeses has also been reported. This bacterium was shown to be viable in cheeses for dif-
ferent periods: in Camembert for 47 [58], 60 [59], and more than 180 [60] days; in Edam for 60 days [59];
in Cheddar for 220 days [60]; in Gruyere for more than 22 days; and in Swiss Tilsitier for more than
305 days [58]. During the ripening of blue cheese made from raw milk with tubercle bacilli (104/mL), a
decrease in numbers was observed during the first and second weeks, but bacilli were still present after
three to four months [61]. Guinea pigs developed TB when inoculated with a 3-month-old Emmental
cheese artificially contaminated with M. bovis [59].

12.7 Genomics
This section provides a short description and also important information related to genome annotation
and the whole genome comparison of some currently available sequenced strains of Mycobacterium spp.,
with special attention to MAP and MTBC. Table 12.1 shows Genbank information and basic features,
such as the chromosome length, %GC, and total number of CDS of the genomes used in this analysis.
M. tuberculosis H37Rv was included as an outgroup in the comparison, since it is closely related with
M. bovis and M. avium spp. and, on the other hand, branched from the parent group before any other one
in this set of genomes.
M. tuberculosis H37Rv and M. bovis AF2122/97 are genomes from the MTBC. The other ones
are genomes from MAP. Specifically, there are two major groups in MAP isolates, known as Type S
(Sheep Type) and Type C (Cattle Type). MapS5, MapS397, and Map104 are of Type S and MpaK10 and
MapMAP4 are of Type C.
Figure 12.1 shows a maximum-likelihood phylogeny of all seven genomes. Even using only MtH37Rv
as the outgroup, the tree clearly shows a separation of MbAF2122 and MtH37Rv from all other

TABLE 12.1
Genbank Information and Some Basic Features of the Genomes Used in This Comparative Analysis
Accession Chromosome %
Taxon Name Short Name Taxon ID Number Length CDS GC References
M. avium subsp. MapK10   262316 NC_002944 4,829,781 4350 69 [35]
paratuberculosis K-10
M. avium subsp. MapMAP4 1199187 CP005928 4,829,424 4326 69 [62]
paratuberculosis MAP4
M. avium subsp. MapS397 1010838 GCA_000219085 4,815,461 4619 69 [63]
paratuberculosis S397
M. avium subsp. MapS5 1247747 GCA_000330785 4,799,927 4288 69 [64]
paratuberculosis S5
M. avium 104 Ma104   243243 NC_008595 5,475,491 5120 69 TIGR
M. bovis AF2122/97 MbAF2122   233413 NC_002945 4,345,492 3918 66 [65]
M. tuberculosis H37Rv MtH37Rv   83332 NC_000962 4,411,532 3906 66 [66]
202 Laboratory Models for Foodborne Infections

M. avium taxa, ratifying the differences among genomes in MTBC and MAP. Besides, strains of Type
C and Type S could not be clearly clustered in the tree, showing that it is not easy to type these strains
using only sequence comparisons in the protein level. The tree was built using first OrthoMCL [67] to
find families with orthologous proteins. From all the 5599 families found by OrthoMCL, 2195 con-
tain exactly one representative member of each genome and at most one member of MtH37Rv (the
outgroup). Each one of these families was aligned by using MUSCLE [68] and filtered by Gblocks [69],
in order to remove noninformative sites. The alignments were concatenated, and the resulting alignment,
with 695,953 columns, was used as input in RAxML [70] software and the PROTCATWAGF evolution
model to build the tree. All bootstrap support values were 100% and were obtained with 100 replicates.
FigTree [71] was used to draw the tree.
In another whole genome comparison method, suggested in Ref. [72], one can calculate a genomic-
distance index, called MUMi, taking into account both criteria of diversity, which are based on DNA
maximal unique matches shared by two genomes. The matrix of MUMi indexes of our seven genomes
is shown in Table 12.2.
A second tree (data not shown) was obtained by using this matrix as input for a neighbor-joining
distance-based phylogeny method [73]. The distance-based topology obtained completely agrees with
the tree shown in Figure 12.1.
By using the software PanGP [74], based on the output families from OrthoMCL, core-genome and
pan-genome rarefaction curves of all five MAP genomes have been built. They are shown in Figure 12.2.

Ma104

MapS397

MapMAP4

MapK10

MapS5

MtH37Rv

MbAF2122
0.03

FIGURE 12.1  Phylogenetic whole genome tree, based on protein families of the seven genomes showed in Table 12.1.

TABLE 12.2
MUMi Indexes of the Seven Genomes Used in Our Comparison
Ma104 MapK10 MapMAP4 MapS397 MapS5 MbAF2122
MapK10 0.1520
MapMAP4 0.1521 0.0010
MapS397 0.1448 0.0252 0.0255
MapS5 0.1558 0.0112 0.0118 0.0296
MbAF2122 0.9060 0.9011 0.9011 0.9008 0.9015
MtH37Rv 0.9061 0.9013 0.9013 0.9010 0.9017 0.0208
Note: Pairs of closely related genomes have small indexes.
Mycobacterium 203

4800

4500

Gene cluster number 4200 Pan-genome size

Core-genome size

3900

3600

3300
0 1 2 3 4 5 6
Genome number

FIGURE 12.2  Core- and pan-genome sizes according to the number of genomes considered in the dataset. For each k in
the X-axis, all possible combinations of k genomes (among five) are taken and, for each one of these combinations, pan and
core numbers are plotted.

Both curves are far from reaching a plateau, suggesting that currently there is little data on the charac-
teristics of isolates of Type C and Type S. Thus, new genome-wide data would improve the identification
of genes involved in functional phenotypes.

12.7.1 Genome Rearrangements
Large genomic rearrangements, like long insertions, deletions, inversions, translocations, and duplica-
tions, are also mutational mechanisms that can cause phenotypic and genotypic differences among spe-
cies and subspecies. It is known that they may lead to distinct virulence and resistance characteristics.
Large inversions, for example, may be used by mycobacteria to modify the expression levels of specific
genes in order to acquire some advantages during infection [75].

12.7.2 Repeat Sequences
Another important source of variation in mycobacteria is repeat sequences. Variable number of tandem
repeat (VNTR) is based on the analysis of DNA segments containing tandem repeated sequences in
which the number of copies of the repeated sequence varies among strains. These repeats are 15–100 bp
long and dispersed at multiple locations in the genome sequence [76]. Mycobacterial Interspersed
Repetitive Units (MIRUs) are a particular case of VNTR. They are intergenic regions and are reported as
12-character designations, each character corresponding to the number of repeats at one of the 12 MIRU
loci. Short-sequence repeats (SSRs) are 2–5 bp long tandem repeats and are also present in mycobacteria.
All of these repeat sequences have been extensively used for typing [77–79] and also for inferring epide-
miologic issues of Mycobacterium isolates [80].

12.8 Conclusion
Several reasons make Mycobacterium an important bacterial model for foodborne infection. The
consumption of contaminated raw dairy products is recognized as a significant cause of transmission of
M. bovis to humans. M. caprae is probably also another species that infects humans by the same means.
M. avium subsp. paratuberculosis is a nontuberculosis mycobacterial human foodborne pathogen, with
the ability to survive pasteurization treatments. In addition, several other Mycobacterium spp. detected
in milk and water have been implicated in foodborne diseases.
204 Laboratory Models for Foodborne Infections

Acknowledgments
We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (grants 443235/2014-7,
305857/2013-4), Fundect-MS (grants TO 0096/12, TO 0085/15, and TO 007/2015) and Embrapa
(03.14.00.054.00.00, 02.13.16.002.00.00) for partial funding.

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13
Staphylococcus

Mar Rodríguez, Alicia Rodríguez, María Jesús Andrade,

Elena Bermúdez, and Juan José Córdoba

CONTENTS
13.1 Introduction................................................................................................................................... 209
13.2 Characteristics and Incidence of Foodborne Intoxications due to Enterotoxin-Producing
Staphylococci................................................................................................................................ 209
13.3 Diagnosis of Staphylococcal Foodborne Poisoning......................................................................211
13.4 Laboratory Models for Study of Staphylococcal Foodborne Poisoning........................................212
13.4.1 Animal Models..................................................................................................................212
13.4.2 Cell Culture Models..........................................................................................................215
13.5 Conclusions....................................................................................................................................216
Acknowledgments....................................................................................................................................217
References................................................................................................................................................217

13.1 Introduction
Staphylococci are Gram-positive and catalase-positive ubiquitous bacteria found on the skin and mucous
membranes of warm-blooded animals and humans. They can also be isolated from environmental
sources such as soil, air, and water and from a wide range of foodstuffs including dry-cured meat prod-
ucts and cheeses.1 To date, 52 species of staphylococci have been described.2,3 Staphylococci are grouped
into coagulase-positive (CPS) and coagulase-negative (CNS) staphylococci, according to their ability to
coagulate rabbit plasma.4 Staphylococcus aureus is the most frequently characterized CPS and a well-
known etiological factor of a variety of infections including superficial skin inflammations, systemic
infections, and septicemia.4,5 Furthermore, S. aureus is a recognized causative agent of staphylococcal
foodborne poisoning and has been considered the only representative of the genus Staphylococcus capa-
ble of producing enterotoxins (SEs).4,6,7 However, some CNS strains showing enterotoxigenic capacity
have been found in sheep and goat milk and cheeses,8,9 Spanish dry-cured hams,10,11 and even in starter
cultures.12 Enterotoxigenicity of these strains could pose considerable risk for food safety as, in some
cases, they are introduced as starter cultures at high levels in many animal-derived products due to their
positive impact on fermentation processes and sensory characteristics of products.4,13

13.2 Characteristics and Incidence of Foodborne Intoxications

due to Enterotoxin-Producing Staphylococci
Staphylococcal foodborne poisoning is an intoxication produced by consumption of foods containing
enough amounts of preformed enterotoxins.14 It is among the leading causes of foodborne outbreaks
in the European Union15 and the United States.16 However, the true incidence of foodborne disease
could be a lot higher as sporadic foodborne illness caused by enterotoxin-producing staphylococci is
not reportable.17 Several reasons such as misdiagnosis, improper laboratory analysis, and lack of routine

209
210 Laboratory Models for Foodborne Infections

surveillance of clinical stool specimens for S. aureus or its enterotoxins have been reported as the cause
of the low incidence of S. aureus foodborne disease.17
Although enterotoxigenic staphylococci are thermally destroyed, the cooked food may contain
SEs because such toxins are thermostable and cannot be eliminated by heat processing.18 Besides
Staphylococcus spp. are usually very tolerant to NaCl and grow well in NaCl concentrations above 10%.19
In addition, Staphylococcus spp. are tolerant to water activity reduction, being able to survive in ripened
products when water activity is higher than 0.87.11 Thus, foods that have been frequently involved in
staphylococcal intoxication include meat and meat products, poultry and egg products, milk and dairy
products, salads, bakery products, particularly cream-filled pastries and cakes, and sandwich fillings.14,20
Symptoms of staphylococcal intoxication appear in patients between 2 and 8 h after food consumption,
and include nausea, vomiting, retching, and abdominal cramping, with or without diarrhea.21,22 Vomiting
is the most frequently observed symptom.23 The disease is usually self-limiting and typically resolves
within 24–48 h after onset.14 Occasionally, it can be severe enough to warrant hospitalization, particularly
when infants, elderly, or debilitated people are concerned.24 The amount of SEs required to produce food-
borne poisoning in humans is difficult to determine. Reliable results from the examination of food impli-
cated in food-poisoning outbreaks are difficult to obtain because normally the enterotoxin is not uniformly
distributed in the food and it is impossible to know how much food have been consumed by each person.23

TABLE 13.1
Biological Characteristics of Staphylococcal Enterotoxins and Enterotoxin-Like Toxins
Emetic Activity
Molecular Superantigenic
Toxin Genetic Element Weight (kDa) Activity Monkey House Musk Shrew
SEA Prophage 27.1 + + +
SEB Chromosome, plasmid, 28.3 + + +
pathogenicity island
SEC1 Pathogenicity island 27.5 + + NE
SEC2 Pathogenicity island 27.6 + NE +
SEC3 Pathogenicity island 27.6 + + NE
SED Plasmid 26.4 + NE +
SEE Prophage 26.4 + NE +
SEG egc, chromosome 27.0 + + +
SEH Transposon 25.2 + + +
SEI egc, chromosome 24.9 + + +
SElJ Plasmids 28.6 + NE NE
SEK Pathogenicity island 25.5 + + NE
SEL Pathogenicity island 25.2 + + NE
SEM egc, chromosome 24.8 + + NE
SEN egc, chromosome 26.1 + + NE
SEO egc, chromosome 26.8 + + NE
SEP Prophage 26.6 + + +
SEQ Pathogenicity island 25.1 + + NE
SER Plasmid 27.1 + + +
SES Plasmid 26.3 + + +
SET Plasmid 22.6 + + +
SElU egc, chromosome 27.2 + NE NE
SElV egc, chromosome 27.6 + NE NE
SElX Chromosome 19.3 + NE NE
Source: Adapted from Hennekinne, J.A., et al., FEMS Microbiol. Rev., 36, 815, 2012; Omoe, K., et al., Infect. Immun., 81,
3627, 2013; Hu, D.L. and Nakane, A. Eur. J. Pharmacol., 722, 95, 2014.
NE, not examined.
Staphylococcus 211

Enterotoxin-producing staphylococci, mainly S. aureus strains, have been reported as producers of

more than 20 different SEs4,25,26 (Table 13.1). SEs were named on the basis of their emetic activities fol-
lowing oral administration in a primate model. Several SEs were designated as SE-like (SEl) since they
either lack emetic properties or their emetic activities have not been tested in this model yet.4,27 To date,
18 new types of SEs and SEl (SEG, SEH, SEI, SElJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SER,
SES, SET, SElU, SElU2, SElV, and SElX) and five classical types [SEA, SEB, SEC (with the SC1, SC2,
and SC3), SED, and SEE] have been reported.4,6,28–31 Recently, the emetic activity of SElK, SElL, SElM,
SElN, SElO, SElP, and SElQ has been demonstrated in a primate model.28
SEs are toxins of 20–30 kDa that have two separate biological activities: they act on the gastrointesti-
nal tract and as a superantigen on the immune system that trigger a strong T-cell activation.26,32 Among
SEs, the SEB is nowadays classified as a potential bioweapon due to the fact that it has all the charac-
teristics of an ideal biological agent.25 Three mechanisms have been proposed to explain how SEs cause
enteric illness: (1) the release of proinflammatory cytokines as a result of SE-induced superantigenic
T-cell proliferation, (2) the binding of SEs to intestinal mast cells that leads to degranulation, and (3) a
direct effect upon the intestinal epithelium affecting gut transit.33
SEs are resistant to environmental conditions (freezing, drying, heat treatment, and low pH), that eas-
ily destroy the enterotoxin-producing staphylococci strain. SEs are also resistant to proteolytic enzymes,
retaining their activity in the digestive tract after ingestion.6,19
Within SEs, the SEA is the most commonly reported enterotoxin in foods, and its predominance (>75%
of foodborne poisoning outbreaks) is well documented in different countries, followed by SED, SEC, and
SEB.14,34 Foodborne outbreaks where the remaining SEs are involved have been rarely reported. One
possible reason could be that techniques to check for enterotoxigenic staphylococci are usually based on
the detection of the SEs using commercial immunological kits, but these methods also have limitations.
Besides, they are only suitable for the classical enterotoxins (SEA–SEE), while kits to detect recently
reported enterotoxins are not available.35,36 Current studies collected evidence of two outbreaks, sug-
gesting that newly described SEs can be involved in foodborne poisoning.36,37 In general, detection of
SEs in foods is often difficult because of the small SE amounts present in foods and their proteic nature.
Different techniques including animal models have been reported to accurately investigate staphylococ-
cal foodborne disease.

13.3 Diagnosis of Staphylococcal Foodborne Poisoning

The diagnosis of staphylococcal foodborne poisoning is generally confirmed by at least one of the fol-
lowing methods: (1) recovery of 105 S. aureus cells/g from food remnants, (2) detection of SEs in food
remnants, or (3) isolation of S. aureus of the same phage type from both patients and food remnants.38
Conclusive diagnosis of staphylococcal foodborne poisoning is mainly based on the demonstration of
SEs in the food.39
Staphylococcus strains are usually enumerated by using conventional microbiological techniques
employed to detect CPS in food samples, according to EN ISO 6888-1,40 EN ISO 6888-2,41 or EN ISO
6888-3.42 However, they are time consuming since they take up to 5–6 days to be performed. Moreover,
it may not be enough for the reliable identification of all the possible enterotoxigenic staphylococci
(atypical S. aureus, other CPS and CNS). Molecular biology methods have been used as an alternative to
conventional microbiological techniques (often PCR and real-time PCR) to detect CPS in food samples.
PCR-based methods detect genes encoding enterotoxins in strains of Staphylococcus spp. However,
these techniques have a major limitation: the results inform the presence or absence of genes encoding
SEs, but do not provide any information on their production. These methods therefore cannot be used
as sole methods for confirming Staphylococcus spp. as the causative agent in an outbreak.25 In addition,
in some cases the confirmation of staphylococcal foodborne poisoning is difficult because S. aureus is
heat or water activity reduction sensitive, whereas SEs are not. Thus, in heat-treated or in ripened food
matrices with less than 0.87 water activity, S. aureus may be eliminated without inactivating SEs. In such
cases, it is not possible to characterize a foodborne poisoning outbreak by detecting and enumerating
Staphylococcus spp. in food remnants.6
212 Laboratory Models for Foodborne Infections

There are three types of methods to detect bacterial toxins in foods—immunological tools, mass
­spectrometry-based methods, and bioassays.
Immunological tools are the most commonly used methods for detecting SEs in foods. Commercially
available kits have been developed according to two different principles: (1) enzyme immunoassay com-
prising ELISA (TECRA Kit, TRANSIA PLATE, and RIDASCREEN SET) and enzyme-linked fluo-
rescent assay (ELFA) (VIDAS SET and VIDASTM SET2), and (2) latex agglutination (SET-RPLA). It
is widely recognized that the use of immunological methods to detect contaminants in food matrices is
a difficult task, mainly because of the lack of specificity and sensitivity of the marketed kits. Moreover,
only antibodies against SEA to SEE, SEG, SEH, and SElQ were available until recently. The immuno-
logical tools do not detect the other SEs.25 The main drawback of current methods to detect enterotoxins
based on specific polyclonal or monoclonal antibodies remains their high cost.43
As an alternative to immunological tools, other strategies based on physicochemical techniques have
been developed. Among these, mass spectrometry method,44 based on the use of immunoaffinity capture
and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or
electrospray ionization (ESI),45,46 biomolecular interaction analysis mass spectrometry (BIA-MS),47 and
high-performance liquid chromatography (HPLC)48 have been proposed. These methods allow an accu-
rate detection of SEs in foods but require investing in expensive HPLC, MALDI-TOF-MS, or BIA-MS
equipments.
Bioassays are based on the ability of a suspected food extract to induce symptoms such as vomiting or
gastrointestinal signs in laboratory animal models and/or superantigenic action in cell culture models.25
These methods offer a very valuable alternative for foodborne staphylococcal investigation and will be
explorated in a separate section.

13.4 Laboratory Models for Study of Staphylococcal Foodborne Poisoning

13.4.1 Animal Models
Although humans are ideal model to study human pathogens, it is not possible to use humans owing
to safety, ethical, and expense-related concerns. However, human volunteers have been used in certain
nonfatal diseases. Animal models are frequently utilized as a substitute. Among them, nonhuman pri-
mates (monkey, baboon, and chimpanzee) are ideal to mimic many diseases. Again, ethical and expense-
related considerations limit their widespread applications.
Rodents are most commonly used as animal models and include mice, rats, rabbits, hamsters, and
guinea pigs. Other animals including ferrets, pig or piglets, dogs, and cats are also utilized. Careful
considerations while choosing an animal model include pathogens should infect animals by the same
route as humans and exhibit a similar colonization pattern, similar tissue distribution patterns, and same
degree of virulence as in humans.49
As previously described, the emetic activity of SEs is one of the main factors to consider it in the “cat-
egory of SEs.” The International Nomenclature Committee for Staphylococcal Superantigens (INCSS)
emphasizes the relevance of staphylococcal foodborne poisoning (emetic activity). To be categorized as
SEs, the toxins must demonstrate emetic activity via the oral route in a primate model. Lack of progress
in elucidating the mechanism of the emetic activity of SEs can be partially attributed to the lack of con-
venient and appropriate animal models.50 To date, the emetic activity of 17 SE types has been tested in
monkey and only 12 SE types in house musk shrew models (Table 13.1). This is because the commonly
used animal models, with the exception of monkeys, house musk shrews, and ferrets, are relatively insen-
sitive to SEs, unless the toxin is injected intraperitoneally or intravenously. Emesis is the most readily
observable reaction to SEs and animals without a vomiting mechanism, such as rodents, were of little
value as test subjects.51
The most susceptible animal species to develop human-like enterotoxigenic disease are nonhuman
primate models, mainly cynomolgus and rhesus monkeys (Table 13.2). Assays are performed by admin-
istering solutions of the enterotoxins in 10 mL of sterile distilled water to monkeys (2–3 kg) and fed to
monkeys at a dose of 10 or 100 μg/kg by nasogastric intubation. The animals are observed for 5 h after
Staphylococcus 213

TABLE 13.2
Animal Models Used for the Detection of Staphylococcal Enterotoxins
Animal Symptoms, Pathology SE Tested References
Cynomolgus monkey Emetic activity A, B, C, D, E, K, L, M, N, O, P, 28,52,55,107,108
(Macaca fascicularis) Q, R, S, T
Cytokine and chemokine B 109
response
Rhesus monkey (Macaca Emetic activity A, B, C, D, E, G, H, I 53–55,110,111
mulatta) Cytokine response A, B 112
Pigtail monkey (Macaca Emetic activity C1 80,81
nemestrina)
House musk shrew (Suncus Emetic activity A, B, C, C2, D, E, G, H, I, P, R, 52,59–61
murinus) S, T,
Intestinal loop assay A, C 61
Ferret (Mustela putorius furo) Emetic activity B, C2 66,67
Pig/piglet Emetic activity A, B 68,69
Cytokine response B 70
Superantigen activity B 71
Goat Clinical symptomatology B 72
Dog Acute hemodynamic and Unknown 73
gastrointestinal changes
Cat Emetic activity A, B, C2 74,75
Pyrogenic activity A, B 76
Rabbit Intestinal loop assay A, C 61
Pyrogenic activity A, C2, K, Q, L 75,78–81
Superantigenicity
Lethality K 79
Rat Gastrointestinal changes A, B 84,85
Superantigen activity B 86,87
Mouse Cytokine response A, B, C3, K, Q 89–93,95
Toxic shock B 82

the oral administration of the toxin in parallel with real-time recording using a video camera. The num-
ber of vomiting events, the time until the first vomiting event (latency period), and behavioral changes
are recorded. To minimize the effect of previous intoxication, there was at the minimum a 2-week inter-
val between the intoxication experiments. A response in at least two animals is accepted as a positive
reaction.28,51 Monkeys have been considered to be the primary animal model.
Regarding the sensitivity of this method, the emetic dose for monkeys is somewhat variable for the
different SEs. The least amount of SEA that was observed to produce an emetic reaction when given
intragastrically was 5 μg/kg of weight, with SEB, SEC, and SEE requiring 10 μg/kg and SED requiring
20 μg/kg. The minimum dose required for the other SEs was not determined.51 Studies with the new SEs
reveal that higher concentrations are needed to induced emetic reactions in monkeys, with values rang-
ing from 30 to 150 μg/kg.28,52–54
In addition, it has also been found that some monkeys responded only to some SEs, while other mon-
keys were sensitive to almost all the SEs, suggesting that the emetic responses of monkeys vary on an
individual basis.28,55
Other researchers have shown that monkeys have developed tolerance to the emetic effects of SEs if
repeated doses are given during a short period of time.51,56
Finally, this animal model has been used to demonstrate that the abdominal viscera is the site of
action for the induction of the emetic reaction and that the SEs stimulate the vomiting center of the
medulla oblongata through the vagus and sympathetic nerves.57 The use of monkeys in researching SEs
214 Laboratory Models for Foodborne Infections

is severely restricted by the high cost, availability of these animals, and ethical considerations.50,58 All
these reasons have limited their use for routine testing.
The house musk shrew has been described as a suitable small animal model for research on the emetic
response to various emetic SEs52,59–61 (Table 13.2). The emetic assays are performed with house musk
shrews weighing from 40 to 70 g. The shrews are housed under controlled conditions of illumination
(12/12 h light/dark cycle). Purified SEs are diluted in 0.01 M phosphate-buffered saline (PBS; pH 7.2),
and 200 μL volumes of SEs at an appropriate dilution were administered perorally or intraperitoneally to
the house musk shrews. The animals are observed for emesis for 3 h after the administration of SEs. The
number and times of vomiting, the time to the first vomiting episode, and any behavioral changes were
recorded.62 Vomiting occurred within 14–130 min after administration.
SEA provoked more potent emetic response in vivo. The 50% emetic dose of SEA by peroral adminis-
tration is 10 times higher than by intraperitoneal administration (32 and 3 μg/kg of body weight, respec-
tively).61,62 Studies with other SEs reveal that higher concentrations are needed to induce emetic reactions
in house musk shrews, with values ranging from 10 to 1000 μg/animal.52,59,60 It is noteworthy that differ-
ent types of SEs have different emetic activities in house musk shrews.50
Interestingly, in contrast to the emetic responses, diarrhea was observed in none of the animals tested,
although SEA and SEC induced emetic responses. The results showed that the administration of SEA
provoked a potent emetic response in vivo and showed high superantigenic activity in vitro, but did not
induce diarrheal symptom in the animal model.61
This model has been useful for vaccine development against SEA, using a recombinantly attenuated
SEA devoid of superantigen and emetic activity.63 Furthermore, sera from these vaccinated animals
inhibit SEA-induced proliferation of naïve shrew splenocytes (in vitro) as well as emesis.52
Another animal used as a model system for SEs detection has been the ferret (Table 13.2). Ferrets were
used as the emetic model for finding antagonists to treat emesis induced by anticancer therapy64 and
respond to the full spectrum of agents known to induce emesis in humans.65 An advantage of the ferret
for the investigation of foodborne poisoning is that the morphology and physiology of its gastrointestinal
tract have many features in common with the human gastrointestinal tract.66
For foodborne staphylococcal investigation, the emetic assays are performed with ferrets weighing
700–735 g. Prior to dosing with SEs, animals are deprived of food for 24 h, but are allowed free access
to water. SEs are given to groups of five animals at doses of 1–5 mg into the stomach via an oral dosing
tube.66,67 Changes in the body temperature and the activity and the incidence of retching, vomiting, and
defecation were monitored over a period of 3 h. This ferret model has been used to check oral SEB and
SEC2 intoxications.66,67 However, in the ferret, the dose of SEs required to elicit emesis is higher than
that required to elicit it in primates. Possible explanations for requiring this large dose of SEs include
receptor differences and/or more efficient degradation of SEs in the gut of ferrets versus humans or
monkeys.52
Pig68 and piglet69 models have also been used to reproduce some of the features associated with staph-
ylococcal intoxication in humans. However, it is not a widely used animal model. It has been only applied
for SEA and SEB foodborne investigation (Table 13.2). In this animal model, several breeds (Yorkshire
pig, Hampshire pig), crossbreeds, and pigs of different ages (piglets, weanling pigs—0.9 to 9.1-kg weight
range) have been used.68–71 For the analysis, each pig is placed in a separate cage with commercial pig
feed and water provided ad libitum. Pigs are maintained under controlled lighting (15-h light and 9-h
dark cycle) and temperature (20.5°C ± 0.75°C) conditions. The SE can be administered orally, by duode-
nal catheterization, or intravenously. In these conditions, vomiting occurs 90–180 min after emetic doses
of SEs. The 50% emetic dose of SEA was between 20 and 50 μg. The lowest dose inducing emesis in
weanling pigs was from 10 to 20 μg.68–71 Pigs are somewhat more resistant than monkeys to the emetic
effects of SEs.68 This animal model has been used to determine the efficacy of immunization with vac-
cine against SEB too.70,71
Goats are used for studying in vivo gastrointestinal effects caused by SEB72 (Table 13.2). This entero-
toxin induces colic and watery diarrhea and a more pronounced increase in blood urea nitrogen and tem-
perature in this animal.52,72 For analysis, goats weighing between 21 and 45 kg are kept indoors and fed a
diet of hay and pelleted concentrate. Water is provided ad libitum. Dose levels of SEB are between 0.02
Staphylococcus 215

and 0.5 μg/kg and are administered by nasal catheters into the rumen. This animal does not have emetic
activity, but SEB-induced inhibition of rumen contractions, which is a consequence of internal vomiting.72
Dogs and cats have been used too, but only in some of the staphylococcal toxin investigations
(Table 13.2). The dog model system has been used to study gastrointestinal and hemodynamic changes
caused by intravenous and intraintestinal administration of SEs. They were observed by an autopsy 24 h
after injection to make a histological study of injuries.73
The cat model has been widely used to study emetic74,75 and pyrogenic76 activities. For these analyses,
cats aged about 8–10 weeks old and, with a mean body weight of 500 g, are caged separately in 12-h light/
dark cycle at a temperature of 22°C–26°C with food and water available at all times. Two-milliliter vol-
umes of SEA, SEB, and SEC2 are administered intraperitoneally to the cats. The animals are observed
for emesis and diarrhea for up to 6 h after the intraperitoneal administration. The number and times of
vomiting and diarrhea and the time to the first response episode are recorded for the foodborne staphy-
lococcal investigation.75
The main disadvantage of using those models remain similar to the monkey model—high cost and
short supply in the available tools for the study of SEs-associated immunopathology.77
Other animals like rabbits, rats, and mice also have low susceptibility to SEs; their response to SEs are
not specific or they are not vomit-competent species.50 These animals are used mainly as a model for the
study of superantigenicity, pyrogenicity, capacity to enhance endotoxin shock, and lethality (Table 13.2).
The rabbit model was used to compare in vivo toxicity induced by SEC2 using intravenous injection
to test the pyrogenic activity. Rectal temperatures of rabbits were measured with indwelling rectal ther-
mometers and recorded for 4 h after pyrogen administration.75 Pyrogen assays have also been used in
rabbits that are administered with SEA,78 SEK,79 SEL,80 and SEQ.81
Rabbit intestinal loop assay has been used to test the diarrheagenic activity of SEA and SEC.61 New
Zealand white and Dutch belted are the most frequently utilized rabbit strains for testing SEs.52
Rodents are frequently used as models because of their inbred homogeneity. In addition, large num-
bers of animals with the corresponding results are available in a relatively short time. However, mice are
poor responders to SEs as the affinity of these toxins to mouse major histocompatibility complex class II
(MHC class II) is much lower than that for human MHC class II.82 Moreover, rat- and mouse-based
models are regarded as being substantially less sensitive to SEB intoxication than monkey models.83
Therefore, the use of potentiating agents such as d-galactosamine, actinomycin D, lipopolysaccharide
(LPS), viruses, or even protozoa is required. Thus, lower amounts of these protein toxins elicit a quantifi-
able form of toxic shock useful for therapeutic and vaccine developments.52
Rat has been reported in a model of intestinal inflammation postweaning, based on the systemic
administration of SEB84 and SEA85 (Table 13.2) and for evaluating the SEB superantigenic activity by
intravenous administration.86,87
Mice are useful for basic toxin studies and discovery of therapeutics/vaccines for combating staphy-
lococcal superantigen-induced shock. However, mice lack an emetic response and are thus not very
appropriate for investigation of SEs foodborne poisoning.82
Transgenic mice with human MHC class II were found to be an ideal animal model for examining the
biological effects of superantigens, as they responded to much lower doses of toxins due to the higher
affinity binding of SEs to human MHC class II molecules.82,88,89 For this purpose, several mouse strains
(BALB/c, NMRI, C57BL/6, C3H/HeJ, C3H/OuJ, HLA-DR3, knockout mouse, etc.) weighing 18–20 g
are used.52
Major studies using mice as animal model have focused on comparing the responses of T-cells in the
gut-associated lymphoid tissue to different SEs administered by oral, intragastric, or intraperitoneal routes.
SEA, SEB, SEC3 and SEK, and SEQ have been assessed by using this animal model89–95 (Table 13.2).

13.4.2 Cell Culture Models

Cells derived from animal tissues are attractive models for studying pathogenesis. There are two types of
cells: primary and secondary. Primary cells are mortal, consist of mixed cell types, and are short-lived.
Secondary cells are immortal and consist of one type of cell.
216 Laboratory Models for Foodborne Infections

There are several advantages of cell culture models over animal models: (1) it is a simple and con-
trolled model to study host–SEs interaction, (2) it is easy to run experiments, (3) cells are able to
multiply rapidly—thus the experiment can be conducted quickly, (4) the secondary cells are immortal
if nutrients and proper culturing conditions are provided, (5) it is relatively inexpensive compared
to animal model, specially secondary cells, and (6) it is ethically more acceptable than assays using
animals.49
However, there are several limitations: (1) cultured mammalian cells are generally derived from tumor
cells, and therefore, genetic aberrations have occurred in these cells, may lose traits of original tissue and
lose tissue-specific receptor, (2) cultured cells consist of only one type of cell; therefore, interaction with
concerted host cell cannot be studied with cell culture models, and (3) lack of mucus and other secretory
components, which normally interact. Thus, animal models are often used to confirm or verify the find-
ings from the in vitro cell culture models.49
The gastrointestinal injuries associated with SEs foodborne poisoning have been extensively studied
for a number of years using various animal models (Section 13.4.1), but in particular, insight into how
SEs breach the epithelial barrier is scarce and has been mainly studied in epithelial cell lines.33 The bind-
ing of SEA and SEB toxins onto the surface of the enterocyte microvillus, mediated by binding to diga-
lactosylceramide residues, has been demonstrated. This research further confirmed that the toxin entry
into the enterocytes via apical endocytosis within the endosomes and the pathological events following
SEs introduction into the intestinal area are due to the combined effect of SEs that disrupt the epithelial
barrier by inducing enterocyte-cytopathic toxins produced by S. aureus.96,97
SEs act as superantigens that target the immune system, inducing massive T-cell activation, cyto-
kine release, and systemic shock.98 Examination of these SEs for the ability of causing mammalian cell
damage provides a means to assay these toxins. Recognition of the superantigen–MHC II complex by
T-cell receptor (TCR) results in cell signaling, proliferation, and subsequent release of cytokines/chemo-
kines.33,52 Immune cell activation by superantigens and subsequent cellular changes are similar to those
of conventional antigens and require three important signals: (1) from superantigen interaction with
TCR and activation of protein tyrosine kinases; (2) engagement of costimulatory molecules on antigen-­
presenting cells and T-cells, upon superantigen binding that optimizes T-cell activation; and (3) inter-
leukin (IL)-1, tumor necrosis factor α (TNFα), interferon gamma (IFNγ), IL-2, IL-6, and chemokines,
specifically monocyte chemoattractant protein-1 (MCP-1), which are induced directly by superantigens
and represent the third signal for T-cell activation.52 Clearly, SEs-based activation of cells involves a
multifactorial event encompassing multiple host molecules.
Several types of cells are available for superantigenic activity bioassays induced by SEs. Thus, human
peripheral blood mononuclear cells (PBMCs) and human B lymphoblastoid cells have been used for SEB,
SEC2, SEP, SER, SES, SET, and SElX-induced immune response analysis.31,52,60,67,83,99–101 Superantigenic
effect was explored by treating T-lymphocytes isolated from thymus of rats or mice using various doses
of SEA, SEG, SEI, SEK, SEM, SEN, SEO, and SEQ.30,95,102,103 Splenocyte assays have been used for
measuring the superantigenic activity of SEA, SEH, and SEL.32,80,104,105
Moreover, the interaction between SEs and MHC II molecules was studied using secondary cell mod-
els as the human B cell lymphoma Raji and human colorectal cancer cells.67,106

13.5 Conclusions
SEs, produced by the Staphylococcus genus, mainly S. aureus, belong to the family of superantigens
that induce potent emesis and are involved in foodborne poisoning. Staphylococcal foodborne poisoning
occurs as a consequence of consumption of food containing enough amounts of preformed enterotoxins.
Vomiting is the most frequently observed symptom.
Enterotoxin-producing staphylococci have been reported as producers of 23 types of SEs. They are
characterized by superantigenic activity and by induction of emesis. Emetic activity is the main factor that
has to be met so as to be considered in the “category of SEs.” The diagnosis of staphylococcal foodborne
poisoning could be confirmed by detecting bacterial toxins in foods by immunological tools or mass
spectrometry-based methods. However, additional laboratory models are needed to evaluate the emetic
Staphylococcus 217

activity (just animal models) as superantigenic activity (both animal and cell models). Lack of progress
in elucidating the mechanism of the emetic activity of SEs can be attributed to the lack of convenient and
appropriate animal models. Thus, the commonly used animal models are relatively insensitive to the SEs.
The most susceptible animal species are monkeys, followed by house musk shrews and ferrets.
Moreover, pigs, piglets, goats, dogs, and cats have been used. The main disadvantage of using these later
models remains similar to the monkey models—high cost and short supply in the available tools for the
study of SE-associated immunopathology.
Others animals such as rabbits, rats, and mice are less susceptible to SEs or their response to SEs are not
specific or they are not vomit-competent species. These animals are mainly used as a model for the study of
superantigenicity, pyrogenicity, capacity to enhance endotoxin shock, and lethality. The house musk shrew
and the ferret appear to be valuable animal models for studying the emetic activity of SEs. Comparative
studies using monkeys, house musk shrews, and ferrets to assess the emetic activity of SEs will lead to an
understanding of the molecular basis of the emesis caused by SEs.59
Cells derived from animal tissue are attractive models for studying superantigenic activity. There are
several advantages of cell culture over animal model: simple host–SEs interaction, easy, rapid, relatively
inexpensive compared to animal models, and ethically more acceptable to assays using animals. But this
is not an appropriate model for testing the emetic activity of SEs.
Finally, animal models could be useful for additional identification of SEs or the identification of
staphylococcal strains that produce an unidentified enterotoxin. For this reason, when other methods
become available for all SEs involved in staphylococcal foodborne poisoning, experimental models will
not be employed for routine analysis, but only in special cases to confirm outbreak due to SEs.

Acknowledgments
This work has been funded by the Spanish Instituto Nacional de Investigación Agraria y Agroalimentaria
(INIA) and the Spanish Comisión Interministerial de Ciencia y Tecnología with the projects RTA-2013-
00070-C03-03 and Carnisenusa CSD2007-00016, Consolider Ingenio 2010, and GRU09162 of the Junta
de Extremadura and FEDER.

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14
Streptococcus

Dongyou Liu

CONTENTS
14.1 Introduction................................................................................................................................... 223
14.1.1 Classification and Morphology......................................................................................... 224
14.1.1.1 Classification..................................................................................................... 224
14.1.1.2 Morphology....................................................................................................... 226
14.1.1.3 Genomics.......................................................................................................... 226
14.1.2 Biology and Epidemiology............................................................................................... 226
14.1.3 Clinical Features and Pathogenesis.................................................................................. 227
14.1.3.1 Pharyngitis........................................................................................................ 227
14.1.3.2 Scarlet Fever...................................................................................................... 228
14.1.3.3 Impetigo............................................................................................................ 228
14.1.3.4 Acute Rheumatic Fever (ARF)......................................................................... 228
14.1.3.5 Acute Poststreptococcal Glomerulonephritis (APSGN).................................. 228
14.1.3.6 Pediatric Autoimmune Neuropsychiatric Disorders Associated with
Streptococcal Infections (PANDAS)��������������������������������������������������������������� 228
14.1.4 Diagnosis.......................................................................................................................... 229
14.1.5 Treatment and Prevention................................................................................................. 229
14.2 Laboratory Models........................................................................................................................ 230
14.2.1 Animal Models................................................................................................................. 230
14.2.1.1 Rodents............................................................................................................. 230
14.2.1.2 Chinchilla.......................................................................................................... 232
14.2.1.3 Nonhuman Primates......................................................................................... 232
14.2.1.4 Insects............................................................................................................... 232
14.2.1.5 Caenorhabditis elegans Nematode.................................................................. 232
14.2.1.6 Zebrafish........................................................................................................... 232
14.2.2 In Vitro Models................................................................................................................. 232
14.3 C onclusion..................................................................................................................................... 233
References............................................................................................................................................... 233

14.1 Introduction
The genus Streptococcus comprises a large group of Gram-positive bacteria that have been known to
cause human diseases from ancient times. A quick review of historical records indicates that in 1553, a
rash (then termed “rossalia,” with the whole body covered by numerous spots of large and small, fiery,
and red) that differs from measles was first described by Giovanni Filippo Ingrassias of Italy; in 1565, a
sore throat epidemic was mentioned by Johann Weyer of the Netherlands; in 1578, scarlet fever (show-
ing general weariness, headache, redness of the eyes, sore throat, and fever) was noted by Jean Cottyar;
in 1628, an epidemic showing scarlatinal desquamation, arthritis, and postscarlatinal dropsy and asci-
tes was documented by Daniel Sennert; in 1675, the term “scarlatina” was first used by Sydenham to

223
224 Laboratory Models for Foodborne Infections

describe a disease that is distinct from other exanthemas (e.g., measles); in the 1840s, childbed fever
was linked by Ignac Semmelweis of Hungary to medical personnel failing to wash their hands and who
then transmitted the disease to patients; in 1874, an organism isolated from patients with erysipelas and
wound infections was named “streptococcus” (Greek streptos, a chain; coccos, a berry) by Theodor
Billroth of Austria due to its formation of short chains; in 1879, streptococcus was confirmed by Louis
Pasteur as the etiological agent of puerperal fever, which caused the highest mortality rates of women
and newborns at that time; in 1884, the bacterium isolated from suppurative lesions was defined by
Friedrich Julius Rosenbach as Streptococcus pyogenes (Greek pyo, pus, and genes, forming); shortly
afterward, the previously proposed species names of pyogenes, eryespaltis, scarlatinae, and puerperalis
were united under the single name Streptococcus pyogenes by Andrews and Christie; in 1909, serotype-
specific immunity against streptococci was reported by Meakins; in the 1920s, hemolytic streptococci
that produced a secreted toxin (known as scarlet fever toxin or Dick toxin) were identified by George and
Gladys Dick as the causative agent of sore throat that is accompanied by scarlet fever [1].
To date, more than 50 species have been recognized in the genus Streptococcus, of which nine
(S.  pyogenes, S. agalactiae, S. equisimilis, S. bovis, S. anginosus, S. sanguinis, S. mitis, S. mutans,
and S. pneumoniae) have been implicated in human infections. In particular, S. pyogenes (commonly
referred to as group A Streptococcus or GAS) is responsible for a majority of human streptococcal dis-
eases, with clinical manifestations ranging from pharyngitis, impetigo, cellulitis, scarlet fever, puerperal
sepsis, bacteremia, pneumonia, streptococcal toxic shock syndrome (STSS), necrotizing fasciitis, acute
rheumatic fever (ARF), rheumatic heart disease (RHD), to acute poststreptococcal glomerulonephritis
(APSGN). All together, over half a million deaths per year worldwide are attributable to S. pyogenes [2].
This chapter will focus on S. pyogenes, beginning with a brief overview on its classification, morphology,
genomics, biology, epidemiology, clinical features, pathogenesis, diagnosis, treatment, and prevention,
followed by discussion on laboratory models applied to S. pyogenes research.

14.1.1 Classification and Morphology

14.1.1.1 Classification
Classified in the family Streptococcaceae, order Lactobacillales, class Bacilli, phylum Firmicutes, domain
Bacteria, the genus Streptococcus consists of more than 50 recognized species of facultative anaero-
bic Gram-positive cocci (GPC), namely, S. agalactiae, S. anginosus, S. bovis, S. canis, S. constellatus,
S. downei, S. dysgalactiae, S. equinus, S. ferus, S. infantarius, S. iniae, S. intermedius, S. milleri, S. mitis,
S. mutans, S. oralis, S. orisratti, S. parasanguinis, S. peroris, S. pneumoniae, S. pseudopneumoniae, S. pyo-
genes, S.  ratti, S. salivarius, S. tigurinus, S. thermophilus, S. sanguinis, S. sobrinus, S.  suis, S.  uberis,
S. vestibularis, S. viridans, and S. zooepidemicus [3].
Based on the hemolytic properties detected on blood agar plates, members of the genus Streptococcus
are distinguished into three categories: α-, β-, and γ-hemolysis. α-Hemolytic species produce hydrogen
peroxide that oxidizes hemoglobin within red blood cells to become green methemoglobin, leading to a
greenish and dark zone surrounding and underneath the colonies with short chains (as in the case of S.
pneumoniae and S. viridans or viridans streptococci; viridans, from Latin vĭrĭdis, green). β-Hemolytic
species produce streptolysins (e.g., streptolysin O and streptolysin S) that cause complete lysis of red
blood cells in blood agar plate, giving a clear (lightened/yellow and transparent) zone around and under-
neath the colonies with short chains (as in the case of S. haemolyticus). More specifically, streptolysin O
(SLO, an oxygen-sensitive cytotoxin secreted by most GAS) interacts with cholesterol in the membrane
of eukaryotic cells (red and white blood cells, macrophages, and platelets), causing β-hemolysis under the
surface of blood agar. Streptolysin S (SLS, an oxygen-stable cytotoxin also secreted by most GAS strains)
induces clearing on the surface of blood agar. It is notable that some weakly β-hemolytic species (e.g.,
S. agalactiae, Clostridium perfringens, and Listeria monocytogenes) cause intense β-hemolysis when
grown together with a strain of Staphylococcus (so called the CAMP test). γ-Hemolytic species cause no
hemolysis (no change) on blood agar (as in the case of enterococci) [3].
Streptococcus isolates may also be subgrouped by serological typing schemes targeting M protein,
T-antigens (from pili), and serum opacity factor (SOF), which are LPXTG-linked (or similar) surface
Streptococcus 225

proteins with high levels of antigenic heterogeneity and are direct determinants of host tissue site prefer-
ences of infection [4].
Developed by Lancefield in 1933, serological typing scheme using group-specific antisera to ­M pro-
tein (a surface protein that is responsible for colony’s matte appearance) differentiates Streptococcus
β-hemolytic species into Lancefield groups A to X (excluding I and J), with strains from human diseases
classified as group A, those from bovine and dairy sources as group B, those from other animal sources
as group C, and so on. GAS consists of S. pyogenes, which causes both noninvasive and invasive infec-
tions in humans. The noninvasive infections are more common but less severe, as exemplified by strepto-
coccal pharyngitis (strep throat), impetigo, and scarlet fever. The invasive infections are more severe but
less common, as exemplified by STSS, necrotizing fasciitis, pneumonia, and bacteremia. Complications
of GAS infections include ARF and acute glomerulonephritis. Affecting the joints, kidneys, and heart
valves, rheumatic fever results from damages caused by the antibodies that are generated by the host
immune system against the untreated GAS infection. These antibodies cross-react with other proteins
in the body, leading to self-inflicted attack and damage. Indeed, the group A S. pyogenes strains can be
further subdivided into >200 M-types using a combination of serological and molecular typing methods.
Group B Streptococcus (GBS) consists of S. agalactiae, which causes pneumonia, meningitis, and occa-
sional systemic bacteremia in neonates and the elderly. As it also colonizes the intestines and the female
reproductive tract, this bacterium may contribute to premature rupture of membranes during pregnancy
and subsequent transmission to the infant. Group C Streptococci include S. equi (causing strangles in
horses), S. zooepidemicus (infecting cattle and horses, with S. equi being a clonal descendent or biovar
of the ancestral S. zooepidemicus), and S. dysgalactiae (causing pharyngitis and other pyogenic infec-
tions similar to GAS). Group D streptococci consist of enterococcal and nonenterococcal strains. The
former have been reclassified and placed in the genus Enterococcus (including E. faecalis, E. faecium,
E. durans, and E. avium, with E. faecalis being sometimes α-hemolytic and E. faecium being sometimes
β-hemolytic) (see Chapter 10); the latter include S. bovis and S. equinus. Group F streptococci (so-called
“minute hemolytic streptococci”) are represented by S. anginosus or the S. milleri group. Group G strep-
tococci are usually, but not exclusively, β-hemolytic and represented by S. canis that typically occurs in
animals, but may cause infection in humans. Group H streptococci are found in canines and rarely cause
illness unless humans have direct contact with a canine (mouth-to-mouth or canine licking a human
hand) [3].
The serotyping scheme based on T-antigens (trypsin-resistant surface antigens, which are contained
within extended surface pili composed of covalently linked polymers of two or three distinct gene prod-
ucts) divides S. pyogenes strains into ∼20 T-serotypes. However, many S. pyogenes strains have multiple
T-types (e.g., T3/13/B, T8/25/Imp19) [4].
Another classification scheme proposed by Sherman in 1937 incorporating Lancefield grouping and
other criteria divides streptococci into four groups: pyogenic, viridans, lactic, and enterococci. The pyo-
genic division comprises β-hemolytic strains of Lancefield groups A, B, C, E, F, and G. The viridans
division consists of non-β-hemolytic streptococci that are not tolerant to high-pH growth conditions and
salt and do not grow at 10°C. The lactic division includes strains of dairy origin, which are nonhuman
pathogenic. This group differs from the pyogenic group by being non-β-hemolytic, growing at 10°C
but not at 45°C, and failure to grow in broth with 6.5% NaCl. The lactic division was reclassified as
the Lactococcus genus in the mid-1980s. The enterococci division includes Lancefield group D strains,
which are now known as the genus Enterococcus. Members of this genus have the capacity to grow in
broths at high pH (9.6), high salt concentrations (6.5% NaCl), and a wide temperature range (10°C–45°C),
with some enterococci demonstrating β-hemolytic property (see Chapter 10) [3].
Examination of Streptococcus 16S rRNA sequences also permits discrimination of streptococci into
six groups: S. anginosus, S. bovis, S. mitis (including S. pneumoniae), S. mutans, S. pyogenes, and
S. salivarius.
Further, sequencing analysis of the emm gene that encodes the mature M protein molecule provides
additional confirmation on the validity of the traditional M serological typing scheme. Specifically, the
emm sequence-based typing divides S. pyogenes strains into five pattern groups (A–E), with emm pattern
A–C groups accounting for 47% of pharyngitis isolates, but only 8% of impetigo isolates; emm pattern D
group accounting for 50% of impetigo isolates, but only <2% of pharyngitis isolates; and emm pattern E
226 Laboratory Models for Foodborne Infections

group accounting for almost equal fractions of throat and skin infections (52% and 42%, respectively).
Not surprisingly, emm pattern A–C groups are referred to as “throat specialists,” emm pattern D group
as “skin specialists,” and emm pattern E group as “generalists” [4].
Moreover, genetic analysis of pilus genes (particularly the FCT region, with FCT standing for
Fibronectin- and Collagen-binding proteins and T-antigen) uncovers the molecular basis of the serotyp-
ing scheme targeting T-antigens. However, not all S. pyogenes strains express pili, and those with a nega-
tive regulator may be T-nontypable [4].
Multilocus sequence typing (MLST) targeting seven core housekeeping genes (glucose kinase, gki;
glutamine transporter protein, gtr; glutamate racemase, murI; DNA mismatch repair protein, mutS;
transketolase, recP; xanthine phosphoribosyl transferase, xpt; and acetyl coenzyme A acetyltransfer-
ase, yqiL) offers another valuable approach for determination of S. pyogenes strains, with clones of
S.  pyogenes being defined by their sequence type (ST) (http://pubmlst.org/spyogenes/). Interestingly,
while most emm pattern A–C strains correspond to same clone (ST) or clonal complex, emm patterns D
and E strains correlate with distant ST [4].

14.1.1.2 Morphology
Streptococci are Gram-positive, facultative anaerobic cocci that often grow in chains or in pairs (due to
the fact that cell division occurs along a single axis). Streptococcus colonies on agar plates are small,
smooth, and moist in appearance. Streptococcal cell wall is largely composed of group-specific carbohy-
drate (the M protein), with Lancefield group A being a polymer of rhamnose and N-acetylglucosamine.
Apart from the M protein, other proteins present on the cell surface include T-protein, SOF, C5a pepti-
dase, collagen-like protein Scl1, GRAB, and protein F. In GAS, the pili (fimbriae) appear as long, flexible
rods protruding up to 3 μm from the cell surface. Structurally, GAS pili are heteropolymers forming a
pilus shaft, which is composed of the major pilin protein subunit (Spy0128) and assembled through a
series of transpeptidase reactions catalyzed by a class B accessory sortase, SrtC (Spy0129) [3].

14.1.1.3 Genomics
The genomes of Streptococcus species are of 1.8–2.3 Mb in size with GC content of about 38.5%,
1700–2300 protein-coding sequences (CDSs), 5–6 rRNA, and 57–67 tRNA encoding genes. Specifically,
S. pyogenes, S. agalactiae, S. pneumoniae, and S. mutans possess genomes of 1,852,442, 2,211,488,
2,160,837, and 2,030,921 bp, respectively. The genome of GAS contains genes that encode proteins
secreted into the extracellular fluid during growth (e.g., erythrogenic toxin, streptolysin O, streptoly-
sin S, proteinase, streptokinase, DNase, RNase, hyaluronidase, CAMP factor, streptococcal inhibitor
of complement, immunogenic secreted protein, and superantigens). These proteins are mostly virulence
factors with important roles in colonization, invasion, spreading, and pathogenesis. The genome of
pathogenic GAS strains also harbors a pathogenicity island located in the FCT region, which encodes
fibronectin-binding proteins, collagen-binding proteins, and T-antigens (pilus subunit genes). Among
GAS isolates, the FCT region displays considerable genetic diversity, with nine different FCT variants
identified [4]. Additionally, the S. pyogenes genome consists of some metabolic pathways (e.g., complete
glycolytic pathway, fatty acid synthesis, nucleotide synthesis and transport, and carbohydrate transport
and metabolism), but lacks a complete tricarboxylic acid cycle pathway. Many Streptococcus species
contain bacteriophages, with 18 prophages (ranging from 38 to 41 kb in size, encoding from 42 to 66
genes each) being described in S. pneumoniae.

14.1.2 Biology and Epidemiology

Apart from nine Streptococcus species (S. pyogenes, S. agalactiae, S. equisimilis, S. bovis, S. anginosus,
S. sanguinis, S. mitis, S. mutans, and S. pneumoniae) that are implicated in human infections, most
other streptococcal species are nonpathogenic and form part of the commensal human microbiota of the
mouth, skin, intestine, and upper respiratory tract.
Streptococcus 227

S. pyogenes (GAS) is not considered part of normal flora in humans, despite its presence in 5%–20%
of healthy/asymptomatic individuals. As an exclusively human pathogen, GAS enters into human host
via oral cavity, skin, and wounds, and typically attaches to the epithelial surfaces of the throat and
skin, as well as the vagina and rectum, on which it forms large microcolony aggregates. Additionally,
GAS may organize as biofilms and hide inside nonphagocytic cells. These enhance its survival under
unfavorable or stressful conditions including antimicrobial therapy, and contribute to its spread to other
individuals [5,6].
GAS is commonly transmitted through two routes: foodborne or airborne. Foodborne GAS epidemics
often result from consumption of inappropriately prepared/cooked raw milk, cold salads, eggs, mayon-
naise, tuna, potatoes, cheese, conch, and other ingredients that are inadvertently contaminated by GAS
from infected animals (e.g., cows with streptococcal mastitis) or food handlers (who have sore throat, or
have infected skin lesions on hands/arms, or are asymptomatic carriers) [7–9]. Airborne GAS epidem-
ics are due to inhalation of respiratory/saliva droplets from carriers who disseminate the bacterium via
sneeze or cough. Another means for GAS to spread is through person-to-person skin contact. External
factors favoring GAS disease outbreaks include crowded settings (e.g., military training centers), mass
consumption of contaminated foods, and hospital acquisition (e.g., puerperal sepsis). Intrinsic factors
include the emergence of dominant clones [e.g., M1T1 clone that acquires three regions of heterologous
DNA: a 36-kb chromosomal region encoding the toxins SLO and NAD-glycohydrolase and two bacte-
riophages encoding the DNase Sda1 and the superantigen SpeA (streptococcal pyrogenic exotoxin A);
M3 clone that acquires prophages encoding phospholipase A2 and SpeA and the duplication of four
amino acids in the N-terminal region of the M3 protein].
Interestingly, S. pyogenes pharyngitis tends to predominate in temperate regions, with seasonal
peak in winter, whereas S. pyogenes impetigo is mostly present in tropical and subtropical regions,
with seasonal peak in summer. In addition, there are notable links of certain emm types to particular
clinical diseases, including the association of emm types 1, 3, 5, 6, 12, 14, 17, 19, and 24 with pharyn-
gitis; emm types 33, 41, 42, 52, 53, and 70 with impetigo; emm types 1, 3, 5, 6, 11, 12, 14, 17, 18, 19,
24, 27, 29, 30, 32, and 41 with ARF; emm types 1, 4, 12, 49, 55, 57, and 60 with APSGN; emm type 28
with puerperal sepsis; emm types 1, 3, and 28 with necrotizing fasciitis; and emm types 1 and 3 with
STSS [2].

14.1.3 Clinical Features and Pathogenesis

Human pathogenic species within the genus Streptococcus include S. pyogenes (GAS, pharyngitis, scar-
let fever, impetigo, pyoderma), S. agalactiae (GBS, neonatal meningitis, sepsis), S. equisimilis (endo-
carditis, bacteremia, pneumonia, meningitis, respiratory infections), S. bovis (biliary or urinary tract
infections, endocarditis), S. anginosus (subcutaneous/organ abscesses, meningitis, respiratory infec-
tions), S. sanguinis (endocarditis, dental caries), S. mitis (endocarditis), S. mutans (dental caries), and
S. pneumoniae (pneumonia) [10–13].
In individuals with competent immune functions, S. pyogenes (GAS) often induces mild and self-
healing purulent symptoms in mucosal membranes and skin (e.g., pharyngitis, impetigo) that may dis-
appear in 7–14 days. In patients with predisposed conditions [e.g., immune-suppression, diabetes, or
specific MHC class II cell surface receptor (HLA-DR) subtypes], GAS may breach the epithelial barrier,
and cause invasive and life-threatening diseases [e.g., cellulitis, puerperal sepsis (childbed fever), necro-
tizing fasciitis, STSS, RHD, septic arthritis, pneumonia, meningitis, abscess, osteomyelitis, endocardi-
tis, peritonitis], with 8%–23% of patients succumbing within 7 days of infection [2].

14.1.3.1 Pharyngitis
Commonly known as “strep sore throat,” GAS pharyngitis presents with sudden-onset fever accompa-
nied by sore throat, and obvious inflammation in the pharynx and tonsils. Other symptoms may include
general malaise, headache, nausea, abdominal pain, vomiting, patchy exudates, and cervical lymph node
adenopathy. Patients with uncomplicated GAS pharyngitis usually recover within 7–14 days [2].
228 Laboratory Models for Foodborne Infections

14.1.3.2 Scarlet Fever
When pharyngitis involves a GAS strain that generates bacteriophage-encoded streptococcal pyogenic
exotoxins (notably SpeA), scarlet fever (also known as scarlatina) may emerge, with a deep red, finely
papular, erythematous rash (strawberry tongue) and exudative pharyngitis. Scarlet fever tends to occur in
children of 4–8 years but rarely in adults. Pharyngitis and soft tissue infection at a surgical site (surgical
scarlet fever) are most commonly associated with scarlet fever [2].

14.1.3.3 Impetigo
As a contagious GAS infection of the skin, impetigo (or pyoderma, which is a term for a localized
purulent infection of the skin and is used synonymously with streptococcal impetigo and impetigo con-
tagiosa) shows pustules that enlarge and rupture to form thick, honey-colored scabs. Spread through
direct skin contact, impetigo mainly affects children with poor hygiene and crowded living conditions
in tropical and subtropical climate. When GAS moves to deeper layers of the skin, erysipelas and cel-
lulitis may appear. Further spreading of GAS to the fascia may lead to necrotizing fasciitis (flesh-eating
disease), which is severely invasive, potentially fatal disease in the absence of immediate surgical and
medical intervention. Another severe invasive disease due to GAS is STSS, which often develops within
24–72 h of minor nonpenetrating trauma resulting in hematoma, deep bruise to the calf, or following
muscle strain, in addition to suction lipectomy, hysterectomy, vaginal delivery, bunionectomy, and bone
pinning [2].

14.1.3.4 Acute Rheumatic Fever (ARF)

Being a sequela of untreated GAS pharyngeal infection, ARF is a systemic disease that shows inflam-
mation of the joints (arthritis) and heart (carditis, also known as rheumatic heart disease or RHD), along
with neurologic manifestation (i.e., Sydenham chorea). Other manifestations may include erythema mar-
ginatum and subcutaneous nodules. ARF/RHD largely results from inflammatory changes occurring in
cardiac tissue, joints, brain, blood vessels, and skin, with hemodynamic changes in cardiac tissue leading
to decompensatory cardiac failure. Once closely linked to childbed fever, nonpasteurized milk, surgical
wards, schools, and day care centers, the incidence of ARF has decreased dramatically with the routine
use of penicillin in the treatment of GAS nowadays [14].

14.1.3.5  Acute Poststreptococcal Glomerulonephritis (APSGN)

Another sequela of GAS infection is APSGN, which is an immune-complex-mediated disorder of
the kidneys, with symptoms ranging from edema, hypertension, urinary sediment abnormalities, and
reduced serum complement components. APSGN is particularly prevalent in children in less developed
countries, but rarely causes long-term renal damage when proper supportive care is provided [14].

14.1.3.6 Pediatric Autoimmune Neuropsychiatric Disorders Associated with

Streptococcal Infections (PANDAS)
PANDAS is a rare child disease tentatively linked to GAS infection, in the form of obsessive–compulsive
disorders and Tourette’s syndrome, due possibly to the effects of GAS-induced antineuronal antibodies.
PANDAS symptoms appear to be similar to neuropsychiatric symptoms of Sydenham’s chorea, which is
a disease associated with a prior group A streptococcal infection [14].
GAS expresses a number of surface proteins and virulence factors that facilitate its attachment and
entry into human host cells, and escape from human immune surveillance. For example, GAS produces
M protein, Cpa, Eno, Epf, and firbronectin-binding proteins for extracellular matrix and serum protein
binding via microbial surface components recognizing adhesive matrix molecules (MSCRAMMS);
secretes EndoS, Mac, C5a peptidase, capsule protein, and Sic for degradation/inhibition of host immu-
noglobulin and complement factor; utilizes plasminogen/plasmin binding and streptokinase Ska
Streptococcus 229

activity for dysregulation of coagulation; generates SLS, SLO, streptococcal pyrogenic exotoxins A
and C (SpeA and SpeC, which are superantigens responsible for scarlet fever and STSS), and Nga for
cytotoxic and cytolytic activity toward various host cell types, including neutrophils, platelets, and
subcellular organelles [15,16].

14.1.4 Diagnosis
Isolation of GAS from throat, food specimens, or swabs from kitchen utensils and surfaces represents a
useful initial step for the diagnosis of streptococcal pharyngitis and hemolytic streptococci.
GAS may be grown in a highly nutritious medium consisting of meat extract, peptones, dextrose, and
salts (e.g., 5% horse-blood agar), with the formation of colonies showing β-hemolysis. The medium may
be modified to enhance the production of some GAS proteins. For instance, addition of a digested RNA
fraction increases streptolysin S production; supplement with reducing agents (e.g., glutathione) favors
streptolysin O production; a slightly acidic pH strengthens the production of the cysteine proteinase pre-
cursor; and addition of hyaluronate improves the production of hyaluronidase [3].
The resulting GAS isolates are then typed by using serotype-specific antisera raised against the M pro-
tein (an immunodominant surface antigen and key virulence determinant), T-antigens (trypsin-resistant
surface antigens, which are contained within extended surface pili), or SOF (an LPXTG-anchored,
multifunctional surface protein that binds fibronectin and enzymatically disrupts the structure of high-
density lipoproteins present in blood). Further analysis with pulsed-field gel electrophoresis (PFGE)
using SmaI is also valuable.
More recently, typing of GAS strains is conducted according to the sequence of the 5′ variable region
of the emm gene encoding the M protein, leading to the identification of >200 emm types. Of these, emm
pattern A–C strains are considered as “throat specialists” (causing pharyngitis), emm pattern D strains
as “skin specialists” (causing impetigo), and emm pattern E strains as “generalists” (causing both phar-
yngitis and impetigo) [4].
Use of MLST technique allows definition of S. pyogenes clones by their ST (http://pubmlst.org/
spyogenes/). Interestingly, most emm pattern A–C strains closely correspond to a single clone (ST) or
clonal complex [4].

14.1.5 Treatment and Prevention

To avoid the development of secondary autoimmune sequelae (e.g., RHD or glomerulonephritis), it is
necessary to implement antibiotic therapy for GAS infections. Despite continued use over the past few
decades, GAS remains extremely sensitive to penicillin-based antibiotics. However, GAS resistance to
macrolides, clindamycin, and lincosamide has been observed in some parts of the world. The recom-
mended treatment regimen for GAS infections consists of a 10-day course of oral penicillin or intra-
muscular benzathine penicillin. For penicillin-allergic individuals, erythromycin may be administered.
For the prevention of foodborne streptococcal pharyngitis, individuals with acute pharyngitis (with
sneeze or cough) or skin lesions should be excluded from food handling, use of bare hands in food
handling should be banned, food (especially prepared in large quantities) must be properly cooked and
stored, and unpasteurized milk or milk products should not be used.
Owing to the fact that GAS strains demonstrate considerable serotype diversity and antigenic vari-
ability, a safe and effective vaccine for human streptococcal infections is still unavailable. To date, GAS
proteins and molecules that have been assessed as vaccine candidates consist of cell-wall-anchored
proteins, membrane-associated proteins, secreted proteins, anchorless proteins, and the group A car-
bohydrate molecule. A number of protein antigens have shown promise in vaccine trials involving
various animal models. These include fibronectin-binding protein A (FbaA), protein F1/SfbI, serum
opacity factor (SOF/SfbII), fibronectin-binding protein 54 (FBP54), R28 protein, streptococcal protec-
tive antigen (Spa), C5a peptidase (ScpA), streptococcal hemoprotein receptor (Shr), streptococcal pyro-
genic exotoxin B (SpeB), streptococcal secreted esterase (Sse), streptococcal immunoglobulin protein
35 (Sib35), Streptococcus pyogenes cell envelope protease (SpyCEP), arginine deiminase (ADI), and
trigger factor (TF).
230 Laboratory Models for Foodborne Infections

14.2 Laboratory Models
As a strictly human pathogen, S. pyogenes is implicated in a range of clinical diseases, including
(1) local, lesional diseases (inflammation) in soft tissue, (2) both local and systemic diseases associated
with streptococcal toxins, and (3) immune dysfunction due to streptococcal antigens. To complicate the
situation further, S. pyogenes encompasses a diversity of strains with varied virulence potential. In spite
of our long-standing efforts to uncover the secrets about this ever-present pathogen, our understanding
of GAS and its pathogenesis and immune evasion is far from being adequate. Use of laboratory models
is vital in helping address this conundrum. Indeed, over the past two decades, a number of in vivo and
in vitro models based on both laboratory animals and cell lines have been exploited, with the ultimate
goal of elucidating the molecular basis of S. pyogenes pathogenesis and other perplexing issues [17–20].

14.2.1 Animal Models
14.2.1.1 Rodents
Being small, relatively inexpensive to maintain, easy to handle, along with rapid reproduction and short
life span, rodents (e.g., mice and rats) represent ideal animal models for disease investigations includ-
ing GAS infections. It is no surprise that a diverse range of model systems have been established using
rodents.

14.2.1.1.1 Murine Subcutaneous Ulcer Model

Murine subcutaneous ulcer model involves injection of 106 –108 CFU of S. pyogenes strain into the sub-
cutaneous tissue of a mouse flank (inbred BALB/c C57BL6 or outbred hairless SKH1), leading to a
localized inflammatory lesion (characterized by the recruitment of neutrophils and macrophages and the
production of cytokines IL-12, interferon-γ, and TNF-α) in soft tissue within 8–12 h, with development
of ulceration and eschar by 18–24 h and resolution of lesion by 14 days postinfection. This model offers
a useful platform to determine the virulence potential of individual S. pyogenes strains according to the
variations in lesion size and resolution time, weight change, and numbers of colony forming units (CFU)
from recovered bacteria [20].

14.2.1.1.2 Murine Subcutaneous Air Sac Model

Being a derivative of murine subcutaneous ulcer model, murine subcutaneous air sac model involves
injection of air under the skin (to create an air pouch) followed by injection of S. pyogenes bacteria.
This model permits easy recovery of host inflammatory cells in the sac lumen by lavage for subsequent
analysis by FACS and other in vitro methods, and facilitates analysis of host innate immune response to
S. pyogenes [20].

14.2.1.1.3 Murine Impetigo Model

This model relies on a humanized mouse (hu-mouse) that is created by engrafting human epidermal tis-
sue from neonatal foreskin onto the flanks of the SCID mouse (which is incapable of rejecting the tissue
grafts due to the absence of an adaptive immune response). S. pyogenes bacteria are topically applied
to the grafts that are superficially damaged by cross-wise cuts with a scalpel blade, and then occluded
with a bandage, leading to an impetigo-like lesion [characterized by erosion of the stratum corneum,
infiltration of murine polymorphonuclear leukocytes (PMNs), formation of pus surrounded by clumps of
proliferating streptococci]. Through a semiquantitative visual assessment of histopathology or enumera-
tion of CFU, the virulence of S. pyogenes is ascertained [20].

14.2.1.1.4 Murine Systemic Disease Model

Inoculation of mice with 105–106 CFU of S. pyogenes bacteria via intravenously (IV), intraperitoneally
(IP), intranasally (IN), or intratracheally (IT) facilitates examination of different types of S. pyogenes-
induced systemic disease. Compared to IN infection, IT inoculation bypasses the initial site of upper
Streptococcus 231

airway colonization and allows quick development of clinical signs in infected mice. S. pyogenes viru-
lence is determined by lethality and the kinetics of clearance in the spleen, liver, and lungs (via CFU
enumeration) [5].

14.2.1.1.5 Murine Implanted Chamber Model

Murine implanted chamber model involves surgical implantation of a “cage” made of steel or Teflon into
the subcutaneous tissues on mouse flank. The healing of the incision over the course of several weeks
results in the formation of fibrous capsule around the implant. Subsequent injection of S. pyogenes bacte-
ria through the skin and into the inside of the cage allows the influx of nutrients and the release of strep-
tococcal products without streptococcal cells. This scenario resembles a glomerulonephritis-like disease
(characterized by deposition of streptokinase and host complement components in the glomerular base-
ment membrane), facilitating the monitoring of toxin production in vivo. A rabbit implanted chamber
model has been also described for modeling STSS [20].

14.2.1.1.6 Murine Footpad Model

In this model, S. pyogenes bacteria labeled with fluorescent dyes are injected into LysM-eGFP mouse
footpad, whose macrophages and neutrophils express eGFP; the velocity and meandering of neutrophils
are immediately monitored by two-photon microscopy [20].

14.2.1.1.7 Mouse Oropharyngeal Colonization Model

In this model, mice are infected by IN inoculation into one nostril with 107 CFU of mouse-pathogenic,
streptomycin-resistant S. pyogenes strain in a 2.5 μL normal saline drop; colonization of the upper air-
way and oropharynx is then monitored by throat swabs after resuspension in saline and plating onto
selective media [(Todd Hewitt yeast extract) THY agar with 1000 μg/mL streptomycin] [20].

14.2.1.1.8 Murine Nasopharyngeal-Associated Lymphatic Tissue Colonization Model

This model exploits the ability of murine nasal-associated lymphoid tissue (NALT) (in the form of lym-
phoid lobes located in the lateral nasopharyngeal wall) to allow colonization of S. pyogenes strain 591
following intranasal inoculation and to develop a specific immune response [20].

14.2.1.1.9 Murine Vaginal Colonization Model

In this model, C57BL/6 or BALB/c mice are administered with up to 0.5 mg ethinyl estradiol dissolved
in sterile sesame oil 24–48 h (to synchronize mice into the estrous phase of the estrous cycle, which
enhances epithelial cell proliferation and minimal inflammatory cell infiltrate and renders mice recep-
tive to bacterial colonization) prior to vaginal inoculation of ∼1 × 106 CFU of streptomycin-resistant
S.  pyogenes in 20 μL of PBS. Vaginal lavages with sterile PBS are stained with a modified Wright–
Giemsa stain, and epithelial cells and leukocytes are counted under microscope. In addition, dilution and
plating of vaginal lavages onto selective media (e.g., THY agar, supplemented with 1000 μg/mL strepto-
mycin) allows determination of streptococcal colony counts. Further, cytokine and other inflammatory
cell markers in vaginal lavages are assessed by ELISA [20].

14.2.1.1.10 Rat Oropharyngeal Colonization Model

This model involves treatment of Fischer CDF344 rats with streptomycin (1000 μg/mL) in drinking
water (to disrupt their normal flora) before IN (20 μL) or oral (50 μL) inoculation with streptomycin-
resistant S. pyogenes for pharyngeal colonization; the bacteria are then recovered from throat swabs and
CFU enumerated [20].

14.2.1.1.11 Rat Autoimmune Carditis Model

In this model, Lewis rats immunized with S. pyogenes M protein (which cross-reacts with rat heart
tissue) develop a myocarditis and a valvulitis, with the formation of Anitschkow cell-containing granu-
lomas (called Aschoff bodies) and infiltration of CD4+ and CD8+ T cells into the valvular lesions, that
resemble human rheumatic disease [20].
232 Laboratory Models for Foodborne Infections

In addition, sera from Lewis rats immunized with a whole cell lysate of S. pyogenes (showing quantifi-
able alterations in behavioral and motor functions) cross-react with brain tissue in vivo and with dopa-
mine and serotonin receptors in vitro. Direct perfusion of purified IgG from these cross-reacting sera
into the striatum region of the brain could reproduce symptomatology of PANDAS [17,18,21].

14.2.1.2 Chinchilla
Chinchilla otitis media model involves inoculation of 105 CFU S. pyogenes via transbullar injection into
the ear of a chinchilla, with tympanic membrane and inner ear inflammation as well as serous middle ear
effusion evident by day 2 as examined by otoscopy. The infection can be monitored through enumera-
tion of CFUs recovered from macroscopic structures and also serous effusion, and observation of animal
mortality over the 7-day period [20].

14.2.1.3 Nonhuman Primates
Nonhuman primate pharyngitis model offers a more reliable approach for assessing S. pyogene-induced
pharyngitis than other animals such as rodents. Several nonhuman primate species (e.g., baboon, rhesus
monkey, chimpanzee, and cynomolgus macaque) appear to be susceptible to S. pyogenes colonization in
the oropharynx, with the production of type-specific M protein antibodies in serum [20].
Nonhuman primate sepsis model involves intravenous infusion of baboons (Papio cynocephalus cyno-
cephalus) under light anesthesia with 1010 CFU S. pyogenes over a 2-h period followed by analyses of
physiology and blood chemistry over a 10-h period. The infection is evaluated by body temperature,
heart rate, mean systemic arterial blood pressure, serum chemistry, and cytokine profiles. It appears that
blocking the cytokine TNF-α with a therapeutic monoclonal antibody helps improve both mean arterial
blood pressure and survival and reduce hypotension and multiorgan failure. This model is valuable for
the study of streptococcal toxic-shock syndrome [20].

14.2.1.4 Insects
Silkworm model involves injection of approximately 9 × 108 CFU S. pyogenes into the hemolymph (com-
parable to the bloodstream) of invertebrate silkworm (Bombyx mori) followed by monitoring survival for
5 days at 27°C. Wax worm model differs from silkworm model in that wax worm (Galleria mellonella)
allows infection to be conducted at 37°C [20].

14.2.1.5  C aenorhabditis elegans Nematode

C. elegans is an invertebrate animal that can be used to analyze S. pyogenes virulence in plate or liquid for-
mat, as >80% of C. elegans organisms are killed by S. pyogenes-produced hydrogen peroxide within 24 h.

14.2.1.6 Zebrafish
Zebrafish myonecrosis model involves inoculation with S. pyogenes via IM injection into the dorsal
muscle of zebrafish (Danio rerio), leading to the formation of a hypopigmented lesion at the site of injec-
tion within 24 h (indicative of extensive muscle necrosis as revealed by histology) and the death (resulting
from toxic shock) of the animal at 36–96 h postinjection. Colonization may be evaluated by dissecting
the muscle tissue and the spleen for serial dilution plating [22].

14.2.2  I n Vitro Models

Using several mammalian cell lines (e.g., mouse fibroblast cell line, L cells, as well as HeLa and HEp-2
cells), Miyoshi-Akiyama et al. [23] noticed that high-virulence GAS isolates associated with STSS have
lower ability to adhere to mammalian cells than low-virulence isolates.
Streptococcus 233

In a separate study, human keratinocytes are shown to allow in vitro formation of GAS biofilms that
have superior capacity to colonize the oropharynx in vivo compared to broth-grown GAS and provide an
effective means to evaluate GAS colonization, invasive disease, and natural transformation [5].

14.3 Conclusion
Constituting a key member of the Gram-positive bacterial genus Streptococcus, S. pyogenes (commonly
called group A Streptococcus or GAS) is renowned for its serotype diversity, virulence variation, and
ferocious infectivity. Adapted exclusively to human host, pathogenic S. pyogenes strains are capable of
inducing localized inflammatory lesions (e.g., pharyngitis, impetigo), secreting streptococcal toxins that
cause both local and systemic diseases (e.g., pneumonia, sepsis, STSS), and producing streptococcal
antigens that confuse host immune systems, leading to autoimmune diseases (e.g., ARF, RHD, APSGN).
Given its medical and public health significance, S. pyogenes has been extensively studied, and a plethora
of laboratory models (both in vitro and in vivo) have been applied to elucidate the molecular mechanisms
of its pathogenicity and to aid in the design of innovative measures for its control and prevention.

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 Section III

Foodborne Infections due to

Gram-Negative Bacteria
15
Aeromonas

Dongyou Liu

CONTENTS
15.1 Introduction................................................................................................................................... 237
15.1.1 Classification, Morphology, and Genomics..................................................................... 238
15.1.1.1 Classification..................................................................................................... 238
15.1.1.2 Morphology....................................................................................................... 238
15.1.1.3 Genomics.......................................................................................................... 239
15.1.2 Biology and Epidemiology............................................................................................... 240
15.1.3 Clinical Features and Pathogenesis.................................................................................. 240
15.1.3.1 Gastrointestinal Infections................................................................................ 240
15.1.3.2 Extraintestinal Infections.................................................................................. 240
15.1.4 Diagnosis.......................................................................................................................... 241
15.1.5 Treatment and Prevention................................................................................................. 242
15.2 Laboratory Models........................................................................................................................ 242
15.2.1 Animal Models................................................................................................................. 242
15.2.1.1 Rodents............................................................................................................. 242
15.2.1.2 Zebrafish........................................................................................................... 243
15.2.1.3 Caenorhabditis elegans Nematode.................................................................. 243
15.2.1.4 Tetrahymena Protozoa...................................................................................... 243
15.2.2 In Vitro Models................................................................................................................. 243
15.3 Conclusion..................................................................................................................................... 244
References............................................................................................................................................... 244

15.1 Introduction
Aeromonas was first described in 1890 by Zimmermann as part of the genus Bacillus (Bacillus punc-
tatus). In the following year, an Aeromonas strain was isolated (then named Bacillus hydrophilus fus-
cus, which is now known as Aeromonas hydrophila) by Sanarelli from the blood of infected frog with
hemorrhagic exudation in the abdominal and peritoneal cavities (so-called “red leg” disease). In 1936,
the genus Aeromonas (Greek aer aeros, air, gas; Greek monas, unit, monad; Aeromonas, gas-­producing
unit) was created by Kluyver and van Neil to cover this group of bacteria. In 1951, association of the
genus Aeromonas with human infection (fulminant metastatic myositis) was confirmed with the recov-
ery of Aeromonas from autopsy samples. In 1968, involvement of the genus Aeromonas in other human
diseases (e.g., septicemia associated with Laennec’s cirrhosis) was documented. In 1986, the genus
Aeromonas was shown to be phylogenetically distinct from vibrios, and was transferred from the family
Vibrionaceae to a newly established family Aeromonadaceae. To date, about 30 species have been iden-
tified in the genus Aeromonas, and some of them are responsible for a variety of infections in humans
and animals.

237
238 Laboratory Models for Foodborne Infections

15.1.1 Classification, Morphology, and Genomics

15.1.1.1 Classification
Encompassing a diverse group of Gram-negative, non-spore-forming, cocco bacillary species, the
genus Aeromonas is classified taxonomically in the family Aeromonadaceae, order Aeromonadales,
class Gammaproteobacteria, phylum Proteobacteria, and domain Bacteria. Being one of the five gen-
era (i.e., Aeromonas, Oceanimonas, Oceanisphaera, Tolumonas, and Zobellella) within the family
Aeromonadaceae, the genus Aeromonas shares remarkable biochemical (e.g., cytochrome oxidase), eco-
logical (aquatic), and pathological similarities to members of the families Vibrionaceae (Vibrio) and
Enterobacteriaceae (Plesiomonas), and indeed was once included in the family Vibrionaceae. It was only
in 1986 when the genus Aeromonas became a member of the newly established Aeromonodaceae family
after detailed examination of rRNA and housekeeping gene sequences [1].
Currently, about 30 species are recognized in the genus Aeromonas, including Aeromonas aquatica,
Aeromonas australiensis, Aeromonas bestiarum (HG2, formerly Aeromonas hydrophila genomospe-
cies 2), Aeromonas bivalvium, Aeromonas cavernicola, Aeromonas caviae (HG4, synonym Aeromonas
punctata), Aeromonas dhakensis (synonyms Aeromonas aquariorum, Aeromonas hydrophila subsp.
dhakensis), Aeromonas diversa (HG13, synonym Aeromonas group 501), Aeromonas encheleia
(HG16), Aeromonas eucrenophila (HG6), Aeromonas finlandensis, Aeromonas fluvialis, Aeromonas
hydrophila (HG1, synonyms Bacillus hydrophilus fuscus, Bacillus hydrophilus, Proteus hydrophilus,
Bacterium hydrophilum, Pseudomonas hydrophila), Aeromonas jandaei (HG9), Aeromonas lacus,
Aeromonas media (HG5A, HG5B), Aeromonas molluscorum, Aeromonas piscicola, Aeromonas popof-
fii (HG17), Aeromonas rivuli, Aeromonas salmonicida (HG3), Aeromonas sanarellii, Aeromonas
schubertii (HG12), Aeromonas simiae, Aeromonas sobria (HG7), Aeromonas taiwanensis, Aeromonas
tecta, Aeromonas trota (HG14, synonym Aeromonas enteropelogenes), and Aeromonas veronii (HG8,
HG10, synonyms Aeromonas ichthiosmia, Aeromonas allosaccharophila, Aeromonas culicicola) [1–3].
Interestingly, Aeromonas sharmana (which unlike other members of the genus, is negative for
nitrate reductase, lysine or ornithine decarboxylase or arginine dihydrolase, and lacks deoxyribonu-
clease activity) is now considered to be non-Aeromonas although it may still fall within the family
Aeromonadaceae [1].
Based on their growth and biochemical characteristics, members of the genus Aeromonas may
be distinguished into two major groupings: mesophilic and psychrophilic. The mesophilic group
(e.g., A. hydrophila) contains motile strains that grow optimally at 35°C–37°C and cause diseases in humans.
The psychrophilic group (e.g., A. salmonicida) includes nonmotile strains that grow optimally at 22°C–25°C
and cause diseases in fish. Further genetic analysis led to the subdivision of the mesophilic group into
three biochemically distinct phenospecies (A. hydrophila, A. sobria, and A. caviae). Additionally, con-
sidering their biochemical and genetic resemblances, A. hydrophila sensu stricto, A. bestiarum, and
A. salmonicida are collectively referred to as the A. hydrophila complex; and A. caviae sensu stricto,
A. media, and A. eucrenophila are known as the A. caviae complex. Further, using DNA–DNA hybrid-
ization techniques, Aeromonas species may be separated into 16 hybridization groups (HG, which refers
to phenotypically distinguishable species) in direct contrast to phenospecies (which refers to a single
heterogeneous species that may be composed of multiple HGs). Moreover, 16S rRNA restriction frag-
ment length polymorphism (RFLP) methods have been developed to classify Aeromonas strains into
genomospecies [4].

15.1.1.2 Morphology
Aeromonas is a Gram-negative, cocco bacillary or rod-shaped bacterium 0.3–1.0 μm × 1.0–3.5 μm
in size, which appears singly, in pairs, and occasionally in short chains. On blood agar, Aeromonas
forms round, raised, opaque colonies of 1–3 mm in diameter, with color changing from grayish (due to
β-hemolysis) to dark green after 3 days (except A. caviae). Being a facultative anaerobe, Aeromonas is
oxidase and catalase positive (best tested on media without a fermentable sugar, such as MacConkey
agar).
Aeromonas 239

Other morphological features include the possession of a single polar flagellum (especially motile
strains, although some species may form peritrichous or lateral flagella or no flagella in solid media) and
pili (fimbriae, which are surface appendages that facilitate attachment to host cells), which are either
short rigid (S/R type) or long wavy flexible (L/W type). Most motile aeromonads do not have capsule,
although A. hydrophila serotypes O:11 and O:34 are known to produce capsule when grown in a glucose-
rich medium, which has a potential role in virulence.

15.1.1.3 Genomics
The genome sequences of a number of Aeromonas species are available in GenBank. The sizes of
Aeromonas genomes appear to be in the range of 3.9–5.18 Mb, with GC contents of 57.6%–63.1% and
3609–4794 genes (Table 15.1). Many Aeromonas species harbor pseudogenes, while a few also possess
plasmids (e.g., A. salmonicida subsp. salmonicida strain A449). In addition, the genomes of Aeromonas
spp. encode a variety of virulence factors (including structural components, extracellular factors, secre-
tion systems, iron-acquisition, and quorum-sensing mechanisms) that actively participate in host adher-
ence, colonization, and infection [5–8]. Phylogenomic network analysis of three clinically important
Aeromonas species (A. hydrophila, A. veronii, A. caviae) highlights the influence of homologous recom-
bination and lateral gene transfer in the evolution of Aeromonas spp. [9].

TABLE 15.1
Genomic Features of Aeromonas Species
Species Strain HG Genome (Mb) GC Content (%) Genes Plasmid (kb)
A. aquatica 4.58 61.2 4163
A. australiensis 4.11 58.1 3735
A. bestiarum HG2 4.69 60.6 4153
A. bivalvium 4.3 62.2 3912
A. caviae 8LM HG4 4.47 61.8 4076 30
A. dhakensis AAK1 4.76 61.8 4268
A. encheleia HG16 4.47 61.9 4065
A. eucrenophila HG6 4.54 61.1 4081
A. finlandiensis 4.72 58.6 4376
A. fluvialis 3.9 58.3 3609
A. hydrophila ATCC 7966 HG1 4.74 61.5 4284
A. lacus 4.39 59 4005
A. media WS HG5 4.78 60.7 4340
A. molluscorum 848 4.24 59.2 4033
A. piscicola 5.18 59.2 4794
A. popoffii HG17 4.76 58.6 4329
A. rivuli 4.52 60 4230
A. salmonicida A449 HG3 4.7 58.5 4320 168 and 175
subsp.
salmonicida
A. sanarellii 4.19 63.1 3823
A. schubertii HG12 4.4 61.5 4287
A. simiae 3.99 61.3 3756
A. sobria HG7 4.68 57.6 4212
A. tecta 4.76 60.1 4329
A. veronii B565 HG8/HG10 4.66 58.6 4057
240 Laboratory Models for Foodborne Infections

15.1.2 Biology and Epidemiology

Typically, aeromonads establish infections in humans through oral ingestion of contaminated food or
water (intestinal infection) [10]. Direct mucocutaneous contact with or exposure of open wound to envi-
ronmental mud or water offers another way for aeromonads to enter human hosts (extraintestinal infec-
tions). In Aeromonas gastroenteritis, after enduring the detrimental effects of gastric acidity, ingested
bacteria settle in the small or large intestine, and release enterotoxigenic molecules (leading to enteritis),
or invade the gastrointestinal epithelium (causing dysentery or colitis).
With the capacity to tolerate the temperature range of 0°C–45°C, pH range of 4.5–9.0, and sodium
chloride concentrations of 0%–4%, Aeromonas species are ubiquitously distributed in the environment
(freshwater, estuarine, brackish, and saltwaters, and soil) and in foods (beef, pork, lamb, poultry, fish,
shellfish, raw milk, dairy products, and fresh produce), and they have been isolated from humans, horses,
pigs, sheep, cows, domesticated pets, birds, invertebrate, ticks, and insects [11,12]. It is of interest to note
that A. veronii is commonly found in raw surface water, A. hydrophila in ozonated water, and A. caviae
(A. puntacta) and A. media in waste water.
Aeromonad infections often occur in the community settings, as well as in health-care facility settings
(e.g., indwelling-device-related infections). While all people are susceptible to Aeromonas-associated
gastroenteritis, young children (<5 years of age) and older adults (>60 years of age) are particularly vul-
nerable. Individuals with underlying diseases (e.g., leukemia, carcinoma, diabetes, hepatitis, cirrhosis)
or suppressed immune functions are notably prone to Aeromonas systemic infections [13]. Additional
risk factors for Aeromonas infections include traumas and near-drowning events related to recreational
activities (e.g., boating, fishing, and diving) and bites from reptiles, snakes, and bears [14,15].

15.1.3 Clinical Features and Pathogenesis

Aeromonads have been implicated in (1) gastrointestinal infections (secretory, dysentery, chronic, and
choleraic) and (2) extraintestinal infections (wound and soft tissue infections, blood-borne infections,
and miscellaneous infections), although the bacteria may be found in the stools of up to 4% of asymp-
tomatic individuals [16–18].

15.1.3.1 Gastrointestinal Infections
The most common gastrointestinal infection is gastroenteritis, with symptoms ranging from fever,
vomiting, and abdominal cramp to diarrhea (secretory, dysentery, chronic, and choleric). The secretory
form of Aeromonas gastroenteritis is most common and manifests with low-grade fever and abdomi-
nal pain, and watery diarrhea, with mild to moderate dehydration. The watery diarrhea is generally
a self-limiting illness, lasting a few days to a few weeks. The dysenteric form of Aeromonas gas-
troenteritis is less common and shows cramping abdominal pain and mucus (leukocytes) and blood
in stools (similar to shigellosis). The chronic form of Aeromonas gastroenteritis lasts for more than
2 months and is f­ requently associated with A. caviae and A. hydrophila infections. The choleraic form
of Aeromonas gastroenteritis is a rare, cholera-like disease. With the spread of gastrointestinal infec-
tions, appendicitis, peritonitis, pancreatitis, and acute cholangitis may emerge [19]. Complications
linked to Aeromonas gastroenteritis include small bowel obstruction, ileal ulceration, intramural intes-
tinal hemorrhage with small bowel obstruction, refractory inflammatory bowel disease, acute renal
failure, and hemolytic-uremic syndrome (HUS). Among 14 Aeromonas species involved in human
illness, six (A. hydrophila, A. caviae, A. veronii, A. schubertii, A. jandaei, and A. trota) are shown to
cause human diarrhea [1].

15.1.3.2 Extraintestinal Infections
Extraintestinal infections include wound and soft tissue infections (cellulitis, abscesses), blood-borne
infections (bacteremia/septicemia, pneumonia, empyema, septic arthritis, necrotizing fasciitis, myo-
necrosis, endocarditis, meningitis), and miscellaneous infections (hepatobiliary tract infections,
Aeromonas 241

osteomyelitis, endophthalmitis, keratitis, corneal ulcer, infections of bones and joints, the respiratory and
urogenital tracts) [20–23]. Application of leeches (which may harbor aeromonads symbiotically) to tis-
sue flaps or replantation areas during plastic or reconstructive surgery to relieve venous congestion may
be a potential cause of Aeromonas infections (e.g., cellulitis) [24,25]. Aeromonas bacteremia/septicemia
(attributed mainly to A. hydrophila, A. dhakensis, A. veronii, or A. caviae) often displays fever, jaundice,
abdominal pain, septic shock, and dyspnea and tends to occur in severely immunocompromised individ-
uals (e.g., those with myeloproliferative disorders, Laennec’s cirrhosis, chronic liver disease, neoplasia,
biliary disease, acute myeloid leukemia, myelodysplastic syndromes, non-Hodgkin’s lymphoma, acute
lymphocytic leukemia, diabetes mellitus, renal problems, cardiac anomalies, aplastic anemia, thalas-
semia, multiple myeloma, and Waldenstrom’s macroglobulinemia) [26]. Aeromonas biliary tract infec-
tions (due to A. hydrophila and to a lesser extent, A. caviae and A. veronii) often occur in patients with
biliary tract obstruction or stasis due to hepatobiliary cancer or stones, and most of them are polymicro-
bial in nature (e.g., Escherichia coli, Klebsiella pneumoniae, Enterococcus, and Staphylococcus aureus).
A. hydrophila and A. veronii may be also a cause of hemorrhagic septicemia in carp, tilapia, perch,
catfish, and salmon; red sore disease in bass and carp; ulcerative infections in catfish, cod, carp, and
goby; ulcerative stomatitis in snakes and lizards; “red leg” disease in frogs; septicemia in dogs; and
septic arthritis in calves. A. salmonicida sensu stricto is linked to fish furunculosis, particularly in sal-
monids, with presentation ranging from septicemia, hemorrhages at the bases of fins, inappetence, mela-
nosis, lethargy, and slight exophthalmia to hemorrhaging in muscle and internal organs.
Aeromonas spp. produce a number of virulence factors and proteins that enhance their pathogenicity.
These include invasins, adhesins, outer membrane proteins, S-layer proteins, lipopolysaccharide (LPS),
proteases [thermostable metalloprotease (TSMP) and thermolabile serine protease (TLSP)], lipases,
glycerophospholipid cholesterol acyl-transferase (GCAT), superoxide dismutase, hemolysins, cytotoxic
and cytotonic enterotoxins [Aeromonas cytotoxic enterotoxin (Act), Aeromonas heat-labile (56°C) cyto-
tonic enterotoxin (Alt), and Aeromonas heat-stable cytotonic enterotoxin (Ast)], as well as type III secre-
tion system (AscV and AscF-G) [27–31].

15.1.4 Diagnosis
Laboratory identification of aeromonads is facilitated by in vitro isolation of Aeromonas spp. from stool or
other gastrointestinal samples. Aeromonads grow readily on routine enteric isolation media (MacConkey,
xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and deoxycholate-
citrate (DC) agars). Most (90%) of the Aeromonas species from humans produce β-hemolysis on sheep
blood agar, with the exception of A. popoffii and A. trota (0% and 50%, respectively) [32]. Identification at
the genus level is achieved by positive oxidase test, fermentation of ­d-glucose, motility (most Aeromonas
species are motile apart from A. salmonicida and A. media), the absence of growth in 6.5% sodium chlo-
ride, and resistance to the vibriostatic agent O/129 (150 μg). However, phenotypic identification to species
level may require additional work involving the use of selective and differential media [33].
A number of selective media (supplemented with ampicillin and/or inhibitors such as bile salts, ­brilliant
green, and sodium lauryl sulfate) have been used for recovery of Aeromonas species. For example, ampi-
cillin dextrin agar (ADA) is useful for isolating most Aeromonas spp. apart from A. trota and certain
strains of A. caviae, due to their sensitivity to ampicillin. Comparative analysis indicated that enrichment
in alkaline peptone water (APW) and consecutive plating in two selective media [­ampicillin–sheep-blood
agar supplemented with 30 μg/mL of ampicillin ASBA 30 or bile salts–Irgasan–brilliant green (BIBG)
agar] enable qualitative isolation of Aeromonas species from meat and fish. In BIBG, bile salts and brilliant
green inhibit the growth of Gram-positive bacteria while Irgasan inhibits the growth of Gram-negative
bacteria, which possess a type A nitratase. In addition, BIBG also detects the fermentation of xylose in
some aeromonads, as production of acid during xylose fermentation retains BIBG’s original, purple-red
color (instead of green-yellow color). Other selective media include Aeromonas agar (AA,  containing
irgasan and d-xylose, which aeromonads do not ferment), and cefsulodin–irgasan–­novobiocin (CIN) agar
(in which Aeromonas forms a bull’s-eye-like colony due to fermentation of d-mannitol) [33].
Aeromonas isolates surviving the selective process can be further distinguished via differentiation
media (e.g., detection of amylase and pattern of carbohydrate fermentation in Aeromonas species). For
242 Laboratory Models for Foodborne Infections

example, the modified BIBG medium (mBIBG) has a higher pH (up to 8.7), together with the replace-
ment of xylose by soluble starch as a carbon source, allowing identification of mesophilic aeromonads
that are not able to ferment xylose. Further differentiation of aeromonads from vibrios and plesiomo-
nads is their resistance to the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine), growth on
nutrient agar without salt supplementation, and inability to grow on media containing 6%–6.5% sodium
chloride. A combination of selective and differential media enables to classify clinical Aeromonas
isolates into the A. hydrophila group (A. hydrophila, A. bestiarum, and A. salmonicida), the A. caviae
group (A. caviae, A. media, and A. eucrenophila), and the A. sobria group (A. veronii, A. jandei,
A. schubertii, and A. trota). Although use of the Aerokey II system allows an accurate and reliable
phenotypic identification of A. hydrophila, A. caviae, A. veronii biovar sobria, A. veronii biovar veronii,
A. schubertii, A. jandaei, and A. trota, it is unable to detect many newly described taxa. Similarly, the
Vitek 2 does not differentiate between A. hydrophila and A. caviae [33].
Molecular methods targeting 16S rRNA, hemolysin (ahh1), aerolysin (aerA), and asa1 genes allow
precise determination of Aeromonas species identity and virulence potential [34–39]. Analysis of house-
keeping genes (e.g., dnaK, gltA, gyrB, radA, rpoB, tsf, and zipA) provides an effective tool for study
of taxonomic and phylogenetic relationships among Aeromonas species [4,5,40–43]. Indeed, use of a
multiplex PCR directed toward the gyrB and rpoB genes enabled simultaneous identification of four
Aeromonas species (A. hydrophila, A. media, A. veronii, and A. caviae) [44].

15.1.5 Treatment and Prevention

Treatment is s necessary for patients with chronic diarrheal disease or systemic infection. Fortunately,
major Aeromonas species (e.g., A. hydrophila, A. caviae, A. veronii) appear to be susceptible to flu-
oroquinolones (e.g., levofloxacin, gatifloxacin, ciprofloxacin, and moxifloxacin), cefotaxime, and
trimethoprim–sulfamethoxazole, although some Aeromonas isolates show resistance to penicillins, most
cephalosporins, erythromycin, nalidixic acid, ciprofloxacin, norfloxacin, chloramphenicol, tetracycline,
cotrimoxazole, ampicillin, ceftazidime, and meropenem [45–50].
For specific diseases associated with Aeromonas infections, therapeutic approaches may be con-
sidered for those presented with: chronic diarrhea (trimethoprim–sulfamethoxazole or ciprofloxacin),
biliary tract infections (drainage and fluoroquinolones), spontaneous bacterial peritonitis (cefotaxime,
fluoroquinolone, cephalosporin, and tetracycline), soft-tissue infections (cephalosporin with or without
doxycycline, aztreonam, or fluoroquinolone as well as surgical debridement for invasive soft-tissue infec-
tions), bacteremia (cefotaxime, ceftriaxone, or ceftazidime, with or without minocycline; alternatively,
aztreonam, cefepime, or fluoroquinolone such as ciprofloxacin or levofloxacin), pneumonia (cephalo-
sporins, aminoglycosides, fluoroquinolones, and imipenem), Aeromonas meningitis (cefotaxime, ceftri-
axone, or meropenem), endocarditis (an aminoglycoside plus a β-lactam; alternatively, a cephalosporin
such as cefotaxime or ceftriaxone, in conjunction with an aminoglycoside such as gentamicin), eye
infections (chloramphenicol, trimethoprim-sulfamethoxazole, and tetracyclines), and severe, invasive
eye infections (trimethoprim-sulfamethoxazole) [51,52].
Prevention of Aeromonas infections should focus on avoiding physical contact with marine microorgan-
isms or wild water or incidental ingestion of contaminated food or water, and eating uncooked seafood.

15.2 Laboratory Models
15.2.1 Animal Models
15.2.1.1 Rodents
Intramuscular inoculation into BALB/c mice provides a useful way to assess the pathogenicity of
Aeromonas isolates (e.g., A. hydrophila, A. veronii, and A. caviae) in soft tissue infections. It appears
that A. hydrophila is capable of causing more severe muscle damage (e.g., fragmentation of muscle fibers,
Aeromonas 243

edema of myocytes, and infiltration of inflammatory cells) than A. caviae. Additionally, mice undergoing
streptomycin pretreatment allow transient Aeromonas colonization and enable determination of the colo-
nization rates of different isolates. Indeed, A. hydrophila, A. veronii, and A. caviae exhibit relatively high
rates of mouse colon tissue colonization than A. salmonicida, A. encheleia, and A. allosaccharophila [53].
Furthermore, intraperitoneal injection of immunocompromised mice or gastric lavage of neonatal mice
offer alternative approach for investigating of Aeromonas pathogenicity relating to Aeromonas-induced
gastroenteritis as well as extraintestinal infections [54,55].
Rats (Rattus norvegicus) undergoing clindamycin pretreatment develop a self-limited, loose stool
(evidence of enteritis) after oral feeding with A. hydrophila, suggesting that antibiotic usage represents a
predisposing risk factor to Aeromonas infection [56].

15.2.1.2 Zebrafish
Zebrafish represents a useful model for assessing the virulence of and host immune responses against
Aeromonas strains, and its value for examining the host–pathogen interactions at the molecular level
may be limited. Zebrafish succumbing to Aeromonas infection often displays clinical signs typical of
hemorrhagic septicemia [57].

15.2.1.3  C aenorhabditis elegans Nematode

C. elegans is an invertebrate that has simple immune systems, is genetic tractability, is amenable to high-
throughput experiments, and can be used in liquid-toxic (LT) assay to assess the virulence of Aeromonas
species [58]. In C. elegans model, wound isolates of A. dhakensis demonstrates a higher virulence than
wound isolates of A. hydrophila. The main advantages of C. elegans model include rapid generation
time, large progeny, and ease of observation.

15.2.1.4  Tetrahymena Protozoa

Tetrahymena spp. (T. thermophila and T. pyriformis) are freshwater free-living ciliate protozoa that
grow readily in culture media between 12°C and 41°C without a CO2-enriched atmosphere, and thus
offer an economical and permissive model to evaluate the virulence is aquatic Aeromonas isolates.
Aeromonas isolate is considered virulent when relative survival is >60%, whereas Aeromonas isolate
is considered avirulent when relative survival is <40%. Similarly, Aeromonas isolate is regarded
as virulent when relative survival of T. thermophila is >40% after coculture with Aeromonas,
whereas Aeromonas isolate is classified as avirulent when relative survival of T. thermophila is
>50% after coculture with Aeromonas [57]. In addition, highly virulent Aeromonas strains grow
well in T. thermophila, causing deformation to and even lysis of T. thermophila, whereas aviru-
lent Aeromonas strains are largely phagocytozed by T. thermophila, causing no obvious damage to
T. thermophila [59].
Other animal models used to study Aeromonas strains include crayfish (Pacifastacus leniusculus),
mealworm larvae (Tenebrio molitor), leech, and blue gourami. Using crayfish and mealworm larvae
models, the role of LPS (O-antigen and external core) in the virulence of A. hydrophila was con-
firmed [60].

15.2.2  In Vitro Models

Various cell lines (mouse C2C12 fibroblast cell and human intestinal epithelial cell Caco-2) have been
used to assess the virulence and cytotoxicity of Aeromonas species. Using Caco-2 cells, it was shown
that c-jun and c-fos are upregulated upon incubation with virulent Aeromonas isolates and thus provide a
predictive indicator of Aeromonas virulence [61]. Further, it was observed that A. dhakensis isolates
exhibited more potent cytotoxicity in human normal skin fibroblast cells than A. hydrophila, while
A. veronii isolates showed higher cytotoxicity in C2C12 cells than A. caviae [58].
244 Laboratory Models for Foodborne Infections

15.3 Conclusion
The genus Aeromonas encompasses a large group of Gram-negative, rod-shaped bacteria that are com-
monly present in aquatic environments (marine water, fresh water, and sediments), vegetation, and
various animals. Besides causing diseases in fish and other animals, Aeromonas species are also respon-
sible for opportunistic foodborne or wound-related infections in humans, including gastroenteritis and
extraintestinal infections (e.g., serious wound infections in healthy individuals, primary and secondary
septicemia in immunocompromised individual, as well as peritonitis, meningitis, and infections of the
eye, joints, and bones). Pathogenic Aeromonas species are known to generate a number of virulence
factors that aid their invasion of host cells and sabotage host immune surveillance. Molecular assays
targeting the virulence genes, housekeeping genes, and 16S rRNA genes of Aeromonas spp. provide a
rapid and precise means for their detection, identification, and epidemiological tracking and contribute
to the early implementation of antimicrobial therapy. Nonetheless, despite our concerted efforts in the
past, an effective vaccine against Aeromonas infections is still unavailable. Therefore, further research
is urgently required to reveal the intricacy of host–bacterial interactions, and pinpoint the weak links in
Aeromonas physiobiology and pathogenicity. Undoubtedly, laboratory models will form an indispens-
able part of this endeavor.

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tions, and phenotypic identification schemes. J Clin Microbiol 2003;41(6):2348–57.
34. Balakrishna K, Murali HS, Batra HV. Detection of toxigenic strains of Aeromonas species in foods by
a multiplex PCR assay. Indian J Microbiol 2010;50(2):139–44.
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characterization of Aeromonas spp. and Vibrio cholerae O1 isolated during a diarrhea outbreak. Rev
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Israel. PLoS One 2012;7(2):e30070.
38. Sreedharan K, Philip R, Singh IS. Characterization and virulence potential of phenotypically diverse
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39. Meng S, Wang Y, Wang Y, Liu D, Ye C. Development of cross-priming amplification assays for rapid
and sensitive detection of Aeromonas hydrophila. Lett Appl Microbiol 2015;61(2):171–8.
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42. Rather MA, Willayat MM, Wani SA, Munshi ZH, Hussain SA. A multiplex PCR for detection of entero-
toxin genes in Aeromonas species isolated from foods of animal origin and human diarrhoeal samples.
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quinolones against Aeromonas hydrophila. Antimicrob Agents Chemother 2003;47(7):2217–22.
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imported shrimp. Appl Environ Microbiol 2012;78(22):8137–41.
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16
Bacteroides

Mario Julio Avila-Campos

CONTENTS
16.1 Introduction................................................................................................................................... 247
16.2 Taxonomic and Ecological Aspects.............................................................................................. 249
16.3 Biology and Virulence Factors...................................................................................................... 249
16.3.1 Capsule............................................................................................................................ 250
16.3.2 Evasion from the Host’s Immune Response................................................................... 250
16.3.3 Enzymes.......................................................................................................................... 250
16.3.4 Enterotoxin.......................................................................................................................251
16.3.5 Endotoxin/LPS.................................................................................................................251
16.4 P athogenesis.................................................................................................................................. 252
16.5 D iagnosis....................................................................................................................................... 252
16.6 S usceptibility to Antimicrobials................................................................................................... 253
16.6.1 Mechanisms of Antimicrobial Resistance...................................................................... 253
16.6.2 β-Lactam Agents............................................................................................................. 253
16.6.3 Carbapenems................................................................................................................... 253
16.6.4 PBP.................................................................................................................................. 253
16.6.5 Outer Membrane Proteins or Porins............................................................................... 255
16.6.6 Aminoglycosides............................................................................................................. 255
16.6.7 M  acrolides, Lincosamides, and Chloramphenicol......................................................... 255
16.6.8 Tetracycline..................................................................................................................... 255
16.6.9 Nitroimidazoles............................................................................................................... 255
16.6.10 Quinolones...................................................................................................................... 255
16.6.11 P lasmids.......................................................................................................................... 256
16.6.12 Multidrug Resistance...................................................................................................... 256
16.7 Laboratory Models........................................................................................................................ 256
16.8 C  onclusions................................................................................................................................... 257
References............................................................................................................................................... 257

16.1 Introduction
Taxonomically, the genus Bacteroides belongs to the phylum Bacteroidetes, class Bacteroidia, order
Bacteroidales, and family Bacteroidaceae. This genus covers a large number of species pathogenic to
humans and animals. Bacteroides species are strictly anaerobes and some are aerotolerant, and are
considered the most important constituent of the human resident microbiota. Species of this genus are
Gram-negative, non-spore-forming rods, living in intestines and giving the host several nutrients pro-
duced by the breaking down of food. In disequilibrium in the gastrointestinal ecosystem, they can pro-
duce several infections of endogenous nature, such as periodontal diseases, lung and brain infections,
urinary or genital infections, and intraabdominal abscesses, and recently they have been linked to colon

247
 248
TABLE 16.1
Phenotypic Characteristics of the Bacteroides fragilis Group and Parabacteroides
Fermentation
Species Growth in 20% Bile Indol Catalase Esculin Hydrolysis Arabinose Cellobiose Rhamnose Sucrose Trehalose
B. fragilis group + V V + −+ + V + +
B. fragilis + − + + − +− − + −
B. caccae + − + + + +− +− + +
B. ovatus + + + + + + + + +
B. stercoris + + − + −+ −+ + + −
+ + + + + +− + + +

Laboratory Models for Foodborne Infections

B. thetaiotaomicron
B. uniformis +w + V + + + −w + −w
B. vulgatus + − −+ − + − + +−
P. merdae + − −+ + −+ V + + +
P. distasonis + − −+ + −+ + V + +
+, positive; −, negative; −+, some species positive; +−, some species negative; +w or −w, some species weak positive or weak negative; V, variable.
Bacteroides 249

cancer. Bacteroides spp. show a great variability to the antimicrobial susceptibility, and several species
are resistant to various antimicrobial drugs expressing resistance genes or other mechanisms, such as
efflux pumps.1 The key phenotypic characteristics of the species of the B. fragilis group are summarized
in Table 16.1.

16.2 Taxonomic and Ecological Aspects

The genus Bacteroides has undergone major taxonomic revisions in the past few years, and most species
suffer changes frequently. Suitable taxonomic changes have become important to clinicians and micro-
biologists, since these changes can be used as an indicator of virulence or antimicrobial resistance. It is
known that some species of the Bacteroides genus, such as B. thetaiotaomicron, are much more resistant
to antimicrobials than B. fragilis, for example. In 1990, various species were moved to other genera,
including Porphyromonas and Prevotella, and others were included, such as Dialister, Megamonas,
Mitsuokella, Tannerella, Tissierella, and Alistipes. By using culture-independent techniques such as
16S rRNA gene sequencing, a number of new species have been added to the genus Bacteroides, achiev-
ing more than 20 bacterial species.2,3 In 2005, new species added to this genus included Bacteroides
goldsteinii, Bacteroides nordii, and Bacteroides salyersai, Bacteroides plebeius and Bacteroides copro-
cola (isolated from human feces), and Bacteroides massiliensis (isolated from the blood of a newborn).
Subsequently, B. goldsteinii, Bacteroides distasonis, and Bacteroides merdae were moved to the genus
Parabacteroides.4 The occurrence of species of the B. fragilis group in the fecal microbiota of children
with diarrhea is shown in Table 16.2.

16.3 Biology and Virulence Factors

Most Bacteroides spp. have a G-C composition of 40%–48%; for example, B. fragilis NCTC 9343 shows
43.19% G-C content, and sequencing studies have demonstrated an influence of environmental factors
on biology of these microorganisms.
B. fragilis constitutes 0.5% of the human colonic microbiota, but it is the most commonly isolated
anaerobic pathogen. Virulence factors produced by B. fragilis can be divided into three categories:
(1) adherence to epithelial tissues, (2) protection from the host immune response, and (3) destruction of
tissues. The presence of fimbriae and agglutinins causes B. fragilis allows its colonization on the host’s
tissue; the polysaccharide capsule, LPS, and various bacterial enzymes work against the host’s immune
response, and its histolytic enzymes collaborate in tissue destruction.5 In addition, Bacteroides spp. are
able to produce bacteriocin-like substances that inhibit the growth of other species or genera, as observed
in Figure 16.1.

TABLE 16.2
Occurrence of the Bacteroides fragilis Group in 170 Stool Samples from Children
with and without Acute Diarrhea
Diarrhea No Diarrhea
Bacterial Isolates No. % No. %
Non-ETBF 67 72.82 42 53.8
ETBF 2   2.17  0  0
B. uniformis 7   7.6  1   1.3
B. vulgatus 4   4.3 13 16.7
B. thetaiotaomicron 1   1.1  1   1.3
B. ovatus 0  0  4   5.1
Parabacteroides distasonis 11 11.9 17 21.8
250 Laboratory Models for Foodborne Infections

FIGURE 16.1  Bacteriocin-producing B. fragilis against B. vulgatus. Arrow shows the inhibition halo.

16.3.1  C apsule
The capsule of B. fragilis is largely responsible for abscess formation due to its protection afforded to the
bacteria against the host’s immune response. These abscesses can produce intestinal obstruction, fistula,
bacteremia, and disseminated infection. Encapsulated B. fragilis has been used to produce experimental
abscess in animal model, and it has been reported that responses to other polysaccharide antigens are
T-cell independent, but abscess formation induced by B. fragilis is dependent on the involvement of
T cells. B. fragilis produces a polysaccharide capsule of varying molecular weights (PS-A, PS-B, and
PS-C). By using electron microscopy, three capsule variants within an individual strain of B. fragilis
might be observed: large and small capsules, and noncapsulate strain.

16.3.2 Evasion from the Host’s Immune Response

The ability to evade the host’s immune response contributes to the bacterial virulence. Encapsulated
B. fragilis is more resistant to the action of peritoneal macrophages, the first host immunologic defense
response to the rupture of the intestine or any other compromise of the peritoneal cavity. The production
of nitric oxide (NO) is an important biological mediator in the living organism that is synthesized from
l-arginine using NADPH and molecular oxygen. However, the overproduction of NO, which is catalyzed
by the inducible nitric oxide synthase (iNOS), a soluble enzyme active in its dimeric form, is cytotoxic,
and after exposure to cytokines such as gamma interferon it has a microbicidal, cell necrosis, or death role.

16.3.3 Enzymes
Histolytic enzymes, such as hyaluronidase and chondroitin sulfatase present in some B. fragilis, can
attack the host’s extracellular matrix. Proteases of B. fragilis have also been implicated in destroying
Bacteroides 251

brush border enzymes acting on the microvillus membranes for the final digestion of food and absorp-
tion of nutrients. Other enzymes, hemolysins (HlyA and HlyB), and neuraminidase encoded by the nanH
gene are also important. Neuraminidase is found in many pathogenic bacteria and is generally con-
sidered as a virulence factor; this enzyme catalyzes the removal of the sialic acid from epithelial cells
and immunoactive proteins such as IgG. Several B. fragilis strains have neuraminidase activity, and it
has been suggested that this activity plays a role in the bacterial attachment to animal cells and to the
hemagglutination.5

16.3.4 Enterotoxin
The B. fragilis enterotoxin (BFT) is a zinc metalloprotease that destroys the adherence tight junctions in
intestinal epithelium by cleaving E-cadherin, resulting in rearrangements of the actin cytoskeleton of the
epithelial cells, which causes diarrhea. Enterotoxigenic B. fragilis (ETBF) strains encode three isotypes
of BFT on distinct bft loci, carried on a 6-kb genome segment that is unique and called the B. fragilis
pathogenicity island, and the presence of the bft gene is generally detected by PCR techniques.6–9 This
pathogenicity island is flanked by genes encoding mobilization proteins and may be transmissible to
nontoxigenic strains (non-ETBF) (Figure 16.2). Recent studies have shown that the BFT has a possible
role as a carcinogen in colorectal cancer.10

16.3.5 Endotoxin/LPS
LPS in B. fragilis is 10–1000 times less toxic than that in Escherichia coli. This endotoxin displays
toxicity, and the exposure to antibiotics enhances its production many times higher in B. fragilis than in
the other species of the B. fragilis group. This may partly explain its association with clinical infections
and mortality.

18-kb region

6-kb region

Pattern I (ETBF)
bft (isotypes bft-1, bft-2, and bft-3)

12-kb region

Pattern III (NTBF)

Pattern II (NTBF)

BfPAI DNA flanking the BfPAI Chromosome segment

FIGURE 16.2  Pathogenicity island (6-kb region) anchored in chromosomal DNA from enterotoxigenic B. fragilis, and
their genetic profile. (Adapted from Franco, A.A. et al., Mol. Microbiol., 45, 1067–1077, 2002.)
252 Laboratory Models for Foodborne Infections

16.4 Pathogenesis
Capsule present in anaerobes is considered an important virulence factor. Other factors related to the
virulence of anaerobes include mucosal damage and resistance against the host immune response. ETBF
has been implicated in inflammatory intestinal diseases.11–13 Studies show that intestinal colonization
with ETBF leads to acute or chronic intestinal inflammations explaining its potential pathogenicity. The
BFT alters the F-actin structure, resulting in disruption of the epithelial barrier-producing diarrhea.14,15
Studies have implicated the presence of E. coli and Bacteroides vulgatus in the development of Crohn’s
disease and ulcerative colitis; however, the relationship of this bacterial association with these diseases
remains unclear.
Adhesion to the epithelial surface is considered a prerequisite for pathogenicity in most bacteria, and
this attachment may be selective for different cell types.5,16 Establishing a site in the host is critical to the
role of Bacteroides spp. and Parabacteroides spp., both as commensals on the mucosal surfaces of the
intestinal epithelium and as pathogens causing abscesses or other infectious processes. The adhesion and
invasion processes of intestinal Bacteroidales isolated from fecal microbiota of children with diarrhea
are shown in Table 16.3.
These organisms contain a variety of cell surface molecules that are either critical or advantageous
for colonization, including adhesins, hemagglutinins, a polysaccharide capsule, fimbriae, and proteases.
B. fragilis is one of the most important species of intestinal Bacteroidales and possesses a complex
capsular polysaccharide composed of at least eight different polysaccharides, which appear to be anti-
genically diverse. Also, B. fragilis may express three different types of capsules, large or small, and an
electron-dense layer. This heterogeneous nature of encapsulating structures within individual strains
could explain the controversial observations in the literature with respect to the B. fragilis surface struc-
tures related to adhesion. Similar surface structures have been seen in other intestinal species, such as
B. thetaiotaomicron.

16.5 Diagnosis
Species of Bacteroides are considered resident members in intestinal microbiota, and they are mostly
recovered in intra- and extra-abdominal infections. Most anaerobic infections originate from the host’s
resident microbiota and are considered infections of an endogenous nature. Conditions such as low blood
supply into an affected site can predispose the host to anaerobic infection, as well as trauma, foreign
body, malignancy, surgery, edema, shock, colitis, and vascular disease. Mixed infections with aerobic
or facultative organisms make the local tissue conditions favorable for the growth of anaerobic bacteria.
Anaerobic bacteria are the most common residents of the skin and mucous membrane surfaces and
natural cavities’ microbiota. Anaerobes belonging to the resident microbiota of the oral cavity can be
recovered from various infections adjacent to that area, such as cervical lymphadenitis, subcutaneous

TABLE 16.3
Adhesion and Invasion Assays of 114 Strains of the Intestinal Bacteroidales Isolated
from Children with Acute Diarrhea
Adhesion-Positive Invasion-Positive
Species No. of Isolates (%) (%)
Bacteroides fragilis 39 30 (77) 13 (33.3)
B. vulgatus  8 6 (75) 4 (50)
B. uniformis  6 5 (83.3) 5 (83.3)
B. ovatus  5 4 (80) 0
B. eggerthii  0 0 0
B. thetatiotaomicron  0 0 0
Parabacteroides distasonis  6 4 (66.6) 2 (33.3)
Bacteroides 253

abscesses, and burns in proximity to the oral cavity, human and animal bites, tonsillar and retropharyn-
geal abscesses, chronic sinusitis, chronic otitis media, and periodontal abscess. Species of anaerobic
Gram-negative rods including pigmented Prevotella, Porphyromonas, B. fragilis group, Fusobacterium,
and Gram-positive anaerobic cocci are associated with several infections, such as peritonitis, liver
abscess, intra-abdominal abscesses, neonatal infections, and recently to colorectal cancer.17,18 In addi-
tion, quantitative real-time PCR has provided a convenient, dependable, and rapid method to study the
diversity of the presence of the bft subtypes and the significance of ETBF in clinical infections.12

16.6 Susceptibility to Antimicrobials
Because Bacteroides spp. are most commonly found in mixed infections, β-lactams added or not with
β-lactamase inhibitors, carbapenems, clindamycin, and metronidazole are often the antibiotics of
choice.19,20 Clindamycin and metronidazole along with or in combination with some fluoroquinolones
are also used. These species are highly resistant to a broad range of antibiotics, including heavy metals,21
and their resistance to antimicrobials can vary in accordance with geographical locations. The resistance
to active drugs, such as imipenem, piperacillin-tazobactam, ampicillin-sulbactam, and metronidazole, is
observed in some strains.17,22 Considering that a distinct profile of drug resistance is observed in different
countries, it is possible that this resistance is geographically dependent. The resistance profiles of some
Bacteroidales species are shown in Table 16.4, and the distribution of resistance genes and β-lactamase
production in Bacteroides spp. and P. distasonis are shown in Table 16.5.

16.6.1 Mechanisms of Antimicrobial Resistance

Species of the genus Bacteroides display varied resistance mechanisms to antibiotics. B. fragilis is intrin-
sically resistant to several classes of antibiotics, but the mechanisms are poorly understood. The antimi-
crobial resistance can be due to altered target binding affinity, decreased penetration due to permeability
or efflux changes, or by the presence of inactivating enzymes.1,23 Bacteroides species isolated from clini-
cal samples have exhibited increasing resistance to many antibiotics, including cefoxitin, clindamycin,
metronidazole, carbapenems, and fluoroquinolones, such as gatifloxacin, levofloxacin, and moxifloxacin,
but the newer ones, including sitafloxacin, clinafloxacin, and garenoxacin, have shown a good activity.1

16.6.2  β
 -Lactam Agents
Most Bacteroides spp. are resistant to β-lactam agents as they produce β-lactamase commonly medi-
ated by the chromosomal cepA gene. However, this enzyme is inhibited by the most commonly used
β-lactamase inhibitors (sulbactam, clavulanic acid, and tazobactam).24

16.6.3 Carbapenems
Resistance to carbapenems remains rare, and the genes cfiA and ccrA, both encoding class B metallo-β-
lactamases, are able to degrade these agents. Some strains of Bacteroides contain “silent” carbapenemase
genes, and expression levels are dependent on promoter-containing insertion sequences (IS). Alterations
in penicillin-binding proteins (PBP) or porins, and efflux pumps are other bacterial resistance mecha-
nisms for carbapenems.

16.6.4 PBP
Penicillin and other β-lactams are cell-wall-active drugs. The structure of the β-lactam antibiotics facili-
tates their binding to the active site of PBP in an irreversible inhibition of proteins blocking the trans-
peptidation of the peptidoglycan layer. In B. fragilis, the sequenced genome showed the presence of
seven putative PBP genes, and it was difficult to correlate the MIC50 since this is not the only mechanism
responsible for the resistance.25
 254
TABLE 16.4
Resistance Profiles of Intestinal Bacteroidales Species to Eight Antibiotics
% Resistanceb
Bacteroides spp. and
B. fragilis B. vulgatus B. uniformis B. ovatus B. thetaiotaomicron P. distasonis Parabacteroides sp.
Antibioticsa (n = 66) (n = 14) (n = 7) (n = 7) (n = 2) (n = 16) (n = 144)
Amoxicillin 92.4 85.7 100 100 100 93.7 93
Amoxicillin/clavulanic acid 40.9 42.8 0 85.7 100 68.7 47.3
Ampicillin 98.4 92.8 100 100 100 87.7 96.4
Cephalexin 100 92.8 100 100 100 100 99

Laboratory Models for Foodborne Infections

Cefoxitin 7.5 0 14.2 0 0 12.5 23
Clindamycin 31.8 100 0 71.4 0 43.7 34.2
Penicillin 100 92.8 100 100 100 100 99
Tetracycline 59 50 28.5 71.4 0 43.7 53.5
a Breakpoints in accordance with CLSI (2007). Amoxicillin, amoxicillin/clavulanic acid, cephalexin, cefoxitin, clindamycin, penicillin, and tetracycline: 8 μg/ml; ampicillin: 4 μg/ml.
b All strains were susceptible to imipenem and metronidazole.
Bacteroides 255

TABLE 16.5
Distribution of Resistance Genes and β-Lactamase Production in Intestinal Bacteroides spp. and
Parabacteroides distasonis
Genes
β-Lactamase
cfiA cepA ermF tetQ nim Production
Species (No.) No. (%) No. (%) No. (%) No. (%) No. (%) No. (%)
B. fragilis (64) 51 (77.2) 53 (80.3) 16 (24.2) 54 (81.8) 5 (7.5) 60 (90.9)
B. vulgatus (14) 5 (35.7) 11 (78.5) 5 (35.7) 7 (50) 1 (7.14) 13 (92.8)
B. uniformis (7) 6 (85.7) 7 (100) 0 (0) 5 (71.4) 2 (28.5) 7 (100)
B. ovatus (7) 1 (14.2) 1 (14.2) 1 (14.2) 6 (85.7) 0 (0) 7 (100)
B. eggerthii (2) 0 (0) 2 (100) 1 (50) 2 (100) 0 (0) 2 (100)
B. thetaiotaomicron (2) 2 (100) 2 (100) 2 (100) 2 (100) 0 (0) 1 (50)
P. distasonis (16) 6 (37.5) 11 (68.7) 6 (37.5) 15 (93.7) 1 (6.25) 15 (93.7)

16.6.5 Outer Membrane Proteins or Porins

Some data have shown a correlation between alterations in porins and antimicrobial resistance in
Bacteroides spp., but it is not totally elucidated.

16.6.6 Aminoglycosides
Bacteroides species are intrinsically resistant to aminoglycosides since the uptake of this drug requires
free oxygen or a nitrate-dependent electron transport chain, and it is lacking in anaerobes.

16.6.7 Macrolides, Lincosamides, and Chloramphenicol

These antimicrobials inhibit the protein synthesis by binding to the 50S subunit of the bacterial ribo-
some. Genes conferring resistance to clindamycin and erythromycin in Bacteroides are similar.

16.6.8 Tetracycline
Tetracyclines inhibit bacterial protein synthesis by blocking the attachment of the tRNA-amino acid
to the ribosome. Studies show that 80%–90% of the clinical isolates of Bacteroides are resistant to
this drug, due to the presence of the gene tetQ that is the most commonly involved one in this resis-
tance. Also, the efflux pump for tetracycline has also been described as a resistance mechanism in this
microorganism.

16.6.9 Nitroimidazoles
Metronidazole is the most commonly used drug for anaerobic infections, and resistance to this drug is
rare. Metronidazole-resistant B. fragilis strains have been reported, and their resistance is due to the
presence of nim genes or associated genes to IS; however, this gene can be “silent” unless activated by
IS elements.26,27 In Bacteroides strains, seven nim genes (nimA to nimG) can be found, and each one is
associated with both a distinct mobile genetic element and a specific activating IS element.28

16.6.10 Quinolones
Quinolones inhibit DNA gyrase and DNA topoisomerase IV, which act in bacterial DNA replication,
and gene mutations producing these enzymes are the most common causes of quinolone resistance.29
256 Laboratory Models for Foodborne Infections

Mutations in gyrA causing fluoroquinolone resistance in B. fragilis have been identified. Studies have
shown that the quinolone resistance in clinical isolates and in mutants is due to the increased activity of
the efflux pumps belonging to the RND family.

16.6.11 Plasmids
These genetic elements are very common in Bacteroides species and are found in 20%–50% of strains.
Genes conferring resistance to different classes of antibiotics have been found on plasmids in Bacteroides.
Resistance genes nimA to nim-F and cfiA have been observed in clinical isolates and identified on trans-
ferable plasmids. Intestinal Bacteroidales species often display genetic elements, such as plasmids and/
or transposons, because of their predominant number and a close contact with other intestinal bacteria.
Clinical isolates of Bacteroidales often harbor cryptic plasmids (from 3 to 7 kb of size) with no defined
phenotypic or genotypic association.25

16.6.12 Multidrug Resistance
Little is known about efflux pumps in anaerobic bacteria. In Bacteroides spp., particularly in B. fra-
gilis, 16 homologs of RND pumps (bmeABC1 to bmeABC 16) called B. fragilis multidrug efflux have
been described. A MATE-type efflux system has also been characterized in B. thetaiotaomicron.28 This
system (B. fragilis multidrug efflux) shows that (1) each operon is formed for three components, (2) the
bmeC10 outer membrane component forms part of a contiguous gene with the bmeB10 pump gene,
(3) there are two functional pump genes (bmeB11 and bmeB11) which are transcribed separately in
bme11, and (4) at least 15 of the 16 genes are transcribed.1

16.7 Laboratory Models
Laboratories studying anaerobic bacteria use some principles for isolating and identifying Bacteroides
species. Most of the laboratories refer to the Wadsworth-KTL Anaerobic Bacteriology Manual and the
Manual of Clinical Microbiology. The processing for clinical specimens is important to the success of
isolation, and they are as follows: (1) aseptic collection of the specimen avoiding contamination with resi-
dent microbiota, (2) oxygen-free transport medium system, (3) species of Bacteroides grown on selective
medium Bacteroides bile esculin agar, and (4) B. fragilis is resistant to 20% bile, kanamycin, vancomy-
cin, and colistin. Several PCR schemes are also used to identify Bacteroides species according to the
genus and/or species level, such as using group-specific primers to the β-isopropylmalate dehydrogenase
gene leuB for rapid diagnosis from infections.30 In addition, a multiplex PCR assay with group- and
species-specific primers to identify rapidly 10 species of the B. fragilis group has also been developed.31
This bacterium can cause a wide range of human diseases including abscesses, diabetic foot, diarrhea,
and sepsis. Studies in vivo have shown that B. fragilis toxin (BFT) produced by ETBF induces both a
fluid secretion and exfoliation of intestinal epithelial cells. In certain human intestinal carcinoma cell
lines, particularly HT29/C1, this toxin causes the rounding-up of cells and the rearrangement of the
F-actin cytoskeleton.32,33
Inoculation of ETBF strains harboring different gene subtypes in germ-free mice produced histo-
pathological alterations mainly in the distal ileum–cecum–colon transition area. Moreover, inoculation
in the cecum can produce a superficial erosion and inflammatory infiltration in the lamina propria. It is
known that BFT toxin stimulates morphological changes in intestinal epithelial cell lines, such as HT29,
HT29/C1, Caco-2, T84, MDCK, and HCT-8.
Studies have suggested that strains carrying different bft gene subtypes may have a different patho-
genic potential; however, more studies are needed to evaluate the pathogenesis of each subtype in differ-
ent animal hosts.34 Certainly, they would provide a better understanding of these organisms in ecological
and pathogenic terms.
Bacteroides 257

16.8 Conclusions
Species of Bacteroides, including Bacteroides fragilis, in an unbalanced gastrointestinal ecosystem, can
produce several infections of endogenous nature, and recently have been linked to colon cancer. The
B. fragilis group is considered as the most commonly isolated anaerobic pathogen. Several virulence
factors are produced by B. fragilis. The presence of adhesins allows its colonization of the host’s tissues,
and Bacteroides spp. are able to produce bacteriocin-like substances that inhibit the growth of other
bacterial species or genera.
ETBF has been implicated in inflammatory intestinal diseases, and its intestinal colonization results
in acute or chronic intestinal inflammations. Species of Bacteroides are considered resident intestinal
microbiota, but they are often recovered from intra- and extra-abdominal infections. Bacteroides spp.
show great resistance to various antimicrobial drugs expressing resistance genes. B. fragilis is intrin-
sically resistant to several classes of antibiotics, but the mechanisms are poorly understood. Most
Bacteroides spp. are resistant to β-lactam by producing β-lactamase commonly mediated by the chro-
mosomal cepA gene.
Studies in vivo have shown that the inoculation of ETBF strains harboring different gene subtypes in
germ-free mice produced histopathological alterations, since the BFT toxin stimulates morphological
changes in different intestinal epithelial cells. The in vivo evaluation of pathogenic potentials of the sub-
types of B. fragilis could uncover important details about the biology and pathogenesis of this important
anaerobic bacterial group.

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12. Merino, V. R. C.; Nakano, V.; Liu, C.; Song, Y.; Finegold, S. M.; Avila-Campos, M. J. 2011. Quantitative
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13. Nakano, V.; Gomes, T. A. T.; Vieira, M. A. M.; Ferreira, R. C.; Avila-Campos, M. J. 2007. bft gene
subtyping in enterotoxigenic Bacteroides fragilis isolated from children with acute diarrhea. Anaerobe
13: 1–5.
14. Franco, A. A.; Cheng, R. K.; Goodman, A.; Sears, C. L. 2002. Modulation of bft expression of the
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15. Wexler, H. M.; Read, E. K.; Tomzynski, T. J. 2002. Identification of an OmpA protein from Bacteroides
fragilis: ompA gene sequence, OmpA amino acid sequence and predictions of protein structure.
Anaerobe 8: 180–191.
16. Nakano, V.; et al. 2008. Adherence and invasion of Bacteroidales isolated from the human intestinal
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17. Diniz, C. G.; Farias, L. M.; Carvalho, M. A.; Rocha, E. R.; Smith, C. J. 2004. Differential gene expres-
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18. Fukugaiti, M. H.; Ignacio, A.; Fernandes, M. R.; Júnior, U. R.; Nakano, V.; Avila-Campos, M. J. 2015.
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19. Behra-Miellet, J.; Calvet, L.; Dubreuil, L. A. 2004. A Bacteroides thetaiotamicron porin that could take
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20. Brazier, J. S.; Stubbs, S. L.; Duerden, B. I. 1999. Metronidazole resistance among clinical isolates belong-
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21. Ignacio, A.; Nakano, V.; Avila-Campos, M. J. 2015. Intestinal Bacteroides vulgatus showing resistance
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Antimicrobial resistance and prevalence of resistance genes in intestinal Bacteroidales strains. Clinics
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26. Pumbwe, L.; Chang, A.; Smith, R. L.; Wexler, H. M. 2007. BmeRABC5 is a multidrug efflux system that
can confer metronidazole resistance in Bacteroides fragilis. Microb. Drug Resist. 13: 96–101.
27. Pumbwe, L.; Glass, D.; Wexler, H. M. 2006. Efflux pumps over expression in multiple-antibiotic-
resistant mutants of Bacteroides fragilis. Antimicrob. Agents Chemother. 50: 3150–3153.
28. Ueda, O.; Wexler, H. M.; Hirai, K.; Shibata, Y.; Yoshimura, F.; Fujimura, S. 2005. Sixteen homologs of
the mex-type multidrug resistance efflux pump in Bacteroides fragilis. Antimicrob. Agents Chemother.
49: 2807–2815.
29. Miyamae, S.; Ueda, O.; Yoshimura, F.; Hwang, J.; Tanaka, Y.; Nikaido, H. 2001. A MATE family
multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron. Antimicrob.
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30. Miki, T.; et al. 2005. Simultaneous detection of Bacteroides fragilis group species by leuB-directed
PCR. J. Med. Invest. 52: 101–108.
31. Liu, C.; Song, Y.; McTeague, M.; Vu, A. W.; Wexler, H.; Finegold, S. M. 2003. Rapid identification of
the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific
primers. FEMS Microbiol. Lett. 222: 9–16.
32. Chambers, F. G.; Koshy, S. S.; Saidi, R. F.; Clark, D. P.; Moore, R. D.; Sears, C. L. 1997. Bacteroides fra-
gilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro.
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33. Donelli, G.; Fabbri, A.; Fiorentini, C. 1996. Bacteroides fragilis enterotoxin induces cytoskeletal
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pathogenicity of the Bacteroides fragilis toxin gene subtypes in gnotobiotic mice. Curr. Microbiol. 53:
113–117.
17
Brucella

S.C. Olsen and B. Bricker

CONTENTS
17.1 The Pathogen................................................................................................................................. 259
17.2 Brucella Species Associated with Foodborne Infections............................................................. 260
17.3 Microbiological Characteristics of Brucella..................................................................................261
17.4 Invasion and Cellular Pathogenesis...............................................................................................261
17.5 Clinical Disease in Humans.......................................................................................................... 263
17.6 Immunity....................................................................................................................................... 263
17.7 Foodborne Brucellosis.................................................................................................................. 264
17.7.1 Inactivation in Food Products.......................................................................................... 264
17.7.2 Transmission by Milk or Cheese Products....................................................................... 264
17.7.3 Meat or Fish Products....................................................................................................... 264
17.7.4 Laboratory Models........................................................................................................... 265
17.8 Conclusion..................................................................................................................................... 265
References............................................................................................................................................... 266

Clinical disease caused by infection with bacteria in the genus Brucella, brucellosis, remains one of
the most important zoonotic diseases worldwide and is considered to be reemerging in some countries.
Although the bacteria causing “Malta fever” was reported in human spleens by Bruce in 1887,1 the link
between human disease and infection in animal reservoirs was not correlated until 1918.2 The highest
prevalence of human disease is currently found in areas of Africa, Asia, Latin America, and the Middle
East and is associated with significant public health costs. Although transmission to humans can occur
through direct contact with abortions or birth materials from infected animals, indirect contact through
consumption of nonpasteurized dairy products is a common route for human infection.
Over half a million human cases are reported worldwide annually. Due to the wide-ranging and non-
specific nature of clinical signs, the true number may be much higher.3 Estimates of disease prevalence
range from <1 per 100,000 people in the United Kingdom, United States, and Australia, to >70 per
100,000 people in some countries in the Middle East and Central Asia.4 Of particular concern is the
observation that children appear to represent a high proportion of human brucellosis cases. Multiple
studies have demonstrated that addressing brucellosis in animal reservoirs is the most cost-efficient
mechanism for controlling human brucellosis.5–7 Therefore, most control strategies involve control of
livestock brucellosis through vaccination and use of test and removal strategies. Livestock vaccina-
tion has proved to successfully decrease human brucellosis and can be especially helpful in developing
nations. After initiation of a vaccination in Mongolia,8,9 the incidence rate of human brucellosis declined
from 4.8/10,000 to 0.23/100,000.

17.1 The Pathogen
Members of the genus Brucella are small (0.4–3 μm), Gram-negative, coccobacillary organisms within
alpha Proteobacteria that exist as intracellular pathogens in mammalian hosts. Most species of Brucella

259
260 Laboratory Models for Foodborne Infections

are obligate pathogens in that they do not exist as commensals nor are they found free-living in the envi-
ronment. However, a recently identified species of Brucella in voles, Brucella microti, may have genetic
differences that support a free-living as compared to a host-associated life style.10 Although traditional
species of Brucella may temporarily be recovered from environmental samples associated with infected
animals, environmental persistence is generally not believed to be of epidemiologic importance. Because
direct or close contact is generally believed to be required for transmission, maintenance of brucellosis in
animal populations generally requires continual infection of susceptible hosts. As humans are essentially
dead-end hosts, infection in humans requires exposure to brucellosis from animal hosts.
The Brucella genus has traditionally been characterized as having six species: Brucella abortus,
Brucella melitensis, Brucella suis, Brucella canis, Brucella ovis, and Brucella neotomae. The high
degree of homology at the genomic level has led to the proposition that the genus is actually composed
of only one species, B. melitensis, with the other classical species proposed as strains of B. meliten-
sis.11 However, host preference, epidemiologic features, and diagnostic benefits obtained by assigning
Brucella strains to separate “nomenspecies” based on their distinctive phenotypic characteristics is more
compelling and has supported retention of the current nomenclature. In the past 20 years, new Brucella
species from sea mammals,12–14 voles,15,16 and a prosthetic breast implant17 have been added to the genus.
Recent isolations from Austrian foxes,18 African bullfrogs (Pyxicephalus edulis),19 and baboons (Papio
spp.)20 may eventually further expand the number of species in the Brucella genus. Some of the classical
species (B. melitensis, B. abortus, and B. suis) are divided into biovars based on biochemical, pheno-
typic, and antigenic properties. Although division into biovars has been used for epidemiologic purposes,
biotyping can be somewhat subjective because it is based on subtle differences such as requirements for
higher CO2 tensions for growth, production of hydrogen sulfide, growth on media containing dyes (thio-
nin or basic fuchsin), and agglutination with monospecific A and M antisera.
One of the important characteristics of Brucella is the number of species in the genus that are capable
of causing zoonotic infections in humans. Most virulent strains of Brucella express the O-polysaccharide
(homopolymer of 4,6-dideoxy-4-formamido-α-d-mannopyranosyl units) on their lipopolysaccharide (LPS)
and are known as “smooth” strains. With the exception of B. canis which does not express the O side-chain
(a “rough” strain), most zoonotic strains of Brucella are smooth strains. Cumulative data suggests dosages
of virulent Brucella species required for 50% infection rates across mucosal surfaces of hosts other than
guinea pigs or mice are most likely in the 103–104 CFU range,21,22 Although virulent strains of Brucella
have a predilection for lymphoreticular tissues and the reproductive tract, most also frequently localize in
mammary tissues and are shed in milk.

17.2  B rucella Species Associated with Foodborne Infections

The preferred host of B. abortus is cattle but it can also naturally infect numerous other species includ-
ing: bison (Bison bison), elk (Cervus elaphus), camels (Camelus dromedarius and Camelus bactrianus),
yaks (Bos grunniens), African buffalo (Syncerus caffer), goats, swine, and other species.23–25 Biovars 1
and 2 have widespread worldwide distribution, while biovar 3 is predominantly found in India, Egypt,
and Africa. Several countries in Northern and Central Europe, Canada, Australia, Japan, and New
Zealand are considered to be free of this pathogen.
Sheep and goats are the preferred hosts of B. melitensis, but this species of Brucella is also known to
infect cattle,26 camels,27 wild ibex (Capra ibex),28 chamois (Rupicapra rupicapra),28 Nile catfish (Clarias
gariepinus),29 and other species. B. melitensis is considered to be endemic in parts of Central and South
America, Africa, Asia, the Middle East, and countries in the Mediterranean region. Areas considered
to be free of B. melitensis include Canada, the United States, South-East Asia, Northern and Central
Europe, Australia, and New Zealand.
Domestic and feral swine are the preferred hosts for biovars 1, 2, and 3 of B. suis. Biovars 1 and 3 of
B. suis can also infect cattle and horses.22,30 Biovar 2 can establish infection in hares (Lepus capensis)
and has a geographical distribution in a broad range between Scandinavia and the Balkans. Biovar 4
is predominantly associated with infection in caribou (Rangifer tarandus) but has also been found in
moose, arctic foxes, and wolves in subarctic areas. Biovar 5 has been exclusively isolated from wild
Brucella 261

rodents in the former USSR. Porcine brucellosis (biovars 1, 2, and 3) has widespread distribution in
domestic and feral swine globally, but prevalence is higher in some areas such as South-East Asia and
South America. It should be noted that unlike biovars 1, 3, and 4, biovar 2 of B. suis is not considered to
be important for causing human brucellosis. The increasing isolations of B. suis in cattle in the south-
eastern U.S. has public health significance due to the shedding of high numbers of bacteria within milk
of infected cattle.31
Isolates of marine mammal Brucella have been recovered from the Mysticeti and Odontoceti sub-
orders of cetaceans. Genomic data have led to taxonomic classification of these isolates into Brucella
ceti (predominantly porpoises and dolphins) and Brucella pennipedalis (predominantly seals) strains,
each of which has several subgroups.32 Bacteriologic studies have indicated these strains are phenotypi-
cally smooth; like the classical Brucella strains. Seropositive responses to marine brucellae have been
observed in 53 cetaceans, with isolation or identification of the bacteria having occurred in samples from
18 marine mammal species.14 It is suspected that cetacean brucellosis may be distributed worldwide in
the oceans. The ST27 genotype in Pacific B. ceti and B. pinnipedalis strains, characterized by a specific
genetic location for the mobile genetic element IS711, has been associated with zoonotic infections in
Peru and New Zealand.33

17.3 Microbiological Characteristics of Brucella

With the exception of B. suis biovar 3 with a single chromosome, most Brucella strains have two cir-
cular chromosomes encoding approximately 3.2 kb with two replicons. Bacteriophages have been iso-
lated from Brucella strains,34 but plasmids have not. The extent to which Brucella are able to undergo
exchange of genetic material among themselves or with other bacteria is unknown. It has been proposed
that intracellular niches inhabited by Brucella during residence in the host limit their opportunities for
genetic exchange with other bacteria. It should be emphasized that Brucella infections are polyclonal
rather than clonal infections, and clinical infection is generally associated with multiple bacteria crossing
mucosal barriers and colonizing tissues under in vivo conditions.23
Brucella are nonmotile and do not have spores. The cell wall of Brucella is typical for Gram-negative
bacteria. The outer membrane, approximately 4–5 nm in thickness, is composed of asymmetric layers of
LPS and phospholipids and is supported by an underlying 3–5 nm layer of peptidoglycan. Some proteins,
such as OmpA, are covalently bound to the peptidoglycan layer and stabilize the outer membrane. The
hydrophobic region of the membrane provides an anchor for proteins and forms a functional and struc-
tural barrier between the periplasm and the exterior of the cell. The periplasmic space varies from 3 to
30 nm. Porins in the outer membrane function as channels to the interior of the cell. Other proteins, such
as lipoproteins, are also embedded in the outer membrane.
The LPS of Brucella is composed of lipid A, core oligosaccharides, and O-polysaccharides. The struc-
ture of LPS in Brucella differs from Gram-negative enteric bacteria in that the backbone sugar of the
lipid A is different and the LPS has a low phosphate content. The LPS protects the bacteria from cationic
peptides, oxygen metabolites, and complement-mediated lysis. The O-polysaccharide on the LPS,
expressed by smooth strains, is very immunogenic and can express A and/or M antigens dependent upon
the species of Brucella. The Brucella cell envelope, LPS, lipoproteins, and flagellin display reduced
pathogen-associated molecular pattern (PAMP) for recognition by innate immunity, most likely due to
hydrophobic moieties of the outer membrane including ornithine lipids. The altered PAMP of Brucella
contributes to its stealthiness in vivo and its failure to induce robust innate immune responses.35–37

17.4 Invasion and Cellular Pathogenesis

Brucella penetrate the mucosal epithelium and are transported as free bacteria, or within phagocytic
cells, to regional lymph nodes. If bacteria are not localized and killed within regional lymph nodes
draining the site of infection, they replicate and spread via blood or lymph to other lymphoreticular
262 Laboratory Models for Foodborne Infections

tissues and organs such as the spleen, reproductive tract, and/or mammary gland. The bacteremia
associated with most Brucella species is short, and live bacteria are not readily isolated from blood
samples.
For cellular entry, smooth Brucella that have not been opsonized by antibodies use the cytoskeleton of
the host cell and interact with cholesterol-rich microdomains (lipid rafts) within the plasma membrane
that facilitate contact with the host cell and mediate internalization. Lipid rafts contain glycosphin-
golipids, cholesterol, and glycosyl-phosphatidylinositol-anchored proteins38 and facilitate membrane-
associated sorting events such as the formation of multi-subunit membrane complexes and signaling
across membranes and membrane fusion. Besides the plasma membrane, lipid rafts are also found in
intracellular organelles and vesicles. The Brucella LPS O-polysaccharide appears to be a key molecule
for interaction with lipid rafts on host cells, but also prevents complement-mediated bacterial lysis and
prevents host cell apoptosis.39 Opsonization of smooth strains of Brucella increases entry 10-fold and
occurs through IgG (Fc) and complement (C3b and 4b) receptors on the surface of phagocytes, which
diverts smooth bacteria from lipid rafts and targets entry to the phagolysosomal compartment. Receptor-
mediated phagocytosis leads to greater killing of internalized Brucella by monocytes. In contrast to
smooth Brucella, rough strains of Brucella cannot sustain interactions with lipid rafts and are phago-
cytosed by either toll-like receptor 4 or mannose receptor recognition of the LPS-deficient bacterial
surface leading to rapid targeting to the phagolysosomal compartment where they are generally unable
to replicate.
Smooth Brucella that enter via lipid rafts quickly traffic through the early endosomal compartment
and depart the phagosome to form the modified phagosome (termed brucellosome) by acquiring com-
ponents of endoplasmic reticulum in a manner similar to autophagosome biogenesis. Brucella initially
localize within acidified phagosomes40 where they are exposed to free oxygen radicals generated by the
respiratory burst. Brucella require acidification of the phagosomal compartment to a pH < 4.5 before
they display wild-type intracellular replication. The requirement for low pH is transient and only extends
through the initial stages of intracellular infection. Localization in an acidified environment induces
expression of the VirB operon (virB 1–10), which controls expression of genes associated with a type IV
secretion system. The VirB operon interacts with the endoplasmic reticulum to neutralize the pH of the
phagosome.41 Brucella-induced modifications of the phagosome leads to inhibition of phagosome matu-
ration and prevention of fusion with lysosomes. The brucellosome environment provides Brucella with
conditions of nutrient depletion and limited oxygen availability. It should be noted that under in vitro
conditions, up to 90% of virulent Brucella, and 99% of nonvirulent Brucella, may be killed following
intracellular entry.42
Brucella have multiple mechanisms to detoxify free radicals produced by host phagocytes, cationic
peptides, and oxygen metabolites. Brucella probably use both stationary and exponential stages for
intracellular survival, with stationary-phase physiology providing Brucella with benefits for adapting
to the harsh conditions encountered in the phagosome and exponential stages associated with repli-
cation under favorable conditions. The presence of cytochromes (cytochrome bc1 complex or quinol
oxidase) with a high oxygen affinity may represent an important adaptation of Brucella to their intra-
cellular survival. Several Brucella have been shown to utilize heme as an iron source in vitro, and have
a critical need for heme during residence in the phagosomal compartment43 and during replication
in trophoblasts.44 Brucella scavenge iron through siderophores such as 2,3-dihydroxybenzoic acid or
brucebactin.45
In accordance with its stealthy nature, Brucella has developed mechanisms to minimize stimulation of
pattern recognition receptors (PRRs) by the innate immune system of the host. The Brucella cell enve-
lope has high hydrophobicity, and its LPS has a noncanonical structure that elicits a reduced and delayed
inflammatory response as compared to other Gram-negative bacteria,40 and has lower stimulatory activity
on TLR4 receptors.46 The O side-chain on the LPS can form complexes with the MHC Class II molecules
that interfere with the ability of macrophages to present exogenous proteins. Brucella ornithine-containing
lipids and lipoproteins in the outer membrane are poor activators of innate immunity. Brucella bacteria
are also devoid of many classical structures involved in virulence such as pili, fimbria, capsules, and
plasmids that stimulate PRRs. The ability of Brucella to prevent phagosome maturation and fusion with
Brucella 263

lysosomes may interfere with other innate and adaptive immune processes. As proteins have been identi-
fied in Brucella that demonstrate significant homology with toll-like receptor (TLR) adaptor molecules,
these peptides may be a mechanism to interfere with, or subvert TLR signaling.47 Compared to other
Gram-negative bacteria, Brucella induces a reduced innate immune response, and a lower rate of matu-
ration and activation of dendritic cells, which may impair development of adaptive immune responses.

17.5 Clinical Disease in Humans

Human infection with B. abortus can cause chronic, debilitating clinical illness with nonpathogno-
monic symptoms48,49 including fever, night sweats, anorexia, polyarthritis, meningitis, and pneumonia.
Incubation periods are variable and can range from a week to months (up to 6 months). Although mor-
tality is uncommon, it primarily results from endocarditis associated with B. melitensis infection. The
most common manifestations of localized disease are osteoarticular (i.e., peripheral arthritis, sacroili-
itis, spondylitis).50 Human infection occurs across mucosal surfaces by aerosolization into respiratory
tissues, oral consumption, or penetration through breaks in the epidermis. Inadvertent exposure to live
vaccine strains, most commonly via needle sticks, has also been a frequent source of human infections,
especially in the veterinarian profession. In the absence of specific treatment, infection may persist for
weeks or months.51 Relapse of infection after treatment is not uncommon, with relapse occurring within
6 months after therapy and not usually associated with emergence of antibiotic-resistant strains.52 The
duration of the human illness and its long convalescence makes brucellosis both a medical and an eco-
nomic problem due to costs associated with treatment and the time lost from normal activities.52 The
disease can be insidious and may present in many atypical forms.52 While rare, human-to-human dis-
semination of brucellosis through breast milk has occurred.53–55
Currently in the United States, human brucellosis is predominantly a disease associated with interna-
tional travel, or as a foodborne disease caused by ingestion of nonpasteurized dairy products originating
from other countries.56,57

17.6 Immunity
Currently, no vaccines are available to protect humans from brucellosis. Currently available livestock
vaccines are composed of attenuated live bacteria, which can cause clinical disease in humans in
the event of inadvertent infection. Long-term protection against brucellosis in livestock is associ-
ated with stimulation of cellular immunity, while antibodies are considered to play a minor role
in protection. Although specific correlates of protective immunity are currently not known, it is
believed that protection is mediated by the Th1 subset of CD4+ lymphocytes and is associated with
the production of IFN-γ and other cytokines associated with cellular immunity. CD4+ cells play a
central role in coordinating and culminating the adaptive immune response by differentiation into
functional subsets, such as the Th1 type, which is associated with the production of interferon-γ
(IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α). CD4+ cells also provide growth
factors and signals for the generation and maintenance of CD8+ T cells. CD8+ cells are considered to
be of importance for protective immunity because of their ability to lyse or kill infected cells, thereby
releasing Brucella from intracellular hiding and exposing it to extracellular bactericidal mechanisms.
Cytokines released by CD4+ and CD8+ cells may activate macrophages and dendritic cells, thereby
increasing their bactericidal activity against Brucella.58 Although many studies have used qualita-
tive measurements of IFN-γ production as an indication that vaccination induces protective Th1
responses, recent data with other pathogens have suggested that a better indicator of the quality of
the immune response after immunization may be through the measurement of increases in antigen-
specific polyfunctional T cells capable of producing a traid of relevant cytokines.59 In addition, the
type of memory cell produced may influence the capability for protective responses, cytokine pro-
duction, and cytotoxic activity.60
264 Laboratory Models for Foodborne Infections

17.7 Foodborne Brucellosis
17.7.1 Inactivation in Food Products
In regard to survival in food materials, Brucella is killed by pasteurization61 or heat treatment (up to
71°C), but environmental studies indicate the bacteria can survive drying, freezing, or long-term stor-
age at cold temperatures.62 Souring of milk has negligible effects on the survival of Brucella63 and the
bacteria can survive in milk for up to 87 days, water for up to 60 days, less than a week in yogurt, and in
ripened cheese for up to 50 days despite the reduction in pH associated with ripening.64–67 Ripening of
cheese at 24°C for 20 days was associated with the inability to recover B. melitensis, whereas the bacteria
were recovered when cheese was ripened at 4°C.68 In meat products, processes for preparation of cured/
smoked meat can reduce, but not completely kill, Brucella.69

17.7.2 Transmission by Milk or Cheese Products

Foodborne transmission of Brucella to humans is almost exclusively associated with B. abortus, B. suis,
and B. melitensis infection. As these strains of Brucella have a predilection for the mammary gland with
intermittent shedding by caprine, bovine, or water buffalo hosts,2,70–73 the predominant route for human
infection occurs through consumption of dairy products made from nonpasteurized milk. Data suggest
that shedding of B. melitensis bacteria in goat milk is generally greater (up to 2.5 × 105 bacteria/mL)71 as
compared to shedding of B. abortus bacteria in milk from cattle.2 As chronically infected animals fre-
quently do not demonstrate any clinical signs, shedding of Brucella in milk can be intermittent and occur
in the absence of symptoms of infection. In cattle, excretion of B. abortus in milk was more consistent
and abundant during later parts of lactation.73 It should be noted that similar localization in mammary tis-
sue occurs in humans, as shedding of B. melitensis in human breast milk has been noted in a number of
reports.53–55
Although swine are not a source for milk products, B. suis frequently infects cattle and is shed in high
numbers of milk. Transmission of B. suis from feral swine to cattle is a significant and growing issue in
the south and southeastern parts of the United States. Consumption of nonpasteurized bovine milk con-
taining B. suis has caused clinical disease in numerous people on several occasions.74,75 This is a concern
due to recent efforts in many states to allow the sale of nonpasteurized milk products. It should be noted
that The American Academy of Pediatrics has issued a policy statement that raw or unpasteurized milk
or milk products pose a health risk for foodborne infection by numerous agents (including Brucella),
and scientific evidence does not support the argument that pasteurization alters the nutritional content
of milk.76
In most countries in which brucellosis has been effectively controlled in domestic livestock, human
infection is associated with international travel to countries with endemic disease, or importation of
nonpasteurized milk products.77–79 However, in the absence of travel for tourism, ethnic or cultural habits
are a very important factor for influencing human infections with Brucella spp., with epidemiologic data
from some countries indicating higher prevalence of brucellosis in certain ethnic groups.80–82 Although
some infections in these ethnic groups may be related to foodborne infection in the country of origin,
many appear to be related to unregulated movement of unpasteurized milk products into a country.83–86
Unregulated international movement of unpasteurized milk products may to some extent reflect cultural
food preferences.84–86

17.7.3 Meat or Fish Products

As Brucella can be recovered from raw meat,87 foodborne infection could also occur by handling or con-
sumption of raw or undercooked meat products. In general, bacterial loads in animal muscle are at lower
levels as compared to levels in unpasteurized milk products.88 This route of foodborne transmission is of
greatest concern in cultures where consumption of raw or undercooked meat occurs.89,90
Brucella 265

Ingestion of fish or marine products may also be a source for foodborne infection with Brucella.
Three cases of human infection with the ST27 genotype of Pacific B. ceti and B. pinnipedalis strains
have occurred in individuals with a history of consumption of raw shellfish or fish.33,91 The isolation of
B. melitensis biovar 3 from Nile catfish may also indicate a possible route for foodborne transmission
to humans. The presence of B. melitensis in this species was hypothesized to have spilled over from
endemic infection in ruminants in the area.29

17.7.4 Laboratory Models
Due to their high susceptibility to infection with Brucella, guinea pigs have historically been used for
isolation. For some samples such as cheeses, animal inoculation may be the only means for detecting
the presence of brucellae.92 Guinea pigs are also an accepted model for testing the virulence of Brucella
strains by assessment of spleen pathology and quantification of infection at 8–12 weeks after inocula-
tion. Lesions of brucellosis are observed in the liver, spleen, lungs, and lymph nodes.93 Guinea pigs have
been used to evaluate effectiveness of antibiotic treatments, and for evaluation of efficacy of vaccines.94
Currently, inbred mice are the most common laboratory animal model used to study chronic infec-
tion or disease pathogenesis.95 This is partly because Brucella colonize and induce pathologic lesions in
liver and spleen tissues of mice that to some extent mimics human infection. Inbred mouse strains differ
in susceptibility to infection, with BALB/c and CBA/H being considered relatively more susceptible
to infection and C57BL/6 and C57BL/10 more resistant.95 Intraperitoneal is the most common route of
infection for mouse models, but aerosol, oral, and intranasal routes have also been used. It should be
noted that murine models generally do not accurately replicate disease pathogenesis in natural hosts
where Brucella spp. more frequently localize in lymphatic tissues, mammary gland, and reproductive
organs, and reproductive losses (i.e., abortion in females and orchitis/epididymitis in males) are common.
With the exception of abortion, natural hosts generally do not demonstrate clinical disease. In compari-
son, humans appear to be aberrant hosts for Brucella and often have significant symptoms of clinical
disease that are frequently chronic.
Other laboratory models that have been used include rabbits, rats, and nonhuman primates although
each has limitations regarding the study of Brucella pathogenesis. Brucella-induced abortion is rare,
but rats can venerally transmit B. abortus and offspring can be latently infected.93 Lesions of necrosis
in periplacentomal chorionic epithelium and metritis have been observed in the uterus of pregnant rats
infected with B. abortus. Rabbits are partially susceptible to Brucella, with susceptibility increasing
with pregnancy. Nonhuman primate models (Macaca arctoides and Macaca mulatta) have been infected
through oral, subcutaneous, and aerosol routes with virulent strains of Brucella. Bacteremia was dem-
onstrated for up to 8 weeks, and lesions of focal granulomatous hepatitis, splenitis, lymphadenitis, and
occasional lesions of granulomatous orchitis, epididymitis, and endometritis were reported, similar to
lesions in humans infected with Brucella reviewed by Silva et al.95 Current knowledge would suggest
that primate models most closely mimic pathogenesis of disease in humans. Although laboratory models
continue to have value for basic research, they do not accurately replicate disease pathogenesis in natural
hosts of Brucella.

17.8 Conclusion
Brucellosis in humans remains a significant zoonosis in many parts of the world and frequently occurs
through international travel. In the absence of direct contact with infected animal hosts, transmission of
infection to humans almost exclusively occurs through products made from nonpasteurized milk. Brucella
appear to have a long shelf life in these types of products. In some regions or countries, human brucellosis
is associated with certain ethnic groups and may be related to dietary preferences. Although addressing
the disease in its natural hosts is the most economic approach, regulatory efforts to prevent the sale or con-
sumption of nonpasteurized milk products would also reduce human brucellosis and benefit public health.
266 Laboratory Models for Foodborne Infections

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perception and knowledge in French Haji pilgrims. Vector Borne Zoonotic Dis. 13, 425, 2013.
79. Di Pierdomenico, A., Borgia, S.M., Richardson, D., & Baqi, M. Brucellosis in a returned traveller. Can.
Med. Assoc. J. 183, E690, 2011.
80. Chommel, B.B., et al. Changing trends in the epidemiology of human brucellosis in California from
1973 to 1992. A shift toward foodborne transmission. J. Infect. Dis. 178, 1216, 1994.
81. Taylor, J.P., & Perdue, J.N. The changing epidemiology of human brucellosis in Texas 1977–1986. Am.
J. Epidemiol. 130, 160, 1989.
82. Al Dahouk, S., et al. Human brucellosis in a nonendemic country: a report from Germany, 2002 and
2003. Eur. J. Clin. Microbiol. Infect. Dis. 24, 450, 2005.
83. Galbraith, N.S., Ross, M.S., DeMowbray, R.R., & Payne, D.J. Outbreak of Brucella melitensis type 2
infection in London. Br. Med. J. 1, 612, 1969.
84. Thaper, M.K., & Young, E.J. Urban outbreak of goat cheese brucellosis. Pediatr. Infect. Dis. 640, 1986.
85. Beutlich, J., et al. Characterization of illegal food items and identification of foodborne pathogens
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2015, doi: 10.1016/j.ijfoodmicro.2014.10.017.
86. Fosgate, G.T., Carpenter, T.E., Chomel, B.B., Case, J.T., DeBess, E.E., & Reilly, K.F. Time-space clus-
tering of human brucellosis, California, 1973–1992. Emerg. Infect. Dis. 8, 672, 2002.
87. Hassan Ali, N., Farooqui, A., Khan, A., Khan, A.Y., & Kazmi, S.U. Microbial contamination of raw
meat and its environment in retail shops in Karachi, Pakistan. J. Infect. Dev. Countries 4, 382, 2010.
88. Pappas, G., Akritidis, N., Bosilkovski, M., & Tsianos, E. Brucellosis. New Engl. J. Med. 352, 2325,
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89. Mohd, M.G. Brucellosis in the Gezira area, Central Sudan. J. Trop. Med. Hyg. 92, 86, 1989.
90. Chan, J., Baxter, C., & Wenman, W.M. Brucellosis in an inuit child, probably related to caribou meat
consumption. Scand. J. Infect. Dis. 21, 337, 1989.
91. Whatmore, A.M., et al. Marine mammal Brucella genotype associated with zoonotic infection. Emerg.
Infect. Dis. 14, 517, 2008.
92. Alton, G.G., Jones, L.M., Angus, R.D., & Verger, J.M. Techniques for the brucellosis laboratory. Institut
National de la Recherche Agronomique, Paris, pp. 33, 154, 1988.
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cellosis research. J. Biomed. Biotechnol. 2011, 518323, 2011.
18
Burkholderia

Danielle L. Peters, Fatima Kamal, and Jonathan J. Dennis

CONTENTS
18.1 Introduction................................................................................................................................... 271
18.2 B. mallei........................................................................................................................................ 272
18.2.1 Overview.......................................................................................................................... 272
18.2.2 Laboratory Infection Models............................................................................................ 272
18.3 B. pseudomallei............................................................................................................................. 273
18.3.1 Overview.......................................................................................................................... 273
18.3.2 Laboratory Infection Models.............................................................................................274
18.4 Burkholderia cepacia Complex (Bcc).......................................................................................... 275
18.4.1 Overview.......................................................................................................................... 275
18.4.2 Laboratory Infection Models............................................................................................ 276
18.5 B. gladioli pv. cocovenenans........................................................................................................ 278
18.5.1 Overview.......................................................................................................................... 278
18.5.2 Laboratory Infection Models............................................................................................ 278
18.6 Conclusions................................................................................................................................... 279
References............................................................................................................................................... 279

18.1 Introduction
The genus Burkholderia covers a diverse group of Gram-negative β-proteobacteria. Although currently
at least 60 species and proposed species exist in the genus Burkholderia, very few have been studied
extensively. Much of the research to date has focused on the bacteria of the Burkholderia cepacia com-
plex (Bcc), Burkholderia mallei, Burkholderia pseudomallei, and more recently, Burkholderia gladioli.
The bacteria of the Bcc are pathogens that typically cause serious infections in plants, animals, and
humans.1–3 However, they can also be beneficial in the environment as they fix nitrogen symbiotically
for plants, produce antibiotics and antifungals, and have the capacity to degrade organic and xenobiotic
compounds.4–6 B. mallei causes “glanders,” a rare condition usually associated with horses, but that can
also affect humans, whereas B. pseudomallei, endemic in Southeast Asia, causes “melioidosis,” a serious
disease in humans with a wide variety of symptoms.7 The species B. gladioli has been divided into four
pathovars: gladioli, alliicola, agaricicola, and cocovenenans.8 The first three B. gladioli pathovars listed
are primarily plant pathogens,9–11 but members of these pathovars can occasionally also infect immu-
nocompromised patients with chronic granulomatous disease (CGD), cystic fibrosis (CF), or acquired
immune deficiency syndrome (AIDS).8,12,13 The taxonomic description of B. gladioli pvs. gladioli, alli-
icola, and agaricicola, published in 2003, suggests that they do not produce toxins that are harmful to
humans, although some strains have since been shown to synthesize toxoflavin.8,14 The fourth B. gladioli
pathovar, B. gladioli pv. cocovenenans, is distinct from the other pathovars with regards to its epidemiol-
ogy and pathogenicity. Although the other Burkholderia species can be found as contaminants in food
and water supplies (including the Bcc, B. mallei, and B. pseudomallei), B. gladioli pv. cocovenenans is
the only bacterium of the Burkholderia genus that is traditionally characterized as a foodborne pathogen.

271
272 Laboratory Models for Foodborne Infections

Strains of B. gladioli pv. cocovenenans do not cause disease directly, but instead produce toxins that
contaminate foods before ingestion by humans, similar to Clostridium botulinum. The laboratory infec-
tion models used to test Burkholderia pathogenicity and virulence are generally useful across the genera,
whereas specific models have been devised to measure B. gladioli toxin activity. This chapter will briefly
present the literature regarding the presence of B. mallei, B. pseudomallei, the Bcc, and B. gladioli in
food and water supplies, and subsequently review the laboratory infection models that have been devel-
oped for each group of Burkholderia pathogenic bacteria.

18.2  B . mallei
18.2.1 Overview
B. mallei causes glanders, a zoonosis disease of horses that can also be transmitted to humans. The
symptoms vary depending on the route of transmission, but can include pneumonia, skin lesions, and
septic shock.15 Horses generally become infected following ingestion of B. mallei introduced into their
food and water by other infected horses.15 Humans may also become infected via ingestion, typically due
to drinking untreated water.16 Glanders is difficult to diagnose, there is no vaccine available to protect
against the bacterium, and any potentially successful therapy requires prolonged antibiotic treatment.
However, as a result of stringent measures implemented to control the spread of glanders, the disease
has not been seen in the Western hemisphere since 1939 (except following accidental laboratory expo-
sure).17,18 However, because both B. mallei and B. pseudomallei have been identified as potential food
contaminants, there is concern that these species could be released as bioterrorism agents into a coun-
try’s food supply.19,20

18.2.2 Laboratory Infection Models

Several animals have been investigated as possible infection models of glanders. Because B. mallei natu-
rally exists in an equid reservoir, and equine glanders share many features with human glanders, disease
progression has been well studied in equine models.15,21 However, working with equids is cost and space
prohibitive. To address this problem, the US Army Medical Research Institute of Infectious Diseases
(USAMRIID) conducted a comprehensive study in the late 1940s evaluating the susceptibility of several
common laboratory animals to B. mallei infection.22 This study, as well as others, showed that cattle,
hogs, birds, rats, and rabbits are relatively resistant to B. mallei infection, that mice, sheep, monkeys, and
camels are moderately susceptible, that guinea pigs are variably susceptible, and that cats, dogs, ferrets,
goats, and hamsters are highly susceptible to infection.23–25 Although the infectious dose of B. mallei in
humans has not been established, the low incidence of human glanders throughout history suggests that
humans are moderately resistant to B. mallei infection. The Syrian golden hamster is one of the most
common laboratory animals used to experimentally model acute glanders infection and assess B. mallei
strain virulence.23,26–30 Although the pathological characteristics of glanders disease progression in ham-
sters are comparable to that observed in humans, hamsters are significantly more sensitive to B. mallei
infection, with an approximate LD50 of 10 CFU via aerosol or IP routes of infection. Therefore, hamsters
are often chosen to model the acute phase of glanders, whereas BALB/c mice, which like humans are
only moderately susceptible to B. mallei infection, are commonly used to model chronic B. mallei infec-
tion and disease progression.23,31,32
More recently, experiments were performed to establish a nonhuman primate marmoset model of
intranasal infection to study B. mallei in order to develop effective medical countermeasures.33 In mar-
mosets inoculated intranasally with moderate doses (104 –105 CFU) of B. mallei, 83% developed acute
lethal infection within 3–4 days and exhibited clinical signs such as lethargy, rough coat, and conjunc-
tivitis, and B. mallei was cultured from the lungs, spleen, and liver of these animals, with lesion foci
similar to that characteristic of human glanders.33 Similarly, Nelson et al.34 demonstrated that the com-
mon marmoset could also be used to model B. mallei subcutaneous infection. Marmosets inoculated in
the inner thigh with a lower dose 102 CFU B. mallei reached endpoints 7–10 days postchallenge,34 with
Burkholderia 273

typical clinical signs and tissue histopathology, whereas intranasal inoculation at this dosage did not
produce any obvious symptoms of disease. The differences observed in bacterial dosage and survival
reflect the different routes of inoculation used: subcutaneous injection likely circumvents physical bar-
riers used to combat infection, such as the respiratory system’s mucosal barrier, and allows immediate
access to host tissues. These results suggest that, depending on the site of inoculation and dosage used,
marmosets can be used to model effectively different aspects of B. mallei infection and pathogenesis,
producing results similar to infections observed in humans and equids.
Also recently, researchers have begun to utilize low-cost alternative infection models in order to study
various aspects of B. mallei infection. For example, in order to evaluate 650 potential gene targets in
the B. mallei “virulome,” Schell et al.35 utilized Galleria mellonella (wax moth) larvae as a surrogate
host. B. mallei was highly pathogenic in this host, and importantly, four previously identified mutants
avirulent in hamster and mouse models also proved to be avirulent in the wax moth larvae, thus vali-
dating this approach.35 Similarly, Madagascar hissing (MH) cockroaches possess a number of qualities
that make them desirable for use as B. mallei surrogate hosts, including low cost, ease of breeding and
handling, a competent innate immune system, and the ability to thrive at 37°C.36 MH cockroaches are
highly susceptible to B. mallei infection, the LD50 being <10 CFU. Similar to the results obtained for
G. mellonella, B. mallei avirulent Type VI secretion system mutants originally identified in rodents were
also attenuated in MH cockroaches.36 Again, this indicates that alternative infection models can be effec-
tive and may be useful as an alternative to mammals to study some aspects of host–pathogen interactions
and bacterial virulence determinants.

18.3  B . pseudomallei
18.3.1 Overview
B. pseudomallei causes melioidosis, a potentially fatal condition with a variety of symptoms including
pneumonia, skin lesions, and septic shock.7 Most melioidosis cases are seen in Southeast Asia (especially
Thailand) and northern Australia, although incidents also occur in nonendemic areas such as Brazil.7,37 In
Southeast Asia, approximately 80% of the population tests seropositive by the age of 4 years,38 although
only a small percentage of these seropositive individuals go on to develop melioidosis. This finding sug-
gests that the dose and frequency of inoculum influences the infectivity and severity of the symptoms. In
support of this idea, increased rates of melioidosis follow the monsoon and typhoon seasons,39,40 when
prolonged inhalation of storm-generated aerosols of B. pseudomallei-contaminated particles would be
highest.7
Although the first cases of melioidosis were described in 1913, there remains some debate as to how
B. pseudomallei is transmitted to humans.41,42 It has been established that infections can occur following
inoculation of broken skin or inhalation of water aerosols containing B. pseudomallei.43 Although there
is some evidence of infection occurring following ingestion, it is by no means conclusive. Historically,
it was thought that melioidosis was spread through the consumption of contaminated food and water,
as it can be transmitted in this manner to guinea pigs, rabbits, and rats.41,44 However, B. pseudomallei
has not been definitively shown to spread to humans by this route, although a report published in 1998
described the development of melioidosis in five adults in northwestern Australia.45 Using pulsed-field
gel electrophoresis (PFGE) typing, it was determined that all five patients were infected with the same
PFGE type and that this type was identical to that of B. pseudomallei isolates found in the area’s drink-
ing water, suggesting that these cases developed due to ingestion of a single source of B. pseudomallei.45
B. pseudomallei can also be isolated from drinking water in areas where melioidosis is not endemic.
Zanetti et al.46 showed that B. pseudomallei could be isolated from 7.1% of drinking water samples
taken in Bologna, Italy, and that the levels of B. pseudomallei were relatively high, ranging from 1020
to 15,000 CFU/100 mL sample. Treatment of water with chlorine will kill B. pseudomallei (as well as
B. mallei) in an experimental setting, but various factors in the environment (including attachment and
nutrient limitation) may affect this susceptibility.47 Antibiotic treatment for acute and chronic melioi-
dosis includes ceftazidime followed by trimethoprim–sulfamethoxazole (TMP–SMX) with or without
274 Laboratory Models for Foodborne Infections

doxycycline.48 Despite this, melioidosis has a high mortality rate and is the third most common cause of
death from an infectious disease in endemic areas.49 Probable causes for this include late diagnosis due
to nonspecific symptomatology and a high recurrence rate when antibiotics are discontinued early. Given
the severity of the disease, it is understandable that there is significant interest in the development of a
B. pseudomallei vaccine, and the identification of an accurate laboratory infection model for melioidosis.

18.3.2 Laboratory Infection Models

Historically, the most heavily utilized animal model for melioidosis has been rodents.50 Animal studies
have revealed that the severity of melioidosis and the resulting proinflammatory cytokine involvement
stimulated by B. pseudomallei infection are mouse strain dependent, and mice are typically classified
as either models of acute infection (BALB/c) or chronic infection (C57BL/6) based on their sensitivity
to infection and inflammatory responses.50–52 However, as mentioned above, dose and route of infection
may also affect the nature of the melioidosis presentation.53 In either mouse strain, B. pseudomallei
presents the same lung, liver, and spleen abscess foci as those observed in humans. However, despite
these parallels, early studies in mice have been based on intravenous or intraperitoneal exposure, which
does not represent the natural route of human infection.50,54 In contrast, subcutaneous and aerosol mouse
infection models both result in a systemic infection with similar clinical presentation as percutaneous
inoculation and inhalational melioidosis in humans.50,55,56
Similarly, rats have been successfully used to model pulmonary and chronic melioidosis, and have
been instrumental in understanding how underlying diabetes increases the risk of B. pseudomallei infec-
tion.57 Syrian golden hamsters have also been evaluated as a small animal model of melioidosis but
do not closely mimic human infection due to their extreme sensitivity to B. pseudomallei infection.26
However, they have also been instrumental in evaluating B. pseudomallei virulence factors, such as
which Type VI secretion systems are essential for virulence and intracellularity.58
There are limited published data characterizing B. pseudomallei infections in animal models other
than mice and rats. Miller et al.22 evaluated the susceptibility and natural environment of B. pseudomal-
lei infection in different animal models, particularly rhesus macaques, following subcutaneous infec-
tion with graded doses of B. pseudomallei. Only the primate receiving the highest dose (106 CFU) had
clinical and immunological signs of infection, including abscess formation at the site of infection, fever,
and elevated white blood cell counts. However, the infection was cleared after 1–2 weeks, suggesting
that rhesus macaques are resistant to B. pseudomallei.22 Similarly, an evaluation of intraperitoneal and
subcutaneous B. pseudomallei infection in Hamadryas baboons resulted in the same lung, liver, spleen,
and lymph node tropisms observed in humans.59 More recently, the pathology of inhaled B. pseudomal-
lei in the marmoset, rhesus macaque, and African green monkeys was evaluated.60,61 The marmoset is
exquisitely sensitive to aerosols of B. pseudomallei strain K96243; 102 CFU was consistently lethal.60
Disease onset occurred within 24 h and was characterized by fever, decreased activity, and a rapidly
progressive septicemia similar to the acute form of melioidosis observed in humans. The disease pro-
gression of melioidosis following inhalation in rhesus macaques and African green monkeys is similar
to the marmoset.61 All of these monkeys exhibit a nonspecific fever 24–40 h postinfection, followed by
dyspnea and septicemia. Gross pathology was also similar as both rhesus macaques and African green
monkeys presented with lung lesions, lymph node enlargement, splenomegaly, and bone marrow lesions.
These reports highlight the comparable pathology of B. pseudomallei infection in nonhuman primates
and humans.
Because melioidosis is well modeled in rodents and primates, initially few alternative infection mod-
els were developed to study B. pseudomallei. However, with the influx of funding to support research
into “medical countermeasures,” several new B. pseudomallei alternative infection models have been
recently developed. One of these models is a measure of invasion and cytotoxicity toward macrophages.
Murine macrophages were used to identify B. pseudomallei virulence factors that were expressed using
an in vivo expression technology (IVET) experiment.62 Researchers found that the B. pseudomallei
Cluster 1 Type 6 secretion system was expressed 12-fold over cell culture medium, when cultured in the
presence of murine macrophages. Interestingly, the T6SS-1 genes appeared to play no role in the sur-
vival or growth of B. pseudomallei when cultured inside the macrophages. In contrast, B. pseudomallei
Burkholderia 275

T6SS-1 Hcp proteins, integral membrane proteins of the T6SS mechanism that are expressed dur-
ing functional operation of the system, were found to be important in the intracellular behavior of
B. pseudomallei in RAW 264.7 macrophages. Mutants unable to express the Hcp1 protein were only
weakly cytotoxic toward murine macrophages, exhibited a growth defect inside of macrophages, and
were unable to form multinucleated macrophage cells like wild-type B. pseudomallei.58 In a different
murine macrophage cell line (J774A.1), it was shown that different strains of B. pseudomallei exhibit
different levels of pathogenicity and that these differing strain-dependent levels of pathogenicity were
conserved across mice infection models, murine macrophages, and a G. mellonella (wax moth larvae)
alternative infection model.63 It was also shown that in these same models, related species B. oklaho-
mensis and B. thailandensis were significantly less virulent than B. pseudomallei strains.63 Interestingly,
another study demonstrated an inverse relationship between B. pseudomallei virulence in a murine mac-
rophage J774 A1 phagocytosis assay as compared to a mouse infection model.54 However, in this case,
the mouse model used was an intraperitoneal infection model, which, as mentioned above, does not
accurately mimic the human condition, even though it is reproducible.54
Besides G. mellonella, which has also been used to identify virulence factors of and antibiotic phar-
macokinetics against B. pseudomallei,64,65 another invertebrate alternative infection model that has been
successfully used to characterize B. pseudomallei infection is Caenorhabditis elegans.66 At least five dif-
ferent bacterial mechanisms of killing C. elegans have been described, including toxin-mediated killing,
persistent or transient killing, intestinal infection, direct invasion, and biofilm formation.67 Originally,
studies suggested that a B. pseudomallei toxin was involved in C. elegans killing68; however, more recent
studies suggest that no toxin-mediated killing is involved, but rather direct prolonged contact with the
bacterium.69 How these putative B. pseudomallei virulence mechanisms identified in C. elegans relate
to the human infection condition remains to be determined. Unlike the G. mellonella infection model,
both C. elegans and the tomato leaf 70 infection models are unable to differentiate between B. pseudo-
mallei and B. thailandensis virulence levels, which may be an important consideration with respect to
accurately modeling human melioidosis.

18.4  B
 urkholderia cepacia Complex (Bcc)
18.4.1 Overview
The Bcc is currently made up of 20 closely related Burkholderia species.71,72 These bacteria cause poten-
tially fatal infections in immunocompromised patients, particularly those with CF and CGD.73,74 Whereas
both melioidosis and glanders can have a wide range of symptoms that affect many different systems of
the body, Bcc infections tend to be limited to the lungs. Bcc infections may be asymptomatic, may cause
a patient’s condition to worsen over time, or may develop into a rapidly fatal pneumonia accompanied by
damage to the lung tissue and septicemia.3
The bacteria of the B. cepacia complex are found more commonly in food and water supplies than
either B. pseudomallei or B. mallei. They can be isolated from the rhizospheres of rice, corn, wheat, soy-
bean, and alfalfa plants.75,76 Bcc bacteria have also been found associated with Pleurotus ostreatus, the
oyster mushroom, which is the second-most cultivated mushroom in the United States.77,78 Bcc organisms
may cause spoilage in foods as diverse as onions, Swiss cheese, and Parma ham.1,79,80 However, these
bacteria can also prevent food spoilage: a Bcc strain isolated from Washington navel oranges was found
to prevent fungal infection of these fruits during storage.81 Because of the ability of the Bcc to produce
novel antifungals and antibiotic compounds and fix nitrogen for certain crops, there has been a great deal
of interest in developing these species into agricultural product growth promoters. However, because of
the potential risk to immunocompromised persons, the United States Environmental Protection Agency
(EPA) has placed a moratorium upon the use of Bcc bacteria for agricultural purposes since 2003.82
Despite their occasional identification in food products, including bakery items, onions, cheese, ham,
and oranges, in an extensive examination of different foods, Moore et al.83 were only able to isolate Bcc
from unpasteurized milk. Similarly, Bcc species were isolated from unpasteurized milk by Uraz and
Çitak84,85 and Munsch-Alatossava and Alatossava.86,87 It is unknown how milk becomes contaminated,
276 Laboratory Models for Foodborne Infections

but possibly it is through transfer of Bcc organisms from soil to the cow udder and/or to milk stor-
age tanks.88 However, the Bcc are not found in commercially available dairy products and are effec-
tively killed by pasteurization.83,88 Berriatua et al.2 found that the Bcc species Burkholderia cenocepacia
and Burkholderia vietnamiensis were responsible for causing subclinical mastitis (inflammation of the
udder) in milking sheep. However, when Moore et al.88 examined milk samples taken from cows with
mastitis to determine whether this condition was responsible for the contamination, they were unable to
isolate Bcc organisms.
Like B. pseudomallei, Bcc organisms can also be isolated from drinking water. Zanetti et al.46 found
that 3.5% of drinking water samples in Bologna, Italy, contained Bcc bacteria. The counts in these
samples were very low compared to B. pseudomallei, containing between 1 and 19 CFU/100 mL sample.
Pegues et al.89 examined a Bcc isolate from a water jug at a CF summer camp and found that the ribo-
type of this isolate was different from the ribotypes taken from patients infected at the camp, indicating
that this water was not the source of infection. In contrast to these findings, samples isolated by Moore
et al.83 and Vermis et al.90 from a variety of drinking water samples, including bottled, tap, well, and
spring water, were found to not contain Bcc organisms. However, the major contaminants of the NASA
space shuttle water systems for more than 24 missions have been bacteria of the Bcc, with more than
20 CFU/100 mL detected.91
To date, only one study has suggested a link between food consumption and Bcc infection. Fisher
et al.92 found that Bcc isolates taken from cucumber, lettuce, and cauliflower purchased from salad bars
and food stores near two CF centers were of the same ribotype that was most commonly isolated from
the patients in these centers. However, it is not possible to discern from these results whether or not these
foods acted as vectors of Bcc infection.

18.4.2 Laboratory Infection Models

Like B. pseudomallei, various aspects of B. cepacia complex bacteria pathogenicity have been modeled
using mammals (rodents), including mice and rats. In particular, the intranasal application of Bcc bac-
teria to mice93–95 has been used to introduce and assess Bcc lung infections. However, more consistent
results have been achieved using bacterial aerosols,96 and for chronic infections, by implanting Bcc
encapsulated in agar beads into the lungs of mice97 and rats95,98–100 by the method of Cash et al.101 Each
of these models has provided insights into different aspects of Bcc virulence, with different virulence
factors being identified using each different approach.102
The rat agar bead model for studying chronic respiratory infections has been used extensively to
study virulence properties of B. cenocepacia strains.99,103–105 An avirulent B. cenocepacia strain H111,
isolated from a human patient without symptoms for 5 years, was also similarly tested in the rat agar
bead model.102 Interestingly, there was no difference in the ability of H111 versus that of K56-2 to cause
persistent infection in the rat. However, there was a marked qualitative difference between the strains in
the lung histopathology observed in the infected animals. K56-2-infected lungs had extensive inflam-
matory infiltrates with predominantly polymorphonuclear cells, whereas H111-infected lungs showed
only slight signs of inflammation. At higher doses of H111, infections did result in higher levels of lung
histopathology, although inflammation was still limited. Taken together, these results suggest that the
virulence of Bcc strains in mammalian infection models is strain dependent and that even mouse and
rat infection models do not always accurately model the Bcc strain pathogenicity observed in humans.
Similarly, in immunocompromised intranasal mouse lung infections, it was observed that strains of
B. cenocepacia caused what was described as an acute infection, which stimulated an intense immune
response and relatively early clearance of the infection, whereas the presence of B. multivorans strains
did not induce such an immune response and thus produced a longer-lasting, persistent type of infec-
tion.94 Therefore, not only is the strain of Bcc important in a particular infection model, so too is the Bcc
species. Presumably, each different Bcc strain and species has a subset of virulence factors evolved to
defend against different types of hosts, and some Bcc strains or species have better developed weaponry
against some hosts than others.
Relating to these findings, a large number of alternative infection models have been developed for the
analysis of Bcc virulence, including plants, invertebrates, insects, and fish. It is thought that bacterial
Burkholderia 277

virulence factors evolved to combat natural predators in the environment and that different factors
might be effective against more than one type of organism. Because the innate immune systems of
invertebrates and vertebrates share many common well-conserved features, a bacterium able to infect
and/or cause disease in one type of host may use similar mechanisms and strategies to infect and/or
cause disease in another.106,107
Because the Bcc are natural pathogens of onions, causing soft rot,1 one of the earliest alternative infec-
tion models developed for the Bcc permitted the observation of the maceration of onion tissue.108–110 Such
studies identified a plasmid-encoded pectate hydrolase as the enzyme responsible for maceration in type
strain B. cenocepacia ATCC 25416.108 But B. cepacia and B. cenocepacia strains lacking this enzyme
can also produce a tissue water soaking phenotype through expression of a plasmid-encoded Type IV
secretion system gene cluster.111 It was also discovered that quorum sensing appears to be involved in
onion tissue maceration, as the quorum sensing signal synthase gene cepI and regulator gene cepR
mutants are attenuated for maceration.112
Another simple model used to assess Bcc virulence against plants is the alfalfa seedling infection
model,103 in which the yellowing of leaves (chlorosis), stunted root development, and brown tissue
destruction on leaves and roots were used as a measure of bacterial virulence. This model has been used
with success to ascertain the role of various virulence factors expressed by the Bcc.103 More recently,
the common duckweed (Lemna minor) was used for similar purposes, the advantage of duckweed
being that it can be grown axenically. Several different virulent factors discovered in other Bcc hosts
were also found to be virulent factors in duckweed,113 confirming duckweed’s validity as an infection
model. Interestingly, duckweed was also found to be an effective infection model for enteropathogenic
Escherichia coli.113
A large number of Bcc researchers are also now using insect animal models such as wax moth larvae
(G. mellonella) or fruit flies (Drosophila melanogaster) as substitutes for mammalian infection mod-
els.114,115 Such “lower” animal infection models hold the advantage that they typically possess at least
an innate immunity system, but do not involve all of the regulatory and ethical requirements needed for
“higher” animal experimentation. Although each alternative infection host may not exactly model the
same types of tissue found in humans, it is thought that for each different host, a subset of the bacterium’s
total armamentarium is expressed to overcome that host’s defenses. Therefore, at least some of the cel-
lular weapons used by the Bcc to defeat G. mellonella would also be expressed when the Bcc bacterium
finds itself in the human lung. Since its development,114 the G. mellonella infection model has been
used to identify a novel hemolytic toxin,116 as well as several other Bcc virulence factors,117–119 and also
show that the larvae can be rescued from lethal bacterial infection by the action of bacteriophages.120
Similarly, D. melanogaster has been successfully employed to demonstrate that different strains of the
Bcc can kill by infection. Unlike results found for Pseudomonas aeruginosa in D. melanogaster, species
of the B. cepacia complex were only able to kill flies by infection after pricking, rather than through
ingestion.115 This suggests that the Bcc are less toxic to fruit flies than other opportunistic CF bacterial
pathogens. Nevertheless, the results obtained with the fruit fly model are comparable with those obtained
using mammalian infection models, and B. cenocepacia K56–2 mutants less virulent in murine hosts or
other alternative infection models than wild type are also less virulent in the fruit fly, even when tested
using competitive index analyses.115
Caenorhabditis elegans, a roundworm nematode, has also been used successfully as a Bcc model
for mammalian infection.121 These studies have shown that there are two modes of killing, a fast mode
due to paralysis by an extracellular Bcc toxin and a slow mode due to ingestion and accumulation of
bacteria in the nematode intestine.121 The Bcc quorum sensing system appears to be required for both
types of killing,121 but only the slow mode was shown to be partially due to certain Bcc virulence fac-
tors such as AidA.122 Several different environmental parameters can influence the performance of this
model, including worm age,123 prior diet,124 diet,125 Bcc strain,102,126,127 and Bcc strain mucoidy status.128
Nevertheless, a number of Bcc virulence factors effective against C. elegans have been discovered or
confirmed using this model, including the type III secretion system,129 the phenylacetic acid catabolic
pathway,130 two different Hfq proteins,1321,132 the pleiotropic regulator of phenazines Pbr,133 a small regu-
latory RNA MtvR,134 a Type 2 secretion system pseudopilin GspJ,135 and the third chromosome/virulence
plasmid pC3 in many Bcc species.136,137
278 Laboratory Models for Foodborne Infections

The final Bcc alternative infection model to be discussed is perhaps the most recently developed
zebrafish embryos.138 Bcc bacteria survive and multiply in zebrafish macrophages, later disseminating
and causing bacteremia and death within 3 days. B. cenocepacia strains that are highly virulent in
other animal models are also highly virulent for the embryos, whereas less virulent Bcc species were
controlled by the fish innate immune system.138 This model was used to confirm that both the quorum
sensing system and the third chromosome are required for full virulence of B. cenocepacia in the zebraf-
ish embryos.136,138

18.5  B . gladioli pv. cocovenenans

18.5.1 Overview
B. gladioli pv. cocovenenans cells are motile, aerobic, non-spore-forming, nonencapsulated Gram-negative
rods. These bacteria form smooth, round colonies (on potato dextrose agar) that are yellow pigmented
due to the production of toxoflavin. Growth can occur between 6°C and 41°C, but is optimal between
30°C and 37°C, and toxin production occurs between 22°C and 30°C.139,140
The taxonomical classification of the bacteria named B. gladioli pv. cocovenenans is historically com-
plicated. van Veen and Mertens141 published the first studies on this bacterium, which they isolated from
contaminated “tempe bongkrek,” an Indonesian fermented coconut cake. This bacterium was named
Pseudomonas cocovenenans (cocos = coconut, veneno = to poison), and this nomenclature was retained
in early studies.142–144 In 1963, a similar bacterium was isolated from contaminated fermented corn flour in
China145 and eventually named in English as Flavobacterium farinofermentans sp. nov.146 After assessing
a variety of cellular characteristics, Zhao et al.147,148 determined that P. cocovenenans and P. farinofermen-
tans belonged to the same species, suggesting that P. farinofermentans should be renamed P. cocoven-
enans biovar farinofermentans. In 1992, seven Pseudomonas species were reclassified and assigned to the
new genus Burkholderia (including B. pseudomallei, B. mallei, B. cepacia, and B. gladioli).149 Following
analysis of the type strain isolated from tempe bongkrek, in 1995 P. cocovenenans was subsequently reas-
signed as Burkholderia cocovenenans comb. nov.150 In 1997, Vandamme et al.151 found that SDS–PAGE
protein patterns of whole-cell proteins of B. gladioli and B. cocovenenans strains were similar, and fur-
ther experimentation confirmed this relationship.152 This led to the reclassification of B. cocovenenans as
a member of B. gladioli, now called B. gladioli pv. cocovenenans, to join three existing phytopathogenic
pathovars of B. gladioli (gladioli, alliicola, and agaricicola).9–11

18.5.2 Laboratory Infection Models

For nontoxigenic B. gladioli, it is most commonly isolated from the respiratory tract.153–155 These infec-
tions are much less serious than Bcc infections, and the impact on patient health is often minimal.153,154
Septicemia has also been reported in patients with CF, CGD, AIDS, and diabetes.12,13,156,157 Laboratory
infection models would be similar to those used for bacteria of the Bcc, though few studies have been
carried out thus far.
In contrast, there are few laboratory models available for toxigenic B. gladioli. Unlike most other food-
borne illnesses (except perhaps C. botulinum), the pathogenic effects of B. gladioli pv. cocovenenans
are mediated solely by toxins and not by the bacteria themselves. When mice are administered either
live or heat-killed bacteria, no adverse effects are observed, as opposed to when mice are administered
culture supernatants or partially purified toxins.8,146 B. gladioli pv. cocovenenans produces two toxins:
toxoflavin (also called xanthothricin) and bongkrekic acid or BA (also called bongkrek acid or flavotoxin
A). Production of toxoflavin is not restricted to B. gladioli pv. cocovenenans, as it is also produced by
Streptomyces, Burkholderia glumae, Burkholderia plantarii, and phytopathogenic B. gladioli.14,158–160
Although toxoflavin is highly toxic, it is generally not the cause of B. gladioli pv. cocovenenans intoxi-
cation. Instead, BA is both more toxic and present in higher concentrations in contaminated foods.161,162
The genes encoding BA have recently been identified, and were found to be part of a Type I polyketide
synthase (PKS) operon, involving the action of a cytochrome P450 monooxygenase.163
Burkholderia 279

BA toxin is one of the most potent respiratory poisons known. Following human consumption of food
contaminated with B. gladioli pv. cocovenenans, intoxication occurs rapidly, usually within 1–10 h.164
The classical symptoms are hyperglycemia followed by hypoglycemia, spasms, loss of consciousness,
and death.165 Following autopsy, the liver, kidneys, and brain all show signs of damage, and in some
cases, there is also damage to the heart, lungs, gastrointestinal tract, and spleen.164 Laboratory animal
models of infection are generally too sensitive to BA toxin to be of much experimental value. When food
contaminated with B. gladioli pv. cocovenenans is fed to dogs, they develop spasms, enter comas, and
die within 2–3 h.164 Dogs and rhesus monkeys fed with B. gladioli pv. cocovenenans culture supernatants
die within 6–33 h and 15.5–35 h, respectively.146 When mice were given B. gladioli pv. cocovenenans
culture supernatants (0.4 mL/mouse) partially purified by the method of Hu et al.,166,167 all mice died
within 45 min with stiff tail and feet.8
With respect to a mechanism of action, BA inhibits oxidative phosphorylation by binding to the ade-
nine nucleotide translocator, which normally shuttles ADP and ATP across the inner mitochondrial
membrane.168 The activity of this transporter is thought to inhibit cellular apoptosis.169 Because BA
inhibits oxidative phosphorylation, the only way for cells to generate ATP is through anaerobic glycoly-
sis.170 This metabolic changeover is responsible for the symptoms of intoxication. Glycogen stores are
broken down to allow for an increased level of glycolysis, leading to hyperglycemia and increased lactic
acid levels, but these stores are soon depleted, resulting in hypoglycemia and an inability to regenerate
ATP.171 It is the ATP depletion that is fatal and not the hypoglycemia, as the injection of glucose is insuf-
ficient to prevent death.171

18.6 Conclusions
The major pathogenic bacteria of the genus Burkholderia include the species B. mallei, B. pseudomal-
lei, the B. cepacia complex, and B. gladioli. Surprisingly, the diseases that each of these bacteria causes
are diverse in terms of host, mechanism of action, prognosis, and outcome. Coordinately, the laboratory
systems used to model these diseases and assess the virulence and toxicity of each of these bacterial spe-
cies are diverse as well. For B. mallei, the causative agent of glanders, the primary hosts are equine, and
thus, a variety of equine animal models can be used to study glanders disease progression. For B. pseu-
domallei, the modeling of the disease melioidosis is much more difficult, as the disease progression is
more complex, including variable human immune interactions, a latency period, and multifactorial local
and systemic virulence effects. Moreover, as humans appear to be a primary host, different aspects of
the disease are best studied in different animal models. Similarly, the B. cepacia complex is best studied
using a variety of animal models, due to its multifactorial virulence factor production. In contrast to
B. pseudomallei, however, modeling B. cepacia complex disease typically requires the animals to be
immunocompromised in some way, as healthy animals (and humans) are generally able to resist Bcc
infections. Finally, nontoxigenic B. gladioli disease modeling can be performed with systems similar to
those used for the Bcc, whereas toxigenic B. gladioli can be studied at the organismal level, or through
the purification of the toxin and with the use of specialized cellular or molecular assays. Historically,
murine and equine animal models have been used for the study of Burkholderia infections and toxicol-
ogy. In recent years, however, there has been a great deal of interest in alternative host infection models
to analyze all or specific components of the diseases caused by Burkholderia species. The development
of new and better laboratory models of disease caused by Burkholderia will bring about a better under-
standing of the disease progression and virulence factors utilized by each individual Burkholderia spe-
cies, leading to the identification of drug targets and the development of antibacterial strategies against
pathogenic Burkholderia species.

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19
Campylobacter

Martin Stahl and Bruce A. Vallance

CONTENTS
19.1 Introduction................................................................................................................................... 289
19.2 General Features of C. jejuni Colonization of Animal Models.................................................... 290
19.2.1 Cellular Models of Infection............................................................................................ 292
19.2.2 Three-Day-Old Chick Model........................................................................................... 292
19.2.3 Colostrum-Deprived Piglets............................................................................................. 294
19.2.4 Ferret Model..................................................................................................................... 294
19.2.5 Galleria mellonella.......................................................................................................... 295
19.2.6 Mouse Models.................................................................................................................. 295
19.3 Conclusions................................................................................................................................... 298
References............................................................................................................................................... 299

19.1 Introduction
Campylobacter jejuni is a Gram-negative, curved-rod shaped, microaerophilic, enteric bacterial patho-
gen, and one of the leading causes of gastroenteritis in both the developing and the developed world. This
highly infectious bacterium is often acquired through the consumption of contaminated water, raw milk,
or meat products (particularly contaminated poultry).1 Once infected, an individual’s symptoms can vary
significantly. Milder cases can be largely asymptomatic or involve watery diarrhea. In more severe cases,
fever, bloody diarrhea, and abdominal pain can be evident. In most cases, the disease resolves within
a few days or in up to 2 weeks; however, under rare and poorly understood circumstances, the infec-
tion can trigger the development of autoimmune disorders such as reactive arthritis or Guillain–Barré
syndrome,1 or other complications such as postinfective irritable bowel syndrome.2 Despite the broad
prevalence of this pathogen, the mechanisms by which this organism causes disease in humans remain
poorly understood.
Aside from assessing the severity of symptoms, the details of how C. jejuni triggers disease symp-
toms remain poorly defined. We know that a very small infectious dose in humans is capable of tran-
siting the stomach and establishing infection, largely within the colon. Pathogen burdens can be very
high, although they drop significantly as the adaptive immune response becomes engaged. The infection
usually responds to antibiotic treatment, such as either fluoroquinolones (e.g., ciprofloxacin) or macro-
lides (e.g., erythromycin), although antibiotic resistance rates in C. jejuni have been steadily increasing
in recent years.3 The nature of intestinal colonization by C. jejuni in humans is poorly characterized,
although numerous in vitro studies, as well as in vivo animal work, would suggest it is primarily local-
ized to intestinal crypts as well as to the mucus layer that overlies the mucosa. Moreover it is capable
of adhering to the intestinal epithelial surface and translocating across the epithelium.1 Cell invasion by
C. jejuni has been indicated in vitro, in vivo, and in clinical samples1,4; however, what role cell invasion
plays in C. jejuni pathogenesis remains unknown. The spread of C. jejuni into the blood stream and to
systemic sites has been documented in some patients, but it remains relatively atypical.5 Additionally,
long-term complications such as reactive arthritis and Guillain–Barré syndrome are rare and result from

289
290 Laboratory Models for Foodborne Infections

antigen mimicry between C. jejuni lipooligosaccharides and host gangliosides. To date, no proven ani-
mal models exist that mimic these long-term complications.
One of the difficulties in improving our understanding of campylobacteriosis has been the fact that
C. jejuni behaves quite differently depending on its surroundings. In the wider environment, birds are the
main reservoirs for the organism and its preferred hosts1,6; however, C. jejuni remains a relatively harm-
less commensal in avian hosts, even as it is capable of growing to high numbers in birds’ cecum.6 Various
Campylobacter species, including those that are infectious to humans such as C. jejuni, are commonly
recovered from wild birds,7,8 although the main reservoirs that serve as a source for human infection
are poultry farms.9 In the densely packed environment of a modern poultry barn, any Campylobacter
contamination can quickly spread between birds until the entire flock becomes colonized, making
Campylobacter contamination a significant health concern within the poultry industry.10 Precisely how
C. jejuni is able to spread so rapidly and so easily has been somewhat of a mystery, especially consider-
ing its rather fastidious growth requirements, including warm, low-oxygen conditions. Evidence suggests
that flies11 or wild birds6 may serve as sources of contamination, while other sources include runoff from
other livestock such as pigs or cattle,10,12 which themselves can become asymptomatically colonized.12
Coprophagous behavior of chickens, along with a high density of birds can quickly spread contamina-
tion throughout a flock.13 It has also been suggested that amoeba may serve as a temporary host for
Campylobacter, allowing for its prolonged survival within contaminated water sources.14,15 Additional
survival mechanisms within the C. jejuni cells themselves, such as their observed ability to convert to a
nonculturable coccoid form, may also aid in prolonging their survival in the low-nutrient, high-oxygen
conditions commonly found in the environment outside of a host,16 but these mechanisms remain poorly
understood.
Besides the commensal-like colonization of poultry, few other animals are susceptible to C. jejuni
colonization and infection in a fashion similar to human campylobacteriosis.17 Following exposure to
C. jejuni, most animals either exhibit only brief, transient colonization, or are colonized asymptomati-
cally. An exception to this involves a connection between spontaneous abortion in sheep and other live-
stock, and infection by certain Campylobacter species, including Campylobacter fetus and C. jejuni.18
Humans remain one of the relatively few hosts that exhibit enteric disease in response to exposure to
C. jejuni, making the use of animal models in the study of C. jejuni infection more problematic. Those
animal models that are employed usually come with the caveats that they do not entirely replicate the
human disease (e.g., poultry and insect models), or that they require some significant modification of the
host, such as the construction of mice deficient in key genes designed to make them more susceptible to
infection. Some of the more promising infection models currently being used include neonatal piglets,
ferrets, and certain knockout mouse strains, whereas models of commensal colonization are typically
newly hatched chicks and wild-type mice. Table 19.1 summarizes the current animal models available,
along with a brief description of each. In this chapter, we will outline the animal models currently
employed for the study of C. jejuni in published research, and discuss the advantages and drawbacks of
each one, while providing an overview as to what the research community has learned about C. jejuni
infection from these research models.

19.2 General Features of C. jejuni Colonization of Animal Models

Before discussing each animal model in detail, we will start by giving an overview of several common
characteristics of C. jejuni colonization. Firstly, unless forced into an unusual mode of infection, such
as via intraperitoneal (IP) injection for example, the standard route of infection for C. jejuni is by oral
inoculation. Following inoculation, C. jejuni proceeds quickly to the intestine, where it can establish
itself. Although there are Campylobacter species that can colonize the mouth,19 and the relatively closely
related Helicobacter pylori primarily lives within the stomach,20 C. jejuni does not typically remain for
long in the upper regions of the GI tract. Even in the lower portions of the small intestine, it does not
generally reach high numbers, even if it can establish itself as a stable population. Rather, it proceeds
to the large bowel or cecum, which also supports the bulk of the intestinal commensal microbiota. In
animals with a cecum (birds, rodents, and pigs), this is the primary site of C. jejuni colonization, with
Campylobacter 291

TABLE 19.1
Animal Models for the Study of Campylobacter jejuni Infections
Animal Model Colonization Pathology References
Chick Strong commensal-like Little to no pathology 21,35,49,50–56,60
colonization of the cecum and
colon, with a very small
inoculum
Piglet Colonization of the small Significant variation in pathology. 37,42,64,6–71
intestine, cecum, and colon Inflammation, distension with gas, and
immune cell infiltration
Ferret Colonization principally in the Significant inflammation and immune cell 22,72,75–78
colon infiltration in colonized areas
Galleria No real colonization, although The mortality and melanization of infected 80,81
C. jejuni may replicate and larvae is an indicator of pathology
spread following injection
Wild-type mouse Inconsistent colonization Minimal and inconsistent pathology 84–91
Antibiotic-pretreated Consistent colonization of the Minimal pathology in wild-type mice 26,86,88
or germfree mouse cecum and colon
IL-10−/− mouse Consistent colonization of the Chronic infection, severe inflammation, and 99,102–107
cecum and colon immune cell infiltration in the colon and
cecum
SIGIRR−/− mouse Consistent colonization of the Acute infection, moderate inflammation, 26
cecum and colon and immune cell infiltration

the microbes reaching very high numbers (often over 109) of colony forming units (CFUs) per gram of
luminal content.21 In animals without a developed cecum (ferrets) and in humans, the primary site of
colonization becomes the colon.22,23
Focusing more closely, current research suggests that C. jejuni’s preferred location within the intes-
tine is the mucus layer,24,25 which segregates the host epithelium from the bacteria and other contents
of the lumen. Histological images of a number of C. jejuni animal models, including both chicks21
and mice,26 have identified C. jejuni as being localized primarily within the mucus layer, particularly
within the mucus-filled crypts that line the intestine. Mechanistically, this observation fits with several
known aspects of C. jejuni colonization and cell biology. Firstly, C. jejuni is microaerophilic, meaning
it requires low levels of oxygen to grow. This reflects it having only a single class I-type ribonucleotide
reductase, which requires oxygen to function.27 However, C. jejuni is also dependent on numerous
metabolic enzymes which contain oxygen-sensitive iron–sulfur complexes.28 Together, these make it
unable to survive for extended periods under anaerobic conditions (such as the intestinal lumen), or
under higher oxygen conditions. This makes the low oxygen conditions of the intestinal lining its ideal
environment as it is distant from the anaerobic lumen, where the commensal anaerobes thrive, yet close
enough to be able to access the diffuse oxygen from the highly vascularized epithelium. Furthermore,
early research on C. jejuni chemotaxis indicated that it was attracted to mucins,29 which are the primary
glycoprotein component of intestinal mucus. Additionally, at least some strains are chemotactic toward
l-fucose,29 a common terminal glycan on mucins. The current hypothesis is that C. jejuni recognizes
mucin 2 as a chemoattractant to find its way to the mucus layer, using its flagellar motility and cork-
screw shape to push its way through intestinal contents to reach its destination.30,31 In all animal models
where the intestine is colonized, mutants in chemotaxis or flagella are either completely or severely
impaired for colonization, making motility toward the mucus layer one of the most critical factors for
colonization.32,33
The second critical factor that governs C. jejuni colonization, in almost any host it encounters, is how it
deals with the common stresses it almost universally encounters. For example, in a mammalian or avian
digestive system, orally inoculated C. jejuni would be exposed to the low pH of the stomach, antimi-
crobial effects of bile salts, oxidative stress, nitrosative stress, competition with the host and microbiota
292 Laboratory Models for Foodborne Infections

for resources, not to mention the innate immune defenses of the host organism. While many of these
stressors may depend on the specific host and situation in which C. jejuni finds itself, and thus require
more of an explanation than can be addressed here, a few common mechanisms appear to be present in
most potential animal hosts that C. jejuni can colonize. For example, mutants in the CmeABC multidrug
efflux pump, as well as several related pumps and regulators are more sensitive to a number of factors,
including bile salts, heavy metals, antimicrobials, and antibiotics, and are significantly impaired for
survival inside a living host.34 Oxidative stress is also a major factor at several stages of colonization,
and deletion mutants in key oxidative-stress-related genes including katA, cj1386, sodB, ahpC, as well as
strains lacking other defense and regulatory genes are severely impaired in gut colonization in a number
of C. jejuni animal models.35,36
Finally, the acquisition of nutrients is a common feature of C. jejuni’s survival in almost any environ-
ment. A curious feature of C. jejuni is its relatively limited repertoire of potential carbon sources. With
the exception of certain strains that have acquired an l-fucose metabolic pathway,37 C. jejuni is limited
to only a handful of amino acids, 4C-dicarboxylates, and a few molecules that can be easily plugged into
the citric acid cycle.38,39 This makes mutants in almost any of these key metabolic pathways exceptionally
poor colonizers, due to a lack of alternate metabolic pathways to draw upon.40,41 In particular, mutants
impaired in the acquisition or metabolism of two of its preferred amino acids, glutamate and serine,
are exceptionally poor colonizers.40,41 Furthermore, the acquisition of a number of metals and other key
micronutrients, especially iron, is critical for C. jejuni, particularly in a competitive environment such as
an animal host. In the case of iron, which is tightly regulated by the host and required by the microbiota,
the loss of any of C. jejuni’s major iron acquisition pathways or their regulators can result in a substantial
decrease in its colonization potential.42,43

19.2.1 Cellular Models of Infection

Simple cellular infection models strip away most of the complexity of the whole organism, and allow
for the precise study of cellular–bacterial interactions. Due to their simplicity and availability, cellular
infection models have been one of the preferred methods for the study of C. jejuni. They remain useful,
particularly for the study of C. jejuni interactions with host cells, but the degree to which they model
host colonization and infection is debatable. The most common assays for the study of C. jejuni in cel-
lular systems are adherence or invasion assays of colonic epithelial cell lines. The preferred cell lines are
usually the polarized Caco-2 or HT29 cells44; however, nonpolarized INT 407 cells have been frequently
used as well,45 along with other varieties of established cell lines, depending on the experimental design.
The standard assay involves either measuring bacterial adherence to the host cell surface, or using a gen-
tamicin protection assay to quantify the number of internalized bacteria. Increases in either adherence or
invasion are usually taken as a sign of increased virulence; however, precise links between these in vitro
results and infection in vivo can often be difficult to establish.46
Upon contact with most intestinal epithelial cells, including chicken, mouse, and human cell lines,47
C. jejuni will readily adhere, invade, and persist; however, no intracellular replication has been previ-
ously observed.48 These experimental models are also useful for establishing the mechanisms of cell
invasion, as well as for measuring cytokine expression in response to C. jejuni, although precisely how it
extrapolates to infection of human or animal intestines remains a matter of some debate.

19.2.2 Three-Day-Old Chick Model

One of the most prominently used animal models for C. jejuni colonization has been newly hatched
chicks.49 Although early studies indicated some signs of diarrhea in chicks if infections were estab-
lished early enough (<1 day) after hatching,50,51 it is generally accepted that the colonization of chicks by
C. jejuni is commensal in nature, with little detriment to the host.21,52,53 An infectious dose of approxi-
mately 107–108 CFUs is considered more than sufficient to guarantee infection,21 with even significantly
lower numbers (104 –106 CFUs) often being sufficient.54 The common age of chicks used for this model is
between 1- and 3-days after hatching, although older chicks have been used as well.54 After inoculation,
most studies have found that chicks remain colonized for a few days to a few weeks.35,54 The number of
Campylobacter 293

CFUs per gram of luminal contents remains relatively constant over this period in the absence of any
external manipulation.21 Any limitations on the duration of an experiment are dependent on the facility
and the growth of the birds. While infected chicks are quite small at the beginning of the experiment,
they will grow and gain weight rapidly, making housing and handling more problematic over time.
While it has been demonstrated that C. jejuni will quickly establish itself within the GI tract of an
infected bird, our knowledge of exactly how C. jejuni colonizes chicks is relatively limited. The main
site of colonization is the cecum and to a lesser extent the cloaca,54 with relatively low numbers found
throughout the rest of the avian GI tract, although there has been little research performed to determine
whether other locations of the GI tract can be colonized, or if C. jejuni recovered from these regions is
just a transient population. Interestingly, several early studies were conducted to better characterize how
C. jejuni established itself within the cecum. Beery et al. compared histological sections of infected and
uninfected 8-day-old chicks.21 Their primary observation was the presence of large numbers of C. jejuni
at the base of the chick cecal crypts. A closer examination using transmission electron microscopy
clearly enabled the visualization of the helical C. jejuni cells in the mucus layer filling the crypts, but
they were not associated closely with the microvilli or glycocalyx, suggesting that they were not adher-
ing to the intestinal epithelial cells themselves. In contrast, other studies have identified cell adhesion not
only taking place in the chick cecum, but also being an important step in proper colonization.55 Whether
or not cell invasion takes place in the chicken cecum in vivo remains uncertain owing to conflicting
studies and evidence53,56; however, it has been observed to occur in chicken cells in vitro.57 This has led
to the belief that C. jejuni has the capacity to invade chick cecal epithelial cells; however, additional
factors may intervene to prevent this from occurring routinely in an in vivo situation.58 Notwithstanding
the lack of conclusive evidence, the cecal mucus layer has been implicated as a modulator of C. jejuni
pathogenicity. In cell culture experiments, the addition of chicken cecal mucus has been found to reduce
cell invasion of both chicken and human epithelial cells, although a mechanism to explain this obser-
vation remains unknown.57,58 It has been suggested that cell invasion may still take place in vivo, but
infrequently, as an explanation for observations regarding the stimulation of the avian immune response;
however, this remains only a hypothesis.56
Conflicting information also exists as to whether C. jejuni can be found outside of the chick intestinal
tract. Some studies have not recovered any C. jejuni from other sites throughout the body following
inoculation,21 however, other studies have been able to recover C. jejuni from other organs, including the
spleen and liver up to 7 days postinoculation.47 Despite the presence of C. jejuni at systemic sites, the
chick immune system remains largely unresponsive to C. jejuni, with virtually no signs of disease devel-
opment.53 The degree to which the chick immune system reacts to C. jejuni remains largely unknown,
despite the number of studies that have attempted to answer this question. It is of particular interest for
researchers attempting to develop a vaccine against C. jejuni chicken colonization, which would rely
heavily on the ability to stimulate the chicken’s own immune system to recognize and clear the coloniz-
ing C. jejuni, something the immune system does not seem inclined to do under normal circumstances.59
Despite the seeming lack of an immune response to C. jejuni, evidence would suggest that the chick
immune system is not entirely oblivious to the presence of C. jejuni. A transcriptomic study identified
270 genes differentially regulated in chicks inoculated by C. jejuni, as compared to uninfected controls,
with some of these genes involved in inflammatory pathways.60 Furthermore, studies have identified
chTLR4 and chTLR21 expressions as being particularly sensitive to C. jejuni colonization.61,62 Evidence
suggests that prior to exposure to C. jejuni, the avian innate immune system remains at least somewhat
sensitive to C. jejuni. Upon initial exposure to C. jejuni, there is a measurable spike in production of the
chicken equivalent of the human chemokine IL-8 (CXCLi1 and CXCLi2), accompanied by an increase
in the numbers of monocytes/macrophages within the cecal tissues.63 Curiously, this does not lead to an
increase in heterophil numbers (roughly the equivalent of a human neutrophil), as one would expect with
IL-8. This increase in a key proinflammatory chemokine appears to be temporary, and immune toler-
ance to C. jejuni appears to develop,53 allowing for a high pathogen burden, but virtually no immune/
inflammatory response.
In spite of the lack of immunological responses that resemble human disease, the chick has been
widely used as a model for C. jejuni colonization. This remains quite relevant due to similarities in basic
colonization factors, even in the absence of an immune response, as well as the primary role of poultry
294 Laboratory Models for Foodborne Infections

as a common source of human infection, making the colonization and spread of C. jejuni in chickens
relevant in its own right.

19.2.3 Colostrum-Deprived Piglets
The use of colostrum-deprived piglets as a model for C. jejuni infection was first defined by Babakhani
et al.64 Although conventionally reared piglets may be susceptible to Campylobacter infection early
in life, maternal antibodies are usually sufficient to prevent infection. Adult pigs are also known to be
frequent carriers of both C. jejuni and Campylobacter coli65; however, the development of symptoms
of gastroenteritis in response to these pathogens is rare. Colostrum-deprived piglets lack the protection
of maternal antibodies that usually helps prevent infection in piglets. Additionally, at very young ages,
they lack a fully mature immune response or a protective, fully developed intestinal microbiota, making
them particularly susceptible to infections by enteric pathogens. This vulnerability has been previously
exploited for the study of Escherichia coli66 and Salmonella67 infection, among others, and in a number
of studies, it has been successfully used as a model for C. jejuni infection.37,42,68
In the initial characterization of the model by Babakhani et al., the M129 strain of C. jejuni was orally
inoculated into newborn, colostrum-deprived piglets.64 Symptomology was somewhat varied, but all the
piglets became colonized, and exhibited symptoms including diarrhea, with blood and mucus in their
stool. These symptoms were often evident within a day of inoculation, and pathogen burden and disease
severity peaked within 3 days postinoculation. This first study is also the only study to undertake a
histological analysis of infected piglets. In their analyses, they found signs of inflammation, goblet cell
depletion, and immune cell infiltration primarily in the cecum and colon, with minimal signs of damage
in the small intestine. Electron microscopy also successfully identified C. jejuni cells adherent to the
epithelial surface as well as bacteria internalized within epithelial cells.
Continuing studies using the colostrum-deprived model have focused primarily on pathogen burdens,
with less attention paid to histology or immune responses within the host piglets. The most common
application has been to compare changes in pathogen burden between wild-type C. jejuni and isogenic
mutants lacking key pathogenicity genes. These have included genes related to nutrient uptake,37,42
stress-response regulators,68,69 capsule-modifying enzymes,70 as well as other genes of interest.71 In most
cases, the strain of choice has been the commonly used NCTC11168 lab strain, which induces limited
pathology and mortality in infected piglets.42 Impaired pathogenesis in a mutant strain is usually con-
cluded based on a decrease in pathogen burdens either by comparison to control piglets infected with
the wild-type strain, or through a competitive index from a piglet infected with both the mutant and
wild-type strains. In contrast to the NCTC11168 strain, the 81-176 strain is much more pathogenic in the
piglet model,70 and is less frequently used. On the occasions it has been studied, piglets often suffer high
mortality within 1–2 days postinoculation.

19.2.4 Ferret Model
Although they are a relatively uncommon and difficult animal species to use as a laboratory animal
infection model, domestic ferrets have been used in several studies as a model for human C. jejuni
infection.22,72 The first use of these animals as a model for C. jejuni infection was published by Fox
et al.22 Prior to this, Campylobacter-like organisms (likely Helicobacter), had been recovered from fer-
ret gastric lesions,73 leading to their common use as an animal model in the early study of Helicobacter
infection.74 Campylobacter infection was further described using domesticated ferrets, and has subse-
quently been used in several studies to assess the pathogenicity of isogenic C. jejuni mutant strains.
Recently, a more detailed evaluation of the immune responses and histopathology of C. jejuni infection
in ferrets has been published.75
Despite the difficulty in using ferrets as an animal model of infection, including cost, availability, and
a relative lack of experimental tools and expertise relating to ferrets, the ferret model is attractive due
to the ability of C. jejuni to trigger an acute, self-limiting diarrheal disease without substantial manipu-
lations of the host to render them more susceptible to infection.22 The exception to this is the need to
anaesthetize the animals prior to inoculation, and in some studies treatment with sodium bicarbonate
Campylobacter 295

was used prior to inoculation to reduce stomach acidity, and thereby increase the survival of C. jejuni
through the stomach.72,75–78 Additionally, some studies used a postinoculation treatment with opium to
reduce intestinal peristalsis to give more time for the infection to take hold.
As an infection model, the ferret remains somewhat understudied due to the relatively few researchers
who have actually employed it. The initial characterization of the model described an acute diarrheal
disease, with mild to moderate diarrhea observed within the first few days postinoculation.72 Symptoms
resolved within a few days, and the infection was cleared within 1–2 weeks. A more detailed descrip-
tion of the ferret immune response to C. jejuni was published in 2009.75 Researchers found a relatively
high pathogen burden (107–1010 CFUs/g) and more pronounced pathology in the colon relative to the
small intestine. The pathology observed included substantial increases in immune cell infiltration into
the mucosa, and the detection of lactoferrin and blood in the stool. Elevated fecal and systemic IgG
levels were detected and the authors linked their presence to the successful clearance of the infection.
Furthermore, immunohistochemistry and electron microscopy detected C. jejuni adherent to the surface
of epithelial cells, with possible evidence of epithelial cell invasion.

19.2.5  Galleria mellonella

One of the less intuitive animal models for C. jejuni involves the use of the larva of the greater wax moth,
Galleria mellonella. The use of this animal model has so far been uncommon, and the mechanisms
behind Galleria infection by C. jejuni are poorly characterized; however, the plentiful and low-cost lar-
vae, which are also a commonly used fish bait, make for an attractive infection model.79,80
These insects have long been employed for the study of a wide variety of bacterial infections. The
bacterial pathogen of choice is injected into the foreleg, where it can infect the insect hemocoel. The rela-
tive pathogenicity of the inoculated bacteria is then measured by counting the numbers of resulting dead
Galleria, accompanied by the development of a distinctive black melanization of the dead and dying
larvae.80,81 In this scenario, the pathogenicity of the bacteria is assessed based on their ability to prolif-
erate and infect the insect’s hemocytes, which function in a similar fashion to mammalian neutrophils
by phagocytosing and killing engulfed bacteria through a superoxide burst. This makes the Galleria
insect model a useful means to test a bacterium’s ability to defend against a host inflammatory response
comprised of neutrophils and/or superoxide production. However, given the fundamental differences
between most other aspects of insect biology and a mammalian host, other insights into pathogenesis
will likely be limited.
Precisely what can be learned about C. jejuni infection using this model is probably limited in its
scope. The first uses of this model were published by Champion et al.81 and by Senior et al.80 After test-
ing a variety of C. jejuni strains and deletion mutants, the mutants showing the largest attenuation in
the insect model were those in O-methyl phosphoramidate capsule modification, with mutants defective
in the MeOPN capsule modification gene cj1416 being largely nonvirulent.82 This observation was sup-
ported by further studies by van Alphen et al.70 An additional study by Gundogdu et al. assessed how a
mutant in an oxidative stress regulatory protein (cj1556) fared in the insect host, and found them to be
much less virulent.83 Together, these previous studies suggest that assessing bacterial capsule function as
well as susceptibility to stresses may be the most effective use of the C. jejuni insect model.

19.2.6 Mouse Models
For researchers using animals to model human infections, mice have long been the species of choice.
Mice have the advantage of being relatively small, thereby allowing one to house large numbers in a rela-
tively small space, and thus keeping maintenance costs manageable. Mice breed and mature relatively
quickly, allowing for the establishment of whole colonies within a matter of months, and for mice to be
generated for new experiments relatively quickly. Finally, in many respects, mice are physiologically
similar to humans, making them fairly relevant for studying human biology and diseases.
With these advantages in their favor, a variety of research tools have sprung up surrounding mouse
research, which are unmatched in any other animal model. Commercial companies have been established
to breed mice, providing them on order, thereby eliminating the need for all researchers to maintain their
296 Laboratory Models for Foodborne Infections

own mouse colonies. Genetic tools have allowed for the creation of mouse strains lacking almost any
gene that can be feasibly knocked out. Finally, commercial reagents and assays have been developed
specifically for use with mouse models. Many antibodies and diagnostic tools are tailored to mouse biol-
ogy, allowing for accurate tests and experiments. Although many of the other animal models have some
of these tools available for them, none comes close to matching what is available for mice.
With mice already firmly established as research tools, very early experiments on Campylobacter
sought to apply them to existing mouse infection models. As early as the 1970s, when Campylobacter
was just starting to be identified as a distinct genus of its own, some initial characterizations of
Campylobacter spp. colonization in mice were performed.84 Results from these experiments were often
limited and conflicting due largely to a lack of standard tools and techniques at the time. Field et al.
reported susceptibility in neonatal mice to intragastric Campylobacter infection;85 however, adult mice
were found to be resistant to colonization and infection, unless pretreated with antibiotics.86 Conversely,
using adult HA-ICR mice and a high inoculating dose, Blaser et al. successfully colonized the mice and
observed some degree of colonic inflammation and anti-Campylobacter IgG production.87 As further
studies were performed, results remained mixed using mice as models for C. jejuni infection. Germfree
mice, mice with limited flora, or antibiotic pretreated mice were found to be highly susceptible to colo-
nization.26,86,88 Untreated, conventional mice carrying a full microbiota were found to be colonized in
some studies, but in others, colonization either failed or was sporadic, making reproducibility problem-
atic.89 Symptomology was equally inconsistent between studies. Some studies reported the development
of mild to severe diarrhea, and even mortality, in infected mouse strains90; however, in other studies,
colonization was largely asymptomatic with no significant signs of diarrhea or overt pathology.86,88 These
significant inconsistencies between studies dampened the enthusiasm for the use of mice as a model for
C. jejuni infection, and by the end of the 1980s, conventional, wild-type mice were used only infrequently
as an infection model for C. jejuni. Some researchers with better success at colonization continued to
use mice as a simple colonization model,91 but, the success of chicks as an animal model for colonization
made them more attractive to those able to procure them. Mouse models, however, remained tempting,
once again due to the plethora of tools available, so researchers began trying more creative means of
infection, or began testing genetically modified knockout mouse strains.
With regards to the route of infection, IP injection and intranasal challenge have both been employed
in mice. IP injection is a common technique, often applied for the study of Salmonella Typhimurium,
or other pathogens that can cause systemic infections in mice. This technique with Campylobacter
has been applied on several occasions in mice and it serves the purpose of exposing C. jejuni directly
to the mouse’s immune system.92,93 Intravenous injection of C. jejuni in mice has also been tried, with
a similar effect.92,94 While the relevance of these infection routes has been questioned, they are not
entirely artificial, as C. jejuni has been found on occasion to cause systemic infection in humans.95
However, Campylobacter-associated bacteremia is rare, and not typical for human infection. Despite
this, these models can prove useful in understanding interactions between C. jejuni and the mouse
immune system.
The intranasal challenge model is perhaps more unusual in concept since C. jejuni does not normally
affect or even come into contact with the respiratory system. The inspiration for this model came from
the effective use of an intranasal challenge using Shigella, which proved effective in evaluating potential
vaccine candidates.96 In the case of C. jejuni, an intranasal challenge not only results in exposure of the
respiratory system, but also quickly leads to C. jejuni colonization at systemic sites around the body
within a few days, including persistent colonization of the intestine, mesenteric lymph nodes, liver, and
spleen.96 In the years since the first study utilized this technique, it has been repeated on several occa-
sions, including being used to assess the colonization potential of the cell-adhesion-deficient Peb1A
mutant and it was used to study cytokine production at colonized systemic sites.91,97
Some of the most promising results using mice to study Campylobacter infection have come from
the use of knockout mice, particularly those deficient in key aspects of their immune systems. An early
example of this was a study that assessed C. jejuni infection in both SCID- and RAG2-deficient mice,
both of which are deficient in cellular and humoral immunity.88,98 In these cases, pathogen burdens were
significantly enhanced compared to immunocompetent BALB/c mice; however, in neither case did the
infection trigger diarrhea or significant pathology in a reproducible fashion.
Campylobacter 297

A more successful and widely used approach utilized IL-10 deficient (IL-10−/−) mice to mimic the
intestinal inflammation caused by a C. jejuni infection, in order to assess C. jejuni virulence, rather
than the simple colonization potential.99 IL-10 is one of the key cytokines that regulates inflammation.100
Typically, once inflammation has been induced by infection or injury, the upregulation of IL-10 plays a
critical role in its subsequent resolution. High levels of IL-10 can also prevent inflammation from begin-
ning in the first place, making it an important cytokine for the control of unwanted inflammation and
the collateral damage it can cause. As expected, mice deficient in this cytokine are very susceptible to
developing intestinal inflammation, either from injury, a chemical trigger-like dextran sodium sulfate
(DSS),101 or in response to pathogenic bacteria such as C. jejuni. Several studies have used this infection
model to assess the ability of different C. jejuni isolates from human, animal, and environmental sources
to induce inflammation and pathology.99,102–104 A number of C. jejuni mutants, including those lacking
Campylobacter invasion antigen (Cia) proteins,105 showed a reduced potential to trigger inflammation
in these mice. Additionally, mice doubly deficient in IL-10 as well as one of the several innate immune
receptors such as NOD2, TLR2, and TLR4 were recently used to link signaling by these receptors to the
development of inflammation in this model.106,107
The increased susceptibility of the IL-10−/− mice has proven very useful as a means of overcoming the
seemingly high immune tolerance of mice to C. jejuni; however, it comes with a number of complica-
tions. With IL-10 being one of the linchpins for controlling and resolving inflammation, IL-10−/− mice
are often unable to recover from infection, making C. jejuni infection a chronic and ultimately terminal
infection, rather than the acute, self-limiting infection typically observed in humans. Furthermore, the
sensitivity of these mice to almost any inflammatory trigger often results in the spontaneous develop-
ment of inflammation, likely in response to their own intestinal microbiota. To limit the development of
spontaneous inflammation, IL-10−/− mice are typically kept under germfree conditions,108 adding to the
cost and difficulty of maintaining colonies.
Modifications to the innate immune system have also been investigated as a means of generating an
improved mouse infection model for C. jejuni, as well as for studying the interactions between C. jejuni
and innate immune receptors. When Watson et al. infected single and double knockouts of Nramp1 and
MyD88 with C. jejuni,109 they found that all of them were highly susceptible to C. jejuni colonization,
with these mouse strains carrying a much higher pathogen burden than wild-type mice, including higher
numbers recovered from systemic sites following oral inoculation. However, once again, neither mouse
strain developed any particular signs of inflammation in response to this higher pathogen burden.
With a MyD88 knockout being susceptible to asymptomatic colonization, we took the opposite approach,
and infected mice lacking single IgG IL-1 related receptor (SIGIRR), a protein highly expressed by the
intestinal epithelium, and known to act as a repressor of MyD88-dependent signaling.26 The resulting
mouse exhibits enhanced signaling via MyD88-dependent receptors such as TLRs and IL-1R. Previous
work infecting these mice with Salmonella Typhimurium and Citrobacter rodentium found that their
increased innate signaling impaired microbiota-dependent colonization resistance, resulting in increased
susceptibility to infection along with increased pathogen burdens and the development of more severe
intestinal inflammation in response to infection.110 Unlike IL-10−/− mice, SIGIRR−/− mice do not develop
spontaneous colitis, although they do maintain a slightly higher intestinal inflammatory “tone,” reflect-
ing increased basal levels of several key cytokines.110 Despite being prone to developing more significant
pathology in response to infection or DSS colitis, they are still capable of resolving inflammation after a
bacterial infection has been cleared. This makes them more relevant for studying the type of acute infec-
tion that is normally associated with C. jejuni.
When orally inoculated with C. jejuni, colonized SIGIRR−/− mice developed noticeable signs of
inflammation, primarily within the cecum and proximal colon.26 This corresponded with the primary
site of colonization for C. jejuni, with relatively few microbes being recovered from the small bowel
or at systemic sites. Inflammation was typically characterized by inflammatory and immune cell
infiltration into both the mucosa and submucosa of infected tissues, along with crypt hyperplasia,
increased sloughing of epithelial cells, submucosal edema, and in more severe cases the develop-
ment of ulcers and the loss of crypt structure as the epithelium was damaged/destroyed. Mortality
or severe morbidity was not observed, likely due to C. jejuni’s inability to reach systemic sites in
significant numbers. The development of diarrhea was not typical, but the appearance of mucoid or
298 Laboratory Models for Foodborne Infections

soft stools was common, which differs from typical human infection, in which diarrhea is one of the
most prominent sequelae.
Further investigation into C. jejuni infection of SIGIRR−/− mice linked most of the inflammatory sig-
naling being triggered by C. jejuni to TLR4.26 TLR4−/− and TLR4/SIGIRR double knockouts were found
to display only minimal inflammation and pathology in response to the presence of C. jejuni. This was
also fairly consistent with other results published with mice lacking both IL-10 and TLR4, where sub-
stantially decreased pathology was observed in the absence of TLR4.106 Conversely, although TLR2 had
been previously linked to C. jejuni-induced colitis, in SIGIRR−/− mice, TLR2 appeared to have a protec-
tive role, with TLR2−/− mice and TLR2/SIGIRR double knockouts both developing more severe colitis
following C. jejuni infection.26
A common refrain among those who use mouse models of C. jejuni infection has been the diffi-
culty of establishing consistent infections and colonization numbers, even in knockout mice with more
susceptible immune systems. This is believed to reflect the colonization resistance provided by the
mouse intestinal microbiota. While most birds, ferrets, and humans can become readily infected by
C. jejuni without any manipulation to their intestinal microbiota, the piglet model requires a neonatal
piglet, lacking a fully developed microbiota. Most other animal species, including mice, can harbor
Campylobacter within their intestines transiently, but persistent colonization is largely limited by com-
petition from the intestinal microbiota. In order to allow for reliable and consistent mouse colonization,
some form of disruption to their intestinal microbiota is necessary. Germfree mice, or mice with a
limited microflora are well-known to be very susceptible to C. jejuni colonization, even if they typi-
cally do not develop significant pathology.88 The downside of germfree mice, aside from the difficulty
of maintaining germfree conditions, is the key role microbiota play in the proper development of the
immune system, often leaving germfree mice with an underdeveloped immune system. Fortunately,
a temporary disruption of the microbiota through a single antibiotic treatment was sufficient to allow
for consistent colonization. We treated mice with a single gavage dose of vancomycin 4 h prior to
inoculation with C. jejuni and achieved reliable colonization and consistent pathogen burdens follow-
ing infection with a relatively modest 107 CFU inoculating dose.26 Other studies have used a variety of
antibiotic cocktails to achieve similar results.86 In one study, Bereswill et al. “humanized” mice with
a representative human microbiota, by clearing out the existing mouse microbiota with a cocktail of
antibiotics, followed by inoculation with human fecal samples.111 The newly “humanized” mice became
easily susceptible to C. jejuni colonization by several different strains. The authors characterized a
number of differences between the mouse and human microbial composition; however, precisely how
shifts between mouse and human microbiota opened intestinal niches for C. jejuni colonization remains
unknown. Stahl et al. also carried out a basic characterization of microbiota shifts following vancomy-
cin pretreatment.26 Aside from a dramatic drop in overall bacterial numbers, there were dramatic shifts
in the relative proportions of Firmicutes and Bacteroidetes, however, how those shifts may influence
C. jejuni colonization is presently unknown.
With better knowledge regarding the mechanisms underlying the colonization resistance of the murine
microbiota and new means for increasing the sensitivity of mice to C. jejuni infection, mice may finally
become a relevant infection model for C. jejuni. What is needed now is new research describing the inter-
actions between C. jejuni and the mouse microbiota and immune system to better describe how infection
occurs in mice. Once this is accomplished, we can start drawing more parallels between C. jejuni infec-
tion in mice and humans and employ mouse models to their full potential in understanding the human
disease better.

19.3 Conclusions
Campylobacter jejuni is a common foodborne pathogen that can trigger serious diarrhea in infected
patients. Proper laboratory models for infection have been difficult to establish due to C. jejuni being a
commensal or transient colonizer in most potential animal models of infection. Cellular infection mod-
els, such as the Caco-2 and HT-29 cell lines have proven to be an effective model for studying cellular
interactions. Chicks and germfree mice are useful models for commensal colonization, whereas ferrets,
Campylobacter 299

piglets, and certain knockout mouse strains can be used as effective models for human disease. Herein,
we have discussed how each of these models has been used, the advantages and disadvantages, and some
of what has been learned about Campylobacter from each model.
Many of the mechanisms of pathogenesis for C. jejuni remain either completely unknown or, at best,
poorly characterized. When the first C. jejuni genome sequences failed to reveal identifiable pathogenic-
ity factors akin to those already identified in pathogenic E. coli and Salmonella, a renewed effort was
placed on using known animal models to try and elucidate how C. jejuni colonizes its hosts and how it in
fact causes disease. The lack of animal models that fully replicate the human disease has hindered this
process; however, as a better understanding of the interactions between C. jejuni and the immune system
has started to unfold, better and more relevant animal models are being explored.

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20
Cronobacter: Virulence and Pathogenesis

Nemani V. Prasadarao

CONTENTS
20.1 Introduction................................................................................................................................... 305
20.2 Necrotizing Enterocolitis.............................................................................................................. 306
20.2.1 A Rat Model of C. muytjensii-Induced Necrotizing Enterocolitis.................................. 306
20.2.2 C. muytjensii Interaction with Epithelial Cells in Culture............................................... 307
20.2.3 C. muytjensii Interaction with Dendritic Cells................................................................ 308
20.2.4 The Role of Neutrophils and Macrophages in Cronobacter Infection............................ 309
20.2.5 Prevention of Cronobacter-Induced NEC by Lactobacillus........................................... 309
20.3 Septicemia and Meningitis.............................................................................................................310
20.3.1 Serum Tolerance of Cronobacter spp...............................................................................310
20.3.2 A Neonatal Rat Model of Meningitis................................................................................310
20.3.3 Cronobacter Interaction with Brain Endothelial Cells.....................................................311
20.4 C. sakazakii: Motility and Biofilm Formation...............................................................................312
20.5 C. sakazakii Interaction with Caenorhabditis elegans.................................................................313
20.6 Conclusions....................................................................................................................................313
References................................................................................................................................................314

20.1 Introduction
Originally referred to as yellow-pigmented Enterobacter cloacae, it was later classified as a new species
Enterobacter sakazakii. Subsequent characterization enabled the reclassification of these bacteria into a
new genus called Cronobacter [1]. Cronobacter is composed of a diverse group of Gram-negative bacilli,
which includes Cronobacter sakazakii, Cronobacter muytjensii, Cronobacter malonaticus, Cronobacter
dublinensis, Cronobacter turicensis, Cronobacter universalis, and Cronobacter condimenti [2]. Except
Cronobacter condimenti, all other Cronobacter spp. are associated with human infections. They cause
life-threatening infections in neonates due to the consumption of powdered infant formula contami-
nated with Cronobacter [3,4]. Outbreaks of Cronobacter infections in neonatal intensive care units have
resulted in several CDC warnings, and so efforts are in place to improve health care. Considered as an
opportunistic pathogen, Cronobacter causes severe illness in neonates, such as necrotizing enterocolitis,
bacteremia, and meningitis, often in low-birth-weight preterm infants [5]. Infections due to Cronobacter
in normal and immunocompromised adults have also been noted, but these are less severe. Based on
partial 16S rRNA and hsp60 sequencing, four cluster groups of C. sakazakii have been identified among
this diverse group of pathogens [6]. The bacterium can be found in a variety of foods, including dairy-
based foods (cheese), dried meats, and rice. Furthermore, it was also detected in environmental sources
such as soil, livestock facilities, and food preparation units. Compared to other Enterobacteriaceae fam-
ily members, Cronobacter is highly resistant to heat, dryness, and acidic conditions [7]. It also forms
biofilms that function as a protective barrier to withstand environmental stress and obviate immune
surveillance of the host.
Several selective media for detecting Cronobacter have been developed [8,9]. However, these
media are insufficient to support the growth of all strains of Cronobacter [10]. To detect Cronobacter

305
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spp. in powdered infant formula, a one-step enrichment protocol using a chromogenic medium has been
designed [11]. More useful molecular-based detection techniques for understanding the epidemiology
of Cronobacter have also been developed by targeting a number of genes such as 16S rRNA, 16S-23S
rRNA intergenic regions, ompA, zinc-containing metalloprotease, dnaG and gluA genes by real-time
polymerase chain reaction (PCR) [12–15]. Genes that are unique to different species of Cronobacter are
particularly useful as candidate markers in molecular detection protocols [16–18]. Due to limitations of
individual gene-based techniques, whole-genome sequencing may help identify Cronobacter species
and assist in comparing the genotypic and phenotypic features of the pathogen being studied.
Although powdered infant formula has been found to be the main source of Cronobacter for infecting
newborns, transmission from mother to child during the delivery cannot be excluded. It was also reported
that plant material may be a natural source of Cronobacter spp. [19]. In a study performed to characterize
genotypic and phenotypic features of C. sakazakii strains obtained in an outbreak in intensive care units
in France in 1994 [20], a total of 31 stains were collected, and on the basis of 16S rRNA analysis, 30
strains were confirmed as C. sakazakii. Pulsed-field gel electrophoresis (PFGE) typing recognized four
different pulsotypes and clearly demonstrated that C. sakazakii strains exhibit different genotypic and
phenotypic characteristics. This reinforces the necessity for rigorous and careful testing to identify the
strains. Genomic sequencing of C. sakazakii strain ATCC BAA-894 isolated from contaminated infant
formula revealed a single 4.4 Mb chromosome and two plasmids, pESA2 (31 kb) and pESA3 (131 kb) [21].
ATCC BAA-894 strain contains 4392 genes in the core genome, and 21 genes are found to be unique in
5 other C. sakazakii strains. Similar to ATCC BAA-894 strain, C. turicensis contains a similar genome
and 2 plasmids, which also encodes open reading frames (ORFs) for 223 virulence-associated genes [22].
Comparison of pESA3 and pCTU1 (C. turicensis plasmid) by in silico analysis revealed the presence of
two iron-acquisition systems, which may be essential for pathogenesis.
For the pathogenesis of Cronobacter spp., the bacteria must attach to intestinal epithelial cells of
infants fed with contaminated formula, followed by invasion and hematogenous spread. The adherence
capacities of 50 C. sakazakii strains have been studied using HEp2 and Caco-2 cell lines and brain
microvascular endothelial cells, and two distinctive patterns of binding were observed, diffuse adhesion
and localized cluster formation [23]. The adherence of these Cronobacter strains appears to be non-
fimbrial mediated. Of note, some fimbrial clusters have been identified in the genomes of Cronobacter,
although C. sakazakii is the only bacterium that expresses β-fimbriae, but its role in the pathogenesis
is still to be evaluated. The putative virulence factors produced, such as enterotoxin-like compounds,
by Cronobacter were first assessed by the suckling mouse assay [24]. Analysis of various Cronobacter
strains from different brands of infant formula revealed that they contain high levels (500-fold) of heat-
stable endotoxin (lipopolysaccharide or LPS) [20]. Since these formulae contain a variety of bacterial
species, it is not clear what the source of LPS is. However, its presence enhances the translocation of
C. sakazakii across the gut and blood–brain barrier. Surveillance of microbial burden in food products,
growth conditions, and epidemiology would provide clues to developing methods that reduce health
risks in vulnerable individuals. Furthermore, understanding the pathophysiology of the diseases caused
by Cronobacter spp. is also desirable to identify long-term risks in infected neonates and infants. To
gain insights into the pathophysiology of Cronobacter-induced diseases, a careful selection and usage
of animal models is clearly required. Therefore, the purpose of this chapter is to present the animal and
tissue culture models used thus far to identify bacterial and host factors that contribute to Cronobacter
pathogenesis.

20.2 Necrotizing Enterocolitis
20.2.1 A Rat Model of C. muytjensii-Induced Necrotizing Enterocolitis
Premature infants are prone to a variety of diseases including necrotizing enterocolitis (NEC), which
is a serious gastrointestinal disease and occurs in ∼1 in 1000 live births [25]. The etiology of NEC is
multifactorial, and the risk factors include prematurity, formula feeding, and abnormal bacterial coloni-
zation of intestinal tract. To replicate histopathological manifestations of NEC that mimic human NEC,
Cronobacter: Virulence and Pathogenesis 307

investigators have used several animal models to study the disease. The pathological features of NEC
show the presence of mucosal edema, pneumatosis intestinalis (i.e., the presence of gas within the wall
of the intestine), epithelial sloughing/villous atrophy, enterocyte apoptosis, vascular thrombosis, and dis-
continuous necrotic segments of intestine. A variety of bacterial species are thought to cause NEC such
as Enterobacter, Clostridium, and Staphylococcus spp. Since milk-based infant formulas contaminated
with C. sakazakii have been linked to many NEC outbreaks, a newborn rat model of Cronobacter-
induced NEC using the combination of hypoxia and formula feeding has been developed [26]. The strain
used in these studies, obtained from ATCC (strain 51329), was later classified as C. muytjensii. Newborn
rats fed with infant formula mixed with 105 CFU of C. muytjensii produced clinical symptoms similar
to Grade 3 NEC by day 4 postinfection (Figure 20.1). The pathological analysis of intestinal sections
obtained from infected rats showed macroscopic and microscopic features analogous to that seen in
human NEC [27]. The formula-fed and hypoxic group of rats only showed lower pathological symptoms
and had a mortality rate of 40%, whereas the mortality rate increased to 70% with Cronobacter infec-
tion. Scanning electron microscopy of intestines of newborn rats revealed that specimens from formula-
fed and hypoxic rats exhibited intact villi. C. muytjensii infection increased the binding of the bacteria
to villi tips and showed enterocyte blebbing and gap formation in the epithelium.

20.2.2  C . muytjensii Interaction with Epithelial Cells in Culture

To understand the molecular events induced by C. muytjensii in intestinal epithelial cells, an in vitro
model of infection using IEC-6 cells has been developed. Studies from these experiments revealed that
the bacteria bind to IEC-6 cells efficiently in a dose-dependent manner; however, the entry of the bac-
teria is negligible [28]. Furthermore, the binding of C. muytjensii to IEC-6 cells induced apoptosis at
4 h postinfection as assessed by ApopTag staining. Corroborating with the in vitro results, newborn rats
whose intestines were infected also showed a greater number of apoptotic epithelial cells, whereas the
intestines of those rats that were formula fed showed very few apoptotic cells. Follow-up studies revealed
that C. muytjensii interaction of IEC-6 cells also increased the production of nitric oxide (NO) by 4 h
postinfection. This NO production is due to the activation of inducible nitric oxide synthase (iNOS),
not endothelial nitric oxide synthase (eNOS), as transfection of IEC-6 cells with siRNA to iNOS sig-
nificantly inhibited the NO generation. The increased iNOS activity was transcriptionally regulated,
which in turn is responsible for the enhanced production of iNOS at the protein level. Furthermore,
IEC-6 cells transfected with siRNA to iNOS, not eNOS, and infected with C. muytjensii showed no or
very few apoptotic cells compared to control or eNOS-transfected cells. Examination of the binding
capacities of 24 C. sakazakii strains obtained from human and environmental sources exhibited varied
binding patterns to IEC-6 cells [29]. Interestingly, C. sakazakii strains isolated from humans had higher
binding to IEC-6 cells at a frequency of 4%–5% compared to other environmental strains. High-binding

(A) (B) (D)

(C)

FIGURE 20.1  Neonatal rat model of Cronobacter muytjensii-induced necrotizing enterocolitis: Newborn rat pups were
fed with Esbilac formula two times and three times daily and were placed under hypoxic conditions with 5% carbon dioxide
(FF+H) with (A, left) or without (A, right) C. muytjensii infection. FF+H treated rats showed a normal abdominal wall
with a milk spot (B). Abdominal discoloration and evidence of clinical peritonitis were observed in FF+H+C. muytjensii
rats (C). In contrast, control rat pups showed normal intestine (D). (Reprinted with permission from Hunter, C.J., et al., J.
Infect. Dis., 198, 586–593, 2008.)
308 Laboratory Models for Foodborne Infections

C. sakazakii strains induced more apoptosis in Caco-2 cells than the low-binding strains. Approximately
25% of infected Caco-2 cells were apoptotic. Abnormal colonization of bacteria usually results in the
loss of gut barrier integrity, which is an important pathophysiological feature in the development of
NEC. Similar to that in human epithelial cells, high-binding C. sakazakii infection of Caco-2 cells
caused an increase in permeability of the monolayers as assessed by horseradish peroxidase leakage.
C. sakazakii-induced leakage of the tight junctions in Caco-2 cells were due to the disruption of ZO1 at
the periphery of the cells. Moreover, these permeability changes of Caco-2 cells require the activation of
PKC, which in turn activates NO production upon C. sakazakii infection.
Outer membrane protein A (OmpA) is a 35-kDa surface protein of Gram-negative bacteria whose
structure is highly conserved throughout the evolution [30]. OmpA contains eight transmembrane
domains and four extracellular loops. However, some minor changes are present in the extracellular
loops that help differentiate whether the bacterium is pathogenic or nonpathogenic [31]. Comparison
of OmpA sequences among several Gram-negative strains revealed that the extracellular loops of
C. sakazakii are significantly different from other sequences [30]. OmpA of C. sakazakii has been shown
to play a critical role in the invasion of INT407 cells with an invasion frequency of 0.08 + 0.002 at a mul-
tiplicity of infection of 100 [32,33]. This invasion frequency in epithelial cells is considerably negligible
compared to the invasion frequencies of other gut pathogens such as Salmonella or Shigella [34,35]. The
invasion into epithelial cells depends on both microfilament and microtubules [36]. Fibronectin appears
to be critical for the binding of C. sakazakii to epithelial cells [37]. However, deletion of OmpA in this
strain reduced the invasion by 80% but did not reduce the attachment to the cells. Subsequently, a mouse
model of infection has been developed in which oral feeding of 103 CFU C. muytjensii to 3-day-old mice
induces NEC-like pathology such as intestinal dilation and bowel discoloration by 48 h postinfection [38].
In contrast, OmpA− C. muytjensii does not cause such injury and the mice survived. In this model, wild-
type C. muytjensii binds to the intestine efficiently, whereas OmpA− C. sakazakii could not associate
with epithelial cells. Of note, prebiotic galacto-oligosaccharides and polydextrose inhibit the binding of
C. sakazakii 4603 to HEp-2 cells and Caco-2 cells [39].

20.2.3  C . muytjensii Interaction with Dendritic Cells

Systemic inflammatory response and local cytokine production contribute to the pathogenesis of NEC.
IEC-6 cells infected with C. muytjensii led to IL-6 production in a time-dependent manner, which is in
agreement with the cytokines observed in the serum of infected rat pups [26]. Initiation and regulation of
required immune response to microbial pathogens is a critical function of specialized antigen-presenting
cells such as dendritic cells (DCs). The immature DCs in the peripheral tissues recognize pathogenic
organisms by capturing antigens or whole bacteria upon contact and undergo maturation by expressing
major histocompatibility complex class II, CD40, and CD86 molecules [40]. Supporting the role of DCs,
C. muytjensii infection of 3-day-old mice resulted in recruitment of a greater number of DCs to the
lamina propria, whereas the number of polymorphonuclear cells (PMNs) and macrophages were slightly
increased [41]. OmpA− C. muytjensii infection also caused similar DC migration to the lamina propria.
The recruited DCs are positive for CD11c/CD103 and negative for F4/80/Gr-1. DCs recruited by wild-
type C. muytjensii are not mature as they do not express CD40, MHC class II, and CD86 markers, while
OmpA− C. muytjensii-infected mice showed mature DCs [38]. How does C. muytjensii manipulate DCs
to suppress the immune response? The entry of wild-type C. muytjensii is critical for suppression of mat-
uration markers and is mediated by DC-specific ICAM grabbing integrin (DC-SIGN) [42]. Concurrent
with the requirement of DC-SIGN, DCs pretreated with mannan, which binds mannose-related receptor,
and C. muytjensii pretreated with His-Mermaid, a C-type lectin, which competes with DC-SIGN, pre-
vented bacterial entry into DCs. Overexpression of DC-SIGN in HeLa cells enabled the cells to take up
50-fold more bacteria, both wild type, and OmpA− C. muytjensii, compared to plasmid-alone transfected
cells. The DC-SIGN-mediated entry of C. muytjensii did not require the expression of OmpA. However,
only wild-type bacteria survived in DCs, indicating that OmpA is necessary for the survival of the bacte-
ria inside DCs. Analysis of signaling events modulated in DCs after infection with C. muytjensii revealed
that the bacterium suppresses MAP kinases for preventing maturation.
Cronobacter: Virulence and Pathogenesis 309

Furthermore, supernatants of myeloid-derived DCs infected with wild-type C. muytjensii when incu-
bated with Caco-2 cells grown in Transwell inserts increased the permeability of the monolayers [38]. In
contrast, OmpA− C. muytjensii-infected DC supernatants showed only minor permeability changes. The
supernatant of the wild-type strain also caused disruption of tight junctions, which was further increased
by infection. It appears that tight junction disruption is the first step in the loss of epithelial cells in
the pathogenesis of NEC. In support of this concept, incubation of Caco-2 cells with the supernatants
obtained from DCs infected with C. muytjensii enhanced apoptosis of the cells in the presence of the
bacteria, which is prevented by the presence of apoptosis inhibitor, ZVAD. The responsible soluble factor
in C. muytjensii-infected DC supernatants is an anti-inflammatory cytokine, TGFβ. Wild-type strains
produced robust quantities of TGFβ from bone-marrow-derived DCs, while OmpA− C. muytjensii gener-
ated basal levels of the cytokine. Mucosal scrapings obtained from the intestines of infected mice also
showed increased tgfβ transcript levels. Similarly, intestinal homogenates from C. muytjensii-infected
mice also revealed higher concentrations of TGFβ. Anti-TGFβ antibodies, but not anit-IL-10 antibodies,
when added to the supernatants of C. muytjensii-infected DCs prevented the bacteria-induced monolayer
permeability of Caco-2 cells and apoptosis. This inhibitory effect was due to the inhibition of iNOS
expression in the cells. Upon infection, DCs are recruited to the intestinal lamina propria, which extend
their protrusions between enterocytes to sample the pathogen or macromolecules. Using a double-layer
model by culturing monocyte-derived macrophages on the top of a Transwell filter insert and bone-
marrow-derived DCs at the bottom, it was demonstrated that the presence of DCs increased the produc-
tion of TGFβ, thereby increasing the permeability of the Caco-2 monolayers. Corroborating the in vitro
results, depletion of DCs in newborn mice by injecting anti-CD11c antibody at day 1 after birth resulted
in resistance to C. muytjensii-induced NEC and survival as normal, uninfected mice. DC-depleted
mice showed no signs of pathology in intestines. Adoptive transfer of DCs into these mice rendered the
animals susceptible to infection again.

20.2.4 The Role of Neutrophils and Macrophages in Cronobacter Infection

Neutrophils and macrophages are critical for clearing bacterial pathogens in tissues; however, bacteria
may be equipped with strategies to manipulate these two immune cells. In newborn mice infected with
C. muytjensii, both neutrophils and macrophages are recruited to the intestine in higher numbers com-
pared to the number of cells in uninfected mice; however, these numbers are lower than the number of
dendritic cells. Depletion of either neutrophils or macrophages from 2-day-old mice followed by infec-
tion with C. muytjensii rendered the animals susceptible to infection sooner than normally infected
littermates [41]. The intestinal bacterial load was greater and the pathology severe. In the absence of neu-
trophils or macrophages, the recruitment of DCs increased by 50%, which appears to be responsible for
increased severity of the disease. Consistent with the bacterial load and intestinal pathology, the levels
of proinflammatory cytokines TNF-α, IL-1β, IL-2, and IL-6 also increased. Furthermore, flagellin, the
major protein constituent of flagella, recognizes Toll-like receptor 5 (TLR5) and triggers the production
of various cytokines [43]. Flagellins from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii,
C. turicensis, and C. dublinensis have been tested for the production of cytokines in monocyte-derived
macrophages, and the results have shown that similar levels of IL-8, TNF-α, and IL-10 are produced.
Both anti-flagellin and anti-TLR5 antibodies prevented the production of proinflammatory cytokines in
these cells, thus confirming the importance of their interaction in the pathogenesis by these bacterial
strains.

20.2.5 Prevention of Cronobacter-Induced NEC by Lactobacillus

Several randomized human clinical trials revealed that oral administration of certain probiotic species
can reduce the incidence of NEC although the exact mechanism of protection is unknown [44]. Yogurt
is used widely by humans, and its health-promoting effects are ascertained to be due to the presence
of probiotics. Pre- and coincubation of IEC-6 cells with Lactobacillus bulgaricus and C. muytjensii
demonstrated that the preincubation of the probiotic inhibited C. muytjensii-induced NO production by
310 Laboratory Models for Foodborne Infections

suppressing the iNOS expression [28]. This effect was due to prevention of the binding of C. muytjensii
to IEC-6 cell by L. bulgaricus. Moreover, the probiotic also inhibited C. muytjensii-induced apoptosis
of IEC-6 cells. The observations made in IEC-6 were also reflected in infant rats that were orally fed
with L. bulgaricus and then infected with C. muytjensii as they maintained the structural integrity of
enterocytes by preventing the attachment of Cronobacter under hypoxic conditions. Recent studies have
also shown that the conditioned medium of the probiotic Bifidobacterium infantis protects neonatal mice
from intestinal inflammation when infected with C. sakazakii [45]. The probiotic conditioned medium
reduced enterocyte apoptosis and mucin production and maintained the integrity of the ileal structure
when compared with intestines of mice treated with wild-type bacteria.

20.3 Septicemia and Meningitis

20.3.1 Serum Tolerance of Cronobacter spp.
As a foodborne pathogen, Cronobacter spp. cause systemic infections by crossing the gastrointestinal
barrier, which is followed by invasion of the blood–brain barrier. Therefore, these bacteria should have
virulence factors that help to avoid serum killing. Transposon mutagenesis was used to generate a library
of C. sakazakii ES5, which was then screened for serum tolerance [46]. Several genes encoding surface
and membrane proteins, chaperones, and regulatory proteins have been identified. Of these genes, an
undescribed Wzt_C superfamily domain consisting a coding region for serum tolerance was identified
and confirmed by experiments. Other genes that enhance serum tolerance were also identified, among
them YbaJ. Deletion of ybaJ element affected the expression of the fimA gene, which encodes a struc-
tural component of fimbriae.

20.3.2 A Neonatal Rat Model of Meningitis

C. sakazakii strains also cause meningitis in rare situations in newborn infants due to consumption of
contaminated infant formula [47,48]. The bacterium must cross the gut and the blood–brain barrier and
evade host defenses to cause meningitis [49]. Infection of newborn rats with distinct C. sakazakii (E.
sakazakii) strains via intracranial injection leads to sepsis and chronic inflammation between 6 and
9 days postinfection [50]. Although two different cluster groups of C. sakazakii strains have been used,
the occurrence of meningitis and the inflammation pattern varies from 30% to 80% except for one strain
NTU2, which induced 80% incidence of meningitis and inflammation. Newborn rat brains infected with
NTU2 strains showed severe bilateral ventriculitis, astrocytosis, and microhemorrhage. Furthermore,
these animals also showed vascular permeability, leading to hydrocephalus. Electron microscopy studies
further revealed that C. sakazakii NTU57 strain was found in infiltrated neutrophils and macrophages
in the brain.
Since the natural route of infection of this pathogen is by gut colonization, the information obtained
from the intracranial infection model may have a limitation as regards the extrapolation of these data
to human disease. Using 2-day-old rats that were fed C. muytjensii orally, which mimics the natural
route of infection with the wild-type strain and its OmpA mutant, it was demonstrated that wild-type
C. muytjensii induced meningitis [50]. Histopathological analysis of brain sections from the infected
mice showed that wild-type strain induced migration of glial cells into the cortex and also neutrophil
infiltration (Figure 20.2). Focal hemorrhage and neuronal apoptosis were also observed in several areas
of the brain. On the other hand, although OmpA− C. muytjensii entered the circulation by 6 h postinfec-
tion, the bacterial load was significantly lower compared to wild-type bacteria and could not cause men-
ingitis. The role of OmpA in C. muytjensii-induced meningitis was confirmed by introducing a plasmid
containing ompA gene into OmpA− C. muytjensii, which then regained the capacity to cause disease.
OmpA expressing wild-type C. muytjensii crossed both the gut and blood–brain barrier very efficiently,
whereas OmpA− C. muytjensii is defective in these translocations. Lack of efficient translocation of
OmpA− C. muytjensii across the gut barrier is due to inefficient binding of the bacteria to intestinal cells.
Intestinal pathology after being fed with wild-type strains showed dilated lumen and focal bacterial
Cronobacter: Virulence and Pathogenesis 311

White matter Cortex Leptomeninges

(A) (B) (C)

Control

X 20 X 40 X 40
(D) (E) (F)
OmpA+ CS

X 20 X 20 X 40

FIGURE 20.2  The presence of OmpA+ C. muytjensii in infected newborn rats: C. muytjensii-fed newborn rat brains were
harvested 48 h postinfection, fixed, sectioned, and stained with the anti-OmpA antibody. Mayer’s hematoxylin was used to
counterstain the sections. Control pups received saline. Arrows indicate the presence of clusters of bacteria in white matter
and the cortex. (Reprinted with permission from Mittal, R., et al., Lab. Invest., 89, 263–277, 2009.)

growth along with microvilli flattening. Furthermore, epithelial degeneration was observed in mucosa
along with focal ulceration, reactive stromal changes, and suppurative inflammation. The binding of
C. muytjensii also caused apoptosis of epithelial cells. Infection of mouse pups with wild-type strain
produced TNF-α, IL-1β, IL-6, and IL-10, and chemokine MIP-2 at highest levels by 48 h postinfection.
Of note, OmpA− C. muytjensii-infected animals showed very low levels of these cytokines and chemo-
kines except for IL-10, the levels of which increased between 96 and 120 h postinfection. Despite a small
number of OmpA− C. muytjensii entering the circulation, further multiplication of the bacteria was not
observed. Follow-up serum bactericidal assays showed that wild-type C. muytjensii was resistant to
killing compared to OmpA− C. muytjensii, which are killed within 1 h postincubation with neonatal rat
serum. Other investigators demonstrated that a plasminogen activator (Cpa) mutant of C. sakazakii was
serum sensitive in comparison with its wild-type strain [51]. Cpa activates plasminogen by inactivat-
ing α2-AP and also proteolytically cleaves complement components C3, C3a, and C4b. Lack of serum
resistance in OmpA− C. muytjensii could be due to alteration of Cpa expression because of downstream
effects of ompA deletion. Taken together, it appears that newborn rats infected with C. muytjensii without
hypoxia also results in NEC-like symptoms followed by penetration of the organism into the blood to
cause meningitis.

20.3.3  Cronobacter Interaction with Brain Endothelial Cells

The onset of meningitis occurs by hematogenous spread to the central nervous system following the
invasion of the blood–brain barrier. Several investigators have used brain microvascular endothelial cells
isolated from different sources as an in vitro model of the blood–brain barrier. Using rat brain capil-
lary endothelial cells (rBCEC4) in gentamicin protection assays, it was demonstrated that C. sakazakii
strains NTU2, NTU658, and NTU57 invade the cells with a frequency of 0.43%–1% of input. This inva-
sion frequency is similar to that of the invasion levels exhibited by E. coli K1, a meningitic bacteria in
these cells. Similar to these studies, C. muytjensii also invades human brain microvascular endothelial
cells (HBMECs) at a frequency of 0.5%, which increases in a time-dependent manner [52]. In con-
trast, both epithelial cells and human umbilical vein endothelial cells (HUVECs) were invaded less by
C. muytjensii. Confocal imaging of HBMECs infected with GFP-expressing C. muytjensii (Figure 20.3)
and transmission electron microscopy revealed that the bacterium enters and survives inside the cells with
a single intact bacterium enclosed in vacuoles. The entry of C. muytjensii into HBMECs also requires
312 Laboratory Models for Foodborne Infections

(A) (B)

(C) (D)

(E) (F)

FIGURE 20.3  Invasion of C. muytjensii into human brain microvascular endothelial cells (HBMEC): Green-fluorescent-
protein-expressing C. muytjensii were incubated with HBMEC monolayers for 4 h, washed, and fixed with 2% parafor-
maldehyde. The monolayers were imaged, and Z-stacks of confocal images were acquired using a Leica laser scanning
microscope. The images were taken at 63× magnification. (Reprinted with permission from Singamsetty, V.K., et al.,
Microb. Pathog., 45, 181–191, 2008.)

the expression of OmpA and microtubule reorganization in the cells. In this entry process, microtubule
aggregation is observed beneath the bacterial binding sites but not actin accumulation. Although many
bacterial strains utilize microfilament reorganization in eukaryotic cells for internalization, C. muytjensii
entry appears to be a specialized mechanism. PKC-α plays a central role in cytoskeletal reorganization
in the entry of many bacterial pathogens. C. muytjensii invasion of HBMECs also depends on PKC-α
as overexpression of a dominant negative form of PKC-α, PKC-CAT/KR, in HBMECs prevented the
entry of the bacteria. Studies have demonstrated that both PKC-α and PI3-Kinase stabilize microtubules,
and in agreement, PI3-kinase activation is also necessary for C. muytjensii entry into HBMECs. In
contrast, the invasion of C. sakazakii strain (ATCC 29544) into HBMECs was prevented by cytochala-
sin D, an inhibitor of actin microfilaments [53]. However, the invasion also depended on PI3-kinase as
C. sakazakii ATCC 29544 induced activation of Akt, a downstream substrate for PI3-Kinase.

20.4  C . sakazakii: Motility and Biofilm Formation

Cronobacter spp. form biofilms on a variety of surfaces including enteral feeding tubes, which could
be a source of infections in low-birth-weight infants [54]. The formation of Cronobacter biofilms
requires multiple nutritional factors and environments, which increases its antibiotic resistance
Cronobacter: Virulence and Pathogenesis 313

capacity [55]. A mutant strain of C. sakazakii, LWW02, was constructed by deleting a gene encoding
heptosyltransferase I [56]. This mutant exhibited slower growth, higher permeability of the mem-
brane, and surface hydrophobicity. The biofilm formation by this mutant was stronger compared
to wild-type strains. Furthermore, studies have shown that flagella of Cronobacter is responsible
for the formation of biofilms and for binding to Caco-2 cells [57]. To study the virulence genes of
C. sakazakii ATCC 29544, a transposon-mediated random mutant library was generated and screened,
leading to the identification of a mutant deficient in invasion in Caco-2 cells [58]. A novel plasmid
pCSA2 that contains six ORFs and 4938 bp has been identified. Of the six ORFs, one was predicted
to encode methyl-accepting chemotaxis protein (MCP), which contains one MCP domain and two
sensor PAS (Per-Arnt-Sim sensory) domains that show similarity with biofilm dispersion protein
BdlA of Pseudomonas aeruginosa. Deletion of mcp gene significantly reduced the invasion in Caco-2
cells, and complementation with a plasmid-containing mcp gene enabled the mutant to invade the
cells. Lack of invasion into Caco-2 cells is due to reduced adherence to the cells. Corroborating with
the role in adherence and invasion for MCP, 3-day-old rats fed with the mcp-deleted mutant showed
100-fold reduced efficiency to translocate across the gut and deep into liver and spleen. Interestingly,
the deletion of the mcp gene in C. sakazakii resulted in organisms becoming hypermotile (∼31.5 mm
in 8 h on 0.3% TSA agar) compared to the wild-type strain (∼17.3 mm). As flagella are important
for the motility of Cronobacter, analysis of genes involved in flagellar assembly revealed that mcp
deletion enhanced the mRNA levels of fliA and fliC genes. In addition to the role of the mcp gene in
motility, it is also required for biofilm formation. Thus, MCP plays a critical role in the virulence of
C. sakazakii by regulating multiple functions.

20.5  C
 . sakazakii Interaction with Caenorhabditis elegans
Besides rat and murine models, several other models have also been used to study bacterial patho-
genesis, such as zebrafish and C. elegans [59,60]. C. elegans is a nonparasitic free-living nematode
that is very useful for studying bacterial interaction due to its well-developed genetic and molecular
tools to manipulate the organism. Studies by Shivamurthy et al. revealed that C. elegans fed with
C. sakazakii died in liquid conditions with LT50 of 134 + 2.8 h, whereas feeding on E. coli OP50
(food source of C. elegans) had no effect [61]. C. sakazakii binding to intestines of C. elegans is
responsible for killing the organism, which increased with time of infection. Only live Cronobacter,
not heat-killed ones, were able to colonize and multiply in the intestines, thereby killing the organ-
ism. The death of C. elegans by Cronobacter was due to the apoptosis of the cells of the intestinal
lumen, a mechanism that appears to be similar to that of human NEC pathogenesis. Subsequent stud-
ies by this group revealed that exposure to C. sakazakii LPS led to paralysis, and eventually death,
of C. elegans [61]. Interestingly, the bacterium modifies the structure of LPS upon interaction with
the host to avoid immune response. Map Kinase pathway plays an important role in the immunity of
C. elegans against C. sakazakii LPS.

20.6 Conclusions
Application of suitable animal models to study bacterial infections in such a way that replicates the
pathogenesis in humans is critical to developing therapeutic strategies. To date, investigators have used
a variety of animal species to understand the pathogenic mechanisms. Commonly present in various
food sources and soil, Cronobacter spp. have been linked to the onset of NEC. Both newborn rat mod-
els under hypoxic conditions and mouse models used to explore the pathogenesis of NEC revealed that
C. sakazakii and C. muytjensii can induce NEC-like symptoms. Further studies are clearly required to
fully elucidate the molecular mechanisms involved in the onset of NEC. C. sakazakii-induced neonatal
meningitis is infrequent but often devastating. The neonatal rat model, which mimics human disease, is
extremely useful to identify the bacterial virulence factors and host response to infection, thereby help-
ing to create appropriate treatment strategies.
314 Laboratory Models for Foodborne Infections

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21
Escherichia

Dongyou Liu

CONTENTS
21.1 Introduction....................................................................................................................................317
21.1.1 Classification, Morphology, and Genomics......................................................................318
21.1.1.1  Classification....................................................................................................318
21.1.1.2  Morphology......................................................................................................318
21.1.1.3  Genomics........................................................................................................ 320
21.1.2 Biology and Epidemiology................................................................................................321
21.1.3 Clinical Features and Pathogenesis...................................................................................321
21.1.3.1  ETEC...............................................................................................................321
21.1.3.2  EPEC............................................................................................................... 322
21.1.3.3  EHEC.............................................................................................................. 322
21.1.3.4  EAEC.............................................................................................................. 322
21.1.3.5  STEAEC......................................................................................................... 323
21.1.3.6  EIEC................................................................................................................ 323
21.1.3.7  DAEC.............................................................................................................. 323
21.1.3.8  AIEC............................................................................................................... 323
21.1.3.9  SCEC............................................................................................................... 323
21.1.3.10   NMEC............................................................................................................. 323
21.1.3.11   UPEC.............................................................................................................. 324
21.1.4 Diagnosis.......................................................................................................................... 324
21.1.5 Treatment and Prevention................................................................................................. 324
21.2 Laboratory Models........................................................................................................................ 325
21.2.1 Animal Models................................................................................................................. 325
21.2.1.1  Rodents........................................................................................................... 325
21.2.1.2  Rabbits............................................................................................................ 325
21.2.1.3  Pigs.................................................................................................................. 326
21.2.1.4  Caenorhabditis elegans Nematode................................................................ 326
21.2.2 In Vitro Models................................................................................................................. 326
21.3 Conclusion..................................................................................................................................... 327
References............................................................................................................................................... 327

21.1 Introduction
The genus Escherichia comprises a small group of Gram-negative, rod-shaped bacterial species that
form a part of gut microbiota in mammalian hosts including humans. As part of the well-known and yet
still incompletely understood Escherichia species, Escherichia coli (represented by O157:H4 strain) is an
important cause of intestinal (enteritis, diarrhea, and dysentery), or extraintestinal diseases (urinary tract
infection, intra-abdominal infection, pneumonia, neonatal meningitis, and sepsis). By first presenting a
brief overview on E. coli classification, biology, epidemiology, clinical features, diagnosis, treatment,

317
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and prevention, this chapter then discusses laboratory models that have been applied to the study of
E. coli, with the goal of unlocking the secrets of its pathogenic mechanisms and aiding in the develop-
ment of improved anti-infection strategies.

21.1.1 Classification, Morphology, and Genomics

21.1.1.1 Classification
Covering a group of Gram-negative, rod-shaped, motile, non-spore-forming bacilli (informally known
as “coliforms”), the genus Escherichia (named after its discoverer Theodor Escherich) is classified taxo-
nomically in the family Enterobacteriaceae, order Enterobacteriales, class Gammaproteobacteria, phy-
lum Proteobacteria. To date, six species are recognized within the genus Escherichia, i.e., Escherichia
albertii, Escherichia coli (the type-species, obsolete synonyms: Bacterium coli and Bacillus coli),
Escherichia fergusonii, Escherichia hermannii, Escherichia marmotae, and Escherichia vulneris, with
two former Escherichia species, Escherichia adecarboxylata and Escherichia blattae, being redesig-
nated as Leclercia adecarboxylata and Shimwellia blattae, respectively [1,2].
As a key member of the genus, E. coli represents a predominant facultative anaerobe in the gastrointes-
tinal tract of warm-blooded animals including humans. Although E. coli is largely considered as a harm-
less commensal species and has been extensively used in molecular biology as a cloning host, some of its
strains are known to be involved in various human diseases, such as (1) intestinal disease (gastroenteritis),
(2) urinary tract infection (UTI, which may sometimes evolve to hemolytic uremic syndrome or HUS), and
(3) blood and central nervous system infections (sepsis/meningitis, especially neonatal meningitis) [3,4].
Nonetheless, other members of the genus Escherichia may be occasionally involved in human infections.
On the basis of their unique interactions with eukaryotic cells, adhesion/colonization mechanisms,
toxin/virulence factor production, and clinical disease profiles, pathogenic E. coli strains may be divided
into two categories: intestinal and extraintestinal [1]. The category causing intestinal infections consists
of 10 pathotypes: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic
E. coli (EHEC), enteroaggregative E. coli (EAEC), Shiga-toxin-producing enteroaggregative E. coli
(STEAEC), enteroinvasive E. coli (EIEC), diffusely adhering E. coli (DAEC), cell detaching E. coli
(CDEC), necrotoxic E. coli (NTEC), and adherent invasive E. coli (AIEC). Of these, ETEC, EPEC,
EHEC, EAEC, STEAEC, EIEC, and DAEC are major diarrheagenic E. coli pathotypes (Table 21.1);
CDEC and NTEC are implicated in a very small number of diarrhea-related cases, and AIEC is an
enteric pathogen causing Crohn disease in which diarrhea may present as one of the symptoms [5,6]. The
category responsible for extraintestinal infections includes three pathotypes: septicemia-causing E. coli
(SCEC), neonatal-meningitis-causing E. coli (NMEC), and uropathogenic E. coli (UPEC), leading to
septicemia, pediatric meningitis, and UTIs, respectively [1].
Besides pathotype classification, serological analysis of E. coli surface antigens (i.e., somatic O and
flagellar H antigens, as well as capsular K and fimbrial F antigens) allows distinction of >200 sero-
groups (serotypes), which are designated by specific combinations of O and H antigens (e.g., O157:H7).
Occasionally, the O-antigen [forming part of lipopolysaccharide (LPS) layer] is “masked” by a heat-
labile, acidic polysaccharide capsule (K-antigen). While specific serogroups are often associated with
certain clinical syndromes, some may belong to more than one category of E. coli pathotypes.
More recently, application of genotyping techniques has helped clearly differentiate E. coli strains into
six phylogenetic groups (A, B1, B2, D, E, and Shigella). Nonetheless, due to historic considerations, the
phylogenetic group Shigella has continually been treated as a separate genus Shigella (which consists of
the species Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei) despite its close phylo-
genetic relationship with other members of E. coli and possibly representing some of the clones of E. coli.

21.1.1.2 Morphology
E. coli is a rod-shaped bacterium of about 0.6 μm in diameter and 2 μm in length. The bacterium forms
nonspreading black colonies with a characteristic greenish-black metallic sheen on eosin methylene blue
(EMB) agar, and deep red colonies on MacConkey agar. Other morphological features of note include:
 Escherichia
TABLE 21.1
Characteristics of Major Diarrheagenic E. coli Pathotypes
Pathotype Serogroupa Human Disease Other Features
Enterotoxigenic (ETEC) O6, O8, O15, O20, O25, O27, O63, Watery diarrhea (without Colonizing the small intestine, ETEC employs fimbrial adhesins to bind
O78, O80, O85, O115, O128ac, fever) in children enterocytes and produces two proteinaceous enterotoxins: heat-labile LT
O139, O148, O153, O159, O167 <5 years old, and enterotoxin (with similarity to cholera toxin in structure and function) and
travelers’ diarrhea heat-stable ST enterotoxin (inducing cGMP accumulation in the target cells
and a subsequent secretion of fluid and electrolytes into the intestinal lumen).
ETEC strains are noninvasive and do not leave the small intestinal lumen.
Enteropathogenic (EPEC) O26, O55, O86, O111, O114, O119, Profuse watery diarrhea Colonizing the small intestine, EPEC uses an adhesin known as intimin to
O125, O126, O127, O128, O142, O158 in infants bind host intestinal cells, leading to rearrangement of actin, A/E lesion, and
subsequent diarrhea. Being moderately invasive (with the ability to enter
host cells), EPEC produces a number of virulence factors that are similar to
those found in Shigella and that can elicit an inflammatory response.
Enterohemorrhagic (EHEC) O26, O91, O111, O157 among others Watery diarrhea, Colonizing the distal ileum and colon, EHEC induces A/E lesion and employs
hemorrhagic colitis/ fimbriae for attachment (E. coli common pilus, ECP). EHEC produces a
bloody diarrhea, HUS phage-encoded Shiga toxin that elicits an intense inflammatory response.
Being moderately invasive, EHEC (particularly O157:H7 strain) causes
more serious diarrhea (bloody diarrhea without fever) than EPEC, in
addition to HUS and sudden kidney failure.
Enteroaggregative (EAEC) O3, O15, O44, O77, O104, O126 Travelers’ diarrhea, HUS Colonizing the small intestine and/or colon, EAEC (whose fimbriae aggregate
(stx+), and persistent tissue culture cells) produces a hemolysin and an ST enterotoxin similar to
diarrhea (infants) that of ETEC. EAEC is noninvasive and binds to the intestinal mucosa,
leading to watery diarrhea without fever.
Shiga-toxin-producing O104 Food poisoning STEAEC is an EAEC strain (O104:H4) showing typical EHEC phenotypes
enteroaggregative (STEAEC) (Stx production and strong cell adherence) that causes HUS.
Enteroinvasive (EIEC) O28ac, O29, O112ac, O124, O136, Shigellosis/bacillary Colonizing the colon, EIEC causes a syndrome with profuse diarrhea and
O143, O144, O152, O164, O167 dysentery high fever, which is identical to shigellosis.
Diffusely adherent (DAEC) O86, O127, O142, O158 Persistent watery diarrhea Colonizing the intestine, DAEC shows a distinct diffuse adherence to epithelial
(<5 years old) cells (HEp-2 or HeLa). Isolated from children, DAEC harbors Afa/Dr genes.
Some serogroups are associated with more than one diarrheagenic E. coli pathotype. For example, serogroup O86 is mainly considered as EPEC, but it has also been implicated in ETEC,
a

EAEC, and DAEC infections.

319
320 Laboratory Models for Foodborne Infections

1. Fimbriae (pili), which are of two kinds, common or conjugative. Common fimbriae (about
100–1000 per cell) comprise mainly an acidic hydrophobic protein called fimbrin and may
be divided into seven groups according to the amino acid sequence of their major fimbrin.
Conjugative fimbriae (also known as sex pili, usually number one or a few copies per cell)
facilitate contact between the donor and recipient bacteria, permitting transfer of DNA during
conjugation. Fimbriae are highly antigenic and contain many F antigens.
2. Flagella, which are responsible for bacterial mobility. E. coli flagella (about 5–10 copies per
cell; 5–10 μm in length) are composed of a long filament, a hook, and a basal body and are
arranged randomly around the cell surface (a pattern known as peritrichous flagellation). The
principal component of E. coli flagella is an N-methyl-lysine-rich protein (55 kDa in size)
known as flagellin, and around 20,000 subunits of flagellin are needed to make the flagellar
filament. Flagella are highly antigenic and include a large number of H antigens.
3. Capsule and outer membrane. The outer membrane of E. coli is composed of a lipid bilayer,
which in turn consists of a phospholipid inner leaflet and an LPS outer leaflet, together with
several kinds of membrane proteins in the periplasm (the space between the inner and outer
leaflets/membranes). In the outer membrane, a glycolipid (called the enterobacterial common
antigen or ECA) is found in E. coli. In some E. coli strains, the outer membrane is covered
by a polysaccharide capsule containing K antigens. Under conditions of high osmolarity, low
temperature, and low humidity, other polysaccharides such as M antigens (colanic acids, which
are polymers of glucose, galactose, fucose, and galacturonic acid) are synthesized.
4. Periplasm and cell wall. The periplasm of E. coli is osmotically active and contains over 60
known proteins, including binding proteins for amino acids, sugars, vitamins, and ions; deg-
radative enzymes (phosphatases, proteases, and endonucleases); and antibiotic detoxifying
enzymes (β-lactamases, alkyl sulfodehydrases, and aminoglycoside phosphorylating enzymes).
The cell wall of E. coli is responsible for cell shape and rigidity and is composed of a pepti-
doglycan layer (of one or a few molecules thick) that is anchored to the outer membrane via
covalent links to the major membrane lipoprotein and noncovalent links to porins.
5. Cytoplasmic membrane. The cytoplasmic membrane of E. coli is made up of about 200 distinct
proteins (including those involved in peptidoglycan biosynthesis, cell wall elongation, and cell
division, as well as penicillin-binding proteins that covalently bind β-lactam antibiotics) and
four kinds of phospholipids.
6. Cytoplasm. The cytoplasm of E. coli contains ribosomes and other organelles in which biologi-
cal/biochemical activities necessary for growth are carried out. These activities include meta-
bolic fueling (production of energy, reducing power, and precursor metabolites), biosynthesis
of building blocks, polymerization into macromolecules, and assembly of cell structures [1].

21.1.1.3 Genomics
The genome of E. coli strain MG1655 (a laboratory strain K-12 derivative) consists of a circular DNA
molecule 4.64 Mb in length, with 4288 protein-coding genes (organized into 2584 operons), 7 rRNA
operons, and 86 tRNA genes, together with a number of transposable genetic elements, repeat ele-
ments, cryptic prophages, and bacteriophage remnants. Some pathogenic E. coli strains possess larger
genomes than that of the commensal K-12 strain, such as enterohemorrhagic E. coli strain O157:H7
Sakai (5.50 Mb); enteroaggregative E. coli strain O42 (5.36 Mb); and UPEC isolates CFT073 (5.23 Mb),
536 (4.94 Mb), and UTI89 (5.07 Mb).
While many strains (e.g., strain MG1655) do not have plasmid, others possess one to five plasmids,
which along with chromosomal pathogenicity islands contribute to the plasticity of the E. coli genome.
It is notable that all major diarrheagenic E. coli pathotypes carry at least one virulence-related property
on a plasmid. The plasmid is typically large (>60 megadalton or MDa), of low copy number, of either
conjugative or transmissible incompatibility group, and encodes multiple virulence factors. In addition
to plasmid-encoded clusters of virulence traits and chromosomal pathogenicity islands, transposon-
encoded individual traits (e.g., Shiga toxin) may be also present [7,8].
Escherichia 321

21.1.2 Biology and Epidemiology

E. coli is a chemoheterotroph with the ability to utilize a variety of sugars or amino acids, and grows rapidly
in nutrient rich broths (containing amino acids, nucleosides, sugars, and vitamin precursors, etc.). While
some strains found in nature have a single auxotrophic requirement such as thiamin, the growth of many
strains may be inhibited by the presence of single amino acids such as serine, valine, or cysteine. E. coli tol-
erates temperature between 8°C and 48°C and grows optimally at 39°C. The bacterium survives between
pH 6.0 and 8.0, but is unable to grow in media containing >0.65 M NaCl. In response to changes in the
osmotic pressure of the medium, E. coli increases its concentration of ions, especially K+ and glutamate.
E. coli is well adapted to the mammalian intestine, including the large intestine, the ileum, and the
distal segment of the small intestine. Interestingly, some diarrheagenic E. coli strains infect the sparsely
colonized small bowel (ETEC, EPEC), while others inhabit in the colonic mucosa (EIEC, EHEC,
and probably EAEC), with close proximity to the enterocytes. In addition, some pathotypes (EHEC,
EPEC, and EIEC) utilize type 3 secretion system (T3SS) for translocating bacterial proteins (or effec-
tors) directly into the eukaryotic host cell to subvert host cell processes, while others (ETEC, EAEC,
STEAEC, DAEC, and AIEC) are non-T3SS dependent and require effective colonization factors and
secreted toxins for virulence [9].
Discharged from the intestine into soil and water, E. coli does not survive long (a few days). Mammals
(typically cattle, chickens, deer, sheep, and pigs) usually become colonized with E. coli shortly after
birth, possibly during passage through the birth canal, or via the fecal–oral route from the mother or
other attendants [10]. In humans, E. coli gains entry after exposure to contaminated food, beverages,
water, animals, or other persons. Upon ingestion, E. coli multiplies rapidly in the large intestine and
binds tightly to cells in the intestinal lining, facilitating absorption of the toxin into the small capillaries
within the bowel wall and subsequently attaching to globotriaosylceramide (Gb3) receptors.
It is noteworthy that of >200 E. coli serotypes identified to date, those producing Shiga toxin (Stx),
which is indistinguishable from that produced by S. dysenteriae type 1, are frequently found in contami-
nated foods and beverages (e.g., ground beef, venison, sausages, dried salami, unpasteurized milk and
cheese, unpasteurized apple juice and cider, orange juice, alfalfa, parsley, radish sprouts, lettuce, cabbage,
spinach, fruit nuts, berry, cookie dough, and water). The most notorious Stx-producing E. coli strains
are O157:H7 and, to a lesser extent, O121:H19. Strain O157:H7 is responsible for the majority of E. coli-
related human illnesses such as bloody diarrhea and HUS, the latter being defined by the trilogy of hemo-
lytic anemia (destruction of red blood cells), thrombocytopenia (low platelet count), and acute kidney
failure. First recognized as a human pathogen in foodborne outbreaks associated with hamburgers from
a fast food chain restaurant in 1982, E. coli O157:H7 was later shown to be responsible for a 2006 out-
break in Iowa and Minnesota following consumption of lettuce grown in close proximity to a dairy farm
from which wastewater contaminated with animal feces was used for irrigation. Stx-producing E. coli
strains are hardy organisms that can survive several weeks on surfaces (e.g., countertops) and up to a year
in compost. In addition, Stx-producing E. coli strains have a very low infectious dose (<50 organisms).
Together, these attributes make Stx-producing E. coli strains highly dangerous foodborne pathogens.

21.1.3 Clinical Features and Pathogenesis

Pathogenic E. coli is implicated in a large number of clinical diseases in humans, including intestinal
infection (watery diarrhea or dysentery), UTI, blood infection (septicemia), and meningitis, in addition to
pneumonia and intra-abdominal infection (cholecystitis, cholangitis, and peritonitis) [11].

21.1.3.1 ETEC
ETEC is usually acquired from food or water contaminated with human or animal feces. This pathotype
causes watery diarrhea of varied severity (ranging from mild, self-limiting disease to severe cholera-like,
life-threatening illness). With a sudden onset of watery stool (without blood or inflammatory cells) and
vomiting, ETEC infection may lead to dry mouth, rapid pulse, lethargy, decreased skin turgor, decreased
blood pressure, muscle cramps, and shock due to progressive loss of fluids (dehydration) and electrolytes
322 Laboratory Models for Foodborne Infections

(sodium, potassium, chloride, and bicarbonate). The diarrhea is self-limited and generally lasts for only
3–4 days. If hydration is maintained, the patient may recover without any sequelae. ETEC is a major
cause of diarrhea in infants younger than 2 years of age in the developing world, and it is also associated
with travelers’ diarrhea [12].

21.1.3.2 EPEC
EPEC is an attaching and effacing (A/E) pathogen that induces a characteristic A/E lesion (or pedestal-
like structure) on the lumenal surfaces of host small intestine, without the production of Shiga toxins.
In addition to watery diarrhea containing mucus but not blood, other symptoms associated with EPEC
infection include vomiting, fever, malaise, and dehydration. While these symptoms often persist for
several days, chronic EPEC infection is sometimes observed. Occasionally, EPEC may cause bloody
diarrhea after the colonization of the mid-distal small intestine (ileum). EPEC is separated into two
groups according to the presence/absence of EPEC adherence factor plasmid (or pEAF): typical EPEC
(tEPEC) and atypical EPEC (aEPEC). tEPEC possesses pEAF, forms localized adherence pattern,
and includes classical O26, O55, O86, O111, O119, O125, O126, O127, O128ac, O142, O158, whereas
aEPEC lacks pEAF, but harbors other virulence factors and may form diffuse or aggregative adherence
patterns. EPEC is a common cause of watery diarrhea among infants in the developing world and is
also implicated in sporadic diarrheal outbreaks among infants (e.g., day-care facilities) in the developed
world [13].

21.1.3.3 EHEC
EHEC is characterized by the production of Shiga toxins (Stx1 and/or Stx2, leading to its alternative
name of STEC) and the formation of A/E lesion, notably in the cecum and ascending colon [14–16].
EHEC typically causes an afebrile bloody colitis (bloody stools with ulcerations of the bowel) known as
hemorrhagic colitis (HC), which is characterized by the sudden onset of abdominal pain, severe cramps,
and diarrhea within 24 h. In about 10% of patients (e.g., children and the elderly), infection with EHEC
O157 may result in HUS, which is defined by acute renal failure, hemolytic anemia, and thrombocytope-
nia. Colonoscopy examination reveals the presence of edema, erythema (redness), hemorrhage, erosion,
and, occasionally, a long ulcer-like lesion, with a marked narrowing of the luminal space. Histologic
examination indicates destruction of the surface epithelium, neutrophil infiltration of the lamina propria,
and the formation of crypt abscesses. In patients (especially children of <5 years of age and the elderly)
with diarrhea and HUS, renal pathology consists of endothelial swelling and glomerular thrombosis
with congested rather than ischemic glomeruli. The most prevalent EHEC serotype causing outbreaks in
North America and other parts of the world is O157:H7 [17,18]. Besides Shiga toxin, which may account
for the severe complications including HUS, O157:H7 expresses several other virulence factors including
intimin, translocated intimin receptor (Tir), a T3SS, and enterohemolysin [19]. The genes encoding many
of these factors are located on a 44 kb pathogenicity island (also known as the locus of enterocyte efface-
ment or the LEE locus) [20–22]. Cattle act as a primary reservoir for EHEC, although vegetables (let-
tuce, spinach, and sprouts) and fruits may also serve as vehicles for EHEC outbreaks [23].

21.1.3.4 EAEC
EAEC is noted for forming an AA pattern (which is characterized by prominent, “stacked brick” auto-
agglutination of the bacterial cells to each other) during its adhesion to HEp-2 cells [24,25]. EAEC
infection manifests clinically as watery diarrhea with or without blood and mucus, abdominal pain,
nausea, vomiting, and low-grade fever. Since 1980s, EAEC has been recognized as a causative agent of
persistent diarrhea in malnourished children in the developing world, and it also causes both outbreaks
and sporadic diarrhea among travelers and immunocompromised individuals (specifically HIV-infected
patients) in the developed world. Individuals with single-nucleotide polymorphisms (SNPs) in the IL-8
gene promoter and lactoferrin gene may show higher susceptibility to EAEC infection [26]. EAEC is
Escherichia 323

primarily transmitted through contaminated food and water. Food handlers (especially those working in
tourist hotels) are important carriers of EAEC. Interestingly, EAEC-induced travelers’ diarrhea occurs
constantly throughout winter and summer, whereas other diarrheagenic E. coli strains such as ETEC,
EPEC, and EIEC show lower rates of infection in winter [27].

21.1.3.5 STEAEC
STEAEC is an EAEC strain (O104:H4) that has acquired typical EHEC phenotypes such as Stx pro-
duction and strong cell adherence. STEAEC O104:H4 is responsible for HUS in a high percentage of
patients, with a mortality rate of 1%.

21.1.3.6 EIEC
EIEC is pathogenetically, biochemically, and genetically related to Shigella spp. and principally resides in
the large intestine. EIEC causes an invasive inflammatory colitis with a watery diarrhea syndrome that is
indistinguishable from those caused by other E. coli pathotypes and Shigella spp. In severe cases, scanty
dysenteric stools may contain blood and mucus, and this may occur together with fever and severe cramps.

21.1.3.7 DAEC
DAEC is differentiated from other diarrheic E. coli by its diffuse adherence to epithelial cells in the clas-
sical laboratory assay of adherence to HEp-2 or HeLa cells. This diffuse adherence pattern is essentially
linked to the production of adhesins encoded by a family of afa/dra/daa-related operons. DAEC causes
a watery diarrhea in adults and children (with increased risk in those aged 18 months to 5 years) [28,29].

21.1.3.8 AIEC
AIEC adheres to intestinal epithelial cells through type 1 pili (especially FimH adhesin variant) that
interacts with host glycoprotein CEACAM6 in a mannose-associated manner, gains access to the lam-
ina propria, and replicates in macrophages (which represent an environment with acidic pH, oxidative
stress, active proteolytic enzymes, and antimicrobial compounds) [30]. Transient AIEC colonization
elicits intestinal inflammation and alters microbiota composition leading to Crohn’s disease (CD), with
symptoms ranging from fever, fatigue, abdominal pain, cramping, nausea, vomiting, bloody stool, mouth
sores, reduced appetite, weight loss, and perianal disease to diarrhea. Host deficiencies in CD patients
linked with the increased ability of AIEC LF82 to cause infection include the overexpression of the
CEACAM6 and Gp96 receptors in the apical membrane of intestinal epithelial cells (which facilitates
AIEC adhesion and invasion), defects in autophagy related to NOD2, ATG16L1, and IRGM function
and expression (which impair the ability of host cells to resolve infections), altered bile salts metabolism
(which enhances the expression of long polar fimbriae in AIEC, permitting better translocation via
M cells), and decreased levels of protease meprin (which degrades type 1 pili) [31].

21.1.3.9 SCEC
SCEC is associated with sudden high fever with chills, nausea, vomiting, diarrhea, abdominal pain,
hypotension, confusion, anxiety, tachypnea (short of breath), tachycardia (rapid heart rate), and uremia.

21.1.3.10 NMEC
NMEC is responsible for fever, failure to thrive, neurologic signs, jaundice, decreased feeding, periods of
apnea, and listlessness in neonates. Infants of <1 month of age may also show irritability, lethargy, vomiting,
lack of appetite, and seizures, while those >4 months of age display neck rigidity, tense fontanels, and fever.
Older children and adults tend to present with headache, vomiting, confusion, lethargy, seizures, and fever.
324 Laboratory Models for Foodborne Infections

21.1.3.11 UPEC
UPEC utilizes type 1 fimbriae (particularly FimH) to interact with urinary tract host epithelia and is
among the most prevalent extraintestinal bacteria accounting for 90% of all UTIs, including both cystitis
and pyelonephritis. This pathotype has evolved a multitude of virulence factors and strategies to facili-
tate its growth and persistence within the adverse settings of the host urinary tract [32,33].

21.1.4 Diagnosis
E. coli grown on EMB agar produces black colonies with a diagnostic greenish-black metallic sheen. In
addition, being lactose positive, E. coli generates deep red colonies on MacConkey agar, as fermentation
of lactose decreases the medium’s pH and darkens the medium. Other biochemical features of E. coli
include the ability to reduce nitrates to nitrites and to generate succinate, ethanol, acetate, and carbon
dioxide. While most E. coli strains are positive for catalase, they are negative for oxidase, citrate, urease,
and hydrogen sulfide. Further, E. coli is positive for indole production and the methyl red test. Given
that about 98% of E. coli strains are positive in the indole test, it offers a useful approach to differentiate
E. coli from other members of the family Enterobacteriaceae.
Use of epithelial cell lines (e.g., HEp-2 and HeLa) provides another means to characterize E. coli
pathotypes. For instance, HEp-2 cell-adherence assay enables differentiation among EPEC, EAEC,
and DAEC isolates, and HeLa cell monolayers facilitate identification of plaque-forming EIEC isolates.
Further, Y1 adrenal and Chinese hamster ovarian cells may display morphological changes in the pres-
ence of heat-labile enterotoxin (LT)-producing E. coli. Infant mouse physiological assay is applicable
for heat-stable enterotoxin (ST) identification. Guinea pig may be used for detecting keratoconjunctivitis
caused by EIEC isolates. Moreover, Stx-producing E. coli may be verified by mammalian cell cultures,
which are rapidly killed by the Stx.
By targeting the O (lipopolysaccharide), H (flagellar), K (capsular), and F (fimbrial) antigens, sero-
logical procedures (e.g., mannose-resistant agglutination of erythrocytes, passive latex agglutination,
enzyme-linked immunosorbent assay, immunoprecipitation in agar, and Biken test) employing poly-
clonal or monoclonal antibodies offer a more specific way to define E. coli strains and pathotypes.
Recent development of nucleic-acid-based techniques makes rapid, sensitive, and specific identi-
fication and typing of E. coli strains and pathotypes possible. These new-generation methods detect
E. coli genes that encode toxins, pili, and other virulence factors. Indeed, polymerase chain reaction
(PCR) procedures targeting the pap, afa, and sfa genes have proven valuable for UPEC identification.
Application of multiplex and real-time PCR allows for simultaneous detection of several diarrheagenic
E. coli pathotypes [34–38].

21.1.5 Treatment and Prevention

As most patients with E. coli infection recover in a week or two without any long-term sequelae, good
supportive care (fluid and electrolyte balance, nutrition) is all that is needed. However, patients who
develop HUS (usually 2 weeks after E. coli infection) often require hospitalization (3 days to 3 months for
children, and even longer for adults). Apart from supportive care, antibiotics may be administered. These
range from amoxicillin, cephalosporins, carbapenems, aztreonam, trimethoprim–sulfamethoxazole,
ciprofloxacin, and nitrofurantoin to aminoglycosides. It should be noted that some E. coli strains may
be resistant to β-lactam antibiotics such as penicillins and cephalosporins as well as carbapenem [39].
Considering the widespread dissemination of Shiga-toxin-producing E. coli strains nowadays, and
their possible transmission via contaminated foods, as well as direct person-to-person/animal-to-person
contact, prevention of E. coli infection at the individual level should focus on (1) practicing meticulous
personal hygiene; (2) proper cleaning, processing, and cooking of fruits or vegetables; (3) avoiding cross-
contamination during preparation and cooking of food; (4) avoiding touching/petting farm animals; and
(5) avoid drinking any nonchlorinated water.
Application of enterobacteria phage T4 in a mist, spray, or wash on live animals helps reduce
potential E. coli O157:H7 contamination of meat products. Furthermore, development of effective
Escherichia 325

vaccines will contribute to the control and prevention of E. coli infection. In this regard, an E. coli
O157:H7 O-specific polysaccharide conjugated to recombinant exotoxin A of Pseudomonas aerugi-
nosa (O157-rEPA) has been shown to elicit a mitigating immune response in both adults and children
2–5 years of age [40].

21.2 Laboratory Models
In order to uncover the virulence mechanisms of pathogenic E. coli, understand bacteria–host cell inter-
actions, determine colonization and attachment factors, investigate host immune response, and evaluate
candidate vaccines, use of laboratory models (both in vivo and in vitro) is critical [41–43].

21.2.1 Animal Models
A variety of animals have been utilized as models for investigating E. coli infection and disease. These
range from mice, rats, rabbits, chickens, pigs, cows, and dogs to baboons and macaques. For example,
gnotobiotic piglets, infant rabbits, calves, chickens, and macaques have proven valuable for assessing
characteristic A/E lesions associated with E. coli O157:H7. Greyhounds and rabbits are useful for exam-
ining naturally occurring HUS-like diseases [i.e., idiopathic cutaneous and renal glomerular vasculopa-
thy of greyhounds (CRVGs) or Alabama rot] [21,44].

21.2.1.1 Rodents
Mice do not show signs of intestinal disease following oral infection with EHEC, although they do
develop kidney damage (acute tubular necrosis instead of prothrombotic condition seen in humans) and
subsequently die [45]. Pretreatment with antibiotics (e.g., streptomycin), use of germfree animals, or
dietary-induced changes (e.g., protein–calorie malnutrition) appears to reduce the natural resistance of
mice to EHEC colonization [46,47]. Another useful mouse model is the intact commensal flora (ICF)
model for which mice harboring normal flora are utilized. ICF mice infected with O157:H7 enable moni-
toring of both colonization and disease (e.g., ruffled fur, lethargy, weight loss, renal tubular damage, and
mortality) [21,48]. A natural mouse pathogen Citrobacter rodentium (which carries a homolog of the
LEE locus of EPEC and EHEC and has the capacity to evoke A/E lesions) may be exploited as a sur-
rogate for E. coli O157:H7 for evaluation of the virulence mechanisms of EHEC. A mouse intoxication
model involving parenteral injection of Stx facilitates in vivo can be used for assessment of the effects of
Shiga toxin (especially Stx2), which are responsible for damage to renal cortical tubule epithelial cells,
paralysis, and mortality (i.e., symptoms associated with HC and HUS) [21].
A neonatal rat model provides a useful method to evaluate systemic infection due to neuropathogenic
E. coli, as colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-
lymph-blood-brain course of infection. Similar to human host, neonatal rat shows local inflammation
upon E. coli penetration of the central nervous system [49]. Additionally, use of a rat model of unilateral
E. coli epididymitis reveals that despite the eradication of the pathogen by antimicrobial agents accom-
panied by the reduction of epididymal damage, inflammation in the contralateral epididymis is neverthe-
less present, which may contribute to impaired fertility [50]. On the whole, rodent models are relatively
inexpensive and easy to care for and handle.

21.2.1.2 Rabbits
Suckling New Zealand White (NZW) rabbits are susceptible to oral infection with E. coli O157:H7, lead-
ing to watery diarrhea in young (5–10 days old) but not older (20 days old) rabbits. Histological abnor-
malities (e.g., edema, hemorrhage, the presence of an inflammatory infiltrate, and mucosal epithelial
apoptosis) are observed in the colon. However, suckling rabbits do not develop signs of renal disease [51].
These data suggest that suckling rabbits may be a useful model for studying the intestinal manifestations
of EHEC infection. Nevertheless, older rabbits appear to be recalcitrant to colonization by EHEC [52].
326 Laboratory Models for Foodborne Infections

21.2.1.3 Pigs
The intestine of gnotobiotic piglets [i.e., cesarean-derived colostrum-deprived (CDCD) piglets] is per-
missive for EHEC replication, as oral infection of CDCD piglets with 1010 CFU of E. coli O157:H7
leads to watery diarrhea without blood by 2–4 days postinoculation. Histologic abnormalities include
destruction of the mucosal brush border and inflammation (so called A/E lesions) in the cecum and
colon. Apart from intestinal disease, gnotobiotic piglets also exhibit signs of central nervous system
disease (e.g., vascular damage in the cerebellum, ataxia) and renal lesion upon oral or intraperitoneal
administration of O157:H7. The kidneys of infected gnotobiotic piglets show signs of diffuse glomeru-
lar endothelial swelling and congestion as well as a narrowing of the capillary vessels. In addition,
morphological signs of thrombotic microangiopathy, which is characteristic of HUS in humans, are
also noticeable. Naturally born piglets are found to exhibit more severe neurological disease and more
quickly succumb to EHEC infection than traditional CDCD animals, although no signs of kidney dam-
age are found. Nonetheless, maintenance of gnotobiotic piglets requires considerable veterinary skill,
space, and expense [21].

21.2.1.4  C aenorhabditis elegans Nematode

C. elegans is susceptible to EPEC infection and succumbs to the disease via a “slow killing” mechanism
that results from bacterial accumulation in the nematode intestine and formation of microcolonies simi-
lar to those formed during EPEC infection of cultured epithelial cells.

21.2.2  In Vitro Models

In vitro systems [e.g., cell monolayers, transwells, organoids, in vitro organ culture (IVOC), and ex vivo
cultures of biopsies] provide excellent models for elucidation of E. coli pathogenesis, including aspects
of cell adherence and Stx toxicity. Due to their convenience, rapid growth, uniformity, availability of
antibody reagents, and variation in genetic backgrounds, established cell lines represent particularly
valuable tools for the study of E. coli.
Epithelial cells (e.g., MDCK, Caco-2, T84, and HT29) facilitate evaluation of E. coli adherence mech-
anism and disruption of epithelial barrier function during infection, as they have the ability to mimic
polarization by forming tight junctions and brush border. Endothelial cells [human umbilical vein endo-
thelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs)] are valuable
for assessing the effect of Stx on vascular cell integrity and cytokine response. Macrophage-like cell
lines (e.g., J774 and U937) enable assessment of E. coli uptake by professional phagocytes and identifica-
tion of EPEC EspF, EspJ, and EspH effector functions. In addition, differing from polarized cells of the
intestinal epithelium, nonpolarized cell lines (e.g., HeLa cells) are extensively used to study basic aspects
of EPEC–host interaction and T3SS function [42].
Transwell system allows for the development of polarized cell monolayers with features characteristic
of differentiated cells (e.g., the formation of tight junctions and the expression of unique cellular factors),
which are useful for exploring the mechanisms of both E. coli O157:H7 binding to and Stx transit across
the epithelium.
Organoid system involves cells grown on a scaffold under microgravity conditions that form pieces of
tissue-like material. This permits detailed investigation of E. coli O157:H7 adherence, formation of A/E
lesion, and other forms of host cell damage.
IVOCs involve infection of freshly obtained human intestinal biopsies kept in tissue culture media
under high oxygen pressure to delay ischemia and cell death. As the infected tissue is as close as one
can get in vitro to native live tissue, IVOCs helps uncover insight on E. coli O157:H7 adherence to gut
mucosal cells and subsequent damage to the intestinal cells. Furthermore, the development of the polar-
ized IVOC model allows better mimicking of the in vivo situation during specific apical EPEC infection.
Nonetheless, IVOC is limited to several hours until the tissue dies, is technically challenging, and shows
sample variability.
Escherichia 327

21.3 Conclusion
Constituting a key member within the genus Escherichia, E. coli is a remarkable bacterium that encom-
passes a large number of strains of varied pathogenic potential. Although many E. coli strains are com-
mon symbiotic organisms in the gut, some demonstrate propensity to cause intestinal and extraintestinal
diseases in humans. To date, 10 pathotypes have been identified among E. coli strains involved in intes-
tinal diseases, including ETEC, EPEC, EHEC, EAEC, STEAEC, EIEC, DAEC, CDEC, NTEC, and
AIEC [53], whereas three pathotypes have been recognized among E. coli strains causing extraintestinal
diseases, i.e., SCEC, NMEC, and UPEC. Extensive past research has uncovered valuable insights in
the biology, epidemiology, and pathogenesis of E. coli infections and contributed to the development
of rapid and precise diagnostic techniques [54]. However, much remains unknown or unclear about
the molecular and immunological basis of E. coli pathogenicity. The application of various laboratory
models is unquestionably necessary in generating new clues to virulence strategies employed by E. coli
organisms [55].

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tion of six diarrheagenic E. coli pathotypes and Salmonella. Int J Med Microbiol. 2013;303(4):210–216.
35. Wiles TJ, Mulvey MA. The RTX pore-forming toxin α-hemolysin of uropathogenic Escherichia coli:
progress and perspectives. Future Microbiol. 2013;8:73–84.
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col. J Clin Lab Anal. 2013;27(2):155–161.
37. Fujioka M, Otomo Y, Ahsan CR. A novel single-step multiplex polymerase chain reaction assay for the
detection of diarrheagenic Escherichia coli. J Microbiol Methods. 2013;92(3):289–292.
38. Souza TB, Lozer DM, Kitagawa SM, Spano LC, Silva NP, Scaletsky IC. Real-time multiplex PCR
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42. Law RJ, Gur-Arie L, Rosenshine I, Finlay BB. In vitro and in vivo model systems for studying entero-
pathogenic Escherichia coli infections. Cold Spring Harb Perspect Med. 2013;3(3):a009977.
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22
Helicobacter

Tetsuya Tsukamoto, Yuka Kiriyama, and Masae Tatematsu

CONTENTS
22.1 Introduction..................................................................................................................................331
22.2 Discovery of Gastric Microorganisms.........................................................................................331
22.3 Gastric Microenvironment of H. pylori.......................................................................................332
22.4 Characteristics and Pathogenesis of H. pylori.............................................................................332
22.5 Infection Route and Childhood Acquisition of H. pylori............................................................333
22.6 Role of H. pylori Infection in Gastric Carcinogenesis................................................................333
22.7 H. pylori Infection Induces No Neoplastic but Hyperplastic Lesions in Mongolian Gerbils...... 334
22.8 Prevention of Gastric Carcinogenesis by Eradication of H. pylori............................................ 336
22.9 Intestinal Metaplasia and Intestinalization of Gastric Cancer................................................... 336
22.10 Exacerbating Factors for Gastric Carcinogenesis: Synergistic Effects of H. pylori
and High-Salt Diet��������������������������������������������������������������������������������������������������������������������� 336
22.11 Chemoprevention of Gastric Carcinogenesis.............................................................................. 337
22.12 Conclusion................................................................................................................................... 337
References............................................................................................................................................... 337

22.1 Introduction
Since Helicobacter pylori was discovered to play a major causative role in chronic gastritis and gastric
carcinogenesis, several attempts have been made to treat and prevent the lesions. In parallel, many exper-
imental studies have confirmed the importance of H. pylori in gastric disease processes. Among animal
models, mice and Mongolian gerbils offer powerful tools for the analysis of H. pylori-associated gastric
lesions.1,2 The Helicobacter genus nowadays contains more than 30 species; this chapter focuses on
H. pylori within the genus and reviews its characteristics, interaction with host environment, and patho-
genesis in gastric disease. As gastric cancer remains a significant contributor to cancer-related death,
prevention of infection and treatment of gastric cancer represent important aspects of cancer control.

22.2 Discovery of Gastric Microorganisms

When Warren and Marshall successfully isolated a hitherto unidentified Gram-negative bacillus from
patients with chronic active gastritis, the role of bacteria as a causative factor in gastric disorders was
established.3,4 Initially named Campylobacter pyloridis, this flagellated microorganism thrives under
microaerophilic condition. Following the description of several features (including the presence of
flagellar sheath and bulb, glycocalyx, urease and catalase, and other biochemical characteristics) that
discriminate this bacillus from the Campylobacter or Wolinella genuses, Goodwin et al.5 renamed it
“Helicobacter pylori.” According to the recent literature,6,7 the Helicobacter genus currently consists of
over 30 gastric and enterohepatic species. Readers may refer to the taxonomy database provided by the
National Center for Biotechnology Information for most updated and detailed information about this

331
332 Laboratory Models for Foodborne Infections

H. pylori
(A) (B)

CagA
Type IV Flagella
Src
secretion
system
SHP-2

Urease
VacA

(H2N)2C=O + H2O 2NH3 + CO2

FIGURE 22.1  H. pylori and its schematic view. (A) Immunohistochemical view of H. pylori in the human stomach colo-
nized in the human gastric surface mucin. Top inset: active bacillary form. Bottom inset: coccoid form in the unsatisfactory
condition. The center square is enlarged in (B) as a schematic view. Immunohistochemistry against H. pylori. Original
magnification, 630×. (B) H. pylori utilize flagella to move into gastric surface mucin. CagA is injected through type IV
secretion system and tyrosine phosphorylated with Src family kinase. Phosphorylated CagA binds and activates SHP-2
tyrosine phosphatase. VacA is a vacuolating toxin. Bacterial urease catalyzes urea to ammonia to neutralize hydrogen
chloride in the acidic environment of the stomach.

bacterial genus.8 Here, we concentrate mainly on H. pylori, since most of the laboratory models were
established with this species (Figure 22.1A).

22.3 Gastric Microenvironment of H. pylori

In the stomach, gastric glands are made up of surface mucous cells (foveolar epithelium) in the top part
and pyloric glands at the bottom region in the antral mucosa. These two kinds of cells produce multilay-
ered histochemically different kinds of mucins, surface mucous cell mucin (SMCM) and pyloric gland
mucous cell mucin (GMCM), respectively, in humans9,10 and Mongolian gerbils.11 H. pylori prefers the
SMCM since GMCM contains terminal α1,4-GlcNAc residues attached to core O-glycans, which inhibit
biosynthesis of cholesteryl-α-d-glucopyranoside, a major cell wall component of the bacteria.12 H. pylori
shows morphologic conversion from bacillary form to coccoid form to adapt to certain worse conditions,
as in human stomach possessing adenocarcinomas,13 by lowering metabolic activity with 1000-fold less
adenosine triphosphate (ATP) level14 (Figure 22.1A).

22.4 Characteristics and Pathogenesis of H. pylori

Sequencing analysis revealed that H. pylori strain 26695 has a circular genome of 1,667,867 bp. Of
these, 91.0% are coding regions with 1590 predicted coding sequences.15 Global transcriptomic analysis
revealed transcriptional organization of the same strain; acid stimulus induced major H. pylori virulence
loci such as urease (ure) operon or the cag pathogenicity island (cagPAI).16
CagA is injected into gastric surface epithelial cells through the bacterial type IV secretion system
and is tyrosine phosphorylated with Src and Abl17 at variable EPIYA (Glu-Pro-Ile-Tyr-Ala) motif repeat
regions, which show structural diversity among East-Asian and Western countries.18 H. pylori found
Helicobacter 333

in the former possess EPIYA-A, B, and D motifs and may contribute to the geographical differences
observed in gastric carcinogenesis. Phosphorylated CagA then activates SHP-2 phosphatase, which
then dephosphorylates FAK kinase, resulting in impairment of epithelial cell adhesion and morphol-
ogy.19 Studies in wild-type CagA transgenic mice have confirmed the oncogenicity of this bacterial pro-
tein by demonstrating the development of gastrointestinal epithelial hyperplasia and adenocarcinomas
as well as hematopoietic malignancies, whereas phosphorylation-resistant CagA mice did not develop
disease.20
Broth culture supernatant of H. pylori was known to induce vacuolization of the mammalian cells,
which was characterized as VacA.21,22 VacA binds to receptor protein tyrosine phosphatases (RPTPs) α
and β on the surface of target cells.23 Mice deficient in RPTP type Z (RPTPζ or RPTPβ) revealed toler-
ance to VacA for mucosal damage.24 Oligomerized VacA enters into the cytoplasm through endocytosis
and shows multifunctional toxicity including interference of T cell activation and proliferation, reduction
of mitochondria membrane potential, and formation of vacuolation in endosomal/lysosomal compart-
ments25 (Figure 22.1B).

22.5 Infection Route and Childhood Acquisition of H. pylori

It has been demonstrated that transmission of the bacteria in the early childhood and interaction with
an infected sibling is an important risk factor. The presence of H. pylori in the oral cavity substantiated
the oral–oral route of spread. It has also supported the oral–fecal route of spread through contaminated
drinking water, especially in areas where living condition and sanitation are poor.26
Childhood infection with H. pylori is a major concern in the pediatrics field. In the H. pylori inocu-
lated and carcinogen administered model, early acquisition of H. pylori significantly increased gastric
inflammation and carcinogenesis compared to the late counterpart (Figure 22.2).27 H. pylori infection
in childhood (mean age 12.1 years) induced chronic gastritis, but this was mostly limited to moderate
atrophy without intestinal metaplasia in contrast to that seen in the elderly patients.28 Thus, earlier eradi-
cation of the bacteria should be considered, i.e., before development of atrophy and intestinal metaplasia,
for the prevention of gastric cancer later in adult life.29,30

22.6 Role of H. pylori Infection in Gastric Carcinogenesis

Epidemiological evidence has been accumulated indicating a significant relationship between H. pylori
infection and chronic atrophic gastritis, intestinal metaplasia, and gastric malignancies including can-
cer and lymphoma.31–41 The World Health Organization/International Agency for Research on Cancer
(WHO/IARC) defined H. pylori as a “definite biological carcinogen” in 1994 in spite of lack of evidence
in experimental animals.42
Various experimental animal models have been attempted to help clarify the role of H. pylori. Lee
et al.43 isolated Helicobacter felis (H. felis) from cat stomach and inoculated it in germ-free mice, result-
ing in colonization of the glandular stomach and induction of acute and chronic inflammation. A type of
H. pylori, designated Sydney strain (SS1), established by screening of fresh clinical isolates, showed high
colonizing ability and is currently widely used for mice experiments.44 The Mongolian gerbil (Meriones
unguiculatus) model was successfully established to mimic human H. pylori infection, resulting in
chronic active gastritis, peptic ulcers, and intestinal metaplasia resembling human lesions.45
For the gastric carcinogenesis models, chemical carcinogens such as N-methyl-N′-nitro-N-
nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea (MNU) were found to induce adenocarcinomas
in rat,46 mouse,47 and gerbils.48 H. pylori infection was found to exacerbate the incidence of MNU- 49 and
MNNG-50,51 induced adenocarcinomas in Mongolian gerbils, in which all histological types including
differentiated and undifferentiated adenocarcinomas and signet-ring cell carcinoma were found as in
human histopathology. Thus, H. pylori was confirmed as a strong promoter of gastric carcinogenesis
(Figure 22.2).
334 Laboratory Models for Foodborne Infections

Incidence of
gastric cancer
(A) S

(B) S

(C) S

(D) S
Early
S
Late
(E) Early S

Late S

(F) High-salt diet S

Chemopreventive agents S

: Chemical carcinogens
: Inoculation of H. pylori : Eradication S : Sacrifice

FIGURE 22.2  Modifying factors for H. pylori-associated stomach carcinogenesis in the Mongolian gerbil models.
(A) H. pylori infection alone is rarely carcinogenic. (B) Drinking water containing chemical carcinogens including
MNU or MNNG induces stomach cancers. (C) When combined, H. pylori become a strong promoter. (D) Earlier infec-
tion increases the risk compared with a later event. (E) Earlier eradication of H. pylori reduces risk of stomach cancers.
(F) A high-salt diet exacerbates inflammation and increases the incidence of H. pylori-associated cancer. On the other
hand, various natural products and pure chemicals appear to have chemopreventive potential.

Besides the gerbil models, mouse models feature advantages especially for genetic approaches.
Transgenic mice (K19-C2mE transgenic mouse) simultaneously expressing cyclooxygenase-2 (COX-2)
and microsomal prostaglandin E synthase (mPGES)-1 as well as Wnt1 revealed the importance of these
pathways in gastric tumorigenesis with Helicobacter infection.52,53 K19-C2mE mice developed adeno-
carcinomas not only in pyloric mucosa but also in fundic glands with MNU treatment as well as H. pylori
infection, and so are considered as a model for increasing proximal malignancies.54 Interleukin 1β (−/−)
knockout mice showed that IL1β induced by H. pylori infection affected both inflammatory and epithe-
lial cells and enhanced gastric carcinogenesis.55
Long-term infection of H. pylori frequently induced neuroendocrine tumors (NETs) (endocrine cell
hyperplasia/dysplasia and carcinoid tumors) in a Mongolian gerbil model in association with increased
serum gastrin level. Eradication of H. pylori prevents development of NETs in the glandular stomach,
being associated with reduction in serum gastrin levels.56 Proton pump inhibitors (PPIs), routinely used
for treatment of upper gastrointestinal disorders, have raised some concerns regarding the long-term
safety and development of NETs in the stomach. When PPI was administered to H. pylori-infected
Mongolian gerbils, high dose increased NET development with higher serum gastrin in contrast to no
influence at low dose.57

22.7  H. pylori Infection Induces No Neoplastic but Hyperplastic Lesions

in Mongolian Gerbils
Several studies reported that H. pylori infection alone induced gastric adenocarcinomas in gerbils with
a very high incidence without carcinogen exposure.58–60 However, several other studies rarely showed
development of carcinomas in animals inoculated only with H. pylori.48–51,61 Mongolian gerbils are char-
acteristic for the development of expanded cystic glands in mucosa and submucosa, sometime as deep as
Helicobacter 335

subserosa (several months after inoculation of H. pylori), and these are designated as heterotopic prolif-
erative glands (HPGs).62 HPGs consisted of gastric and/or intestinal metaplastic epithelial cells, the latter
progressed depending on the duration of H. pylori-induced inflammation. HPGs, in turn, dramatically
diminished with the eradication of the bacteria, indicating that HPG is not neoplastic but rather hyper-
plastic. HPGs often resemble differentiated or mucinous adenocarcinomas showing structural abnor-
mality, but lack obvious cellular atypia. Characteristics of HPG include (1) organized polarity of their
component cells; (2) differentiation from gastric phenotypic HPG into intestinal phenotypic HPG some-
times associated with mature Paneth cells; (3) formation of large cystic dilatations containing mucin,
often with calcification; (4) shedding of epithelial cells and necrosis at the tips of lesions; (5) high-grade
inflammation with infiltration of inflammatory cells (neutrophils in acute phase and mononuclear cells in
chronic phase); and (6) organized polarity of proliferating zones. These features are quite different from
those of well-differentiated adenocarcinomas, which are characterized by obvious cellular atypia. HPGs
need to be precisely distinguished from adenocarcinomas (Figure 22.3 and Table 22.1).1,2

(A) (B)

(C) (D)

FIGURE 22.3  Gastric lesions in H. pylori-infected Mongolian gerbils. (A) Submucosal hyperplastic proliferating glands
called HPG after 44-week long inflammation. (B) HPG has drastically disappeared 20 weeks after eradication of the bac-
teria. Well- (C) and poorly (D) differentiated adenocarcinomas developed with carcinogen treatment. (A–D) Hematoxylin
and eosin staining. Original magnification, 25× (A and B); 200× (C); and 400× (D).

TABLE 22.1
Comparison of Submucosal Lesions in H. pylori-Infected Mongolian Gerbils
HPG Tubular Adenocarcinoma
Gland distribution Dispersed Compactly proliferated
Gland shape Large and cystic Relatively small
Intracystic material Eosinophilic, sometimes with calcification Usually transparent
Lining epithelium of cysts Shedding of epithelial cells with necrosis at the tip of Fully lined with atypical cells.
the cyst
Intestinal metaplasia Frequent, sometimes with Paneth cells Relatively infrequent
Stroma Severe inflammatory cell infiltrates. Neutrophils in acute Desmoplastic reaction
phase. Lymphocytes and plasma cells in chronic phase
with lymphoid follicle formation.
Atypia No atypia. Organized polarity. Partly degenerated. Enlarged nuclei with increased
chromatin. Loss of polarity.
336 Laboratory Models for Foodborne Infections

22.8 Prevention of Gastric Carcinogenesis by Eradication of H. pylori

Several studies have been performed to assess whether gastric cancer could be prevented by eradica-
tion of H. pylori in patients with chronic atrophic gastritis. Some investigated the prophylactic effect
of H. pylori eradication on the development of gastric carcinomas after endoscopic resection for initial
lesions and revealed significant reduction up to 3 years.63,64 In contrast, longer follow-up studies (more
than 7.5 years) in China65 and Japan66 showed similar overall incidences in both H. pylori eradicated
and non-eradicated groups. However, eradication significantly decreased the development of gastric
cancer in the subgroup of H. pylori carriers without precancerous lesions in the former Chinese trial.65
Furthermore, in the latter trial, cancer incidence was significantly reduced after eradication only in
pepsinogen-test-negative subjects with mild gastritis.66
In H. pylori-infected, carcinogen-treated Mongolian gerbil models, incidences of gastric cancers after
curative treatment for H. pylori were significantly lower than in those without eradication.61 The inci-
dences of gastric cancer were significantly reduced to 6.7%, 27.3%, and 38.2% with eradication at early,
middle, and late time points, respectively, compared to 56.3% incidence in the noneradicated group67
(Figure 22.2).

22.9 Intestinal Metaplasia and Intestinalization of Gastric Cancer

H. pylori infection plays a causative role in the development of chronic atrophic gastritis and intesti-
nal metaplasia. Using gastric and intestinal epithelial cell markers, intestinal metaplasia was divided
into two major types: a gastric-and-intestinal mixed type and a solely intestinal type.68,69 In the former,
gastric and intestinal phenotypic markers are localized within a glandular level as well as in a cellular
level.70 In the Mongolian gerbil model, gastric-and-intestinal mixed-type intestinal metaplasia was found
to appear first, followed by the solely intestinal type with appearance of Paneth cells during the overall
course of H. pylori infection in the HPG.62 It indicated that intestinal metaplasia could be caused by the
gradual intestinalization from proper gastric gland cells toward completely intestinal type cells through
the intermittent gastric-and-intestinal mixed phenotype.
Stomach cancers could also be classified with gastric and intestinal markers: gastric, gastric-and-
intestinal mixed, intestinal, and null types. Although intestinal metaplasia has been considered as a
preneoplastic lesion of intestinal type cancer, detailed mechanisms have not yet been clarified.71 In
H. pylori-infected gerbils, adenocarcinomas were divided phenotypically into 21 gastric, 24 gastric-and-
intestinal mixed, 4 intestinal, and 1 null types. In contrast, noninfected gerbils harbored only gastric-
type cancers. Thus, H. pylori infection was considered to trigger intestinalization of stomach cancers in
parallel with progression of intestinal metaplasia.2

22.10 Exacerbating Factors for Gastric Carcinogenesis: Synergistic Effects

of H. pylori and High-Salt Diet
A large number of case–control and ecological studies have shown salt and salted foods, among various
other food ingredients, as probable risk factors for gastric cancer.72–75 In an experimental study on rats,
sodium chloride (NaCl) had been thought to enhance the carcinogenic effects by hindering the gastric
mucous barrier.76 Later, it was revealed that high-salt diet exacerbated H. pylori-induced inflammation
and gastric carcinogenesis in a Mongolian gerbil model.77 Food containing various concentrations of
salt (2.5%, 5%, and 10% NaCl) increased the incidence of gastric cancer in a dose-dependent manner
(33%, 36%, and 63%, respectively) compared to the normal diet group (15%). In contrast, intermittent
intragastric injection of saturated NaCl solution induced gastric erosion but did not promote gastric
carcinogenesis.11 A high-salt diet was associated with a more severe inflammatory response including
elevation of anti-H. pylori antibody titers, serum gastrin levels, and inflammatory cell infiltration in a
Helicobacter 337

dose-dependent fashion (Figure 22.2). A high-salt diet upregulated the amount of SMCM, suitable for
H. pylori colonization, despite no increment in the corresponding mucin core protein, MUC5AC mRNA.
Although GMCM with its mucin core protein MUC6 was induced against H. pylori infection, high-salt
diet decreased the amount of GMCM, which acts against H. pylori infection by inhibiting the bacterial
cell wall component.12 In an H. pylori-infected MNU-treated mouse model, microarray analysis revealed
overexpression of CD177 and Reg3g in a high-salt diet group, the former being associated with better
prognosis in human cases.78 Reduction of salt intake could thus be one of the most important chemopre-
ventive methods for human gastric carcinogenesis.

22.11 Chemoprevention of Gastric Carcinogenesis

Eradication of H. pylori is most effective in the earlier stages of infection, as evidenced by studies on
the prevention of gastric carcinogenesis carried out in both humans and animals. However, eradica-
tion may not be efficient for ones with severe chronic atrophic gastritis. A selective cyclooxygenase
2 (COX-2) inhibitor, etodolac, was attempted to prevent H. pylori-infected and MNU-induced gastric
carcinogenesis in a Mongolian gerbil model. Etodolac inhibited the development of epithelial cell pro-
liferation, intestinal metaplasia, and gastric cancer in a dose-dependent manner although inflammatory
cell infiltration or oxidative DNA damage was not alleviated.79 In humans, Yanaoka et al.80 investigated
the preventive effects of etodolac on metachronous cancer development after endoscopic resection of
early gastric cancer. Etodolac significantly reduced emergence of gastric cancer even with extensive
metaplastic gastritis, which indicated that controlling COX-2 function could be an alternative method
(Figure 22.2).
Oxidative stress is also a major concern for gastric carcinogenesis because of its ability to damage
DNA. A potent antioxidative compound derived from canola oil, 4-vinyl-2,6-dimethoxyphenol (can-
olol), was effective in reduction of inflammatory cytokines and serum 8-hydroxy-2′-deoxyguanosine
(8-OHdG) as well as reduction of the incidence of gastric adenocarcinomas in H. pylori-infected and
MNU-treated Mongolian gerbils. Canolol was also effective in inhibition of gastric tumorigenesis in
K19-C2mE transgenic mice.81 Thus, the suppression of oxidative stress rather than disappearance of the
bacteria would be effective in prevention of gastric cancer (Figure 22.2).

22.12 Conclusion
Discovery of H. pylori from the human stomach opened a new avenue to clarify the mechanism of
gastric lesions including chronic atrophic gastritis, intestinal metaplasia, and gastric carcinogenesis.
A large number of epidemiological studies revealed the positive correlation of H. pylori with gastric can-
cer development. In Mongolian gerbil models, H. pylori inoculation caused severe hyperplastic lesions
called HPG and showed strong promoting effects in carcinogen-treated animals; H. pylori infection alone
rarely induced adenocarcinomas. Mouse models are also effective for genetic approach. Eradication of
the bacteria was effective in prevention of gastric carcinogenesis, especially in cases of milder gastritis
without severe atrophy or intestinal metaplasia, both in human and rodent models. Besides eradication
of the bacteria, chemopreventive approaches have been attempted in prevention of gastric neoplasia. It is
necessary to establish effective approaches for application in human trials.

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23
Klebsiella: Caenorhabditis elegans as a Laboratory
Model for Klebsiella pneumoniae Infection

Arumugam Kamaladevi and Krishnaswamy Balamurugan

CONTENTS
23.1 Foodborne Infection: A Global Burden...................................................................................... 344
23.2 K. pneumoniae: A Potent Agent for Foodborne Diseases.......................................................... 344
23.3 Importance of Model Animals in Research............................................................................... 345
23.4 Discovery and Exploration of C. elegans................................................................................... 345
23.5 Other Model Animals to Study K. pneumoniae Infection......................................................... 345
23.6 Characteristic Features of C. elegans......................................................................................... 346
23.7 C. elegans as a Model for Host–Pathogen Interaction Studies................................................... 346
23.8 Key Differences between Innate Immune System of C. elegans and Mammals....................... 347
23.9 p38 MAPK Pathway in C. elegans............................................................................................. 347
23.10 Live K. pneumoniae Was More Pathogenic to C. elegans�������������������������������������������������������� 347
23.11 K. pneumoniae Proliferates inside the Host Intestine and Causes Persistent Infection������������ 347
23.12 Role of p38 MAPK in Immune Defense of Nematode against K. pneumoniae Infection�������� 349
23.13 Role of Toll-Dependent p38 MAPK Pathway in C. elegans against
K. ­pneumoniae Infection .....������������������������������������������������������������������������������������������������������ 350
23.14 Upregulation of Virulence Gene in K. pneumoniae during Infection........................................ 351
23.15 Conclusion................................................................................................................................... 351
Acknowledgments................................................................................................................................... 351
References............................................................................................................................................... 351

Klebsiella pneumoniae has emerged as one of the most frequent and potent pathogens causing foodborne
infection in humans since 1980. Despite its clinical significance, the absence of appropriate animal mod-
els has hampered efforts in identifying the virulence and host factors that determine the pathogenesis
and susceptibility of host to gastrointestinal infection. For the past few decades, Caenorhabditis elegans
has been recognized as a better laboratory animal model based on its potential to closely recapitulate
human disease. Additionally, owing to its conserved immune pathways with higher animals and avail-
ability of developed transgenic, knockout, and knockdown strains, C. elegans has attracted the atten-
tion of researchers in various fields as a powerful and versatile model system. C. elegans has played a
crucial role in elucidating the pathophysiology and immunology of microbial infections and has thus
provided valuable insights into the pathogenesis and response of host system, from chronic to acute
infection. The information derived from animal model aids researchers to characterize the host response
and helps design chemotherapeutic treatment for the public risk associated with infection caused by
K. pneumoniae-contaminated food commodities. This chapter focuses on C. elegans as a model system
that has been instrumental in uncovering the clues on host–pathogen interactions during K. pneumoniae
infection.

343
344 Laboratory Models for Foodborne Infections

23.1 Foodborne Infection: A Global Burden

Foodborne diseases are currently recognized as a global burden by the World Health Organization.
The alarming increase in the annual morbidity and mortality has highlighted the importance of proper
hygiene in public places. In 2005, about 1.8 million people succumbed to diarrheal diseases follow-
ing consumption of contaminated food and water [1]. This is not only reported in underdeveloped
countries—the same fate of massive death due to foodborne illness has also been reported in developed
countries. Annually, there are 76 million cases of foodborne diseases, resulting in 325,000 hospitaliza-
tions, and 5000 deaths have been estimated to occur in USA [2,3].
Generally, foodborne infections induce a wide spectrum of symptoms. Although the actual inci-
dence of gastrointestinal infection is largely unknown, the risk of getting infection through heavily
contaminated food is probably high [4]. To eradicate the foodborne illness, public health awareness is
critical. Since the study based on volunteers feeding does not represent a feasible option, the under-
standing of K. pneumoniae infection is chiefly based on epidemiological data, infection case reports,
and use of animal models. An ideal animal model should mimic all phases of the human disease
condition, as the host–pathogen interaction in a natural biological condition reveals the host’s resis-
tance mechanism and virulence properties of pathogen that promote colonization and subsequent
dissemination [4].

23.2  K
 . pneumoniae: A Potent Agent for Foodborne Diseases
Foodborne illness represents an important public health hazard and has a significant economical
impact on developing countries worldwide. Approximately 1.5 million neonatal deaths are caused
by foodborne diarrheal disease alone. Viruses, bacteria, fungi, and parasites are the causative agents
for foodborne infections [5]. Among them, bacteria are implicated in sporadic illness among differ-
ent populations. Prior to 1990s, the species of Salmonella and Campylobacter and Escherichia coli
accounted for most of the outbreaks. Subsequently, K. pneumoniae was shown to cause foodborne
diseases in humans through contaminated vegetables [6] and hamburgers [7]. Given the increased
number and severity of infection, K. pneumoniae has been recognized as one of the most important
foodborne bacterial pathogens.
K. pneumoniae is one of the most versatile and clinically significant pathogens in causing both
nosocomial- and community-acquired infections worldwide. It is a Gram-negative, rod-shaped bac-
terium belonging to the family Enterobacteriaceae [8]. Although it is found as a common component
of normal microflora in the nasopharyngeal and gastrointestinal regions of humans, K. pneumoniae
can cause a wide variety of diseases, ranging from mild infections to life-threatening diseases, in
immunocompromised patients. The diseases caused by K. pneumoniae are bowel infections, pneu-
monia, suppurative infections, bacteremia, septicemia, and inflammation of the urinary tract. In
severe infection cases, K. pneumoniae causes liver abscess along with meningitis and endophthal-
mitis, which accounts for a mortality rate of 20%–55% [8]. Due to its severity in causing infection
in immunocompromised individuals, it is classified as an opportunistic pathogen. As far as hospital-
acquired infection by K. pneumoniae is concerned, the prime significance is its ability to evade the
innate immune response and cause colonization in the intestinal tract of patients and cause symp-
toms. Infection with K. pneumoniae begins with its initial colonization in the gastrointestinal tract,
irrespective of its site of infection [9]. The degree of colonization mainly depends on the potential
of the bacterial strain to form biofilms [10] and emerge resistant to antibiotics. The resistance of K.
pneumoniae toward third-generation cephalosporins and carbapenem were identified in 1983 [11–13].
Since, K. pneumoniae is undergoing multidrug resistance, it is necessary to study its pathogenesis
in a multicellular host system to approach the infection using an alternate therapy. Thus, research-
ers paid a great deal of attention to choose a suitable animal model to decipher the host perspectives
during bacterial infections.
Klebsiella: C. elegans as a Laboratory Model for K. pneumoniae Infection 345

23.3 Importance of Model Animals in Research

During the 18th century, researchers were fascinated with the availability of huge diversity between
model organisms with a variety of purposes. Based on their objectives and aim, they chose the
potential model organism. This scientific exploration around the globe paved the way for a variety
of theories and classifications (e.g., Linnaean classification and cell theory). Later in the 19th cen-
tury, researchers understood the various complex processes like metabolism, embryogenesis, neu-
ronal behavior, and disorder by experimental validation using model systems. In addition, much of
our knowledge on heredity, development, metabolism, physiology, and several molecular mecha-
nisms have been gained from the studies of model organisms [14]. A model organism is selected
on the basis of its amenability for experimental procedure and its potential for extracting a wealth
of information on common mechanisms among various life forms. Furthermore, most of the model
organisms have been sequenced and provide deep knowledge in respective fields, which enable the
researcher to address the serious query raised [14]. Using model organisms for studying pathogenic-
ity of human pathogens did not gain serious interest till Frederick Ausubel used a surrogate model to
study host–pathogen interactions [15,16]. The use of model organisms for understanding pathogen-
esis is not surprising since several of the underlying processes are conserved from yeast to mammals.
Additionally, the genes that contribute to important biological pathways have also been found to be
conserved across phylogeny. Since most of the pathogens share a common mode of pathogenesis
mechanisms in a wide variety of hosts, it is necessary to employ a model host to identify the com-
mon and novel virulence factors of pathogen and also to understand the cross talk between a host
and pathogen.

23.4 Discovery and Exploration of C. elegans

C. elegans was first discovered and described by Emile Maupas, a French librarian zoologist and bota-
nist, in 1900 from soil in Algeria [17]. Initially, Maupas classified this nematode under the Rhabditis
genus rather than Caenorhabditis. Later, in 1952, the new subgenus Caenorhabditis was coined by
Osche [18], which was later given generic status by Dougherty in 1955 [19]. Surprisingly, the sequence
of C. elegans has high homology with human genes and many of the genes can code for the function
similar to mammalian genes. C. elegans is a eukaryotic, free-living soil nematode. It has a complex
developmental process including embryogenesis, morphogenesis, and growth mechanism. Besides
showing about 35% homology with the human at the genome level, C. elegans shares 83% similar-
ity in protein sequence [20]. Its genome has been completely sequenced, and the genome size is
approximately 100 Mb. Due to this significant property and smaller genome size, C. elegans was
introduced to the scientific community by Sydney Brenner as an experimental model in 1960s to
explore developmental biology and report his findings on the behavior of the nematode [21]; this
attracted huge attention from the researchers. Later, it was used for the study of host–pathogen inter-
actions against Mycobacterium nematophilum [22–25], Drechmeria coniospora [26], Pseudomonas
aeruginosa [27–29], Serratia marcescens [25,30], Escherichia coli [31,32], Enterococcus faeca-
lis [33,34], Staphylococcus aureus [29,35], Salmonella spp. [36–38], Shigella spp. [39–41], and Vibrio
spp. [42–45]. Beside C.  elegans, other animals have also been used as a model organism to study
K. pneumoniae pathogenesis and host defense mechanisms.

23.5 Other Model Animals to Study K. pneumoniae Infection

Most of the studies on K. pneumoniae pathogenesis have been focused on the murine model [46–48].
As K. pneumoniae is a prime infectious agent of the respiratory tract, murine models having well-
developed respiratory systems have been suggested to be the best option to decipher the response of
346 Laboratory Models for Foodborne Infections

the host immune system. Injection of K. pneumoniae into the murine model alters the level of interleu-
kin-17 (IL-17) [49], tumor necrosis factor alpha, keratinocyte-derived chemokines and bronchoalveolar
fluid, and IL-10 [50] during the course of infection. Furthermore, interaction of K. pneumoniae with
the lung epithelial cells [51] markedly increased the recruitment of neutrophils to the lungs as well as
proinflammatory cytokines and chemokines. A recent study has used the wax moth Galleria mellonella
as a surrogate host model to understand the pathogenesis of K. pneumoniae at the molecular level for
testing new therapies [52]. In that study, it was reported that G. mellonella could distinguish pathogenic
and nonpathogenic Klebsiella strains. Moreover, infection of K. pneumoniae in G. mellonella attenu-
ates the host innate immune response by inhibiting the antimicrobial peptides. Furthermore, the study
by Wu et al. in HepG2 cells of humans suggested that infection with K. pneumoniae attenuates the p38
mitogen-activated protein kinase (p38 MAPK) pathway of host system and that it required lipopolysac-
charide for complete pathogenesis during host–pathogen interaction [53]. A recent study by us has used
C. elegans as a model and reinforced the essential role of Toll-dependent p38 MAPK pathway during
K. pneumoniae infection [54].

23.6 Characteristic Features of C. elegans

Easy maintenance: C. elegans is propagated and maintained on agar plate with lawns of non-
pathogenic E. coli OP50 as food source. Maintenance is less expensive.
Short life cycle: The life cycle of C. elegans is temperature dependent and is approximately 3.5–7
days; within this period, each animal can produce around 300 genetically identical progenies.
Easy handling: Stocks can be frozen at −80°C and stored for many years and can be revived when-
ever required for research purpose and propagated continuously.
Sequenced genome: C. elegans is the first complex model organism to have its entire genome
sequenced and available on the Wormbase.
Availability of genetic tools: RNAi mechanism is well established in C. elegans, and more than
90% of the genome is available as a dsRNA expression library. Generation of transgenic
C. elegans is made easy by injecting the dsRNA by microinjection method.
Transparency: The transparent body of the C. elegans facilitates the characterization of gene
expression and enables easy localization of pathogens tagged with fluorescent labels.
Simple anatomy: C. elegans has a simple anatomy. It possesses 302 neurons and 959 somatic cells
corresponding to the development of lineage, and all these cells have been thoroughly tracked
back to the zygote. Hence, it is considered as a simple organism with complex structures such
as nervous system, digestive system, and extensive germ line.

23.7  C . elegans as a Model for Host–Pathogen Interaction Studies

C. elegans has the ability to survive even after repeated infections as it evokes several immune path-
ways that activate the host defense against several bacterial infections. Studies in C. elegans have shed
more light on many pathways of innate immune system that are evolutionary conserved from worms to
mammals [55]. Thus, C. elegans is a very good model to study both the host immune response and the
pathogen’s virulence mechanism that facilitate the host–pathogen interface [56]. The orchestrated regu-
lation of immune defense is an important criterion to combat microbial infection and prevent deleterious
effects on host cells. Hence, it is particularly important for the mammalian intestine where pathogenic
bacteria must be harbored to enable differentiation from bacterial species that could be a part of com-
mon flora of humans. One approach to understand the mechanisms of immune defense at the epithelial
surface is to use invertebrate hosts like C. elegans to decipher the evolutionarily conserved aspects of
innate immune defense and pathogen virulence [57].
Klebsiella: C. elegans as a Laboratory Model for K. pneumoniae Infection 347

23.8 Key Differences between Innate Immune System of C. elegans

and Mammals
Generally, some of the mammalian’s innate immune response is not encoded in the C. elegans genome,
such as homologues of the transcription factor, NF-κB, and adaptor protein MYD88. Additionally,
C. elegans does not produce any molecules that have homology with the known vertebrate cytokines.
Even though the nematode lacks NF-κB, MYD88, and other secretary molecules of the TLR signaling
pathway, it mounts an innate immune defense that employ several evolutionarily conserved signaling
pathways, including p38 MAPK, β-catenin, and FOXO transcription factors, which interestingly appear
to function in synergy to activate effector genes.

23.9 p38 MAPK Pathway in C. elegans

The p38 MAPK is a multistep mechanism that includes immune regulators like NSY-1/SEK-1/PMK-1
MAP kinase pathway, which was identified through forward genetics using mutants that had enhanced
susceptibility to infection with Gram-negative bacteria P. aeruginosa [58]. This pathway is orthologous
to the ASK1 (MAP kinase kinase kinase)/MKK3/6 (MAP kinase kinase)/p38 (MAP kinase) pathway
in mammals, and its identification in C. elegans provided a deeper knowledge on host perspective. The
p38 MAP kinase pathway acts cell-autonomously in the intestinal epithelium [59] to coordinate immune
defense against a wide variety of ingested pathogens. C. elegans having a loss-of-function mutations
in pmk-1 are hypersusceptible to infection by Gram-negative pathogens such as P. aeruginosa [58,60],
S. entrica [61], Y. pestis [62], and S. marcescens [63]; Gram-positive pathogens E. faecalis [63] and
Staphylococcus aureus [33]; and the fungus C. albicans [64,65]. Nematodes’ global transcriptional pro-
filing analyses revealed that PMK-1 regulates the expression putative antimicrobial effectors, including
C-type lectins, Shk toxins, and CUB-like domain [58], in the absence of microbial challenge, and this is
termed as “basal regulation.”

23.10 Live K. pneumoniae Was More Pathogenic to C. elegans

The pathogenicity of K. pneumoniae is determined by its ability to affect normal physiological activities
like survival, reproduction, and feeding in nematodes. In this study, the physiological activities of the
C. elegans infected with K. pneumoniae were examined and compared with the control nematodes fed
with E. coli OP50. The control C. elegans fed on E. coli OP50 showed an intact pharynx [Figure 23.1A(i)]
and normal egg formation [Figure 23.1A(ii)], whereas the nematodes fed on K. pneumoniae were severely
infected, which was evidenced by distended pharynx [Figure 23.1A(iii, iv)], abnormal egg formation and
accumulation [Figure 23.1A(v)], and internal hatching with accumulation of bacterial load inside the gas-
trointestinal region of the exposed nematodes [Figure 23.1A(vi)]. In addition, the severity of K. pneumoniae
pathogenicity was determined by assessing the rate of killing in nematodes. As shown in Figure 23.1B,
K. pneumoniae displayed a significant (p < 0.05) reduction in the lifespan of nematodes when compared
to that of OP50 control. Nematodes fed on K. pneumoniae displayed a significantly (p < 0.05) decreased
mean lifespan of 48 ± 5 h when compared to that of nematodes fed with OP50, which had a mean lifespan
of 21 ± 0.5 days (Figure 23.1B). Infection with K. pneumoniae also significantly (p < 0.05) inhibited egg
laying (Figure 23.1C) and feeding (Figure 23.1D) in C. elegans [54].

23.11  K . pneumoniae Proliferates inside the Host Intestine and Causes

Persistent Infection
In confocal laser scanning microscopy (CLSM) images, the intense profile of the pathogen-exposed
nematode showed increased colonization in the intestine of the C. elegans with longer residence time
348 Laboratory Models for Foodborne Infections

(A) (i) (ii) (iii) (iv)

(v) (vi)

(B) 120 (C) OP50 control

100 K. pneumoniae
100
80
Percentage survival (%)

80
No. of eggs/worm

60
60
40
40
20
20
OP50
K. pneumoniae 0
0
0 10 20 30 40 50 0 4 8 12 24
Time (h) Time of exposure (h)

OP50 control
(D) 60 K. pneumoniae

50
No. of flings/worm/30 s

40

30

20

10

0 4 8 12 24
Time of exposure (h)

FIGURE 23.1  (A) Microscopic images of C. elegans exposed to K. pneumoniae for 12 h at 20°C are shown. Wild-type
C. elegans fed with OP50 showed intact pharynx (i) and (ii) normal eggs. In contrast, nematodes exposed to K. pneumoniae
showed severe physiological defects such as (iii, iv) distended pharynx (indicated by arrowhead), (v) accumulation of eggs
inside the intestine (indicated by arrowhead), (vi) internal hatching, and intestinal colonization (indicated by arrowhead).
Physiological assays showing the impact of K. pneumoniae infection in C. elegans. (B) Survival of C. elegans exposed to
live K. pneumoniae. (C) Cessation of egg laying in nematodes exposed to K. pneumoniae. (D) Inhibition of feeding dur-
ing K. pneumoniae infection. Data are presented as mean ± standard deviation of three biological replicates. Statistical
analysis was performed by one-way ANOVA followed by Duncan’s post hoc analysis.

(Figure 23.2). Measuring the bacterial load inside the intestine of nematodes by colony forming unit (CFU)
assay revealed that the number of bacterial cells in C. elegans exposed for 4 h was 2.7 × 103, and it was
found to be increased to 3.6 × 104, 4.7 × 105, and 5.3 × 106 at 6, 12, and 24 h, respectively (Figure 23.2A).
The results of CFU assay suggested that K. pneumoniae colonized and proliferated inside the host intes-
tine with an increase in residence time.
Klebsiella: C. elegans as a Laboratory Model for K. pneumoniae Infection 349

(A) 5

4
Log CFU/worm

4 6 12 24
Time (h)

(B) 35
30
Fold change in expression

25 pmk-1
tol-1
20
clec-60
15 clec-67
10 lys-1
nlp-29
5

0
KP 6 h KP 12 h KP 24 h KP 36 h

FIGURE 23.2  The histogram showing the CFU of K. pneumoniae inside the intestine of C. elegans in response to time
of infection (A). Changes in the expression profile of N2 nematodes during infection with K. pneumoniae (B). (From
Kamaladevi, A. and Balamurugan, K., Pathog. Dis., 73(5), ftv021, 2015.)

23.12 Role of p38 MAPK in Immune Defense of Nematode against

K. pneumoniae Infection
Initial defense of host immune response is the recognition of the pathogen by toll-like receptor (Toll).
Toll is encoded by the gene tol-1 in C. elegans. In nematodes exposed to K. pneumoniae, the tol-1 was
observed to be significantly (p < 0.005) increased. Additionally, the genes pmk-1, clec-60, clec-67,
clec-85, lys-1, and nlp-29 were upregulated in the initial hours of infection and downregulated gradu-
ally during the later hours of infection (Figure 23.2B). The cluster of clec-60/67 increased the host
resistance to infection and increased the survival of nematodes [29,30]. However, the downregulation
of clec-60 and clec-67 during exposure to K. pneumoniae indicated the surrendering of host immune
defense to K. pneumoniae infection. In particular, lys-1 and nlp-29 were reported to be regulated by
p38 MAPK pathway during bacterial and fungal infection [62,63]. The increased expression of pmk-1
along with the increased level of lys-1 and nlp-29 in the initial hours of infection and gradual down-
regulation in the later hours revealed that p38 MAPK pathway may have a role against K. pneumoniae
(Figure 23.2B).
350 Laboratory Models for Foodborne Infections

23.13 Role of Toll-Dependent p38 MAPK Pathway in C. elegans against

K. pneumoniae Infection
To establish the role of p38 MAPK in C. elegans against K. pneumoniae infection, mutants of this
pathway such as AU37 (sek-1), KU25 (pmk-1), and IG10 (tol-1) were examined for their survival during
K. pneumoniae infection. The mutants of sek-1, pmk-1, and tol-1 exhibited a significantly shorter mean
lifespan of 24 ± 5, 33 ± 3, and 30 ± 7 h, respectively, than the N2 nematodes. The hypersusceptibility of
mutants to K. pneumoniae infection suggested the role of p38 MAPK pathway and toll-like receptor in
nematode defense (Figure 23.3A).
To explore the involvement of p38 MAPK pathway and Toll receptor during infection with K. pneu-
moniae, an expression analysis was done in p38 MAPK pathway mutants (sek-1 and pmk-1) and tol-1
mutant. The expression of tol-1 was significantly (p < 0.005) upregulated in sek-1 and pmk-1 mutants
(Figure 23.3), suggesting the requirement of toll-like receptor during K. pneumoniae exposure. Exposing
AU37 to K. pneumoniae significantly (p < 0.005) downregulated the expression of pmk-1 during the
initial hour of infection itself. However, in tol-1 mutant IG10, pmk-1 was not observed to be activated
(Figure 23.3B). This indicated that the lack of toll receptor in IG10 during K. pneumoniae infection failed
to activate the expression of pmk-1. These data suggested that C. elegans require toll-dependent p38
MAPK pathway to act against K. pneumoniae infection. Furthermore, the nonactivation of antimicrobial

(A) (B) 30 IG10 (tol-1)

100 KP+AU37 (sek-1) *
KP+KU25 (pmk-1)
KP+IG10 (tol-1) 25
Percentage survival (%)

80
Fold change in expression

pmk-1
20
clec-60
60
15 clec-67
40 lys-1
10 clec-87
20 clec-85
5
** ** * nlp-29
* * * ** ** * **
0 0 ** **
0 5 10 15 20 25 30 35 40 45 50 KP 6 h KP 12 h KP 24 h
Time (h) –5

(C) 10 AU37 (sek-1) (D)2.5 KU25 (pmk-1)

* pmk-1
Fold change in expression

clec-60
Fold change in expression

8 *
clec-67 2 clec-60
**
clec-67
6 lys-1
1.5 * * lys-1
* tol-1 ** * tol-1
** ** *
4 ** clec-87 *
1 * clec-87
* clec-85 clec-85
2 ** nlp-29
**
** ** ** nlp-29
** ** 0.5 **
* *
0 * **
KP 6 h KP 12 h KP 24 h ** ** ** ** **
0
–2 KP 6 h KP 12 h KP 24 h

(E) 7
**
6 **
Fold change in expression

5
4
3 *
2
1
0
rmpA oxyR uge

FIGURE 23.3  The increased susceptibility of p38 MAPK pathway mutants against K. pneumoniae infection (A).
Changes in the expression profile of the tol-1 mutant IG10 (B), sek-1 mutant AU37 (C), pmk-1 mutant KU25 (D), and
regulation of selective virulence factors in K. pneumoniae during interaction with host C. elegans (E). (Figure 23.3A–D
were adapted from Kamaladevi, A. and Balamurugan, K., Pathog. Dis., 73(5), ftv021, 2015; Figure 23.3E unpublished
data.)
Klebsiella: C. elegans as a Laboratory Model for K. pneumoniae Infection 351

genes, such as nlp-29 and lys-1 (Figure 23.3B), that are primarily regulated by p38 MAPK also strongly
suggests the role of toll-dependent p38 MAPK pathway against K. pneumoniae infection. The antimi-
crobial genes clec-60, clec-67, clec-85, and clec-87 were significantly (p < 0.005) upregulated in the
early hours and downregulated at the later hours in the AU37 (sek-1) and KU25 (pmk-1) mutants during
infection, suggesting that K. pneumoniae thwarts the host defense and causes a persistent lethal infection
(Figure 23.3C and D).

23.14 Upregulation of Virulence Gene in K. pneumoniae during Infection

On the other hand, to check the hypothesis that K. pneumoniae utilizes the same virulence strategies
to infect nematode and mammalian hosts, real-time polymerase chain reaction (PCR) analysis was per-
formed for selective candidate virulent genes. The virulent genes including rmpA, uge, and oxyR have
been well established as being essential for colonization [46,47] and to withstand the oxidative stress [48].
Here, the transcriptional analysis showed that the interaction of K. pneumoniae with C. elegans signifi-
cantly increased the expression of virulence genes such as rmpA (p < 0.005), oxyR (p < 0.005), and uge
(p < 0.05) compare with their respective controls (Figure 23.3E). The overexpression of these reported
genes in K. pneumoniae during host–pathogen interaction suggested that the K. pneumoniae shares a
common mode of pathogenesis between nematodes and mammals (unpublished data).

23.15 Conclusion
C. elegans has been considered as an excellent animal model to study the host immune factors and patho-
genesis of K. pneumoniae under in vivo conditions. Concerning the pathogenicity of K. pneumoniae to
C. elegans, the killing of C. elegans by K. pneumoniae is mediated by the colonization and prolifera-
tion inside the host intestine. While C. elegans utilizes the Toll-dependent p38 MAPK pathways against
K. pneumoniae infection, K. pneumoniae increases the expression of rmpA, oxyR, and uge genes during
infection. Overall, these interactions underscore the host–pathogen relationship between C. elegans and
K. pneumoniae and will form the basis for identification of potential therapeutic targets against K. pneu-
moniae and for formulation of effective immune therapy in hosts.

Acknowledgments
We thank Caenorhabditis Genetics Center, which is funded by the National Institutes of Health, National
Center for Research Resources, for providing C. elegans N2 WT, mutant strains, and E. coli  OP50.
Dr. K. Balamurugan thankfully acknowledges the DBT (BT/PR14932/MED/29/233/2010), ICMR (Sanction
No: 5/3/3/13/2010-ECD-I), DST-SERC Fast Track Young Scientist Scheme [No. SR/FT/LS-83/2009 (G)],
DST-SERB (No. SR/SO/AS-80/2010), UGC Major Research Project [No. 42-222/2013 (SR)], CSIR [No.
37(1460)/11/EMR-II], DST-PURSE [Grant No. SR/S9Z-23/2010/42(G)], FIST (Grant No. SR-FST/LSI-
087/2008), and UGC-SAP-DRS-I [Grant No. F.3-28/2011 (SAP-II)], New Delhi, India, for financial assis-
tances. Financial support to AK by DBT in the form of SRF is thankfully acknowledged. The authors
gratefully acknowledge the computational and bioinformatics help provided by the Alagappa University
Bioinformatics Infrastructure Facility (funded by DBT, GOI; Grant No. BT/BI/25/001/2006).

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24
Proteus

Paola Scavone, Victoria Iribarnegaray, and Pablo Zunino

CONTENTS
24.1 Introduction....................................................................................................................................355
24.2 Proteus mirabilis............................................................................................................................355
24.3 Virulence Factors and Biofilm...................................................................................................... 356
24.3.1 Virulence Factors............................................................................................................. 356
24.3.2 Techniques for the Study of Virulence Factors................................................................ 358
24.3.3 Biofilm Formation and Cell-to-Cell Communication...................................................... 359
24.3.4 Techniques for Biofilm Evaluation....................................................................................361
24.4 Host–Pathogen Interaction.............................................................................................................361
24.5 Immunity and Vaccines................................................................................................................ 364
24.6 Conclusion..................................................................................................................................... 365
References............................................................................................................................................... 365

24.1 Introduction
The genus Proteus was named in 1885 by Hauser, inspired by the character of Proteus in Homer’s
Odyssey as he had the power to change his shape to escape different threats. Members of the genus
Proteus are ubiquitously distributed in the environment, and many of them are human pathogens. In
particular, P. mirabilis is an important etiological agent of opportunistic and nosocomial infections.
It causes urinary tract infections (UTIs), which can be severe in patients under catheterization or with
abnormalities in the urinary tract. Pyelonephritis, bacteremia, renal and bladder stones, and catheter
obstruction are among the most common complications in P. mirabilis infection. Occasionally, it can
cause foodborne illness as a result of mishandling during food processing.
In order to uncover its pathogenicity, several virulence factors were expressed in vitro and different
strategies have been developed to assess the contributions of these factors. For example, application of
laboratory models, from pioneer work with cell culture to animal model, has revealed valuable insights
into various aspects of P. mirabilis infection. Indeed, several conserved antigens have been evaluated as
vaccine components using a mouse model of infection. There is no doubt that a thorough understanding
of the fundamental aspects of pathogenicity and host–pathogen interaction of P. mirabilis infection will
be crucial for the formulation of new treatments and preventive strategies.

24.2  P
 roteus mirabilis
Proteus mirabilis is a Gram-negative bacterium, a dimorphic and motile member of the Enterobacteriaceae
family. It has the ability to differentiate from short rods into elongated, multinucleate swarm cells that
overexpress flagella [1]. It is a commensal bacterium found in the intestinal microbiota and in the envi-
ronment, including in soil and contaminated water. Generally associated with complicated UTIs, Proteus

355
356 Laboratory Models for Foodborne Infections

may also cause a wide variety of nosocomial infections, including respiratory infections, affecting dif-
ferent organs (eyes, ears, nose, and skin, among others) [2,3].
Contamination of food by enteric pathogens can occur from the farm if human or animal sewage is
used to fertilize soil or if sewage water is used to irrigate crops. Such risks are further increased if the
food is mishandled during processing and preparation, a stage where pathogens could multiply exponen-
tially under favorable conditions [4].
Members of the family Enterobacteriaceae have been considered a potent cause of foodborne out-
breaks [5]. P. mirabilis has been frequently implicated in food deterioration and spoilage. Besides oppor-
tunistic infections, it can also cause food poisoning when consumed in contaminated food such as meat,
vegetables, and seafood [6]. Indeed, P. mirabilis is regularly detected in food. For example, the bacte-
rium was found in 7% of the beef, 10% of the chicken, and 17% of the rice from a cafeteria in Alice Town,
South Africa [7]. In China, P. mirabilis isolated from chicken products showed a high level of antibiotic
resistance, posing a potential threat to public health [8]. Moreover, the presence of blaVEB was confirmed
in a nonhuman P. mirabilis strain isolated from poultry meat in Bern, Switzerland, in addition to the
detection of blaCMY-2 in a sample imported from Austria [9]. CMY-2-positive P. mirabilis isolates are
commonly found in humans, while VEB-6 isolates are less common. Several reports showed that VEB-6
P. mirabilis is responsible for infection in other countries [9]. It is worth noting that P. mirabilis strains
harboring blaCTXM-2 were recently identified in chicken meat sold in markets in southeast Brazil [10].

24.3 Virulence Factors and Biofilm

24.3.1 Virulence Factors
Proteus mirabilis expresses several virulence factors that are related to pathogenicity and to biofilm
formation. Among them are fimbriae that mediate adhesion to different surfaces, flagella-based motility,
outer membrane proteins, urease production, hemolysin, proteus toxic agglutinin, and iron acquisition
systems [11].
Fimbriae are proteinaceous appendages that protrude from the bacterial cell wall and mediate adhe-
sion to both biotic and abiotic surfaces. P. mirabilis genome encodes 17 different fimbrial operons, so far
the highest number seen in any bacteria [12]. The most studied fimbriae are MR/P, UCA, PMF, and ATF,
while the roles of the other 13 potential fimbriae in P. mirabilis pathogenesis are scarcely known. MR/P
fimbria is important in UTIs [13], exhibiting reported roles in adhesion [14], in biofilm formation [15],
and in eukaryotic cell cytotoxicity and genotoxicity [16]. The MR/P operon includes 10 genes that show
several interesting features including an invertible element that modulates the ON/OFF phase variation
(MrpI) and a transcriptional regulator (MrpJ) that represses motility, thus allowing the bacteria to con-
trol motility and adherence [17]. UCA fimbria facilitates adherence to uroepithelial cells, contributing to
infection and also playing a role in biofilm development [18], whereas PMF fimbriae contribute to kidney
colonization [19]. While all these fimbriae are involved in infection, ATF, whose expression is optimal at
ambient temperature, does not have a role in colonization of the mouse urinary tract. However, it could
play a part in catheter colonization, in survival in the environment, and probably in food contamination
[20,21]. It has been recently reported that there is a tight regulation of fimbrial expression, due in part
to MrpJ, a global regulator of P. mirabilis virulence [22]. These authors reported that MrpJ directly
represses flhDC and other fimbriae (PMF, Fim8), T6SS, and protease expression. Although it is not well
understood how this regulation works, it is clear that a controlled expression of fimbriae is needed to
maintain bacterial fitness.
Flagella-mediated motility is related to P. mirabilis swarming motility. This behavior is based on
sequential rounds of swarm cell differentiation, swarming colony migration, and consolidation with
dedifferentiation to a swimmer-cell morphology [23]. Swarmer cells are multinucleate, are elongated
(more than 10 μm long), and express several flagella, while swimmer cells are rod shaped, are 1–2 μm
long, and have peritrichous flagella [23]. Several studies have revealed a complex regulatory network
during swarm cell differentiation. There is a reciprocal regulation between fimbrial and flagella expres-
sion. There are 14 additional mrpJ paralogs associated with other fimbrial operons [23,24], and most of
Proteus 357

them repress motility, inhibiting swarm cell differentiation [24]. Bacteria adopt a filamentous morphol-
ogy as a result of the sensor activities of flagella in contact, for example, with a urinary catheter. Contact
to solid surfaces creates a torsional change in the outer membrane, and this is sensed by an upregulator
of the flagellar master operon (Umo) protein, which induces the expression of flagella to produce the
highly flagellated cells that are required for swarming [11]. This motility is related with migration across
different abiotic surfaces including catheters.
Iron acquisition systems are an important virulence determinant that enhances bacterial colonization
of the host cells and survival in the environment [25]. Iron is an essential micronutrient for all liv-
ing organisms, and its acquisition is vital for bacteria considering that only a minor fraction is avail-
able (10 −18 M). In order to capture iron, bacteria have evolved high-affinity iron-scavenging and uptake
systems [25]. The main strategies used by bacteria are the production and uptake of siderophores and
the direct utilization of host iron compounds such as transferrin, lactoferrin, or heme-containing mol-
ecules [25]. Gram-negative Fe(III) acquisition systems usually consist of an outer membrane receptor,
with transport across the outer membrane by TonB/ExbB/ExbD complex, a periplasmic binding protein,
and an inner membrane ABC transporter. In P. mirabilis, there are at least two gene clusters related to
siderophore biosynthesis and ABC transport, three outer membrane proteins induced by iron starvation
involved in heme uptake, and a heme receptor [26,27]. One of the clusters related to siderophore biosyn-
thesis is a novel nonribosomal peptide syntheses (NRPS)-independent siderophore (NIS) named proteo-
bactin, as this was first described in a bacterium [28]. The other one contains the nrp operon, which has
been previously described to be upregulated during iron limitation [29]. This operon is encoded within
the high pathogenicity island (HPI) in P. mirabilis HI4320 that shows homology compared to the HPI of
Yersinia spp. [30]. Infection challenges with mutant strains in different genes involved in yersiniabactin-
related siderophore showed that it contributes to P. mirabilis fitness in vivo [31].
Quorum sensing describes the communication between bacterial cells, whereby a coordinated popula-
tion response is controlled by diffusible molecules produced by individuals, often known as autoinduc-
ers. The influence of quorum-sensing molecules in Proteus strains is much less known compared to
Pseudomonas or Escherichia. N-acyl homoserine lactone (AHL) signaling molecules are utilized by
several Gram-negative species to sense population density and coordinate gene expression [32]. P. mira-
bilis lacks a clear AHL synthase (LuxI) homologue and does not seem to produce this type of signaling
molecule [12,33]. However, P. mirabilis encodes a LuxR family transcriptional regulator and seems to
produce compounds with AHL-like activity. Previous studies found that the addition of exogenous AHL
to a P. mirabilis population had a strain-specific impact on virulence factor expression, swarming, and
biofilm formation [34,35]. The quorum-sensing molecule autoinducer 2 (AI-2), encoded by luxS, can
mediate both intra- and interspecies interactions. P. mirabilis possesses a luxS homologue and produces
AI-2 [36]. However, mutation of luxS in P. mirabilis str. BB2000 does not significantly affect swarming,
virulence factor production, or survival in a mouse model, suggesting that AI-2 does not contribute to
pathogenicity [36]. This lack of phenotype might indicate that P. mirabilis uses LuxS strictly as part of
the activated methyl cycle, particularly as P. mirabilis str. HI4320 contains no clear homologue of the
Lsr system for sensing and responding to AI-2. However, AI-2 produced by P. mirabilis might influence
gene expression in other species that use this signaling molecule. Other studies have reported that the
presence of a quorum-sensing molecule (N-butanoyl homoserine lactone, BHL) increased P. mirabilis
biofilm thickness and ureolytic activity. Laser interferometric determination of diffusion showed that
urea easily diffuses through P. mirabilis biofilm, while acetohydroxamic acid (AHA) is blocked. This
may suggest that the use of urease inhibitors in catheter-associated UTIs (CAUTIs) may be less effective
than in other urease-associated infections [37].
Urease is produced by P. mirabilis and is responsible for the hydrolysis of urea to carbon dioxide and
ammonia [38]. Urease production is induced by urea and is expressed during growth in urine where it
is highly active [39]. In urine, this activity elevates environmental pH and induces crystal formation
(calcium crystals and magnesium ammonium phosphate) [39]. These crystals become trapped within
the polysaccharides produced by attached bacteria, forming the characteristic crystalline biofilms on
catheters [40]. Formation of these crystalline biofilms by P. mirabilis could be considered as a protective
structure as the host immune system and antibiotics cannot penetrate the biofilm [40]. The activity of
this enzyme also causes indirect tissue damage as ammonia becomes toxic for the uroepithelial cells [41].
358 Laboratory Models for Foodborne Infections

P. mirabilis produces two toxins that are implicated in tissue damage and dissemination to the kid-
neys. Hemolysin (HpmA) is a Ca+ -dependent pore-forming cytolytic agent that destabilizes the host cell
by inserting itself into the cell membrane and causing a Na+ efflux [42]. The other is a surface-associated
cytotoxic protease proteus toxic agglutinin (Pta) that is functional only in an alkaline pH; it punctures
the host cell membrane, causing leakage of the cytosol, osmotic stress, and depolymerization of actin
filaments, affecting the integrity of the cell and inducing bladder and kidney damage in the urinary
tract [23,43].
One important virulence factor that protects bacteria against immune response is the metalloprotein-
ase ZapA, which cleaves serum and secretory immunoglobulins A1 (IgA1), IgA2, and IgG [44]. ZapA
might also cleave complement components C1q and C3; cell matrix components such as collagen, fibro-
nectin, and laminin; and cytoskeletal proteins such as actin and tubulin [45].

24.3.2 Techniques for the Study of Virulence Factors

Generation of mutants with interrupted or deleted genes is a powerful tool for studying the role of
virulence factors. One of the most common mutagenesis strategies is to interrupt the gene of interest
with an antibiotic cassette by allelic replacement. This has been used in P. mirabilis with high success
[13,16,19–21,46], and some of the most relevant mutagenesis techniques and genes identified are shown
in Table 24.1. The comparison of the mutants with the wild-type strain allows for the study of the effect
of the absence of the gene in UTI.
Signature-tagged mutagenesis (STM), described by Hensel et al. in 1995, was designed to detect new
virulence genes of the target organism Salmonella typhimurium in a murine model of typhoid fever.
This technique is a negative selection method in which unique identification tags allow analysis of pools
of mutants in mixed populations. In STM, each mutant is tagged with a unique DNA sequence, these
allow for coamplification of the DNA tags of a mixed population of mutants by a single polymerase chain
reaction (PCR) [56,57]. The tag consists of a short DNA sequence of 40 bp, inserted in a transposon
system. DNA tags can be included during allelic replacement (signature-tagged allele replacement) on
a systematic genome-wide scale [58]. Detection of the signature tags could be done by dot blot, PCR, or
polymorphic tag-length transposon mutagenesis.
In P. mirabilis, several articles have described the use of this technique. Burall et al. 2004 [50] used
STM in uropathogenic P. mirabilis in conjunction with a murine model of ascending UTI to identify

TABLE 24.1
Identification of P. mirabilis Microbial Factors Using Different Molecular Tools
Gene Genetic Tool References
pmfA Allelic replacement [20]
Flagellin genes Allelic replacement [46]
Protease STM [47]
Mutants with increased sensitivity to Mini-Tn5 mutagenesis [48]
antimicrobial peptides
Swarming-deficient mutants Random transposon mutagenesis [49]
Genes that contribute to pathogenesis of UTI STM [50]
64-kDa iron-regulated OMP; heme receptor Transposon mutagenesis [27]
Fimbrial mutants Allelic replacement [51]
mrpJ Site-directed mutagenesis [28]
Virulence genes STM [52]
qnrC and qnrA1 Site-directed mutagenesis [53]
fliL Null mutations [54]
mrpI; glnE; fimbrial subunit; putative MuA-like Random transposon mutagenesis [55]
DNA-binding protein; rsbA; bcr; putative
transmembrane protein; gltS; lrp; nirB; putative
lipoprotein
Proteus 359

virulence determinants required for colonization of the urinary tract (Table 24.1). They could iden-
tify genes that affected motility, iron acquisition, transcriptional regulation, phosphate transport, urease
activity, cell surface structure, and key metabolic pathways [50].
A traditional tool that allows for the identification of gene functions is random transposon mutagen-
esis. The basis of this technique is in the generation of mutant libraries that harbor a transposon insert,
which abolishes the function of the affected gene. Transposons are mobile genetic elements that can
move within the genome and can affect the function of gene expression. One of the limitations in iden-
tification and separation of nonvirulent mutants from a pool of mutants is that it is time consuming. In
P. mirabilis, there are many studies that report the use of this technique. Recently, Holling et al. [55]
used random transposon mutagenesis to identify genes involved in biofilm formation. They used a mini-
Tn5Km2 transposon that was introduced into the wild-type P. mirabilis strain B4 by conjugal transfer
from the donor organism, Escherichia coli S17.1λpir (Table 24.1). The mutants were screened by the
crystal violet microtiter plate assay to identify bacterial impairment in catheter blockage. The end adja-
cent to the transposon was sequenced in order to identify the mutated gene.
The use of null mutations is also an important strategy as it consists of the insertion of a reprogrammed
intron carried into a plasmid that finally results in a lack of function of a particular gene. Lee et al. [54]
used a fliL knockout mutant to gain further insight into the function of FliL (Table 24.1). The mutant
was generated by a reprogrammed intron, the intron was inserted between nucleotides (nt) 30 and 31 in
fliL. The loss of FliL results in cells that cannot swarm on agar surfaces but can swim in broth cultures.

24.3.3 Biofilm Formation and Cell-to-Cell Communication

During the past few decades, biofilms have been widely studied because they cause 65% or more of all
infections, being particularly prevalent in device-related infections, infections on body surfaces, and
chronic infections [59–61]. Bacterial biofilms are problematic for many food industries including dairy
processing, poultry and red meat processing, and brewing among others [62]. This may become a risk of
food contamination and transmission of foodborne pathogens [63]. Several other problems are related to
biofilm formation in food industry such as slime production, decrease of heat transfer in heat exchanger
or condensers, corrosion problems, and hygienic concerns about the sanitation efficacy [64]. Bacterial
biofilms are difficult to eliminate from food processing environments and make the design of control
strategies a big challenge in the industry [62].
Bacteria are usually regarded as free-living unicellular organisms, but we now know that they pre-
dominantly exist as adherent multicellular biofilms in diverse environmental niches [59–61]. The tran-
sition from the planktonic state to biofilm growth occurs as a consequence of environmental changes
that triggers the disruption of multiple regulatory networks [59–61]. Thus, upon sensing a signal, free
planktonic cells will initiate attachment to a surface that will lead to biofilm formation. During this
process, dramatic changes occur in gene expression when compared with its planktonic counterparts.
Several steps in biofilm formation are common to most bacterial species, but this process can vary from
one organism to another. The first stage comprises the attachment to a surface, either biotic or abiotic.
Here, flagella play a key role as motility and biofilm development are mutually exclusive events and the
transition to sessile occurs in this first stage [65]. Flagella are not only required for propulsion but also
have a critical mechanosensory role in surface sensing and thus on the initial stages of surface adhesion
that will lead to the formation of biofilm [66]. Lipopolysaccharide (LPS) and O-antigen play a role in
P. mirabilis surface sensing [67,68].
Five stages in P. mirabilis biofilm formation have been described using confocal laser scanning micros-
copy (CLSM) and different morphologic and topologic descriptors. P. mirabilis biofilms on coverslips
and Luria-Bertani (LB) broth showed (1) reversible bacterial adhesion to the surface characterized by a
slow growth, presence of elongated bacteria, and absence of extracellular matrix (day 1); (2) irreversible
bacterial adhesion concomitant to decreasing elongation, and the beginning of extracellular polymer pro-
duction (days 2–3); (3) accelerated bacterial growth concomitant to continuously decreasing elongation
and halting of extracellular polymer production (days 3–4); (4) maturation of biofilm defined by maxi-
mum bacterial density and volume, minimum elongation, maximum extracellular material, and highest
compaction (day 5); and (5) decreased bacterial density and extracellular material through detachment
360 Laboratory Models for Foodborne Infections

and dispersion (day 7) [69]. Our group has observed that different P. mirabilis mutant strains express fla-
gella in different stages of biofilm formation generally associated with the dispersion step (Figure 24.1).
In other works, the structure of biofilms in artificial urine (AU) and LB broth, a standard laboratory
medium, has been studied. Different authors have shown that P. mirabilis biofilm formation occurs
rapidly in both LB broth and AU. When grown in LB broth, P. mirabilis produces large and structurally
complex biofilms. In AU, biofilms were less well structurally organized, contained crystalline precipi-
tates that are associated with the urease activity definitive of P. mirabilis, and contained significantly
higher numbers of the swarmer cell form of P. mirabilis [70].
Biofilm development and quorum sensing are closely interconnected processes. Signal molecules
(quorum sensing) are important factors in biofilm formation and development [71]. Different studies
have demonstrated that quorum-sensing molecules, normally associated with the regulation of virulence
factors, could also regulate the development of complex mushroom structures in a P. aeruginosa PAO1
biofilm [72]. Disruption of QS systems has been reported to affect the dynamics of biofilm formation
in some cases, but the specific mechanisms downstream of the QS regulators are, for the most part,
poorly understood [73]. Also, QS antagonists, p-nitrophenyl glycerol and tannic acid, have been shown
to inhibit the quorum-sensing system and subsequently inhibit P. mirabilis biofilm formation in artificial
urine [74].
On the other hand, Stankowska et al. [75] identified the luxS gene coding S-ribosylhomocysteine
lyase responsible for AI-2 synthesis, proving that P. mirabilis uses a quorum-sensing communication
system.
As mentioned before, P. mirabilis can live as single cells (planktonic) or as members of organized
microbial communities called biofilms, which are composed of microorganisms and the extracellular
matrix-forming polymers they produce [73]. Proteus has been shown to produce biofilms in diverse
environments, including industrial and clinical settings [76]. Urinary catheters are the medical devices
that are most frequently colonized by Proteus biofilms. Due to the deposition of crystals within these
biofilms, blockage of the urinary catheter can occur. These episodes of bacteriuria can lead to septicemia
and shock [77]. In addition, the encrustation of urinary catheters can cause trauma to the bladder mucosa
and urethra upon removal of the catheter [77]. Biofilm-based P. mirabilis lifestyle provides a protected
environment against stresses, such as desiccation, attack by the immune system, protozoa ingestion, and
antimicrobials. In the host, biofilm bacteria are protected from the host immune system and treatment
by antibiotics [77]. Other works have demonstrated the biofilm ability to induce host hyperinflammation,
as shown by elevated levels of proinflammatory cytokines [78] and matrix metalloproteases [79], and
excessive numbers of neutrophils [80]. Taking into account that several infections are associated with
biofilm formation, we can consider biofilm as another virulence factor.

FIGURE 24.1  3D reconstruction of P. mirabilis mutant strain that overexpress flagella during biofilm development. Left
panel represents 3D model of bacteria, and right panel shows flagella and extracellular matrix. Images were taken in a
FV300 Olympus Confocal Microscope.
Proteus 361

24.3.4 Techniques for Biofilm Evaluation

A semiquantitative biofilm formation assay was described by O’Toole and Kolter [81]. This assay
is based on the ability of cells to adhere to 96-well microtiter dishes made of poly(vinylchloride)
(PVC). The assay consists in the inoculation of bacteria in adequate culture medium (100 μL/well)
and incubation for 24–48 h at the specific temperature. After that, planktonic bacteria are removed
and the bacteria adhered to the well are stained with 1% crystal violet. The plate is subjected to sub-
sequent washes in order to eliminate all the unspecific staining and only the crystal violet associated
with bacteria will be solubilized in 95% ethanol, and the absorbance is then determined at 600 nm.
The use of polystyrene microtiter plates as a substrate for the formation of biofilms, and subsequent
staining with crystal violet, has allowed the isolation of a large number of mutants unable to form
biofilms [15,82–85].
Microscopy has evolved as a powerful technique to study microorganisms, and in particular in bio-
films, the use of confocal and scanning electron microscopy has become important for understanding
such dynamic process.
Confocal laser scanning microscopy (CLSM) has been employed since 1990s and represents one of
the most significant advances in optical microscopy ever developed. It is a very useful tool for imag-
ing microbiological samples. It has several advantages that allow many options for sample mounting,
high resolution, optical sectioning, analytical precision by virtually eliminating interference from out-of-
focus objects, and multichannel imaging [86,87]. Also, the main advantage of CLSM is the reconstruc-
tion of 3-dimensional models.
CLSM allows the study of architecture and identification of stages of P. mirabilis biofilm formation
in a nondestructive manner. Jones et al. [70] compared the structure of P. mirabilis biofilms in artificial
urine and LB broth. Biofilms in LB formed mushroom structures at 24 h and contained nutrient channels,
while AU biofilms were a flat layer without nutrient channels. The authors also observed the presence of
swarmer cells protruding out of the biofilm. In another study, the 3D biofilm architecture development
in P. mirabilis batch culture has been described [69]. Confocal images were used in order to perform 3D
reconstruction and calculation of different morphotopological descriptors. Authors stated that P. mirabi-
lis biofilm formation followed a five-stage process as mentioned above.
Scanning electron microscopy (SEM) relies on a beam of electrons with high energy generated by a
suitable source, typically a tungsten filament. The electrons pass through a system of apertures and elec-
tromagnetic lenses to produce a thin beam of electrons. Electrons are emitted from the specimen by the
action of the scanning beam and are collected by a detector. This tool was used to study bacterial surface
and biofilms and was used by Cox and Hukins [88] to assess the morphology of mineral deposits on
encrusted urinary catheters. SEM allows for observation of biofilm formation over catheters of different
materials (Figure 24.2). Silicon and latex are common materials for the manufacturing of urinary cath-
eters. SEM also allows the observation of differences among the surface of each material. While silicon
is smooth with a repeated pattern, latex seems to be more wrinkled, and this could influence bacterial
colonization of the catheter surfaces. However, and taking into account these differences, P. mirabilis is
able to colonize both surfaces (Figure 24.2). Differences in biofilms generated over both surfaces could
be observed, including a brick-shaped biofilm formed over the latex surface.
The difference between SEM and environmental scanning electron microscopy (ESEM) is that ESEM
allows the examination of practically any specimen under any gaseous conditions and allows for obtain-
ing images without prior specimen preparation. Holling et al. [55] used this tool to study the surface
of catheters colonized by P. mirabilis biofilms. This tool permits the imaging of unprocessed, fully
hydrated samples, which may provide much insight into the development of P. mirabilis biofilms.

24.4 Host–Pathogen Interaction
In vivo approaches are still very useful for the study of bacterial pathogens. Although in the last years
efforts have been successful in reducing the use of animals and in refining in vivo-based experimental
methods, the host environment cannot be easily replaced by in vitro techniques.
362 Laboratory Models for Foodborne Infections

(A) (B)

×600 20µm

(C) (D)

×1, 700 10µm ×1, 900 10µm

FIGURE 24.2  Scanning electron microscopy of P. mirabilis biofilm over catheter material. (Panel A) Latex catheter
without bacteria. The round balls were over all the latex surface and correspond to zinc depositions due to manufacturing.
(Panel B) Silicon catheter without bacteria. (Panel C and D) P. mirabilis growth over latex (C) and silicon (D). In order to
allow bacteria to colonize the surface, the catheter bridge technique was employed. Briefly, small sections of the catheter
were cut and used as bridges in an agar plate (small pictures in Panel C and D). Images were presented in the 115th General
Meeting of the American Society of Microbiology, New Orleans, LA.

Although P. mirabilis is a very ubiquitous bacterium, its capacity to induce uropathogenesis is remark-
able. Animal models have proven valuable in this regard. A classic ascending UTI described initially by
Hagberg et al. [89] for the study of E. coli is still being used by several researchers. In this case, bacte-
ria are slowly introduced into the mouse bladder through a transurethral soft catheter so that different
aspects of urinary infections can be studied.
This model has been traditionally used to assess the ability of different strains to colonize bladders
and kidneys after ascending infection through the urinary tract, typically measured by bacterial count-
ing [21]. Infection of different groups of mice with the different strains or even coinfection of the same
animals with the wild type and isogenic mutants is frequently performed to assess bacterial colonization
competence.
Different studies have been developed to assess the potential of different P. mirabilis UTI treatment-
or prevention-based strategies, including vaccination, using this model. A wide range of vaccination
strategies to prevent UTI, based on different antigens or immunization routes, have been proposed using
this model [90,91].
This model has been recently refined for modern approaches like STM [52], morphological analysis
of kidneys colonization by laser confocal microscopy [92], or transcriptome analysis to compare in vitro
and in vivo P. mirabilis gene expression [93].
Also, different modifications have been performed to increase this animal model input. For example,
an infection model of permanently catheterized mice has been recently developed, which was initially
proposed for the study of Enterococcus faecalis biofilms associated with urinary catheters [94]. This
new approach will help to elucidate the molecular basis and prevention alternatives of in vivo biofilm
formation.
The adhesion of pathogenic bacteria to host cells is crucial for the establishment of the infec-
tion. Subsequent events include tissue colonization and, in some cases, cellular invasion fol-
lowed by intracellular multiplication and/or persistence [95]. Colonization of the epithelium is the
first step in the pathogenesis of P. mirabilis and has been demonstrated by in vitro and in vivo
approaches [3]. However, the mechanisms by which P. mirabilis adhere to epithelial cells are not
fully elucidated  [3,96].
Pioneer work by Chippendale et al. showed that P. mirabilis could survive in the epithelial cells, but
no significant replication was noticed [97]. Oelschlaeger and Tall found that P. mirabilis strains isolated
from different sources had varied internalization efficiencies and that microfilaments were important for
Proteus 363

the process [98]. Bacteria were detected in membrane-bound vacuoles after 3 h of infection using human
renal proximal tubular epithelial cells [97].
Torzewska et al. demonstrated that not only could P. mirabilis produce urease with the concomitant
formation of calcium crystals and magnesium ammonium phosphate precipitates but it also could cause
crystallization of urine components inside the cells [99]. The production of crystals inside the cells could
induce cell damage or death, but this was not evaluated by the authors. However, in another work, it was
demonstrated for the first time that both MR/P fimbriae and flagella mediate genotoxic and cytotoxic
effects on eukaryotic cells (Figure 24.3) [16]. It has also been reported that flagella have a role in P. mira-
bilis adhesion [16] (Figure 24.3), as reported in other pathogens [100].
Considering all the information available on eukaryotic cell–Proteus interaction, we can conclude that
P. mirabilis produces several virulence factors that induce damage in cell. The lack of knowledge on the
intracellular life of P. mirabilis could be explained in part because of the wide range of virulence factors
that the bacteria produce. This should be taken into account when cell culture experiments are going to
be set up. These factors include toxins such as Pta and HpmA (bacterial toxin hemolysin) that cause cell
damage, the expression of MR/P fimbriae that mediate adhesion and cytotoxic/genotoxic damage, and
flagella that mediate adhesion and even cell death, among others.
One of the main techniques that allows for visualization of host–pathogen interaction is immunofluo-
rescence. It involves the staining of different cellular structures with antibodies coupled to fluorescent
molecules followed by observation with a fluorescent or confocal microscope. The development of this
type of technique and the use of time-lapse imaging will contribute to the understanding of infection and
to the increase of knowledge of the mechanisms involved in cell invasion.

(A) 7,00 µm
X (B) 7,00 µm
X

Y Y Y Y
7,00 µm

7,00 µm
7,00 µm

7,00 µm

X X
7,00 µm 7,00 µm

(C) 7,00 µm
X
(D) 7,00 µm
X

Y Y Y Y
7,00 µm

7,00 µm
7,00 µm

7,00 µm

X X
7,00 µm 7,00 µm

FIGURE 24.3  Confocal microscopy image of P. mirabilis wild-type adhesion to Vero cell line (kidney); bacteria is
stained with a polyclonal anti-proteus antibody (A), actin is stained with phalloidin-rhodamine (B), and DNA with DAPI
(C). Panel D shows a superimposition of the three images (A–C).
364 Laboratory Models for Foodborne Infections

24.5 Immunity and Vaccines

Several efforts have been made to generate vaccines against P. mirabilis infections. UTIs represent an
important problem in health and result in high economical losses; so the development of vaccines is not
worth without mentioning that the quality of life of afflicted individuals is also affected. Only in USA,
UTIs cause an estimated 11 million doctor visits with an annual cost of 6 billion dollars [11].
UTIs are commonly treated with antibiotics that are effective in noncomplicated infections. The most
recommended antibiotics for UTI are trimethoprim–sulfamethoxazole, ciprofloxacin, and ampicillin [101].
In the case of resistant strains, recurrent UTIs, catheterized patients, or those with abnormalities in the
urinary tract or with stones, the success of the antibiotic therapy is less effective [102]. The main problem
of the antibiotic-based therapy is the fast increase in the resistance to antimicrobial molecules.
This worldwide situation and the magnitude of UTIs clearly justify the efforts to develop effective
vaccines.
Early studies showed that initial infection with P. mirabilis does not prevent reinfection. Moreover,
recovery from a UTI does not generate a protective immune response, and it was also demonstrated that
the same strain can be isolated from successive infection episodes [103]. Unlike other mucosal infections
such as cholera, where a single exposure to the etiological agent results in protective immunity for life,
the resolution of UTIs did not confer protection in case of another episode. In particular, P. mirabilis has
a high number of conserved surface antigens that do not differ significantly between strains of differ-
ent origin, which could contribute to the successful development of such strategies [104,105]. There are
some vaccines for the prevention of UTIs available in Europe, and these are recommended in patients
with uncomplicated recurrent UTI. They are formulated from complex protein extracts from different
bacterial strains, producing significant side effects, and are not able to produce strong mucosal responses
against antigens related to virulence [106]. Moreover, so far, there are no licenses for UTI vaccines avail-
able in the USA. Extensive clinical studies are needed to determine the effectiveness of these vaccines.
Various strategies have been developed in recent years to prevent UTIs caused by P. mirabilis, directed
mainly against bacterial defined subunits. In early studies developed by Pazin and Braude, purified
flagella were used to immunize rats by injection. These authors demonstrated that the serum against
flagella immobilized the bacteria and prevent the spread of the disease to the other kidney [107]. Moayeri
and coworkers showed that immunization of BALB/c mice with outer membrane protein preparations
protected the animals against an intravesical homologous challenge [108]. Legnani-Fajardo et al. showed
that parenteral immunization with fimbrial preparations protected mice after a hematogenous challenge,
both homologous and heterologous [109] strains. In more recent studies carried out in our department,
Pellegrino et al. vaccinated mice with purified recombinant antigens of P. mirabilis fimbriae (MrpA,
PmfA, and UcaA) and observed that MrpA-protected mice when immunized subcutaneously faced
hematogenous and ascending challenges [110]. Furthermore, intranasal immunization with the adhesin
MrpH or the N-terminal receptor binding domain protects mice against experimental UTI [111]. These
authors evaluated four different immunization routes and concluded that the intranasal route produces
a response to broader antibodies and specific antibody titers in serum, urine, vaginal douching, and in
bile [111,112]. A more recent work showed that a MrpH-FimH recombinant protein induces a significant
increase in IgG and IgA in serum, nasal wash, vaginal wash, and urine samples. However, vaccinated
mice showed a reduction in bladder and kidney bacterial load, but the differences were not statistically
significant [91].
Mucosal vaccines should induce the production of systemic and mucosal antibodies and cellular
immune responses. They have the advantage of being safe and inexpensive, and have fewer side effects
than systemic vaccines [113]. In our department, we evaluated two mucosal immunization routes (nasal
and vesical), the induction of specific antibodies MrpA, UcaA, and PmfA in serum and urine, and
protection against an upward experimental UTI in mice. From these results, it was determined that the
nasally immunized animals developed a strong antibody response in serum IgG and IgA when immu-
nized with MrpA. Also, a significant decrease in the number of bacteria recovered in kidneys and blad-
ders after experimental infection was reported compared with nonvaccinated animals [114]. However,
a correlation between the induction of specific antibodies and reduced bacterial colonization could not
Proteus 365

be observed. On the other hand, the immune response that develops from entering uropathogens in the
urinary tract is particularly complex. The mechanisms of innate immunity would have an important
role in the UTI. The role of different mechanisms in the adaptive immune response in resolving urinary
infection is currently a matter of debate [11]. During the entrance of uropathogens, an early response
in the host that includes recruitment and activation of effector cells at the site of infection results, lead-
ing to an inflammatory response whose magnitude and location explains many of the clinical mani-
festations of UTIs [115]. This response depends on cell receptors present on the uroepithelium that
recognize pathogen-associated molecular patterns (PAMPs), which are highly conserved in different
organisms, [116] and these can be recognized by the innate immune system that then limits the action
of microbial evasion mechanisms and allows for detection of infectious agents from a limited number of
cellular receptors. It has been determined that the detection of PAMPs by toll-like receptors (TLRs) is
crucial for activation of the innate immune response and control of the adaptive immune response that
occurs later [117]. Several studies have shown that bladder epithelial cells, like macrophages, express
TLR4, which mediates a rapid cytokine response against low concentrations of LPS [116,118]. In mac-
rophages, the recognition of LPS requires a CD14 coreceptor [119], which is not present in epithelial
cells. It has also been demonstrated that TLR4 recognizes fimbriae regardless of the presence of the
CD14 [120] coreceptor. The control of UTIs in the bladder depends on TLR4 expression on urothelial
and stromal cells and is needed to drive inflammatory responses [121]. Studies of TLR4 expression in
humans with UTIs demonstrated that lower TLR4 expression attenuates host responses and promotes
asymptomatic carriage of UPEC [122]. TLR5 is essential to mediate flagellin-induced inflammatory
responses; it plays a role in control of infection in both the bladder and kidneys [123]. In humans, a
polymorphism in TLR5 gene abrogates the flagellin signaling cascade and has been associated with an
increased susceptibility to recurrent UTIs [124].
The last TLR identified and characterized is the TLR11, which is specifically expressed in murine
kidney cells [125]. The PAMP that the TLR recognizes was not identified but was observed in an in vitro
assay as a motif present in E. coli uropathogenic strains [126]. Furthermore, in TLR11 knockout mice,
a massive kidney infection has been observed by challenging the animals with a uropathogenic strain of
E. coli. These authors suggest that TLR11 has a role in the innate immune response specifically against
uropathogenic bacteria [125]. Human TLR11 gene is present but not expressed due to the existence of a
stop codon in the reading frame. This finding opens new directions such as the absence of TLR being
one reason for the particular susceptibility of humans to the ITU.

24.6 Conclusion
P. mirabilis has become an interesting model microorganism since it expresses an impressive variety
of virulence factors. Its relation with infection, particularly with UTIs, has increased the necessity for
vaccines and new therapeutic agents. Antimicrobial resistance and biofilm development over different
surfaces including food make P. mirabilis an excellent model for the study of bacterial behavior in differ-
ent conditions. The study of the basic mechanisms used by this bacterium for survival and multiplication
in different environments could contribute to the prevention of infection and even food contamination.

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stromal and hematopoietic cells mediates innate resistance to uropathogenic Escherichia coli, Proc Natl
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Trends Immunol., 26, 509–511, 2005.
25
Pseudomonas aeruginosa

Stavria Panayidou and Yiorgos Apidianakis

CONTENTS
25.1 Introduction................................................................................................................................... 373
25.2 Vertebrate Models..........................................................................................................................374
25.2.1 Cystic Fibrosis Mouse Models of Chronic Lung Infection...............................................374
25.2.2 Acute Lung Infection Mouse Models............................................................................... 377
25.2.3 P. aeruginosa Induced Gut-Derived Sepsis Mouse Models............................................. 377
25.2.4 Burn- and Open-Wound Infection Mouse Models........................................................... 378
25.2.5 P. aeruginosa Keratitis Models (Mice, Rabbits, and Guinea Pigs).................................. 378
25.2.6 Otitis Media Models (Mice, Rats, Guinea Pigs, and Chinchilla)..................................... 379
25.2.7 Zebrafish (Danio rerio)..................................................................................................... 379
25.3 Invertebrate Models....................................................................................................................... 379
25.3.1 Drosophila melanogaster................................................................................................. 379
25.3.2 Caenorhabditis elegans.................................................................................................... 380
25.3.3 Dictyostelium discoideum..................................................................................................381
25.3.4 Galleria mellonella (Wax Moth).......................................................................................381
25.3.5 Bombyx mori (Silkworm)................................................................................................. 382
25.4 Plants............................................................................................................................................. 382
25.5 Conclusions................................................................................................................................... 383
References............................................................................................................................................... 383

25.1 Introduction
Classified in the family Pseudomonadaceae, order Pseudomonadales, class Gammaproteobacteria,
the genus Pseudomonas comprises some of the most ubiquitous and diverse Gram-negative bacterial
species in nature that are capable of utilizing a wide range of organic compounds and colonizing a vari-
ety of ecological niches. Among the members of this genus, Pseudomonas aeruginosa is remarkable
for its capacity to inhabit diverse environments, including soil and water, and infect multiple organisms,
such as insects, plants, and animals.1–6 P. aeruginosa is an important opportunistic human pathogen
inflicting predominantly burn, cystic fibrosis (CF), and otherwise immunocompromised patients. It is
a frequent cause of nosocomial infections, being the most common pathogen isolated from patients
hospitalized for longer than 1 week. One reason for its high prevalence is that it is foodborne—found,
for example, in hospital water, food, and feeding tubes—and an efficient intestinal colonizer, especially
upon antibiotic treatment and surgical stress.7 Another reason is its high virulence repertoire, which
includes biofilm formation and quorum-sensing controlled factors.8,9 A third reason is its resistance to
antibiotics.
The multifaceted pathogenicity of P. aeruginosa in humans necessitates the use of various models
of infection and alternative model organisms. Due to ethical considerations and high cost of experi-
menting with vertebrate animals, invertebrates are widely used as alternative model hosts. In the
following sections of this chapter, we describe the mammalian models that recapitulate pivotal aspects

373
374 Laboratory Models for Foodborne Infections

Mouse
Caenorhabditis elegans
Rat
Ear
- Mouse/rat
- Guinea pig
Drosophila melanogaster
- Chinchilla
Eye
- Mouse/rat
- Rabbit Lung
Rabbit - Mouse/rat
- Guigea pig
- Zebrafish

Silkworm (Bombyx mori),

Wax moth (Galleria mellonella)
Blood (systemic
infections)
Guinea pig
- Mouse/rat
- Zebrafish
- D. melanogaster Plants
- Silkworm
- Wax moth Wounds
- D. discoidum - Mouse/rat
Chinchilla - D. melanogaster
Gut - Plants
- C. elegans
- D. melanogaster
- Mouse/rat Dictyostelium discoideum
- Zebrafish
- Silkworm
- Wax moth
Zebrafish (Danio rerio)

FIGURE 25.1  Model organisms recapitulating aspects of human blood, wound, lung, gut, eye, or ear infection with
P. aeruginosa.

of severe P. aeruginosa pathogenicity, namely, burn and open wound, acute and persistent lung infec-
tion, and bacteremia, as well as less severe but potentially dangerous infections of the ear, eye, and
intestinal tract. Zebrafish and invertebrate models also recapitulate aspects of wound, systemic, or
intestinal/epithelial barrier infection. All established models, including those based on plants, are use-
ful for assessing virulence, the efficacy of various treatments, and the role of host defense to infection
(Figure 25.1 and Table 25.1).

25.2 Vertebrate Models
25.2.1 Cystic Fibrosis Mouse Models of Chronic Lung Infection
CF is an inherited disease of the secretory glands that is caused by mutations in the cystic fibrosis trans-
membrane conductance regulator (CFTR) gene.10 CFTR gene mutations prevent the ion channels of the
lung and other tissues from moving salt and water into and out of cells. As a result, mucus accumulates in
the lungs, trapping bacteria that cause chronic infections.10,11 P. aeruginosa is a common cause of chronic
CF lung infection that may persist for decades12 and leads to mortality in the majority of the cases, due
to progressive lung damage. In 1992, only 3 years after the identification of the CFTR gene, Snouwaert
et al. generated the first CF mouse model.10,13 Since then, several mouse models of CF and the P. aerugi-
nosa lung infection have been developed.14 Although none of them are ideal, mouse models may provide
significant information about the CF pathogenesis and are essential for the preclinical assessment of
new therapeutics.14 A method that mimics very well the human chronic lung infection of CF disease is
based on the introduction of P. aeruginosa-laden agar/agarose or alginate beads in the mouse lung by
transtracheal injection. This model was first described by Cash et al. in 1979 using rats.15 In this model,
agarose beads act as artificial biofilms and protect bacteria from a direct neutrophil attack, facilitating
Pseudomonas aeruginosa 375

TABLE 25.1
P. aeruginosa Models of Infection and Indicative Findings Using Each Model
P. aeruginosa Infection Models Indicative Findings Using the Model

Vertebrate Models
Cystic fibrosis mouse models of • Suitable model for testing anti-inflammatory compounds e.g., BIIL 284.
chronic lung infection • P. aeruginosa and B. cenocepacia interaction in the mouse lung induces
bacterial virulence and concomitant inflammatory response.
Acute lung infection mouse • Increased expression of IL-6 is associated with edema formation and
models decreased lung function.
• IL-17 facilitates neutrophil recruitment in the infected lung areas of
P. aeruginosa-infected mice.
• Immunosenescence leads to impaired neutrophil response in the lungs.
• IL-27 is implicated in sepsis-induced immunosuppression.
• A leukopenic mouse model was developed for testing novel drugs.
P. aeruginosa gut-derived sepsis • The probiotic bacterium B. longum strain BB536 suppresses the intestinal
mouse models colonization of P. aeruginosa.
• IL-1 is critical during gut-derived sepsis.
• The bacteriophage strain KPP10 decreases P. aeruginosa burden and
inflammatory response in the infected mice.
• Surgical stress induces P. aeruginosa PA-I lectin in the mouse intestine,
causing lethal sepsis.
• PA-I lectin and cytotoxic exoproducts compromise the intestinal barrier.
• PA-I lectin is highly expressed during intestinal ischemia/reperfusion injury
in mice and contributes to lethal sepsis.
• Surgical hepatectomy leads to low phosphate levels in the mouse intestine,
which is sensed by P. aeruginosa, which then, enhances its virulence causing
gut-derived sepsis.
Burn-wound and open-wound • P. aeruginosa strain PAO1 quorum-sensing mutants exhibit reduced virulence
sepsis mouse models in this infection model.
• Suitable model for testing new antibacterial agents and treatments.
• Targeting of fragellin protein can be a promising approach for the treatment
of P. aeruginosa-infected burns.
• Negative-pressure wound therapy can prevent sepsis.
• Low GstA4 expression in the muscle causes susceptibility to infection.
P. aeruginosa keratitis models Studies in rabbits:
(mice, rabbits, guinea pigs) • Numerous antimicrobial treatments against P. aeruginosa keratitis have been
assessed.
• The P. aeruginosa proteases, elastase B, and PASP contribute significantly to
the pathogenesis of keratitis, whereas alkaline protease has a lesser contribution.
Studies in mice:
• LiCl and β-catenin promote host resistance against P. aeruginosa keratitis.
• Mouse TREM-2 suppresses corneal inflammation and promotes resistance to
P. aeruginosa infection.
• MRP8/MRP14 signaling amplify the inflammatory responses and increase
corneal susceptibility.
• Extracellular matrix protein Lumican, surfactant protein SP-D, and chemokine
CXCL10 have protective roles against P. aeruginosa keratitis.
• P. aeruginosa proteases, PASP, and MucD contribute significantly to keratitis
pathogenesis.
Studies in guinea pigs:
• Evaluation of P. aeruginosa virulence factors, antimicrobial drugs,
treatments, and host defense in the course of infection.
Otitis media models (mice, rats, • Evaluation of P. aeruginosa virulence factors, inflammatory responses, and
guinea pigs, and chinchilla) treatments.
Zebrafish (Danio rerio) (injection • P. aeruginosa strains PA14 and PAO1 are pathogenic to zebrafish embryos.
or feeding) • Infection is influenced by the developmental stage of the host.
• CFTR mediates resistance against P. aeruginosa infection.
(Continued)
376 Laboratory Models for Foodborne Infections

TABLE 25.1 (Continued)

P. aeruginosa Models of Infection and Indicative Findings Using Each Model
P. aeruginosa Infection Models Indicative Findings Using the Model

Invertebrate Models
Drosophila melanogaster (feeding, • Humoral and cellular innate immunity is important against infection.
wounding, or injection) • PA14 escapes from the host defenses by suppressing the expression of
antimicrobial peptides and muscle genes at the wound site.
• Selection of virulence-attenuated mutants, e.g., KerV, which is a conserved
virulence factor in Proteobacteria.
• Expression of the human lactonase PON1 protects flies from P. aeruginosa
infection.
• CHD1 is important for fly intestinal resistance against P. aeruginosa.
• JNK signaling pathway synergizes with Ras1 oncogene to induce stem-cell-
mediated tumorigenesis and invasion/dissemination in the fly midgut and
hindgut, respectively.
• The RhlIR and LasIR quorum-sensing systems are important for the full
virulence in orally infected flies.
Dictyostelium discoideum (feeding) • The rhl quorum-sensing system is required for the full virulence of
P. aeruginosa strain PAO1.
• P. aeruginosa strain PA14 is more virulent than PAO1.
• trpD, pchH, and pchI mutants are attenuated in virulence not only in
D. discoideum but also in flies and mice.
Caenorhabditis elegans (feeding) • P. aeruginosa pathogenesis (slow or fast killing) against C. elegans depends
on the bacterial culture media.
• The transcription factor DAF-19, the bZIP transcription factor zip-2, and the
small organic molecule RPW-24 are important for C. elegans immune
response against P. aeruginosa strain PA14.
• HTS assays are developed for identifying novel antimicrobials.
Galleria mellonella (Wax Moth) • Different P. aeruginosa strains exhibit different strategies of evading the
(feeding or injection) immune system of G. mellonella larvae.
• P. aeruginosa metalloproteinase elastase B stimulates the humoral immune
responses in G. mellonella.
• G. mellonella oral infection model can be useful in investigating
P. aeruginosa virulence mechanisms.
Silkworm (Bombyx mori) • Screening for P. aeruginosa virulence factors in the silkworm larvae.
(feeding or injection) • The NO detoxification enzyme NO reductase is important for full virulence
of P. aeruginosa in the silkworm.
• PvdE and ExoS are important virulence factors for P. aeruginosa to cross
epithelial barriers.
Plants
• Arabidopsis thaliana • P. aeruginosa strains PA14 and PAO1 are pathogenic to A. thaliana and sweet
• Sweet basil (injection) basil.
• The antimicrobial compound rosmarinic acid protects the sweet basil root
only from P. aeruginosa quorum-sensing mutants unable to form biofilms.
• The P. aeruginosa virulence factor pyocyanin inhibits the development of
A. thaliana roots though ethylene-dependent signaling.
• PA14 pathogenicity islands, PAPI-1, and PAPI-2 carry many genes, 11 of
which are necessary for full virulence in both A. thaliana and mice.
• Salicylic acid attenuates the virulence of PA14 against A. thaliana and
C. elegans.
• P. aeruginosa-secreted proteases activate a novel A. thaliana-immune
signaling pathway.
• P. aeruginosa alkaline protease AprA cleaves the bacterial fragellin
monomers in order to prevent immune recognition in both plants and
mammals
Pseudomonas aeruginosa 377

the modeling of delayed bacterial clearance. As a consequence, there is a prolonged neutrophil influx
into the lung as well as accumulation of cytokines that resembles the Pseudomonas lung infection seen
in CF patients.16 Neutrophils are the protagonists of the excessive inflammatory response observed due to
bacterial infections in CF patients,17 but their role in pathogenicity is not clear. Accordingly, many stud-
ies are exploring the effect of specific anti-inflammatory compounds, such as BIIL 284, an antagonist of
the leukotriene B4 (LTB4)-receptor.18 LTB4 is a product of activated neutrophils and macrophages, and
once it makes a complex with its receptor, it triggers NF-κB-dependent inflammatory responses.18 BIIL
284 had previously caused adverse pulmonary reactions when given to CF patients. Therefore, Döring
et al. examined the effect of BIIL 284 treatment in mice infected intratracheally by injection with the
P. aeruginosa strain PAO1 embedded in agar beads.18 Interestingly, BIIL 284 treatment led to decreased
numbers of neutrophils, and consequently increased bacterial numbers, in the mouse lungs.18 These
observations were accompanied with strong presence of bacteria in the blood of the treated mice, com-
pared with the untreated animals, indicating the important role of neutrophils in controlling bacterial
lung infection and preventing sepsis.18 The data also show the importance of mouse infection models for
testing anti-inflammatory drugs before further assessment in clinical trials. Although most studies
are focused on a single pathogen each time, CF disease is more complicated and often includes a number
of different pathogens. Accordingly, the agar beads model of chronic lung infection has also been used
to investigate the interaction between strains of two different opportunistic pathogens, P. aeruginosa
and Burkholderia cenocepacia, in the mouse lung.8 This coinfection increases the inflammatory
response, as compared to the single infections, without any increase in the bacterial load, indicating that
P. aeruginosa interacts with other bacterial species to increase bacterial virulence.8

25.2.2 Acute Lung Infection Mouse Models

Acute microbial lung infection, which mimics the human acute bacterial pneumonia, can occur in mice
upon exposure to infectious aerosols or directly by intranasal or intratracheal instillation.19 Intranasal
infection allows the spreading of the bacteria from the upper airways to the intestine and the lower air-
ways.20 This protocol has been widely used for P. aeruginosa virulence factor assessment. Nevertheless,
intratracheal instillation delivers much more bacteria into the distal bronchi.20 Both P. aeruginosa acute
lung infection and intratracheal instillation murine models can be used to gain insights about the immune
responses and the lung function of the infected animals.21 In mice infected intratracheally with P. aeru-
ginosa, there is a correlation among increased interleukin-6 (IL-6) expression, edema formation, and
decreased lung function.21 In addition, the proinflammatory cytokine IL-17 facilitates the recruitment of
neutrophils in the infected lung areas of infected mice.22 On the other hand, immunosenescence leads to
impaired neutrophil response, as observed in aged versus young mice subjected to intratracheal infec-
tion.23 Septic mice infected intratracheally lead to the induction of IL-27, which in turn induces immuno-
suppression.24 Toward standardization of new therapeutic approaches against human P. aeruginosa lung
infections, Lawrenz et al. proposed recently a leukopenic (cyclophosphamide-treated) mouse model of
lung intratracheal instillation for therapeutic testing of novel drugs against multidrug resistant strains.25

25.2.3  P. aeruginosa Induced Gut-Derived Sepsis Mouse Models

P. aeruginosa-induced gut-derived sepsis models mimic the pathophysiology of humans, because they
involve intestinal colonization, proliferation, and invasion of other host tissues.26 In order to produce
murine gut-derived sepsis, mice receive bacteria in their drinking water as well as antibiotics e.g., ampi-
cillin and streptomycin for a few days. Antibiotics disrupt the intestinal flora of the mice enabling gut
colonization with P. aeruginosa.27 To facilitate translocation of P. aeruginosa away from the gut, the
immunosuppressant cyclophosphamide is administered during infection.27 Interestingly, intestinal colo-
nization with P. aeruginosa is reduced by a major member of probiotic bacterial species, Bifidobacterium
longum strain BB536, which inhibits P. aeruginosa adherence to the intestinal epithelial cells in a murine
model of gut-derived sepsis, encouraging its further assessment as a probiotic for immunocompromised
378 Laboratory Models for Foodborne Infections

patients.28 Moreover, in IL-1-deficient mice, P. aeruginosa load and the inflammatory response are sig-
nificantly higher in the liver during gut-derived sepsis.29 This effect is reversed when mice are treated
with the bacteriophage strain KPP10, as compared with the phage-untreated mice.27
Another way to induce lethal gut-derived sepsis is by surgical stress (30% hepatectomy).30 Following
surgical hepatectomy in mice, P. aeruginosa expresses PA-I lectin/adhesin in the intestine of the ani-
mals, indicating that pathogens may sense host stress and respond by expressing specific virulence effec-
tors that promote lethal sepsis.30 The PA-I lectin contributes to damaging the intestinal epithelium barrier
by compromising enterocyte tight junctions.31 Moreover, P. aeruginosa senses low phosphate (Pi) levels
in the mouse intestine following surgical hepatectomy, promoting lethal gut-derived sepsis.32 Similarly,
mice subjected to intestinal ischemia/reperfusion injury exhibit P. aeruginosa PA-I lectin-dependent
translocation from the cecum to other organs including liver, lung, and kidney causing lethal sepsis.33
These, and other studies, suggest that P. aeruginosa exhibits enhanced virulence upon stress, surgery,
and trauma, all of which may promote intestinal pathologies and systemic bacterial spreading.7,30–33

25.2.4 Burn- and Open-Wound Infection Mouse Models

The P. aeruginosa-infected burn-wound sepsis model is used to mimic the human burn wound sepsis.9,34
In 1975, Stieritz and Holder developed a nonlethal thermal injury to examine the pathogenesis of
P. aeruginosa infection by injecting viable bacteria into the burn skin area.34 Injection in the burn
area caused rapid sepsis,34 systemic inflammatory response syndrome, and multiple organ dysfunction
syndrome.35 This model allows the investigation of the pathogenicity of various P. aeruginosa strains
and the identification of virulence factors. For example, Rumbaugh et al. demonstrated that the single
quorum-sensing mutants, lasI, lasR, rhlI, exhibited reduced virulence compared to the wild-type PAO1
strain, while the double-mutant lasI rhlI was even more attenuated in virulence, suggesting the important
role of quorum sensing in virulence in this infection model.9 Additionally, the burn-wound sepsis model
is useful for testing antibacterial agents and evaluating treatments for P. aeruginosa-infected burn
patients.36
P. aeruginosa fragellin is a structural component of flagella and a potent immunostimulant. Barnea
et al. examined the effect of anti-fragellin subtype A monoclonal antibody (anti-fla-a) in a P. aeruginosa-
infected burn-wound mouse sepsis model.37 Anti-fla-a reduced the mortality and morbidity of the
infected mice, showing that targeting fragellin protein can be a promising approach for the treatment
of P. aeruginosa-infected burns.37 Moreover, negative-pressure wound therapy may prevent sepsis and
decrease mortality by inhibiting the invasion and proliferation of P. aeruginosa in the injured tissue of
burn-wound septic mice.38
In addition to burn-wound, open wound infection models can be used to evaluate and treat infections
in the absence of severe systemic stress caused by burns. It involves the removal of ∼1 cm2 of skin from
the mouse back and the application of luminescent or GFP-expressing P. aeruginosa cells that can be
followed longitudinally along with the assessment of mouse survival.39,40 In a pivotal study, glutathione
S-transferase A4 (GstA4), a detoxification enzyme against lipid peroxidation byproducts, was found
downregulated in human and mouse muscles following burns, while low muscle expression postburn in
humans predicted their susceptibility to infection.41 Moreover open wound infection with P. aeruginosa
of wild-type and GstA4 mutant mice shows that the mutant mice are more susceptible to infection, indi-
cating the usefulness of the open-wound model to pinpoint genes relevant to both burn- and open-wound
infections.41

P. aeruginosa Keratitis Models (Mice, Rabbits, and Guinea Pigs)

25.2.5 
Keratitis is a disease of the cornea that can be due to the infection with various microbes, including
bacteria.42 The characteristics of bacterial keratitis include inflammation with concomitant pain and
redness.42 P. aeruginosa is a common cause of bacterial keratitis in humans,42 but it can also infect
the cornea of other mammals including mice, rabbits, and guinea pigs. For example, the guinea pig
is a model for evaluating not only antimicrobial drugs, identifying treatments,43–48 and studying the
Pseudomonas aeruginosa 379

pathogenesis of P. aeruginosa keratitis but also host defense.49–51 Rabbits, on the other hand, have large
eyes similar in size to those of humans and can be used to evaluate several parameters of the disease.42
The strain most commonly used to model bacterial keratitis and the efficacy of multiple treatments
against P. aeruginosa is the New Zealand white rabbit.42,52–59 For example, Chen et  al. demonstrated
in two separate studies that lithium chloride (LiCl) and β-catenin promote host resistance against
P. aeruginosa keratitis by reducing the inflammatory responses of the host and by decreasing the
bacterial burden.60,61
Moreover, P. aeruginosa elastase B, protease PASP, MucD, and, to a lesser degree, alkaline protease
contribute significantly to the pathogenesis of keratitis.62–64 Furthermore, induction of the triggering
receptor expressed on myeloid cells-2 (TREM-2) upon infection in cornea scrapes triggers PI3K/Akt
signaling to confer resistance against P. aeruginosa infection.65 In contrast, induction of myeloid-related
protein-8 (MPR8) and MRP14 upon infection in cornea scrapes, despite promoting bacterial clearance,
induces inflammation and concomitant susceptibility to infection.66 Three additional proteins, the extra-
cellular matrix protein Lumican, the Surfactant Protein D (SP-D), and the C-X-C motif chemokine 10
(CXCL10) protect against P. aeruginosa keratitis in mice.67–69

25.2.6 Otitis Media Models (Mice, Rats, Guinea Pigs, and Chinchilla)

Otitis media (OM) includes a group of inflammatory diseases of the middle ear that can be caused by
various conditions including infections by pathogens. Several models have been developed in various
animals, including mice, rats, guinea pigs, and chinchilla, to evaluate P. aeruginosa biofilm formation,
virulence factors, and the role of inflammatory responses and ciprofloxacin-hydrocortisone treatments
against OM.70–75

25.2.7 Zebrafish (Danio rerio)

The zebrafish (Danio rerio) is an attractive vertebrate animal model for studying host–pathogen interac-
tions.5,76 One of its advantages is that, unlike invertebrates, it has an adaptive immune system similar to
that of mammals, although most infection models are based on injecting embryos that only have innate
immune system.5,76 Moreover, zebrafish embryos are transparent, which allows the visualization of bacte-
rial infections in real time by using microbes that express fluorescent proteins.5,76 Rawls et al. took advan-
tage of this transparency to monitor the motility defects of P. aeruginosa flagellar mutants within the
intestine in vivo and in real time and to assess the impact on host immune responses.77 Another important
advantage of zebrafish model is the availability of a wide range of genetic tools that permit generation
of ∼200 progeny following a single mating.5,76 Therefore, zebrafish models could help clarify important
aspects of the host innate immunity upon bacterial infection. Live bacteria of the wild-type P. aerugi-
nosa strains PA14 and PAO1 can kill injected embryos, while quorum sensing and type three secretion
system mutants are attenuated in virulence.76 However, strains significantly attenuated in virulence in late
developmental stages are highly lethal in early embryos that lack pivotal innate immunity mechanisms.76
Furthermore, embryos with reduced CFTR gene expression (Cftr morphants) produce less reactive oxy-
gen species (ROS) in their phagocytes and sustain more bacteria during infection.78 ROS production by
phagocytes is known as respiratory burst response and is an important host defense mechanism. Thus,
Cftr morphants indicate a connection between the CFTR function and the innate immune response.78

25.3 Invertebrate Models
25.3.1  D rosophila melanogaster
Drosophila melanogaster (the fruit fly), despite its small size (∼2 mm in length), is a great invertebrate
model organism that adequately reflects some aspects of the mammalian pathogenesis of infection.79,80
Its short life cycle and easy rearing allows the production of up to ∼50 adult progeny per female fly within
380 Laboratory Models for Foodborne Infections

2 weeks, which facilitates the large-scale in vivo screening of bacterial mutants. Many human bacte-
rial, fungal, and viral infections can be studied in Drosophila.81 Notwithstanding the lack of adaptive
immunity as we know it in mammals, Drosophila has similar innate immunity, disease-related signaling
pathways, and cellular types to those of mammals. Thus, it is a good model for studying the pathogenic-
ity of microbial infections, including those caused by P. aeruginosa.79,80,82
There are three most common methods to infect Drosophila with P. aeruginosa2,82,83: (1) the feeding
method involves mixing of bacteria with the fly food, which causes intestinal colonization and fly lethal-
ity within a few days; (2) the thoracic or abdominal needle pricking infection, that is, an injury being
caused using a tungsten needle dipped into a bacterial suspension. Accordingly, bacteria are introduced
locally at the wound site and later on spread systemically, killing the flies within 2–4 days; and (3) the
injector pumping, which appears similar to the pricking method, but is actually a method of systemic
infection and involves the injection of a controlled dose of bacteria directly into the fly hemocoel with a
thin glass capillary tip.83
The latter two methods have been used to screen D. melanogaster for virulence-related mutants of
the P. aeruginosa strain PA14, for example the virulence-attenuating factor hudA84 and the hypotheti-
cal methyltransferase KerV, which is conserved among Proteobacteria.85 Moreover, NF-κB and JNK
signaling pathways are important for flies to resist P. aeruginosa infection,86 although highly virulent
P. aeruginosa escapes host defenses by suppressing or evading the induction by these pathways that
would normally activate antimicrobial peptides systemically and muscle genes at the wound site.40,87 In
addition, transgenic expression of the human lactonase paraoxonase-1 (PON1) in flies protects them from
P. aeruginosa wound infection by interfering with the bacterial quorum sensing.88 Thus, human innate
immunity factors such as PON1 can be introduced and studied in Drosophila.89
Using the oral infection model, which recapitulates intestinal colonization and systemic dissemination
of P. aeruginosa, new aspects of bacterial quorum sensing and intestinal pathology have been revealed.
For example, the Drosophila chromatin remodeling factor chromo helicase domain protein 1 (CHD1)
contributes to fly intestinal resistance to P. aeruginosa infection90 and the quorum-sensing factor rhlR
contributes to circumvent the fly cellular immune response when bacteria escape the intestine and spread
systemically.91 Actually, both RhlIR and LasIR quorum-sensing systems are required for full virulence
in orally infected flies.92 In addition, intestinal P. aeruginosa senses Gram-positive bacterial peptido-
glycan to enhance its quorum-sensing-mediated virulence.93 Strikingly, intestinal P. aeruginosa and the
quorum-sensing-produced virulence factor pyocyanin induce intestinal stem-cell-mediated regeneration,
which facilitates tumorigenesis in the presence of oncogenes or in the absence of tumor suppressor
genes.94 Moreover, the activation of the JNK innate immune signaling pathway in the adult Drosophila
hindgut cells during P. aeruginosa infection synergizes with Ras1V12 oncogene expression to induce
enterocyte invasion and dissemination to distant sites.95,96

25.3.2  Caenorhabditis elegans

Caenorhabditis elegans is a small (∼1 mm in length) transparent nematode living in the soil that feeds
on bacteria. Its life cycle starts with the embryonic stage, followed by four larval stages (L1–L4) and
adulthood. Its cellular simplicity and its small generation time facilitates screens related to human
pathogens,97 including P. aeruginosa, for the identification of virulence-related genes,98 host defense
factors,99–101 and antimicrobials.102,103
P. aeruginosa can cause different pathologies in C. elegans depending on the culture media it grows
in.104–106 C. elegans dies slower when exposed to P. aeruginosa strain PA14 grown on nematode growth
(NG) media due to the accumulation of the bacteria in the gut of the worms.104 This is known as “slow
killing assay” because C. elegans succumbs after a few days.104 In contrast, the nematode dies within a
few hours when PA14 is cultured in media of high osmotic strength. This is referred to as “fast killing
assay,” according to which worms die as a result of diffusible bacterial toxins in their food rather than
bacterial growth within them.105 P. aeruginosa strain PAO1 quickly paralyzes and then kills C. elegans
by using hydrogen cyanide, a poison that could also inflict tissue damage in cystic fibrosis patients.107
Additionally, the digestive tubes of nematodes fed on PAO1 grown in low-phosphate media become red
Pseudomonas aeruginosa 381

before they die.106 This phenomenon named “red death” occurs due to the activation of three systems:
the phosphate signaling (PhoB), the MvfR-PQS quorum-sensing system, and the pyoverdine iron acqui-
sition system.106 Recently, Kirienko et al. established a liquid-based killing assay to show that pyover-
dine causes hypoxia-related toxicity to C. elegans and that pyoverdine production by the PA14 strain is
necessary for killing the worms.108
Regarding host defense, many factors important for C. elegans innate immunity against P. aeruginosa
were identified within the last decade, including DAF-19, the ortholog of the human RFX, zip-2, a bZIP
transcription factor and a small organic molecule, 2N(3chloro-4methylphenyl)-quinazoline-2,4diamine
(or RPW-24).99–101 Moreover, Conery et al. established a high-throughput screening (HTS) protocol in
C. elegans for the identification of novel anti-infectives against P. aeruginosa.102 Similarly, Zhou et al.
developed an HTS assay for secondary metabolites of endophytic fungi using extracts of medicinal
plants associated with these fungi to identify bioactive molecules that prolong the survival of C. elegans
after P. aeruginosa infection.103 With the caveat that HTS hits may not be validated in other systems and
that extracts do not provide information on specific chemicals, such studies might serve as a starting
point for the discovery of novel therapeutics.

25.3.3  D ictyostelium discoideum

Dictyostelium discoideum is a slime mould109 with two remarkable multistage life cycles.110,111 One is
the asexual cycle, known as social cycle, characterized by the formation of fruiting bodies that release
spores.110,112 Spores give rise to haploid amoebae, which need to feed on bacteria to undergo mitosis.110
Starving amoebas aggregate, forming new fruiting bodies. If starvation is combined with darkness and
humidity, the sexual cycle starts with the fusion of two haploid amoebas of the opposite mate types, which
attract and cannibalize surrounding cells forming a macrocyst that releases recombined new amoe-
bas.110,113 Dictyostelium cells naturally live in forest soil, and, by obligingly feeding on bacteria, they can
be a natural host of pathogenic bacteria. Thus, they can serve as a great model organism for studying the
mechanisms of bacteria–phagocyte interaction. Indeed, D. discoideum has been used to investigate the
virulence of many human bacterial pathogens, including P. aeruginosa.114 For example, the wild-type
P. aeruginosa strain PAO1 inhibits D. discoideum growth, while rhl quorum-sensing system is required
for full virulence.115 Interestingly, the P. aeruginosa strain PA14 is more virulent than PAO1 against
D. discoideum.116 This is probably because 169 genes are differentially expressed between the 2 strains.116
Of note, a random mutagenesis screen of the P. aeruginosa strain 22D10 identified anthranilate phospho-
ribosyltransferase gene trpD to be important for quorum-sensing function and the siderophore pyochelin
genes pchH and pchI for the induction of the type III secretion system. Importantly, trpD, pchH, and
pchI mutants are also attenuated in virulence in the Drosophila pricking and feeding assays and the
mouse lung acute infection assay.117

25.3.4  Galleria mellonella (Wax Moth)

The greater wax moth, the lepidopteran Galleria mellonella, is a widely used model host for investigat-
ing microbial pathogenesis.118,119 Similar to other insects, its life cycle progresses from egg to larva, pupa,
and, finally, adult (moth).119 The larvae of G. mellonella are relatively large in size (1–3 cm), facilitating
the injection of bacterial pathogens and antimicrobial compounds.118,119 An additional asset compared
to other invertebrate model hosts is that it survives at the physiologic mammalian temperature (37°C),
which favors the growth of many human pathogens.3
While G. mellonella can be infected orally by feeding, most infection studies use the injection
method.118–120 For example, Andrejko et al. have shown that different strains of P. aeruginosa, including
two clinical isolates induce innate immunity genes to different extents when injected into hemolymph.121
Additional data show that injected P. aeruginosa escapes the cellular immune responses of G. mellonella
larvae by causing destruction of their hemocytes.122 The virulence factors responsible for this strategy
are still unknown. However, other studies implicate the P. aeruginosa metalloproteinase elastase B in
virulence and counteraction of G. mellonella immune responses.123,124
382 Laboratory Models for Foodborne Infections

25.3.5  B ombyx mori (Silkworm)

The Silkworm, Bombyx mori, is the best-known species of the lepidopteran superfamily Bombycoidea.
The life cycle of this insect mainly consists of four developmental stages: egg, larva, pupa, and adult.125
Numerous immunological, microbiological, and pharmacological studies of many pathogens have been
carried out using this simple model showing, for example, that the overactivation of innate immunity in
silkworms induces tissue damage followed by host death, resembling sepsis-induced multiorgan failure in
humans.126 Moreover, P. aeruginosa mutants and toxins, namely, pyocyanin, gacA, superoxide dismutase
(sodB and sodM), nitric oxide (NO)-detoxification enzymes (NO reductase and flavohemoglobin), exotoxin A,
and pyoverdine pvdE, have been assessed for virulence against silkworm larvae.127–133 Among those, pvdE
induces ExoS production, which is a bifunctional protein with GAP and ADP-ribosyltransferase activity
that facilitates the translocation of the bacteria from the lumen to the hemolymph.132 Interestingly, the
ADP-ribosyltransferase activity of ExoS acts on mouse lung pneumocytes to disrupt the pulmonary–
vascular barrier during P. aeruginosa acute pneumonia, leading to bacterial dissemination.134

25.4 Plants
Arabidopsis thaliana is a popular model plant due to its small size, its short life cycle of only 6 weeks—
required for each seed to germinate into a mature plant and produce ∼5000 new seeds—and the large number
of mutant lines and genetic tools available.135 It is also a model host for studying various aspects of infection,
including host immune responses and microbial virulence strategies.4 P. aeruginosa pathogenicity against
A. thaliana involves various steps.136 Infection starts with the syringe-mediated application of the bacteria
on the leaf surface. The bacteria then attach and congregate and enter the plant tissues via stomatal open-
ings.136 Next, bacteria proliferate locally in the substomatal cavity and the intercellular space and destroy
the local plant cells before dispersing systemically.136 P. aeruginosa strains PA14 and PAO1 can infect the
roots of both A. thaliana and sweet basil (Ocimum basilium), killing these plants within a week.137 At the
initial stage of infection, the two strains form biofilm structures on the root surfaces of the plants, which is
reminiscent of the condition of the lungs of cystic fibrosis patients.137 Biofilms confer antibiotic resistance
and persistent pathogenicity to the organism. Accordingly, the antimicrobial compound rosmarinic acid,
a multifunctional caffeic acid ester secreted from the sweet basil root, shows some in vitro activity only
against P. aeruginosa strains unable to form biofilms.137 Also, A. thaliana root development is inflicted by
the multihost P. aeruginosa virulence factor pyocyanin, which induces the production of ROS and sub-
sequent A. thaliana ethylene-dependent signaling.138 The disaccharide trehalose of P. aeruginosa strain
PA14 is a virulence factor that promotes pathogenesis only in A. thaliana and not in other hosts including
mice.139 However, He et al., demonstrated that PA14 carries two pathogenicity islands, PAPI-1 and PAPI-2,
that harbor virulence genes, 11 of which are necessary for full virulence in both A. thaliana and in mice.140
The majority of those genes are present in P. aeruginosa clinical isolates,140 indicating that PA14 could
survive in evolutionarily diverse hosts by using conserved functions. Accordingly, Starkey and Rahme
have published infectivity protocols of A. thaliana and lettuce for screening P. aeruginosa bacterial strains
to identify virulence factors potentially conserved for pathogenicity against other hosts.141 A high through
put (HTP) P. aeruginosa-infection system of Arabidopsis seedlings was also proposed by Gopalan and
Ausubel for the discovery of potent anti-infective agents.142 Furthermore, the A. thaliana pathogenicity
model was used for the identification of host defense mechanisms and factors that repress P. aeruginosa
virulence. For example, plant-derived salicylic acid, which is a phenolic metabolite, attenuates P. aerugi-
nosa virulence against A. thaliana and C. elegans by downregulating the production of several virulence
factors, including pyocyanin, protease, and elastase and by reducing biofilm formation.143 Remarkably,
Cheng et al., discovered a novel A. thaliana immune signaling pathway, namely, a mitogen-activated pro-
tein kinase (MAPK) cascade that is activated by proteases secreted by P. aeruginosa.144 Nevertheless,
P. aeruginosa has developed mechanisms to escape from the immune system of both plants and mammals.
For example, P. aeruginosa alkaline protease AprA cleaves the bacterial fragellin monomers, which are
ligands of pattern-recognition receptors in both plants and mammals, preventing the recognition and clear-
ance of the bacteria from the host.145
Pseudomonas aeruginosa 383

25.5 Conclusions
While all described models are useful, none is ideal. The main asset of mammalian models lies in
the close recapitulation of the human disease, especially regarding host response. Nevertheless, zebraf-
ish and invertebrate models also mimic, to a lesser degree, the pathophysiology of systemic, wound,
or intestinal/barrier epithelial infections. All models, including plants, are able to elucidate aspects of
host defense, including adaptive and innate immunity in the case of mammals and innate immunity in
the case of all the rest. Importantly, all models are suitable for screening P. aeruginosa virulence fac-
tors. As different models may reflect different aspects of infections, involve different virulence factors
and require different treatments may be relevant in each case, it is not clear if some models are always
superior to the others. Therefore, it is valuable to use as many different models of infection as possible
in order to generate the strongest evidence for the relevance of a P. aeruginosa gene, and to provide the
soundest rationale for the implementation of an anti-P. aeruginosa measure.

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26
Salmonella

Dongyou Liu

CONTENTS
26.1 Introduction....................................................................................................................................391
26.1.1 Classification, Morphology, and Genomics..................................................................... 392
26.1.1.1 Classification..................................................................................................... 392
26.1.1.2 Morphology....................................................................................................... 392
26.1.1.3 Genomics.......................................................................................................... 393
26.1.2 Biology and Epidemiology............................................................................................... 393
26.1.3 Clinical Features and Pathogenesis.................................................................................. 394
26.1.3.1 Clinical Features............................................................................................... 394
26.1.3.2 Pathogenesis...................................................................................................... 395
26.1.4 Diagnosis.......................................................................................................................... 395
26.1.4.1 Serotyping and Biotyping................................................................................. 395
26.1.4.2 Virulence and Resistance Gene Profiling......................................................... 395
26.1.4.3 Plasmid Profiling.............................................................................................. 396
26.1.4.4 Epidemiological Tracking................................................................................. 396
26.1.5 Treatment and Prevention................................................................................................. 396
26.1.5.1 Treatment.......................................................................................................... 396
26.1.5.2 Prevention......................................................................................................... 396
26.2 Laboratory Models........................................................................................................................ 397
26.2.1 Animal Models................................................................................................................. 397
26.2.1.1 Mice.................................................................................................................. 397
26.2.1.2 Guinea Pigs....................................................................................................... 398
26.2.1.3 Rabbits.............................................................................................................. 398
26.2.1.4 Calves................................................................................................................ 398
26.2.1.5 Chicken............................................................................................................. 398
26.2.1.6 Nonhuman Primates......................................................................................... 398
26.2.1.7 Zebrafish........................................................................................................... 398
26.2.1.8 C . elegans Nematode........................................................................................ 398
26.2.2 In Vitro Models................................................................................................................. 398
26.3 Conclusion..................................................................................................................................... 399
References............................................................................................................................................... 399

26.1 Introduction
Salmonella is a Gram-negative bacterium that was first observed in the Peyer’s patches and spleens of
typhoid patients by Karl Eberth in 1880, and the first culture was obtained by Georg Theodor Gaffky in
1884. Following the isolation of an organism (then called “hog-cholera bacillus”) from the intestine of a
pig by Theobald Smith and Daniel Salmon in 1885, the name Salmonella enterica (Choleraesuis) was pro-
posed by Joseph Leon Lignières in 1900 to honor the contribution by the Daniel Salmon’s group. In 1892,

391
392 Laboratory Models for Foodborne Infections

a pathogen (termed Bacillus typhimurium, now known as nontyphoidal Salmonella typhimurium) asso-
ciated with typhoid fever-like disease in mice was isolated by Friedrich Loeffler. Since then, a large
number of Salmonella serovars have been identified and their roles in the causation of human restricted,
invasive, and systemic disease (typhoid fever) and noninvasive gastroenteritis (nontyphoidal Salmonella
infection) have been clearly determined.

26.1.1 Classification, Morphology, and Genomics

26.1.1.1 Classification
The genus Salmonella encompasses a large group of Gram-negative, rod-shaped, facultative anaer-
obes in the f­ amily Enterobacteriaceae, order Enterobacteriales, class Gammaproteobacteria, phylum
Proteobacteria, and domain Bacteria. Besides Salmonella, the family Enterobacteriaceae includes sev-
eral medically important pathogens (e.g., Klebsiella, Shigella, and Yersinia).
Based on their differences in genome sequences, two species (Salmonella bongori and Salmonella
enterica) are recognized in the genus Salmonella. While only one subspecies (serotype V) exists in the
S. bongori species (which is found mainly in cold-blooded animals such as reptiles, but may be occa-
sionally involved in human infection), six subspecies [i.e., enterica (serotype I), salamae (serotype II),
arizonae (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI)] are identified in the S. enterica spe-
cies, with the subspecies enterica affecting warm-blooded animals (e.g., mammals) and other subspecies
being specific for cold-blooded animals (e.g., reptiles).
Upon examination of the somatic (O) and flagellar (H) antigens on the bacterial surface using sero-
logical techniques, 2600 serovars are identified in the S. enterica species. Additionally, according to
their host specificity and disease manifestations in humans, Salmonella serovars are separated into two
categories: typhoidal and nontyphoidal. Whereas typhoidal Salmonella serovars (TS) demonstrate host
specificity and cause invasive bacteremia only in humans, nontyphoidal Salmonella serovars (NTS) are
present ubiquitously in the environment and cause noninvasive, self-limiting, cross-species gastrointes-
tinal disease.
It is noteworthy that S. enterica subsp. enterica comprises >2000 genetically similar serovars and
accounts for almost all Salmonella infections in humans and animals. Belonging to the TS category,
S. enterica subsp. enterica serovars Dublin (bovine), Choleraesuis (swine), and Typhi (abbreviated to
S.  Dublin, S. Choleraesuis, and S. Typhi, respectively) are host-specific and responsible for systemic
(invasive) disease in immunocompetent hosts. Indeed, S. Typhi induces typhoid fever in humans (and
higher primates), but not in any other mammals. On the other hand, those of the NTS category, S. enterica
subsp. enterica serovars Typhimurium and Enteritidis (abbreviated to S. Typhimurium and S. Enteritidis,
respectively), produce a localized (noninvasive) infection in the intestine (gastroenteritis) of humans and
various animal species (e.g., rodents, cattle, and mammals) and are occasionally linked to severe sys-
temic disease in immune-deficient individuals [1].

26.1.1.2 Morphology
Salmonella is rod-shaped, non-spore-forming, predominantly motile enterobacterium of approximately
0.7–1.5 μm in diameter and 2–5 μm in length, with peritrichous flagella around the entire bacterial sur-
face. Typically, Salmonella forms transparent and colorless colonies of 1–3 mm, sometimes with dark
center, on MacConkey agar (in mixed culture, Salmonella colonies tend to clear areas of precipitated
bile produced by other organisms); red colonies with or without black centers on xylose lysine desoxy-
cholate (XLD) agar; blue-green to blue colonies with or without large, glossy black centers on Hektoen
enteric (HE) agar; and brown, gray, or black colonies on bismuth sulfite (BS) agar, with surrounding
medium being brown at first, and turning black with longer incubation, producing the so-called halo
effect. Sometimes, Salmonella may produce atypical yellow colonies with or without black centers on
HE and XLD agars or green colonies with little or no darkening of the surrounding medium on BS agar.
Interestingly, colonies formed by Proteus may appear similar to those of Salmonella; however, Proteus
colonies may swarm on blood agar (trypticase soy agar + 5% sheep blood), while Salmonella colonies
Salmonella 393

do not. Apart from Gallinarum and Pallorum serovars, most Salmonella isolates produce motile colo-
nies. It is notable that nonmotile Salmonella primary cultures may switch to the motile phase using a
Craigie tube or ditch plate. As a chemoorganotroph, Salmonella generates energy from organic sources
via oxidation and reduction reaction, and has the capacity to survive with or without oxygen (facultative
anaerobe). Salmonella is positive for catalase, nitrate reduction, methyl red, acid production from glu-
cose, mannitol, and d-mannose [1].

26.1.1.3 Genomics
The genome of S. bongori is 4.46 Mb in size, with GC content of 51.3%, and 4010 protein-coding genes.
This species possesses only a basic set of ancestral Salmonella virulence functions and lacks several
S. enterica-defining metabolic pathways. However, the S. bongori genome contains genes encoding novel
effector proteins (sboA-L) that may have originated from enteropathogenic Escherichia coli (EPEC).
Thus, by inheriting the ancestral Salmonella virulence gene set, and incorporating virulence determi-
nants that resemble those in EPEC, S. bongori may represent an evolutionary link between E. coli and
S. enterica [2].
The genome of typhoid-fever-causing S. Typhi strain CT18 consists of a 4.81-Mb chromosome (with
GC content of 52%) and two plasmids [including a 218-kb multiple-drug-resistance (MDR) incH1 plas-
mid (pHCM1), and a 106-kb cryptic plasmid (pHCM2, possibly derived from a virulence plasmid of
Yersinia pestis)]. Within the chromosome, hundreds of insertions and deletions (ranging in size from
single genes to large islands), 4455 coding genes, and 233 pseudogenes (with some corresponding to
those related to virulence in S. Typhimurium) are found [3].
The genome of gastroenteritis and food-poisoning-inducing S. Typhimurium strain LT2 is composed
of a 4.86-Mb chromosome (with GC content of 52.2%) and a 94-kb virulence plasmid (pSLT). Within the
chromosome, 4631 coding genes and 54 pseudogenes are identified. Compared to those in the S. typhi
genome, 11% of the coding genes in the S. Typhimurium LT2 genome are unique [4].
Salmonella serovars harbor large discrete genomic islands that contain prophage elements and spe-
cialized loci termed Salmonella pathogenicity islands (SPIs). Many Salmonella-specific functions [e.g.,
complete type III secretion systems encoded by SPI-1 (T3SS-1) and SPI-2 (T3SS-2)] may be traced to
the genes located in these large discrete genomic islands, and some of these functions may have been
acquired by S. enterica after splitting from S. bongori. For example, S. bongori lacks SPI-2 (essential for
optimal replication within macrophages), SPI-6 (encoding a type VI secretion system), SPI-13 (required
for survival in chicken macrophages), SPI-14 (encoding an electron transport system), and SPI-16
(bacteriophage remnant carrying genes associated with LPS modification), all of which are present in
S. enterica [2].

26.1.2 Biology and Epidemiology

As a facultative intracellular pathogen, Salmonella usually enters the host via ingestion of contaminated
food. After enduring gastric acidity in the stomach, the surviving Salmonella migrates to the small
intestine and infects epithelial and M cells of Peyer’s patches, in which Salmonella is phagocytosed by
macrophages and dendritic cells and then disseminated to the organs of the reticuloendothelial system
(RES) (spleen, liver, bone marrow, etc.) via the lymphatic system and blood. Salmonella replication takes
place in macrophages within the spleen and liver. Additionally, in the event of intravascular hemolysis,
Salmonella can replicate in neutrophils and reach high concentrations in the blood. Inside macrophages,
Salmonella often forms an intracellular vacuole (termed as Salmonella-containing vacuole or SCV),
which gets juxtaposed to the nucleus by utilizing the microtubule meshwork of the host cell and derives
nutrition from the Golgi apparatus. The endotoxins released from the dead salmonellae induce enteritis,
gastrointestinal disorder, systemic febrile illness, and ultimately death [1].
Salmonella is commonly found in various water sources (which act as reservoirs), and also in the
digestive tracts of humans and animals (especially reptiles) [5]. Feces from infected animals or people
represent an important source of water or food contamination. Salmonella on the skin of reptiles or
394 Laboratory Models for Foodborne Infections

amphibians may be passed to animal handlers. Salmonella may persist in a contaminated bathroom for
weeks, and tolerates freezing, although it is sensitive to UV light and heating at 55°C for 90 min, or 60°C
for 12 min.
Both typhoid and nontyphoid Salmonella serovars are distributed throughout the world. The etio-
logic agents of typhoid and paratyphoid fever, S. Typhi and S. Paratyphi A and B (and occasionally C),
collectively known as the enteric fever serovars, thrive in places with a limited sanitation infrastructure
that permits transmission between infected humans without involvement of animal hosts. With the abil-
ity to spread to the deeper tissues of humans (liver, spleen, and bone marrow), S. Typhi [serologically O
(lipopolysaccharide) type 09, 012; H (flagellin) type d; and Vi (extracellular capsule) positive] is respon-
sible for a serious invasive bacterial disease of humans, with an annual global burden of approximately
22 × 107 infections and >200,000 deaths [1,6].
Although infection with NTS usually occurs after ingestion of foods that contain a high concentra-
tion of bacteria, infection through inhalation of bacteria-laden dust is possible in infants. Important
nontyphoidal Salmonella serovars include S. Typhimurium and S. Enteritidis, which are associated with
rapid-onset gastroenteritis as well as serious systemic/invasive diseases (e.g., bacteremia, meningitis)
in immune-deficient individuals, young children, HIV-positive individuals, or patients who happen to
be coinfected with malaria worldwide. In industrialized countries, NTS infection in young infants or
the elderly may sometimes lead to severe invasive diseases accompanied by high case fatality rates.
In the sub-Saharan Africa, NTS diseases are associated with severe invasive disease in the absence of
gastroenteritis [7,8].

26.1.3 Clinical Features and Pathogenesis

26.1.3.1 Clinical Features
Salmonella infection (salmonellosis) in humans is associated with gastrointestinal tract infection, enteric
fever, bacteremia, local infection, and the chronic reservoir state. The initial nonspecific symptoms
include fever, general weakness, and myalgia. Once the bacteria spread to other parts of the body (bacte-
remia), localized infections (e.g., arthritis, urinary tract, central nervous system, and bone and soft-tissue
infections) or abscesses may appear. Along with deterioration of the function of reticular endothelial
cells (after acute salmonellosis or bone infection), back pain or spondylosis may emerge [1].
With an incubation period of about 2 weeks, typhoid fever caused by Salmonella Typhi, Paratyphi A,
Paratyphi B and Paratyphi C is an invasive disease that tends to occur in immunocompetent indi-
viduals. Its main symptoms include fever, headache, a slowed heart rate (bradycardia), and sepsis, but
diarrhea is largely absent [9]. During the infection, Salmonella goes through the lymphatic system
of the intestine into the blood (typhoid form) and migrates to various organs (liver, spleen, kidneys)
to form secondary foci (septic form). Histologically, inflammatory infiltrates in the intestine are pre-
dominantly mononuclear cells, with few neutrophils. The bacterium is found in histiocytic granulo-
mas (or typhoid nodules) located in the bone marrow, the liver, and the spleen. In a small proportion
(approximately 4%) of patients, the bacterium remains in the gallbladder for a period of 1 year after
disease resolution. These chronic carriers (or “typhoid Marys”) can transmit the disease for the rest
of their lives [1,10,11].
Often linked to food poisoning, noninvasive, NTS infection due to S. Typhimurium or S. Enteritidis is
characterized by acute enterocolitis (enteric fever) and gastroenteritis (inflammatory diarrhea), mainly
involving immunocompetent individuals in developed countries. With a short incubation period (<1
day), NTS gastroenteritis is localized in the terminal ileum, colon, and mesenteric lymph node, leading
to diarrhea, fever, and intestinal inflammatory infiltrates (composed of neutrophils). However, there is
evidence that NTS infection may be invasive, presenting with fever, bacteremia, hepatosplenomegaly,
and respiratory signs, often without gastroenteritis, involving immunocompromised individuals (as
well as those suffering from malnutrition, anemia, and severe malaria), as noted in sub-Saharan Africa.
Asymptomatic carriage of NTS may occur infrequently (0.15% in healthy adults, 3.9% in children);
therefore, the role of asymptomatic carriers in the maintenance and spread of the disease cannot be
ignored [1].
Salmonella 395

26.1.3.2 Pathogenesis
Typhoidal Salmonella serovars breach the intestinal epithelial barrier via phagocytosis and elicit intesti-
nal inflammatory infiltrates consisting primarily of mononuclear cells. After entry into macrophages via
macropinocytosis, typhoidal Salmonella serovars disseminate via the mononuclear phagocyte system
(a network of connective tissue containing immune cells) to other parts of the body, including the liver,
spleen, and bone marrow. During the systemic phase of typhoid fever, Salmonella may transfer from the
liver into the gallbladder, resulting in chronic carrier state [1,12].
On the other hand, NTS serovars enter into M cells through bacterial-mediated endocytosis, which is
accompanied by intestinal inflammation (with a massive influx of neutrophils into the terminal ileum
and colon). Gaining a growth advantage in the inflamed gut due to their ability to respire on tetrathionate
and to grow anaerobically on ethanolamine, NTS serovars disrupt tight junctions between the cells of
the intestinal wall, leading to the increased traffic of ions, water, and immune cells into and out of the
intestine. A combination of inflammation and the disruption of tight junctions ultimately contributes to
vomiting and diarrhea [13].
Salmonella genomes contain many virulence and antimicrobial resistance genes that play crucial parts
in host cell invasion (bapA, siiE, sopB), motility (fliC), intracellular survival (sseF, sseG), plasmids
(spvB, spvC), and ion acquisition (corA, mgtA, mgtB). The T3SS (encoded by genes in pathogenicity
islands SPI-1 and SPI-2) aids the invasion of nonphagocytic cells, colonization of the intestine, induc-
tion of intestinal inflammatory responses and diarrhea, survival and proliferation in macrophages, and
establishment of systemic disease [14–16].

26.1.4 Diagnosis
Conventional laboratory identification of Salmonella organisms is based on phenotyping methods that
assess their morphological, biochemical, and serological characteristics. In particular, the serotyping
technique (targeting somatic and flagellar antigens on the bacterial surface) separates Salmonella into
>2600 serovars in the White–Kauffmann–Le Minor scheme. This provides a useful tool to diagnose
and track Salmonella implicated in outbreaks, despite its obvious limitations (e.g., slow turnaround, skill
demand, and result variability, especially in dealing with rough, monophasic, and nonmotile strains). In
addition, on the basis of their ability to ferment certain substrates, Salmonella strains and serovars (e.g.,
Typhi, Paratyphi A, Paratyphi B, Typhimurium) can be biotyped via the phenotypic lead acetate test [17].
With the ongoing development and refinement in molecular detection technologies, a variety of highly
efficient and specific procedures have become available for identification, subtyping, and tracking of
Salmonella strains. These include serotyping and biotyping, virulence and resistance gene profiling,
plasmid profiling, and epidemiological tracking of clonality and genetic relatedness [1,18].

26.1.4.1 Serotyping and Biotyping

Polymerase chain reaction (PCR) detection of genes encoding surface antigens (e.g., flagellar antigens)
offers a rapid and accurate alternative to conventional serotyping for determination of Salmonella
strains [19]. Similarly, PCR-based methods allow rapid and reliable identification of S. paratyphi B and
other biotypes.

26.1.4.2 Virulence and Resistance Gene Profiling

PCR amplification of virulence-associated genes (e.g., invA and spvC from pathogenicity islands that
are involved in adhesion, invasion, intracellular survival, colonization, and systemic infection) facilitates
detection, characterization, and monitoring of Salmonella serovars, subtypes, and strains [20]. Further,
PCR detection of genes associated with antimicrobial resistance (e.g., aac, aad, aph, strA/B, blaTEM,
blaCMY, sulI, tet(A,B,C,D), dfrA, etc.) enables accurate profiling of resistance to tetracycline, sulfonamide,
streptomycin, nalidixic acid, trimethoprim–sulfamethoxazole, ampicillin, chloramphenicol, cephalo-
thin, kanamycin, ciprofloxacin, gentamicin, cefoxitin, amoxicillin–clavulanate, and amikacin [18].
396 Laboratory Models for Foodborne Infections

26.1.4.3 Plasmid Profiling
On the basis of the specific banding patterns of plasmid DNA molecules formed in electrophoretic gel,
plasmid profiling reveals details on the mechanisms and modes of transmission of the virulence or anti-
microbial resistance determinants among Salmonella strains.

26.1.4.4 Epidemiological Tracking
Various gel- and sequence-based genotyping methods may be employed for epidemiological tracking
of Salmonella strain clonality and genetic relatedness. These include pulsed-field gel electrophoresis
(PFGE), PCR-based DNA fingerprinting, multilocus variable-number tandem repeat analysis (MLVA),
multilocus sequence typing (MLST), next-generation sequencing (NGS), etc. [18].
PFGE involves digestion of bacterial chromosomal DNA with a rare cutting enzyme (XbaI or AvrII)
and electrophoretic separation of digested DNA fragments using an alternating electric current (pulsed
field), resulting in a distinct pattern for each Salmonella strain. With sufficiently high discriminative
power, PFGE remains the reference method for subtyping Salmonella organisms.
PCR-based DNA fingerprinting methods examine the genomic contents of Salmonella organisms,
with a goal to differentiate among strains and identify the source of the outbreaks. The commonly
used methods include random amplified polymorphic DNA (RAPD), enterobacterial repetitive inter-
genic consensus (ERIC)-PCR, repetitive extragenic palindromic sequences (REP)-PCR, and PCR-based
single-nucleotide polymorphism (SNP) typing.
MLVA assesses the variability (in terms of nucleotide sequence and unit size) of the genetic entity
called variable number tandem repeat (VNTR), which is present at multiple loci in the Salmonella
genome. MLVA subtyping of Salmonella strains demonstrates serovar specificity, in comparison with
PFGE, which is applicable to all serotypes.
MLST exploits the polymorphisms in selected housekeeping genes (aroC, dnaN, hemD, hisD, purE,
sucA, and thrA) among Salmonella strains to determine the sequence types of the organism. MLST sub-
typing represents a robust technique for epidemiological and evolutionary analyses of Salmonella strains.
PCR-based SNP typing is capable of differentiating Salmonella enteritidis strains that are indistin-
guishable by PFGE and phage typing.
NGS is a high-throughput genome sequencing technique that generates whole-genome sequences of
Salmonella strains in a timely and cost-effective fashion. NGS has proven superior to PFGE for subtyp-
ing Salmonella.

26.1.5 Treatment and Prevention

26.1.5.1 Treatment
Since many patients infected with Salmonella (especially uncomplicated nontyphoidal Salmonella gas-
troenteritis) recover in 3–7 days without treatment, only persons with severe diarrhea may require fluid
and electrolyte replacement. However, antibiotic therapy (10–14 day duration) is recommended for persons
at increased risk of invasive disease, including infants of <3 months of age and individuals with sup-
pressed immune function. Under this circumstance, patients with typhoid fever may be given antibiotics
like chloramphenicol, ampicillin, amoxicillin, and trimethoprim–sulfamethoxazole (if susceptible); or
fluoroquinolones (not approved for persons <18 years of age), cefotaxime, and ceftriaxone as alternatives.
Surgical intervention may be needed for typhoid fever patients with complication of intestinal perforation
or hemorrhage, cholecystitis, endocarditis, arteritis, osteomyelitis, or soft-tissue or spleen abscess. Patients
suffering NTS gastroenteritis may be administered antibiotics ampicillin, amoxicillin, and trimethoprim–
sulfamethoxazole (if susceptible); or ciprofloxacin, chloramphenicol, and ceftriaxone as alternatives [1].

26.1.5.2 Prevention
Proper preparation and cooking of food offers an effective means to prevent the spreading of Salmonella
infection. These include washing hands before preparing food; heating food (chicken and beef) to an
Salmonella 397

internal temperature of 75°C for at least 10 min; avoiding drinking raw (unpasteurized) milk; avoiding
cross-contamination; washing hands with soap after handling reptiles, amphibians, or birds, or after
contact with pet feces.
A number of killed Salmonella whole-cell vaccines (through thermal inactivation, or chemical inacti-
vation with acetone, deoxycholate, or formalin) have been tested in mice. In addition, candidate subunit
vaccines [targeting protein (purified porins, bulk outer membrane proteins, flagellin protein), lipopoly-
saccharide, or O-polysaccharide (OPS)] are being developed. Conjugate vaccines that link Salmonella
OPS to a protein carrier have been shown to enhance the immunogenicity of polysaccharide hapten and
increase functional immunological memory [21–25].

26.2 Laboratory Models
Laboratory models offer valuable approaches for elucidating the pathogenesis of human Salmonella dis-
ease, uncovering insights into immunity to infection, and assessing the efficacy of vaccine preparations.

26.2.1 Animal Models
Due to the fact many Salmonella serovars demonstrate broad host range and that the sources of human
Salmonella infections often come from meat, eggs, and related products, warm-blooded animals of any
kind would be suitable models for foodborne Salmonella enteritis and systemic typhoid [26].

26.2.1.1 Mice
Mouse models of Salmonella infections have a number of obvious advantages, such as relatively low
cost, ready availability, and easy access to host genetics and immunological reagents [27].
Mouse colitis model involves streptomycin pretreatment (which reduces the intestinal microbiota in
mice) followed by oral administration of NTS Salmonella infection, leading to inflammatory colitis and
subsequent systemic infection. Interestingly, different mouse strains demonstrate varied susceptibility
to S. Typhimurium infection, with mouse strain 129sv being resistant and mouse strains C57BL6/J and
BALB/c being susceptible (which lack allele of the Slc11a1 or Nramp1 gene). Mouse colitis model allows
for mechanistic study of intestinal inflammation and Salmonella growth in the intestinal lumen. The
use of this model has helped clarify the role of flagella, T3SS-1, and T3SS-2 in intestinal inflammation
during S. Typhimurium infection; highlighted the contribution of intestinal inflammation to luminal
outgrowth of S. Typhimurium; and revealed the importance of luminal outgrowth of S. Typhimurium to
transmission of the pathogen [28,29].
The mouse typhoid model involves oral administration with NTS serovar S. Typhimurium without
streptomycin pretreatment, since oral challenge of mice with the human host-restricted serovars S. Typhi
and S. Paratyphi does not lead to a productive invasive infection [30]. This oral administration leads to
an invasive, systemic infection (characterized by a predominantly mononuclear leukocyte infiltrate, with
follicular hyperplasia, capillary thrombosis, hemorrhage, and ulcerations at areas of Peyer’s patches
in moribund animals) of the gut-associated lymphoid tissue and RES that resembles typhoid fever in
humans (in terms of entry route, tissue tropism, cellular location of bacteria, immune responses, and
profound hepatosplenomegaly) [31]. Nevertheless, without a capsule, S. Typhimurium does not show any
“stealth” incubation period before the clinical signs and does not express the typhoid toxin and the Vi
capsule polysaccharide (ViCPS) that can reduce toll receptor (TLR)-dependent inflammatory responses
and prevent complement deposition during Salmonella infection. This model has helped verify the role
of SPI-1 effector protein GtgE (present in S. Typhimurium, but absent in S. Typhi) in the proteolyti-
cal inactivation of the Rab29 GTPase, permitting intracellular replication. The absence of this effector
results in the rapid killing of S. typhi inside nonhuman cells [25,28].
Creation of humanized mice provides a sensitive and susceptible model to assess S. Typhi infection that
resembles the pathogenesis of typhoid in humans. The most commonly used humanized mice include
398 Laboratory Models for Foodborne Infections

immunodeficient mice (either Rag2−/− γc−/− or nonobese diabetic scid IL2rγnull) and TLR11-deficient
mice that are engrafted with human hematopoietic stem and progenitor cells (from fetal liver or cord
blood) [26].

26.2.1.2 Guinea Pigs
Guinea pigs represent a useful model for studying intestinal inflammation that results from Salmonella infec-
tion, although preconditioning through starvation and opium treatment is necessary prior to experimentation.

26.2.1.3 Rabbits
Based on New Zealand white rabbits, this model is useful for evaluating immunogenicity and reactoge-
nicity after oral administration of Salmonella serovars. It can also be applied for assessing S. Typhi and
S. Paratyphi A vaccine strains.

26.2.1.4 Calves
Calves infected orally or subjected to surgical ligation of ileal loops are useful for examining the intestinal
inflammation (a severe diffuse infiltrate composed predominantly of neutrophils, necrosis of the upper
mucosa, and formation of a pseudomembrane) and fluid secretion that are associated with S. Typhimurium-
induced gastroenteritis. Application of this model confirmed the ability of S. Typhimurium T3SS-1 in
inducing intestinal inflammation and diarrhea, and the contribution of flagella to neutrophil recruitment
in the intestinal mucosa during S. Typhimurium infection [28,32].

26.2.1.5 Chicken
The chick model has been employed to demonstrate the role of tviA in enhancing S. Typhimurium dis-
semination to internal organs.

26.2.1.6 Nonhuman Primates
Nonhuman primates such as rhesus macaque are susceptible to S. Typhimurium infection, which leads to
gastroenteritis resembling that in humans. Chimpanzees may develop typhoid fever after oral infection
with S. Typhi.

26.2.1.7 Zebrafish
Being strongly susceptible to S. Typhimurium, zebrafish embryos undergo pathological changes (bacte-
remia and proinflammatory response or cytokine storm) within hours after intravenous injection, resem-
bling those seen in humans [33].

26.2.1.8 
C . elegans Nematode
This animal model may be utilized for investigation in Salmonella pathogenicity and host-pathogen
interactions.

26.2.2 In Vitro Models

A number of in vitro cell models based on established cell lines as well as primary cells have been utilized
to unravel the mechanisms of Salmonella entry to the host cells and disease outcome. For example, epi-
thelial cells (HeLa, Caco-2, HT-29, Intestine 407, and primary cells), which engulf Salmonella by mac-
ropinocytosis, provide an excellent platform for determining the essential processes during Salmonella
Salmonella 399

invasion of, proliferation in, and apoptosis of epithelial cells. Resembling M cells (which lack glyco-
calyx) in Peyer’s patches, a combination of Caco-2 cells and Raji B cells helps uncover critical details
to caveolae-mediated endocytosis that is employed by Salmonella in its adhesion and entry into host
reticuloendothelial system before being taken up by macrophages. Macrophages that are found in the
gut-associated lymphoid tissue may be simulated by murine macrophage like cell line RAW 264.7 and
murine macrophages J774-A.1, yielding clues on Salmonella intracellular survival. Primary dendritic
cells (isolated from bone marrow of animal models or healthy humans) may be employed to investigate
the uptake (phagocytosis) and passive dissemination of Salmonella to systemic sites. In addition, use of
monocytes and granulocytes (such as human monocyte cell line THP-1 and primary cells isolated from
animal models) allows study of survival of Salmonella in unconventional targets [25].

26.3 Conclusion
The genus Salmonella contains some important foodborne pathogens that have the capability to cause
both localized infection (gastroenteritis) and systemic infection (typhoid fever) in humans and other ani-
mals. With the application of biochemical, serological, and molecular techniques together with the use of
various laboratory models (including in vivo animal models and in vitro epithelial, phagocytic, and other
cell models) in the past few decades, the molecular mechanisms of Salmonella invasion and intracellular
survival in host cells have been largely elucidated, and many rapid and accurate diagnostic techniques
for identification and subtyping of Salmonella strains have been developed. Nevertheless, much still has
to be learnt about the most efficient way to vaccinate humans against Salmonella infections. For this
reason, laboratory models will continue to play a vital part in helping decipher the host–bacterium inter-
actions, and design improved control strategies against Salmonella infections.

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2. Fookes M, et al. Salmonella bongori provides insights into the evolution of the Salmonellae. PLoS
Pathog. 2011;7(8):e1002191.
3. Parkhill J, et al. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar
Typhi CT18. Nature. 2001;413(6858):848–52.
4. McClelland M, et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2.
Nature. 2001;413(6858):852–6.
5. Calenge F, Kaiser P, Vignal A, Beaumont C. Genetic control of resistance to salmonellosis and to
Salmonella carrier-state in fowl: a review. Genet Sel Evol. 2010;42:11.
6. de Jong HK, Parry CM, van der Poll T, Wiersinga WJ. Host–pathogen interaction in invasive salmonel-
losis. PLoS Pathog. 2012;8(10):e1002933.
7. Majowicz SE, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis.
2010;50:882–9.
8. Garai P, Gnanadhas DP, Chakravortty D. Salmonella enterica serovars Typhimurium and Typhi as
model organisms: revealing paradigm of host–pathogen interactions. Virulence. 2012;3(4):377–88.
9. Bhan MK, Bahl R, Bhatnagar S. Typhoid and paratyphoid fever. Lancet. 2005;366:749–62.
10. Menendez A, et al. Salmonella infection of gallbladder epithelial cells drives local inflammation and
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11. Gonzalez-Escobedo G, Marshall JM, Gunn JS. Chronic and acute infection of the gall bladder by
Salmonella Typhi: understanding the carrier state. Nat Rev Microbiol. 2011;9(1):9–14.
12. Gunn JS, et al. Salmonella chronic carriage: epidemiology, diagnosis, and gallbladder persistence.
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by Salmonella. Gut Microbes. 2013;4(2):140–5.
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14. Coburn B, Li Y, Owen D, Vallance BA, Finlay BB. Salmonella enterica serovar Typhimurium pathoge-
nicity island 2 is necessary for complete virulence in a mouse model of infectious enterocolitis. Infect
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pathogen. Gut Microbes. 2012;3(2):62–70.
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directions. Vaccine. 2015;33(Suppl 3):C8–15.
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Salmonella enterica in Malaysia. Biomed Environ Sci. 2015;28(10):751–64.
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tion of Salmonella. Crit Rev Microbiol. 2015;41(3):309–25.
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a systemic agenda. Mucosal Immunol. 2011;4(4):371–82.
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27
Shigella

Soumik Barman and Yoshifumi Takeda

CONTENTS
27.1 Shigellosis..................................................................................................................................... 401
27.1.1 Incidence........................................................................................................................... 401
27.1.2 Bacteriology...................................................................................................................... 402
27.1.3 Transmission..................................................................................................................... 402
27.1.4 Pathogenesis..................................................................................................................... 402
27.2 Animal Models............................................................................................................................. 402
27.2.1 Guinea Pigs....................................................................................................................... 402
27.2.2 Mice.................................................................................................................................. 403
27.2.3 Rabbits.............................................................................................................................. 404
27.2.4 Pigs and Piglets................................................................................................................. 404
27.2.5 Chicken............................................................................................................................. 405
27.2.6 Caenorhabditis elegans.................................................................................................... 405
27.2.7 Monkeys........................................................................................................................... 405
27.2.8 Zebrafish........................................................................................................................... 405
27.2.9 Human Cell Lines............................................................................................................. 405
27.3 C onclusion..................................................................................................................................... 406
References............................................................................................................................................... 406

27.1 Shigellosis
27.1.1 Incidence
At the end of the 19th century, young microbiologist Kiyoshi Shiga discovered a unique bacillus causing
dysentery during the dysentery epidemic in Japan, and this bacterium, now known as Shigella dysente-
riae 1, was the first acknowledged member of the genus Shigella.1
Shigella species are crucial causes of traveler’s diarrhea in individuals from industrialized countries
visiting developing areas.2,3 Through accidental hygiene or sanitation breaches, endemic outbreaks of
shigellosis may also occur in developed countries.4 Very recently, the global burden of shigellosis has
been reevaluated.5 Shigellosis generally affects poor populations, especially young children, in the
developing world.5–8
By invading the large intestinal mucosa, Shigella spp. induce major inflammatory destruction and gen-
erate dysenteric symptoms in humans and in nonhuman primates.9 In a few cases, only watery diarrhea
is present.10,11 In humans, remarkably low bacterial loads are able to trigger severe shigellosis.12
Suitable antimicrobial therapy of shigellosis reduces the duration of fever and diarrhea.
Azithromycin and fluoroquinolones are the first-line choice for treatment of shigellosis.13 Currently,
the emergence of multidrug-resistant Shigella variants is the greatest concern among global health
problems.14,15 During the last five decades, extensive research has yielded a large number of vaccine
candidates, but no effective vaccines.16–20 The goal of an effective vaccine against Shigella remains
elusive.

401
402 Laboratory Models for Foodborne Infections

27.1.2 Bacteriology
The Shigella genus includes four species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and
Shigella sonnei. Based on the antigenicity of the O-specific polysaccharide chain of their lipopolysac-
charide (LPS) molecules, Shigella species are further divided into serotypes: 15 for S. dysenteriae, 15
for S. flexneri (serotypes and subserotypes comprising the recently entitled 7a and 7b subtypes), 20 for
S. boydii, and 1 for S. sonnei.21 In most tropical countries, shigellosis is endemic and is dominated by a
few S. flexneri serotypes, as well as S. dysenteriae 1.19 The dominant isolates in Europe and the United
States are S. sonnei and S. flexneri.19
Shigella is nonmotile and nonflagellated in nature.21 Most shigellae are capable of fermenting glucose,
with a few exceptions (S. flexneri 6, S. boydii 13 and 14, and S. dysenteriae 3).22 S. dysenteriae is inca-
pable of fermenting mannitol, and this property is used as a key feature for its identification.21
For daily laboratory screening of shigellae, two different selective media are generally used, namely
Hektoen enteric (HE) agar and xylose lysine desoxycholate (XLD) agar. Shigella colonies on XLD agar
appear translucent, pink, or red and are smooth in nature. S. dysenteriae 1 colonies on XLD agar are
commonly very small, unlike those of other Shigella species.23 Colonies of shigellae on HE agar appear
green. Serological identification by slide agglutination with polyvalent somatic antigen grouping sera
further confirms the serotypes and subserotypes.

27.1.3 Transmission
The most common route of Shigella transmission is via fecal–oral route. The bacteria may also be spread
by direct contact with an infected person, as shigellae have the ability to survive on human skin for up to
1 h.21,24 Shigella infection requires only a very small inoculum,12 making person-to-person transmission
favorable. Shigella can spread rapidly by foodborne transmission and cause large outbreaks.25–28 Flies
may play a role in spreading shigellae from human excrement to foods.29,30 Shigellae are able to survive
for at least 6 months in water at room temperature.21

27.1.4 Pathogenesis
The cellular pathogenesis of shigellosis is the result of the complicated actions of a large number of bac-
terial virulence factors. The essential machinery for bacterial penetration and intracellular endurance is
usually encoded in a large “virulence plasmid.”31,32 Loss of the virulence plasmid gives rise to avirulent
phenotypes. A type III secretion system (T3SS) is another key factor involved in Shigella invasion.33,34
Shigella bacilli invade the distal region of the colon and rectum,35 where they become imprisoned by
specialized M-cells. The M-cells deliver the bacterial antigens, LPS, and invasive plasmid antigen (Ipa)
proteins to antigen-presenting macrophages and dendritic cells.36 Shigella bacilli are phagocytized by
macrophages, but subsequently escape through apoptosis.37 Before death, the macrophages release pro-
inflammatory cytokines interleukin-1b and interleukin-18.38
These cytokines trigger a strong inflammatory response and stimulate the migration of neutrophils,35
which infiltrate the infected site and destabilize the epithelium.39 The death of infected macrophages and
subsequent destabilization allow penetration of more shigellae into the tissue and invasion of epithelial
cells through the basal membrane. Survival and replication in macrophages represent the fundamental
key to extensive colonization of the intestinal epithelium.40

27.2 Animal Models
27.2.1 Guinea Pigs
The oldest animal model for assaying Shigella invasion is the guinea pig keratoconjunctivitis model,
known as the Sereny test.41 In this invasion assay, a suspension of live shigellae is generally inocu-
lated into the keratoconjunctival sac of guinea pigs. Invasive shigellae disrupt the conjunctival wall and
Shigella 403

cause severe conjunctivitis, which is characterized by redness of the eye and corneal opaqueness. Highly
pathogenic clinical isolates usually cause acute purulent inflammation, which leads to closing of the
eyelids. Noninvasive Shigella isolates cannot invade the ocular mucosa. This assay can determine the
invasive phenotypes of Shigella.
The Sereny test was used to assess immunization efficacy, i.e., determination of protection against
ocular infection, by various research groups.42–48 Although the keratoconjunctivitis assay is regarded as
the gold standard for measuring Shigella invasion, it does not allow accurate quantification of the inflam-
matory response.
A lethal enteric infection in the colon was established in 4-day-starved guinea pigs by the combina-
tion of calcium carbonate and opium.49 In a series of experiments, guinea pigs were inoculated orally
with live S. flexneri 2a ranging from 8 × 105 to 1.4 × 107 CFU, which induced a fatal enteric infection
with ulcerative lesions in the colon. However, this model might not be ideal for the purpose of measuring
protective efficacy, because the fatal effects appear at a relatively early stage of infection.20
The guinea pig colitis model usually induces typical bacillary dysentery after rectal inoculation of
Shigella species (S. flexneri 2a and 5a) without any preparatory treatment like starvation or antibiotic
treatment.50 In this model, guinea pigs were inoculated with four different doses (108, 109, 1010, and 1011
CFU) of Shigella through the intrarectal route. Within 24 h of challenge, all doses conferred significant
signs of bacillary dysentery. This colitis model has been proven successful in many protective effi-
cacy studies.50–52 However, recent reports suggested that it has several limitations53 and methodological
complications.54
The recently developed guinea pig luminal model was found to be sensitive as it mimics human bacil-
lary dysentery.54 In this model, the lumen of the colon was separately infected with virulent S. dysente-
riae 1 and S. flexneri 2a (109 CFU) after ligation of the distal cecum. The ligation was placed at 4-cm
distance from the ileocecal junction. The placement of the surgical tie may facilitate the colonization
of shigellae in the distal colon by controlling the flow of the stool from the cecum. Bacillary dysenteric
symptoms were recorded within 24 h of infection. The luminal model can be used to demonstrate protec-
tive efficacy,54 but it has become untenable because of a surfeit of surgical complications.
To understand the adhesion and invasion of Shigella, in vitro studies were performed on colonic
explants from guinea pigs, wherein bacterial suspensions were incubated with luminary cuts of the cecal
mucosa.55–57

27.2.2 Mice
Mice have been the most frequently used small animal species in preclinical studies of shigellosis. The
major advantages of mice are the ease of handling, availability of a broad spectrum of reagents, and fea-
sibility of genetic manipulation. Mice are also cost-effective for protective efficacy studies.
During oral inoculation studies, Shigella was unintentionally inoculated into the respiratory pas-
sage, leading to pulmonary Shigella infection.58,59 Over the last five decades, the murine pulmonary
model was used to demonstrate protective efficacy against Shigella infection.59–65 Typically, after
immunization through the external nares, mice were generally inculcated with a challenge strain
(10 6 –107 CFU) through the intranasal pathway. Protection was usually demonstrated by the degree
of Shigella invasion into the lungs and the induction of pulmonary pneumonia. The major lacuna
of the pulmonary model is the lack of clinical relevance with respect to the infection site of the
pathogen.20,53
Another murine infection model was demonstrated via oral inculcation of invasive shigellae in new-
born mice.66 This murine model is extremely sensitive because the neonatal gut is only susceptible to
Shigella infection by oral inculcation for 3–4 days after birth.66 Usually, 4-day-old newborn mice were
orally inculcated with S. flexneri 5a (5 × 109 CFU) for experimental shigellosis. Effective Shigella inva-
sion was achieved in the intestinal mucosa of 4-day-old newborn mice, while 5-day-old mice were found
to be resistant.
It has been assumed that murine models are inadequate for determination of protective efficacy.20
However, recent studies have helped overcome this shortcoming.67,68 The offspring of immunized dams
were orally inoculated with invasive shigellae and the level of protection measured by the degree of
404 Laboratory Models for Foodborne Infections

colonization in the bowel. The murine model developed in these studies can be used as a tool for deter-
mination of passive immunization.
To study the interactions of Shigella with the human intestine in vivo, one group of researchers placed
human intestinal xenografts into the subscapular region of 6–8-week-old severe combined immunode-
ficient (SCID) mice.69 Inoculation of Shigella into these human intestinal xenografts led to the develop-
ment of severe inflammation and mucosal destruction at 4 h after infection. This xenograft model is
useful for understanding intracellular communications, but is somewhat limited and strenuous for the
purpose of measuring protective efficacy.20
A recent study showed that intraperitoneal inoculation of S. flexneri 2a (5 × 108 CFU) into mice
provoked acute shigellosis and mimicked human dysentery.70 The inoculated mice had diarrhea within
2 h of infection. This intraperitoneal model was also shown to be useful for evaluating Shigella vaccine
candidates.72

27.2.3 Rabbits
Rabbits are not naturally susceptible to shigellosis, but may acquire the infection under preparatory con-
ditions. New Zealand white rabbits are usually preferred in the Shigella research field because they are
commonly available and inexpensive.
For investigations of Shigella pathogenesis at the cellular level, the ileal-ligated loops assay has
proven beneficial.71–74 For this, ligated segments of the rabbit small intestine are generally inoculated
with various serotypes of S. dysenteriae, S. flexneri, S. boydii, and S. sonnei.71 Within 12–18 h of inoc-
ulation (109 –1010 CFU), bacterial invasion and ulceration can be achieved.71,72 Furthermore, immune
responses can be measured in a quantitative manner after infection of the ligated loops.74 Surgical
complications are one of the drawbacks of the ligated loops assay, and it is not useful for protective
efficacy studies.
Several studies indicated that acute necrosis occurred in the rabbit ileum after oral inoculation of
Shigella, under preparatory treatments like starvation, antibiotic treatment, and stomach acid neutraliza-
tion.75,76 However, there has been no prominent follow-up of such orogastric “conditioned” rabbit models
to date.
Adult rabbits exposed to direct colonic inoculation by cecal bypass also progress to shigellosis.77 Cecal
bypass was achieved by blockade of the cecal lumen above the ileocecal junction with a silk thread liga-
ture, while the ileo-ceco-colic communication was properly maintained. After cecal bypass, bacteria
(107 CFU) were inoculated into the proximal colon. Onset of dysentery occurred within 24 h of inocula-
tion. The colonic infection model was shown to be worthwhile in various Shigella vaccine candidate
screenings.62,78,79 A very recent study clearly demonstrated the usefulness of this model in the assessment
of anti-shigella vaccine candidates.80 Despite encouraging results, the surgical obstacle appears to be the
major lacuna of this model.

27.2.4 Pigs and Piglets

Domestic pigs are not susceptible to shigellosis.81 However, 4-week-old antibiotic-treated pigs were sub-
jected to oral inoculation with invasive S. dysenteriae 1 and S. flexneri 2a (1 × 1011 CFU). Most of the
pigs had no dysenteric symptoms throughout the duration of infection, although signs of bacteria were
detected in rectal cultures within 24 h of infection. Histopathological analysis detected tonsillar lesions,
which are not clinically relevant for assaying shigellosis.
Jeong et al.82 developed a unique model for assaying shigellosis using gnotobiotic piglets. Piglets
were delivered by cesarean section and kept aseptically in a plastic sterile isolator to maintain their gut
sterility. An oral inoculum of S. dysenteriae 1 (5 × 109 CFU) induced acute shigellosis within 24 h of
infection. The sterile gut of these piglets at 1–3 days of age allowed rapid colonization by Shigella organ-
isms. Despite promising results, the piglet model has some limitations. The model became less efficient
as maintaining piglets in sterile conditions was not cost-effective, and the model was also incapable of
quantifying protective efficacy.
Shigella 405

27.2.5 Chicken
Very recently, a research group in China reported invasion of clinically isolated S. flexneri 2a into spe-
cific pathogen-free (SPF) chicken.83 SPF chickens at 3 days of age were inoculated by intraperitoneal
injection or crop injection. Inoculation of 3 × 109 CFU resulted in death in 100% of the chickens, while
crop injection did not cause any intestinal clinical signs of dysentery. The ligated intestinal loop of
1-day-old SPF chicken also showed severe congestion and edema. This study has clinical significance
regarding human–poultry cross-infection, although it has a shortcoming regarding vaccine candidate
screening.

27.2.6  Caenorhabditis elegans

In recent years, the free-living nematode Caenorhabditis elegans has been acknowledged as a valuable
in vivo model for studying host–pathogen interactions. Two independent research groups suggested that
C. elegans could be used as an in vivo model for evaluating the pathogenesis of shigellae.84,85 These stud-
ies established that S. flexneri 2a and S. flexneri 2b killed C. elegans on solid media and in liquid culture,
although the exact mechanism remains unknown.
An S. boydii-mediated infection assay in C. elegans followed by bioinformatics analysis revealed vari-
ous immune regulators that are important for Shigella species-mediated immune responses.86
A very recent study by George et al.87 demonstrated that invasive S. flexneri 3a accumulated in the
nematode intestinal lumen, produced outer membrane vesicles, and invaded nematode intestinal cells.
Further studies revealed that S. flexneri 3a interrupted iron homeostasis in nematodes and potentially
stimulated a hypoxic response, which could cause death.

27.2.7 Monkeys
In 1965, Formal et al.88 successfully demonstrated immunogenicity and protective efficacy of attenu-
ated Shigella strains against S. flexneri 1b, 2a, and 6 infections in rhesus monkeys (Macaca mulatta).
Both immunization and challenge (5 × 1010 CFU) were performed by the oral route. Later studies by the
same group revealed that typical bacillary dysentery could be induced in rhesus monkeys by oral infec-
tion with S. flexneri without starvation and/or pretreatment with antibiotics.89,90 Subsequent protective
efficacy studies for Shigella vaccine candidates also became effective in rhesus monkeys.91–95 The rhesus
monkey model mimics human shigellosis and the human immune response, making it ideal for assem-
bling preclinical data before human trials of vaccine candidates.
Cynomolgus monkeys (Macaca fascicularis)96,97 and Aotus nancymaae monkeys98 were also estab-
lished as tools for screening Shigella vaccine candidates. Despite auspicious results, the cost of using
these animals can be prohibitive, especially in developing countries where shigellosis is endemic.

27.2.8 Zebrafish
The zebrafish model opens a new avenue to investigate the significance of innate immunity. It provides a
framework where no adaptive responses have yet been developed, because early lymphocytes make their
first appearance in 4-day-old larvae and full adaptive immunity requires several weeks to be displayed.99
An in vivo study of S. flexneri interactions with phagocytes and bacterial autophagy was reported very
recently.100 An inoculum of 4 × 103 S. flexneri 5a resulted in the death of most zebrafish larvae within 48 h
postinfection. Intravenously administered bacteria could survive and replicate in both macrophages and
nonimmune cells. The zebrafish larva represents a valuable new host for analysis of Shigella infection,
although this model cannot define the protective efficacy.

27.2.9 Human Cell Lines

Human cell lines are broadly used as a naive model for understanding the interactions of Shigella with
its host. The advantages of cell lines are their ease of handling and relatively low costs.
406 Laboratory Models for Foodborne Infections

The primary step of shigellosis is invasion of the human colonic mucosa. Host–pathogen interactions
can easily be examined in vitro using cell lines before animal trials are carried out. Several inves-
tigations were performed to disclose host–Shigella interactions at the cellular level using HeLa,101–105
T84,106–108 THP-1,109,110 macrophage J774,111–113 HT-29,114,115 monoblastic U937,113,116,117 human embryonic
kidney (HEK) 293,118,119 human colonic Caco-2 epithelial,120–122 and human CD4+ T (Jurkat)123 cells. The
results identified the relative contributions of antigens, which need to be taken into account for vaccine
development.

27.3 Conclusion
The use of various animal models has already improved our understanding of the in vivo consequences
of intracellular defense mechanisms as well as protective measures against Shigella spp. We expect that
further application of these powerful models will continue to unravel the intricacy of the virulence strat-
egies employed by shigellae to mitigate host’s innate and acquired immune defenses.

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105. Lum, M. & Morona, R. Myosin IIA is essential for Shigella flexneri cell-to-cell spread. Pathog Dis. 72,
174–187 (2014).
106. Zurawski, D.V., Mitsuhata, C., Mumy, K.L., McCormick, B.A. & Maurelli, A.T. OspF and OspC1 are
Shigella flexneri type III secretion system effectors that are required for postinvasion aspects of viru-
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107. Zurawski, D.V., Mumy, K.L., Faherty, C.S., McCormick, B.A. & Maurelli, A.T. Shigella flexneri type
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108. Nandakumar, N.S., Pugazhendhi, S. & Ramakrishna, B.S. Effects of enteropathogenic bacteria & lac-
tobacilli on chemokine secretion & Toll like receptor gene expression in two human colonic epithelial
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by Shiga toxin 1 and/or lipopolysaccharides in the human monocytic cell line THP-1. Infect Immun. 72,
2618–2627 (2004).
110. Harrison, L.M., van den Hoogen, C., van Haaften, W.C. & Tesh, V.L. Chemokine expression in the
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111. Clerc, P.L., Ryter, A., Mounier, J. & Sansonetti, P.J. Plasmid-mediated early killing of eukaryotic cells
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endocytic vacuoles of mouse and human macrophages. Infect Immun. 68, 3608–3619.
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115. Kuehl, C.J., Dragoi, A.M. & Agaisse, H. The Shigella flexneri type 3 secretion system is required for
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116. Nonaka, T., Kuwae, A., Sasakawa, C. & Imajoh-Ohmi, S. Shigella flexneri YSH6000 induces two types
of cell death, apoptosis and oncosis, in the differentiated human monoblastic cell line U937. FEMS
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28
Vibrio: Caenorhabditis elegans as a Laboratory
Model for Vibrio Infections

Sellegounder Durai and Krishnaswamy Balamurugan

CONTENTS
28.1 V. cholerae....................................................................................................................................413
28.2 Models to Study V. cholerae.........................................................................................................414
28.3 V. alginolyticus..............................................................................................................................414
28.4 Models to Study V. alginolyticus..................................................................................................415
28.5 V. parahaemolyticus.....................................................................................................................415
28.6 Models to Study V. parahaemolyticus..........................................................................................416
28.7 C. elegans as Model for Studying Bacterial Infection..................................................................417
28.8 C. elegans for Host–Pathogen Interaction Studies.......................................................................418
28.9  C. elegans as a Model for Vibrio spp. Infection...........................................................................418
28.10  Conclusion....................................................................................................................................421
Acknowledgements..................................................................................................................................421
References............................................................................................................................................... 422

Vibriosis is a common illness among seafood consumers that is caused by bacteria of the family
Vibrionaceae. Among many species within the Vibrionaceae family, Vibrio cholerae, Vibrio parahae-
molyticus, Vibrio vulnificus, and Vibrio alginolyticus are important foodborne pathogens in humans.
Transmitted through uncooked or partially cooked seafood, vibriosis produces a range of symptoms,
including cholera, dysentery, diarrhea, gastroenteritis, sepsis, and fasciitis. The virulence, mode of infec-
tion, symptoms, and treatment are different for each of these species. Showing variations in virulent
factors as well as differences between the wild-type and clinical isolates, the pathogenicity of Vibrio spe-
cies is complex. The nematode Caenorhabditis elegans has been the preferred model system for many
investigators to study pathogenesis of this disease. The current chapter summarizes the available models
for studying Vibrio spp. infections, with an emphasis on the utility of C. elegans model.

28.1  V
 . cholerae
Vibrio cholerae is facultative anaerobic, Gram-negative, non-spore-forming, curved rod-shaped bac-
terium measuring 1.4–2.6 µm with the ability for respiratory and fermentative metabolism, which can
be defined on the basis of biochemical test and DNA sequence homology analysis. The bacterium is
oxidase-positive, reduces nitrate, and moves using a single sheathed polar flagellum. The growth of the
bacterium is regulated by altering sodium chloride (NaCl) concentration.1 V. cholerae moves by means
of its single polar flagellum, which is driven by the sodium-motive force.2 The ability of the bacterium to
grow in nutrient broth without NaCl makes it unique among Vibrio spp. V. cholera is a causative agent
for the deadly disease cholera, which is considered as a life-threatening intestinal infection. Millions
of people are affected with cholera every year, and the disease accounts for thousands of causalities

413
414 Laboratory Models for Foodborne Infections

worldwide, most commonly in developing countries, and the number of cholera cases increases day by
day.3,4 Infections spread through contaminated water and seafood. In the lumen of the small intestine, the
bacterium begins to multiply persistently, leading to colonization. Recent studies reported the ability of
this pathogen to successfully metabolize glucose, thereby facilitating its proliferation in a glucose-rich
environment like the human intestine.5
V. cholerae initiates infection by adhering to the epithelium while swimming, which is the first step
of colonization. Quorum sensing plays an important role in the colonization of the host system by
V. cholerae.6 Evolution, growth, and virulence of V. cholerae is rigidly controlled by the location of
ribosomal protein gene locus and its distance from the origin of replication.7 During antibiotic treatment,
the pathogen modulates its shape from a rod to a sphere, thereby protecting itself from the antibiotics.8
The availability of model organisms and information about the bacterial genetics has enabled the under-
standing of the few aspects of V. cholerae pathogenicity in humans; however, complete information
about the natural habitat of the bacterium and its interaction with host system remains unknown.

28.2 Models to Study V. cholerae

Several model organisms have been used to identify the various virulent factors of V. cholerae and under-
stand the host immune response against V. cholerae infection and effect of secreted cholera toxin. Studied
models include rabbit,9 infant mice,10,11 adult mouse,11 Danio rerio12 (Zebrafish), and Caenorhabditis
elegans,1 each having its own advantages and disadvantages. The bacterium was administered by par-
enteral injection as well as oral administration. None of the animal models was found to be successful
and reproducible in mimicking the clinical status of cholera infection until the development of the rabbit
model.9,13,14 The model was first developed in India by ligating the intestinal loop of adult and infant rab-
bit, thus providing insights into the colonization and pathological effect of the cholera toxin.13 Although
this method was extensively used, it is different from the original infection status. Infant animals are
easily prone to infection and do not develop immunity; in other words, a model to better study the enteric
infection should have a patent intestine and its immune system would be functioning and noncompro-
mised. Two models were developed to overcome the above hurdle using dogs and chinchillas. But, both
these models were not extensively used due to high cost in dogs15 and scarce availability of chinchillas.16
The other models in which lethal watery diarrhea can be reproduced include the removable intestinal tie
adult rabbit diarrhea (RITARD) in dogs and rabbits. These two systems were extensively used for sev-
eral years and can provide information on the pathogenesis as well as the host immunity status against
infection. The direct inoculation of cholera bacteria into the small intestine of rabbits was done in the
mid-1950s by Dutta and Habbu to provide reproducible results. This model was very rarely used due to
the reluctance of investigators to carry out surgery. In recent years, a nonsurgical method was developed
using infant rabbit models. The diarrheal fluid developed in this model was found to be chemically
similar to that in human infections but was absent in rabbits infected with mutant strains lacking genes
responsible for establishing infection in humans, suggesting that this system is the more reliable for the
study of human cholerae infection.17 Histological, confocal, and scanning electron microscope results in
the model revealed the possibility for analyzing the cholera-toxin-mediated mucin secretion from goblet
cells in the small intestine. The ability of V. cholerae ctxAB mutant to cause mild diarrhea in this model
further emphasizes the use of such models to study reactogenicity of live attenuated cholera vaccines.18

28.3  V
 . alginolyticus
Vibrio alginolyticus is a common inhabitant of the marine environment in both temperate and tropical
waters.19,20 V. alginolyticus is also present in estuarine environments and is frequently isolated from bath-
ing areas as free-living bacteria and is also associated with biotic and abiotic surfaces.21 V. alginolyticus
is one of the most common and important pathogen that causes vibriosis in human and marine species,
and infection in humans was first recognized in 1973. The complete genome sequence of V. alginolyticus
ATCC 17749 was revealed recently.22 Infection of fish by V. alginolyticus results in biofilm formation
Vibrio: C. elegans as a Laboratory Model for Vibrio Infections 415

in their intestine, which leads to mortality and potentially significant economic losses. V. alginolyticus
has been described to cause human infections including wound infection (71%), gastroenteritis (12%),
ear infection, and septicemia.23 Other clinical syndromes reported in association with V. alginolyticus
infection include chronic diarrhea in a patient with acquired immunodeficiency syndrome (AIDS), con-
junctivitis, and posttraumatic intracranial infection.
V. alginolyticus produces many extracellular proteases responsible for interaction between the bacte-
rium and cell hosts (human and fish) and plays an important role in infections.24,25 The mechanism of
pathogenicity induced by V. alginolyticus infections is complex and related to several factors, including
cytotoxins, enterotoxins, and lytic enzymes.26,27 The adhesive properties of Vibrio spp. are considered
as the key factor of bacterial pathogenicity and have been implicated as a risk factor for infection in
humans and stressed aquatic animals.21,28,29 A set of genes regulated during environmental stress are
known for their key role in regulating the adhesion process of V. alginolyticus.30 Larger numbers of
Vibrio spp. strains were frequently isolated from zooplankton, especially from copepods.21 The ability
to form biofilms on biotic and abiotic surfaces and to activate a survival state called “Viable but not
cultivable” allow V. alginolyticus strains to persist in seawater under environmental stress conditions.31
In V. alginolyticus, genetic analysis reveals that there is no correlation between the virulence of strains
and the presence of general virulent genes such as tlh, trh, toxR, toxRS, ctxA, and VPI, which are present
and contribute to the virulence in other Vibrio spp. pathogens.32,33
Production of multiple virulent extracellular products (ECP), mainly protease, is used to characterize
the virulent strains of V. alginolyticus.34,35 Extracellular alkaline serine proteases (Asp) have been dem-
onstrated to be important exotoxins of V. alginolyticus strains pathogenic to prawn35 and fish,36 and their
expressions are subjected to the regulation of a LuxO–LuxR quorum-sensing regulatory circuit.37 At
present, little is known about the relationship between the secretion systems and ECP production as well
as their contribution to the pathogenesis in V. alginolyticus. A recent study identified and characterized
the Tat translocation system in V. alginolyticus. The Tat system was strongly involved in the swarming
ability and biofilm formation in this bacterium. In addition, the Tat system in V. alginolyticus was also
required for virulence against the fish model and epithelial cells. These findings suggest that Tat secre-
tion is related to the extracellular protease activity as well as virulence in V. alginolyticus.38

28.4 Models to Study V. alginolyticus

Many studies on V. alginolyticus pathogenicity have been focused on marine animals, such as Epinephelus
awoara, shrimp,39 Pagrus majori,40 Pseudosciaena crocea,41 Epinephelus bruneus,42 Haliotis diversi-
color supertexta,43 Lutjanus sanguineus,44 and Lutjanus erythropterus,45 while few studies also reported
pathogenicity of this microbe in humans. Lack of an appropriate model to study V. alginolyticus hin-
dered the understanding of the mechanism for many years. A recent study by Liu et al. has established
mice as a model for studying V. alginolyticus. Results of the study revealed pathogenicity of two different
strains of V. alginolyticus ATCC 17749 and E0666. Intraperitoneal injection of V. alginolyticus caused
severe lung and liver damage in mice with high bacterial counts found in heart, lung, and blood of the
dead mice. The data suggest that high dose of V. alginolyticus injected intraperitoneally absorbs bacteria
directly into the blood. IL-1β and IL-6 levels were significantly higher in serum of the V. alginolyticus-
infected group compared to those in the control. Results suggested that IL-6 and IL-1β are important
inflammatory factors that may play a role in the inflammation induced by V. alginolyticus infection.46
A very recent study used high-throughput sequencing to identify differentially expressed genes between
normal grouper larvae and larvae with vibriosis.47

28.5  V. parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that causes acute gastroenteritis in
humans.48 Infection occurs through the consumption of raw or inadequately cooked seafood. Since its
discovery about 50 years ago, V. parahaemolyticus has been implicated as a major cause of foodborne
416 Laboratory Models for Foodborne Infections

illness around the world.49 Clinical manifestations of V. parahaemolyticus infections include gastroen-
teritis, wound infection, and septicemia. Almost all clinical V. parahaemolyticus show β-hemolysis in
a Wagatsuma agar, and this hemolytic activity is termed the Kanagawa phenomenon (KP).50 The KP
is considered as a good marker to differentiate human pathogenic V. parahaemolyticus strains from
the nonpathogenic strains. Almost all clinical isolates carry tdh, trh, or both genes, whereas 1%–2% of
­environmental isolates may likely possess these genes.50 Isolates lacking tdh and trh genes are also iso-
lated from patients with V. parahaemolyticus infection symptoms. Both virulent and ­avirulent strains of
V. parahaemolyticus are known to occur in a host, leading to in vivo serotype diversity.51 The complete
genome sequence of clinical and environmental isolates were reported recently52,53 followed by draft
genome sequence of 14 Canadian V. parahaemolyticus clinical isolates that were serologically identified
as K group II using polyvalent antisera but were not specifically K serogrouped using monovalent anti-
sera.53 The use of whole-genome sequencing coupled with phylogeny and multiplex polymerase chain
reaction (PCR) assay is also under development for identifying sequence of several V. parahaemolyticus
isolates.54 The complete genome sequence followed by pulsed-field electrophoresis showed that some
V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates,
which enables them to make the transition from normal aquatic state to a pathogen during infection.55
Most of the studies exploring the pathogenesis of V. parahaemolyticus on the host focus on TDH and
TRH. TDH is responsible for various cytotoxicity effects that include erythrocyte lysis, disruption of
the microtubule cytoskeleton, and ion flux disturbance in cultured cells.56–58 Very less is known about
the targets of TRH; a study on the purified TRH toxin indicates their effect on cultured erythrocytes
and also result in the fluid accumulation in rabbit ileal loop model.59 Infection by V. parahaemolyticus
bacteria results in cell rounding and disruption of epithelial barrier function, which is mediated through
the effect of TDH on the ion flow; this loss of barrier results in the excessive loss of body fluid and the
condition is termed diarrhea.50
The genetic approach for identifying genes regulating the virulent pathway in V. parahaemolyticus
showed a close association between the urease gene and the TRH gene.60 Hence, it is hypothesized that
possession of the gene for TRH coincides with the presence of the urease gene among many clinical
V. parahaemolyticus strains, making urease gene a marker for identifying clinically pathogenic trh posi-
tive strains.61 Urease is an enzyme involved in the hydrolysis of urea into ammonia and carbon dioxide
and is present across kingdoms in plants, fungi, algae, and bacteria.62 Many bacterial ureases have been
characterized, and the organization of this gene is found to be similar among bacteria with regard to the
structural and accessory genes involved in processing nickel ions.63 Comparing with other Vibrio spp.,
only a small portion of clinical V. parahaemolyticus isolates possess the urease gene.61 But the presence
of urease gene is always associated with presence of trh gene. Specialized PCR techniques such as the
long and accurate PCR have revealed that the distance between these two genes is <8.5 kb.64
Type III secretion systems (T3SSs) are considered as one among the major virulent pathways in many
Gram-negative pathogens; bacteria use this system to translocate the produced virulent factors to the
cytosol of the host cells.65 The T3SS apparatus is found to be highly conserved across Gram-negative
bacteria, and the specific property of the effectors brings about symptomatic effects on the host organ-
ism that vary widely.65 Genome sequence of TDH-positive V. parahaemolyticus strains displayed the
presence of gene sequences that are similar to T3SS present in chromosome 1 and 2, which are named
T3SS1 and T3SS2, respectively.66 T3SS1 gene sequence was found to be present in most or all of the
V. parahaemolyticus strains, whereas the T3SS2 was found only in KP-positive strains. Intensive study on
the T3SS of V. parahaemolyticus revealed that these two systems confer specific phenotypes to the bacteria.
Understanding the effector proteins necessary for the activation of T3SS and environmental influence on
the regulation of T3SS will provide deeper insight into V. parahaemolyticus infection in humans.64

28.6 Models to Study V. parahaemolyticus

The rabbit or rat ileal loop model has been considered as the best method to detect fluid accumulation
due to the enterotoxic activity of V. parahaemolyticus TDH in vivo.67,68 The role of the Type III ­secretion
system 1 and 2 was tested using in vitro cell lines. Results of the in vitro studies have shown that T3SS1
Vibrio: C. elegans as a Laboratory Model for Vibrio Infections 417

induces cytotoxicity in several cell lines, while T3SS2 can induce cytotoxicity in Caco-2 cells and entero-
toxicity in vivo.69,70 The importance of TDH and T3SSs in V. parahaemolyticus virulence was recently
assessed using a mouse model. These results clarified that T3SS2 rather than TDH was the major con-
tributor to enterotoxicity; however, both T3SS1 and TDH contributed to lethality in a mouse model, sug-
gesting that TDH and/or other proteins(s) that are part of T3SS1 are also likely to contribute to virulence.71
Two different animal models were used to understand the contributions of T3SS1 and T3SS2 to V. para-
haemolyticus virulence.72 Orogastric inoculation of V. parahaemolyticus in neonatal pigs demonstrated
that T3SS2, but not T3SS1, was required for gastrointestinal disease. The study also showed that T3SS1
was necessary to cause mortality during intrapulmonary inoculation, which raises questions on role of
unknown factors related to T3SS1 responsible for lethality.72 Host intestine damage during V. parahae-
molyticus infection was studied using infant rabbit model.73 Enteroinvasiveness of the bacterium has been
reported for a rabbit model, in which the organism invaded, colonized, and produced inflammation in the
small intestine. In another study, an adult zebrafish model was investigated to assess the overall virulence
of V. parahaemolyticus strains.74 The model detected differences in the virulence potential of strains when
animals were challenged intraperitoneally, and this was based on survival time. Differences in survival
were noted irrespective of the source of isolation of the strain (environmental or clinical) and regardless
of the presence or absence of the known virulence factors TDH and TRH, suggesting the influence of
additional virulence factors. The model was also effective in comparing differences in virulence between
the wild-type V. parahaemolyticus strain RIMD2210633 and isogenic pilin mutants.

28.7  C
 . elegans as Model for Studying Bacterial Infection
Caenorhabditis elegans was first discovered and described by Emile Maupas, a French zoologist and
botanist, in 1900 from soil in Algeria.75 Earlier, Maupas used Rhabditis as genus name rather than
Caenorhabditis. In 1952, the new subgenus Caenorhabditis was coined,76 which was later raised
into generic status by Dougherty in 1955. The definition of genera is somewhat arbitrary. Even now,
Caenorhabditis is a subgenus name, one among many in the Rhabditis genus, and C. elegans are formally
called Rhabditis (Caenorhabditis) elegans. C. elegans is a eukaryotic and free-living soil nematode. It
has a complex developmental process and includes stages of embryogenesis, morphogenesis, and growth
to an adult. C. elegans demonstrates about 35% of homology to human at genome level and 83% similar-
ity in protein sequence.77 C. elegans was introduced to the scientific community by Sydney Bernner as
an experimental model in 1960s in order to explore animal developmental biology when he published
his first report on the behavior of the nematode.78 According to the C. elegans Sequencing Consortium,
its genome has been completely sequenced, and the genome size is around 100.2 Mb. The availability of
the entire genome sequence is useful in genetic studies, allowing researchers to pick a gene of interest
to study. Surprisingly, it has been shown frequently that human genes replace their C. elegans homologs
when introduced into C. elegans, and many genes in C. elegans can function similar to mammalian genes.
In contrast to other invertebrates, immunity in C. elegans solely depends on the innate immune system
as it lacks the sophisticated adaptive immune signaling.79 With this primary innate immune system, the
nematode’s immune response involves the production of numerous antimicrobial proteins that are con-
served in higher vertebrates and humans. Exploring the innate immune signaling pathways in C. elegans
revealed that many pathways important for development in early stages were reused in the adult stage
for immune signaling. The nematode’s defense involves three major mechanisms against any microbial
infection:80 (1) avoidance behavior, (2) physical barrier, and (3) inducible signaling pathways.
The neuronal system in C. elegans is well equipped in differentiating between the food source and
pathogenic bacterium.81 The repelling behavior against Serratia marcescens shed light on the role of sev-
eral neurons, G-protein-coupled receptors, and TOL-1.82,83 The cuticle of C. elegans is made of collagen
and chitin that act as a strong physical barrier that cannot be pierced easily, thereby protecting from the
entry of pathogens via the dermal layer.84,85 The immune system includes pathogen recognition by PRR
and other signaling cascades like MAPK, DAF-2, UPR, TGF-β, apoptosis, and necrosis pathways.86–88
Forward and reverse genetic studies have identified genes that belong to six signaling cascades, which
are activated upon infection with Gram-positive and Gram-negative bacteria, fungi, and bacterial toxins.
418 Laboratory Models for Foodborne Infections

The six pathways include ERK MAP kinase,89 p38 MAP kinase,90 TGF-β,91 programmed cell death,92
DAF-2/DAF-16 insulin-like receptor signaling,93 and JNK-like MAP kinase.94

28.8  C
 . elegans for Host–Pathogen Interaction Studies
Studies in C. elegans have shed light on many pathways of the innate immune system that are phyloge-
netically conserved from worms to humans.95 C. elegans is a very good model to study both the host and
the microbe factors that underscore the host–pathogen interaction.96
M. nematophilum is one among many pathogens that infect C. elegans in its natural environment.
It infects C. elegans and leads to an unusual and visible tail swelling or deformed anal region (Dar),
formerly believed to be a spontaneous heritable and morphological mutation that arises during a regular
genetic cross. Later, Hodgkin et al. revealed that M. nematophilum establishes a specific rectal infection
due to its strong extracellular adherence to the cuticle, resulting in the swelling response and the Dar
phenotype.97,98 The tail-swelling phenotype observed during M. nematophilum infection is related to the
enhancement of immune pathways ERK and MAPK in C. elegans as a response of the innate immune
system against the bacterial infection.89
Studies with mutant strains that lack the bacterially unswollen (Bus) phenotype revealed that the
role of several genes responsible for the Dar phenotype have implications on both the host (capabil-
ity to elicit a swelling response) and the bacterium (ability to adhere and colonize).99 The role of genes
responsible for normal cuticle development and prevention of bacterial adherence have been reported in
previous studies.100 Quantitative molecular studies identified nearly 68 host genes that are induced upon
M. nematophilum infection; most of these genes are pathogen receptor molecules including C-type lectin
domains and lysozymes.101
Drechmeria coniospora is an agriculturally important nematode parasite that infects C. elegans
through the mouth and penetrates the body by means of the proteinaceous hyphae.102 C. elegans triggers
an immune response against the infection through the induction of neuropeptide-like proteins (NLPs).103
Nematocida parisii is a microsporidian parasite of C. elegans, and its infection did not induce basic
immune-responsible genes known to be vital for other pathogenic infections but alters the components of
the immune-signaling pathways (p38 MAPK and insulin signaling/DAF-2 pathways), thereby affecting
animal survival.104 Recent studies have reported the interaction between C. elegans and Orsay virus, a
small, positive-sense RNA virus belonging to the family Nodaviridae.105

28.9  C . elegans as a Model for Vibrio spp. Infection

Studies with C. elegans and Vibrio spp. started with the most dreadful pathogen V. cholerae.106 In the
marine environment, the bacterium secretes well-characterized toxins including cholera toxin (CT) and
toxin coregulated pili (Tcp). Bacterial cell-to-cell communication has a major role in regulating the
virulence of the bacterium.107 Studies with C. elegans shed light on the V. cholerae metalloprotease
prtV, which is involved in establishing infection in higher vertebrates, and this also proved by studying
its effect on a monolayer of mammalian intestinal cell lines. Since this protein (PrtV) is found to be
essential for infection in humans and C. elegans, the results support the use of C. elegans as model to
study V. cholera infection.1 Another study explored V. cholerae cytolysin (VCC), which is one among
the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC,
encoded by hlyA gene, belongs to the most common class of bacterial toxins known as pore-forming
toxins (PFTs). V. cholerae infects and kills C. elegans in a cholerae-toxin-independent manner. VCC is
required for lethality, growth retardation, and intestinal cell vacuolation during infection. A microar-
ray analysis was performed in C. elegans exposed to V. cholerae strains with intact and deleted hlyA
genes. Many of the VCC-regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/
asparagine [N]-rich)-domain containing genes, genes regulated by insulin/IGF-1-mediated signaling
(IIS) pathway, were previously reported as mediators of innate immune response against other bacteria
in C. elegans. The protective function of the subset of the genes upregulated by VCC was confirmed
Vibrio: C. elegans as a Laboratory Model for Vibrio Infections 419

using RNAi. By means of a machine learning algorithm called FastMEDUSA, the study identified sev-
eral putative VCC-induced immune regulatory transcriptional factors and transcription factor binding
motifs. Results from the experiments suggest that VCC induces a wide variety of immune-response-
related genes during V. cholerae infection in C. elegans.108
A recent study by Durai et al. established C. elegans as a model for studying phenotypic changes and
regulation of immune-responsible genes in the host against bacterial infection. Preference of C. elegans
between nonpathogenic E. coli OP50 and pathogenic V. alginolyticus was well documented in the study.
The infection was localized using green fluorescent protein (GFP)-tagged pathogen, and intestinal colo-
nization was reported as the major reason for mortality in C. elegans.109 Since C. elegans displayed lawn
avoidance behavior to V. alginolyticus, a liquid killing assay was developed that comprised both M9
buffer and pathogen liquid culture (in a 3:1 ratio). Infection with V. alginolyticus in liquid assay reduced
the lifespan of the nematode significantly. However, earlier reports revealed a reduction in lifespan of
C. elegans exposed to V. cholerae, with complete killing of nematode in approximately 5 days,1 while
with V. vulnificus infection, the TD50 (toxic dose) of the nematode was found to be 9 days.110 Microscopic
image analysis of the infected worm showed colonization of GFP-tagged V. alginolyticus in the pharyn-
geal region and in the intestine of the pathogen-exposed animals. V. alginolyticus was able to colonize
the gut of C. elegans within a short period of time, i.e., after 8 h of infection, and the bacterial load from
the colonized animals were found to be ∼3.7 ± 0.3 × 103 CFU. It was also observed that a bacterial load
of at least 6.5 × 104 CFU of V. alginolyticus was required to cause and maintain infection in C. elegans.
It was also found that V. alginolyticus induced a strong inflammatory response in the intestine, which
appeared to be more severe than the infection caused by the V. cholerae. The persistence of V. alginolyti-
cus leads to the damage of the pharyngeal bulb and distention of intestinal lumen, which was confirmed
by confocal laser scanning microcopy (CLSM) image analyses.
C. elegans are attracted toward the smell of bacteria for a short period of time irrespective of the bacte-
rial nature. On sensing the pathogens, in this case V. alginolyticus, the worms learned to avoid the smell
of the bacterium and defended themselves from infection. The lawn-avoidance behavior of C. elegans
observed in the study could be attributed to its chemosensory response to the pathogen (Figure 28.1).

Zone A E. coli OP50

Zone A
V.alginolyticus

Zone B V.alginolyticus
E. coli OP50 Zone B

(a) (b)

Zone A
E. coli OP50

Zone B V.alginolyticus

(c)

FIGURE 28.1  Chemotaxis behavior: in all cases, animals were tracked on an agar plate. (a) Animals in the presence of
food source, E. coli OP50 marked at zone A and zone B. (b) Animals in presence of V. alginolyticus marked at zone A and
zone B. (c) Animals in presence of food source, E. coli OP50 marked as zone A and pathogen source, V. alginolyticus
marked as zone B. (Adapted from Durai, S. et al., J. Basic Microbiol., 51, 243–52, 2011. With permission.)
420 Laboratory Models for Foodborne Infections

The immune response of C. elegans against V. alginolyticus infection was analyzed using quantitative
PCR (qPCR). Analogous to the previous studies in M. nematophilum, three candidate genes, viz., lys-7,
clec-60, and clec-87, were analyzed for their expression during infection by having animals treated with a
food source as control.101 The level of expression of the immune effector gene(s) was significantly higher
when compared to the control animals. During the initial stage of infection, the C-type lectin genes clec-60
and clec-87 seem to be responsible for the enhancement in adhesion and antibacterial activity.101 The role
of lysozyme gene lys-7 as a presumptive antimicrobial gene and in induction of immune response as well
as its encoded protein in the upregulation to infection with Gram-negative bacterium S. marcescens has
previously been reported.111
Proteome regulation in C. elegans against V. alginolyticus was also explored by Durai et al.112 Proteins
were separated using two-dimensional differential gel electrophoresis (2D-DIGE), and the differentially
regulated proteins were identified using positive matrix factorization (PMF) and matrix-assisted laser
desorption/ionization–time of flight (MALDI–TOF)/TOF analysis. The results thus obtained were vali-
dated using western blotting for candidate proteins. The corresponding transcriptional regulation was
quantified subsequently using real-time PCR. Interaction network for candidate proteins was predicted
using the search tool for the retrieval of interacting genes/proteins (STRING), and functional validation
was performed using respective mutant strains. Out of the 25 proteins identified, 21 proteins appeared to
be upregulated, while 4 were downregulated (Table 28.1). Upregulated proteins included those involved
in stress response (PDI-2, HSP-6), immune response (KIN-18, GST-8), and energy production (ATP-2),
while proteins involved in structural maintenance (IFB-2) and lipid metabolism (SODH-1) were down-
regulated. The roles of these players in the host system during Vibrio infection were analyzed in vivo
using wild-type and mutant C. elegans. Survival assays using mutants lacking pdi-2, ire-1, and xbp-1
displayed enhanced susceptibility to V. alginolyticus. Cellular stress generated by V. alginolyticus was
determined using reactive oxygen species (ROS) assay. This is the first report of proteome changes in

TABLE 28.1
Differentially Expressed Early Responsive Proteins of C. elegans against V. alginolyticus
Infection
S.No. Protein Gene
A. Upregulated C. elegans Protein against V. alginolyticus Infection
1 Alpha integrin Vab-20
2 Serine/threonine protein kinase kin-18
3 Mitochondrial ribosomal protein, small mrp-25
4 Heat shock protein hsp-6
5 Protein BCCIP homolog ZK1127.4
6 COP9/signalosome and eIF3 complex shared subunit K08F11.3
7 H1 histone hil-1
8 Glucose-6-phosphate 1 dehydrogenase gspd-1
9 Protein disulfide isomerase pdi-2
10 DNA-directed RNA polymerase subunit II phi-14
11 ATP synthase subunit-2 atp-2
12 Glutathione S-transferase 8 gst-8
13 cGMP-dependent protein kinase pkg-1
14 Eukaryotic translation initiation factor 3 subunit eif-3.G
15 Putative aminopeptidase lap-2
16 Nuclear hormone receptor family nhr-44
17 Probable signal recognition protein F55C5.8
B. Downregulated C. elegans Protein against V. alginolyticus Infection
1 Intermediate filament protein ifb-2
2 Sorbitol dehydrogenase sodh-1
3 Putative selenium-binding protein R11G10.2
Vibrio: C. elegans as a Laboratory Model for Vibrio Infections 421

50 µm
50 µm
(a) 50 µm (b) (c)

FIGURE 28.2  CLSM images of C. elegans exposed to GFP-tagged (a) E. coli OP50, (b) V. parahaemolyticus CM2, and
(c) V. parahaemolyticus ATCC.

C. elegans against V. alginolyticus challenge and highlights the significance of unfolded protein response
(UPR) pathway during bacterial infection.
Changes in C. elegans against V. parahaemolyticus infection were reported by Durai et al.113 The study
compared the virulence of two V. parahaemolyticus strains and proved that intestinal colonization of the
bacterium was associated with pharyngeal damage, leading to the death of the nematode in 48 h. The
colonization of V. parahaemolyticus in C. elegans intestine was localized using CLSM, and the intensity
of the colonization was measured and compared with respective controls. The 2.5-dimensional topog-
raphy of the colonized animal clearly showed the colonization of V. parahaemolyticus CM2 throughout
the intestinal lumen of C. elegans (Figure 28.2). The study also showed that transferring the nematodes
to a benign food source cleared the intestinal colonization of V. parahaemolyticus, which was similar
to the earlier observations during Staphylococcus aureus infections.114 The virulence of V. parahaemo-
lyticus is associated with the production of two major toxins, TDH and TRH.115–118 In addition, few
strains lacking these virulence genes/factors were also found to be pathogenic, indicating that infection
was independent of toxin production.119 Results from experiments showed that strain CM2 is negative
to hemolysin test performed in the Wagatsuma agar using human erythrocytes. Molecular studies for
the tdh and trh supported the hemolysin test. However, the presence of tlh favored the notion that CM2
could be a pathogenic strain of V. parahaemolyticus. Previous reports show that tlh has been used for the
molecular-level identifications of the Vibrio species.119–121 C. elegans shows a selective response to food
source and pathogenic bacteria with the phenomenon of olfactory learning.

28.10 Conclusion
With a mode of transmission through seafood contaminants, vibriosis in humans represents a challenge
to researchers. Given that many reoccurring Vibrio species containing various virulent factors (including
antibiotic resistance genes) are being identified every year, these bacteria pose a significant public risk
to human populations. Precautions and proper identification of Vibrio contamination in seafood using
advanced tools provide an effective way to control and reduce risk of infection. Further elucidation of the
molecular mechanisms of Vibrio infections and virulent determinants using model organism will help
identify potent anti-infective compounds that can be used for combating vibriosis.

Acknowledgements
This work was supported in part by the Grants from UGC, DBT, and Department of Science and
Technology to Dr. K. Balamurugan. Financial support to Durai by Lady Tata Memorial Trust’s Senior
Scholarship is thankfully acknowledged.
422 Laboratory Models for Foodborne Infections

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101. O’Rourke, D., Baban, D., Demidova, M., Mott, R. & Hodgkin, J. Genomic clusters, putative pathogen
recognition molecules, and antimicrobial genes are induced by infection of C. elegans with M. nema-
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102. Jansson, H.B. Adhesion of conidia of Drechmeria coniospora to Caenorhabditis elegans wild type and
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103. Couillault, C. et al. TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR
domain adaptor protein TIR-1, an ortholog of human SARM. Nat Immunol 5, 488–94 (2004).
104. Troemel, E.R. et al. p38 MAPK regulates expression of immune response genes and contributes to
longevity in C. elegans. PLoS Genet 2, e183 (2006).
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105. Félix, M.A. et al. Natural and experimental infection of Caenorhabditis nematodes by novel viruses
related to nodaviruses. PLoS Biol 9, e1000586 (2011).
106. Félix, M.A. & Braendle, C. The natural history of Caenorhabditis elegans. Curr Biol 20, R965–9 (2010).
107. Joelsson, A., Liu, Z. & Zhu, J. Genetic and phenotypic diversity of quorum-sensing systems in clinical
and environmental isolates of Vibrio cholerae. Infect Immun 74, 1141–7 (2006).
108. Sahu, S.N. et al. Genomic analysis of immune response against Vibrio cholerae hemolysin in
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109. Durai, S., Pandian, S.K. & Balamurugan, K. Establishment of a Caenorhabditis elegans infection
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Genome Biol 8, R194 (2007).
29
Yersinia

Xin Wang, Ran Duan, Junrong Liang, Wenpeng Gu, and Huaiqi Jing

CONTENTS
29.1 Mouse Model................................................................................................................................. 428
29.1.1 Lifestyle and Interaction with Host.................................................................................. 428
29.1.2 Virulence of Pathogen...................................................................................................... 428
29.2 Pig Model...................................................................................................................................... 430
29.3 Caenorhabditis elegans Model......................................................................................................431
29.4 Rabbit Model..................................................................................................................................431
29.5 Cell Models....................................................................................................................................431
29.5.1 Adhesion and Invasion of Mammalian Cells....................................................................432
29.5.2 Modulation of Host Immune Defense Mechanisms..........................................................432
29.6 Conclusion......................................................................................................................................433
References................................................................................................................................................433

Yersinia is a genus of Gram-negative, rod-shaped, facultative anaerobes in the family Enterobacteriaceae.

Of the 17 species identified within the genus, three are pathogenic to humans, namely, the causative
agent of plague (or black death)—Yersinia pestis—and two enteropathogens—Yersinia enterocolitica
and Yersinia pseudotuberculosis.1 While Y. pestis is generally transmitted by a flea bite, the other two are
foodborne pathogens, with Y. enterocolitica associated with gastrointestinal syndromes (ranging from
acute enteritis to mesenteric lymphadenitis) and Y. pseudotuberculosis implicated in mesenteric adenitis
and septicemia. Despite their differences in transmission routes and disease types or severity, these three
pathogens demonstrate a common tropism for lymphoid tissues and the capacity to resist the nonspecific
immune response of the host, in particular phagocytosis and killing by macrophages and polymorpho-
nuclear leukocytes (PMNs).2–4 Y. enterocolitica is recovered from diverse animal sources, from farm
animals and domestic pets to free-living and captive wild animals.1,5 Pigs and dogs are considered to be
the primary reservoir of human pathogenic Y. enterocolitica strains in the world, and the isolation rate
from tonsil samples is the highest.6–9
The virulence of pathogenic Y. enterocolitica is attributed to a 70-kb pYV (plasmid for Yersinia vir-
ulence) plasmid and some chromosome-encoded factors. A similarly sized plasmid, termed pCD, is
found in Y. pestis,10 and pYV is also present in Y. pseudotuberculosis.11 Despite this common feature,
the diseases caused by the two enteropathogenic yersiniae are chronic in nature and obviously differ-
ent from plague. The virulent plasmid pYV encodes Yersinia adhesin A (YadA) and the Yersinia outer
protein (Yop) virulon, a system consisting of secreted proteins called Yops and their dedicated type III
­secretion system (T3SS) apparatus called Ysc.12 The Ysc apparatus forms a channel composed of several
proteins.13 Yop proteins fall into two categories. Some are intracellular effectors, while the others are
“translocators” needed to deliver the effectors across the eukaryotic plasma membrane into eukaryotic
cells. The translocators (YopB, YopD, LcrV) form a pore in the eukaryotic cell’s plasma membrane.
The effector Yops are YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT.12 The chromosome-
encoded virulence factors include ail (attachment and invasion locus), inv (invasin), and yst (Yersinia
stable toxin). The high pathogenicity island (HPI) bearing an iron acquisition system only exists in the

427
428 Laboratory Models for Foodborne Infections

highly pathogenic biotype 1B strains.14 Many studies have reported the role of biotype 1A strains in
foodborne outbreaks, in which the pYV plasmid is absent.15

29.1 Mouse Model
29.1.1 Lifestyle and Interaction with Host
After orogastric inoculation of mice, Y. enterocolitica selectively invades the Peyer’s patches (PP) via M
cells.16–18 This invasion leads to recruitment of PMNs, formation of microabscesses comprising extra-
cellular Yersinia, and, finally, complete destruction of the cytoarchitecture of the PP. Subsequently yer-
siniae disseminate to lymph nodes, spleen, and liver where they form microcolonies/microabscesses,
suggesting that Y. enterocolitica disseminates via the lymphatic vessels.16 Similar to in vivo observa-
tions, Yersinia manifests in vitro resistance to phagocytosis by macrophages19,20 and by PMNs.21–24 Once
phagocytosed, Y. pseudotuberculosis and Y. enterocolitica generally do not survive long. These findings
suggest that Y. enterocolitica is an extracellular pathogen and that its survival strategy is based on avoid-
ing the nonspecific immune response.
Recent studies showed that in the oral mouse infection model using RFP- (red fluorescent protein) and
GFP- (green fluorescent protein) expressing yersiniae, the oral Y. enterocolitica infection of mice leads
to monoclonal microabscess formation in PP, spleen, and liver. Furthermore, experiments with red and
green fluorescing yersiniae revealed that only very few yersiniae were able to invade PP from the gut
lumen and that both Yersinia and the host contribute to this phenomenon. In contrast to other enteric
pathogens such as Salmonella and Shigella, Yersinia is predominantly an extracellular pathogen.18,25,26
Initially, innate host defenses such as PMNs, macrophages, and natural killer (NK) cells are involved
in controlling Yersinia infection. But subsequently, a robust adaptive immune response is required to
overcome Yersinia infection. Specific Abs27,28 as well as IFN-γ-producing CD4 and CD8 T cells play
an essential role in clearing Yersinia infection and have been shown to mediate protection in adoptive
transfer experiments.29
Wang et al. investigated the lethality, cytokine alterations, and histopathological changes of patho-
genic Y. enterocolitica bioserotypes 1B/O:8 and 2/O:9 in susceptible BALB/c and resistant C57BL/6
mice. They found that the 50% lethal dose (LD50) for the pathogenic Y. enterocolitica bioserotype 1B/O:8
was 103 CFU in both BALB/c and C57BL/6 mice; while the LD50 for the bioserotype 2/O:9 was 108
CFU in BALB/c mice and 109 CFU in C57BL/6 mice. In BALB/c mice, the bioserotype 2/O:9 induced
a higher level of GM-CSF, IL-6, and TNF-α than the bioserotype 1B/O:8, but the status was reversed
subsequently. Levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-10, and IL-12 following infection with the
bioserotype 1B/O:8 were always higher than those with the bioserotype 2/O:9 (Figure 29.1). The histo-
pathological changes in the liver and spleen in BALB/c mice infected with the two strains were similar at
different times and doses. These observations indicate the different immunological effects and changes
in the mouse model due to pathogenic Y. enterocolitica 1B/O:8 and 2/O:9 infections.30

29.1.2 Virulence of Pathogen
In an oral mouse model, a phospholipase-negative strain was constructed to ascertain whether phospho-
lipase A plays a role in pathogenesis. At a dose of 108 CFU/mouse, fewer mutant strains were recovered
and less flamed tissue was found from the mesenteric lymph nodes and PP. When given extremely high
dose (109 CFU/mouse), the numbers of foci and the extent of inflammation were noticeably less as well,
confirming the role of phospholipase A in pathogenesis.31
The T3SS is a complex system used to deliver bacterial proteins into the cytosol of host cells.32 The
proteins secreted by T3SSs are known as effectors working together to establish an extracellular infec-
tion that disrupts the actin cytoskeleton and deregulates signal pathways.33
YopM was originally described as Yop48 in Y. enterocolitica.34 According to the LD50 test,
the yopM mutant has a strongly reduced virulence in mice. Bacterial counts in liver and spleen showed the
yopM mutant had a reduced ability to multiply in the host.35 When three challenge routes were done for YopE
mutant, it was avirulent after oral or intraperitoneal infection but virulent after intravenous injection.36
Yersinia 429

80 3000
1000
70 900
Cytokine GM-CSF(pg/ml)

2500

Cytokine IFN-γ(pg/ml)
800

Cytokine IL-1β(pg/ml)
60
700 2000
50 high O:8 high O:8 high O:8
600
high O:9 high O:9 high O:9
40 500 1500
medium O:8 medium O:8 medium O:8
30 medium O:9 400 medium O:9 medium O:9
1000 low O:8
low O:8 300 low O:8
20 low O:9
low O:9 200 low O:9
500
10 100
0 0 0
3 6 9 12 15 24 48 72 h 3 6 9 12 15 24 48 72 h 3 6 9 12 15 24 48 72 h

(A) (B) (C)

–500

200
180 1000 1800

Cytokine IFN-4(pg/ml)
900
160 1600
Cytokine IL-2(pg/ml)

Cytokine IL-5(pg/ml)
800
140 high O:8 high O:8 1400 high O:8
high O:9 700 high O:9 high O:9
120 1200
medium O:8 600 medium O:8 medium O:8
100 medium O:9 1000
medium O:9 500 medium O:9
80 low O:8 400 low O:8 800 low O:8
60 low O:9 low O:9 600 low O:9
300
40 200 400
20 100 200
0 0 0
3 6 9 12 15 24 48 72 h 3 6 9 12 15 24 48 72 h 3 6 9 12 15 24 48 72 h

120000
(D) (E) (F)
3000
250
100000 2500
Cytokine IL-10(pg/ml)

Cytokine IL-12(pg/ml)
Cytokine IL-6(pg/ml)

200
80000 high O:8 2000 high O:8 high O:8
high O:9 high O:9 high O:9
150
60000 medium O:8 1500 medium O:8 medium O:8
medium O:9 medium O:9 medium O:9
40000 100 low O:8
low O:8 1000 low O:8
low O:9 low O:9 low O:9
20000 500 50

0 0 0
3 6 9 12 15 24 48 72 h 3 6 9 12 15 24 48 72 h 3 6 9 12 15 24 48 72 h
–500 –50
700
(G) (H) (I)
600
Cytokine TNF-α(pg/ml)

high O:8
500
high O:9
400 medium O:8
medium O:9
300 low O:8
low O:9
200

100

0
3 6 9 12 15 24 48 72 h
–100
(J)

FIGURE 29.1  Comparison of cytokines between two strains in BALB/C mice. (A) GM-CSF, (B) IFN-γ, (C) IL-1β,
(D) IL-2, (E) IL-4, (F) IL-5, (G) IL-6, (H) IL-10, (I) IL-12, and (J) TNF-α. *, The cytokine values were statistically signifi-
cant comparing the two strains in different groups (p < 0.05). ▲, All of the cytokine values were statistically significant
comparing the two strains in every group (p < 0.05). This figure demonstrates the comparison for each cytokine changes
between two strains. (From Wang, X. et al., Mol. Immunol., 55, 365–371, 2013. With permission from Elsevier.)

Compared to extensive study of YopP effects in vitro, the role of YopP in vivo is inadequately inves-
tigated. Monack et al. have shown that Y. pseudotuberculosis induces apoptosis of Mac-1 cells in mice,
which is probably responsible for the attenuated phenotype of a yopJ mutant.37 In 2015, a study using
Listeria model shows for the first time that YopP inhibits the development of an effective CD8 T cell
response in mice.38 In vitro, this is due to the rapid induction of dendritic cell (DC) apoptosis and matura-
tion,39 whereas in vivo a possible mechanism for this could be the inhibition of Ag presentation by DC.38
By using mouse model infected with Y. pseudotuberculosis, Rosqvist et al. showed that a strain unable to
express YopH has a reduced ability to resist phagocytosis,20 while the ability to resist phagocytosis could be
complemented by introduction of a plasmid carrying only the yopH gene, demonstrating that YopH is indeed
involved in the antiphagocytic effect. However, mutation of yopH did not completely abolish the resistance to
phagocytosis, suggesting another virulence factor was involved in this phenomenon.
A systematic study of Yops pathogenicity of the highly virulent mouse O:8 strain administered by oral
and intravenous routes in mice showed the yopH, yopO, yopP, yopE, yopM, and yopQ mutants had only
modest defects in colonization of the small intestine (SI) and PP,40 consistent with that of Y. pseudotuber-
culosis,41 indicating that no single effector is absolutely necessary for the colonizing. YopH, YopQ, and
YopM are important for inducing systemic disease, whereas YopO, YopP, and YopE are dispensable to
reach the spleen and liver. On the other hand, YopT seems to slightly decrease the virulence in both oral
and intravenous model.40
430 Laboratory Models for Foodborne Infections

With the help of the mouse model, the yersinia T3SS has been studied in detail using molecular, cellular,
genetic, and biochemical techniques, yielding insight into the efficiency and sophistication of the complex.
Progression of Y. enterocolitica infection in mouse model closely mirrors that in the human. In natural
infection, Y. enterocolitica enters by the oral route.42 Ruiz-Bravo et al. conducted a comparative experiment
with three challenge routes (oral, intraperitoneal, and intravenous) using moderate–virulent O:9 strains with
or without pYV and noted considerable differences between oral and intraperitoneal infections by the two
strains.43 This suggests the virulence potential of Y. enterocolitica may be influenced by the route of infec-
tion. Therefore, at the beginning of an experiment, the route of infection should be chosen prudently.

29.2 Pig Model
In swine, fecal shedding may stop soon after the ingestion of Y. enterocolitica, while tonsils may carry
the bacterium from hours to months,44 indicating that tonsils represent a more reliable tissue for the
indication of an infection/colonization by Y. enterocolitica. As is known, clinical response to Y. entero-
colitica infection in piglets is related to inoculum size.45 A dose of 4 × 1010 CFU could cause death46;
in contrast, a dose of 3 × 108 CFU would provoke a subclinical response as near as possible to a natural
infection.44 The early infection showed a transient bacteremia within the first 3 h postinfection.45 So if
pigs are infected within hours prior to slaughter, carcass of the animal might not be completely cleared
of the microorganism and thus result in widespread contamination.44
The high prevalence of O:3 strains in fattening pigs indicates serotype- and host-specific colonization
strategies. Comparison of O:3 patient isolate and O:8 8081v strain in minipigs and mice models showed
that small genetic variations contribute to profound changes of the virulence gene expression pattern.47 In
the O:3 strain, IS1667 insertion (an additional promoter) as well as the more stable variant of RovA (invA
transcriptional activator protein) toward Lon protease resulted in high expression of invasion at 37°C,
indicating a fine adjustment of pathogenicity to pig, with a higher body temperature (Figure 29.2).47 They

25°C 37°C

InvA InvA

O:8 O:3 O:8 O:3

InvA

RovA RovA

O:8 invA invA

PinvA PinvA
RovA Lon protease

RovA*
RovA*
** ** **
O:3 Transposase invA
PTn PinvA

IS1667

FIGURE 29.2  Different expression of the invasin gene invA in Y. enterocolitica O:3 and O:8. Whole-cell extracts prepared
from overnight cultures of Y. enterocolitica O:3 and O:8 grown at 25°C and 37°C were separated on SDS-polyacrylamide
gels and analyzed by western blotting using polyclonal antibodies directed against InvA. Overview of the invA promoter
region of Y. enterocolitica O:8 (upper panel) and O:3 (lower panel). The transcriptional start site of the invA and the IS1667-
specific promoter are indicated by broken arrows. The blue boxes represent the RovA binding sites. The light blue box
illustrates the RovA binding site in the IS1667 sequence. The RovA dimer is given in red; the stars illustrate the amino acid
substitution that renders the RovA protein less susceptible to the Lon protease. (From Valentin-Weigand, P., et al., Int. J.
Med. Microbiol, 304, 824–834, 2014. With permission from Elsevier.)
Yersinia 431

further uncovered why the cell adhesion and invasion properties were restricted to pig-associated O:3
strains. Coinfection studies with O:3 wild and mutant strains demonstrated that small variations within
the O:3 genome improved colonization/survival in swine but had only a minor effect on the colonization
in mice. In addition, a deletion of the invA abolished long-term colonization in the pigs.48
As the most important reservoir and asymptomatic carriers of Y. enterocolitica prevalent serotype,49,50
the pig (model) is useful for studies ranging from colonization tropisms to research on the virulence
properties of O:3 strains.

29.3  C aenorhabditis elegans Model

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. It has been suggested
that bacterial–invertebrate interaction has shaped the evolution of microbial pathogenicity.51 Spanier
et al. demonstrated that Y. enterocolitica strains W22703 (2/O:9) and WA314 (1B/O:8) colonize and kill
C. elegans following oral infection and that the insecticidal toxin TcaA, which is expressed only at ambi-
ent temperature, is required for full nematocidal activity but alone is not sufficient.52 In contrast, with
Y. pestis and Y. pseudotuberculosis, both of which use biofilm-dependent mechanisms, biofilm formation
is not responsible for the nematocidal activity in Y. enterocolitica.53
Temperature is a key environmental clue for the expression of yersinia genes.54,55 Several genetic deter-
minants of Y. enterocolitica are repressed at 37°C but induced at a low temperature, suggesting a role in
invertebrates.56,57 However, Yop effectors and YadA are produced only at 37°C, while C. elegans cannot
be cultivated above 25°C. This is perhaps why Inv, YopE, and YadA did not remarkably contribute to
C. elegans killing.52
Together, these data do not support the feasibility of the C. elegans infection model for Y. enteroco-
litica pathogenicity toward mammals.

29.4 Rabbit Model
Rabbits have been a suitable and reproducible animal model to examine the course of intestinal Yersinia
infection.58–62 In mouse model, diarrhea is not a major symptom.63,64 But the infection of the young rabbit
resembles that in children: the oral inoculation induces both a clear diarrhea and a systemic invasion.59,60,65–67
Thus, Delor and Cornells constructed one for establishment of the virulent role of Yst toxin.68
The pathogenic and immunogenic potential of parental and mutant Y. enterocolitica strains have been
successfully evaluated in oral rabbit model, showing O-antigen is required for full virulence. The strain
that lacks Wzz protein (the O-antigen chain length determinant) is more attenuated than one that lacks
Wzy protein (expressing only one repeat unit of lipopolyasccharide [LPS]).69
On the basis of sodA finding in mouse model,70 Najdenski et al.71 established a rabbit model to show
that the sodA mutant strain not only attenuated in dissemination into various organs but also showed
an improved antibody response. In contrast to previous LPS mutant strains, this mutant was noticeably
more sensitive to leukocytes. Besides, they first observed yersiniae dissemination to brain in experimen-
tal infection. More surprising were the pathomorphological changes not established in tonsils with the
virulent strain but with sodA mutant strain. Together, these showed the potential of sodA mutant to be a
candidate for immunization.
Overall, the susceptibility of rabbits to Y. enterocolitica appeared to be more variable in this study
focused on diarrhea manifestation and immunomorphology.

29.5 Cell Models
In cell models, innate host defenses such as PMNs, macrophages, and NK cells are involved in con-
trolling Yersinia infection initially, but a robust adaptive immune response is required to overcome
Yersinia infection subsequently. Specific Abs27,28 as well as IFN-γ-producing CD4 and CD8 T cells play
432 Laboratory Models for Foodborne Infections

an essential role in clearing Yersinia infection and have been shown to mediate protection in adoptive
transfer experiments.29 Some of the Yops (including YopE, YopH, YopO, and YopM) are delivered into
the host cell cytosol where they damage the cytoskeleton and disrupt the signaling network. Infected
macrophages displayed general features of apoptosis such as membrane blebbing (apoptotic body forma-
tion) and nuclear and cellular shrinkage.

29.5.1 Adhesion and Invasion of Mammalian Cells

The ability to penetrate host cells can be considered crucial to infectious processes caused by enteric
pathogens. Specifically, two chromosomal genes, inv and ail, and one plasmidial gene, yadA, of
Y. enterocolitica are associated with ability to adhere and invade mammalian cells.72 Invasin (Inv) is the
first adhesin required for Yersinia to cause infection. Inv is optimally expressed at 26°C or under acidic
conditions at 37°C. This protein is thought to be expressed in the cell wall of the bacteria when they
reach the small intestine and is required for the initial steps of host colonization and penetration into
the intestinal epithelium cells.73 Another virulence-associated adhesin of enteropathogenic yersiniae is
Ail (for attachment and invasion locus). Ail is expressed at 37°C under aerobic conditions. Like Inv, Ail
is chromosomally encoded and also mediates the binding and invasion of epithelial cells.74,75 The major
adhesin from enteropathogenic yersiniae is YadA. Different from Inv and Ail, YadA is encoded by the
virulence plasmid pYV. YadA can bind to collagen, fibronectin, and laminin and mediate adhesion to
epithelial cells and macrophages. Beyond adhesion, YadA can act as an autoagglutinin, has antiphago-
cytic properties, and serum resistance.76–78 The heat-stable enterotoxin Yst, encoded by the chromosomal
ystA gene, is another factor related to Y. enterocolitica virulence. Reports suggest that it may cause diar-
rhea.79 Yst affects binding and activation of cell-associated guanylate cyclase and elevation of intracel-
lular concentrations of cyclic GMP. Besides inv, ail, ystA, and virF, other chromosomal and plasmidial
encoded genes are responsible for the expression of virulence determinants in Y. enterocolitica, like iron
uptake, fimbriae, flagella, and urease, among others.

29.5.2 Modulation of Host Immune Defense Mechanisms

To overcome host defense mechanisms, pathogenic yersiniae translocate a mixture of effector proteins
called Yops into the cytosol of eukaryotic cells by their T3SSs to inhibit the innate and adaptive immune
system of the host. These major antihost determinants are located on a 70-kb virulence plasmid pYV,
which encodes a protein microinjection apparatus called the T3SS and at least six effector proteins
(Yops; YopH, YopO/YpkA, YopP/YopJ, YopE, YopM, and YopT).12,80,81 The main function of these
Yops is to inhibit the immune response of the host. At least four Yops (YopH, YopE, YopT, and YopO/
YpkA) are involved in inhibiting phagocytosis of yersiniae by disrupting the cytoskeleton of PMNs and
macrophages.82
A series of studies suggest that Yersinia blocks macrophage TNF-α production by downregulation
of mitogen-activated protein kinase (MAPK) activities.83 A recent report from Orth et al. revealed
that YopP/YopJ binds and inactivates members of the MAPK kinase superfamily, which function as
upstream MAPK activators.84 The NF-κB system is central to innate immunity. It controls the synthesis
of cytokines, acute-phase proteins, and adhesion molecules and mediates cellular survival by prevention
of apoptosis. Certain extracellular stimuli, such as TNF-α, genotoxic agents, and ionizing radiation,
simultaneously activate proapoptotic and antiapoptotic pathways in eukaryotic cells. NF-κB functions
to upregulate synthesis of proteins that counteract the proapoptotic pathways, such as members of the
inhibitor of apoptosis protein (IAP), TNFR-associated factor, and Bcl-2 families.85
Studies using HeLa cells have shown that YopH dephosphorylates p130cas, paxillin, and the focal
adhesion kinase (FAK), leading to disruption of the focal adhesion and a reduced invasin-mediated
engulfment.86,87 Andersson et al. showed that exposure of J774A.1 macrophages to yopH mutant of
Y. pseudotuberculosis infection resulted in a transient increase in tyrosine phosphorylation of a number
of proteins, including paxillin, which is known to be tyrosine phosphorylated upon Fc-receptor-mediated
signaling associated with phagocytosis in macrophages.88
Yersinia 433

Studies involving Y. enterocolitica and Y. pseudotuberculosis demonstrated that Yersinia triggers

apoptosis of cultured macrophages, and infected macrophages displayed general features of apopto-
sis, such as membrane blebbing, cellular shrinkage, and DNA fragmentation.25,89 Monack et al. found
that YopJ, the Y. pseudotuberculosis homologue of YopP, is required for the induction of the cell death
process.25 YopP/YopJ plays an anti-inflammatory role by preventing the activation of the transcription
factor NF-κB.90,91 It also induces rapid apoptosis of macrophages. The apoptosis process is accompanied
by cleavage of the cytosolic protein BID, the release of cytochrome c, and the cleavage of caspase-3
and 7. The release of cytochrome c and the cleavage of BID can both be inhibited by caspase inhibitors,
suggesting that YopP/YopJ interferes with a signaling pathway upstream of the mitochondria.89

29.6 Conclusion
Y. enterocolitica and Y. pseudotuberculosis are foodborne bacteria associated with gastrointestinal
syndromes (from acute enteritis to mesenteric lymphadenitis), or mesenteric adenitis, and septice-
mia. These pathogens generate a number of proteins that help evade host innate immune response and
replicate inside the host. Use of mouse models revealed the interaction between the host and the extracel-
lular lifestyle of the pathogen. Application of molecular, cellular, genetic, and biochemical techniques
in combination with in vitro models yielded valuable insights into efficiency and sophistication of the
Yersinia T3SS. Comparative studies employing minipig and mouse models uncovered that O:3 strains
utilize serotype- and host-specific colonization strategies in fattening pigs. While C. elegans infection
model is not feasible for pathogenicity study due to inappropriate temperature, rabbit model is suscep-
tible to diarrhea manifestation. Cell models are useful for investigating different stages of yersinia infec-
tion: adhesion and invasion, and evasion of host innate and adaptive immune mechanisms.

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 Section IV

Foodborne Infections due to Fungi

30
Alternaria

Alicia Rodríguez, Andrea Patriarca, Mar Rodríguez,

María Jesús Andrade, and Juan José Córdoba

CONTENTS
30.1 Introduction................................................................................................................................... 441
30.2 Foodborne Diseases due to Alternaria Mycotoxins..................................................................... 442
30.3 Investigation and Analysis of Toxigenic Alternaria Strains......................................................... 443
30.4 Laboratory Models for Foodborne Alternaria Analysis............................................................... 445
30.4.1 Animal Laboratory Models.............................................................................................. 447
30.4.2 In Vitro Laboratory Models.............................................................................................. 447
30.4.2.1 Cell Line Systems............................................................................................. 447
30.4.2.2 Other In Vitro Models....................................................................................... 449
30.5 Conclusions................................................................................................................................... 450
Acknowledgments....................................................................................................................................451
References................................................................................................................................................451

30.1 Introduction
Alternaria is a ubiquitous fungal genus covering 275 species,1 and it has been typified as saprobic,
endophytic, and pathogenic. Currently, 24 Alternaria sections, 16 of which are newly described,
and 6 monotypic lineages are recognized.2 Alternaria is associated with a variety of foods. Species
of Alternaria are serious plant pathogens that damage crops in the field and cause postharvest
decays. In addition, with the ability to grow at low temperatures and low water activity (aw),3,4
Alternaria spp. may contaminate and infect fruit and vegetables kept refrigerated.5,6 The most com-
monly reported species include Alternaria alternata, Alternaria tenuissima, Alternaria arbores-
cens, Alternaria radicina, Alternaria brassicae, Alternaria brassicicola, and Alternaria infectoria.
Depending on geographical areas, a specific Alternaria species can predominate over others. For
instance, A. tenuissima is a more frequently isolated species in Argentinean wheat7 than A. alternata
and A. infectoria, which have been reported as the predominant species in cereals in several stud-
ies worldwide.8–12 Alternaria species colonize a range of plants including cereals,13 tomatoes,14 oil-
seeds,15 apples,16 berries, grapes, citrus fruits,17 carrots,18 and others. Some of these moulds may have
a negative impact on foods due to spoilage, causing economic losses and infections.19 It is well known
that Alternaria colonization of ripening ears can result in black pointed grain and impact directly on
flour color of bread-making wheats,20 its spores can be allergenic and cause asthma,21 and some spe-
cies may synthesize mycotoxins whose high toxicity is detrimental to both human health and animal
welfare.

441
442 Laboratory Models for Foodborne Infections

30.2 Foodborne Diseases due to Alternaria Mycotoxins

The main cause of Alternaria foodborne diseases is due to the production and accumulation of mycotox-
ins on foods that could lead to acute and, more commonly, chronic effects. Although growth of moulds
in foods is not necessarily associated with the formation of mycotoxins,22 in many cases their presence
could lead to the accumulation of these metabolites in foods. Only about 30 of the 120 known secondary
metabolites of Alternaria are considered toxic to humans and animals, many of them acting as phyto-
toxins.23 Only a small proportion of such phytotoxins has been chemically characterized and reported
to act as mycotoxins in humans and animals. The most important Alternaria mycotoxins are alternariol
(AOH), alternariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN),
and altertoxins I, II, and III (ATX-I, -II, and -III),24 which belong to three structural classes25: (1) diben-
zopyrone derivatives (AOH, AME, ALT), (2) perylene derivatives (ATX-I, -II, -III), and (3) tetramic acid
derivatives (TeA). The production of important Alternaria mycotoxins by the most common Alternaria
species is shown in Table 30.1.
The environmental conditions in which specific Alternaria mycotoxins may be produced have been
identified. In general, the conditions for mycotoxin production are slightly narrower in terms of tem-
perature and aw conditions relative to those for growth. Studies suggest that mycotoxin production by
Alternaria strains is more variable than their growth.26–28 There are numerous studies on A. alternata
growth and evaluation of mycotoxin production in different matrices such as wheat, soya beans, sor-
ghum, and tomatoes.4,29–32 The optimal conditions for AOH, AME, and TeA production varied with
strains and contaminated food. Thus, e.g., for AOH, in soya beans the optimum conditions are at 25°C
and 0.98 aw,30,31 while in tomatoes the optimum conditions are at 21°C and 0.954 aw after 28 days.32 For
AME, the optimal production in irradiated soya beans is around 25°C and 0.98 aw, and no production
is found at temperatures higher than 30°C.30,31 However, the maximum concentration of AME in toma-
toes is obtained at 0.954 aw and 35°C, although high amounts are also produced at 21°C.32 For TeA, the
optimal conditions for production are 0.98 aw over a range of 25°C–30°C on soya-bean-based media,27
0.99–0.93 aw over 28 days growth periods on sorghum,31 and 0.982 aw at 21°C after 28 days of incuba-
tion on tomato-based media.32 In the latter, this mycotoxin is even detected after 28 days of incubation
at 0.982 aw and 6°C. Recently, a study has been performed about the impact of aw × temperature condi-
tions on ATX-II by using strains of A. tenuissima isolated from Argentinean cereals.28 In this study, the

TABLE 30.1
Toxic Secondary Metabolites Produced by the Most Important Alternaria Species and Foods Involved
Species Toxic Secondary Metabolites Foods References
A. alternata AOH, AME, ALT, TeA, ATX-I, ATX-II, Tomato, red pepper, fresh 37,87,88
ATX-III fruit, vegetables, peanut,
wheat
A. tenuissima AOH, AME, ATX-I, -III, TeA Strawberries 28,88
A. arborescens AOH, AME, TEN Fruits, tomato, barley grain 87,89
A. radicina ATX-I, -II, -III, TeA Umbelliferous plants 88
A. brassicae AOH, AME Mustard, opium 90,91
A. brassicicola Small phytotoxic metabolites (causal Rape 92–94
agent of black spot disease)
A. infectoria 4Z-infectopyrone, novae-zelandin A, Wheat 87
novae-zelandin B, phomapyrone A, B,
E, G, F, D
Source: Ostry, V., World Mycotoxin J., 1, 175, 2008.
AOH, alternariol; AME, alternariol methyl ether; ALT, altenuene; TeA, tenuazonic acid; ATX-I, -II, -III,
altertoxins-I, -II, and -III.
Alternaria 443

optimal conditions for ALTX-II production were 0.98 aw and 30°C, although A. tenuissima strains also
accumulate significant amounts of this toxin at 34°C.
Taking into account the ecological conditions for mycotoxin production by Alternaria species, it is
not surprising that these toxins can contaminate a wide range of food and foodstuffs including, e.g.,
citrus, cereals, olives, tomatoes, apples, seeds, berries, and juices (reviewed by Scott,33 see Table 30.1).
The maximum levels of Alternaria mycotoxins reported in marketed food products are in the range of
1–103 μg/kg.24
Alternaria toxins have recently received increasing attention. The European Commission asked the
European Food Safety Authority (EFSA) to review the safety of Alternaria toxins in food and feed.34
EFSA concluded that Alternaria toxins are of high concern for public health. In fact, exposure to
Alternaria toxins has been linked to esophageal cancer in Linxian Province, China, as well as in areas of
South Africa where high levels of A. alternata contamination have been found in grains.23,35,36 However,
due to the possible co-occurrence of mycotoxins in contaminated grains, this correlation may not have
been exclusively ascribed to Alternaria toxins. For this reason, it is important to have adequate methods
first for analyzing and identifying Alternaria species and their toxins and second to examine the toxicity
for foodborne mycotoxin-producing Alternaria.

30.3 Investigation and Analysis of Toxigenic Alternaria Strains

The isolation and identification of Alternaria species from food products is based on traditional meth-
ods including culturing under standardized conditions and observation of morphological characteristics.
Besides being laborious, time consuming, and sometimes restricted to experts in this field, this identi-
fication scheme is difficult owing to variations within the same species as the morphological charac-
teristics are very sensitive to the cultural conditions. The most recent taxonomic keys, as reviewed by
Simmons1 in his identification manual, provide descriptions and illustrations of at least 296 taxa, based
on the examination of stable isolates in axenic culture. The isolates are cultured in potato carrot agar
(PCA) and V8 agar and incubated for 7 days at 23°C under an alternating light/dark cycle consisting of
8 h of cool-white fluorescent daylight and 16 h darkness so as to observe morphological characteristics
(Figure 30.1).
An obvious drawback of such traditional methods for detecting potential mycotoxin-producing
Alternaria is the need of an evaluation of the production of mycotoxins by foodborne strains. The pro-
duction of secondary metabolites has been used as a means of classification and identification, taking
advantage of the enormous potential of this genus to biosynthesize secondary metabolites. Combinations
of conidial morphology, genetics, and pathogenicity have been shown to be valuable in Alternaria
taxonomy.8
Many published reports have demonstrated the benefits of using in vitro techniques to investigate
mycotoxin production by foodborne Alternaria. Having been used to assess the mycotoxin production
for many years, rice represents an excellent matrix for comparison of toxigenic potential of mould strains
isolated from different foods and geographical regions. Mycotoxin production could be evaluated in
culture media made with sterilized rice or directly in sterilized rice. However, it should be noted that
autoclaved rice has defective mechanical barriers and suppressed defenses because of their thermolabil-
ity. This provides conditions for Alternaria strains to express their whole toxigenic potential, which may
or may not be possible under natural or real-food conditions.37
Mycotoxin production is strain dependent and additionally conditioned by the food in which the mould
strain is growing and by the environmental factors. Thus, it is necessary to evaluate the potential risk
of toxin accumulation in the specific food matrix. Culture media based on the food are frequently used
as it simplifies the extraction of the toxins, such as tomato medium, wheat agar medium, etc.28,32 When
the real food matrix is used, e.g., cereal grains or legumes, it could be autoclaved4 or gamma irradiated31
to eliminate the natural mycobiota. However, as these mild treatments do not destroy mycotoxins, the
natural occurrence of the toxins of interest should be verified before their utilization for analyzing the
Alternaria toxigenic potential.
444 Laboratory Models for Foodborne Infections

FOOD

Isolation of Alternaria

Traditional Molecular
methods methods

Potato carrot and DNA

V8 agar Evaluation of mycotoxin
production
Alternating light/dark (Food-matrix-based media) PCR
cycle incubation Real-time PCR
Organic extracts

Microscopy
(Morphology)
Chromatographic
techniques for
mycotoxin analysis

Mycotoxin
purification

Laboratory models for

toxicity studies

-Animal models
-Cell lines
-Other tests

FIGURE 30.1  Procedures for analyzing foodborne Alternaria.

For the evaluation of mycotoxin production by Alternaria, analytical methods based on chromatogra-
phy are often used for their extraction and detection from the food matrices (Figure 30.1). Usually, a sol-
vent extraction from solid foods with organic solvents, such as dichloromethane, methanol, acetonitrile,
or ethyl acetate, is required, although an acidic extraction or a further acidification step is preferable to
increase the recovery for TeA.33 Cleanup procedures are necessary when a complex matrix is involved.
Successive steps of solvent partitioning, solid phase extraction (SPE) columns, or solid phase microex-
traction are common cleanup techniques used in most food matrices.24
Mycotoxin determination has been traditionally carried out by thin-layer chromatography (TLC),
gas chromatography (GC), and mainly high-performance liquid chromatography (HPLC), usually with
ultraviolet (UV) detection, although fluorescence and electrochemical detection have also been used
for certain toxins. Atmospheric pressure chemical ionization (APCI), LC-mass spectroscopy (MS), and
LC-MS/MS have been applied to the determination and confirmation of AOH and AME identity at
sub ng/mL levels.33,38 Several multimycotoxin methods have been recently developed, many of which
include the detection of Alternaria toxins, most of them based on LC-MS/MS systems. A multimethod
for detection of 33 mycotoxins (including AOH and AME) in various food matrices has been developed
by Spanjer et al.,39 based on LC-MS/MS using an electrospray ionization interface (ESI) for detec-
tion and MS/MS with multiple reaction monitoring (MRM). Rasmussen et al.40 developed a method for
simultaneous detection of 27 mycotoxins (including AOH, AME, TeA, and altersetin) in maize silage.
A simple pH-buffered sample extraction has been developed based on the QuEChERS method, and
the detection is made by LC-MS/MS without further cleanup. Wang et al.41 developed an LC-MS/MS
method for detecting 17 mycotoxins (including AOH and AME) with application in traditional Chinese
medicine products, using a MultiPurification Column for cleanup.
Alternaria 445

Even though several techniques are effective for Alternaria mycotoxin quantification in different
food matrices, there are a number of difficulties related to the performance of these methods, such
as the efficiency of sample cleanup, low or unequal recoveries for some of the toxins, availability of
standards, lack of reference materials for food and feedstuffs, etc. In addition, these methods are time
consuming.
As an alternative to traditional methods, nucleic-acid-based techniques are being increasingly
applied to examine the taxonomic relationships among Alternaria species. Most of them have been
focused on small-spored catenulate Alternaria, which show little resolution in their molecular phy-
logeny. However, cladistics analyses of “housekeeping genes” commonly used for other genera,
such as the mitochondrial large subunit (mtLSU) ribosomal DNA, internal transcribed spacer (ITS),
β-tubulin, translation elongation factor α, calmodulin, actin, chitin synthetase, etc., failed to discrimi-
nate among the small-spored species, except for the A. infectoria species group.42–45 There are also
genomic techniques to detect, identify, and quantify toxigenic moulds in foodstuffs. So far, there are
no molecular methods based on genes involved in the Alternaria mycotoxin biosynthesis pathways;
however, some methods, which have used unique conserved genes that distinguish toxigenic and non-
toxigenic Alternaria spp., have been developed successfully. Several molecular methods have been
developed to detect the presence of Alternaria spores and biomass in foods, such as the one reported
by Zur et al.,46 a polymerase chain reaction (PCR)-based method with primers specific to the ITS1
and ITS2 of the 5.8S rRNA gene of Alternaria to detect its presence in commercial tomato products.
The main inconvenience of these methods is that viable and nonviable cells cannot be distinguished,
thus resulting in an overestimation of the amount of spores that can actually produce mycotoxin in a
food product. More recently, Crespo-Sempere et al.47 developed a method including a pretreatment of
samples with nucleic-acid-intercalating dyes (propidium monoazide, PMA) prior to quantitative PCR.
PMA selectively penetrates cells with a damaged membrane, inhibiting DNA amplification during
PCR. The method, based on a primer pair (Alt4 and Alt5) specific to Alternaria spp., allowed quan-
tifying a detection limit of 102 spores/g on tomatoes. Even though the tomato matrix had a protective
effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and non-
viable cells, the method is still a suitable tool for quantifying viable Alternaria cells, which could be
useful for estimating potential risks of mycotoxin contamination. The main drawback of these nucleic-
acid-based methods as well as of the traditional identification of Alternaria followed by evaluation of
mycotoxin production is that they only give information about the negative potential effect derived
of the Alternaria presence in foods. Although the latter techniques allow taking corrective actions
to avoid presence of mycotoxin-producing Alternaria on foods, for a more appropriate investigation
of foodborne Alternaria it is necessary to use laboratory animals or cell system models in which the
effect of Alternaria extracts or Alternaria mycotoxin contaminated foods can be evaluated. This could
allow estimating the real risk of the presence of Alternaria in foods.

30.4 Laboratory Models for Foodborne Alternaria Analysis

The design of extensive biological tests for foodborne Alternaria analysis based on mycotoxin effects is
limited because of the high number of animals needed and ethical and scientific reasons.48–50 In addi-
tion, such methods have an important limitation related to the toxin amount required for performing
the experiments. For this, the total synthesis of AOH, AME, ALT, and iso-ALT has been already estab-
lished.51,52 However, due mainly to the mycotoxin production and purification costs, the use of high
amounts of toxins for in vivo trials is still difficult. Most of the toxicity tests have thus been performed
using Alternaria culture extracts53–56 or in vitro models focused on cellular systems to obtain data about
the toxicity from the different purified mycotoxins synthesized by Alternaria species. On the other hand,
the extent of in vivo mycotoxin absorption could be predicted using in vitro permeability and solubility
measurements. For this, animal and human cell lines, undifferentiated epithelial cells, have been exten-
sively used for understanding the mechanisms by which some mycotoxins induce different disorders. In
Tables 30.2 and 30.3, the most relevant laboratory models described for analyzing foodborne Alternaria
have been summarized.
446 Laboratory Models for Foodborne Infections

TABLE 30.2
Animal Models for Evaluating the Toxic Effects of Alternaria Extracts and
Produced Mycotoxins
Animals Alternaria Extracts/Mycotoxin References
Chickens Alternaria extract 55
TeA 10
Rats Alternaria extract 55
Chicken embryos Alternaria extract 53
Mice Alternaria extract 53,55
AOH 50
TeA 48
Dogs TeA 10
TeA, tenuazonic acid; AOH, alternariol.

TABLE 30.3
Cell Line Systems and Other In Vitro Tests Used for Studying the Toxic Effects of Alternaria Mycotoxins
Cell Lines/In Vitro Mycotoxins/Alternaria
Assay Extracts Studied Toxic Effects References
Caco-2 AOH/AME Absorption, metabolism, oxidative stress 59–61
HT-29 AOH/AME DNA-damaging properties 62–65
TeA, ATX-I, -II, -III DNA-damaging properties 66
V79 AOH/AME Cell proliferation, clastogenic potential, 63,67,69
mutagenicity
ATX-I, -II Mutagenicity 68,70
H4IIE ATX-I Mutagenicity 70
MLC AOH Mutagenicity 69
CHL/3T3/L-O2 TeA Cytotoxicity 71
RAW 264.7 AOH DNA-damaging properties, autophagy, 72–74
senescence
HCT116 AOH Cell death mode 75
AOH/AME Cytotoxicity 76
Ishikawa AOH Estrogenic potential 67
H295R AOH Alteration in steroid hormone production 77
RGA AOH Alterations in androgen, progestogen, 77
glucocorticoid, estrogen nuclear receptor
Porcine endometrial AOH Oestrogenic effect 78
Hepa-1c1c7/Hepa- AOH/AME Cytotoxicity 79,80
1c1c4/Hepa-1c1c12
HepG2 AOH Cytotoxicity 81
A431 AOH/AME/ALT Viability and cytotoxicity potential 62
MCF-7 AOH DNA-damaging properties 62
Artemia salina test AOH, AME, ALT, ATX-I, Toxicity 82,83
TeA and Alternaria extracts
Ames Salmonella test Alternaria extracts and Mutagenicity 56,84
ATX-I, -II, and -III
ALT, AOH, AME, ATX-I, Mutagenicity 70,85
TEN, TeA
Human digestive tract Alternaria extracts and AOH Bioaccessibility from food 48
simulation and ALT
AOH, alternariol; AME, alternariol methyl ether; TeA, tenuazonic acid; ATX-I, -II, -III, altertoxins-I, -II and -III;
ALT, altenuene.
Alternaria 447

30.4.1 Animal Laboratory Models

Several animals could be used as model for foodborne Alternaria analysis using extracts from Alternaria
growing on culture media. Culture extracts of these moulds show toxic effects toward chickens and
rats,55 and chicken embryos.53 In addition, Alternaria cultures have also been found to be teratogenic
and fetotoxic in mice53,55 (Table 30.2). The main disadvantage of using these kinds of extracts is their
unknown composition, which restricts obtaining of conclusions from the experiments. However, these
tests in animal models give general information on the toxicity of Alternaria extracts, which could be
useful in foodborne Alternaria investigation.
Regarding assays of purified Alternaria toxins in laboratory animal models, few scientific reports
focusing on the combined toxicokinetic and in vivo genotoxicity potential have been published.
Schuchardt et al.50 tested the toxicokinetic behavior and in vivo genotoxicity of AOH in NMRI mice.
The study revealed low systemic absorption, and the target organ toxicity would most likely be restricted
to the gastrointestinal tract. In addition, the above study demonstrated no toxic or genotoxic effect of
AOH in bone marrow, and the comet assay with liver tissue did not indicate systemic genotoxicity too.
TeA has been shown to be subacutely toxic in mice [LD50 (lethal dose 50) i.v. 115 mg/kg]. In fact, mice
consuming feed contaminated with TeA for 10 months developed alterations in the oesophageal mucosa,
suggesting the possibility that progression to esophageal cancer might occur after prolonged exposure.48
ALT and ATX-I are acutely toxic in mice, with LD50 of 50 and 200 mg/kg, respectively22 (Table 30.2).
TeA is acutely toxic for chickens and dogs. In particular, in chicken feed with increasing TeA levels
from sublethal to lethal, the feed efficiency was progressively reduced, the weight gain was suppressed,
and the internal hemorrhaging increased.10 In dogs, a daily ingestion of 10 mg/kg body weight provokes
hemorrhaging in several organs10 (Table 30.2).
Although whichever former animal model could be used for further foodborne Alternaria investiga-
tion, in vitro laboratory models (including cell lines) offer a good alternative to extract adequate conclu-
sions in this kind of investigation. These experiments will be discussed in the next section.

In Vitro Laboratory Models

30.4.2 
30.4.2.1 Cell Line Systems
In vitro cell line models have to satisfy the following two basic requirements: (1) must be available and
easy to handle for high-throughput testing and (2) yield data must support interpretation of results for
in vivo situation.57 Only a few cell lines are utilized to examine the toxic effect of Alternaria myco-
toxins. The main characteristics of these cellular systems and the effect produced by the Alternaria
mycotoxins and Alternaria culture extracts on them will be detailed in this section and summarized
in Table 30.3.
In most of the in vitro studies of the gut, the human colon tumorigenic cell lines Caco-2 and HT-29
have been used for studies related to intestinal cell function and differentiation.57,58 Several experiments
have been performed to examine the absorption, metabolism, and oxidative stress of AOH and AME in
Caco-2 cells.59–61 There are similarities between the structures of the conjugated metabolites of AOH
and AME since they are readily conjugated with glucuronic acid and sulfate.61 In this study, it was also
shown that, based on apparent permeability coefficients, AOH is expected to be extensively and rapidly
absorbed from the intestinal lumen in vivo and reaches the portal blood both as aglycone and as gluc-
uronide and sulfate.61 Oxidative stress due to AOH is confirmed by alteration of glutathione (GSH) and
the enzymes involved in the redox system after 15, 30, and 60 μM of AOH exposure during 24 h.60 AOH
also causes DNA damage after 24 h in Caco-2 cells.60 Cytotoxicity of AOH (from 3.125 to 100 μM) was
determined during 24, 48, and 72 h of exposure by using different end points.59 AOH decreases the cell
viability and induces a strong oxidative stress in Caco-2 cells.59
HT-29 cell line has been mainly used to evaluate the DNA-damaging properties of AOH and AME.
Fehr et al.62 characterized AOH as a poison of topoisomerase I and II, with a certain selectivity for the
IIα isoform playing a role in the DNA strand breakage. On the contrary, several studies63–65 demonstrated
that AOH and AME do not have an apparent negative effect on DNA integrity, or at least they do not play
448 Laboratory Models for Foodborne Infections

a main role in the genotoxic properties. In a toxicity-guided fractionation assay, aimed to identify DNA
strand breaking impact compounds in extracts obtained from rice heavily infested with A. alternata, the
HT-29 cell line was used.66 TeA, AOH, and AME do not cause significant DNA-damaging effects, while
ATX-II contributes to the genotoxic effects of the extracts, showing potent DNA-damaging properties in
HT-29 cells. ATX-II does not provoke oxidative stress, while it does influence the cell cycle distribution
of HT-29 cells. The inhibition of proliferation of HT-29 cells (seen in a sulforhodamine B assay) matched
this interference with the cell cycle, thus arguing for effects on inhibition of cell proliferation rather than
cytotoxicity.66
Chinese hamster cell line V79 has been developed from lung tissue of a young male Chinese ham-
ster. This cell line is used to investigate DNA damage, the effect on cell proliferation, and the clasto-
genic potential of AOH. Pfeiffer et al.63 showed that AOH and AME induce DNA strand breaks in a
concentration-dependent manner in the cell line V79. Lehmann et al.67 demonstrated that AOH inhibits
cell proliferation by interference with the cell cycle and induces kinetochore-negative micronucleus
(MN) in cultured V79 cells. ATX II is a potent mutagen in the cell line V79, inducing a concentration-
dependent increase of mutations at the hypoxanthine guanine phosphoribosyltransferase gene locus at
concentrations similar to that of the established mutagen 4-quinoline-N-oxide.68 However, the muta-
genic potency of AOH is at least 50 times lower than that of ATX II.69 In contrast to AOH and AME,
ATX II does not affect the cell cycle of V79 cells. ATX II also causes DNA strand breaks in V79
cells, being more potent than AOH and AME. Schrader et al.70 demonstrated that nitrosylated ATX-I
is mutagenic to V79 cells too. They also used rat hepatoma H4IIE cells to evaluate the mutagenicity of
ATX-I, since they retain more metabolic activities and have been used as a model for assessing chemi-
cal effects on the induction of cytochrome P450, aryl hydrocarbon hydroxylase, and epoxide hydrolase
activities.70 This study concludes that if nitrosylated ATX-I is similarly toxic to other cell types, in par-
ticular esophageal cells, carcinogenesis would be promoted through cell death and the proliferation of
neighboring cells to generate replacements. The sensitivity of H4IIE cells also suggested that exposure
to nitrosylated ATX-I could lead to liver damage/carcinogenesis.
The mutagenicity of AOH at the thymidine kinase (TK) gene locus in mouse lymphoma L5178 tk+/−
(MLC) cells has also been investigated.69 Concentrations higher than or equal to 10 μM AOH give rise
to a significant and concentration-dependent induction of TK mutations in MLC cells. Discrimination
between small and large colonies in the TK assay reveals the predominant induction of small colonies,
which are indicative for extensive chromosomal deletions and correlate with the induction of micronuclei
in MLC cells.
Chinese hamster lung (CHL) cells are also used to evaluate TeA toxicity to mammalian cells. Cell
proliferation inhibition increases with extension of toxin exposure time.71 Total protein content in culture
of cells was measured and showed that TeA decreases the total protein content after 72 h toxin exposure
with half maximal effective concentration (EC50) value of 56.28 μg/mL. In the same study, 3T3 mouse
fibroblasts (3T3 cells) and human hepatocytes (L-O2 cells) showed a similar response. It was concluded
that TeA is the most cytotoxic to 3T3 cells, followed by CHL cells and L-O2 cells. At lower concentra-
tions, TeA had lower cytotoxicity to the human hepatocytes, which suggests that such cells may tolerate
TeA at low concentrations.
The murine macrophage cell line RAW 264.7 was used to test AOH toxicity as a response to DNA dam-
age and to test if AOH induces autophagy, senescence, abnormal morphology, and cell cycle arrest.72–74
High AOH concentrations block cell proliferation and increase the level of reactive oxygen species
(ROS). However they are not directly linked to each other. It seems that AOH-induced DNA damage and
resulting transcriptional changes in the p21, MDM2, and Cyclin B genes contribute to the reduced cell
proliferation, while the expression of Sestrin 2 gene would contribute to the oxidant defense.74 Besides,
it seems that the AOH-induced cell cycle arrest, most probably due to DNA damage and incomplete
decatenation, is followed by very specific morphological changes.72 Furthermore, Solhaug et al.73 found
that autophagy and senescence using the RAW264.7 macrophage model are related to stress responses
caused by the DNA-damaging AOH.
Human colon carcinoma cells HCT116 are used to evaluate the cell death mode and pathways trig-
gered by AOH 75 and the cytotoxic potential of AOH and AME mixtures.76 Cells treated with AOH show
a loss of cell viability by inducing apoptosis.75 Bensassi et al.76 demonstrated that the exposure of HTC116
Alternaria 449

cells to low cytotoxic AOH doses causes a moderate cytotoxicity. However, when AOH and AME are
combined, they exert a significant increase in their toxic potential.
The cell line Ishikawa allows examining the estrogenic potential of AOH.67 In this study, the estroge-
nicity of AOH was about 10,000-fold weaker than of the endogenous hormone E2. To identify if AOH
may alter steroidogenesis, an in vitro screening assay based on measuring alterations in steroid hormone
production and the expression of several important genes that encode the various enzymes involved
in steroidogenesis using the human adrenocortical carcinoma cell line H295R has been performed by
Frizzel et al.77 They demonstrated that AOH has the ability to interfere with steroidogenesis pathway
since it modifies the expression of important genes in steroidogenesis in H295R cells.
Human breast adenocarcinoma cell line RGA is of great relevance to evaluate the effect of AOH on
the androgen, progestogen, glucocorticoid, and estrogen nuclear receptors present in those cells.77 These
authors concluded that AOH has a weak estrogenic activity when tested in the estrogen-responsive RGA
cells. The sensitivity of AOH to different cell types may be an important factor when considering this
mycotoxin oestrogenic effect. In another study performed by Willemsen et al.,78 the estrogenic influence
of the ER α in porcine endometrial cells was not detected.
The evaluation of AOH and AME toxicity in the mouse hepatoma Hepa-1c1c7 cell line with intact
aryl hydrocarbon receptor (AhR) signaling and the Hepa-1c1c4 and Hepa-1c1c12 cell lines, which are
deficient for the AhR nuclear translocator (ARNT) or the AhR, respectively,79 has been performed. It was
demonstrated that AOH and AME are novel inducers of the AhR/ARNT pathway, which mediates induc-
tion of CYP1A1 and apoptosis and might thus contribute to the toxicity of these mycotoxins. Burkhardt
et al.80 used the same three mouse hepatoma (Hepa-1) cell lines with intact and compromised AhR sig-
naling to compare their activities for hydroxylation, methylation, and glucuronidation.
The liver hepatocellular carcinoma HepG2 cell line is widely used to evaluate the cytotoxic effects
of AOH.81 In this study, HepG2 cells were treated at different concentrations over 24, 48, and 72 h. The
half maximal inhibitory concentration (IC50) values were from 65 to 96 μM for AOH. Pfeiffer et al.63 also
demonstrated that AOH and AME cause a concentration-dependent induction of DNA strand breaks in
HepG2 cells.
The human vulva carcinoma A431 cell line allows testing the viability and cytotoxicity potential of
AOH, AME, and ALT as well as to perform a single-cell gel electrophoresis (comet assay) and inmuno-
band depletion assay.62 They demonstrated that AOH and AME significantly increase the rate of DNA
strand breaks in A431 at micromolar concentrations, whereas ALT does not affect DNA integrity up to
100 μM. The inmunoband depletion assay allowed for the observation that AOH affects topoisomerase,
preferentially the IIα isoform. This latter conclusion was also obtained by the same authors62 when they
utilized the human breast adenocarcinoma MCF-7 cell line to evaluate the effects of AOH on topoisomer-
ase I and II by means of relataxion and cleavage assays and decatenation and cleavage assays, respectively.

30.4.2.2 Other In Vitro Models

In addition to cell model systems, other in vitro models can be used to evaluate the toxicity of foodborne
Alternaria. Thus, Artemia salina is widely used to check the toxicity of purified AOH, AME, ALT,
ATX-I, and TeA and different Alternaria extracts.82 In this model, it has been demonstrated that the
toxicity of culture extracts is not caused by the presence of the purified Alternaria mycotoxins studied.
Panigrahi and Dallin83 evaluated the toxicity of several Alternaria mycotoxins (TeA, AOH, AME, ALT,
and ATX-I) to A. salina by means of a disc assay and scoring larvae mortality by examining under a
microscope in a multiwell plate. TeA, AOH, ATX-I, and ALT produced dose-related mortality in the lar-
vae, with the LC50 well concentration for the toxins being 75, 100, 200, and 375 μg/mL, respectively. No
effect was observed for AME on the larvae, although the authors reported a low solubility of this toxin
in the solvents used in this study.
The Ames Salmonella test is widely used to evaluate toxicity and can be utilized to test mutagenicity
of Alternaria. In this test, the crude Alternaria extract shows mutagenic activity.56,84 When this extract
was partitioned, the mutagenic fractions corresponded to ATX-I, ATX-II, and ATX-III. The results
indicated that all altertoxins were mutagenic in the decreasing order of ATX-III>ATXII>ATX-I,
with the potency of ATX-III approximately 10-fold lower than that of aflatoxin B1. Schrader et al.70
450 Laboratory Models for Foodborne Infections

also examined the major Alternaria toxins (ALT, AOH, AME, ATX-I, TEN, and TeA) by the Ames
Salmonella test, using strains TA98 and TA100 to detect histidine revertants that result from frame-
shift and base pair mutations, respectively. In the absence of nitrosylation, they found that ATX-1
was positive for mutagenicity in TA98, while the all other toxins were negative. In general, nitro-
sylation increases mutagenic potencies, although no effect was observed on TEN and TeA mutage-
nicities. These findings suggest that the presence of mould contamination that contains ATX-I in
foodstuff could pose a potent carcinogenic health hazard when combined with a diet high in nitrites
and nitrates. In a later work, Schrader et al.85 reexamined mutagenicity of these toxins with and
without nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run
of C’s, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more
sensitive to oxidative damage. The results suggested that ATX-I, AME, and AOH induce mutations at
AT sites, possibly through oxidative damage. In addition, nitrosylation enhances ATX-I mutagenicity
at runs of C’s.
Several methods have been developed for simulating the human digestive tract. Dall’Asta et al.48 used a
method proposed by Versantvoort et al.86 for assessing the bioaccessibility of mycotoxins from food, and
another one that involved the use of fecal water obtained as a mixture from healthy volunteers follow-
ing an omnivore diet. Samples from the simulated digestion assay were taken at the end of the duodenal
phase and directly analyzed by LC–MS/MS. Samples from the fecal fermentation assay were taken after
30 min and 24 h of incubation and analyzed by LC–MS/MS after high-speed centrifugation. Their results
showed that both AOH and ALT were totally stable along the simulated human gastrointestinal tract.
Neither digestive conditions nor human gut microbiome seemed to be able to degrade AOH and ALT, as
these were fully recovered in the fecal water after 24 h of incubation.

30.5 Conclusions
Alternaria species are found in a wide range of foods including cereals, fruits, and vegetables due to
their ability to grow at low temperatures and low aw. Although some Alternaria spp. may be allergenic
and cause asthma, the synthesis of mycotoxins when the environmental conditions are adequate is the
most worrying undesirable effect. In spite of the fact that Alternaria species produce a huge number of
secondary metabolites, only a small proportion is considered toxic to humans and animals. The most
important Alternaria mycotoxins are AOH, AME, ALT, TeA, TEN, ATX-I, ATX-II, and ATX-III.
The diagnosis of mycotoxin-producing Alternaria and Alternaria toxins in food could be performed
by morphological, biochemical/analytical, and genetic molecular techniques. However, laboratory mod-
els based on animals or in vitro (cell lines or biological tests) systems are needed to examine the toxic
effects of Alternaria and their toxins on humans and animals, thus completing the investigation in food-
borne Alternaria.
Several animal models based on chickens, rats, dogs, and mice could be used to evaluate toxic effects
of Alternaria culture extracts and some of the main purified Alternaria toxins. They may be adapted for
foodborne analysis.
In addition, cell lines and other models (Ames Salmonella test, A. salina, etc.) are also available
for this purpose. Ethical and scientific reasons due to the use of high number of animals and the toxin
amount required for these experiments are the main limitations on the performance of laboratory toxic-
ity for animal model assays. Cell system models could be a good alternative for foodborne Alternaria
analysis, with human cell lines such as Caco-2 and HT-29, the Chinese hamster V79 cells, rat H4IIE
hepatoma cells, or murine macrophage cell line RAW 264.7 being the most appropriate. Overall, these
assays demonstrated that all Alternaria toxins are cytotoxic and that AOH and AME are also genotoxic
and mutagenic; however, AOH is a weak estrogenic. TeA has been reported to exert cytotoxic properties
as well as being acutely toxic. The altertoxins ATX-I, ATX-II, and ATX-III have been shown to be more
mutagenic and acutely toxic than AOH and AME.
The toxicity of Alternaria mycotoxins still needs a further research. Not enough information is
available on the toxicokinetics, including metabolism, of the most toxicologically relevant foodborne
Alternaria toxins.
Alternaria 451

Acknowledgments
This work has been funded by the Spanish Comisión Interministerial de Ciencia y Tecnología with the
projects AGL2013-45729P and Carnisenusa CSD2007-00016, Consolider Ingenio 2010 and GRU09162
of the Junta de Extremadura and FEDER. Dr. Alicia Rodríguez was supported by a postdoctoral research
fellowship (PO12016) from the Gobierno de Extremadura, Consejería de Empleo, Empresa e Innovación,
and the Fondo Social Europeo (FSE).

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41. Wang, S. et al., Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix
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42. Andrew, M., Peever, T.L. and Pryor, B.M., An expanded multilocus phylogeny does not resolve morpho-
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44. Pryor, B.M. and Gilbertson, R.L., Molecular phylogenetic relationships amongst Alternaria species and
related fungi based upon analysis of nuclear ITS and mt SSU rDNA sequences, Mycol. Res., 104, 1312,
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45. Roberts, R.G., Reymond, S.T. and Andersen, B., RAPD fragment pattern analysis and morphological
segregation of small-spored Alternaria species and species groups, Mycol. Res., 104, 151, 2000.
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in food products, J. Food Protect., 62, 1191, 1999.
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48. Dall’Asta, C., Cirlini, M. and Falavigna, C., Mycotoxins from Alternaria: Toxicological implications,
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Alternaria toxins, EFSA supporting publication, EN-679, 2014.
51. Altemöller, M., Podlech, J. and Fenske, D., Total synthesis of altenuene and isoaltenuene, Eur. J. Org.
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52. Koch, K. et al., Total synthesis of alternariol, J. Org. Chem., 70, 3275, 2005.
53. Griffin, G.F. and Chu, F.S., Toxicity of the Alternaria metabolites alternariol, alternariol monomethyl
ether, altenuene and tenuazonic acid in the chicken embryo assay, Appl. Environ. Microbiol., 46, 1420,
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55. Sauer, D.B. et al., Toxicity of Alternaria metabolites found in weathered sorghum grain at harvest,
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59. Fernández-Blanco, C., Font, G. and Ruiz, M., Oxidative stress of alternariol in Caco-2 cells, Toxicol.
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capacity by alternariol in Caco-2 cells, Toxicol. Lett., 235, 61, 2015.
61. Burkhardt, B., Pfeiffer, E. and Metzler, M., Absorption and metabolism of the mycotoxins alternariol
and alternariol-9-methyl ether in Caco-2 cells in vitro, Mycotoxin Res., 25, 149, 2009.
62. Fehr, M. et al., Alternariol acts as a topoisomerase poison, preferentially affecting the IIα isoform, Mol.
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65. Schwarz, C., Kreutzer, M. and Marko, D., Minor contribution of alternariol, alternariol monomethyl
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31
Aspergillus

László Kredics, János Varga†, Rajagopalaboopathi Jayasudha, Sándor Kocsubé, Nikolett

Baranyi, Coimbatore Subramanian Shobana, Muthusamy Chandrasekaran, Shine
Kadaikunnan, Venkatapathy Narendran, Csaba Vágvölgyi, and Palanisamy Manikandan

CONTENTS
31.1 Introduction....................................................................................................................................455
31.2 The Genus Aspergillus...................................................................................................................455
31.3 Mycotoxins of Aspergillus Species and Mycotoxicoses............................................................... 456
31.3.1 Aflatoxins......................................................................................................................... 456
31.3.2 Ochratoxins...................................................................................................................... 457
31.3.3 Fumonisins....................................................................................................................... 458
31.3.4 Patulin............................................................................................................................... 458
31.3.5 Other Mycotoxins Produced by Aspergilli...................................................................... 458
31.4 Laboratory Models for Foodborne Aspergillus Mycotoxicoses................................................... 459
31.4.1 Invertebrate Models.......................................................................................................... 459
31.4.2 Rodent Models.................................................................................................................. 460
31.4.3 Human and Animal Cell Lines........................................................................................ 479
31.5 Conclusions................................................................................................................................... 487
Acknowledgments................................................................................................................................... 487
References............................................................................................................................................... 487

31.1 Introduction
Members of the genus Aspergillus are well-known for their detrimental effects on human and animal
health. Invasive and superficial infections caused by Aspergillus species are more and more frequently
reported, and the genus is on the top of the list of filamentous fungal genera of clinical importance.
However, clinical infections due to Aspergilli are rarely foodborne. On the other hand, the ability of cer-
tain members of the genus to produce various mycotoxins frequently results in serious health problems,
both in animals and humans, that are commonly known as Aspergillus mycotoxicoses.
This chapter aims to give an overview about the laboratory models used to study Aspergillus myco-
toxicoses, with a special focus on the most recent publications applying rodent models (rats and mice)
and mammalian cell lines, the application of which proved especially fruitful to gain more insight into
the toxic and carcinogenic effects of AFB1, OTA, and other Aspergillus mycotoxins.

31.2 The Genus Aspergillus

The name Aspergillus was first used in 1729 for filamentous fungi with conidial heads in Nova Plantarum
Genera by the Italian mycologist and priest Pietro Antonio Micheli as the spore-bearing structures

† In memoriam Prof. János Varga

455
456 Laboratory Models for Foodborne Infections

characteristic of the genus reminded him an aspergillum (holy water sprinkler) used in Catholic liturgy.
These fungi were later recognized as active decomposers and also as the causal agents of diseases in
animals and humans. Today, Aspergillus is considered one of the most economically important and most
widely distributed filamentous fungal genera on our planet [1]. The genus Aspergillus is taxonomically
divided into 8 subgenera and more than 20 sections consisting of about 300–350 species [2,3].
Aspergillus species can be both beneficial and harmful for mankind. The greatest economic ben-
efit of the genus is the ability to produce industrial enzymes (e.g., amylases, glycosidases, pectinases,
proteases), and organic acids (e.g., citric acid, gluconic acid, itaconic acid) [1]. Aspergillus oryzae,
A. sojae, and A. tamarii are known as the “koji moulds” and have been used for centuries in oriental
food fermentation processes for the production of soy sauce and sake [1]. Aspergilli can also produce
a series of secondary metabolites with useful pharmaceutical and biotechnological properties (e.g., the
cholesterol-lowering drug lovastatin produced by A. terreus and some other species). However, taxa of
this genus may also have serious negative impacts on animal and human health as they cause different
diseases called aspergilloses. The most common human pathogen from the genus is A. fumigatus, which
is responsible for more than 90% of both invasive and noninvasive human aspergilloses; however, other
species of the genus are also capable of causing infections [4]. Various fungal diseases like keratitis or
onychomycosis, allergic bronchopulmonary aspergillosis (ABPA), and asthma have been reported to be
caused by the growth or spores of Aspergillus species (among others). Additionally, Aspergilli produce a
range of mycotoxins, which can be harmful to animals and humans. Aspergillus species can contaminate
foods and feeds at different pre- and postharvest, as well as processing or handling stages. The most
economically important Aspergillus mycotoxins identified as contaminants in foods and feeds are the
aflatoxins, ochratoxins, patulin, and fumonisins.

31.3 Mycotoxins of Aspergillus Species and Mycotoxicoses

31.3.1 Aflatoxins
The most thoroughly studied mycotoxins produced by Aspergillus species are aflatoxins (AFs). Regarding
the discovery of AFs, toxicity of animal feeds containing contaminated peanut meal in the early 1960s
resulted in the death of more than 100,000 turkeys from acute liver necrosis [5,6]. The toxin-producing
fungus was identified as Aspergillus flavus, and the toxic agent as AF, named after the first isolated
producer. AFs form a group of structurally related difuranocoumarin compounds named as AFs B1, B2,
G1, and G2 based on their fluorescence under ultraviolet light (blue or green) and relative mobility during
thin-layer chromatography. Aflatoxin B1 (AFB1) is the most efficient known natural carcinogen [7] and
is generally the major AF produced by toxigenic strains. The hydroxylated metabolite AFM1 is primar-
ily found in animal fluids (milk and urine) and tissues as a metabolic product of AFB1 [8]. Recent data
indicate that AFs are produced by more than 20 species belonging to sections Flavi, Nidulantes, and
Ochraceorosei of the genus Aspergillus [8–11]. Although the ability of AF production was claimed for
a series of other fungal species and genera (actually even for bacteria), these observations could not be
confirmed [8].
Sterigmatocystin (STC) is a penultimate precursor of AFs, which is itself a toxic and carcino-
genic substance produced by many species belonging mainly to sections Nidulantes and Versicolores.
STC production also occurs in the phylogenetically unrelated genera Bipolaris, Chaetomium, and
Humicola [11].
AFs are primarily produced by A. flavus and A. parasiticus on agricultural crops like cereals (e.g.,
corn, rice, wheat), cotton, peanut, pepper, spices, and tree nuts [8]. A. nomius was found to be an impor-
tant producer of AFs in Brazil nuts [12]. The other AF-producing species (e.g., A. ochraceoroseus,
A. venezuelensis, A. astellata) are not regarded as potential health hazards, as they produce only small
amounts of AFs or are encountered in food products rarely or not at all. STC producers are also fre-
quently identified in various substrates including indoor air, food, and feed [13,14].
Aspergillus 457

AFs are hepatotoxic and hepatocarcinogenic and usually considered as belonging to the most
potent naturally occurring carcinogens [15,16]. The diseases caused by AF consumption are known
as aflatoxicoses. Acute aflatoxicosis is the result of moderate-to-high level AF consumption. Several
deaths were attributed to aflatoxicosis in tropical and subtropical regions of the world, primar-
ily due to the consumption of contaminated corn [17]. Chronic aflatoxicosis results from prolonged
ingestion of low-to-moderate levels of AFs and can be characterized by usually subclinical effects that
are difficult to recognize. Comprehensive studies indicated that AF is a risk factor for hepatocellular
carcinoma in humans, especially in Asia and the sub-Saharan part of Africa [18,19]. The International
Agency for Research on Cancer (IARC) classified AFs as group I category carcinogens [20].
Because of its toxicity, the content of AF is restricted in food and feed products in more than 100
countries [21].

31.3.2 Ochratoxins
Ochratoxins (OTs) were discovered in 1965 during a systematic examination of the metabolites of
moulds, including Aspergillus ochraceus [22]. Ochratoxin A (OTA) is a cyclic pentaketide dihydroiso-
coumarin derivative linked to an l-β-phenylalanine by an amide bond. The most toxic member of the
group is OTA which is a chlorinated pentaketide. Several OTA-derived metabolites have also been iden-
tified: OTB is a dechlorinated analogue of OTA, while OTC is a chlorinated ethyl ester derivative. OTα is
the isocoumarin core of OTA, while OTβ is a dechlorinated analogue of OTα. Other derivatives include
4-hydroxy-OTA, 10-hydroxy-OTA, OTA methyl ester, OTB ethyl and methyl esters, and several amino
acid analogues [23,24].
Several nephropathies affecting animals and humans can be attributed to OTA, including the Danish
porcine nephropathy as well as renal disorders detected in other animals [25]. In the case of humans, OTA
is often mentioned as the possible causative agent of the Balkan Endemic Nephropathy [26], although
recently aristolochic acid was suggested to play a major role in the etiology of this disease [27]. OTA also
proved to exhibit immunosuppressive, teratogenic, hepatotoxic, and carcinogenic properties [28]. The
IARC classified OTA as a possible human carcinogen in the Group 2B category [20].
Under cooler, temperate climates, OTs are mainly produced by Penicillium species (P. verrucosum
and P. nordicum), while in warmer and tropical regions they are produced by a series of Aspergillus
spp. Aspergillus isolates usually produce both OTA and its dechlorinated analogue OTB, while
Penicillium spp. produce OTA only. In Aspergillus section Circumdati, the recently described new
species A. affinis, A. fresenii, A. occultus, A. pulvericola, A. sesamicola [29,30], as well as A. cre-
tensis, some isolates of A. ochraceopetaliformis (formerly described as A. flocculosus), A. muricatus,
A. ochraceus, A. pseudoelegans, A. roseoglobulosus, A. steynii, and A. westerdijkiae are able to pro-
duce consistently high or medium amounts of OTA. Seven further species of the genus produce OTA
inconsistently or in trace amounts only, including A. ostianus, A. melleus, A. persii, A. salwaensis, A.
sclerotiorum, A. subramanianii, and A. westlandensis. The most relevant species for potential OTA
production in rice, coffee, beverages, and other foodstuffs are A. ochraceus, A. steynii, and A. west-
erdijkiae. In Aspergillus section Flavi, A. albertensis and A. alliaceus are capable of OTA production
in synthetic media [31]. Among the representatives of Aspergillus section Nigri, A. carbonarius, A.
niger, A. sclerotioniger, and A. ­welwitschiae proved to be potential OTA-producing fungi [32,33]. The
majority of A. carbonarius isolates can produce large amounts of OTA; however, different surveys
reported about only 5%–15% of A. niger and A. welwitschiae isolates producing OTA in smaller
quantities [33].
OTs can be detected in several agricultural products including cereals, cocoa, coffee beans, corn,
figs, grapes, peanut, red pepper, rice, soybeans, and spices [34]. A. westerdijkiae proved to be impor-
tant in the OTA contamination of coffee beans in Thailand [35], while A. alliaceus was found respon-
sible for the OTA contamination of figs in California [36]. Members of Aspergillus section Nigri,
primarily A. carbonarius, are responsible for the OTA contamination of grapes and grape-derived
products [37].
458 Laboratory Models for Foodborne Infections

31.3.3 Fumonisins
Several species of the genera Aspergillus and Fusarium (e.g., Fusarium verticillioides) are able to
produce fumonisins (for details, see Chapter 34 on Fusarium). Sequencing the genome of Aspergillus
niger resulted in the discovery of a region homologous with the fumonisin gene cluster of Fusaria
[38]. The production of fumonisins by A. niger was confirmed later [39], while recent studies revealed
that another black Aspergillus species, A. welwitschiae can also produce fumonisins [33]. Black
Aspergilli produce mainly fumonisins B2 and B4, but other analogues have also been identified in
smaller quantities [40].
Black Aspergilli and fusaria are both among the saprotrophic fungi that are widely distributed. Among
the fumonisin-producing Aspergillus species, A. niger is an important opportunistic pathogen of grapes,
causing raisin mould and bunch or berry rot [41]. It is also able to infect apples, corn, mangoes, onions,
peanuts, and dried meat products [42]. In contrast to fusaria, which produces large amounts of fumoni-
sins in plant-extract- (barley, carrots, malt, oat, potatoes, rice)-containing media, Aspergilli produce
fumonisins in high quantities on substrates with low water activities [39].

31.3.4 Patulin
The water-soluble lactone patulin (PAT; 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one) is produced by
many fungi via the polyketide pathway. It was first isolated in 1943 from Penicillium species during a
screening effort aimed at finding novel antibiotics from fungi [43]. As it was discovered independently
in multiple laboratories, various names (e.g., clavacin, expansine, claviformin, clavatin, gigantic acid,
myocin C) were given [44]. PAT was the first compound which was examined under the brand name
Tercinin in a controlled clinical trial to treat common cold [45]; however, it proved to be unsuccessful,
furthermore, even toxic to animals and humans [46].
Although PAT has been reported from up to 30 fungal genera [47], recent studies revealed that it is
produced only by a limited number of species from the genera Aspergillus, Byssochlamys, Paecilomyces,
and Penicillium. According to our recent knowledge, within the genus Aspergillus only three represen-
tatives of section Clavati (A. clavatus, A. giganteus, and A. longivesica) are capable of producing PAT
[48]. The other PAT-producing Aspergilli are rare species and cannot be considered as health hazards in
foods and feeds.
PAT provokes congestion and edema of hepatic, pulmonary, and gastrointestinal blood vessels and
tissues. Regarding its acute toxicity, PAT mainly induces gastrointestinal disorders with distension,
ulceration, and bleeding [49]. It was suggested to be partly responsible for a series of animal intoxications
in France, Germany, Hungary, and Japan [50]. Regarding the carcinogenicity of PAT, its subcutaneous
injection produced local sarcomas in rats [49].

31.3.5 Other Mycotoxins Produced by Aspergilli

Aspergillus species produce a wide range of other mycotoxins that may contaminate our foods and can
be harmful to humans. Among them, the indole-tetramic acid mycotoxin known as cyclopiazonic acid
was discovered in 1968 as a metabolite of Penicillium cyclopium in groundnuts [51]. A number of further
Penicillium and Aspergillus species are known to produce this mycotoxin, including P. camemberti,
P. commune, P. dipodomyicola, P. griseofulvum, P. palitans [52], A. flavus, A. minisclerotigenes, A. oryzae,
A. pseudotamarii [9], and A. lentulus [53]. These mycotoxin-producing fungi are able to contaminate
different agricultural products including various grains and seeds as well as cheese and meat prod-
ucts. Cyclopiazonic acid specifically inhibits calcium-dependent ATPase in the sarcoplasmic reticulum,
which results in increased muscle contraction.
The pentaketide-derived mycotoxin citrinin is structurally related to OTs. There are a number of
citrinin-producing Aspergilli including members of section Terrei (A. alabamensis, A. allahabadii,
A. carneus, A. flavipes, A. floccosus, A. niveus, and A. terreus) [54,55]. Certain Penicillium species including
P. citrinum and other members of section Citrina [56], P. expansum, P. radicicola, and P. verrucosum,
Aspergillus 459

as well as Monascus species are also known producers of this nephrotoxic compound [52]. Citrinin is a
common contaminant of grains, food, and feedstuffs (e.g., corn, oats, wheat, wheat bran, fruit juices) [57].
If ingested by animals and humans, citrinin can cause chronic diseases.
Several further Aspergillus mycotoxins could also be detected in foods and feeds [16]. The extensive
genome sequencing efforts in Aspergilli resulted in the recent clarification of the molecular background
behind the production of a series of Aspergillus mycotoxins (e.g., gliotoxin [58], kojic acid [59], naph-
thopyrones, asperfuranone, and orsellinic acid [60]). However, further studies are needed to clarify the
possible health effects of these mycotoxins on animals and humans.

31.4 Laboratory Models for Foodborne Aspergillus Mycotoxicoses

31.4.1 Invertebrate Models
There is a series of reports available in the literature about the application of invertebrate models for
studying the effects of Aspergillus mycotoxins. Among the insects, AFBl proved to be harmful to Aedes
aegypti, Corcyra cephalonica, Drosophila melanogaster, Heliothis virescens, Heterotermes indicolas,
Locusta migratoria, Musca domestica, Schistocerca gregaria, and Tenebrio molitor [61]. The use of
Drosophila melanogaster as a model organism to study the effects of Aspergillus mycotoxins has a
long history. Studies were published even in the 1970s about the effects of AFB1 on the development
of D. melanogaster [62], the induction of recessive lethals by AFB1 [63], the insecticidal and larvicidal
activities of AFB1 and PAT [61], the application of AFB1-resistant and -sensitive strains for crossing
experiments [64], the variation in sensitivity to AFB1 among several strains of D. melanogaster [65],
the effect of AFB1 on viability, growth, fertility, and crossing over [66], the applicability of AFB1Cl2
as a model of AFB1 in mutagenesis and carcinogenesis [67], the differences displayed by larvae of dif-
ferent ages in their sensitivity to the toxic effects of AFB1 [68], the genetic background of resistance
to AFB1 toxicity [69], and the alterations in gene expression caused by AFB1 [70]. Chinnici et al. [71]
reported that pretreatments with relatively less toxic mycotoxins, like AFB2 and STC, enhance the
effect of AFB1-induced toxicity in D. melanogaster. Foerster and Würgler [72] carried out in vitro
studies on AFB1 metabolism in testes of genetically different strains of D. melanogaster and identified
AFB2a, AFM1, and AFR0 among the observed metabolites. AFB1 was shown to induce a high level
of somatic mutagenesis in imaginal disk of D. melanogaster larvae, while patulin also elevated the
level of somatic mutations [73]. Melone and Chinnici [74] performed selection in D. melanogaster for
increased resistance to AFB1 toxicity. Shibahara et al. [75] studied the DNA-damaging potency and
genotoxicity of AFM1 in somatic cells of D. melanogaster in vivo and recorded a genotoxic effect for
AFM1 comparable to that of AFB1. Karekar et al. [76] studied the antimutagenic profile of antioxidants
in a Drosophila model where AFB1 was chosen as a positive mutagen. Sidorov et al. [77] applied
chemical carcinogens, including AFB1, for the successful induction of tumors in D. melanogaster.
Sişman [78] reported that hydrated sodium calcium aluminosilicate could effectively inhibit AFB1-
induced abnormalities in the developmental stages of D. melanogaster. Mutlu [79] reported about an
increase in mitochondrial DNA copy number in response to OTA-induced mitochondrial DNA damage
in Drosophila.
Other insect models applied include the navel orangeworm Amyelois transitella and the codling moth
Cydia pomonella, which were used to study the in vitro metabolism of AFB1 [80]. A field strain of
A. transitella was found to produce the AFB1 biotransformation products aflatoxicol (AFL), AFB2a, and
AFM1, while a strain of C. pomonella produced trace amounts of AFL only, but neither of them pro-
duced AFBO, the principal carcinogenic metabolite of AFB1.
Leung et al. [81] applied Caenorhabditis elegans as a model organism to study the role of xenobi-
otic-metabolizing cytochrome P450 (CYP) enzymes in the generation of genotoxic metabolites from
AFB1. The exposure of C. elegans to AFB1 resulted in significant DNA damage and more efficient
inhibition of the growth of xpa-1 nematodes than N2 nematodes. AFB1-mediated growth inhibition was
found to be resulting from CYP-mediated metabolism, as a C. elegans strain deficient in emb-8 (the
460 Laboratory Models for Foodborne Infections

CYP-nicotinamide adenine dinucleotide phosphate reductase gene) proved to be more resistant to the
growth-inhibitory effect of AFB1 exposure than N2. The results indicated that C. elegans can metabolize
AFB1 into DNA-binding metabolites in a CYP-dependent manner. González-Hunt et al. [82] examined
the effects of several known toxins including AFB1 on C. elegans and found more damage caused to the
mitochondrial than to the nuclear DNA, and AFB1 also caused dopaminergic neurodegeneration, which
supports the role for mitochondrial DNA damage in this process.

31.4.2 Rodent Models
As most popular laboratory animal models for the examination of Aspergillus mycotoxins are rodents
(principally rats and mice), a broad search strategy was used to collect full text articles and—in cases
when full text access was not available—abstracts from the PubMed database. The search was performed
in article titles and abstracts using the following keyword combinations in order to find specific litera-
ture: aflatoxin OR ochratoxin OR sterigmatocystin OR patulin AND rat OR mouse OR mice. (Although
certain Aspergillus species are known to produce fumonisins as well, FB1 and related compounds are
discussed in Chapter 34: Fusarium of this book.) The search was restricted to papers published in and
after 2010 in order to focus on recent literature. Articles and abstracts were screened to include studies
with relevant information (exact definition of the rodent model and the mycotoxin studied, as well as
details about the dosage and the way of administration).
Table 31.1 summarizes the studies published since 2010 that have used rat models to study
Aspergillus mycotoxins [83–119]. Among the retrieved records, relevant data were found in 37 publi-
cations. The most popular rat models in these studies were Fisher rats (12 studies), Sprague-Dawley
rats (12 studies), and Wistar rats (11 studies), but other rat lines (Charles Foster rats, Dark Agouti
rats, and hybrids of Sprague-Dawley female rats and a Fisher male rat) were also applied in single
studies. Male rats were used in the majority of the studies, with a few exceptions applying both
male and female, or just female animals. The age of the model animals ranged from 28 days to 15
months. The most commonly studied Aspergillus mycotoxin in rat models was AFB1 (22 studies),
followed by OTA (14 studies), AFG1, and STC (1–1 study). Depending on the aim of the particular
study, the Aspergillus mycotoxins were administered through diet, oral or gastric gavage, intraperi-
toneally, intramuscularly, intratracheally, intravenously, or subcutaneously. The majority of these
studies examined AFB1-induced hepatotoxicity [96,99,102,109,114,115] and hepatocellular carci-
noma development [90,101,104,118], as well as the protective effects of different compounds against
them [83,88,89,92,105,107,108]. In the case of OTA, many studies focused on its renal accumula-
tion, toxicity, and induction of kidney failure [84,86,93,95,96,103,112]. AFG1 was examined for lung
toxicity in male Sprague-Dawley rats [110], while the potential link between STC and esophageal
cancer was studied in Wistar rats [87]. Supriya and Reddy [91] used female Wistar rats to examine
the effects of in utero exposure to graded doses of AFB1 on development, behavior, and reproduction
and found that AFB1 severely compromised postnatal development of neonatal rats, caused a delay in
the descent of testes, and a reduction in steroidogenesis and spermatogenesis that were accomplished
by suppressed reproduction at adulthood. Other studies examined compounds like ginseng extract
[108,116] and lycopene [106] for their protective effects against the genotoxicity of AFB1 and OTA,
respectively.
Table 31.2 summarizes the studies published since 2010 that have used mouse models to study
Aspergillus mycotoxins [120–174]. Among the retrieved records, relevant data were found in 55 pub-
lications. The most popular mouse model for studying Aspergillus mycotoxins is the albino, labora-
tory-bred BALB/c strain (16 studies), followed by the black inbred strain C57BL/6 and its mutant and
hybrid (e.g., B6C3F1) derivatives (8 studies), the largely noninbred Swiss albino mice (7 studies), and
ICR outbred mice (6 studies). Other mouse models used include Kunming, Swiss Webster (CFW),
MF1, CF-1, and A/J mice. Male animals were used in more studies; however, the use of females was
more frequent than in the case of rat models. The age of the treated mice ranged from newborns to
12-month-old animals. Similar to the studies using rat models, the most commonly studied Aspergillus
mycotoxin in mouse models was also AFB1, followed by OTA, PAT, AFM1, AFG1, and STC. The myco-
toxins were administered to the mice through diet, oral gavage, intraperitoneally, intratracheally,  or
TABLE 31.1

Aspergillus
Studies Using Rat Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Dose/Administration Aim of the Study Major Findings References
Male Wistar AFB1 1.5 mg/kg intraperitoneally To study the protective effect of herbal ethanolic The plant extracts showed significant protection [83]
albino extracts of Ixora coccinea, Rhinacanthus nasuta, and against AFB1-induced liver damage by lowering
(200–250 g) Spilanthes ciliata against AFB1-induced the activity of serum enzymes and restoring the
hepatotoxicity decrease in GSH levels
Weaning male OTA 289 μg/kg/d given by gavages To demonstrate that OTA cytotoxicity can cause kidney Exposure to OTA induced significant lesions to [84]
Wistar albino through a stomach tube for 28 d failure the renal corpuscles, which can reduce the
(35–40 g) functional capacity of kidney and lead to kidney
failure
Male Wistar AFB1 0.24 mg/kg by gastric gavage for To investigate the power of conventional primary Conventional hepatocyte cultures mimicked the in [85]
Hannover up to 14 d hepatocyte cultures along with their epigenetically vivo induced changes, which may be suitable for
stabilized counterparts in mimicking in vivo genotoxic the identification of genotoxic carcinogens that
insults do not require extrahepatic activation
Male Wistar OTA 3 mg/kg/d gavage feed To analyze the effect of OTA on three human and two The carcinogenic effects in three human cell [86]
rat renal proximal tubular models models were significantly reflected in a rat
in vivo model and partially in two rat in vitro
models, which clearly evidenced the lack of
species specificity between humans and rats in
the biological perturbations caused by OTA
4-w-old male STC 30 μg/kg intraperitoneally injected To investigate the effects of sterigmatocystin exposure STC exposure downregulated TAP1 and LMP2, [87]
Wistar in rats with reflux esophagitis which directly affected the tumor immunity by
(40–50 g) allowing transformed cells to escape the host
immune surveillance, thereby promoting
esophageal cancer
10–12 w old AFB1 3 mg/kg b.w. intraperitoneally To evaluate whether antioxidant CAPE relieves CAPE induced protective effects on the [88]
male Wistar oxidative stress in AFB1-induced liver injury AFB1-induced hepatotoxicity by modulating free
albino (200 ± radical production, biochemical values, and
20 g) histopathological alterations
2-m-old male AFB1 20 μg/d through gavage for 3 w To investigate the effect of Dialium guineense pulp Free and bound phenolic extract of D. guineense [89]
Wistar albino phenolic extract on AFB1-induced oxidative fruit enhanced the detoxification of AFB1
(150 ± 2.54 g) imbalance in rat liver
Wistar AFB1 100 and 200 μg/kg To explore the effect of EGb on expressions of Cox-2 EGb can regulate the expression of GST-Pi, but it [90]
and GST-Pi in the pathogenesis of HCC does not seem to have an effect on Cox-2
expression in the liver of rats with the risk of HCC

461
(Continued)
TABLE 31.1 (Continued)

462
Studies Using Rat Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Dose/Administration Aim of the Study Major Findings References
Female Wistar AFB1 10, 20, or 50 μg/kg b.w. daily from To examine the embryonic exposure to graded doses of In utero exposure to AFB1 severely compromised [91]
(210–220 g) gestation d 12–19 AFB1 on development, behavior, and reproduction postnatal development of neonatal rats and
intramuscularly caused a delay in testes descent and reduction in
steroidogenesis and spermatogenesis that were
accomplished by suppressed reproduction at
adulthood
3-w-old male AFB1 200 μg/kg b.w. intraperitoneally To investigate genotoxic and antigenotoxic effects of PRBE significantly reduced micronucleus [92]
Wistar (approx. PRBE in rats using liver micronucleus assay formation and mitotic index in the liver of
70 g) AFB1-initiated rats
16-w-old male OTA Fed diets with 3 and 5 μg/g b.w. for To measure OTA concentration in whole kidneys of rats Apparent accumulation of OTA in kidney is due [93]
and female females and males, respectively exposed chronically to dietary toxin to binding to plasma proteins and long half-life
Wistar in plasma
Male Fischer OTA Daily dietary intake of 50 mg/kg To compare the carcinogenic response between the Carcinoma incidence (4/34 rats) through dietary [94]
344 b.w. for up to 2 y NTP carcinogenic gavage dose regimen and exposure is considerably less than in the NTP
administration of the same dosage via artificially study (16/51)
contaminated diet
15-m-old male OTA 6.25 mg by oral gavage To evaluate the urinary metabolomic profile through 1H-NMR metabolomics revealed a progressive [95]
Fischer (445, 1H-NMR spectra cyclic shift in principal components data cluster

Laboratory Models for Foodborne Infections

486 and 517 g) in predose status and insult phase, returning
almost completely to normality after 6 months
10-w-old male AFB1 Single dose of a mixture of 0.5 mg/ To validate UHPLC-FLD method with simultaneous UHPLC-FLD has successfully been used in three [96]
and female and kg b.w. of AFB1 and 0.1 mg/kg extraction and analytical quantification of AFB1 and biological matrices, and important advantages of
F344 OTA b.w. of OTA by oral gavage OTA in rat plasma, kidney, and liver this method are even 100 μL of plasma or 25 mg
of tissue are sufficient for obtaining results
Male F344 AFB1 Gavaged with 25 μg/rat for 5 d per To evaluate 1,4-dioxan-2-one both for its ability to 1,4-dioxan-2-one did not statistically increased [97]
(90–100 g) w for 2 successive w for a induce early putative preneoplastic lesions and to focal number or focal volume percent and is not
cumulative dose of 250 μg/rat enhance or promote the growth of foci that have been a common carcinogen
induced by AFB1
(Continued)
TABLE 31.1 (Continued)

Aspergillus
Studies Using Rat Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Dose/Administration Aim of the Study Major Findings References
Male F344/N AFB1 NTP 2000 feed 1 ppm To check whether a subset of genes from a previously Evaluation of gene signatures in archival tissues [98]
derived AFB1 gene signature would be observed in can be an important toxicological tool for
archival RNA from fresh frozen liver, be replicated in evaluating critical molecular events associated
FFPE liver RNA, and also be present in other FFPE with chemical exposures
tissues like the kidney and lung as secondary or
alternate targets for carcinoma
Male F344 AFB1 0.25, 0.75, and 1.5 mg/kg b.w. by To identify mechanisms and potential biomarkers for p53 signaling pathway induced by oxidative [99]
(80–120 g) oral gavage predicting the development and progression of damage was the crucial step in AFB1-induced
AFB1-induced acute hepatotoxicity through integrated acute hepatotoxicity, whereas gluconeogenesis
analysis of general toxicity studies, transcriptomics, and lipid metabolism disorder were found to be
and metabolomics profiles the major metabolic effects after acute AFB1
exposure
6–7-w-old male OTA 0, 70 or 210 μg/kg b.w. gavage To explore the relationship between OTA-induced OTA caused apparent kidney damage within 13 w [100]
F344 oxidative damage and carcinogenicity in the liver and but exerted limited effect on oxidative stress
kidney parameters
Male F344 AFB1 1.5 mg/kg b.w. by gavages To elucidate the functional complexity of microRNAs Abnormally expressed cancer-related miRNAs [101]
in AFB1-induced hepatocellular tumorigenesis were identified
3-w-old (newly AFB1 Diets containing 0, 1, 5, 10, or To develop an experimental animal model of dietary AFB1-exposed animals exhibited dose-dependent [102]
weaned) male 20 ppm aflatoxin exposure to investigate the relationship wasting and stunting, liver pathology, and
inbred F344 or between AFB1 toxin exposure and growth disturbance suppression of hepatic targets of growth
outbred and to explore candidate mechanisms responsible for hormone signaling
Sprague- this association
Dawley
6–7 w-old male OTA 0, 21, 70, or 210 μg/kg b.w. by oral To evaluate a range of novel biomarkers of renal The histopathological alterations induced by OTA [103]
F344/N gavage toxicity to test their ability in detecting acute and were best reflected by changes in urinary Kim-1
chronic kidney injury marker
(Continued)

463
TABLE 31.1 (Continued)

464
Studies Using Rat Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Dose/Administration Aim of the Study Major Findings References
Male F344/N AFB1 1 ppm in feed for 90 d To define gene expression changes that might relate to 49 novel, differentially-expressed transcripts [104]
carcinogenesis produced by AFB1 exposure prior to including two unique, unannotated, hepatic
onset of malignancy and to begin a high-resolution AFB1-responsive transcripts overexpressed by
map of the rat liver transcriptome 10- to 25-fold were identified
Male 344/NHsd AFB1 200 μg/kg b.w. via oral gavage To assess and characterize the chemoprotective efficacy CDDO-Im completely protected against [105]
daily for 4 w of synthetic oleanane triterpenoid CDDO-Im against AFB1-induced liver cancer compared to a 96%
AFB1-induced HCC incidence in AFB1 group. With CDDO-Im
treatment, integrated level of urinary AFB1-N7-
guanine was significantly reduced and
aflatoxin-NAC, a detoxification product, was
consistently elevated after the first AFB1 dose.
Also, the hepatic burden of GST-P-positive foci
and the toxicogenomic RNA expression
signature were largely absent with CDDO-Im
intervention
Male Sprague- OTA 2.2 mg/kg b.w. orally by gastric To demonstrate the effects of free radical scavengers OTA administration caused oxidative damage, [106]
Dawley gavage MEL and CoQ10 on cells damaged by OTA and MEL or CoQ10 treatment ameliorated the
(230–250 g) OTA-induced tissue injuries

Laboratory Models for Foodborne Infections

4-w-old male AFB1 150 μg/kg/d for 3 d To examine the protective effects of KRG against KRG showed protective effects against [107]
Sprague- intraperitoneally hepatotoxicity induced by AFB1 using liver-specific hepatotoxicity induced by AFB1 through its
Dawley serum marker analysis, histopathology, and terminal antioxidant effects by increasing SOD, CAT, and
(85–100 g) deoxynucleotidyl transferase-mediated dUTP GPX activity and reducing lipid peroxidation
nick-end labeling assay
3-m-old male AFB1 Fed diets with 2.5 mg/kg b.w. To evaluate whether AFB1-induced damage in rats can The genotoxicity of aflatoxins was partly [108]
Sprague- be counteracted by feeding with WPC and KGE prevented by dietary supplementation with
Dawley WPC, KGE, or their combination
(100–120 g)
Male and female AFB1 25 μg intraperitoneally To compare induction of hepatic GSTs by SF and the A dose-dependent relationship was found for both [109]
Sprague- resultant impact on the formation of AFB1-DNA induction of GST and reduction of AFB1-N7-
Dawley and adducts in livers of both sexes of two rat strains guanine in both rats, no gender-specific
Fischer CDF responses were observed
(Continued)
TABLE 31.1 (Continued)

Aspergillus
Studies Using Rat Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Dose/Administration Aim of the Study Major Findings References
Male Sprague- AFG1 30 μg/kg b.w. intratracheally To identify the acute toxicity of AFG1 in AT-II cells AFG1 induces structural and functional [110]
Dawley instilled impairment in AT-II cells involved in the acute
(110–130 g, toxicity of AFG1 in lung tissues
120.2 ± 5.9 g)
Male Sprague- OTA 0.2 mg/kg b.w. single dose by oral To develop combinatorial platform of LC–MS/MS and LC–MS/MS method determined OTA with a run [111]
Dawley (200 ± gavage LC–TOF-MS to investigate in vivo kinetics and time of 7.0 min. Simultaneously, an LC–
20 g) biotransformation of OTA in rats TOF-MS method unambiguously identified the
metabolites of OTA in a total run time of 14 min
Male Sprague- OTA 0.5 mg/kg b.w. for 14 d by To investigate the possible protective effect of lycopene Lycopene might be partially protective against [112]
Dawley intragastric lavage against the renal toxic effects of OTA OTA-induced nephrotoxicity and oxidative stress
(<200 g) as it increases GPx1 activity and GSH levels and
decreases apoptotic cell death
12-w-old male OTA 0.5 mg/kg/d by intragastric lavage To investigate the protective effects of lycopene against Lycopene provided a protective effect against [113]
Sprague- for 7 and 14 d the genotoxicity of OTA in rat tissues using the OTA-induced DNA damage, which was
Dawley alkaline comet assay evidenced by decreased tail moment and
(250–260 g) intensity in both rat kidney and liver cells
2-m-old AFB1 Fed diets with 4, 10, 25, and 50 μg/ To evaluate the toxicological impacts on rats fed with Various negative impacts on liver and kidney [114]
Sprague- kg b.w. AFB1-contaminated feedstuffs functions were observed in AFB1-contaminated
Dawley groups, which was positively correlated with
AFB1 concentrations
6–8-w-old male AFB1 0.3 mg/kg/b.w. oral or Transcriptome profiling of rat liver samples treated The resulting large toxicogenomics dataset can [115]
Sprague- intraperitoneal, intravenous or with 27 chemicals to evaluate the utility of RNA-Seq serve as a resource to characterize various
Dawley subcutaneous injection in safety assessment and toxicity mechanism aspects of transcriptomic changes (e.g.,
elucidation alternative splicing) that are byproducts of
chemical perturbation
3-m-old female AFB1 80 μg/kg b.w. AFB1 or/and 100 μg/ To evaluate the protective role of PGE against the PGE induced potential protective effects against [116]
Sprague- and kg b.w. FB1 orally synergistic effect of subchronic administration of the oxidative stress and genotoxicity of these
Dawley FB1 AFB1 and FB1 on DNA and gene expression in rat mycotoxins
(100–120 g)
(Continued)

465
TABLE 31.1 (Continued)

466
Studies Using Rat Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Dose/Administration Aim of the Study Major Findings References
Hybrids of OTA Dietary intake of 5 ppm OTA To explore whether the tumor promoter sodium Female mammary tumorigenesis was advanced [117]
Sprague- barbiturate could shorten the otherwise long latency by 6 ms in all rats given the OTA-barbiturate
Dawley female between exposure to OTA and tumorigenesis
and a F344 male
18- to 20-w-old AFB1 1 mg/kg b.w. of to the 18-h fasted To describe the histopathological progression of AFB1 toxicity activates the oxidative stress [118]
male Charles rats on 2 consecutive d AFB1-induced HCC in the liver of rats proinflammatory pathway, which induces
Foster albino intraperitoneally hepatocarcinogenesis
(200–220 g)
7-w-old dark OTA Fed with 2.5, 7, 40, and 100 μg/kg To examine OTA metabolism in the liver and kidney of The distribution of OTA in male and female rat [119]
Agouti wheat male and female rats using reduced GSH and NAC kidney is not significantly different, but OTA
conjugates along with nontoxic OTα as biomarkers metabolism is greater in male than female rats
d, day; w, week; m, month; AFB1, aflatoxin B1; AT-II, alveolar type II; CAPE, caffeic acid phenethyl ester; CDDO-Im, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole; CAT,
catalase; CoQ10, coenzyme Q10; Cox-2, cyclooxygenase-2; DNA, desoxyribonucleic acid; EgB, Ginkgo biloba extract; FB1, fumonisin B1; FFPE, formalin-fixed, paraffin-embedded;
GPx1, glutathione peroxidase 1; GSH, glutathione; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; KGE, Korean ginseng extract; KRG, Korean red ginseng; LC–MS/MS,
liquid chromatography–mass spectrometry/mass spectrometry; LC–TOF-MS, liquid chromatography–time of flight-mass spectrometry; LMP2, low molecular weight protein 2; MEL,
melatonin; MT, mycotoxin; NAC, N-acetylcysteine; NMR, nuclear magnetic resonance; NTP, National Toxicology Program; OTA, ochratoxin A; PAT, patulin; PGE, Panas ginseng extract;
PRBE, purple rice bran extract; RNA, ribonucleic acid; SF, sulforaphane; SOD, superoxide dismutase; STC, sterigmatocystin; TAP1, transporter associated with antigen processing 1;

Laboratory Models for Foodborne Infections

UHPLC-FLD, ultrahigh-performance liquid chromatography with fluorescence detection; WPC, whey-protein concentrates.
TABLE 31.2

Aspergillus
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
7-w-old male BALB/c OTA Single dose of 3.5, 7, 35, 70, 289, To test whether the acute exposure to OTA In utero exposure to OTA caused adducts in the [120]
(20 g ± 2 g) 578, 1056 μg/kg/b.w. by oral via food and via exposure in utero causes testicular DNA of male offspring that were
gavage, feed containing increasing adducts in testicular DNA and to test similar to DNA adducts observed in the
amount of OTA (0.5, 1.4, 8, 20 μg/ whether these lesions are identical to those kidney and testis of gavage-fed adults,
kg/b.w.) every day during 4 w, a that can be produced in the kidney and supporting a possible role for OTA in
single intraperitoneal injection of testis by the consumption of OTA testicular cancer
OTA (2.5 mg/kg) at gestation day 17
6- to 8-w-old female PAT 5–20 μg by gastric intubation on days To investigate the adjuvant activity of Patulin increased the allergic immune response [121]
BALB/c ByJ 0, 1, 13, 14, 17, and 18, orally on mycotoxins on allergic airway in mice by modulating the Th1/Th2 balance
days 18, 21, and 22, 100 ng inflammation via direct effects on IL-12 secretion in DCs
intranasally on day 22 and by inducing oxidative stress
4- to 6-w-old BALB/c AFB1 Single dose at 240 μg/kg b.w. once a To evaluate the role of HOC in the Oval cells have produced tumors in liver [122]
athymic, nu/nu, male nude week intraperitoneally and fed in development of hepatocellular carcinoma following transfection with hepatitis B virus
(18.0–22.0 g) the drinking water (400 μg/kg d), x gene (HBx) and treatment with AFB1
continuously for 16 w
Male BALB/c (20–25 g) AFB1 250 μg/kg b.w. intraperitoneally To evaluate the hepatoprotective effect of The treatment of CCE showed a total reduction [123]
CCE on AFB1-induced liver damage in of AFB1-induced oxidative damage markers
mice by measuring MDA level, protein and genotoxicity markers and decreased the
carbonyls generation, and expression of expressions of proapoptotic proteins p53 and
Hsp 70 and Hsp 27 in liver bax

6- to 8-w-old male BALB/c AF 625 μg/kg b.w./d given directly into To investigate the antioxidant effect of AF exhibited adverse effects on most of the [124]
the stomach through a gastric tube pumpkin seed oil against the oxidative- oxidative stress markers, but the
stress-inducing potential of aflatoxin administration of pumpkin seed oil
diminished aflatoxin-induced adverse effects
(Continued)

467
TABLE 31.2 (Continued)

468
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
3-w-old specific pathogen- ST 3, 30, 300, and 3000 μg/kg To explore the short-term immunotoxic The proportion of CD8+ T cells was decreased [125]
free male BALB/c intraperitoneally effects of ST, specifically on FoxP3+ Tregs in the thymus in ST (3 μg/kg) group, while
and pDCs, by observing changes in that of CD4+ and CD8+ T cells was increased
number/expression of FoxP3+ Tregs, in the spleen in two treatment groups (3 and
pDCs, and CD4+, CD8+ T cells 30 μg/kg); the proportion of FoxP3+ Tregs
and FoxP3 expressions were all significantly
increased in mPBMCs, the thymus, and the
spleen. Importantly, the population of pDCs
significantly decreased in the thymus but
increased in the spleen, which is due to a
temporary immune response triggered by the
ST inhibition
BALB/c ST 3 mg/kg intraperitoneally To evaluate the putative effects of ST on the Downregulation of TNF-α, IL-6, and IL-12 [126]
expression of TNF-α, IL-6, and IL-12 at mRNA expression in mPBMCs and peritoneal
mRNA levels in mPBMCs and peritoneal macrophage cells was observed, and serum
macrophage cells and on the serum TNF-α TNF-α and IL-6 levels were also decreased
and IL-6 levels
4- to 6-w-old inbred female AFB1 0.1 mL at a daily dose of 30, 15, and To focus on the immunosuppressor activity AGE has increased the level of INF-γ and IL-4 [127]

Laboratory Models for Foodborne Infections

BALB/c 10 μL/kg intraperitoneally of AFB1 and immunostimulatory activity cytokines produced by splenocytes stimulated
of AGE through the evaluation of Treg by specific tumor antigen and decreased the
counts and the pattern of cytokine number of Treg cells in the spleen; AFB1
production increased the number of Treg cells in the
spleen and decreased cytokine production
6-w-old male BALB/c AFM1 100 μg/kg b.w. orally by gavage To isolate food-grade probiotic bacteria able Lactobacillus plantarum MON03 and [128]
(25 ± 0.3 g) to degrade/bind AFM1 in vitro from dairy L. rhamnosus GAF01 were isolated, and both
products and to evaluate whether the same of them could remove AFM1 in vitro; since,
organism(s) could impart a protective role L. rhamnosus GAF01 showed the highest
against AFM1-induced immunotoxicity in binding capacity, it was selected for the in
exposed mice vivo study. L. rhamnosus GAF01 prevented
AFM1-induced effects on total white and red
blood cells and lymphocyte subtypes after
15 d of treatment
(Continued)
TABLE 31.2 (Continued)

Aspergillus
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
Nude/BALB/c AFB1 1 × 107 cells injected subcutaneously To investigate the underlying mechanisms ATR mediated the DNA damage repair [129]
of AFB1-induced effects on ATR function response and subsequent neoplastic
and neoplastic transformation of the cells transformation in P40 B-2A13 cells
when B-2A13 cells were exposed to low
levels of AFB1 (0.1–10 nM)
10-w-old female BALB/c AFM1 100 mg/kg b.w orally (by gavage) To evaluate a new AFM1-binding/degrading The isolated Lactobacillus plantarum MON03 [130]
(21 ± 2 g) microorganism for biologic detoxification, (LP) was found to display significant binding
to examine its ability to degrade AFM1 in ability to AFM1 in PBS (93%) and was taken
liquid medium and to evaluate its potential for in vivo study, where administration of LP
for in vivo preventive effects against with AFM1 strongly reduced the cytotoxic/
AFM1-induced immunotoxicity and genotoxic adverse effects of AFM1
genotoxicity
4-w-old female BALB/c AFB1, 2.5, 5.0, and 5.0 mg/kg b.w. of AFB1, To assess the individual and combined toxic AFB1, ZEA, and DON induced liver injury and [131]
ZEA, ZEA, and DON, respectively, effects of AFB1, ZEA, and DON within the were associated with induced oxidative stress
DON administered orally via gavage liver and an upregulation of the apoptotic genes
needle Caspase-3 and Bax, along with a
downregulation of the antiapoptotic gene
Bcl-2; AFB1 + DON displayed synergistic
hepatotoxic effects, while AFB1 + ZEA
displayed antagonistic hepatotoxic effects
10-w-old female BALB/c AFB1 0.25 mg/kg b.w. AFB1 and 0.27 mg/kg To evaluate a new AFB1- and AFM1-binding/ Lactobacillus plantarum MON03 (LP) was [132]
and AFM1 administered by oral gavage degrading microorganism for biological isolated and displayed significant binding
AFM1 detoxification, examine its ability to ability to AFB1 and AFM1 in PBS (i.e., 82%
degrade AFB1 and AFM1 in liquid medium, and 89%, respectively); in vivo study was
and evaluate its potential for in vivo conducted where LP strongly reduced the
preventive effects against AFB1- and adverse effects of each mycotoxin
AFM1-induced immunomodulation
BALB/c AFG1 100 μg/kg body weight orally To understand the phenotypic alterations of Increased MHC-II expression in alveolar [133]
AT-II cells, which may provide new insight epithelium was observed and associated with
into the immune function of AT-II cells enhanced Treg infiltration in mouse lung
involved in AFG1-induced pulmonary tissues and also with AFG1-induced
tumorigenesis inflammation
(Continued)

469
TABLE 31.2 (Continued)

470
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
Female BALB/c (18–20 g) AFG1 100 μg/kg b.w. orally using the To examine whether oral gavage of AFG1 Inflammatory responses were heightened in the [134]
gavage technique induces chronic lung inflammation and lung alveolar septum 3 and 6 months after
how it contributes to carcinogenesis oral administration of AFG1; 12 months later,
AFG1 induced alveolar epithelial hyperplasia
and adenocarcinoma; upregulation of NF-κB,
p-STAT3, and Cox-2 was also induced in lung
adenocarcinoma
8- to 12-w-old male OTA 0.85 mg/kg b.w. inoculated To investigate the protective effects of The tested polyphenols reduced the toxicity [135]
BALBb/c (20–25 g) intraperitoneally luteolin, ChlA, and CafA against caused by OTA on different target cells with
cyto-genotoxic effects caused by OTA good protective effect, ChlA being the
compound that showed the best effects
5- to 6-w-old female Swiss PAT Topical dose of 400 nmol To investigate the skin carcinogenic Topical application of PAT resulted in cell [136]
albino potential of topically applied PAT proliferation, which is mediated by
ROS-induced MAPKs signaling pathway,
leading to transcriptional activation of
downstream target proteins c-fos, c-jun, and
transcription factor NFκB, showing that PAT
has dermal tumorigenic potential

Laboratory Models for Foodborne Infections

Male Swiss (37–40 g) AF (B1, Orally administered with AF (B1, B2, To investigate the effect of black tea Significant restoration of AF-induced damages [137]
B2, G1, G1, and G2 in the ratio of 8:3:2:1) infusion on AF-induced hepatotoxicity in body weight, organ weight, serum
and using a feeding needle with a chemistry, and histopathological features was
G 2) dosage of 750 and 1500 μg/kg b.w observed in a dose-dependent manner in mice
in 0.2 mL olive oil/animal/d administered AF plus black tea infusion
Young adult inbred male AF Orally with 25 and 50 μg/0.2 mL To evaluate AF-induced biochemical Administration of AF caused significant, [138]
Swiss albino (32–35 g) olive oil/animal/day (750 and changes in the liver and its possible dose-dependent reduction in DNA, RNA,
1500 μg/kg b.w.), respectively, for amelioration by black tea extract protein, and glycogen contents; however,
30 d cholesterol content and phosphorylase activity
were significantly increased; these changes
were significantly ameliorated on cotreatment
with black tea extract
(Continued)
TABLE 31.2 (Continued)

Aspergillus
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
6- to 7-w-old female Swiss OTA Topical application of 25, 50, 100, To evaluate the effect of a single topical OTA has cell proliferative and tumor- [139]
albino (20 ± 3 g) and 200 nmol/0.2 mL acetone application of OTA at lower doses on skin promoting potential in mouse skin, which
epidermis and to evaluate the skin-tumor- involves EGFR-mediated MAPKs and Akt
promoting potential of OTA pathways along with NF-κB and AP-1
transcription factors and that cyclin-D1 and
Cox-2 are the target genes responsible for
tumor-promoting activity of OTA
Young male inbred Swiss OTA 50 μg and 100 μg in 0.2 mL olive oil/ To evaluate the ameliorative effects of an Administration of Emblica officinalis aqueous [140]
albino (30–33 g) animal/d for 45 d orally aqueous extract of Emblica officinalis on extract (2 mg/animal/d) and OTA caused a
OTA-induced lipid peroxidation in the significant amelioration in the OTA-induced
kidney and liver lipid peroxidation in liver and kidney
60-d-old male Swiss albino AFB1 66.6 μg/kg b.w./d orally by gavage To investigate the protective effect of Nephrotoxicity induced by AFB1 was [141]
(30–40 g) esculin against pro-oxidant AFB1-induced ameliorated by esculin, which is due to its
nephrotoxicity antioxidant, anticarcinogenic, and ROS-
scavenging properties
Male Swiss albino (30 ± AFB1 2 μg/30 g b.w orally To investigate the ability of the Tinospora T. cordifolia showed protection against [142]
5 g) cordifolia to scavenge free radicals AF-induced nephrotoxicity due to the
generated during aflatoxicosis presence of alkaloids such as a choline,
tinosporin, isocolumbin, palmatine,
tetrahydropalmatine, and magnoflorine
6-w-old male ICR AFB1 0.75 mg/kg b.w. orally To examine the biochemical mechanisms Quercetin does not directly protect against [143]
associated with the effects of quercetin on AFB1-mediated liver damage in vivo but
AFB1-mediated liver damage exerts a partial role in promoting antioxidative
defense systems and inhibiting lipid
peroxidation
6-w-old male ICR AFB1 0.75 mg/kg b.w. orally To investigate the biochemical mechanisms RCMF attenuates AFB1-mediated damage to [144]
of the RCMF effects on AFB1-mediated the liver, which is at least partially related to
liver damage the restoration of antioxidant defense systems
and an increase in AFB1–GSH conjugate
formation
4-w-old female ICR AF, Fed diets with 597 μg/kg AF, 729 μg/ To investigate the regulation of multiple Naturally contained mycotoxins are toxic in [145]
ZEN, kg ZEN, and 3.1 mg/kg DON mycotoxins on the oxidative stress vivo and able to induce oxidative stress
DON

471
(Continued)
TABLE 31.2 (Continued)

472
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
ICR OTA 1, 5, or 10 μM To explore the cytotoxic effects exerted by In vitro exposure to OTA triggers apoptosis and [146]
OTA on the blastocyst stage of mouse retards early postimplantation development
embryos, on subsequent embryonic after transfer of embryos to host mice; OTA
attachment, on outgrowth in vitro, and induces apoptosis-mediated injury of mouse
following in vivo implantation via embryo blastocysts via ROS generation and promotes
transfer mitochondrion-dependent apoptotic signaling
processes that impair subsequent embryonic
development
4-w-old female ICR DON, Fed diets with 3.875 mg/kg DON, To investigate the effects of a mycotoxin- Mycotoxin-contaminated diet adversely [147]
ZEN, 1897 μg/kg ZEN, and 806 μg/kg AF contaminated diet on oocyte quality affected the developmental competence of
and ovaries and decreased the oocyte quality
AF
7- to 9-w-old male CD1 OTA For acute tests, 1 mg/kg b.w. and To evaluate OTA-degrading and detoxifying OTA was degraded efficiently by Cupriavidus [148]
10 mg/kg b.w. for 72 h; for chronic potential of Cupriavidus basilensis OR16 basilensis OR1; OTα did not display
test, 0.5 mg/kg b.w. for 21 d via oral strain nephrotoxic effects in vivo
gavage
Male Kunming (22 ± 2 g) PAT 1 mg/kg intraperitoneally To investigate PAT-induced hepatotoxicity GTP exerted antioxidative activity in reducing [149]
and genotoxicity and the antioxidant and hepatic ROS and TBARS level and increasing

Laboratory Models for Foodborne Infections

antigenotoxicity efficiency of GTP against GSH content, it also inhibited PAT-induced
PAT-induced toxicity bone marrow damage, including the
formation of micronucleus and chromosomal
aberration
4-w-old male Kunming PAT 1 mg/kg intraperitoneally To investigate the protective effects of Selenium supplementation increased the [150]
(20–23 g) selenium supplementation on PAT-induced activity and expression of GSH-related
neurotoxicity enzymes and offered significant protection
against brain damage induced by PAT
21- to 28-d-old pathogen- STC 4 × 10–5 mol toxin/kg lung weight by To evaluate lungs intratracheally exposed to Time- and toxin-dependent transcription and [151]
free Carworth Farms intratracheal instillation a single dose of toxin for histological and expression of inflammation-associated genes
white histochemical responses at 4 and 12 h and inflammatory responses were observed
Swiss Webster male postexposure
(23 ± 1.2 g)
(Continued)
TABLE 31.2 (Continued)

Aspergillus
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
Swiss Webster AFB1 — To express the putative proteins tGSTAs in Recombinant tGSTAs detoxified AFBO, [152]
an Escherichia coli heterologous system whereas their hepatic forms did not, which
and to characterize the functional implies that the hepatic forms of these
properties of products of the subunits with enzymes are silenced by one or more
respect to their enzymatic activities toward regulatory mechanisms
prototype GST substrates and toward
enzymatically generated AFBO.
9-w-old male CF-1 (28.5 ± PAT A single dose of 1.0, 2.5, or 3.75 mg/ To investigate the genotoxic effects of PAT induced DNA damage in the brain, liver, [153]
1.27 g) kg b.w. intraperitoneally patulin in multiple organs (brain, kidney, and kidneys and demonstrated a high
liver, and urinary bladder) using an in vivo correlation between decreased GSH content
comet assay and increased lipid peroxidation and DNA
damage, suggesting interrelationship between
the pro-oxidant and genotoxic effects of PAT;
pretreatment administration of N-acetyl-
cysteine reduced PAT-induced DNA damage
Female A/J (16–19 g) AFB1 50 mg/kg To examine the effect of in vivo treatment 2 h posttreatment, AFB1 increased 8-OHdG [154]
with a single tumorigenic dose of AFB1 on levels in mouse lung DNA, but did not alter
the formation and repair of oxidative DNA 8-OHdG levels in liver or 5-OHdC levels in
damage in lung and liver lung or liver; AFB1 treatment also increased
BER activity in mouse lung but did not affect
hepatic BER activity; levels of OGG1
immunoreactive protein were increased in
both lung and liver; the results are consistent
with oxidative DNA damage contributing to
the carcinogenicity of AFB1
Male MF1 (25–30 g) PAT Orally administered with 1 mL of To assess the biochemical and PAT-contaminated apple juice resulted in liver, [155]
apple juice contaminated with histopathological effects of PAT in apple kidney, and neurotoxicological effects
152.5 ppb of PAT for 6 w juice samples collected from different
outlets retailing in Jeddah, Kingdom of
Saudi Arabia
(Continued)

473
TABLE 31.2 (Continued)

474
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
Sexually mature female OTA Single dose at 3.0 mg/kg b.w. To evaluate the possible regulatory effect Dlx5 is a target for OTA and the inhibition of [156]
C57BL/6 intraperitoneally induced by OTA administration during a its function, directly or indirectly, could be
critical moment of gestation on expression the cause of the observed differentiation
of some homeotic related genes in order to defects
understand the possible mechanism of OTA
in the induction of differentiation defects
16 timed-pregnant AFB1 7 μg/g body b.w. intraperitoneally To test whether specific intestinal bacteria Intestinal colonization by Helicobacter [157]
Helicobacter-free C3H/ promote liver cancer in chemical and viral hepaticus was sufficient to promote AF- and
HeN female, C57BL/6 transgenic mouse models Hepatitis C virus transgene-induced HCC;
FL-N/35 crossed with neither bacterial translocation to the liver nor
C3H/HeN induction of hepatitis was necessary
Gnmt−/− and wild-type AFB1 10 mg/kg of b.w. at 7 d of age and To test whether the Gnmt−/− mice are Liver tumor formation in AFB1-treated [158]
129/B6 (129sv X second treatment of 40 μg AFB1 per susceptible to AFB1 carcinogenesis and to Gnmt−/− mice occurred earlier than in solvent -
C57BL/6) mouse at 9 w of age test whether Gnmt deficiency may treated Gnmt−/− mice and AFB1-treated wild
intraperitoneally accelerate AFB1-induced liver type mice; Gnmt deficiency increased the
tumorigenesis susceptibility to AFB1 related HCC; 5
detoxification pathway-related genes: Cyp1a2,
Cyp3a44, Cyp2d22, Gsta4, and Abca8a were

Laboratory Models for Foodborne Infections

downregulated in AFB1-treated Gnmt−/− mice
Pregnant gpt delta AFB1 6 mg/kg for mutation analysis and To study DNA damage by AFB1 during Early-life exposure of mycotoxins, especially [159]
C57BL/6J 5 mg/kg for adduct analysis via prenatal development and the risk of during the embryonic period, is strikingly
intraperitoneal injection or oral genetic disease later in life more mutagenic than exposure later in life
gavage on gestation day 14
(Continued)
TABLE 31.2 (Continued)

Aspergillus
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
AlbCre:Keap1flox/flox and AFB1 0.8 mg/kg b.w. orally To examine whether genetic or The inability to rescue GSTA3 knockout mice [160]
GSTA3 knockout mice pharmacologic activation of Nrf2 signaling from AFB1 genotoxicity through the Nrf2
were crossed to produce could rescue the hypersensitive GSTA3 transcriptional program indicated that Gsta3
compound knockout mouse from the genotoxicity of is unilaterally responsible for the
GSTA3:Alb:Cre:Keap1flox/ AFB1 detoxification of AF in mice
flox DKO

Male C57BL/6JOlaHsd AFB1 1 ppm, ∼0.15 mg/kg b.w./d via feed To characterize the potential genotoxic Supplementation with quercetin at ∼350 mg/kg [161]
for 7 d properties of quercetin in the small bw/d for 12 w in mice showed no
intestine as well as in the liver by upregulation of genotoxicity-related pathways
transcriptome analysis in order to provide in liver and small intestine
new additional information to evaluate the
safety of quercetin
HBsAg transgenic mice, AFB1 Single dose of 6 μg/g b.w. To explore the option of utilizing mouse The utility of these mouse models provided a [162]
C57BL/6J- intraperitoneally models to understand in a systematic and rich resource for the longitudinal analysis of
Tg(Alb1HBV)44Bri/J longitudinal manner the molecular molecular changes and biomarkers associated
were crossed to C57BL/6J pathways that are progressively with HCC
WT to obtain HBsAg and deregulated by various etiological factors
WT in contributing to HCC formation and
report the initial findings in characterizing
their validity
4-d-old B6C3F1 AFB1 Single dose of 6 mg/kg AFB1 on d 4 To assess the capability of AFB1 to generate After 3 w, 10-fold increase in the Spi− mutant [163]
after birth via intraperitoneal DNA sequence changes including those fraction (MF) was observed and after 10 w, a
injection driven by nonhomologous recombination further increase was observed; the MF in the
in vivo by analyzing the Spi− (Sensitive to gpt gene was also increased at 10 w compared
P2 Inhibition) phenotype resulting from to 3 w; no gender-specific differences were
the disruption of red and gam genes in the found in the Spi− or gpt MFs; the Spi−
λEG10 transgene of gpt delta B6C3F1 spectrum was dominated by GC to TA
mice transversions, with one exceptionally strong
hotspot at position 314, which is the dominant
mutation seen in people exposed to AF
(Continued)

475
TABLE 31.2 (Continued)

476
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
4-w-old male ICR mice AFB1 50 μg/kg b.w. via intraperitoneal To determine changes in testicular gene Differential expression of genes was noted [164]
(N 32) injection daily for 45 d expression due to exposure to AFB1 and to during cell differentiation, and extracellular
investigate which cell types were affected space was observed in the testis; renin was
by treatment with AFB1 commonly represented amongst many
clusters and chosen for further analyses; renin
was upregulated in response to AFB1,
especially in males that maintained fertility
despite AFB1 treatment
7- to 9-w-old male AFB1 Fed diets with 0, 0.2 or 1.0 ppm AFB1 To determine if chronic exposure of mice to Chronic exposure to AFB1 increased NER [165]
heterozygous p53 low levels of AFB1 alters NER in lung and activity in wild-type mice, and this response
knockout mice (p53 (+/−), liver and to determine if such alterations was diminished in heterozygous p53-
B6.129-Trp53tm1BrdN5) are affected in p53-deficient mice knockout mice, indicating that loss of one
and wild type controls allele of p53 limits the ability of NER to be
(p53 (+/+)) upregulated in response to DNA damage
5- to 7-w-old male OTA Fed diets containing 1, 15, or 40 mg To investigate OTA’s mode of action (MOA) OTA induced renal damage, but no tumors [166]
p53+/− (P53N5-T) and OTA/kg (w 1 and 2) and 0.5, 2, or in p53 heterozygous (p53 +/− ) and p53 were observed in either strain, indicating that
p53 +/+ (P53N5-W) 10 mg OTA/kg (w 3–26) homozygous (p53 +/+) mouse p53 heterozygosity conferred little additional
sensitivity to OTA; the lowest observed effect

Laboratory Models for Foodborne Infections

level for renal changes in p53 +/− and p53 +/+
mice was 200 mg OTA/kg b.w./day; based on
the lack of tumors and the severity of renal
and body weight changes at a maximum
tolerated dose, the results were interpreted as
suggestive of a primarily nongenotoxic
(epigenetic) mode of action for OTA
carcinogenesis
(Continued)
TABLE 31.2 (Continued)

Aspergillus
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
10-w-old male p53- OTA 5 mg/5 mL/kg orally To investigate the role of p53 in the OTA induced DSBs at the carcinogenic target [167]
proficient and p53- progression from OTA-induced DNA site in the kidneys; p53 prevented formation
deficient mice and damage to gene mutations of DSBs and following gene mutation, due to
p53-proficient and p53/p21-mediated cell cycle control
p53-deficient gpt delta
mice (acquired by
intercrossing between
p53-deficient mice, gpt
delta transgenic mice and
C57BL/6 mice)
7- to 9-w-old male AFB1 Fed diets with 0, 0.2, or 1.0 ppm To determine if chronic exposure of mice to Chronic exposure to AFB1 did not affect BER [168]
heterozygous p53 AFB1 alters BER in lung and liver and to in lungs or livers of heterozygous p53-
knockouts and their determine if p53 heterozygosity affects knockout mice. BER activity was lower in
wild-type controls BER activity livers from p53 wild-type mice exposed to
(B6.129-Trp53tm1Brd N5) 1.0 ppm AFB1 versus those exposed to 0.2 ppm
AFB1, an effect that was not attributable to
liver cell death or altered levels of OGG1
Male and female FVB AFB1 20 μg/kg b.w. diet To develope transgenic mice specifically Transgenic mice specifically expressing ADTZ [169]
expressing ADTZ in parotid glands by gene in the parotid gland were successfully
transgenic technology toward AFB1 developed, and the ADTZ produced in the
detoxification transgenic mice was shown to reduce the
incidence of AFB1 sepsis
7- to 10-w-old 129/ AFB1 50 mg/kg b.w. intraperitoneally To determine the effect of ogg1 deficiency ogg1 status did not have a significant effect on [170]
C57Bl/6 female on AFB1-induced oxidatively damaged AFB1-induced oxidatively damaged DNA or
wild-type, heterozygous DNA and tumorigenesis tumorigenesis, but deletion of one or both
(+/−) or homozygous alleles of ogg1 did increase susceptibility to
(−/−) ogg1 gene mice other aspects of AFB1 toxicity
Adult male albino mice AFB1 9 mg/kg b.w. orally To investigate the effects of AFB1 and AFB1 and ethanol coexposure induced severe [171]
(19.75 + 2.0 g) ethanol coexposure on biomarkers of oxidative damage to the liver; thus, humans
hepatic damage consuming excessive amount of ethanol and
diets contaminated with AFB1 simultaneously
may be at greater risk of the hepatotoxic
effects of these compounds

477
(Continued)
TABLE 31.2 (Continued)

478
Studies Using Mouse Models for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Dose/Administration Aim of the Study Major Findings References
ICR female mice (18–22 g) AF, Fed with low-dose mycotoxin- To identify any epigenetic effects of a Diets containing mycotoxins increased the [172]
ZEA containing diet of DON (581 μg/kg), mycotoxin-containing diet, including levels of DNA methylation, H3K9me3, and
DON ZEA (285 μg/kg), and AF (121 μg/ altered DNA methylation, H3K9 H4K20me3 and decreased the levels of
kg); and fed with high-dose methylation, H3K27 methylation, and H3K27me3 and H4K20me2; these altered
mycotoxin-containing diet of DON H4K20 methylation on reduced oocyte epigenetic modifications may be one of the
(1,163 μg/kg), ZEA (569 μg/kg), and developmental competence reasons for the reduced oocyte developmental
AF (242 μg/kg) competence
Theiler outbred (TO) mice AFB1 20 mg/kg b.w. on GD 13 both To study the toxicokinetics of AF in Large amounts of AFB1 are easily and rapidly [173]
intraperitoneally and orally pregnant mice absorbed both from the gastrointestinal tract
and through the peritoneum
Pdn/Pdn (Polydactyly OTA 2 mg/kg b.w. intraperitoneally on day To investigate gender-dependent differences A high incidence of NTD in male Pdn/Pdn was [174]
Nagoya) mouse (Gli3Pdn) 7.5 of gestation in the incidence of NTD induced by OTA induced by OTA treatment, which might be
in the Pdn/Pdn mouse due to complicated altered gene expressions
among Gli3, Wnt7b, Wnt8b, Fez1, Barx1,
Lim1, Dmrt1, Igf1, Fog2, Dax1, and Sox9,
and in particular, upregulation and gender-
dependent difference in Barx1 and gender-
dependent difference in Sox9 gene

Laboratory Models for Foodborne Infections

expressions was observed
AF, aflatoxin; AFB1, aflatoxin B1; AFG1, aflatoxin G1; AFM1, aflatoxin M1; AGE, aged garlic extract; AT-II, alveolar type II cells; ADTZ, aflatoxin-detoxifizyme; BER, base excision repair;
b.w., body weight; CafA, caffeic acid; CCE, cactus cladode extract; ChlA, chlorogenic acid; Cox-2, cyclooxygenase-2; DC, dendritic cell; DKO, double knockout; DNA, desoxyribonucleic
acid; DON, deoxynivalenol; DSB, DNA double strand break; EGFR, epidermal growth factor receptor; FB1, fumonisin B1; GSH, glutathione; GSTA3, glutathione S-transferase subunit;
GTP, green tea polyphenols; HR, homologous recombination; Hsp, heat shock protein; HCC, hepatocellular carcinoma; HOC, hepatic oval cells; IL, interleukin; MDA, malondialdehyde;
mPBMCs, murine peripheral blood mononuclear cells; MT, mycotoxin; NER, nucleotide excision repair; NTD, neural tube defect; OGG1, 8-oxoguanine-glycosylase; 5-OHdC, 5-hydroxy-
2ʹ-deoxycytidine; OTA, ochratoxin A; PAT, patulin; pDCs, plasmacytoid dendritic cells; RCMF, Rhus verniciflua Stokes chloroform–methanol fraction; ROS, reactive oxygen species; STC,
sterigmatocystin; TBARS, thiobarbituric acid-reactive substances; TNF-α, tumor necrosis factor-α; Treg, CD4+ CD25+ FoxP+ regulator cell; ZEA, zearalenone.
Aspergillus 479

subcutaneously. Besides a series of articles dealing with organotoxicity [131,149,155], genotoxicity

[149,153,154,159,160,163,167], or hepatocellular carcinoma development [157,158,162], a large num-
ber of studies focused on the protective, antioxidant, and immunostimulatory effects of plant-derived
metabolites, extracts, and oils against Aspergillus mycotoxins [123,124,127,135,137,138,140–144,161],
as well as on the protective role of bacteria and their mycotoxin-degrading abilities [128,130,132,148].
In the case of OTA, its cytotoxic effects on embryonic development [146] as well as its possible role in
testicular cancer [120] and skin carcinogenesis [139] were also studied in mouse models. The latter has
also been examined for PAT [139], and the adjuvant activity on allergic airway inflammation was also
studied in the case of this mycotoxin [121]. Zhang et al. [126] examined the negative immunomodula-
tory effects of STC, while Miller et al. [151] focused on the histological and histochemical responses
to STC in lungs. In the case of AFG1, the link between induced chronic inflammation and lung tumori-
genesis was investigated [133,134].
Studies are available also about the application of guinea pigs as laboratory models to study
Aspergillus mycotoxins; however, the lack of records in the PubMed database since 2007 reflects that the
rat and mouse models are more preferred. Earlier studies with guinea pigs examined different aspects
of AFB1 toxicity, including its metabolism [175], the effects of pretreatment with AF on a second AF
treatment [176], the complement, bacteriostatic, and enzymatic activities in sera from guinea pigs
treated with AF [177], the protective effect of ascorbic acid from acute AFB1 toxicity [178], the induc-
tion of liver fibrosis by AFB1 in combination with copper [179], the changes in the gastrointestinal tract,
cardiovascular function, and drug metabolizing processes induced by AFB1 intoxication [180], and the
DNA damage caused by AFB1 [181].
Hamster models were much less frequently used in the study of Aspergillus mycotoxins. In an early
study, Ungar et al. [182] treated 8–10-week-old male Syrian hamsters with AFB1 in N-N-dimethyl-
formamide and studied the pathological effects of both the mycotoxin and the solvent. AFB1 could be
associated with periportal and midzonal necrosis of the liver. More recently, Rajmon et al. [183] studied
the influence of repeated low intragastrically administered doses of AFB1 and T2 toxin on Chinese ham-
ster and found the main liver condition indicators to be influenced by the AFB1-T2 toxin combination
more than by the two mycotoxins alone.

31.4.3 Human and Animal Cell Lines

In order to gain an overview of the recently published articles reporting the application of human and
animal cell lines to study Aspergillus mycotoxins, we collected full-text papers and abstracts published
after 2010 from the PubMed database with the keyword combinations aflatoxin OR ochratoxin OR ste-
rigmatocystin OR patulin AND cell line OR cell culture. Similar to the case of rat and mouse models,
the retrieved records were screened for relevant information (exact definition of the mycotoxin and the
cell line used).
Table 31.3 summarizes the studies published since 2010 that have used human or animal cell lines
as laboratory models for the investigation of Aspergillus mycotoxins [184–213]. Among the retrieved
records, relevant data were found in 31 publications. Eleven studies applied different liver cell lines
(human hepatocellular carcinoma cell lines Huh7, HepG2, Hep3B, or SMMC-7721, HepaRG termi-
nally differentiated hepatic cells derived from a human hepatic progenitor cell line that retains many
characteristics of primary human hepatocytes, the HeLa derivative Chang liver cell line, as well as
H4IIE rat hepatoma cells) for AFB1 research. The major outputs of these studies included the devel-
opment of a stable Huh7 cell line with constitutive expression of human CYP4501A2 [184], the dem-
onstration that AFB1 is able to activate the pregnane X receptor (PXR), a known regulator of liver
xenobiotic metabolism in human hepatocytes [185], the demonstration that AFB1 stimulates hepatoma
cell migration [186], the recognition that AFB1 exposure decreases the replication of the hepatitis B
virus [190], and the detection of an interaction between AFB1 and FB1 on activation and expression of
CYP1A and its transcription factor Ahr [194]. Another group of studies applied various kidney cell
lines (RPTEC/TERT1 immortalized human renal proximal tubular epithelial cells, IHKE immortal-
ized human kidney cells, Vero African green monkey kidney cells, or the porcine kidney cell lines
LLC-PK1 and PK-15) to study Aspergillus mycotoxins, primarily OTA. Among others, these studies
TABLE 31.3

480
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
Huh7 human AFB1 2.5 and 1–10 μM To develope a stable cell line with constitutive expression A cell line, Huh7–1A2-I-E, with high expression [184]
hepatocellular of human CYP1A2 in Huh7 cells level of CYP1A2 was developed and evaluated for
carcinoma cells identification of CYP1A2 inhibitors and for studies
of cytotoxicity resulting from CYP-mediated drug
metabolism; cellular toxicity of AFB1 in Huh7–1A2-
I-E cells could be prevented by furafylline, a
CYP1A2 inhibitor
HepG2 human AFB1 10 μM To perform a screen of mycotoxins that pose a known AFB1 was able to activate PXR, a known regulator of [185]
hepatocellular AFM1 environmental threat to human and animal health for the liver xenobiotic metabolism, in human hepatocytes,
carcinoma cells AFG1 ability to activate PXR function in a human hepatocyte and it could upregulate the expression of PXR-
cell line dependent genes responsible for AFB1
biotransformation, including CYP3A4
HepG2 and AFB1 2.5 μM To investigate the effects of AFB1 on key elements in AFB1 has stimulated hepatoma cell migration through [186]
SMMC-7721 IGF-IR signaling pathway and the effects of AFB1 on IGF-IR/IRS2 axis
hepatoma cells and hepatoma cell migration
immortalized human
Chang liver cells

Laboratory Models for Foodborne Infections

HepG2 hepatocellular AFB1 2.5–40 μM To examine the effects of AFB1 on UGT mRNA AFB1 has induced UGT2B isoforms rather than [187]
carcinoma cells expression in HepG2 cells UGT1A isoforms, which may closely contribute to
the toxicity of AFB1
HepG2 hepatocellular AFB1 1, 2.6, 6.5, 16, To develop genetically manipulated, upgraded HepG2 Upgraded cells (Adv-HepG2) were highly able to [188]
carcinoma cells 41, 102, 256, cells (Adv-HepG2) transiently expressing functional metabolize the toxin studied in contrast to the
640 μM levels of three major enzymes (CYP1A2, CYP2C9, and reduced metabolic capacity of HepG2 cells; hence,
CYP3A4) responsible for oxidative metabolism of drugs Adv-HepG2 cells are suitable to test hepatotoxicity
to confer drug-metabolic competence of bioactivable drugs
HepG2 hepatocellular AFB1 Concentrations To evaluate and establish the role of Eclipta alba as MDR EAE significantly inhibited the cell proliferation, and [189]
carcinoma cells of 35, 75, 150, reversal agent using multidrug-resistant hepatocellular findings revealed that Eclipta alba components are
300, 600, 1250, carcinoma cell line DR-HepG2 effective inhibitors of MDR1/P-gp
2500, and
5000 nM
(Continued)
TABLE 31.3 (Continued)

Aspergillus
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
HepaRG cells AFB1 1 and 5 mM To analyze the effects of HBV infection and replication on AFB1 exposure decreased the HBV replication, [190]
DNA damage by AFB1 and vice-versa in HepaRG, a cell whereas DNA damage by AFB1 and subsequent
line with hepatocyte-like morphology that metabolizes p53 induction was not affected by the presence of
AFB1 and supports HBV infection the virus; thus, in HepaRG cell line, AFB1 and
HBV do not cooperate to increase DNA damage
by AFB1
HepaRG cells AFB1 0.05, 0.1, 0.5, and To describe an improved methodology for the in vitro HepaRG hepatocytes exposed to various genotoxic [191]
1.0 μM micronucleus test using an enriched proportion of compounds requiring or not requiring
HepaRG hepatocyte-like cells and based on a complete bioactivation compared favorably with those
in situ assay, thereby avoiding cell detachment after reported in various other cell types; they supported
chemical exposure and allowing cell division after a the view that metabolically competent HepaRG
mitogenic stimulation cells have unique potential benefits for testing
genotoxic compounds using the in vitro
micronucleus assay
Human hepatocytes AFB1 0, 1, 2, and 3 μM To study the application of IdMOC in the definition of the The three model toxicants showed three distinct [192]
and 3T3-L1 mouse role of hepatic metabolism on the cytotoxicity of three cytotoxic profiles: TMX was cytotoxic to 3T3 cells
fibroblast cells model toxicants: CPA, AFB, and TMX via the in the absence of hepatocytes, with slightly lower
coculturing of the metabolically competent human cytotoxicity towards both 3T3 cells and hepatocytes;
hepatocytes with the metabolically incompetent mouse AFB was selectively toxic toward the human
3T3 fibroblasts hepatocytes and relatively noncytotoxic toward 3T3
cells; CPA cytotoxicity to the 3T3 cells was found to
be significantly enhanced by the presence of the
hepatocytes
Hep3B human STC 10−12–10−6 M To investigate the potential cytogenetic effects of STC, STC alone or in combination with OTA and/or CTN [193]
hepatocellular OTA OTA, and CTN alone or in combination, at pM to μM had a cytotoxic and cytogenetic potential even at
carcinoma cells CTN concentrations, on the human hepatocellular cancer cell picomolar concentrations on human hepatoma
line Hep3B cells in vitro
(Continued)

481
TABLE 31.3 (Continued)

482
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
H4IIE rat hepatoma AFB1 0.31, 0.63, 1.25, To evaluate the effects of mycotoxins alone or combined AFB1 and FB1, either alone or in combination, [194]
cells and spleen FB1 2.50, 5, 10, 20, on activation and expression of Cyp1A and its increased cyp1A transcription and Cyp1A activity,
cells from male 30, 40, and transcription factor Ahr in hepatoma cell line H4IIE and as well as upregulated the Ahr activity, but the
Wistar inbred rats 60 μM AFB1 rat spleen mononuclear cells response potency was significantly higher for the
and 1.56, 3.12, mixture, indicating the existence of an interaction
6.25, 12.50, 25, between both toxins
50, 100, 150,
200, and
300 μM FB1
RPTEC/TERT1 OTA 300 nM To characterize the metabolome of the human RPTEC/ Metabolic profiling of RPTEC/TERT1 cells could [195]
human renal TERT1 cell line and define the response over a 72-hour report the effect of chemical exposure on multiple
proximal tubular period to low-level exposure of several compounds that cellular pathways at low-level exposure and
epithelial cells affect kidney using NMR-based metabolic profiling produced different response profiles for the different
approach compounds, with a greater number of major
metabolic effects observed in the toxin-treated cells
RPTEC/TERT1 OTA 300 nM To investigate the effects of several renal chemical After exposure to two renal carcinogens (OTA and [196]
human renal carcinogens on primary cilium in proximal tubular KBrO3), RPTEC/TERT1 cells lost primary cilium;

Laboratory Models for Foodborne Infections

proximal tubular epithelial cells gene expression analysis demonstrated that loss of
epithelial cells the cilium was correlated with significant
dysregulation of cilia-related genes and further
underlined the divergent mechanisms of OTA- and
KBrO3-induced deciliation
LLC-PK1 porcine OTA 2.5–100 μM To compare AAI and OTA impact on VEGF expression as Both toxins exerted complex effects on various [197]
kidney cells well as transcription factors regulating its expression in transcription factors, resulting in differential
cultured kidney tubulus cells regulation of VEGF expression
PK-15 porcine kidney AFB1 ZEA AFB1: 5 mM, To evaluate the nephrotoxicity of individual mycotoxins Exposure to the mycotoxins AFB1 and DON could [198]
cells DON FB1 ZEA: 10 mM, and combinations of AFB1, ZEA, DON, and FB1 to induce ROS generation and cell apoptosis; DON
DON: 500 μM, livestock using PK-15 cells as a disease model tested via was the most cytotoxic mycotoxin to PK-15 cells
FB1: 2.5 mM biochemical approaches followed by AFB1 and ZEA; ZEA ameliorated
ROS production induced by low-dose AFB1; ZEA
or DON displayed synergetic effects with
high-dose AFB1 on ROS production; ZEA also
ameliorated AFB1-induced apoptosis (Continued)
TABLE 31.3 (Continued)

Aspergillus
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
IHKE immortalized OTA 0.01 nM–50 μM To confirm the proposed structure of OTα amide and OTα amide was less cytotoxic compared to OTA; [199]
human kidney cells OTα investigate if this compound can also be formed during thus, the thermal degradation of OTA to OTα
thermal processing, as it is commonly used in food amide might be a detoxification process; complete
technology structure elucidation of OTα amide was performed
via NMR/MS
Vero African green OTA 10, 20, 30, 40, To investigate the cytoprotective effect of quercetin on Quercetin pretreatment prevented the OTA-induced [200]
monkey kidney cells and 50 μM OTA-induced toxicity, with specific reference to oxidative stress and apoptosis in Vero cell line
oxidative stress, intracellular calcium flux, and levels of
protective antioxidant enzymes
J774A.1 murine AFB1 0.01 and To study the effect of AFB1, AFB2, and AFG1 exposure, Exposure of macrophages to low doses of AF [201]
macrophage cells AFB2 0.1 ng/mL alone and in combination, on the secretion of key resulted in a statistically significant change in the
AFG1 pro- and anti-inflammatory cytokines from J774A.1 cells secretion of a number of cytokines; AF exposure
affected expression levels of key cell surface
markers involved in the inflammatory response
Jurkat human OTA 10 μM To gain insights into the molecular mechanisms Diverse modes of action are involved in direct [202]
lymphoblastic underlying direct immunotoxicity immunotoxicity, and a set of pathways or genes,
T-cells rather than one single gene, could be used to
screen compounds for direct immunotoxicity
Jurkat human AFB1 3, 15, 30, and To investigate effects of AFB1 and AFM1 on immune Both AFB1 and AFM1 produced significant [203]
lymphoblastic AFM1 50 μM function decreases in cell proliferation, whereas only minor
T-cells effects were noted on IL-2 and IFN-γ cytokines
mRNA expression; AFB1, but not AFM1, at the
highest concentration induced a marked increase
in IL-8 mRNA expression
(Continued)

483
TABLE 31.3 (Continued)

484
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
Caco-2 human OTA 1 ng/mL, To improve the assessment of the toxicological impact of Induction of CYP1A1 activity and inhibition of [204]
colorectal 1.6–100 μg/mL, dietary contaminants (OTA , DON, IMA, and BEN) at CYP3A4 activity occurred in Caco-2 cells at a
adenocarcinoma 4, 40, 400, realistic human exposure levels, with a special focus on realistic intestinal concentration of IMA, while
cells 4,000, and the intestinal compartment OTA, DON, and BEN had no effect; some
H4IIE rat hepatoma 40,000 ng/mL bacterial stress genes were induced in a range of
cells, HepG2-Luc realistic concentrations, following exposure to
human hepatoma DON and IMA; BEN clearly provoked an
cells, and MMV-Luc estrogenic receptor agonistic activity in a human
human breast cancer estrogen-sensitive reporter cell line
cells
HCT116, SW620, PAT 1.25, 2.5, 5, 10, To study the effect of PAT on cancer cells and study its A new anticancer mechanism of PAT, suggesting that [205]
and Caco-2 human and 20 μM intracellular mechanism EGR-1 phosphorylation by oxidative stress
colorectal cells following PAT treatment might trigger EGR-1
binding to ATF3 promoter, which leads to the
expression of apoptotic proteins and cell growth
arrest of HCT-116 cells
Caco-2 human AFB1, OTA AFB1: 0.0038 ng/ To evaluate the effects of low levels of each mycotoxin in Individual toxicity showed that OTA was the most [206]

Laboratory Models for Foodborne Infections

colorectal FB1 mL–3.8 μg/mL, combination at their EU regulatory limits cytotoxic mycotoxin in all three cell lines; binary
adenocarcinoma OTA: 0.005 ng/ combinations were cytotoxic to the MDBK cell line
cells, MDBK bovine mL–10 μg/mL, in the order [OTA/FB1] > [AFB1/FB1] >
kidney cells, RAW FB1: 0.005 ng/ [AFB1/OTA]; tertiary combinations of AFB1, FB1,
264.7 murine mL–10 μg/mL) and OTA at the EU regulatory limits were not found
macrophages to exhibit measurable cytotoxicity; in the
concentrations above the legal limits, cytotoxicity
was observed with up to 26% reduction in cell
viability, and synergistic effects were evident with
mitochondrial integrity
Sp2/0 mouse AFB1 25 μg, 100 ng To describe the construction and expression of anti-AFB1 The yield of inclusion body of scFvs2E6 in either [207]
myeloma cells, scFvs 2E6 (scFv 2E6VL-linker-2E6VH and scFv VH/VL orientation was similar; however, only the
2C10 and 2E6 2E6VH-linker-2E6VL) in both VH/VL orientations scFv in VH-linker-VL orientation showed anti-AFB1
mouse hybridoma bioactivity after refolding
cells
(Continued)
TABLE 31.3 (Continued)

Aspergillus
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
SAOS-2 human AFB1 5 and 50 ng/mL To describe an unexpected toxicity of AFB1 toward the The expression of vitamin D receptor in [208]
osteosarcoma cells vitamin D receptors osteosarcoma cell line SAOS-2 is significantly
downmodulated by exposure to AFB1
CYP19- AFB1, 0.1% v/v To evaluate the effect of MTs (AFB1, zeranol, and ZEA) ZEA suppressed exon I.3 and II-specific expression [209]
overexpressing zeranol, ZEA on aromatase using 4 cell lines of aromatase and competitively inhibited the
MCF-7aro human enzyme activity; taken together, ZEA was a
breast cancer cells, potential aromatase inhibitor among the three MTs
JEG-3 human
placental cells,
MCF-7 human
breast
adenocarcinoma
cells, T98G human
Caucasian
glioblastoma cells
B-13 and B-13/H rat AFB1 100, 200, and To establish the utility of B-13 cells in toxicity and B-13 cell has generated hepatocyte-like cells with [210]
pancreatic 400 nM genotoxicity screening, CYP induction, susceptibility to functional drug metabolism and transporter
progenitor cells toxins, and transporter gene expression activities that could alone—or in a humanized
form—be used to screen for hepatotoxic and
genotoxic end points in vitro
A549 human lung AFG1 0.5, 2, 4, and To explore whether AFG1 activates the ROS/MAPK/ AFG1 induced the oxidative DNA damage and [211]
carcinoma cells 10 mg/L apoptosis pathway to cause cell damage in human AT-II triggered apoptosis through ROS-mediated JNK and
cells like the cell line A549 p38 signaling pathways in A549 cells, which
resulted in AFG1-induced AT-II cell damage
(Continued)

485
TABLE 31.3 (Continued)

486
Studies Using Human and Animal Cell Lines for the Examination of Aspergillus Mycotoxins Published Since 2010 Based on PubMed
Applied
Applied Cell Line MT Concentration Aim of the Study Major Findings References
INS-1 rat insulinoma PAT 0, 1, 10, 100, To analyze the effects of the microbial secondary Of the three compounds tested, PAT was the most [212]
cells 1,000, and metabolites pyrrolnitrin, phenazine, and PAT on INS-1 toxic; after both 24 and 72 h exposure,
10,000 ng/mL rat pancreatic β-cells concentrations ≥10 ng/mL caused significant cell
death with complete loss of viability at
10,000 ng/mL; PAT at 10 and 100 ng/mL caused a
significant reduction of insulin secretion per live cell
with 20 mM glucose; treatment of INS-1 cells with
PAT had no effect on insulin mRNA levels; PAT
showed no effect on the transcription of the insulin
gene at any concentration tested
NCTC clone L929 AFB1 100, 500, and To detect the antimutagenic and antiproliferative The isolated fractions obtained from octopus contain [213]
murine 1000 ng/plate compounds in octopus (Paraoctopus limaculatus) compounds with chemoprotective properties that
subcutaneous reduce the mutagenicity of AFB1 and proliferation
connective tissue of cancer cell lines
cells, and M12.
C3.F6 murine B cell
lymphoma cells

Laboratory Models for Foodborne Infections

AAI, aristolochic acid I; AF, aflatoxin; AFB1, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFM1, aflatoxin M1; Ahr, aryl hydrocarbon receptor; AT-II alveolar type II; ATF-3, AMP-
dependent transcription factor 3; BEN, benomyl; CFRD, cystic fibrosis-related diabetes; CTN, citrinin; CPA, cyclophosphamide; CYP, cytochrome P450; DNA, desoxyribonucleic acid;
DON, deoxynivalenol; EAE, Eclipta alba hydroalcoholic extract; EGR-1, early growth response 1; EU, European Commission; FB1, fumonisin B1; HBV, hepatitis B virus; IdMOC, inte-
grated discrete multiple organ co-culture system; IFN, interferon; IGF-IR, insulin-like growth factor receptor; IL, interleukin; IMA, imazalil; IRS2, insulin receptor substrate 2; JNK, Jun
N-terminal kinase; MDR, multidrug resistance; MT, mycotoxin; NMR/MS, nuclear magnetic resonance/mass spectrometry; OTA, ochratoxin A; OTα, ochratoxin α; PAT, patulin; PXR,
pregnane X receptor; RNA, ribonucleic acid; ROS, reactive oxygen species; TMX, tamoxifen; UGT, UDP-glucuronosyltransferase; VEGF, vascular endothelial growth factor; ZEA,
zearalenone.
Aspergillus 487

revealed that OTA causes significant deciliation in a model of the proximal tubule [196], that the
hypoxia inducible factor 2α (but not 1α) plays a crucial role in prevention of diminishment of vascular
endothelial growth factor production evoked by OTA in kidney proximal tubular epithelial cells [197],
and that with its high antioxidant potential, quercetin protects Vero cells from OTA-induced oxida-
tive stress and apoptosis [200]. The immunomodulatory or immunotoxic properties of Aspergillus
mycotoxins were studied on J774A.1 murine macrophages [201] and human T-cell lines [202,203].
Studies on human colon carcinoma cell lines (Caco-2, HCT116, and SW620) revealed important data
about the toxicity of AFB1, OTA, and PAT [204–206]; for the latter, a new anticancer mechanism
was proposed [205]. Further cell lines used since 2010 to study Aspergillus mycotoxins include Sp2/0
mouse myeloma cells and 2C10 and 2E6 mouse hybridoma cells [207]; SAOS-2 human osteosar-
coma cells [208], MCF-7aro human breast cancer cells, JEG-3 human placental cells, MCF-7 human
breast adenocarcinoma cells, and T98G human Caucasian glioblastoma cells [209]; B-13 and B-13/H
rat pancreatic progenitor cells [210]; A549 human lung carcinoma cells [211]; INS-1 rat insulinoma
cells [212]; as well as L929 murine subcutaneous connective tissue cells and M12.C3.F6 murine B cell
lymphoma cells [213].

31.5 Conclusions
Since 2010, the most popular laboratory models to study various aspects of Aspergillus mycotoxins were
animal models—primarily mice and rat lines—and human cell lines—mainly different liver and kid-
ney cells. Studies performed with the application of these laboratory models provided large amount of
important scientific information about the genotoxicity, organotoxicity, carcinogenicity, and metabolism
of Aspergillus mycotoxins, as well as about the protective, antioxidant, and immunostimulatory effects
of plant-derived metabolites, extracts, and oils against mycotoxicoses. The results from the studies dis-
cussed in this chapter clearly indicate that these laboratory models have the potential to further remain
in the frontline of mycotoxin research.

Acknowledgments
This work was supported by the Indian National Science Academy and the Hungarian Academy of
Sciences, as well as by projects NKFI-K115690 (National Research and Innovation Office, Hungary)
and GINOP-2.3.3-15-2016-00006 (Széchenyi 2020 Programme). Csaba Vágvölgyi thanks the visiting
professor program of King Saud University, Riyadh.

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32
Candida

María Jesús Andrade, Mar Rodríguez, Alicia Rodríguez, and Juan José Córdoba

CONTENTS
32.1 Introduction................................................................................................................................... 497
32.2 Foodborne Candida Infections..................................................................................................... 498
32.3 Investigation of Foodborne Candida Cases.................................................................................. 498
32.4 Laboratory Models for Analyzing Foodborne Candida Infections.............................................. 501
32.5 Conclusions................................................................................................................................... 506
Acknowledgements................................................................................................................................. 506
References............................................................................................................................................... 506

32.1 Introduction
The Candida genus comprises more than 200 recognized species1,2 of which more than 40 are impli-
cated in human infections.3 Candida is a natural inhabitant of human skin, intestinal and genitourinary
tracts, and mucosal surfaces. Being an opportunistic pathogen, Candida causes disease when the host
defenses are impaired. With recent increase in immunosuppressed patients, Candida infections have
been described with higher frequency.4,5
Nonetheless, the presence of Candida in foods has obvious positive effects, such as in fermented
foods and beverages. This fungus also contributes to the synthesis of some healthy compounds such as
vitamins and antioxidants.6 On the contrary, its negative effects on food products relate to food spoilage,
but not usually to food safety problems. Thus, Candida species are frequently included in daily food
consumption of human beings, as disorders attributable to pathogenic Candida through foods have been
described as improbable.2,7 Consequently, its effect on human health has been scarcely evaluated in the
literature and more attention has been paid to clinical Candida infections.
Candida albicans is the major species of the genus associated with human disease,1 although it is
not involved in foodborne infection. In addition, other Candida species that are frequently referred
to as “non-albicans” Candida species have been isolated as being responsible for infection including
C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei.2,5 In the recent years, the occurrence of infec-
tions caused by such “non-albicans” Candida species has increased. The abovementioned five species
are the causative agents of more than 90% of Candida infections.2,8 In spite of the fact that other Candida
species like C. kefyr, C. rugosa, C. guilliermondii, and C. famata have been reported as causative agents
of clinical infection, they have been rarely isolated from patients.3,5,9
Candida can cause a wide variety of infections in humans1 that range from superficial infection of skin
and mucosal membranes of intestinal and genital tracts,2 including cutaneous and oropharyngeal candi-
diasis and vaginitis,1 to systematic infections. The latter are of significant concern since they encompass
life-threatening diseases such as candidemia, endophthalmitis, endocarditis, osteomyelitis, and dissemi-
nated and central nervous system infections.1,8 Such severe invasive infections cause high morbidity and
mortality rates.8,10

497
498 Laboratory Models for Foodborne Infections

32.2 Foodborne Candida Infections

Candida species frequently associated with food have emerged as important pathogens in debili-
tated patients,3 whose damaged intestinal mucosa may allow translocation of the yeast from the
gastrointestinal tract to the blood system.2,4,6 After colonizing the gastrointestinal tract, systematic
dissemination causing a life-threatening infection could occur in vulnerable hosts. In spite of the fact
that food contaminated with Candida is not the starting factor in these cases, vulnerable patients are
advised to not consume foods with high yeast contents for preventing the contamination of catheters
and the opportunistic infection.6 It seems that Candida, which can be introduced into the hospital
environment via food, has the ability to grow and form biofilms on the catheters and other medical
invasive devices.4,6,11
Several Candida species are among the most common and widespread yeasts in food, but their dis-
tribution varies in relation to the type of food. C. zeylanoides has thus been reported as one of the main
species during the ripening of Iberian dry-cured ham12–14 and on fresh and processed poultry products.15
This species has also been found in fermented sausages and other meat products,16–18 cheeses,19,20 and
fruit juices.21 Candidemia, endocarditis, arthritis, and other disorders have been reported in association
with C. zeylanoides.1,22
Since the Candida genus is integrated by asexual yeast species, several other genera have Candida
anamorphs. Thus, some Candida anamorphs are frequently found in some food such as Debaryomyces
hansenii (anamorph C. famata) or Kluyveromyces marxianus (anamorph C. kefyr). It should be noted
that both species are used as commercial starter cultures and are included in the QPS (Qualified
Presumption of Safety) list developed by the European Food Safety Authority (EFSA).23 C. kefyr appears
to be an emerging pathogen.3 Nevertheless, until now, there are no safety concerns about the use of
K. marxianus because of the history of its apparent safe use and the rarity of its infections in human
beings.24 C. famata, found in food products such as cheese and meat products,12,18,25 has been associated
with occasional infections.3,8,9,23–25
C. krusei, one of the five most clinically relevant Candida species, has been found to be the predomi-
nant yeast in some African fermented products.26,27
Other Candida species related to infection have also been detected in food products. Specifically,
C. rugosa, C. glabrata, and C. tropicalis have been found in soy sauce koji, accounting for about 40%
of total yeasts, with C. rugosa being most predominant.28 On the other hand, C. tropicalis isolated from
sorghum beer has been proven to be adequate as starter culture for barley beer production.29 C. ­tropicalis
has also been found in orange fruit and juice.21,30 The potential significant role of C. krusei, one of
the dominant yeast species in the cocoa fermentation,31,32 has been reported.32 C. krusei isolated from
fermented maize dough has been selected as a candidate to be a starter culture during fermentation of
maize dough too.33 Nevertheless, due to the fact that the main human pathogenic yeasts are included in
the Candida genus and that several Candida species are considered as emergent pathogens, the genus
cannot be considered suitable for QPS status.24 Further investigations are needed to establish the role of
foods in Candida infections.11

32.3 Investigation of Foodborne Candida Cases

A wide range of procedures to detect Candida can be found in the literature. Most of them have been
specifically designed for clinical diagnosis but could be applied for analysis of Candida related to food
safety (Figure 32.1).
Diagnosis of Candida from clinical and food samples causing infection is of critical importance since
Candida species differ widely, both in their ability to cause infection and in their susceptibility to anti-
fungal agents.34 A rapid and accurate identification of Candida species is necessary to accelerate the
beginning of the proper therapy, which would improve the patient outcomes.
In this section, some of the methods reported to detect and differentiate Candida of clinical and food
origin will be reviewed.
Candida 499

Isolation Identification/detection

Phenotypic methods Molecular methods Proteomic phenotyping

Sabouraud’s
dextrose agar
Biochemical tests Sequencing of rDNA regions MALDI-TOF MS
(API®20 C AUX, API®Candida,
VITEK® 2, AuxaColorTM2, Real-time PCR
Bio-Rad Laboratories)
Multiplex PCR

Seminested/nested PCR
Microscopy
Chromogenic media
(morphology)
(CHROMagar Candida, NASBA
Candida ID 2)

Investigation of foodborne Candida infections

Animal models Cell lines

Caco-2
Mice
IPEC-J2

FIGURE 32.1  Procedures for analyzing foodborne Candida cases.

Diagnosis of Candida infections has traditionally relied on a combination of phenotypic features of the
pathogenic yeast and the clinical signs of the infection.2
When analyzing the clinical presenting features, a combination of tests is necessary to obtain an
adequate diagnosis.2 However, the main problem is that clinical signs are often nonspecific for Candida
disease.
Direct microscopic investigation of appropriate clinical samples and culture is included within the
phenotypic techniques. Microscopic morphology of Candida shows important differences depending on
environmental conditions and yeast species. Thus, Candida can produce round or elongated blastoco-
nidia, pseudohyphae, true hyphae, or chlamydospores.1 However, C. glabrata rarely produces pseudohy-
phae or hyphal structures.2
To study the other phenotypic features, the isolation of the causative agent is necessary. Sabouraud’s
dextrose agar (SDA) is the most commonly used primary isolation medium for Candida. However, it
does not normally allow differentiation between species.34 The colonies of Candida species are cream/
white colored with smooth and wet looking. Nevertheless, C. albicans has the capability to reversibly
change the surface appearance of its colony from smooth to rough as well as from creamy/white to
opaque, a modification that corresponds to an alteration in cell shape from round (oval) to elongate.2
After isolating Candida as the causative agent of an infection, the identification at species level of
the obtained isolates has to be performed. The assimilation and fermentation patterns can be studied
for such purpose35 by using the available commercial kits (API® 20 C AUX, API® Candida, VITEK® 2,
bioMérieux; AuxaColor™ 2, Bio-Rad Laboratories). Such commercial techniques have been used to test
for Candida of both food and clinical origin.
In addition, several chromogenic media for the direct and presumptive identification of Candida on the
basis of the colony color have been developed. Thus, agars such as CHROMagar Candida (CHROMagar
Company) and Candida ID 2 (bioMérieux) allow the differentiation of some of the most significant
pathogenic Candida species.36–38 The use of such media allows reducing the time of analysis in com-
parison with conventional phenotypic and biochemical methods. Despite the fact that the CHROMagar
Candida was originally developed for identifying Candida isolates of clinical origin, it has been used for
differentiation and presumptive identification of the most frequently isolated yeast species from different
ingredients used for salads.39
500 Laboratory Models for Foodborne Infections

Species identification of Candida based on the abovementioned phenotypic methods is not always suf-
ficient in spite of using different tests5 because of their possible ambiguous or inconclusive results. This
fact together with the fact that they are often time consuming cause that infections are often not diag-
nosed or the diagnosis is obtained too late. Consequently, these methods are of limited clinical value, at
least when they are used alone.
Nonculture diagnostic methods are available as alternative and supplementary to the conven-
tional ones.
Thus, serological tests can be performed by detecting antigens and antibodies. Such tests are used
in combination with conventional techniques for improving the diagnosis. The mannan and (1,3)-β-d-
glucan detection is widely used. The mannan constituent of the Candida cell wall is a polysaccharide
used as a target of many serological tests performed in serum or plasma specimens since it induces a
strong antibody response.40 Tests based on the detection of antibodies directed against the mannan anti-
gen are recommended in combination with those based on Candida antigens for improving the sensitiv-
ity and the specificity of the diagnosis.41 The commercial Platelia Candida antigen and antibody tests
(Bio-Rad Laboratories) are the most commonly used serological methods for detecting Candida based
on mannan.41,42 However, since the antibodies could be not produced in immunocompromised patients,
it is very complicated to diagnose Candida infections in them.34,41 (1,3)-β-d-Glucan is also a cell wall
component not only of Candida species but also of most of the other fungi. Several assays have been
developed for quantifying such compounds in blood as a tool in the diagnosis of many pathogenic fungi
including Candida.40,43
The detection of d-arabinitol, a metabolite produced by some of the Candida species with clinical
importance, has also been proposed for diagnosis.40
The serological methods have limitations for the diagnosis of candidal infections due to their sensi-
tivity and specificity. Besides, they cannot be used alone for reliable identification of clinical Candida
isolates as previously described for traditional techniques.
To overcome the limitations of culture and nonculture methods, nucleic-acid-based procedures have
been developed since they are powerful tool because of their rapidity and sensitivity. The literature
contains a lot of data regarding molecular-based technology for clinical diagnosis of Candida. Lewis
et  al.2,35 summarized the polymerase chain reaction (PCR) methods published for detecting Candida
species from clinical samples, with most of them being based on rDNA regions as target. These authors
considered that such methods could also be used for screening food products. However, the type of
sample, enrichment processes, and DNA extraction should be optimized for the particular food and for
removal of any interfering compounds within.2
Identification by sequencing of rRNA genes has been widely used for identifying Candida of both
food and clinical origins.28,44,45 However, such technique despite being a valid method for identifying
Candida is time consuming, expensive, tedious, technically demanding, and inappropriate for routine
applications.46,47
Among various molecular-based methods, real-time PCR (qPCR) has advantages of speed, sensitivity,
and specificity.48–50
Multiplex PCR detecting different targets simultaneously and semi-nested/nested PCR incorporating
two round of amplification reactions have also been developed.51–54
Other molecular-based methods for detecting Candida RNA include nucleic-acid-sequence-based
amplification (NASBA),55 etc.
Despite the vast number of suitable published molecular methods for detecting and/or identifying
Candida species, they are still lacking standardization. Thus, it has been suggested that a combination of
methods, including the molecular ones, may be performed to obtain a certain diagnosis.5,43
Novel technologies such as matrix-assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF MS) have been reported as rapid and accurate methods for routine identification of
Candida associated with human and food.46,47,56,57 However, some of the authors have stated the necessity
of improving the databases of the available systems for such technique.
The former methods can detect Candida involved in foodborne infection or in patients with clinical
symptoms of disease. However, the study of candidal infections has also involved the subsequent inves-
tigation of the yeast in animals and other laboratory model systems.58 They are necessary to provide
Candida 501

new insights for clarifying and fully understanding the mechanisms leading to human Candida disease
independently of whether the yeast is of clinical or food origin. Such powerful systems will be detailed
in the next section.

32.4 Laboratory Models for Analyzing Foodborne Candida Infections

Many laboratory models have been developed for analyzing different aspects of clinical Candida infec-
tions, particularly those relating to C. albicans. Nevertheless, experimental models are not normally
designed for investigating foodborne pathogen Candida species. As mentioned in Section 32.2, food-
borne candidal infections could be found in humans with the altered integrity of the mucosal epithelium
of the gastrointestinal tract or due to Candida biofilm formed in invasive devices. Since Candida spe-
cies frequently associated with food are emerging as causative agents, the development of laboratory
models for such species should be essential for understanding their pathogenicity. This section will thus
be focused on studies using laboratory models related to the gastrointestinal tract (Table 32.1), the most
relevant anatomic route of infection of foodborne Candida. Although the laboratory models reported
until now have been developed for analyzing Candida of clinical origin, most of them could be adapted
for being used with foodborne Candida (Figure 32.1).
A number of nonvertebrate and vertebrate animal models have been described as suitable to study
Candida pathogenesis and virulence, immune response of the host, and efficacy of novel antifungal and

TABLE 32.1
Laboratory Models Used to Investigate Gastrointestinal Candida Infections
Candida Species
Model Type Detected Main Measured Parameters References
Mouse C. albicans Histopathology, yeast morphology and burden, 75
C. glabrata inflammatory response
C. albicans Yeast burden, histopathology 68
C. tropicalis
C. parapsilosis
C. albicans Yeast burden, role of MAPK pathways 71
C. albicans Yeast burden, colonization and invasion, mortality 70
C. tropicalis
C. albicans Yeast burden and morphology, immune response 74
C. albicans Yeast burden and morphology 72
Mouse, piglet C. albicans Yeast burden and morphology, gene expression 73
Drosophila larvae C. albicans Microscopy, systematic response 84
Caco-2 cells C. albicans LDH assay, genetic analysis, immune response 90
C. tropicalis
C. parapsilosis
C. krusei
C. tropicalis Adhesion, cell damage, gene expression 89
C. albicans Yeast burden and morphology, adhesion, topography of 88
cells, transcriptional regulation during adhesion
C. albicans Adhesion, cell damage, invasion, transmigration, yeast 66
morphology, viability, and hydrolases
Caco-2 and IPEC-J2 cell C. albicans Yeast morphology, transepithelial electrical resistance 27
lines C. rugosa
C. tropicalis
C. krusei
502 Laboratory Models for Foodborne Infections

vaccination strategies. The advantages and limitations of both in vivo models have been summarized by
Naglik et al.58 The main advantage of nonvertebrate animal models consists of lower cost and fewer ethi-
cal concerns than vertebrate ones. Vertebrate animal model systems are more adequate tools for studying
the host–Candida interactions than the nonvertebrate animal ones due to the fact that the used animals
and their environment can be precisely controlled in a manner that is more relevant to humans. A good
in vivo model for investigating Candida infections should be as reproducible as possible regarding the
process of colonization and invasion at a specific portal of entry leading to infection as well as the major
clinical symptoms detected in the human disease, relatively easy to set up, and closely match the specific
immune defects or hormonal conditions associated with infection at that particular site.59,60 The cost
of the model should also be considered in the choice.60 In addition, the host, the tested Candida strain,
and the route of its administration are of critical importance to obtain reliable experimental conclusions
when selecting the in vivo experimental model.61
Although many clinical animal models have been developed, rodents (mainly mice) are the most
commonly used laboratory ones (Table 32.1) because of ethical reasons and their relative anatomic and
immunological similarity to human.60,62 Moreover, they are widely available, easy to handle, and rela-
tively inexpensive.63 However, since C. albicans is an opportunistic pathogen of humans but not a natural
inhabitant of rodents, such models could give responses to infections in a different way than human
beings, and so comparison between such models and human studies could be difficult.58,64,65 Such differ-
ence should be considered when interpreting experimental data.61
In the case of modeling gastrointestinal Candida infections, mice are the animals selected by the
majority of researchers,65 and rats have been occasionally used.58 However, most of them are mainly
focused on studying the later dissemination of Candida because systematic dissemination normally
originates from the gastrointestinal tract.60,66
In gastrointestinal models, it has to be taken into account that the colonization of the gastrointesti-
nal tract by Candida seems to be apparently inhibited by the innate defenses of the host and mucosal
immune response and also by competition of Candida with the native microbial population of the tract.67
Thus, for establishing the gastrointestinal Candida infection the normal mucosal microbial population
must be removed or the yeast must become implanted in the gastrointestinal tract before colonization
by this microbial population.67 Gastrointestinal models are thus commonly based on the use of immu-
nosuppressed adult mice or neonatal mice.68 For neonatal models, no pretreatment is necessary as for
adult ones, which have to be treated for removing their natural gastrointestinal microbial population by
using immunocompromising agents (such as by antibiotic administration) prior to Candida infection.60,67
Germ-free or transgenic mice could also be used for this purpose.58 The performed mice manipulations
have to be considered when interpreting the obtained results.58 Adult mice have been considered as more
suitable since their handling and care are easier and less time consuming and their mortality due to the
inoculation is minimal.68 Adult mice can be inoculated either intragastrically or orally by using contami-
nated food or drinking water,60 the second option being the most adequate for studying gastrointestinal
infections related to foodborne Candida as the inoculum should be administered via the physiologically
relevant portal of entry.61 Since mice are coprophagous, the gastrointestinal mucosa of the animal fed
with Candida contaminated food seems to be exposed to high levels of the yeast.67 The majority of the
clinical models based on gastrointestinal infection have carried out the injection of the yeast directly into
the tract. After Candida inoculation, the maintenance of antibacterial antibiotic administration is neces-
sary for achieving sustained gastrointestinal colonization69 and allowing yeast dissemination to occur.60
The level of Candida inoculum used for gastrointestinal models ranges from about 107 to 108 cells
suspended in a sterile saline solution, mainly phosphate buffered saline (PBS), when performing
intragastric administration.68,70–73 The inoculum level ranges from about 106 to 107 cells/mL via drink
administration.74,75
The behavior of non-albicans Candida species, including C. tropicalis, C. glabrata, and C. parapsilo-
sis, has also been analyzed by using gastrointestinal models, but scarcely. Several authors have compared
the virulence of C. albicans and C. tropicalis in a gastrointestinal model in mice, with C. tropicalis
appearing as more virulent than C. albicans.60,70 When comparing the virulence of C. tropicalis, C. gla-
brata, and C. albicans in a gastrointestinal mouse model, the three species colonized the gastrointestinal
tract, with C. tropicalis showing the least ability and the subsequent dissemination was only observed in
Candida 503

C. albicans and C. tropicalis.68 The virulence of C. glabrata was also less than that shown by C. albi-
cans by using this kind of models.75 It seems that virulence mechanisms differ between Candida species,
but more research using experimental animal models is necessary.
The most usually measured parameters in mice Candida gastrointestinal models during the coloniza-
tion and the later dissemination include yeast burden at various sites, organ histopathology, and mortality
of animals (Table 32.1). Candida burden has been determined in fecal and cecal contents and also in
organs such as esophagus, stomach, intestines, cecum and colon, and internal organs (kidney, spleen,
lungs, liver).68,70,72,74,75 Visualization under microscope of stools and histologic examination of different
organs for identifying yeast or filamentous forms have also been reported for most of gastrointestinal
models. It seems that hyphae, but not yeast, are implied in the inflammatory and immune responses dur-
ing colonization of the gastrointestinal tract,74 having a significant role in the pathogenesis by invading
epithelial cells and causing damage of tissues.76 The ability to change morphology from yeast to hyphal
form (dimorphism) in response to environmental changes is one of the best studied virulence attributes
and characteristics of C. albicans.77 However, the conversion of C. albicans to hyphae has been described
by other authors as important but not essential for pathogenesis and tissue invasion.78
Regarding Candida immunity in gastrointestinal models, the host immune response during Candida
colonization of the gastrointestinal tract has been scarcely studied. Vautier et al.74 performed an immu-
noassay for analyzing cytokine levels after inoculating antibiotic-treated mice with C. albicans via their
drinking water containing 107 yeast cells/mL together with antibiotics. Kidney, stomach, intestines, and
caecum were used for such analysis. They reported that colonization of the gastrointestinal tract by
C. albicans was not influenced by Th17 immunity.
The inflammatory response in gastrointestinal tissues was studied by Westwater et al.75 in an assay
based on phenotyping of GR1 granulocytes. C. glabrata and C. albicans were separately administrated
to germ-free mice via drink. C. glabrata did not evoke a granulocytic inflammatory response in the host,
in contrast to C. albicans.
Few studies have investigated the genes expressed in C. albicans during the gastrointestinal coloniza-
tion in mice and also in piglets. White et al.73 extracted RNA from different tissues of infected piglets and
from cecum and ileum contents from mice, which was used for quantitative real-time reverse transcrip-
tase PCR (qRT-PCR) and microarrays. Their aim was the study of the genes expressed by C. albicans
during its growth in the gastrointestinal tract. In a separate work, Prieto et al.71 used a gastrointestinal
model in antibiotic-treated mouse to determine the role of the mitogen-activated protein kinase (MAPK)
pathways of C. albicans in the colonization of the gastrointestinal tract. Such pathways represent one of
the main mechanisms of adaptation to environmental changes in C. albicans and play an important role
in virulence.71
In the above-described gastrointestinal models based on the host immune and inflammatory responses
and genetic studies, Candida loads were also determined in different sites of the gastrointestinal tract
and in stools.
Laboratory models focused on other vertebrates animals such as monkeys,79 piglets,80 rabbits,81 and
guinea pigs82 have also been proposed.60 In spite of the fact that the larger the animal model, the easier
it is to sample repeatedly,58 the use of those animals larger in size than mice implies higher cost and
husbandry requirements, more intense monitoring during experimental infection, and fewer available
molecular reagents.61 In addition, they are not available as genetically defined strains.61
Due to the fact that research involving mammals is generally expensive, complex, and fraught with
ethical concerns, alternative animal models for studying clinical Candida infection have been used.83
Zebrafish (Danio rerio), a vertebrate minihost, and some nonvertebrate minihosts, including wax moth
(Galleria mellonella) and silk worm (Bombyx mori) larvae, the fruit fly (Drosophila melanogaster), and a
nematode (Caenorhabditis elegans), have been thus reported for studying Candida virulence.60 A limita-
tion of experimental models based on minihosts is that many of them lack an adaptive immune system.60
A Drosophila larvae model has been reported as appropriate to assay the host–pathogen interaction due
to an intestinal infection by Candida and the factors required for disseminated infection.84 The similarity
of mammals’ and flies’ immune response allows analyzing the genes involved.84 However, the differ-
ences between fly and mammalian pathophysiology have to also be considered when using Drosophila
intestinal models,83 as previously mentioned for murine models. Since in the developed D. melanogaster
504 Laboratory Models for Foodborne Infections

model the larvae were fed with Candida contaminated food, such experimental model should be well
suited for analyzing foodborne Candida infections. This characteristic is also attributable to the model
described by Pukkila-Worley et al.,85 who studied C. albicans pathogenesis using C. elegans as host,
which was fed with artificially contaminated food, and described the utility of the assay for screening
the genes involved in Candida morphogenesis and virulence. Since both the genome of D. melanogaster
and C. elegans have been sequenced,62 genetic analyses are available when performing experimental
Candida models, which is not possible for wax moth and silk worm models because of lack of genome
sequencing.62 The use of invertebrate animal models has the disadvantage that they have to be kept at
temperatures below the normal human body temperature, except for Galleria, which could affect the
Candida–host interactions.60
On the other hand, Candida, including foodborne species, commonly grow as a biofilms in medi-
cal devices, thereby providing protection from the host immune system and antimicrobial therapies.86
Scarce works based on experimental models for evaluating the formation of biofilms in invasive devices
are available.86
Although animals offer a good model for analyzing Candida infections, they have some limitations,87
such as they are not appropriate for wide laboratory testing and routine analysis since special facilities
and special trained personnel are needed. Besides, ethical considerations should be taken into account
when animals are used. The absence of Candida as native microorganisms in the gastrointestinal tract of
rodent models should also be considered when searching for a good model for Candida analysis.
Several in vitro alternative methods based on the use of cell lines and epithelial organotypic models
are available for such purpose, which are less expensive and time consuming to obtain results than ani-
mal models87 (Table 32.1). They have been developed for Candida of clinical origin but not for foodborne
Candida, as mentioned for animal models. However, such models could be adapted for such purpose.
When selecting an in vitro model, its availability and easy handling for high-throughput testing as well
as its good human predictive power must be checked.87
Most of the performed investigations based on the use of cell lines for host–Candida interaction stud-
ies have been focused on the ability of Candida (mainly C. albicans) to adhere to different epithelia.
For studying foodborne Candida, the most interesting cell lines are those of intestinal origin. Specific
studies must be carried out in gastrointestinal models since the environment of the different anatomic
sites that could be colonized by Candida differs significantly, particularly with respect to pH, epithelium
metabolism, or surface characteristics.88 Due to the fact that the attachment of Candida to intestinal epi-
thelium is the initial step in the infection, it is an essential parameter to be analyzed in a gastrointestinal
model based on cell lines. The ability of C. albicans to adhere to and invade into intestinal cells for its
translocation across the intestinal barrier and the subsequent dissemination through the body have been
demonstrated by using intestinal cell lines.66 To analyze the adhesion of C. albicans to an intestinal
epithelium in vitro, the Caco-2 cell line has been the most commonly used. Caco-2 is a human epithelial
colon adenocarcinoma cell line.87 Intestinal cell models should be functionally similar to the in vivo situ-
ation,87 and such cells have this characteristic since they resemble morphologically as well as biochemi-
cally primary enterocytes in many characteristics.88
Some examples of the use of Caco-2 cell line will be briefly described below.
Sohn et al.88 infected monolayers of Caco-2 grown to confluency with C. albicans. Polystyrene plates
were used for an adhesion assay. After applying C. albicans to the confluent monolayers, adherent
C. albicans cells counting was performed. Scanning electron microscopy (SEM) was also carried out for
visualizing the surface topographies of cells and the presence of hyphae. The Caco-2 cells had a brush
border surface consisting of microvilli structures, which seemed to be tightly linked to C. albicans
hyphae. The transcriptional regulation during adhesion of C. albicans to Caco-2 cells was analyzed by
genome-wide Candida DNA microarray analysis using total RNA isolated from the adhesion assay in
order to identify how C. albicans responds on the transcriptional level to the environmental stimuli dur-
ing adhesion to intestinal cell. They found that the adherence of C. albicans to Caco-2 cells requires the
expression of adhesive structures on the yeast surface with the ability of interacting very tightly with the
host tissues.
Negri et al.89 also evaluated the attachment of C. tropicalis to intestinal epithelial cells by using Caco-2
cells. Besides analyzing adhesion of C. tropicalis to a confluent layer of intestinal cells, the degree of
Candida 505

the epithelial cell damage was assessed by measuring the lactate dehydrogenase (LDH) activity, and the
inhibition of cellular activity was determined by tetrazolium salt reduction. C. tropicalis was able to
adhere to and to damage Caco-2. However, differences were observed in this ability depending on the
type of used cells. This fact was also strain dependent. Since the secretion of hydrolytic enzymes such
as secreted aspartyl proteinases (SAPs) is considered an important factor for C. tropicalis virulence, the
expression of four genes encoding SAPs (SAPT1, SAPT2, SAPT3, and SAPT4) present in C. tropicalis
was also investigated by qPCR after RT-PCR, which was affected by C. tropicalis adhesion to the cells.
The SAP expression was also strain and human cell line dependent. This fact highlights the necessity to
check specific Candida strains in specific anatomic sites.
Dalle et al.66 also investigated the potential of C. albicans to adhere to and invade into Caco-2 cells.
They performed adherence assay by using epifluorescence after calcofluor white staining, SEM, and
measurement of LDH release, among other techniques. These authors found that adhesion, invasion, and
damage of C. albicans depended not only on yeast morphology and activity, but also on the epithelial
cell type and the differentiation stage of the epithelial cells, indicating that epithelial cells differ in their
susceptibility to C. albicans. Besides, they reported that hyphae seem to have a dominant role in penetra-
tion of monolayers of intestinal cells in relation to yeasts.
Intestinal Caco-2 cells have been recently used for analyzing the host immune response to Candida
since intestinal epithelial cells not only represent a physiologic barrier for the yeasts, but also have a
crucial role in the innate immune system as they, for example, produce cytokines and antimicrobial
peptides.90 The effect of C. albicans as well as C. krusei, C. tropicalis, and C. parapsilosis on the pro-
duction of human β-defensin 2 (HBD-2), a key component of the innate immune system, by Caco-2 cell
line has been studied by RT-q PCR.90 To determine the HBD-2 secretion induction and HBD-2 peptide
expression in Caco-2 cells infected with Candida spp., enzyme linked immunosorbent assay (ELISA)
and Western blot analysis were carried out, respectively. Candida spp. stimulated HBD-2 expression in
and release from Caco-2 cells, with C. albicans inducing the highest levels of HBD-2.
Cell lines have been interestingly used for investigating the probiotic potential of some yeasts from
food including Candida species, but their pathogenic potential was also analyzed.27 C. rugosa, C. tropi-
calis, and C. krusei, which are considered causative agents of infections, were studied. They were previ-
ously isolated from Fura, a spontaneously fermented pearl millet product consumed in West Africa. As
control, the authors included a pathogenic yeast strain of C. albicans. These authors used two intestinal
cell lines: Caco-2 and IPEC-J2. In spite of the fact that IPEC-J2 was isolated from the small intestine
of neonatal pig,87 it has been used for studying pathogen/beneficial interactions of microorganisms due
to its similarity to the human intestinal epithelial cells.27 In such study, after growing both cell lines
in different media, the cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air
until a confluent monolayer was obtained. The culture media were changed every second day, and the
cells were passaged every 5–7 days by trypsination with 1% trypsin–EDTA 10×. Cell passages used
were P55–57 and P26–28 for Caco-2 and IPEC-J2, respectively. The transepithelial electrical resistance
(TEER) assay was used for analyzing the potential pathogenic effect (and also the probiotic one) of used
yeasts by means of evaluating the effect of yeast strains on cell polarity. To obtain a monolayer of polar-
ized intestinal epithelial cells, both cell lines were cultured on polycarbonate membrane inserts. After
reaching confluence, Caco-2 cells were cultured on the membranes for 21 days in order to obtain cell
differentiation, while IPEC-J2 cells were cultured for 16 days in order to obtain a proper mucus layer. An
inoculum of 106 cells/mL of yeasts was added and incubated at 37°C in a humidified atmosphere of 5%
CO2 and 95% air. The TEER of monolayers with Caco-2 or IPEC-J2 cell lines was then measured and
the morphology of yeasts observed by microscopy. Moreover, C. krusei, C. tropicalis, C. rugosa, and
C. albicans were able to survive under conditions simulating passage through the human gastrointestinal
tract [37°C, pH 2.5, and 0.3% (w/v) bile salts]. The ability of potential pathogenic foodborne Candida
to survive the conditions in the gastrointestinal tract should also be evaluated such as the survival and
growth at 37°C and the presence of high bile salt concentration and low pH.
Epithelial organotypic models have been described as effective as an in vitro model for evaluating
the invasion of C. albicans too. Such models allow studying the tissue degradation in Candida infec-
tion as well as conducting experiments to characterize pathogenesis-related genes. The mucosal epithe-
lium is very important in host defense and immune surveillance, since it is the primary cell layer that
506 Laboratory Models for Foodborne Infections

first encounters potentially pathogenic microorganisms.91 Reconstituted human epithelium that mimics
human epithelium is commercially available. It has been used as model for oral and vaginal candidiasis,
but not for foodborne Candida infections. After infecting the tissues, their level of colonization and
invasion and morphological characteristics of Candida can be analyzed by microscopy.92 Epithelial dam-
age can be visualized by histological analysis and quantified by the extracellular activity of LDH.91,92
Besides, the expression patterns of several Candida virulence genes, such as agglutinin-like sequence
(ALS), hyphal wall protein (HWP), epithelial adhesion (EPA), phospholipase B (PLB), PLD, and SAP
genes, can be analyzed by RT-PCR when using such in vitro models.91–93 The lack of certain host immune
responses is a limitation of such epithelium models, while proinflammatory cytokine responses by the
tissue infected with Candida are associated with the yeast strain virulence.91,93

32.5 Conclusions
Several Candida species are commonly present in food and beverages. However, candidal infections are
not normally associated with consumption of food containing Candida. In the last years, some Candida
species found in food are emerging as causative agents of infections. Further researches are thus neces-
sary for elucidating the role of foodborne Candida in human infections.
A variety of methods to detect and diagnose Candida infections have been described, most of which
have been designed for clinical purpose but could also be applied for investigation of foodborne cases.
Phenotypic methods have been traditionally used for detecting Candida; however, they are time con-
suming and can lead ambiguous or inconclusive results. Serological methods have limited sensitivity
and specificity. More recently, molecular-based protocols have become available, although they are still
in need of proper standardization. Consequently, a combination of methods for diagnosis of pathogenic
Candida from both clinical and food origins should be performed for obtaining definitive results.
In order to analyze different aspects of clinical Candida infections, several laboratory models based
on using vertebrate or nonvertebrate animals, cell lines, and commercial reconstituted human epithelium
have been developed. Mice are the most commonly used animal models because of ethical reason and
their relative anatomic and immunological similarity to human beings despite the fact that Candida is
not a natural inhabitant of mice. The use of cell lines such as Caco-2 cells has less ethical concerns and
other advantages compared to animal models. In addition, epithelial organotypic models used for analyz-
ing oral and vaginal candidiasis could be adapted for Candida foodborne investigation.
As most currently available methods have focused on Candida of clinical origin and not on foodborne
Candida, further investigations are required to develop adequate laboratory models for investigating
foodborne Candida infections. These include refining the experimental gastrointestinal models designed
for clinical Candida.

Acknowledgements
This work has been funded by the Spanish Comisión Interministerial de Ciencia y Tecnología with the
projects AGL2013-45729P and Carnisenusa CSD2007-00016, Consolider Ingenio 2010 and GRU09162
of the Junta de Extremadura and FEDER.

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model, Mycol. Res., 97, 385, 1993.
68. Mellado, E. et al., Sustained gastrointestinal colonization and systemic dissemination by Candida albi-
cans, Candida tropicalis and Candida parapsilosis in adult mice, Diagn. Microbiol. Infect. Dis., 38,
21, 2000.
69. Koh, A.Y., Murine models of Candida gastrointestinal colonization and dissemination, Eukaryot. Cell,
12, 1416, 2013.
70. De Repentigny, L., Phaneuf, M. and Mathieu, L.G., Gastrointestinal colonization and systemic dissemi-
nation by Candida albicans and Candida tropicalis in intact and immunocompromised mice, Infect.
Immun., 60, 4907, 1992.
71. Prieto, D. et al., The HOG pathway is critical for the colonization of the mouse gastrointestinal tract by
Candida albicans, PLoS One, 9, e87128, 2014.
72. Wiesner, S.M. et al., Gastrointestinal colonization by Candida albicans mutant strains in antibiotic-
treated mice, Clin. Diagn. Lab. Immunol., 8, 192, 2001.
73. White, S.J. et al., Self-regulation of Candida albicans population size during GI colonization, PLoS
Pathog., 3, e184, 2007.
74. Vautier, S. et al., Candida albicans colonization and dissemination from the murine gastrointestinal
tract: the influence of morphology and Th17 immunity, Cell Microbiol., 17, 445, 2015.
75. Westwater, C. et al., Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity
in a gnotobiotic model of mucosal candidiasis, FEMS Immunol. Med. Microbiol., 51, 134, 2007.
76. Yan, L., Yang, C. and Tang, J., Disruption of the intestinal mucosal barrier in Candida albicans infec-
tions, Microbiol. Res., 168, 389, 2013.
77. Wächtler, B. et al., From attachment to damage: defined genes of Candida albicans mediate adhesion,
invasion and damage during interaction with oral epithelial cells, PLoS One, 6, e17046, 2011.
78. Pacheco, M. et al., Attachment and entry of Candida famata in monocytes and epithelial cells, Microsc.
Res. Tech., 70, 975, 2007.
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animal model, J. Clin. Microbiol., 38, 2317, 2000.
81. Filler, S.G. et al., Comparison of fluconazole and amphotericin B for treatment of disseminated candi-
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83. Apidianakis, Y. and Rahme, L.G., Drosophila melanogaster as a model for human intestinal infection
and pathology, Dis. Model. Mech., 4, 21, 2011.
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gastrointestinal infection of Drosophila larvae by Candida albicans, Dis. Model. Mech., 4, 515, 2011.
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Caenorhabditis elegans infection model, Eukaryot. Cell, 8, 1750, 2009.
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microbiology—a review, Int. J. Food Microbiol., 141, S4, 2010.
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albicans to different human epithelia, FEMS Yeast Res., 6, 1085, 2006.
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lia, Nat. Protoc., 1, 2767, 2006.
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stituted human vaginal epithelium, J. Infect., 69, 396, 2014.
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confocal laser scanning microscopy, Oral Microbiol. Immunol., 22, 188, 2007.
33
Enterocytozoon bieneusi

Hirotake Mori and Aongart Mahittikorn

CONTENTS
33.1 Structure and Life Cycle................................................................................................................511
33.2 Biochemistry and Physiology.........................................................................................................512
33.3 Immunology...................................................................................................................................512
33.4 Host Range.....................................................................................................................................513
33.5 Transmission..................................................................................................................................513
33.6 Clinical Symptoms and Pathology.................................................................................................514
33.7 Techniques for Detection and Characterization............................................................................514
33.7.1 Light Microscopy..............................................................................................................514
33.7.2 Electron Microscopy.........................................................................................................514
33.7.3 Immunofluorescence.........................................................................................................515
33.7.4 In Vitro Culture and Animal Models................................................................................515
33.7.5 Nucleic-Acid-Based Detection..........................................................................................515
33.7.6 Detection from Environmental Samples...........................................................................516
33.8 Treatment.......................................................................................................................................516
33.9 Conclusion and Perspectives..........................................................................................................517
References................................................................................................................................................517

Microsporidia are a large group of obligate intracellular parasites that infect almost all types of animals,
including humans, domesticated animals, wildlife, fish, and even insects. To date, over 1400 microspo-
ridian species have been described, with new species being discovered every year.1,2 Of these, 14 species
in 8 genera have been recognized as human pathogens.3 Recently, members of the phylum Microsporidia
have been shown to be related to fungi based on phylogenetic analysis of genes coding for tubulin and
mitochondrial-like HSP70, amongst others.4,5
As the most common cause of intestinal microsporidiosis,6 Enterocytozoon bieneusi was the only
known member of the genus Enterocytozoon until the recent identification of E. hepatopenaei from
shrimp.7,8 Since the mid-1980s, E. bieneusi has become an increasingly important opportunistic patho-
gen, especially in HIV/AIDS patients.9,10 While infective to both immunocompetent and immuno-
compromised hosts, E. bieneusi is associated with more persistent and severe diarrhea and wasting in
immunocompromised individuals.11 Given that E. bieneusi is unresponsive to therapies that are effective
against many microsporidia,12 there is an urgent need to utilize tissue culture and small animal models
to better understand the infection, transmission, and pathogenesis of this parasite and to develop more
efficient treatment strategies against E. bieneusi disease.

33.1 Structure and Life Cycle

Microsporidia possess eukaryotic (a true nucleus, an endomembrane system, and a cytoskeleton), prokary-
otic (genome size and transcriptional apparatus), and fungal (chitin and trehalose) characteristics.1 As shown
in Figure 33.1, the life cycle of E. bieneusi includes several stages. In the early proliferative stage, the nucleus

511
512 Laboratory Models for Foodborne Infections

HO
In host cell HN
N
N
N
HN N
N Early
proliferative HC N

Pt cells N
Sporogonial
ES plasmodium
ENDOGENOUS
stages
HC
Sporoblasts N
GI tract HN N
Spore coat N
EXOGENOUS
Excretion

Ingestion Pt
Spore

FIGURE 33.1  Diagram of Enterocytozoon bieneusi life cycle. HN, host cell nucleus; HC, host cell cytoplasm; N, nucleus
of developing E. bieneusi; Pt, polar tube. (Reprinted from Santin, M. and Fayer, R., Res. Vet. Sci., 90, 363–371, 2011. With
permission from Elsevier.)

begins to divide, resulting in multiple elongated nuclei, and electron-dense inclusions appear in the cell.
In the sporogonial plasmodium stage, polar tube elements are formed in the multinucleate plasmodium.
Later, polar tube complexes and nuclei segregate into sporoblasts, which develop further to become spores
that are shed with the host’s feces. The spores of E. bieneusi are oval, 1–2 μm in size, and resistant to most
environmental stresses. They have a cell wall consisting of three layers (plasma membrane, endospore, and
exospore) and a long coiled tubular extrusion apparatus called the polar tube. The polar tube plays a critical
role in invading and injecting infective material (sporoplasm) into the host cell.6,13 In mature spores, the polar
tubule has five to seven coils in two rows, as determined by transmission electron microscopy.

33.2 Biochemistry and Physiology

Microsporidia can survive outside of their hosts for many years in spore form.14 Under appropriate condi-
tions, the polar tube is expelled from the thin anterior part of the spore and the sporoplasm is transferred
into a host cell.15 The mechanisms of how E. bieneusi invade host enterocytes remain unclear, with the
lack of an efficient in vitro E. bieneusi culture method hampering the development of a host–parasite
interaction model. The rapid infection process of the polar tube has a high energy requirement; there-
fore, the process was assumed to be ATP independent. However, a recent genome survey of E. bieneusi
revealed that the organism has a highly unusual and reduced biochemistry. Most of the glycolysis and
trehalose metabolism, along with the pentose phosphate pathways, are missing, indicating that metabo-
lism is largely dependent on the host. However, genes involved in other biochemical pathways, such as
replication, translation, phosphate signaling, and amino acid metabolism, are intact. Therefore, the ger-
mination process may not be ATP independent.16

33.3 Immunology
Prior to the AIDS era, microsporidia were rarely reported in humans. Cell-mediated host immune
responses seem to be key in defense against E. bieneusi. With the widespread outbreaks of AIDS, a
Enterocytozoon bieneusi 513

significant number of microsporidiosis cases have been reported in HIV patients, especially in per-
sons with ≤100 CD4+ T cells per μL of blood.6,13,17,18 Highly activated antiretroviral therapy (HAART)
has dramatically reduced opportunistic infections in HIV/AIDS patients, including those caused by
microsporidia.19 However, intestinal microsporidiosis is still a major problem in some parts of the world
where access to HAART is limited. Additionally, E. bieneusi is being increasingly reported in non-HIV
immunosuppressed patients, including the elderly, patients with diabetes, those with malignant disease
undergoing chemotherapy, and those undergoing organ transplant.20–22 The lack of an animal model
and in vitro culture methods limit the work that can be carried out on E. bieneusi. As such, the detailed
immunological characteristics and host–organism relationship remain unclear.

33.4 Host Range
E. bieneusi was originally only reported in humans, both immunocompromised and immunocompetent
patients. However, many vertebrate animal hosts have now been shown to harbor E. bieneusi.23 There
remain a number of questions regarding the epidemiology of E. bieneusi, as the major host reservoirs and
routes of transmission are not completely understood. Fortunately, recent development of molecular tools
has allowed us to identify a wide range of animal hosts.23,24 Host specificity and zoonotic potential of an
organism can be evaluated by genotyping and phylogenetic analysis targeting the internal transcribed
spacer of the ribosomal RNA gene.23 Zoonotic and animal-specific E. bieneusi genotypes have been
identified from domesticated animals, companion animals, and wildlife, including primates, marmo-
sets, dogs, cats, pigs, cattle, horses, llamas, kudus, foxes, raccoons, otters, guinea pigs, beavers, rabbits,
muskrats, falcons, and some other birds.23 Cats and pigs are common hosts of zoonotic genotypes in
Asian countries.25–28 Phylogenetic analysis of E. bieneusi divides the ITS genotypes into eight groups.1,29
Group 1 is the largest, containing all human-specific, zoonotic, and animal-specific genotypes. Groups
2–8 are mostly only found in specific hosts and wastewater.1

33.5 Transmission
Transmission routes of E. bieneusi are not fully elucidated. In humans, vertical transmission has not been
reported, and the fecal–oral route is considered the main route of transmission. A report of E. bieneusi
in the respiratory tract also suggests the possibility of airborne transmission.30,31 Additionally, the spore
enables the organism to survive in the environment for long periods of time, contributing to both water-
and foodborne transmission. Risk factors associated with human microsporidiosis are homosexual prac-
tices, intravenous drug use, and water contact.32,33 As summarized in Table 33.1, a foodborne outbreak in
Sweden was attributed to contaminated cucumbers.34 E. bieneusi spores also have been identified on other
fresh food products and in milk.35,36 Waterborne transmission is frequently reported, with E. bieneusi
being isolated from surface water, waste water, irrigation water, swimming pools, and river water.25,37–49
In 1999, there was a confirmed waterborne microsporidium outbreak associated with a water distribu-
tion system.50 Numerous genotypes of E. bieneusi have also been identified from animals.23 Cama and

TABLE 33.1
Enterocytozoon bieneusi Detected in Food
Produce Type Country Detection Method Genotype References
Raspberries, bean Poland Microscopy (chromotrope 2R, Unknown 35
sprouts, lettuce calcofluor white), FISH
Cucumbers in salad and Sweden PCR C 34
sandwich
Milk Korea PCR D, I, J, CEbD, type IV 36
PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization.
514 Laboratory Models for Foodborne Infections

colleagues reported the transmission of E. bieneusi between a child and pet guinea pigs,51 whereby an
unusual genotype was identified in both the guinea pigs and a 2-year-old child in the same household.

33.6 Clinical Symptoms and Pathology

E. bieneusi causes intestinal microsporidiosis. Clinical symptoms include watery and nonbloody diar-
rhea, nausea, abdominal pain, and fever. In immunocompetent patients, the diarrhea is usually self-­
limiting, with symptoms lasting approximately 1 month or less.52 However, the diarrhea can be persistent
and severe in immunocompromised patients.53 Respiratory infection, cholecystitis, and cholangitis are
reported in rare cases,53,54 and E. bieneusi has been linked to malnutrition in children.55 Dissemination is
usually not seen with E. bieneusi infection, distinguishing it from infection caused by Encephalitozoon
species.
In humans, E. bieneusi is usually found in the small intestine, especially in the distal duodenum and
proximal jejunum. The organism can also be found in the ileum, but is rarely present in the colon. The
infection induces villous blunting and crypt hyperplasia, but does not induce ulceration. It also reduces
mucosal surface area, leading to malabsorption.56 E. bieneusi spores and proliferating forms are found
on the apical surface of cells, but not on the basal surface.57,58

33.7 Techniques for Detection and Characterization

Several techniques for detection of E. bieneusi are now available. Stool, duodenal drainage, and biopsy
samples are used for diagnosis. The various detection techniques are described below.

33.7.1 Light Microscopy
Microsporidia spores are difficult to identify in stool specimens by light microscopy because they are
very small (1–2 μm) and can look similar to bacteria. Chromotrope-based techniques, initially described
by Weber and colleagues,59 and then modified and further improved by other groups,60–63 are commonly
applied. Using a 5-min rapid-hot Gram-chromotrope staining method, the microsporidial spores stain
dark violet against a pale-green background, and a characteristic purple belt-like strip or bar in the
middle or at the end of the body is enhanced. Microsporidian spores can also be visualized by ultraviolet
microscopy using chemofluorescent stains such as Calcofluor White 2MR, Fungi-Fluor, or Uvitex 2B,
which bind chitin in the endospore.64 However, these types of stains are nonspecific and readily interact
with some bacteria, small fungi, and artifactual materials, resulting in false-positive results.1 Giemsa
stains microsporidia a light blue color, but the organisms are difficult to differentiate from other stool
elements, meaning this stain may only be of use in intestinal biopsies.17,65 Histological examination, such
as duodenal biopsy, can be used in some clinical situations. Various staining techniques for histological
examination have been studied including fluorescent Uvitex 2B stain, Gram-derived stain, silver stain-
ing, Giemsa stain, and chromotrope 2R stain.66–69

33.7.2 Electron Microscopy
Transmission electron microscopy (TEM) remains the gold standard for genus and species identification
of microsporidia. Structural features of the spores, intracellular proliferative forms, method of division,
and host–parasite interface are required for diagnosis and differentiation of the species. All developmen-
tal stages are observed in infected tissue samples, but not in feces. Therefore, fecal samples alone are
not sufficient for definitive classification of microsporidia.70 The major disadvantages of TEM are low
sensitivity, high cost, and laborious sample preparation. TEM is also not suitable for routine diagnosis.71
The classification of microsporidia by TEM and detailed characteristics of E. bieneusi are described
elsewhere.6,72
Enterocytozoon bieneusi 515

33.7.3 Immunofluorescence
Fluorescein-tagged antibodies are useful for detecting pathogens in clinical biopsy samples from
humans and animals.73,74 Immunofluorescence techniques, especially the use of polyclonal antibodies,
allow visualization of all E. bieneusi development stages, including spores, intracellular developmental
stages, and extruded polar tubes, whereas histochemical methods only stain the walls of sporoblasts
or spores.75,76 However, polyclonal immunofluorescence staining frequently cross-reacts with yeast and
bacteria in fecal specimens, and the sensitivity of the technique is poor compared with chromotrope or
chemofluorescent stains.77 Monoclonal antibodies against E. bieneusi spores do not appear to cross react
with Encephalitozoon species, but are not available for commercial distribution.78,79

33.7.4  In Vitro Culture and Animal Models

Although E. bieneusi infects a wide range of mammals and birds, it does not naturally infect rodents.
Only short-term, self-limiting infections can be induced in severely immunosuppressed mice and rats.80
E. bieneusi can cause intestinal microsporidiosis, especially hepatobiliary infections, in simian immuno-
deficiency virus-infected macaques, and experimental infections have been established.81–83 E. bieneusi
cannot be isolated in vitro in continuous tissue culture, with only short-term propagation reported to
date.84–86 In these experiments, adenovirus coinfection reportedly disturbed the short-term cultures.86
Interestingly, both E. bieneusi and Nucleospora salmonis, a salmon pathogen previously assigned to the
genus Enterocytozoon, were successfully cultured for a short time using rainbow trout kidney cells.85

33.7.5 Nucleic-Acid-Based Detection
Nucleic-acid-based detection methods are more sensitive and specific than microscopy, and therefore
have been widely applied.24,87 The most commonly used method is polymerase chain reaction (PCR).
In PCR, the target pathogen DNA is bound by a specific set of primers, and the original few copies
of DNA are amplified across several orders of magnitude, generating millions of copies of a particu-
lar DNA sequence. Molecular diagnostic tests for microsporidia are not routinely available in clinical
diagnostic laboratories despite being widely used in research settings. PCR diagnosis of E. bieneusi
was first reported by Zhu and colleagues.88 PCR methods can also be used for more in-depth analyses,
such as genotypic identification at the subspecies level. The primers generally used for the diagnosis
of E. bieneusi target the small and large subunits and internal transcribed spacer (ITS) region of the
rRNA gene. The ITS region in particular has been used in many studies for detecting and genotyping
E. bieneusi because of the high degree of sequence diversity in this region. Although the ITS sequence
remains the gold standard for the analysis of E. bieneusi, additional gene markers are being sought and,
more recently, a multilocus sequence typing assay targeting three microsatellite and one minisatellite
markers has been developed.23,89
Real-time PCR detects accumulating amplicons in real time via either nonspecific fluorochrome or
specific fluorescently-labeled probes. Real-time PCR has the advantage of being quantitative over a
broad dynamic range, but is relatively expensive. A few real-time PCR analyses have been reported for
detection of E. bieneusi.90,91
Several studies have used fluorescent in situ hybridization (FISH)-based techniques to detect
E. bieneusi. FISH is a cytogenetic technique using a fluorescently-labeled probe that binds to comple-
mentary nucleic acid (DNA or RNA) in the specimen.92 It was used successfully to detect E. bieneusi
from clinical samples, with probes targeting the small subunit or ITS regions of the rRNA.93,94 FISH has
the advantage of providing general morphological information, as the procedure is performed in situ.
The major disadvantages of FISH are the laborious method and lower sensitivity than PCR.
A recently developed oligonucleotide microarray method can simultaneously detect E. bieneusi and
Encephalitozoon species from clinical samples.95 An array of target-complementary DNA fragments
are spotted on a glass slide, and the nucleic acid from the sample is hybridized to the chip after being
fluorescently labeled. This technique is somewhat quantitative; the abundance of the DNA in the sample
is correlated with the intensity of fluorescence. Additionally, this method is high throughput.
516 Laboratory Models for Foodborne Infections

TABLE 33.2
Enterocytozoon bieneusi Detected in Water
Water Source Country Detection Method Genotype References
River France PCR Unknown 41
Surface water, ground USA PCR Unknown 37
water
River Korea Uvitex 2B, PCR Unknown 42
River Ireland FISH, PCR Unknown 43
Lake France PCR Unknown 44
Recreational water USA FISH, PCR Unknown 45
Surface inland and Ireland FISH Unknown 104
coastal waters
Wetlands Ireland FISH, PCR K 46
Waste water Ireland FISH Unknown 47
Waste water China PCR Type IV, EbpA, EbpC, EbpD, 40
Peru6, Peru8, Peru10, C, D,
BEB6, PtEb IV, PigEBITS7,
PigEBITS8, WL4, WL12, WL14,
WW1–WW9
Waste water Tunisia PCR D, IV, etc. 39
Storm water USA PCR WL4, WL6, SW1 to SW3 48
Drinking water, waste Spain Chromotrope stain, PCR C, D, and D-like 49
water
River water China PCR EbpA, EbpB, EbpC, D, CS-8, 38
PtEb IX, Peru8, Peru11,
PigEBITS4, G, O, RWSH1 to
RWSH6
PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization.

33.7.6 Detection from Environmental Samples

Microscopy or molecular techniques are generally used for detecting E. bieneusi from water samples
after filtration and purification steps. There are no standard methods to purify and enrich microsporidia
from water samples, although a few methods have been reported, including continuous separation chan-
nel centrifugation and continuous flow centrifugation.96 The small size of the organism decreases the
efficacy during the filtering of large volumes of water, as the smaller filter size increases the chance of
clogging (Table 33.2).

33.8 Treatment
Albendazole, a benzimidazole drug used to treat of a variety of parasitic infections, is highly active
against Encephalitozoon species, but has shown only limited efficacy against E. bieneusi. When
albendazole is  used in patients infected with E. bieneusi, diarrhea may improve in some patients,
but the excretion of the organism continues and diarrhea exacerbates rapidly after discontinuation of
the drug.97–99 Albendazole works by binding to β-tubulin, and variations in the amino acid sequence
of E. bieneusi β-tubulin may be related to clinical resistance.100 Fumagillin, originally isolated from
Aspergillus fumigatus, is an antimicrobial agent that is effective against E. bieneusi and Entamoeba his-
tolytica. Fumagillin has successfully treated E. bieneusi infection in AIDS and transplant patients.101,102
The main toxicity is thrombocytopenia, which is reversible after cessation of treatment. However, more
severe side effects, such as aseptic meningoencephalitis, have also been reported.103
Enterocytozoon bieneusi 517

33.9 Conclusion and Perspectives

E. bieneusi is the most common intestinal pathogen causing human microsporidiosis. Diarrhea is a
major symptom of E. bieneusi infection in humans and can be severe and persistent in immunocompro-
mised patients. E. bieneusi is characterized by the presence of a polar tube, which plays a central role in
pathogenicity by injecting infective material into the host cell. The spore, which is the dormant form of
the cell, is resistant to most environmental stresses and can survive outside of the host for many years.
The diagnosis of E. bieneusi is difficult by conventional morphological methods because of its small
size; therefore, molecular techniques have been widely used. However, the epidemiology and mode of
transmission are not completely understood. Fecal–oral infection is thought to be the main route of
transmission, and zoonotic transmission may be responsible in certain cases. An effective treatment for
E. bieneusi has not yet been established. The lack of an animal model and in vitro culture methods lim-
its the investigation of immunology, pathophysiology, and development of treatments for E. bieneusi.
Establishing an animal model and in vitro culture methods will help us gain a better understanding of
the transmission dynamics and help develop a comprehensive epidemiological picture for E. bieneusi
infection.

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34
Fusarium

Palanisamy Manikandan, Coimbatore Subramanian Shobana, Mónika Homa,

Sándor Kocsubé, János Varga†, Muthusamy Chandrasekaran, Naiyf S. Alharbi,
Venkatapathy Narendran, Csaba Vágvölgyi, and László Kredics

CONTENTS
34.1 Introduction................................................................................................................................... 523
34.1.1 The Genus Fusarium........................................................................................................ 523
34.1.2 Mycotoxins of Fusarium Species and Mycotoxicoses..................................................... 524
34.1.2.1 Zearalenone....................................................................................................... 524
34.1.2.2 Fumonisins........................................................................................................ 524
34.1.2.3 Trichothecenes.................................................................................................. 525
34.2 L aboratory Models for Foodborne Fusarium Mycotoxicoses...................................................... 525
34.2.1 Rodents............................................................................................................................. 525
34.2.2 Human and Animal Cell Lines.........................................................................................539
34.3 C onclusions................................................................................................................................... 546
Acknowledgments................................................................................................................................... 546
References............................................................................................................................................... 546

34.1 Introduction
The members of the genus Fusarium are hyaline filamentous fungi and are found largely as saprophytic
organisms in soil. Fusaria cause a range of infections collectively known as fusariosis and have been doc-
umented as etiological agents in localized tissue infections, keratitis, endophthalmitis, septic arthritis,
cystitis, peritonitis, brain abscesses, and breast abscess. The mycotoxins of these fungi are involved in
the infectious processes and may serve as potential virulence factors. Fusarium is also one of the major
fungal genera associated with maize and other cereals throughout the world. Several species are the most
prolific producers of mycotoxins and are frequently associated with mycotoxicoses in both humans and
animals. This chapter intends to provide an overview about the rodents (rats and mice) and mammalian
cell lines that were most recently used as laboratory models to study Fusarium mycotoxicoses.

34.1.1 The Genus Fusarium

The members of the genus Fusarium were first described by Link [1] in 1809 as “Fusisporium.” The name
derives from the Latin word fusus (spindle) and refers to the typical macroconidial shape of Fusarium
species. According to the latest molecular studies, this cosmopolitan genus comprises at least 20 spe-
cies complexes and nine monotypic lineages including soil saprophytes and plant endophytes along with
agriculturally and medically important fungi [2].
Fusaria are among the most important plant pathogens in the world. Just the Fusarium solani species
complex (FSSC) alone affects more than 100 plant genera [3]. Fusarium spp. are responsible for a variety

† In memoriam Prof. János Varga

523
524 Laboratory Models for Foodborne Infections

of devastating diseases, e.g., head blight of wheat and barley, wilt of bananas [4], sudden death syndrome
of soybean [5], crown and root rot of tomato [6], and vascular wilt of tomato [7].
Fusarium spp. are also associated with a broad spectrum of human infections, including superficial
(i.e., keratitis and onychomycosis), invasive localized, and disseminated infections, allergic diseases
(i.e., sinusitis), and mycotoxicosis [8]. Out of the 20 species complexes of the genus, seven comprise
­clinically relevant Fusaria: the F. chlamydosporum species complex (FCSC), the Fusarium dimerum
species complex (FDSC), the F. fujikuroi species complex (FFSC), the F. incarnatum-equiseti ­species
complex (FIESC), the F. oxysporum species complex (FOSC), the F. sambucinum species complex
(FSAMSC, including Fusarium sporotrichioides), and the FSSC. Among them, the FSSC—which
comprises at least 60 haplotypes—is the most common group, accounting for 50%–60% of all fusari-
oses worldwide [9,10].
Certain Fusarium species are also able to produce mycotoxins, which may contribute to plant and
human pathogenesis [11]. Besides the direct negative impact of fungal infections on crop yield, the myco-
toxin contamination of animal feeds also causes significant financial loss and damage to agriculture
worldwide. The major Fusarium mycotoxins frequently occurring in cereal grains and animal feeds are
fumonisins, trichothecenes, and zearalenone.

34.1.2 Mycotoxins of Fusarium Species and Mycotoxicoses

34.1.2.1 Zearalenone
Zearalenone (ZEA, F-2 toxin) is a resorcyclic acid lactone mycotoxin. Its name was created as a
­combination of “zea” (for the main producer’s name, G. zeae), “-ral” (for the initials of resorcyclic
acid lactone), “-ene” (for the presence of a double bond at C1′-2′), and “-one” (for the ketone group
at C6′) [12].
ZEA is produced by various Fusarium species commonly occurring on cereal grains, e.g., F. gra-
minearum (teleomorph: Gibberella zeae), F. culmorum, F. equiseti, F. cerealis (synonym: F. crook-
wellense), F. avenaceum, F. tricinctum, and F. oxysporum [13,14]. ZEA is a frequent field contaminant
of maize, barley, oats, rice, rye, soybeans, and wheat; however, poor storage conditions may contribute
to its postharvest production as well [15,16].
It is assumed that ZEA has a role in sexual reproduction of the producing fungi [17], and it also
affects the growth, development, and photosynthetic apparatus of the contaminated plant [18]. The
nonsteroidal, estrogen-like structure also allows the toxin and its metabolites (α-zearalenol and
β-zearalenol) to compete with mammalian endogenous estrogens for specific binding sites on their
receptors. This estrogenic activity of ZEA causes hyperestrogenism and fertility disorders in farm
animals after consumption of a high dose of affected crops (e.g., >0.25 mg/kg ZEA in maize) [14,19].
Pigs—particularly the prepubertal females—are the most sensitive to the higher concentrations of
ZEA, whereas ruminants and poultry are reported as less sensitive [14]. Piglets can be affected as well,
as ZEA can be excreted into sows’ milk [19]. ZEA also represents a potential risk to human health.
It is assumed that ZEA exposure is associated with precocious pubertal development in girls [15,20],
and it was also mentioned as a possible causative agent of cervical cancer [21]. Besides its endocrine-
disrupting effects on animals and humans, ZEA has been reported to be hepatotoxic, hematotoxic,
immunotoxic, and genotoxic [12].

34.1.2.2 Fumonisins
Fumonisins have been discovered in 1988 in South Africa from cultures of the F. moniliforme strain
MRC 826 (=F. verticillioides, teleomorph: Gibberella moniliformis) [22]. The chemical structure of this
cancer-promoting compound was elucidated in the same by Bezuidenhout et al. [23]. The most impor-
tant ­fumonisin-producing Fusaria are the members of section Liseola, including the widespread maize
pathogens F. verticillioides and F. proliferatum. Other sections containing fumonisin-producing strains
are Dlaminia, Elegans, and Arthrosporiella. In section Elegans, F. oxysporum produces only C-series
fumonisins [24].
Fusarium 525

Fumonisins are polyketide-derived mycotoxins and can be divided into four groups (A, B, C, and P)
based on their chemical structure with several isomers and stereomers identified during the past decade
[25–28]. The most prevalent and naturally occurring fumonisins belong to type B analogues (FB). In
Fusaria, usually FB1 is predominant, accounting for 70%–80% of the total fumonisin content [29]. The
A and B series of fumonisins consist of a 20 carbon atom long backbone, while the C-type fumonisins
are 19 carbon atoms long. Fumonisins are sphingosine-analog compounds, which disrupt the biosyn-
thesis of sphingolipids by the inhibition of the ceramide synthase enzyme [30]. The altered sphingo-
lipid metabolism could lead to neural tube defects through the disrupted folate uptake [31]. Ingestion of
fumonisin-contaminated feeds is associated with several fatal diseases in domestic animals, for example,
equine leukoencephalomalacia (ELEM) and porcine pulmonary edema (PPE). Fumonisins can cause
nephrotoxicity, hepatotoxicity, and hepatocarcinogenicity in laboratory animals [32]. Fumonisins are
the possible causative agents of esophageal cancer in several countries like China and Transkei in South
Africa [33,34]; therefore, fumonisin B1 is considered as possibly carcinogenic to humans (Group 2B) by
the International Agency for Research on Cancer (IARC) [35].

34.1.2.3 Trichothecenes
Fusaria and other mold fungi such as Mycothecium, Trichoderma, Trichothecium, Stachybotrys,
Verticimonosporium, and Cephalosporium produce trichothecenes, which comprise a family of closely
related low-molecular-weight, nonvolatile, and relatively water-insoluble compounds known as sesquiter-
penoids. They are classified into four types—A, B, C, and D, among which Fusaria produce type A (like
T-2 and HT-2 toxin) and type B [like deoxynivalenol (DON) and nivalenol (NIV)] trichothecenes [36].
Fusarium species are probably the most cited and among the most prolific trichothecene-producing
fungi [37]. Trichothecenes contaminate many field crops across the world, and it is necessary to screen
large number of food samples for the presence of these toxic fungal metabolites [38].
Trichothecenes are powerful inhibitors of eukaryotic protein synthesis and are phytotoxic, insecti-
cidal, and toxic to animals, and some are among the most toxic non-nitrogenous compounds known
to man. Several are commonly found in cereal grains, and the potential health risk from contaminated
animal feed and human food is a major factor in stimulating research into this group of compounds
[39–42]. Common manifestations of trichothecene toxicity are depression of immune responses and
nausea, sometimes vomiting [43].
Wang et al. [44] reported human toxicosis in China caused by moldy rice contaminated with T-2 toxin
of F. heterosporum and F. graminearum. The chief symptoms were nausea, dizziness, vomiting, chills,
abdominal distension, abdominal pain, thoracic stuffiness, and diarrhea. T-2 mycotoxicosis—or “moldy
corn disease”—in pigs is characterized by multiple hemorrhages on the serosa of the liver, stomach, and
esophagus (at necropsy). T-2 also has an important impact on reproductive performance in pigs [45]. The
in vivo toxicity of T-2 and H-2 is explicitly described by EFSA Panel on CONTAM [46]. Wu et al. [47]
stated that in human cell lines, HT-2 and neosolaniol (NEO) are the major metabolites of T-2 toxin. The
T-2 degradation products are less cytotoxic compared to T-2 toxin [48].
DON—or vomitoxin—is one of the most common mycotoxins produced by F. graminearum and
F. culmorum found in grains. When ingested in high doses by agricultural animals, it causes nau-
sea, ­vomiting, and diarrhea; at lower doses, pigs and other farm animals exhibit weight loss and food
refusal [49]. Human or animal intoxications with either DON or NIV are much less likely to be fatal than
those with T-2 [50].

34.2 Laboratory Models for Foodborne Fusarium Mycotoxicoses

34.2.1 Rodents
Rodents (rats and mice) are the most popular laboratory animal models for the examination of
Fusarium mycotoxins; therefore, a broad search strategy was applied to retrieve full-text papers
526 Laboratory Models for Foodborne Infections

and—in cases when full-text access was not available—abstracts from the PubMed database. The
search was performed in article titles and abstracts using the following keyword combinations in order
to find specific literature: zearalenone OR fumonisin OR T-2 toxin OR deoxynivalenol AND rat OR
mouse OR mice. The search was limited to publications from the year 2010 onwards in order to focus
on recent literature. Articles and abstracts were screened to include studies with relevant information
(exact definition of the mycotoxin studied and the animal model, details about the way of administra-
tion, and the dosage).
Table 34.1 gives an overview of the analyses of Fusarium mycotoxins utilizing rat models [51–74]. Out
of 24 publications analyzed (1 DON, 10 T-2, 7 ZEA, and 6 FB1), it was found that Sprague Dawley rats
were mostly used, followed by Wistar rats. There is only one study included for DON [58] in 8-week-old
axenic male Sprague Dawley rats, where the concentration of E. coli bacteria decreased in the gut at day
27 after subchronic exposure to DON.
Among the 10 studies analyzed for T-2 toxin, 7 studies utilized Wistar rats to investigate MRI in the
tibial bone abnormalities [66,69], chondrocyte necrosis of Kashin Bede disease (KBD) [67], cardiac
and autonomic nervous effect [68], reproductive toxicity [70], KBD [69,71,72], and apoptotic induction
mechanism [70]. The remaining three studies with Sprague Dawley rats analyzed the effect of T-2 on
lipid peroxidation in brain [53,63] and KBD [57].
Out of seven studies analyzing ZEA in rat models, the most popular model animals were Sprague
Dawley rats in the cases the analyses were related to the effect of ZEA on the reproductive system
[51,52,56,64] and modification of mRNA levels involved in ZEA detoxification [54]. Belli et al. [65]
investigated the involvement of ZEA in breast endocrine disorders using Wistar female rats.
Of six studies analyzing the effects of FB1, four used Sprague Dawley rats for the exploration of serum
enzyme activities and DNA lesions [61], gene expression [62], general toxicity after nixtamalization of
whole kernel corn [59], and gastric ulcer [60]. Riedel et al. [73] reported the effect of FB1 in cancer pro-
motion involving lipid changes utilizing Fischer rats. Pellanda et al. [74] investigated the global histone
modification due to FB1 administration in Wistar female and male rats.
Table 34.2 summarizes a total of 51 studies involving mice as animal models to study the possible
effects of Fusarium mycotoxins viz., DON (22 studies), T-2 (9 studies), ZEA (16 studies), and FB1 (10)
[75–126]. Of 22 mice studies involving DON, B6C3F1 followed by BAL13/3 and C57136/6 mice were
the most popular.
B6C3F1 mice were used for the analysis of the effect of DON on immunomodulation [98,99],
insulin-like growth factor [93], and diet-related issues [94,95,96]. C57BL/6 mice were used to ana-
lyze the effect of DON on inflammation [104], diet issues [102], fetal skeletal development [103],
and mouse thymus [100]. With BALB/c mice, the effects of DON were studied on hepatotoxicity
[87], immunomodulation [76,81], and hemostability [77]. With the ICR mouse model, DON was
analyzed for oocyte development [90,91] and oxidative stress [89]. With Swiss albino mice, Mishra
et al. [115] found that topical application of DON increased cell proliferation, DNA synthesis, and
inflammation, while Kouadio et al. [112] revealed that the oral administration of DON created dis-
orders in systemic targets. Choi et al. [122] reported that DON in drinking water can disrupt the
immune response of porcine parvovirus-vaccinated C3H mice. Behavioral changes due to inflam-
matory cytokines after DON intoxication in PGES wild-type and knockout mice were observed by
Girardet et al. [125].
Chaudhary et al. [108,116] and Agarwal et al. [109] studied the oxidative stress due to T-2, while
Agarwal et al. [110] reported the CC-2 formulation’s effectiveness against topical exposure to T-2 toxin
in Swiss albino mice. Using the BALB/c mouse model, Ahmadi et al. [88] showed that selenium protects
the alteration of B lymphocytes after T-2 toxin exposure, while Maragos et al. [82] developed Mab2–13
antibody detection for T-2 and T-2glc. The harmful effect of T-2 toxin in early embryo development was
explored by Somoskői et al. [123] in BDF1 mice. Yang et al. [118] demonstrated the decrease in the tes-
tosterone biosynthesis due to T-2 toxin in Kunming mice.
BALB/c mice were employed to investigate the hepatotoxicity [79,87] and immunotoxicity [75,78,80]
of ZEA. Zhu et al. [90,92] and Hou et al. [91] explained the reduction in oocyte development, while Hou
et al. [89] studied the oxidative stress induced by ZEA in ICR mice. Boeira et al. [111,113,114] clearly
indicated the impaired testicular functions due to ZEA toxicity among male Swiss albino mice. ZEA was
TABLE 34.1

Fusarium
Studies Using Rat Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Administration/Dose Aim of the Study Major Findings References
10-w-old Sprague ZEA Intraperitoneal, 5 mg/kg To investigate the effects of ZEA on ZEA induces apoptosis in germ cells of male rats; this toxicity [51]
Dawley spermatogenesis and possible mechanisms of ZEA is partially mediated through modulation of Fas and
involved in germ cell injury Fas-L systems, though ERα may not play a significant role
8-w-old male ZEA Intraperitoneal, 300 mg/kg To investigate the effects of KRG extract on Impaired spermatogenesis resulting from ZEA treatment was [52]
Sprague Dawley testicular toxicity induced by ZEA prevented by KRG through Fas-Fas L modulation
1-month-old male T-2 Intragastric, 100 and To compare antioxidant capacity and lipid Increasing TBARS and decreasing antioxidants in serum and [53]
Sprague Dawley 200 ng/g b.w./d peroxidation using a novel model cartilage by T-2 toxin treatment with a selenium-deficient
nutritional status may alter oxidative stress in joint tissues and
contribute to the pathological process of cartilage damage
in KBD
Male Sprague ZEA Intraperitoneal, 25 mg/kg To determine the levels of expression of rat The initial modifications in mRNA levels indicate a close [54]
Dawley b.w. proteins that are involved in the ZEA association with microsomal enzyme activity of the CYP2B,
detoxification pathway upon acute ZEA CYP2C, and CYP3A protein families
treatment
6-w-old female ZEA Intragastric, 3 mg/kg To investigate the effect of ZEA ZEA exposure can cause oxidative stress and change common [55]
Sprague Dawley supplementation on rat metabolism systemic metabolic processes, including cell membrane
metabolism, protein biosynthesis, glycolysis, and gut
microbiota metabolism
Pregnant Sprague ZEA Subcutaneous, 0, 1, 50, or To assess the impact of ZEA in adult rats ZEA neonatal exposure could affect the exposure of testis to [56]
Dawley 100 and 0, 0.75, 1.25, or exposed neonatally ABC transporter substrates and negatively influence
2.5 μg/d spermatogenesis and male fertility
1-month-old male T-2 Intragastric, 100 and To observe pathogenic lesions of joint cartilages Rat can be used as a suitable animal model for studying [57]
Sprague Dawley 200 ng/g b.w./d in rats fed with T-2 toxin under a selenium- etiological factors contributing to the pathogenesis
deficiency nutrition status in order to determine (chondronecrosis) observed in human KBD
possible etiological factors causing KBD
8-w-old axenic male DON Gavage feeding, 100 µg/kg To evaluate the impact of a subchronic NOAEL A significant increase of 0.5 log10 was observed for the [58]
Sprague Dawley b.w. dose exposure of DON on the composition of Bacteroides/Prevotella group during the first 3 w of
human gut microbiota administration, concentration levels for Escherichia coli
decreased at d 27
(Continued)

527
TABLE 34.1 (Continued)

528
Studies Using Rat Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Administration/Dose Aim of the Study Major Findings References
3-w-old male FB1 FB1 contaminated corn, To assess how nixtamalization of whole kernel Nixtamalization is an effective cooking method for reducing [59]
Sprague Dawley 30 mg/kg corn affects fumonisin toxicity, male rats were the potential toxicity of FB1-contaminated corn
fed diets containing low, mid or high levels of
uncooked (LU, MU, HU) or alkaline-cooked
(LC, MC, HC) FB1-contaminated corn for
3 week
Male Sprague FB1 Gastric subserosa, To investigate whether accumulated ceramide The ceramide pathway, in particular the metabolites of [60]
Dawley 0.036–0.09 g/kg b.w. could serve as the effector molecule of ulcer ceramide, significantly contribute to acetic-acid-induced
formation in a rat model of acetic-acid-induced gastric damage, possibly via enhancing apoptosis
gastric ulcer
4- to 5-w-old adult FB1 FB1-contaminated corn, To assess changes in serum biochemical profile In rats consuming diets containing FB1, there is a time- and [61]
male Sprague 50, 100, and 200 mg/kg and DNA fragmentation of growing male rats dose-dependent increase in serum enzyme activities and DNA
Dawley diet fed a diet with FB1-contaminated corn and the lesions
role of a Lactobacillus delbrueckii spp. lactis
and Pediococcus acidilactici supplementation
in counteracting the FB1 effects in
intoxicated rats
3-month-old FB1 Oral feed—corn oil To evaluate the protective role of PGE against the AFB1 and FB1 have synergistic genotoxic effects; PGE induced [62]

Laboratory Models for Foodborne Infections

female 100 µg/kg b.w. synergistic effect of subchronic administration protective effects against their oxidative stress and
Sprague Dawley of AFB1 and FB1 on DNA and gene expression genotoxicity through its antioxidant properties
in rat
Early weaning T-2 Intragastric, 0.1 mg/kg/d To explore the effects of T-2 toxin and it’s The single factor of T-2 toxin can cause lipid peroxidation in [63]
male Sprague 0.2 mg/kg/d synergy with low selenium on lipid brain, lower the activity of GSH-Px, and increase the level
Dawley peroxidation in brain of MDA
Female weanling ZEA Oral, 0 or 6 mg/kg To evaluate the efficacy of an ADC in reducing A long-term consumption of ZEA-contaminated diets [64]
Sprague Dawley the toxic effects of ZEA in the diet of rats and stimulated growth of the reproductive tract in rats and piglets
piglets and the presence of ZEA residue in bile in piglets
(Continued)
TABLE 34.1 (Continued)

Fusarium
Studies Using Rat Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Administration/Dose Aim of the Study Major Findings References
Pregnant female ZEA Subcutaneous injection, (1) Morphometric analysis to evaluate the ZEA could contribute to the induction of breast endocrine [65]
Wistar 0.2 μg/kg to 5 mg/kg development of the mammary glands; disorders
(2) immunohistochemical study of mammary
tissue slides to evaluate differentiation and the
level of cell replication and apoptosis in the
tissue; and (3) to test the presence of possible
lesions in the mammary tissue
4-w-old male and T-2 Intragastric, 0.04 mg/kg/d To investigate magnetic MRI in the tibial The MRI image of the rat epiphyseal plate is altered in the [66]
female weanling epiphyseal growth plate development KBD model rats, and epiphyseal plate MRI appearance has
Wistar been reproduced by using T-2 toxin and KBD-affected feed of
an epidemic-affected district
Male and female T-2 Intragastric, 0.1 mg/kg/d To determine whether giving rats selenium- and This animal model of KBD can be approached by feeding rats [67]
Wistar iodine-deficient food low in protein and made a low-nutrition diet (low levels of selenium, iodine, and
with barley from an area where KBD is protein) and exposing them to T-2 toxin. The pathological and
endemic, in combination with exposing them to radiographic changes observed were very similar to those in
T-2 toxin, would produce the characteristic patients with KBD
chondrocyte necrosis of KBD, to establish an
experimental KBD animal model
10-w-old male T-2 Subcutaneous, 0.1 and To clarify and reevaluate the cardiac and T-2 toxin produced significant cardiac dysfunctions involving [68]
Wistar 0.5 mg/kg autonomic nervous effects of T-2 toxin disturbance of the conduction pathway influenced by the
autonomic nervous activity and also possible direct effects on
cardiac myocytes
4-w-old-male and T-2 0.04 mg/kg/d To characterize the features of radiographic A low-nutrition diet may be involved in the etiology of KBD, [69]
female weanling abnormalities of the tibial bone in rats that have and determination of this should be studied in the future
Wistar been fed T-2 toxin and KBD epidemic district
food
Wistar T-2 0, 1, 10, and 100 nM To investigate the reproductive toxicity and A possible underlying molecular mechanism for T-2 toxin is [70]
cytotoxicity of T-2 toxin and to explore its that it induces the apoptosis signaling pathway in rat
potential apoptotic induction mechanism granulosa cells by regulation of ROS-mediated mitochondrial
pathway
(Continued)

529
TABLE 34.1 (Continued)

530
Studies Using Rat Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Rat Model MT Administration/Dose Aim of the Study Major Findings References
Male and female T-2 Intragastric, 0.04 mg/kg/d To investigate the effect of KBD-affected feed KBD-affected feed rats had less weight gain than T-2 toxin [71]
weanling Wistar and T-2 toxin on bone development intervention rats, which means there were other etiological
factors in KBD-affected feed
Male and female T-2 Intragastric, 1 mg/kg/d To observe early lesions of rat epiphyseal platesT-2 toxin combined with a low-nutrition diet could lead to [72]
Wistar rats and metaphysis caused by T-2 toxin and T-2 more serious chondrocyte necrosis in the epiphyseal plate and
toxin combined with a low-nutrition diet to disturb metaphyseal trabecular bone formation
determine possible pathogenic factors of KBD
Male Fischer FB1 250 mg/kg diet To characterize the involvement of lipid changes A typical lipid phenotype was observed, including increased [73]
during cancer promotion resulting in the membrane PE and cholesterol content, increased levels of
development of preneoplastic lesions, altered C16:0 and monounsaturated fatty acids in PE and PC, as well
lipid phenotype, and to compare FA profiles of as a decrease in C18:0 and long-chain polyunsaturated fatty
two different cancer-promotion regimens acids in the PC fraction
3-month-old FB1 Gavage feeding, 4 μg/kg To investigate the synergistic impact of prenatal Low doses of FB1 interact with MDD, thus contributing to the [74]
sexually mature b.w. methyl donor deficiency and low dosage of FB1 disruption of the epigenetic landscape
virgin female and administration on the pattern of global histone
male Wistar modifications
ABC, ATP-binding cassette; ADC, activated diatomaceous clay; b.w., body weight; CYP, cytochrome P450; d, day; DON, deoxynivalenol; ER, estrogen receptor; FA, fatty acid; FB1, ­fumonisin

Laboratory Models for Foodborne Infections

B1; GSH, glutathione; KBD, Kashin–Beck disease; KRG, Korean red ginseng; MDA, malondialdehyde; MDD, methyl-deficient diet; MRI, magnetic resonance imaging; MT, mycotoxin;
NOAEL, no observable adverse effect level; PC, phosphatidylcholine; PGE, Panax ginseng extract; PE, phosphatidyl ethanolamine; ROS, reactive oxygen species; TBARS, thiobarbituric
acid reactive substances; w, week; ZEA, zearalenone.
TABLE 34.2

Fusarium
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
6-w-old female BALB/c ZEA Oral, 40 mg/kg b.w. To investigate the effects of radish extract on the b.w. Radish extract was effective for the protection of high [75]
gain, the lymphoid relative weight organs, dose ZEA-immunotoxication in mice
hematological parameters, and the expression of
antibody level and cytokines production LPS
stimulation on intoxication
6-w-old female BALB/c DON Intraperitoneal To determine the optimal concentrations of antigen/ The detection limit was 0.01–100 μg/mL, and average [76]
inoculation, antibody for DON detection recovery of DON from contaminated grain was
100–250 μg 82%–93%
BALB/c DON 10 μg/kg To elucidate the reason for blood parameters and Significant decrease of hematocrit value and rise of [77]
hemostatic effect after oral administration of DON blood clotting time and bleeding time; DON is a
potential hematotoxin
5-w-old female BALB/c ZEA Oral, 40 mg/kg b.w. To determine the abilities of the living Lactobacillus Both L. plantarum and TM are safe by themselves and [78]
plantarum MON03 cells, TM clay, and their their composite succeeded to exert a potential
composite to accumulate ZEA from a liquid medium prevention by counteracting ZEA-immunotoxicity
and elucidate the preventive effect of their composite
in ZEA-contaminated mice
BALB/c ZEA Oral, 50,100, 200 μg/kg To establish whether ZEA produced hepatotoxicity via ZEA is a potential hepatotoxin when administered by [79]
b.w. oral administration the oral route
8-w-old female BALB/c ZEA Oral, 40 mg/kg b.w. To isolate a new ZEA-binding microorganism for use ZEA induced toxicity in immunologic and [80]
in biological detoxification and to examine its ability hematologic parameters as indicated by the changes
to remove ZEA in liquid medium and its potential for in lymphocyte cell numbers; Lactobacillus paracasei
prevention of ZEA-induced immunomodulation in BEJ01 treatment prevents weight loss and reduces the
mice immunotoxic effects caused by ZEA
Female BALB/c DON Oral, 0, 0.5, or 2 mg/kg To investigate the differential immunomodulatory DON exposure differentially modulated IL-1, IL-10, [81]
effects of DON and TNF-α production; DON can cause various
immunomodulatory effects
Female BALB/c T-2 Injection, 100 μg To develop an antibody capable of interacting with Mab 2–13 will be useful for the simultaneous [82]
T2G-KLH detection of T-2-Glc detection of T-2 toxin and T-2-Glc
7- to 8-w-old male and FB1 0.1 mg/kg To evaluate the genotoxic potentials of FB1 using a FB1 is nongenotoxic in nature, while the reduced ratio [83]
female BALB/c 10 mg/kg simple micronuclei test in a rodent model of PCE/NCE suggests the cytotoxic nature of FB1
(Continued)

531
TABLE 34.2 (Continued)

532
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
Female BALB/c FB1Subcutaneous injection To obtain a monoclonal antibody against FB1 with high Anti-FB1 mcAb excreted by 4G5 can be used to detect [84]
FB1-BSA (0.2 mL, specificity and affinity FB1 in corn and related samples
100 mg)
10-w-old female FB1 Intraperitoneal and To investigate the effect of silymarin on experimental Silymarin ameliorated toxic liver damage [85]
BALB/c gavage, 1.5 mg/kg FB1 liver toxication induced by FB1
4.5 mg/kg FB1
6-w-old male BALB/c T-2 Intraperitoneal, 1, 2, 3, 4, To check the effect of a sublethal dose of T-2 toxin on Selenium could exert an important effect against the [86]
or 5 mg/kg T lymphocyte subpopulation levels and the potential immunotoxic effects of T-2 toxin against T
protective effects from treatment with selenium or lymphocytes
vitamin E
4-w-old female BALB/c DON, 5.0 mg/kg b.w. To assess the individual and combined toxic effects of The combination of AFB1 + DON displayed a [87]
ZEA AFB1, ZEA, and DON within the liver synergistic hepatotoxic effect, while AFB1 + ZEA
displayed an antagonistic hepatotoxic effect
BALB/c T-2 NA To investigate the toxic effect of T-2 toxin on the Selenium plays a pivotal role on altered B lymphocyte [88]
percentage of peripheral blood B lymphocytes and the subset induced by T-2 toxin compared with vitamin E
potential protective role of selenium and vitamin E
4-w-old female ICR DON, Maize feed DON To investigate the regulation of multiple mycotoxins on MT-contaminated diet could result in liver damage, [89]

Laboratory Models for Foodborne Infections

ZEA (3.1 mg/kg) and ZEA oxidative stress elevated GPx activity in the serum and liver tissues,
(729 mg/kg) and increased MDA level in the serum, indicative of
oxidative stress
4-w-old female ICR DON, Low dose mycotoxin- To identify any epigenetic effects of a mycotoxin- DON affects chromatin compaction and the cell cycle [90]
ZEA containing diet (mass containing diet, including altered DNA methylation, progression of oocytes by reduced H4K20me2 and
percentage: 15%) H3K9 methylation, H3K27 methylation, and H4K20 increased H4K20me3 levels; H4K20 methylations
contaminated with DON methylation, on reduced oocyte developmental play important roles on the cell cycle, mitosis, and
(581 μg/kg) or ZEA competence embryonal development; ZEA affects the level of
(285 μg/kg), high dose H4K20 methylation, mitotic chromatin compaction,
mycotoxin-containing and the cell cycle progression of mouse eggs, which
diet (mass percentage: further affect the egg developmental competence
30%) contaminated with
DON (1.163 μg/kg) or
ZEA (569 μg/kg)
(Continued)
TABLE 34.2 (Continued)

Fusarium
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
4-w-old female ICR DON, Contaminated maize feed To check the effect of DON on oocyte quality DON and ZEA affect oocyte polarity during meiosis; [91]
ZEA 3.875 mg/kg of DON or abnormal mitochondrial distributions in the oocytes
1,897 μg/kg of ZEA
Female ICR ZEA Germinal vesicle-intact To study the effect of ZEA on mouse egg ZEA can affect chromatin compaction and the cell [92]
oocytes harvested from developmental competence cycle progression of oocytes by reduced H4K20me2
ovaries of mice: low and increased H4K20me3 levels
dose group—10 μM
ZEA
High dose group—50 μM
ZEA
3- to 4-w-old female DON Oral, 0.1–12.5 mg/kg b.w. To test the hypothesis that impairment of the GH axis Oral DON exposure perturbs GH axis by suppressing [93]
B6C3F1 precedes DON-induced growth retardation in the two clinically relevant growth-related proteins,
mouse IGFALS and IGF1
11- to 12-w-old female DON Contaminated pellet feed, To determine whether this mouse strain is similarly DON consumption lowered weight gain and produced [94]
B6C3F1 0, 2, 5, or 10 ppm affected by DON during the process of obesity weight loss in diet-induced obese mice at higher
induction or when in the obese state thresholds than that observed previously in normal
B6C3F1 mice
10- to 11-w-old female, DON Oral, 0, 0.1, 0.5, 1.0, or To check whether the acute administration of DON to Mice had partial resistance to feed refusal when [95]
B6C3F1 mice 2.5 mg/kg the mouse causes a rapid, measurable, and exposed to DON subsequently after 2 d but not after
reproducible reduction in food intake 7 d, suggesting that this modest tolerance was
reversible
11-w-old female adult DON Oral, 10 mg/kg To relate DON-induced b.w. loss in HF-induced obese DON induced rapid decreases in b.w. and fat mass, [96]
B6C3F1 mice to food intake, fat mass, lean mass, and which stabilized to those of the LF control within
obesity-related hormones 11 d; DON-mediated effects on b.w., fat mass, food
intake, and hormonal levels were consistent with a
state of chronic energy restriction
3-w-old female DON 0, 1, 2.5, 5, and 10 ppm To explore the feasibility of using plasma IGFALS as a Plasma IGFALS was significantly depressed; it might [97]
B6C3F1 biomarker of effect for DON be a useful biomarker for DON’s adverse effects on
To demonstrate that in mice fed with 15 ppm DON diet growth
there are significantly less plasma IGFALS than in
mice fed identical amounts of control diet
(Continued)

533
TABLE 34.2 (Continued)

534
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
8- to 10-w-old male DON Oral 5 mg/kg b.w. To identify early protein phosphorylation changes in DON impacted phosphorylation of proteins within [98]
B6C3F1 the immune system of DON-exposed mice and relate diverse immune cell populations, including
these to the toxin’s downstream immunomodulatory monocytes, macrophages, T cells, B cells, dendritic
effects cells, and mast cells
10- to 12-w-old female DON Oral, 2.5 mg/kg b.w. To compare the effects of DON and its congeners on Naturally occurring and synthetic DON congeners to [99]
B6C3F1 (C57BL/6 ×, DON, D3G, 3-ADON, splenic TNF-α, interleukin and chemokine mRNA elicit aberrant mRNA upregulation of cytokines
C3HeN) 15-ADON, FX, NIV, expression in the mouse associated with acute and chronic trichothecene
EN139528 or toxicity
EN139544 in 100 μL
PBS
7-w-old male C57BL/6 DON 5, 10, or 25 mg/kg b.w. To understand the mechanism of action of DON in the DON downregulated genes involved in proliferation, [100]
thymus mitochondria, protein synthesis, and ribosomal
proteins
Early precursor thymocytes, particularly at the
double-positive CD4+ CD8+ stage, are more
vulnerable to DON than very early or late precursor
thymocytes
10-w-old C57BL/6 DON Intraperitoneal injection To investigate the toxic effects of DON on fetal skeletal Various skeletal defects in fetuses, including [101]

Laboratory Models for Foodborne Infections

4, 6.5, or 10 mg/kg development misaligned or fused sternebrae and vertebrae,
divided or fused ribs and polydactyly,
hemivertebrae, short toe, and tail anomalies were
observed
10- to 11-w-old DON 10 ppm DON To check the reversibility of DON-induced body weight DON’s effects on food consumption and body [102]
C57BL/6 20 ppm DON loss and anorexia, to investigate the role of PKR weight are not permanent; furthermore, PKR is not
an essential signaling molecule for DON’s
anorectic and weight effects
C57BL/6J ZEA 0, 0.8, 4, and 20 ppm To investigate the potential cumulative effects of Exposure to a 20 ppm ZEA diet promoted female [103]
multiple pregnancy and multigenerational exposure to pubertal onset without obvious cumulative effect
dietary ZEA on female puberty and reproduction and diminished female fertility over generations
Adult male C57BL/6 DON Oral, 1–25 mg/kg b.w. To evaluate the impact of subchronic intoxication with Subchronic administration of low DON doses [104]
DON, given at doses below the NOAEL, on produced a low-grade inflammation
inflammatory status
(Continued)
TABLE 34.2 (Continued)

Fusarium
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
Male and female CD1 ZEA Oral, 1.3, 3.9, and To analyze the effects on the transcriptome in testes Mono-(2-ethylhexyl)-phthalate and ZEA both [105]
6.6 mg/kg exposed to mono-(2-ethylhexyl) phthalate, ZEA, produced specific alterations of gene signatures
lindane, bisphenol-A, or 17β-estradiol irrespective of the concentration of the toxicant or
the developmental period
8-w-old CD1 FB1 0, 25, 50, or 100 mg/mL To examine toxicity on the development of embryos NSP had an unexpected high adsorption capacity [106]
of NSP and its capacity to prevent teratogenesis-induced in vitro; NSP is a feasible and effective agent for
by FB1 supplementary use in reducing the toxicity of FB1
to animals
CD1 outbred male ZEA Oral, lower dose To assess the effect of treatment with a low dose of Animals exposed to low ZEA concentration were [107]
(0.15 μg/L, higher dose ZEA on the male gonadal pathology, sperm quality, affected considerably more than animals exposed to
(150 μg/L) and expression of selected genes thigh ZEA concentration; a low concentration of
ZEA is able to negatively influence the sperm
parameters and testicular gene expression
Female Swiss albino T-2 Dermal and subcuta- To evaluate the acute toxicity of dermal and Percutaneously and subcutaneously applied T-2 toxin [108]
neous, 5.94 mg/kg subcutaneous exposure of T-2 toxin on brain oxidative can cause brain oxidative damage possibly after
1.54 mg/kg stress crossing blood–brain barrier by altering its
permeability
Male Swiss albino T-2 Percutaneous, 5.94 mg/kg To investigate the biochemical and histological Skin inflammation and cutaneous injury are [109]
b.w. 2.97, 5.94 and alterations behind inflammation and cutaneous injury mediated through oxidative stress, activation of
11.88 mg/kg b.w. caused by T-2 toxin myeloperoxidase, MMP activity, increase in
inflammatory cytokines, activation of p38 MAPK,
and apoptosis of epidermal cells, leading to
degenerative skin histological changes
Male Swiss albino T-2 Topical, 11.8 and To evaluate the protective efficacy of CC-2 formulation CC-2 formulation may be an effective decontaminant [110]
23.76 mg/kg against lethal topical doses; to check the effect of against topical exposure to T-2 toxin if treated
dose of T-2 toxin and time of CC-2 application on within 5–15 min of T-2 toxin exposure
lethality; to evaluate the recovery profile of surviving
animals
90-d-old male Swiss ZEA Gavage, 40 mg/kg To investigate the effect of an acute dose of ZEA on ZEA has acute toxic effects mainly in reproductive [111]
albino reproductive and hematological parameters, as well as system of adult male mice, and its effect is
on markers of oxidative stress in liver, kidney, and probably related to a reduced activity of GST and
testes increase in SOD activity in testes
(Continued)

535
TABLE 34.2 (Continued)

536
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
7- to 8-w-old male and DON, Oral 45 μg/kg To determine whether association of DON and FB1 NOAEL of both DON and FB1 should be lower than [112]
female Swiss FB1 cause an additive or synergistic toxic effect on their 45 μg/kg b.w./d and 110 μg/kg b.w./d; the oral
systemic targets repetitive administration of low dose of DON
revealed disorders in lipid metabolism, renal
filtration disturbance, and renal cell DNA
methylation, rhabdomyolysis, and blood lymphocyte
cell deaths
9-d-old male Swiss ZEA ZEA (40 mg/kg—8% To check the effects of lycopene on reproductive, Lycopene prevented the testicular histopathological [113]
albino of LD50) hematological, histopathological, and oxidative stress damage caused by ZEA, as well as its harmful
parameters following acute exposure to ZEA effects on sperm count and motility
Male Swiss albino ZEA Oral, single dose To study ZEA toxicity effects and the mechanism of ZEA impaired testicular functions (spermatozoa [114]
(40 mg/kg—8% of lycopene’s protective effects following ZEA exposure count and motility) and testosterone levels;
LD50) lycopene can be considered a potential therapeutic
nutrient in protection against male reproductive
toxicity induced by ZEA
6- to 7-w-old female DON Topical application in To understand the cellular events leading to DON- Topical application increased cell proliferation, DNA [115]
Swiss albino concentrations of 84, mediated in vivo dermal toxicity and to investigate synthesis, and inflammation, which are mediated
168, 336, and whether DON causes induction of cell proliferation through PI3K/AKT and MAPK signaling pathways

Laboratory Models for Foodborne Infections

672 nmol in 0.2 mL via activation of MAPK pathway, which may further involving transcription factor NFκB and AP-1,
acetone lead to tumor formation further leading to transcriptional activation of
downstream target proteins c-fos, c-jun, cyclin D1,
iNOS, and cox-2
Swiss albino female T-2 Percutaneous, To evaluate the effect of time course and route of T-2 Results showed oxidative stress as major underlying [116]
subcutaneous, toxin exposure on hepatic oxidative damage mechanism for T-2 toxin toxicity in vivo
5.94 mg/kg
1.57 mg/kg
25-d-old male Kunming ZEA Oral, 10 μg To explore the effects of Gynostemma pentaphyllum on Gynostemma pentaphyllum protects against toxicity [117]
ZEA-induced apoptosis in germ cells caused by ZEA through antioxidation and
antiapoptosis via the regulation of Bax and Bcl-2
expression
(Continued)
TABLE 34.2 (Continued)

Fusarium
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
60- to 90-d-old T-2 10 ng/mL To investigate the effects of T-2 toxin on testosterone T-2 toxin can directly decrease the testosterone [118]
Kunming biosynthesis in Leydig cells biosynthesis in the primary Leydig cells derived
from the testis
SWV and LM/Bc FB1 Intraperitoneal, 20 mg/kg To determine the relationship between failure of neural Elevated sphingoid base-1-P after FB1 or FTY720 [119]
tube closure and accumulation of sphingoid bases and suggest a potential role for these bioactive lipid
their phosphorylated metabolites in maternal and ligands and activation of S1P receptor signaling
embryonic tissue after administration of FB1 pathways in the failure of neural tube closure after
FB1 or FTY720; Sa1P may represent a biomarker
for FB1-NTD risk assessment
15- to 18-w-old female FB1 25 mg/kg To investigate whether the consumption of a diet Folate deficiency does not exacerbate NTD induction [120]
acclimated LM/Bc 1 mg/kg deficient in folate and with reduced vitamin B12 would by FB1 in LM/Bc mice; interactions between folate,
exacerbate NTD induction by FB1 other nutritional factors, and FB1 in this mouse
model for NTDs are complex and require further
investigations
Male LM/Bc FB1 Oral gavage, 5, 10, 15, To develop and validate in an animal model, and In both mouse and human, the A270, total protein, [121]
25, and 50 mg/kg b.w. ultimately in humans, a method to estimate the and blood volume were closely correlated and the
volume of blood collected as blood spots on absorbent volume of blood spotted was accurately estimated
paper so as to allow quantification of the molar using only the A270 of the extracts; in mouse blood
concentration of sphingoid base 1-phosphates in spots, as in tissues and embryos, the FB1-induced
blood changes in sphingolipids were correlated with
urinary FB1
6-w-old male C3H DON Contaminated drinking To evaluate immune function of mice exposed to DON; Exposure to DON at 2.0 mg/L via drinking water can [122]
water, 2 mg/mL to study the effects of exposure on the immune disrupt the immune response in vaccinated mice by
response after vaccination with inactivated porcine modulating cytokines and chemokines
parvovirus vaccine
6-w-old BDF1 T-2 0.5, 0.75, and 1 ng/mL To investigate embryotoxicity of T-2 via the in vitro T-2 mycotoxin has a harmful effect on early [123]
development of preimplantation mouse embryos and embryonal development, which results in decreased
its effect on the nuclear chromatin status blastocyst proportion, delayed blastulation, and
increased rate of chromatin damage
(Continued)

537
 538
TABLE 34.2 (Continued)
Studies Using Mouse Models for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Mouse Model MT Administration/Dose Aim of the Study Major Findings References
Female FB1 150 mg/kg To examine the apoptotic and proliferative activity of Oral administration of FB1 caused atrophy in gastric [124]
gastric mucosa following administration of FB1 mucosa both via increasing of apoptosis and
suppressing the mitotic activity of these cells
mPGES-1 wild-type DON Oral, 6.25, 12.5, and To demonstrate that DON-induced sickness-like Behavioral changes observed after DON intoxication [125]
and knock out adult 25 mg/kg behavior is independent of PGE2 action differ from classical sickness behavior evoked by
male inflammatory cytokines
5- to 7-w-old FB1 5, 50, and 150 mg/kg To compare the responses to chronic dietary FB1 Based on similar responses in p53+/– and p53+/+ [126]
p53+/– and p53+/+ exposure in p53+/– and p53+/+ mice to check mice, p53 and related pathways play a secondary
whether FB1 is a nongenotoxic carcinogen; to assess role in responses to FB1 toxicity and carcinogenesis

Laboratory Models for Foodborne Infections

the role of p53 in protecting cells and tissues from
FB1-induced changes, including altered sphingolipid
metabolism
AFB1, aflatoxin B1; b.w., body weight; CC-2, N,N′-dichloro-bis(2,4,6-trichlorophenyl) urea; d, day; DNA, deoxyribonucleic acid; DON, deoxynivalenol; FB1, fumonisin B1; GH, growth
hormone; GPx, glutathione peroxidase; GST, glutathione-S-transferase; HF, high-fat; IGF, insulin-like growth factor; IGFALS, insulin-like growth factor binding protein, acid labile subunit;
LF, low-fat; LPS, lipopolysaccharide; Mab, monoclonal antibody; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; MT, mycotoxin; NCE, normochromatic erythrocytes;
NOAEL, no observable adverse effect level; NSP, nanosilicate platelets; NTD, neural tube defect; TNF, tumor necrosis factor; PCE, polychromatic erythrocytes; PBS, phosphate-buffered
saline; PGE2, prostaglandins E2; PKR, double-stranded RNA-activated protein kinase; SOD, superoxide dismutase; TM, Tunisian montmorillonite; w, week; ZEA, zearalenone.
Fusarium 539

confirmed to diminish female fertility in C57BL/6J mice [103]. Using CD1 males, Zatecka et al. [107]
illustrated that ZEA has considerable effect on male fertility, and López-Casas et al. [105] proved the
alterations of gene signatures due to ZEA. Yuan et al. [117] assessed the ZEA toxicity in male germ cells
of Kunming mice.
FB1-induced changes in sphingoids were analyzed by Riley et al. [121] and Gelineau-van Waes et al.
[119] among LM/Bc mice. Voss et al. [120] established that folate deficiency does not exacerbate NTD
induction by FB1 in LM/Bc mice. Hepatotoxicity [85] and cytotoxicity [83] of FB1 were confirmed using
BALB/c mice. Ling et al. [84] utilized anti-FB1 monoclonal antibody from BALB/c mice to detect FB1
in corn and related samples. Liao et al. [106] evidenced that nanosilicate platelets reduced the toxicity
of FB1 in CD1 mice. Kouadio et al. [112] proved that no-observed-adverse effect level (NOAEL) of FB1
should be lower than 110 μg/kg/b.w./day in Swiss albino mice. FB1-induced carcinogenesis was reported
to be similar in both p53 heterozygous cancer-prone model and p53 homozygous mice [126]. Alizadeh
et al. [124] reported that atrophy in gastric mucosa was due to FB1 in female mice.

34.2.2 Human and Animal Cell Lines

The literature review was restricted to papers published after 2010 and included in the PubMed data-
base, with keyword combinations deoxynivalenol OR T-2 toxin OR zearalenone OR fumonisin AND
cell line OR cell culture. Similar to the case of rat and mouse models, the information on exact defini-
tion of the mycotoxin and the cell line applied were collected from studies that used human and animal
cell lines.
Table 34.3 furnishes the cumulative information of studies published since 2010 that have applied
human or animal cell lines as laboratory models for the exploration of Fusarium mycotoxins [127–170].
Among the 44 relevant studies, the most frequently applied laboratory models were intestinal epithelial
cell lines, primarily the IPEC-J2 nontransformed porcine intestinal epithelial cell line (9 studies), which
was used to examine different aspects of Fusarium mycotoxins, including the effect of DON on func-
tional characteristics of epithelial cells and intestinal barrier integrity [128,131,136], the effects of phytic
acid as a possible inhibitor of cellular damage induced by DON [130], the gene-response profiles of
epithelial cell layers in response to DON [129] and the cytotoxicity and combined effects of 4 Fusarium
mycotoxins, DON, NIV, ZEA, and FB1 [133]. Further intestinal cell lines used as laboratory models
to study Fusarium mycotoxins included HT-29 human colon carcinoma cells for studying T-2 toxin
[147,148] and FB1 [155]; Caco-2 human colorectal adenocarcinoma cells for the examination of different
properties of T-2 toxin [138], DON [139,140], and FB1 [141]; as well as HCT116 and HCT116-Bax-KO
human colon carcinoma cells [157], AGS human gastric epithelial cells, and SW742 human colon adeno-
carcinoma cells [156]; IEC-6 (CRL-1592) rat small intestine epithelium cells [159]; and IPEC-1 cells
derived from the small intestine of a newborn unsuckled piglet [127,137].
Among the kidney cell lines, HepG2 human hepatocellular carcinoma cells were used to evaluate the
role of mitogen-activated protein kinase phosphatases in prevention of DON-induced apoptosis [144], to
investigate the effect of FB1 on cytochrome P450 1B1 modulation [146], and to investigate the effect of
DON on the cytosolic redox state and antioxidative system [145]. HL-7702 normal human liver cells were
used to investigate the effect of FB1 on the cell cycle and the expression of cell-cycle-related genes [169].
H4IIE rat hepatoma cells were used to evaluate the effects of FB1 and AFB1 alone or in combination
on the activation and expression of cytochrome P450 1A and its transcription factor Ahr [168], while
SHK-1 salmon head kidney cells were used to investigate the possible metabolization of ZEA in fish cell
lines [170]. Vero African green monkey kidney cells were used to examine the ROS production induced
by FB1 on renal cells [167], and PK-15 porcine kidney cells were used to evaluate the nephrotoxicity
of individual mycotoxins and combinations of AFB1, ZEA, DON, and FB1 to livestock [165], HEK293
human embryonic kidney cells were used to investigate the mechanisms of ZEA nephrotoxicity and
DNA damage [166]. Further cell lines involved in Fusarium mycotoxin research during the past 5 years
included MTEC1 murine thymic epithelial cells [142,143], Jurkat human T-lymphocyte cells [151,152],
HL60 human promyelocytic leukemia cells [149,150], U937 human monocyte cells [154], CTLL-2
mouse blood lymphoblast cells [153], EL4 murine T-lymphocyte cells [160], STC-1 mouse intestinal
neuroendocrine tumor cells [158], GT1-7 murine hypothalamic cells [161], NPTr newborn pig trachea
TABLE 34.3

540
Studies Using Human and Animal Cell Lines for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Applied Cell Line MT Administration/Dose Aim of the Study Major Findings References
IPEC-1 cells derived from DON 30 μmol/L To investigate the involvement of MAPK in the DON-induced activation of the p44/42 ERK signaling [127]
small intestine of a DON-induced loss of barrier function pathway inhibits the expression of claudin-4 protein,
newborn unsuckled piglet which leads to impaired intestinal barrier function
IPEC-J2 non-transformed DON 200 and 2000 ng/mL To elucidate the impact of the direction of DON Severity of impact of the DON on the intestinal epithelial [128]
porcine intestinal columnar exposure on epithelial cell behavior and barrier is dependent on route of application
epithelial cells intestinal barrier integrity
IPEC-J2 DON 200 ng/mL To detect potential expression differences and to Apical and basolateral challenge of epithelial cell layers [129]
2000 ng/mL identify candidate genes for further trigger different gene-response profiles paralleled with a
investigations on cellular reaction mechanisms higher susceptibility towards basolateral challenge

IPEC-J2 DON To evaluate the effects of IP6 as a possible Phytic acid decreased the negative effects of DON on the [130]
inhibitor of cellular damage induced by toxic membrane integrity
substances such as MTs
IPEC-J2 DON 10 μM To assess short-term effects of DON on functional A significant decrease in ATP levels was seen at 48 h in a [131]
characteristics of the intestinal epithelial cells dose-dependent manner and demonstrated that DON
has a distinct cytotoxic effect on IPEC-J2 cells
IPEC-J2 DON 1 mg/mL To determine the penetration of FOS in the Nontoxic concentration of DON on IPEC-J2 cells after [132]

Laboratory Models for Foodborne Infections

presence and absence of DON short-term exposure interferes with the
pharmacokinetics of the antibiotic
IPEC-J2 DON 2.5–40 μM To investigate the cytotoxicity of four common At noncytotoxic concentrations of individual MTs, all [133]
NIV Fusarium MTs mixtures were cytotoxic with DON-NIV, DON-ZEA,
ZEA DON-NIV-FB1, DON-ZEA-FB1, NIV-ZEA-FB1, with
the mixture of all four causing the greatest loss of cell
FB1
viability
IPEC-J2 DON 10 or 40 μM To elucidate the modulatory capacity of individual Data suggest that individual Fusarium toxins or their [134]
NIV Fusarium toxins and their mixtures on mixtures could cause or exacerbate intestinal
ZEA the mRNA expression of proinflammatory inflammation
FB1 cytokines
IPEC-J2 DON To elucidate the individual and combined effects Fusarium toxins, individually and in mixtures, activate [135]
NIV of four common Fusarium toxins distinct antimicrobial defense mechanisms possessing
ZEA the potential to alter the intestinal microbiota through
FB1 diminished antimicrobial effects
(Continued)
 Fusarium
TABLE 34.3 (Continued)
Studies Using Human and Animal Cell Lines for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Applied Cell Line MT Administration/Dose Aim of the Study Major Findings References
IPEC-J2 DON 2 μg/mL To examine whether Bacillus subtilis could B. subtilis may have potential to enhance epithelial [136]
improve barrier integrity and protection against barrier function and to prevent the cells from
DON-induced barrier disruption DON-induced barrier dysfunction
IPEC-1 cells derived from T-2 0, 0.3, 1, 3, 10, and To investigate how ENN modulates T-2 toxicity in Downmodulation of the gastrointestinal toxicity of T-2 [137]
small intestine of a 30 nM the situation of co-contamination, by analysing toxin by the emerging ENN B1 below toxic
newborn unsuckled piglet the acute toxicity of T-2 on the digestive target concentrations; confirmation of the relevance of the
determinist approach for the analysis of toxin
interactions, giving concordant results with the
probabilistic approach in two different pig intestinal
models
Caco-2 human colorectal T-2 1 ng/mL To elucidate the possible underlying molecular p38 MAPK is responsible for mediating the [138]
adenocarcinoma cells mechanism of conditional T-2-provoked IL-8 inflammatory properties of T-2
induction, a possible involvement of the AHR
and the MAPK pathway
Caco-2 DON 3.2, 16, 80, 400, and To investigate dose-dependent AhR agonistic and Induction of CYP1A1 activity and inhibition of [139]
2000 ng/mL antagonistic activities in luciferase reporter cells CYP3A4 activity occurred in Caco-2 cells at a
and to assess the dose-dependent agonistic and realistic intestinal concentration of IMA, and some
antagonistic activities on hER bacterial stress genes were induced in a range of
realistic concentrations following exposure to DON
Caco-2 DON 0.1, 1, 10 l g/mL To evaluate the intestinal absorption and toxicity 15ADON caused the highest permeability and highest [140]
of the major derivatives of DON: 3ADON and IL-8 secretion among DON, 3ADON, and 15ADON
15ADON
Caco-2, MDBK bovine FB1 0.0038 ng/mL To evaluate the effects of low levels of each MTs Only in the case of MDBK cells was a synergistic [141]
kidney cells and raw 264.7 3.8 mg/mL in combination at their EU regulatory limits effect measured, and this was when all three toxins
murine macrophages 0.005 ng/mle10 mg/mL were present at levels in food above the legal limits
(OTA 3 mg/mL; FB1 8 mg/mL and AFB1 1.28 mg/mL)
(Continued)

541
TABLE 34.3 (Continued)

542
Studies Using Human and Animal Cell Lines for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Applied Cell Line MT Administration/Dose Aim of the Study Major Findings References
MTEC1 murine thymic DON 100, 500, 1000, and To investigate the mRNA expression differences Results provided molecular insights into the gene [142]
epithelial cells 2000 ng/mL after treatment with DON in MTEC1 cells expression differences of DON-induced toxic effects
and suggest that p53 signaling pathway may play an
important role in the inhibition of MTEC1 cell
proliferation
MTEC1 DON 500, 1000, and To investigate if DON could induce apoptosis and DON causes activation of p53, increases levels of ROS, [143]
2000 ng/mL to elucidate the possible mechanism of the action and causes the induction of mitochondrial
dysfunction, which may contribute to DON-induced
apoptosis
HepG2 human DON 1 μM To evaluate the impact of MKPs, particularly DUSP1 is a novel target gene of DON, which is [144]
hepatocellular carcinoma 10 μM DUSP1 essential for the prevention of DON-induced
cells apoptosis
HepG2, THP-1 human DON — To investigate the effect of DON on the cytosolic DON induced accumulation of Trx-1 in HepG2 cells, [145]
monocyte-like cells redox state and antioxidative system which plays one of the key roles in protection against
cytotoxicity caused by DON; the mechanism may be
mediated by the antioxidant properties of Trx-1
HepG2 FB1 0–1000 μM To investigate the effect of FB1 on miR-27b CYP1B1 is posttranscriptionally regulated by miR-27b [146]
suppression and its effect on CYP1B1 after HepG2 exposure to FB1; FB1-induced

Laboratory Models for Foodborne Infections

modulation modulation of miR-27b in hepatic cells may be an
additional mode of hepatic neoplastic transformation
HT-29 human colon T-2 1 nM to 200 μM To analyze the metabolism of T-2 toxin Both cell types metabolized T-2 toxin to a variety of [147]
carcinoma cells and compounds; cell cycle analysis in RPTEC proved the
RPTEC human renal apoptotic effect of T-2 toxin and its metabolites HT-2
proximal tubular epithelial toxin and neosolaniol in micromolar concentrations
cells
HT-29 human colon T-2 1 nM to 200 μM To investigate the cytotoxic properties of T-2 toxin Results emphasize the neurotoxic potential of T-2 toxin [148]
carcinoma cells and NHA on cells derived from brain tissue in human astrocytes at low concentrations after short
normal human astrocytes incubation times
(Continued)
TABLE 34.3 (Continued)

Fusarium
Studies Using Human and Animal Cell Lines for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Applied Cell Line MT Administration/Dose Aim of the Study Major Findings References
HL60 human acute DON — To elucidate the mechanisms underlying the Deoxynivalenol induces the secretion of chemokines, [149]
promyelocytic leukemia NIV toxicities of the trichothecene mycotoxins whereas nivalenol has the opposite effect, clearly
cells deoxynivalenol and nivalenol, their effects on the indicating that the toxicity mechanisms of
secretion of antihematopoietic chemokines, deoxynivalenol and nivalenol differ
macrophage inflammatory protein-1 α (MIP-1α),
and MIP-1β
HL60 T-2 0, 4, 8, 16, and 32 μg/mL To study the effects of T-2 toxin produced by T-2 toxin could inhibit proliferation and induce [150]
Fusarium fungi on proliferation and apoptosis apoptosis in vitro in a dose-dependent manner
Jurkat human DON 0.5 mM Immunocytological and biochemical analyses of Immune cells are more sensitive to DON than other [151]
lymphoblastoid T-cells the effects of DON in Jurkat cells cell types due to the induction of a T-cell activation
response by increased intracellular calcium levels
Jurkat DON 0.5 mM To understand the mechanism of action of DON in Immune cells are more sensitive to DON than other [152]
immune cells; to understand why immune cells cell types due to the induction of a T-cell activation
are more sensitive to DON than most other cell response by increased intracellular calcium levels
types
CTLL-2 murine cytotoxic T DON 1 or 2 mM To assess the usefulness of the mouse CTLL-2 Based on the results for TBTO and DON, the CTLL-2 [153]
lymphocyte cells cell line for immunotoxicity cell line does not yield an added value for
immunotoxicity compared to the human Jurkat T cell
line
U937 human monocyte DON 500–1000 ng/mL To elucidate linkages that exist between the PKR and Hck were critical for DON-induced [154]
cultures ribosome and PKR, Hck, and p38 following ribosomal recruitment of p38, its subsequent
stimulation with DON in human and mouse phosphorylation, and p38-driven proinflammatory
mononuclear phagocytes cytokine expression
HT-29 human colorectal FB1 1.1–69 μM To investigate by dose- and time-dependent Lipid peroxidation followed by modifications to [155]
adenocarcinoma cells experiments the modifications induced by FB1 at membrane microviscosity and inflammatory response
concentrations ranging from 0.25 to 69 μM was the main and most sensitive effect of FB1
AGS gastric epithelial cells FB1 4.5–72 mg/L To evaluate FB1 effects on the production of FB1 increases inflammatory cytokines production [156]
and SW742 human colon inflammatory cytokines
adenocarcinoma cells
HCT116 and HCT116- DON 0.2 mg/mL To analyze the molecular mechanisms of Mitochondria-related caspase-dependent apoptotic [157]
Bax-KO human colon DON-induced toxicity and explore the pathway is involved in this in vitro model of DON
adenocarcinoma cells contribution of mitochondria to cell death induced-cytotoxicity
(Continued)

543
TABLE 34.3 (Continued)

544
Studies Using Human and Animal Cell Lines for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Applied Cell Line MT Administration/Dose Aim of the Study Major Findings References
STC-1 murine invasive DON 0–5 mM To test the hypothesis that DON induces hormone DON evokes CCK and GLP-1 secretion in the STC-1 [158]
small intestinal exocytosis in EEC by GPCR-mediated Ca2+ EEC model by activating CaSR- and TRPA1-
neuroendocrine carcinoma signaling mediated Ca2+ signaling pathways
cells
IEC-6 (CRL-1592) rat small DON 0.5–80 mM To test the effects of variable concentrations of Both nivalenol and deoxynivalenol significantly [159]
intestine epithelial cells NIV and/or DON on the nontumorigenic affected IEC-6 viability through a proapoptotic
intestinal epithelial cells, IEC-6 process
EL4 thymoma cells DON 0.5 μM To investigate whether proteomic analysis of DON stimulated the expression levels of several [160]
thymoma cells treated with DON as compared to proteins in thymoma cells
nontreated control cells would reveal differential
protein expression
GT1-7 murine hypothalamic T-2 0.01–1000 ng/mL To find out at how low-dose T-2 toxin influences Low-dose T-2 toxin stimulates GnRH secretion and [161]
neuronal cells GnRH secretion, especially with the presence of alter the expression of associated proteins in vitro, and
kisspeptins the activation of cell response to T-2 toxin is increased
after pretreatment with kisspeptins
MLTC-1 mouse Leydig ZEA 0–200 μg/mL To demonstrate the involvement of ER stress in The activation of an ER stress pathway plays a key role [162]
cells ZEA-induced cell death in ZEA-induced apoptosis

Laboratory Models for Foodborne Infections

MCF-7aro ZEA 0.1% v/v To investigate two estrogenic MTs for their ZEA was a potential aromatase inhibitor among the [163]
CYP19-overexpressing interference in aromatase by using a cell-based three MTs tested, and this 4-cell line approach could
cells, T98G human brain system be employed in principle to screen for compounds
glioblastoma cells and inhibiting or inducing estrogen synthesis
MCF-7 human breast
adenocarcinoma cells
NPTr newborn pig trachea DON 0, 70, 140, 280, 560, and To evaluate the impact of DON on in vitro and DON had no significant effect on clinical manifestation [164]
epithelial cells 1200 ng/mL in vivo PCV2 pathogenesis of PCVAD in PCV2b-infected animals
PK-15 porcine kidney cells ZEA 10 mM To evaluate the nephrotoxicity of individual MTs The combined effects of MTs acted in a concentration- [165]
DON and combinations of AFB1, ZEA, DON, and FB1 dependent manner
FB1 to livestock
AFB1
(Continued)
TABLE 34.3 (Continued)

Fusarium
Studies Using Human and Animal Cell Lines for the Examination of Fusarium Mycotoxins Published Since 2010 Based on PubMed
Applied Cell Line MT Administration/Dose Aim of the Study Major Findings References
HEK293 human embryonic ZEA 0–20 μM To investigate the mechanisms of ZEA Oxidative stress does not play a key role in DNA strand [166]
kidney cells nephrotoxicity and DNA damage breaks induced by ZEA; lysosomal injury precedes
DNA strand breaks; the lysosome may be a primary
target for ZEA
Vero African green monkey FB1 1, 5, and 10 μM To study the ROS production using a procedure Significant increase of ROS products in Vero cells at 10 [167]
kidney cells based on an antioxidant sensitive fluorescein IM dose; ROS production by FB1 on renal cells is a
probe H2-DCFDA and to detect intracellular mechanism of fumonisin-mediated toxicity
ROS as early-stage marker for toxin-induced
oxidative stress
H4IIE rat hepatoma cells FB1 10 and 20 μM To evaluate the effects of MTs, alone or AHR pathway activation as a toxicity mechanism of [168]
AFB1 combined, on activation and expression of AFB1 and FB1; FB1 may increase AFB1 bioactivation
CYP1A and its transcription factor AHR

HL-7702 normal human FB1 0.0, 0.1, 1.0, 10.0, and To investigate the effect of FB1 on the cell cycle The underlying mechanism of action is associated with [169]
liver cells 100 μmol/L and the expression of cell-cycle-related genes alterations in the expression levels of cyclin E and
P21 and cyclin E P21 induced by FB1
RTL-W1 rainbow trout liver ZEA 300 ng/mL To investigate the possible metabolization of ZEA Results confirm a lysosomal pathway as a main target [170]
cells, RTgill-W1 gill cells in fish cell lines suggesting that mainly of ZEA in fish cells
and SHK-1 salmon head glucuronidation takes place
kidney cells
ADON, acetyldeoxynivalenol; AFB1, aflatoxin B1; AHR, aryl hydrocarbon receptor; CaSR, GPCR Ca(2+)-sensing receptor; CCK, cholecystokinin; CYP, cytochrome P; DNA, ­deoxyribonucleic
acid; DON, deoxynivalenol; DUSP1, dual specific phosphatase 1; EEC, enteroendocrine cell; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; GnRH, gonadotropin-
releasing hormone; GLP-1, glucagon-like peptide-17–36 amide; GPCR, G protein-coupled receptor; ENN, enniatin; FOS, fosfomycin; FB1, fumonisin B1; H2-DCFDA,
­dichloro-dihydro-fluorescein diacetate; Hck, hematopoietic cell kinase; hER, the human oestrogenic receptor; IMA, imazalil; IP6, phytic acid; MAPK, mitogen activated protein kinases;
MKP, mitogen activated protein kinase phosphatase; MT, mycotoxin; NIV, nivalenol; OTA, ochratoxin A; PCV2, porcine circovirus type 2; PCVAD, porcine circovirus-associated disease;
PKR, ­RNA-activated protein kinase; RNA, ribonucleic acid; ROS, reactive oxygen species; TBTO, tributyltin oxide; TRPA1, transient receptor potential ankyrin-1 channel; Trx-1, thiore-
doxin 1; ZEA, zearalenone.

545
546 Laboratory Models for Foodborne Infections

epithelial cells [164], MLTC-1 murine testis Leydig cells [162], as well as CYP19-overexpressing MCF-
7aro human breast cancer cells and T98G human glioblastoma cells [163].

34.3 Conclusions
Based on the recent literature search (since 2010), mainly rats, mice, and cell lines—particularly IPEC-J2
nontransformed porcine intestinal epithelial cells—were extensively involved in Fusarium mycotoxin
research. These laboratory models revealed substantial details of scientific and technical information
related to the mycotoxins DON, T-2, ZEA, and FB1. These available data and the particulars discussed in
this review chapter obviously warrant that laboratory animal models and cell lines have the potential for
the detection of fusarial mycotoxins in food and feed, as well as in the research leading to the reduction
and control of these mycotoxins in the products for consumption by humans and animals, which may
otherwise lead to deleterious effects.

Acknowledgments
This work was supported by the Indian National Science Academy and the Hungarian Academy of
Sciences, by project GINOP-2.3.3-15-2016-00006 (Széchenyi 2020 Programme), and by King Saud
University, Deanship of Scientific Research, College of Sciences Research Center.

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138. Kruber, P., Trump, S., Behrens, J., Lehmann, I., T-2 toxin is a cytochrome P450 1A1 inducer and leads
to MAPK/p38- but not aryl hydrocarbon receptor-dependent interleukin-8 secretion in the human intes-
tinal epithelial cell line Caco-2, Toxicology, 284, 34, 2011.
139. Ribonnet, L. et al., Potential of an in vitro toolbox combined with exposure data as a first step for the risk
assessment of dietary chemical contaminants, Food Addit. Contam. Part A Chem. Anal. Control Expo.
Risk Assess., 28, 1136, 2011.
140. Kadota, T., Furusawa, H., Hirano, S., Tajima, O., Kamata, Y., Sugita-Konishi, Y., Comparative study of
deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol on intestinal transport and IL-8
secretion in the human cell line Caco-2, Toxicol. In Vitro, 27, 1888, 2013.
141. Clarke, R., Connolly, L., Frizzell, C., Elliott, C.T., Cytotoxic assessment of the regulated, co-existing
mycotoxins aflatoxin B1, fumonisin B1 and ochratoxin, in single, binary and tertiary mixtures, Toxicon,
90, 70, 2014.
142. Li, D. et al., Gene expression profiling analysis of deoxynivalenol-induced inhibition of mouse thymic
epithelial cell proliferation, Environ. Toxicol. Pharmacol., 36, 557, 2013.
143. Li, D. et al., Deoxynivalenol induces apoptosis in mouse thymic epithelial cells through mitochondria-
mediated pathway, Environ. Toxicol. Pharmacol., 38, 163, 2014.
144. Casteel, M., Nielsen, C., Kothlow, S., Dietrich, R., Märtlbauer, E., Impact of DUSP1 on the apoptotic
potential of deoxynivalenol in the epithelial cell line HepG2, Toxicol. Lett. 199, 43, 2010.
145. Sugiyama, K., Kinoshita, M., Kamata, Y., Minai, Y., Tani, F., Sugita-Konishi, Y., Thioredoxin-1
­contributes to protection against DON-induced oxidative damage in HepG2 cells, Mycotoxin Res., 28,
163, 2012.
146. Chuturgoon, A.A., Phulukdaree, A., Moodley, D., Fumonisin B1 modulates expression of human cyto-
chrome P450 1b1 in human hepatoma (Hepg2) cells by repressing Mir-27b, Toxicol. Lett., 227, 50, 2014.
147. Weidner, M., Welsch, T., Hübner, F., Schwerdt, G., Gekle, M., Humpf, H.U., Identification and apoptotic
potential of T-2 toxin metabolites in human cells, J. Agric. Food Chem., 60, 5676, 2012.
148. Weidner, M., Lenczyk, M., Schwerdt, G., Gekle, M., Humpf, H.U., Neurotoxic potential and cellular
uptake of T-2 toxin in human astrocytes in primary culture, Chem. Res. Toxicol., 26, 347, 2013.
149. Nagashima, H., Nakagawa, H., Kushiro, M., Opposite effects of two trichothecene mycotoxins, deoxyni-
valenol and nivalenol, on the levels of macrophage inflammatory protein (MIP)-1α and MIP-1β in HL60
cells, Environ. Toxicol. Pharmacol., 34, 1014, 2012.
150. Hou, Y., Yuan, Z., Guo, W., Wang, H., Effects of T-2 toxin of Fusarium on proliferation and apoptosis
of human acute promyelocytic leukemia cell line HL60, Wei Sheng Yan Jiu, 42, 381, 2013.
151. Katika, M.R., Hendriksen, P.J., Shao, J., van Loveren, H., Peijnenburg, A., Transcriptome analysis of
the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to
deoxynivalenol (DON): new mechanistic insights, Toxicol. Appl. Pharmacol., 264, 51, 2012.
152. Katika, M.R., Hendriksen, P.J., van Loveren, H., Peijnenburg, A.C.M., Characterization of the modes of
action of deoxynivalenol (DON) in the human Jurkat T-cell line, J. Immunotoxicol., 12, 206, 2015.
153. Schmeits, P.C., Volger, O.L., Zandvliet, E.T., van Loveren, H., Peijnenburg, A.A., Hendriksen, P.J.,
Assessment of the usefulness of the murine cytotoxic T cell line CTLL-2 for immunotoxicity screening
by transcriptomics, Toxicol. Lett., 217, 1, 2013.
154. Bae, H., Gray, J.S., Li, M., Vines, L., Kim, J., Pestka, J.J., Hematopoietic cell kinase associates with the
40S ribosomal subunit and mediates the ribotoxic stress response to deoxynivalenol in mononuclear
phagocytes, Toxicol. Sci., 115, 444, 2010.
155. Minervini, F. et al., Toxic mechanisms induced by fumonisin B1 mycotoxin on human intestinal cell
line, Arch. Environ. Contam. Toxicol., 67, 115, 2014.
156. Mahmoodi, M. et al., Impact of fumonisin B1 on the production of inflammatory cytokines by gastric
and colon cell lines, Iran. J. Allergy Asthma Immunol., 11, 165, 2012.
157. Bensassi, F., Gallerne, C., Sharaf El Dein, O., Lemaire, C., Hajlaoui, M.R., Bacha, H., Involvement of
mitochondria-mediated apoptosis in deoxynivalenol cytotoxicity, Food Chem. Toxicol., 50, 1680, 2012.
158. Zhou, H.R., Pestka, J.J., Deoxynivalenol (vomitoxin)-induced cholecystokinin and glucagon-like
­peptide-1 release in the STC-1 enteroendocrine cell model is mediated by calcium-sensing receptor and
transient receptor potential ankyrin-1 channel, Toxicol. Sci., 145, 407, 2015.
159. Bianco, G., Fontanella, B., Severino, L., Quaroni, A., Autore, G., Marzocco, S., Nivalenol and deoxyni-
valenol affect rat intestinal epithelial cells: a concentration related study, PLoS One, 7, e52051, 2012.
Fusarium 553

160. Osman, A.M., Pennings, J.L., Blokland, M., Peijnenburg, A., van Loveren, H., Protein expression profil-
ing of mouse thymoma cells upon exposure to the trichothecene deoxynivalenol (DON): implications
for its mechanism of action, J. Immunotoxicol., 7, 147, 2010.
161. Liu, Y. et al., Effects of low dose T-2 toxin on secretion of gonadotropin-releasing hormone in the
immortalized hypothalamic GT1-7 cell line, Toxicon, 100, 67, 2015.
162. Lin, P. et al., Mycotoxin zearalenone induces apoptosis in mouse Leydig cells via an endoplasmic reticu-
lum stress-dependent signalling pathway, Reprod. Toxicol., 52, 71, 2015.
163. Wang, Y., Wong, T.Y., Chan, F.L., Chen, S., Leung, L.K., Assessing the effect of food mycotoxins on
aromatase by using a cell-based system, Toxicol. In Vitro, 28, 640, 2014.
164. Savard, C. et al., Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus
type 2 infections, Vet. Microbiol., 176, 257, 2015.
165. Lei, M., Zhang, N., Qi, D., In vitro investigation of individual and combined cytotoxic effects of afla-
toxin B1 and other selected mycotoxins on the cell line porcine kidney 15, Exp. Toxicol. Pathol., 65,
1149, 2013.
166. Gao, F. et al., Genotoxic effects induced by zearalenone in a human embryonic kidney cell line, Mutat.
Res., 755, 6, 2013.
167. Meca, G., Ruiz, M.J., Fernandez-Franzón, M., Ritieni, A., Manes, J., Isolation, purification, LC-MS/
MS characterization and reactive oxygen species induced by fumonisin B1 in Vero cells, Food Chem.
Toxicol., 48, 2891, 2010.
168. Mary, V.S., Valdehita, A., Navas, J.M., Rubinstein, H.R., Fernández-Cruz, M.L., Effects of aflatoxin B1,
fumonisin B1 and their mixture on the aryl hydrocarbon receptor and cytochrome P450 1A induction,
Food Chem. Toxicol., 75, 104, 2015.
169. Wang, S.K., Liu, S., Yang, L.G., Shi, R.F., Sun, G.J., Effect of fumonisin B1 on the cell cycle of normal
human liver cells, Mol. Med. Rep., 7, 1970, 2013.
170. Pietsch, C., Noser, J., Wettstein, F.E., Burkhardt-Holm, P., Unraveling the mechanisms involved in
zearalenone-mediated toxicity in permanent fish cell cultures, Toxicon, 88, 44, 2014.
35
Penicillium and Talaromyces

Elena Bermúdez, Félix Núñez, Josué Delgado, and Miguel A. Asensio

CONTENTS
35.1 Introduction....................................................................................................................................555
35.2 Pathogens...................................................................................................................................... 556
35.2.1 General Description......................................................................................................... 556
35.2.2 Diagnosis...........................................................................................................................557
35.2.3 Molecular Epidemiology...................................................................................................557
35.2.4 Treatment...........................................................................................................................558
35.3 Mycotoxins.....................................................................................................................................558
35.3.1 General Description..........................................................................................................558
35.3.2 Invertebrate Organisms in Toxicity Bioassays................................................................. 562
35.3.3 Microorganisms in Toxicity Bioassays............................................................................. 563
35.3.4 Vertebrate Animals in Toxicity Bioassays....................................................................... 564
35.3.5 Cell Cultures in Toxicity Bioassays.................................................................................. 565
35.3.6 Culture Media for Mycotoxin Production........................................................................ 565
35.4 Allergens....................................................................................................................................... 566
35.4.1 Penicillium Allergies........................................................................................................ 566
35.4.2 Animal Models to Study Allergies Caused by Penicillium spp. ..................................... 567
35.4.3 Cell Cultures to Study Allergies Caused by Penicillium................................................. 570
35.5 Conclusions................................................................................................................................... 570
References................................................................................................................................................571

35.1 Introduction
The genus Penicillium is a group of anamorphic fungi in the division Ascomycota, with over 250 rec-
ognized species, many of them ubiquitous in warm and moderate climates. The genus Talaromyces
was described as a sexual state of Penicillium that produces soft-walled ascomata. Recently, the
genus Penicillium has been redefined into Penicillium sensu stricto and Talaromyces, with all species
of the former Penicillium subgenus Biverticillium being transferred to Talaromyces.1,2 Species like
Talaromyces marneffei lack a defined sexual cycle but possesses all genes believed to be required for
mating.3
Penicillium species play important roles in the environment, agriculture, and industry. Some species
of genus Penicillium are of economic importance to the food industry because they contribute to food
ripening, while others are postharvest pathogens or cause spoilage. For example, Penicillium camemberti
and Penicillium roqueforti are used for cheese manufacture; Penicillium nalgiovense and Penicillium
chrysogenum contribute to ripening of dry-cured meat products. On the other hand, Penicillium expan-
sum is the causal agent of blue mold postharvest rots of apples and is also able to produce patulin and
other mycotoxins, as discussed later. Penicillium digitatum and Penicillium italicum are responsible for
postharvest citrus decay. Heat-resistant ascospores produced by various Talaromyces spp. cause spoilage
of pasteurized juices and other fruit-based products.4

555
556 Laboratory Models for Foodborne Infections

Penicillia are generally soil saprophytes or plant pathogens with limited invasive ability for animal
tissue, except for Talaromyces marneffei (formerly Penicillium marneffei2). However, production of toxic
secondary metabolites is common in Penicillium spp. In addition, Penicillium is an environmental aller-
gen of variable significance among asthmatic and atopic individuals.

35.2 Pathogens
35.2.1 General Description
The genera Penicillium and Talaromyces comprise animal pathogens. Talaromyces radicus and
Talaromyces helicus have been described as rare causative agents of infectious diseases in dogs.5,6
Penicillium corylophilum infects red snapper (Lutjanus campechanus), but infection seems to be con-
fined to the swim bladder.7
Penicillium infections in humans are still called penicilliosis, even those due to Talaromyces marneffei.
Members of the P. chrysogenum–Penicillium rubens complex can be responsible for rare opportunistic
keratitis8 and Talaromyces piceum has been rarely reported in chronic granulomatous disease.9 However,
the most outstanding infection by Penicillium or Talaromyces spp. is due to T. marneffei.
T. marneffei is responsible for one of the commonest opportunistic infections in humans with HIV-
infected patients, causing fatal systemic mycosis in Southeast Asian countries. However, infection by
T. marneffei has also been described as a result of other immunodeficiencies, immunosuppressive ther-
apy, and genetic diseases leading to lymphocytes exhibiting defective IFN-γ production.10
T. marneffei was first identified as the causative agent of mycosis of the reticuloendothelial system.11
T. marneffei can cause a focal infection that may lead to a progressive disseminated penicilliosis, mainly
in immunocompromised patients. The focal infection can be cutaneous or bronchopulmonary, and it may
spread to cause localized lymphadenopathy. However, T. marneffei can also be responsible for asymp-
tomatic fungemia in HIV-infected patients.12
The mode of transmission is not well understood but is probably via ingestion or inhalation of the
fungus.13 By analogy with other endemic fungal pathogens, infection is presumably initiated by the
inhalation of airborne conidia that are subsequently phagocytized by pulmonary alveolar macrophages.
After an inflammatory response in macrophages, T. marneffei can lead to granulomatous or suppurative
reactions in persons with normal immunity, whereas immunocompromised patients develop necrotiz-
ing reaction.13 It is thought that the fungus is disseminated throughout the body to result in systemic
infection. In some cases, the disease did not appear until immunosuppression allowed for the growth
and dissemination throughout the host. Thus, unique skin lesions like abscesses and ulcers are more
commonly noted in non-HIV-infected patients than in HIV-infected patients.14 T. marneffei dissemi-
nates hematogenously causing generalized lymphadenopathy, even leading to osteolysis, which indicates
severe systemic disturbance.15
Adaptation to the new environmental conditions during infection of human hosts is key to fungal
survival.16 T. marneffei is the only dimorphic species of Penicillium and Talaromyces that is capable of
growing in a filamentous multinucleate hyphal form at 25°C or as a uninucleate pathogenic yeast form
at 37°C.17 When grown in air at 25°C, the saprophytic hyphae produce asexual spores (conidia), the
infectious agents. Upon invasion of a human host, most environmental factors undergo immediate and
drastic changes, including temperature, pH, nutrient sources, carbon dioxide, and oxygen concentrations.
Temperature adaptation of T. marneffei within the host manifests itself as a morphological change from
hyphal mold to budding yeast.
It seems that T. marneffei possesses the ability to evade a normally functioning immune system
until in vivo conditions, due to immunosuppression, are favorable to dissemination throughout the
host.18 Upon switching to 37°C, T. marneffei undergoes a process termed arthroconidiation. Conidia
germinate with the onset of extensive hyphal branching. Fragmentation occurs to generate single, uni-
nucleated yeast cells that divide by fission. Switching from yeast growth at 37°C to hyphal growth at
25°C requires the polarized growth of yeast cells, septation, and branching, to form the characteristic
Penicillium and Talaromyces 557

multinucleate hyphal mycelium. Genes responsible for growth, morphogenesis, and development of
T.  marneffei have been characterized.19 Conidial germination and temperature adaptation require
both HHK and heterotrimeric G-protein-Ras signaling, as well as the establishment of actin-mediated
polarized growth through a Rho GTPase. In addition, the p21-activated kinase pathway is involved in
sensing the environment inside a host cell by T. marneffei, being central to eliciting the appropriate
morphogenetic response to the host environment.20 Systems for detecting and responding to changes in
carbon sources also play a major role in adaptation to the host niche and are essential factors for per-
sistence in a mammalian host. Many of the genes whose expression is upregulated during the mold-to-
yeast transition are related to those genes involved in energy metabolism. The temperature-dependent
regulation of isocitrate lyase, a key component of the glyoxylate cycle, activates the glyoxylate cycle
for utilization of poor carbon sources.16
Other potential virulence factors include yeast phase-specific or upregulated genes that encode
enzymes known to combat oxidative host defense responses. Potential virulence factors related to host-
cell attachment have also been identified.18
Proteins secreted at different developmental stages and noncoding RNAs are suggested to play diverse
roles in mycelium-to-yeast transition. RNA structural transition in response to temperature changes may
be related to the control of thermal dimorphism.21

35.2.2 Diagnosis
The diagnosis of penicilliosis has been reviewed recently.22 Patients suffering from penicilliosis may
have skin, palatal, and pharyngeal lesions such as subcutaneous abscesses, molluscum contagiosum-like
lesions and shallow papule-like ulcers. Respiratory signs include productive cough, dyspnea, and hemop-
tysis. The diagnosis is based on the identification of the organism on microscopy with the confirmation
by the culture method through examination of cytology or biopsy specimens from skin, bone marrow,
or lymph nodes.23 In fungemia, yeast cells may be seen inside monocytes in peripheral blood smear.
Detection of nonbudding yeast cells with characteristic central transverse septum should be confirmed
by isolation of T. marneffei on Sabouraud’s dextrose agar at 25°C. A diffusible dark red pigment that is
produced when grown in the mycelial phase at 37°C is one of the first indications that an isolate may be
T. marneffei.24
Diagnosis may also be made by the detection of antibodies produced against various fungal proteins,
or by the detection of fungal proteins in sera or in urine.25–27 A specific exoantigen test using the immu-
nodiffusion technique was devised to identify T. marneffei cultures.28
Also, specific tests have been developed for detecting circulating T. marneffei antigens in serum and
urine specimens by latex agglutination, enzyme immunoassay, and dot blot enzyme-linked immunosor-
bent assay (ELISA) with polyclonal antibodies; and a monoclonal antibody-based sandwich ELISA.27
Several serologic methods have also been developed for detecting specific antibodies against
T. marneffei antigens in clinical specimens. These include an indirect fluorescent-antibody test for
detecting IgG antibodies produced against germinating conidia and yeast forms, immunoblot assays for
detecting at least two protein antigens produced during the growth phase of the yeast, Western blotting
for three cytoplasmic antigens from yeast-form T. marneffei, and an ELISA-based antibody test devel-
oped with a recombinant T. marneffei mannoprotein.27
Specific oligonucleotide primers from the nuclear ribosomal DNA internal transcribed spacer
region have been designed for the specific and selective amplification of T. marneffei DNA in a
PCR-identification system from clinical material.29 Similarly, oligonucleotide probes based on the
18S rRNA gene of T. marneffei have been used to identify T. marneffei DNA from clinical samples
in PCR-hybridization reactions.27

35.2.3 M
 olecular Epidemiology
T. marneffei isolates have been classified using various methods that randomly sample for genetic varia-
tion, including restriction fragment length polymorphisms (RFLP), randomly amplified polymorphic
558 Laboratory Models for Foodborne Infections

DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) using restriction enzymes.27 A number of
types have been identified, but no correlation between restriction patterns and geographic region was
observed. On the other hand, multilocus microsatellite typing allowed discrimination between isolates
occurring within the “eastern” clade, from mainland China, Hong Kong, Indonesia, and Vietnam, or the
“western” clade, from Thailand and India.30
T. marneffei life cycle has been related to the high prevalence of infection in the organs of the bamboo
rats, including hoary bamboo rat (Rhizomys pruinosus), Chinese bamboo rat (Rhizomys sinensis), large
bamboo rat (Rhizomys sumatrensis), and bay bamboo rat (Cannomys badidus), as well as the soil around
their burrows.14 Bamboo rats are the only known nonhuman host of T. marneffei, even though these
animals showed no signs of illness.25
Bamboo rat and human coinfection can occur from a common environmental source, rather than
the patients being infected from the rats.27 However, T. marneffei has never been isolated from soil,
water, vegetation, or air, other than from bamboo rats and their burrows. Multilocus genotypes show
that T. marneffei isolates from humans are similar to those infecting rats and are in some cases identical,
­suggesting that bamboo rats can be a vector for human infections by acting as amplifiers of infectious
dispersal stages.31
The asexual nature of T. marneffei would have led to the evolution of niche-adapted genotypes, which
could explain the geographically restricted endemicity of this fungus.18

35.2.4 Treatment
Unlike some other emerging fungal pathogens, this organism remains sensitive to many antifungals.32
T. marneffei is sensitive to amphotericin B, itraconazole, and other types of triazoles. However, long-
term secondary prophylaxis with itraconazole has been recommended for HIV-infected patients to pre-
vent reoccurrence.33
However, itraconazole interacts with antiretrovirals, which is an important issue in HIV-positive
patients.34

35.3 Mycotoxins
35.3.1 General Description
The production of secondary metabolites is a characteristic of Penicillium species. Some of the extrolites
produced by Penicillium spp. are toxic (Table 35.1).
Penicillium growth on foods and feeds poses a hazard to consumers due to these toxic metabolites.
Mycotoxins produce toxic effects in different organisms, including mammals, birds, amphibians, arthro-
pods, crustaceans, unicellular organisms, microorganisms, and plants. Taking advantage of this fact,
different bioassays have been developed to study mycotoxin toxicity in foods and feeds.41
The sensitivity of bioassays is generally lower than that of chromatographic methods. However, bioas-
says do not require standards for the detection of mycotoxins. This is a major point, given that only a
small part of the over 300 compounds documented as mycotoxins are commercially available. Therefore,
analyses based on the biological action of mycotoxins allow testing various foods for the presence of
both known and unknown Penicillium mycotoxins. On the other hand, biological methods are generally
less reproducible, less sensitive, and require more time than physicochemical methods.42
The organisms used in biological methods for mycotoxins detection include microorganisms and
invertebrate and vertebrate animals. The use of mammals for toxicity research is rather limited due to
ethical o­ bjections as well as high costs for food, habitat, and animal welfare.
Organs and cell cultures have also been used. Cell lines and tissue cultures are the best alternatives
to experimental animals in testing for toxic or pathogenic microorganisms.43 Cell cultures are essential
when seeking information on the effect on DNA or DNA-binding molecules, cell organelles, or cell
membranes. But such studies are usually not necessary to detect toxicity in molds from foods. For this,
simpler and easier tests are used, such as those with microorganisms or invertebrates.
TABLE 35.1

Penicillium and Talaromyces

Mycotoxins and Potentially Toxic Extrolites Produced by Penicillium spp. and Talaromyces spp.
Penicillium spp. and Talaromyces spp. Metabolites
P. aethiopicum Griseofulvin, viridicatumtoxin
P. albocoremium Cyclopenol, cyclopenin, roquefortine C
P. allii Cyclopenol, cyclopenin, roquefortine C
P. atramentosus Roquefortine C, rugulovasine
P. aurantiogriseum Chaetoglobosin A, penicillic acid, roquefortine C, terrestric acid, verrucosidin, viomellein, xanthomegnin
P. bialowiezense Mycophenolic acid
P. brevicompactum Brevianamide A, mycophenolic acid, patulin
P. camemberti Cyclopiazonic acid
P. canescens Griseofulvin, penitrem A
P. carneum Mycophenolic acid, patulin, penicillic acid, penitrem A, roquefortine C
P. caseifulvum Cyclopenin, rugulovasine
P. chrysogenum Cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, PR-toxin, roquefortine C, secalonic acid
P. clavigerum Cyclopiazonic acid, patulin, penitrem A, xanthomegnin
P. citreonigrum Citreoviridin
P. citrinum Citreoviridin, citrinin
P. citreoviride Citreoviridin
P. commune Cyclopenin, cyclopiazonic acid, ochratoxin A, rugulovasine
P. concentricum Patulin, roquefortine C
P. confertum Asteltoxin
P. coprobium Patulin, roquefortine C
P. coprophilum Griseofulvin
P. corymbiferum Patulin, roquefortine C
P. crustosum Cyclopenol, cyclopenin, cyclopiazonic acid, penitrem A
P. cyaneo-fulvum Patulin
P. cyclopium Cyclopenol, cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, roquefortine C, rugulosin, verrucofortine, viomellein, vioxanthin,
xanthomegnin
P. dipodomyicola Cyclopiazonic acid, griseofulvin, patulin
P. discolor Cyclopenol, chaetoglobosins
(Continued)

559
TABLE 35.1 (Continued)

560
Mycotoxins and Potentially Toxic Extrolites Produced by Penicillium spp. and Talaromyces spp.
Penicillium spp. and Talaromyces spp. Metabolites
P. expansum Chaetoglobosins, citrinin, cyclopiazonic acid, patulin, penitrem A, roquefortine C
P. flavigenum Penicillin, penitrem A, roquefortine C, secalonic acids
P. formosanum Patulin
P. freii Cyclopenol, penicillic acid, xanthomegnin
T. funiculosusa Patulin
P. glandicola Patulin, penitrem A, roquefortine C
P. gladioli Patulin
P. granulatum Patulin
P. griseofulvum Cyclopiazonic acid, griseofulvin, patulin, roquefortine C
P. hirsutum Cyclopenol, cyclopenin, roquefortine C, terrestric acid
P. hordei Roquefortine C, terrestric acid
T. islandicusa Luteoskirin
P. janczewskii Griseofulvin, penitrem A
P. marinum Chaetoglobosins, patulin, roquefortine C
P. melanoconidium Penicillic acid, penitrem A, verrucosidin, xanthomegnin
P. mononematosum Fumitremorgins, isochromantoxin, verrucologen, viriditoxin

Laboratory Models for Foodborne Infections

P. nalgiovense Cyclopiazonic acid
P. neoechinulatum Cyclopenol, penicillic acid
P. nordicum Ochratoxin A, viridic acid
P. oxalicum Secalonic acids
P. palitans Cyclopenin, cyclopiazonic acid, fumigaclavine
P. paneum Patulin, roquefortine C
P. paxilli Verruculogen
P. persicinum Griseofulvin, roquefortine C
P. polonicum Cyclopenol, penicillic acid, verrucofortine. Verrucosidin
P. puberulum Cyclopiazonic acid
T. purpurogenusa Ochratoxin A, rubratoxin B
(Continued)
TABLE 35.1 (Continued)

Penicillium and Talaromyces

Mycotoxins and Potentially Toxic Extrolites Produced by Penicillium spp. and Talaromyces spp.
Penicillium spp. and Talaromyces spp. Metabolites
P. radicicola Citrinin, cyclopenol, cyclopenin, roquefortine C, terrestric acid
P. raistrickii Griseofulvin
P. roqueforti Mycophenolic acid, patulin, penicillic acid, PR-toxin, roquefortine C
P. rubrum Rubratoxin, xanthoviridicatin
T. radicusa Rugulosin, skyrin
T. rugulosusa Ochratoxin A, rugulosin
P. simplicissimum Penicillic acid, verruculogen
P. solitum Cyclopenol
P. sclerotigenum Griseofulvin, patulin, roquefortine C
P. tricolor Asteltoxin, terrestric acid, verrucofortine, xanthomegnin
P. tularense Janthitrem, paspalinine, paxilline
P. tulipae Cyclopenol, cyclopenin, penitrem A, roquefortine C, terrestric acid
P. venetum Cyclopenol, cyclopenin, roquefortine C, terrestric acid
P. verrucosum Citrinin, ochratoxin A
P. viridicatum Brevianamide A, citrinin, cyclopiazonic acid, ochratoxin A, penicillic acid, viomellein, viopurpurin, vioxanthin, viridamine, viridic acid,
viridicatin, viridicatum toxin, xanthomegnin, xanthoviridicatin
P. vulpinum Patulin, roquefortine C
T. wortmanniia Rugulosin, skyrin
Source: Adapted from Yilmaz, N. et al., Stud. Mycol., 78, 175, 2014; Moss, M.O., The environmental factors controlling mycotoxin formation, in Mycotoxins and Animal Foods, p. 37, Smith
J.F. and Henderson R.S. (Eds.), CRC Press, Boca Raton, 1991; Mantle, P.G., Miscellaneous toxigenic fungi, in Mycotoxins and Animal Foods, p. 141, Smith J.E. and Henderson R.S. (Eds.),
CRC Press, Boca Raton, 1991; Bullerman, L.B., Fusaria and toxigenic molds and other Aspergilli and Penicillia, in Food Microbiology: Fundamentals and Frontiers, p. 419, Doyle M.P.,
Beuchat L.R. and Montville T.J. (Eds.), ASM Press, Washington, 1997; Pitt, J.I., Toxigenic Penicillium species, in Food Microbiology: Fundamentals and Frontiers, p. 406, Doyle M.P.,
Beuchat L.R. and Montville T.J. (Eds.), ASM Press, Washington, 1997; Frisvad, J.C. and Samson, R.A., Stud. Mycol., 49, 1, 2004; Barkai-Golan, R., Penicillium mycotoxins, in Mycotoxins
in Fruits and Vegetables, p. 153, Barkai-Golan R. and Paster N. (Eds.), Academic Press, San Diego, 2008.
a Formerly Penicillium.2

561
562 Laboratory Models for Foodborne Infections

35.3.2 Invertebrate Organisms in Toxicity Bioassays

Invertebrate organisms have been frequently used for toxicity tests because of their ease of handling.
They do not need special care, are very prolific, and have a short generation time. These characteris-
tics improve the statistical interpretation of the results and enhance the statistical significance without
increased costs.43
Tests based on the use of aquatic animals, including crustaceans and protozoa, are among the most
extensively used in screening bioassays to detect mycotoxins (Table 35.2). The brine shrimp test using
Artemia salina, an aquatic crustacean, is perhaps one of the most extensively used screening bioassay
to detect mycotoxins. Harwig and Scott44 observed that most of the Penicillium mycotoxins showed low
toxicity to Artemia salina, with mortality rates lower than 50%, whereas rubratoxin B reached over 90%
mortality. Moina macrocopa, another fresh water crustacean used for bioassays, was more sensitive
than Artemia salina to ochratoxin A and rubratoxin B.45 Other crustaceans have been also used, such as
Daphnia magna to detect toxicity of patulin46 and Cyclops fuscus that is highly sensitive to the lethal
effects of rubratoxin B and patulin at low doses.47
Tetrahymena pyriformis is a unicellular ciliate protozoan widely used as a test organism for
Penicillium toxins. This organism has been used in toxicological bioassays because its metabolism is
quite similar to that of higher animals, as was shown in studies on the toxicity of patulin in rats and
T. pyriformis.48,49 Several researchers have used this protozoan to detect the toxic effect of Penicillium
­mycotoxins (Table 35.2). However, T. pyriformis did not detect ochratoxin A at low concentrations.54
Dive et al.53 reported that some mycotoxins were responsible for growth inhibition of the ciliated pro-
tozoan Colpidium ­campylum (Table 35.2).

TABLE 35.2
Invertebrate Organisms Used in Bioassay Tests to Detect Penicillium Mycotoxins
Organism Organism Type Mycotoxins/Mold Detected Test Type References
Artemia salina Aquatic crustacean Patulin, citrinin, griseofulvin, Disc screening 44
penicillic acid, rubratoxin B method
Moina macrocopa Aquatic crustacean Ochratoxin A, rubratoxin B Mortality 45
Daphnia magna Aquatic crustacean Patulin Bioluminescent test 46
Cyclops fuscus Aquatic crustacean Rubratoxin B, patulin Mortality 47
Tetrahymena pyriformis Protozoan Patulin Mortality 48–52
Penicillic acid, gliotoxin
Rubratoxin B
Patulin
Colpidium campylum Protozoan Patulin, ochratoxin B, roquefortin Mortality 53
Anagasta kuehniella Insects Penicillium spp. and Penicillium Mortality 41
Attagenus megatoma mycotoxins Growth inhibition
Corcyra cephalonica Reduced larval
Drosophila melanogaster growth

Glycyphagus domesticus Decrease in larva

Heliothis zea and adult size

Lasioderma serricorne Fertility

Lucilia sericata
Spodoptera frugiperda
Spodoptera littoralis
Tenebrio molitor
Tribolium confusum
Penicillium and Talaromyces 563

Other invertebrate animals used in toxicological bioassays are insects. Panigrahi41 reviewed the tests
using insects to detect the toxicity of mycotoxins produced by Penicillium spp. These tests are based on
the effect of feeds intentionally contaminated with mycotoxins on growth or mortality of adults or larvae
(Table 35.2).

35.3.3 Microorganisms in Toxicity Bioassays

The use of microorganisms to detect the toxicity of different species of Penicillium involves different
tests (Table 35.3). Working with microorganisms is simple and usually faster and less expensive than
using cell cultures or animals. Microorganisms have been used for quite some time to detect mycotox-
ins42,55 and toxic effects from Penicillium spp.56
Tests based on Bacillus thuringiensis growth inhibition were optimized to detect low concentrations
of Penicillium mycotoxins.57,58 Watson and Lindsay42 reviewed the use of Bacillus spp. to detect the toxic
effect of certain mycotoxins, including some produced by Penicillium spp. Skaug et al.59 used Bacillus
subtilis to detect toxicity in conidial extracts from Penicillium verrucosum containing ochratoxin A.

TABLE 35.3
Microorganisms Used in Bioassay Tests to Detect Penicillium Mycotoxins
Organism
Organism Type Mycotoxins Detected Test Type References
Azospirillum Motile Patulin, rubratoxin, penicillic acid, Swarming inhibition 57
brasilense bacteria citrinin assay
Bacillus thuringiensis Bacteria Kojic acid Cup plate assay 57,58
Citrinin, kojic acid, luteoskyrin, Cup plate assay
ochratoxin A, patulin, penicillic acid
Bacillus subtilis Bacteria Ochratoxin A Growth inhibition 59
Patulin, rubratoxin, penicillic acid,
citrinin
Bacillus cereus Bacteria Ochratoxin A 42
Bacillus megaterium Bacteria Patulin, ochratoxin A
Escherichia coli Bacteria Patulin, penicillic acid, ochratoxin A, Genotoxicity in the 60,61
rubratoxin B, kojic acid, citrinin, PR SOS spot test
toxin
Ochratoxin A
Photobacterium Bacteria Rubratoxin B, penicillic acid, citrinin, Bioluminescence 62
phosphoreum ochratoxin A, PR-toxin, patulin assay
Proteus mirabilis Motile Patulin, rubratoxin, penicillic acid, Swarming inhibition 57
bacteria citrinin assay
Pseudomonas Bacteria Patulin Growth inhibition 52
syringae
Saccharomyces Yeast Rubratoxin B, penicillic acid, citrinin, Mutagenicity test 62
cerevisiae ochratoxin A, PR-toxin, patulin
Salmonella Bacteria Ochratoxin A Ames test 61,63,64,65,66
typhimurium Patulin (mutagenicity)

Ochratoxin A, B, citrinin
Citrinin, patulin, cyclopiazonic acid,
luteoskyrin, griseofulvin,
mycophenolic acid, ochratoxin A,
penicillic acid, secalonic acid D
Several Kojic acid Growth inhibition 67
microorganisms
564 Laboratory Models for Foodborne Infections

Another bioassay with bacteria is based on the motility inhibition of Proteus mirabilis and Azospirillum
brasilense caused by some Penicillium toxins.57 Escherichia coli is widely used in bioassays for toxic-
ity and for genotoxic or mutagenic activity detection. Auffray and Boutibones60 evaluated the genotoxic
activity of some mycotoxins produced by strains of Penicillium using Escherichia coli in the SOS spot
test. When comparing the efficiency to detect toxigenic activity between SOS spot test and other tests,
including mutagenicity to Salmonella typhimurium (Ames test), Bacillus subtilis (Rec assay), and in vivo
carcinogenicity, similar results were obtained for ochratoxin A and rubratoxin B, but different results
were observed for patulin, penicillic acid, kojic acid, citrinin, and PR toxin. Tests with microorganisms
have also been used to evidence the toxicity of kojic acid,67 but mutagenicity could only be evidenced at
high concentrations of the mycotoxin.
The Ames test is a classic assay used to assess the mutagenic potential of molds or its metabolites that
produce genetic damage leading to gene mutations.68 The test uses a number of histidine-auxotrophic
Salmonella typhimurium strains carrying different point mutations in genes of the histidine operon.
These mutations can revert to wild type when a mutagen is present. Many strains of Penicillium or their
mycotoxins led to negative results in Ames test, even when rat liver S9 mix was used as an external
metabolizing enzyme system or when a preincubation of toxins in cultured rat hepatocytes was per-
formed. This enzyme treatment is necessary for some compounds to be mutagenic because they need a
metabolic liver transformation that bacterial systems do not possess. Ochratoxin A showed no evidence
of mutagenic activity by SOS spot and Ames tests,61 in contrast to the results obtained with more sensi-
tive bioassays, such as cell cultures or experimental animals, as discussed later.
Penicillium toxins were tested using the bioassay based on the bacterial bioluminescence of
Photobacterium phosphoreum,62 which is a reliable short-term method for assessing the toxicity of
mycotoxins. The order of toxicity determined by bacterial bioluminescence parallels that reported for
mammalian cell cultures.
Growth of Pseudomonas syringae in presence of patulin was evaluated measuring optical density of
culture broth. The sensitivity of this microorganism to the mycotoxin was even higher than that obtained
with rats.52

35.3.4 Vertebrate Animals in Toxicity Bioassays

Different vertebrate animals can be used to test the toxicity of mycotoxins from Penicillium spp. (Table 35.4).
Tests with vertebrate animals to assay mycotoxins toxicity require careful consideration of various factors.41
The different animal species have different sensitivity to mycotoxins. Age is another important factor in bioas-
says, with younger animals being usually more susceptible. Males are generally less resistant to mycotoxins
than females. The route of administration also affects the toxicity observed. Lower mycotoxin doses are
needed when the route of administration is intraperitoneal instead of oral. The nutritional and health sta-
tus of animals may influence toxicity symptoms, with high-protein diets having a protective effect against
mycotoxins.
A classic animal toxicity test is the chicken embryos bioassay, which is very sensitive to numerous
mycotoxins from Penicillium strains.67,72 After the administration of samples into the air sac of eggs,
toxicological effects are detected by observing death and teratogenic abnormalities.
Rats and mice have also been used to evaluate ochratoxin A carcinogenicity.61,69,70 The sensitivity
to ochratoxin A carcinogenicity is higher in rodents (higher in rats than in mice, and in males than in
females). Effects of kojic acid in mammals have been reviewed by Burdock et al.67 Both rodents and dogs
showed convulsions and died in few hours.
The carcinogenic effect of ochratoxin A has been assessed in studies on chicks. Degenerative changes
in liver and kidney were detected after exposure of chicks to this mycotoxin.71
Zebrafish (Danio rerio) succeeded as a vertebrate animal model because of its high developmental
similarity to mammals. The larvae and embryo of this fish have been used in toxicity bioassays for
Penicillium mycotoxins. Larvae proved to be sensitive to ochratoxin A and patulin, but not to penicillic
acid.73 Zebrafish embryos revealed profound nephrotoxicity in histological structure and biological func-
tion after citrinin and patulin exposure, as well as a severe impact on heart development affecting heart
morphogenesis after citrinin exposure.74,75
Penicillium and Talaromyces 565

TABLE 35.4
Vertebrate Animals Used in Several Bioassay Tests to Detect Penicillium Mycotoxins
Organism
Organism Type Mycotoxins Test Type References
Dog Mammals Kojic acid Rate of death 67
Rats and mice Mammals Ochratoxin A Kidney and liver lesions 61,69,70
Kojic acid Rate of death 67
Chicks Birds Ochratoxin A Kidney and liver lesions 71
Chicken embryos Birds Mycotoxins from Rate of death 67,72
Penicillium spp.
Kojic acid Teratogenic abnormalities
Zebrafish Fish Ochratoxin A, Toxicity in larvae 73–75
(Danio rerio) patulin,
penicillic acid
Citrinin, patulin Nephrotoxicity in embryos
Patulin Cardiotoxicity in embryos

35.3.5 Cell Cultures in Toxicity Bioassays

Several mammalian cell lines have been employed to evaluate the ability of some Penicillium
­mycotoxins to induce cytotoxicity and DNA damage (Table 35.5). The neutral red (NR) and the tet-
razolium (MTT) assays are two in vitro cytotoxicity tests that use colorimetric measurements for the
quantification of viable cells after incubation with mycotoxins.76 The NR assay is based on the capture
and accumulation of the neutral red dye in the lysosomes of uninjured living cells. The MTT assay is
based on the reduction of the soluble yellow MTT tetrazolium salt to a blue insoluble MTT formazan
product by mitochondrial succinic dehydrogenase of uninjured living cells. These tests have been
developed with different types of cell lines, such as fibroblasts, liver cells, keratinocytes, neural cells,
corneal epithelium, and tumoral cells.
The sister chromatid exchange method (SCE) is based on the differential staining of sister chromatids
during replication and permits the direct visualization of genetic exchanges between sister c­ hromatids.86
The generation of SCE depends on homologous recombination that increases on DNA exposure to
­damaging agents like mycotoxins.
The in vitro micronucleus MN assay is a robust quantitative assay of chromosome damage and
­discriminates between once-divided cells, which are accumulated and recognized by their binucleated
appearance, and mononucleated cells, which did not divide during the in vitro culturing period.87
The comet assay (single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks
in eukaryotic cells.88

35.3.6 Culture Media for Mycotoxin Production

Growth of toxigenic Penicillium spp. in culture media can result in the production of various toxic
metabolites. Different substrates and culture conditions have been tested to produce mycotoxins in higher
amounts than in foods. Dombrink-Kurtzman and Blackburn89 evaluated six different culture media, both
with and without manganese supplementation, to determine maximum patulin production by eleven
Penicillium species. For most strains, potato dextrose broth supplemented with manganese was optimal
for maximum patulin production.
Kokkonen et al.90 evaluated the effect of substrate on mycotoxin production of selected Penicillium
strains using yeast extract sucrose (YES) agar, as well as cheese and bread analogues media. The compo-
sition of the substrates was adequate to support the growth of Penicillium but not mycotoxin production
in all cases. Cheese analog was not favorable for mycotoxin production by P. verrucosum, but favored
roquerfortin C by P. crustosum. However, P. nordicum synthesized ochratoxin A on all three media.
566 Laboratory Models for Foodborne Infections

TABLE 35.5
Cell Lines Used in Bioassay Tests to Study Penicillium Mycotoxins
Mycotoxins
Cell lines Cell Type Detected Test Type References
CHO-K1-BH4 Chinese hamster ovary cells Ochratoxin A Cytotoxicity (in vitro 77
micronucleus MN and Comet
assays)
TK6 Human lymphoblastoid cells DNA damage
CHO-K1 Chinese hamster ovary cells Patulin Cytotoxicity (NR and MTT 78
assays)
Vero Green monkey kidney cells Ochratoxin A, citrinin Cytotoxicity (MTT assay) 79
and DNA fragmentation
A-549 Human lung cancer cells Sterigmatocystin, Cytotoxicity (NR assay) 80
Hep-G2 Human hepatocellular verruculogen,
carcinoma cells roquefortine C,
L-929 Murine fibroblasts penitrem A,
mycophenolic acid
Neuro-2a Murine neuroblastoma cells
Hep G2 Human hepatocellular Ochratoxin A Cytotoxicity (MTT assay) 81,82
carcinoma cells Patulin Comet assay
LLC-PK1 Porcine renal cells Ochratoxin A and B, Cytotoxicity (MTT assay) 83
patulin, citrinin
CHOK1 Chinese hamster ovary cells Patulin, citrinin Sister chromatid exchange test 84
HEK293 Human embryonic kidney Cytotoxicity (MTT assay)
cells
V79 cells Chinese hamster lung Ochratoxin A, citrinin Cytotoxicity (NR assay and 85
fibroblasts cells In vitro micronucleus MN
assays)

Rao et al.91 evaluated the effect of 19 synthetic and flour media of several Penicillium species in the
ochratoxin A production. A rice-flour-based medium supported maximum amount of OTA production.
OTA production was stimulated by addition of Zn2+ and Mg2+ to the medium. However, when Fe3+ was
added, OTA production was suppressed.

35.4 Allergens
35.4.1  Penicillium Allergies
Penicillium is among the four major genera of allergenic fungi, and its species are commonly isolated
from indoor environments.92 Some Penicillium species pose a respiratory health hazard in susceptible
populations, and the increasing exacerbation of current asthma symptoms has been associated with
increased levels of Penicillium and other fungal genera.93 According to allergen database of the World
Health Organization and International Union of Immunological Societies94 five Penicillium species pro-
duce a total of 17 allergens (Table 35.6), including proteases, ribosomal proteins, membrane proteins,
enolases, and heat shock proteins. Moreover, some fungal volatile metabolites could act as irritants in
susceptible individuals.95
Penicillia are common in a wide variety of food, mainly those of low and intermediate water activ-
ity, such as dry-cured ham, dry sausages, and ripened cheese. Also, airborne spores can be found in all
environments where these foods are manufactured, handled, or stored. Some operations, such as brush-
ing and coating, spread a high amount of Penicillium spores into the air, which may cause occupational
respiratory diseases or urticaria to food industry workers, including those at pork butcheries,96–99 and
cheese factories.100 The most frequent problems associated with fungal allergy are respiratory disorders,
Penicillium and Talaromyces 567

TABLE 35.6
Allergens from Penicillium Species According to WHO/IUIS Allergen Standardization Committee
Species Allergen Biochemical Name
Penicillium brevicompactum Pen b 13 Alkaline serine protease
Pen b 26 Acidic ribosomal protein P1
Penicillium chrysogenum Pen ch 13 Alkaline serine protease
Pen ch 18 Vacuolar serine protease
Pen ch 20 N-acetyl-glucosaminidase
Pen ch 31 Calreticulin
Pen ch 33 16 kDa allergen
Pen ch 35 Transaldolase
Penicillium citrinum Pen c 3 Peroxisomal membrane protein
Pen c 13 Alkaline serine protease
Pen c 19 Heat shock protein Hsp70
Pen c 22 Enolase
Pen c 24 Elongation factor 1 β
Pen c 30 Catalase
Pen c 32 Pectate lyase
Penicillium crustosum Pen cr 26 60S acidic ribosomal phosphoprotein P1
Penicillium oxalicum Pen o 18 Vacuolar serine protease
Source: WHO/IUIS, Allergen nomenclature. http://www.allergen.org/index.php

TABLE 35.7
Penicillium Species Causing Food Allergies
Species Food References
Penicillium camemberti Cheese 102
Penicillium chrysogenum Dry-sausage 103
Penicillium italicum Dry-sausage and cheese 104
Penicillium spp. Dry-sausage 101,105

including rhinitis and asthma, atopic dermatitis, allergic bronchopulmonary mycosis, allergic fungal
sinusitis, and hypersensitivity pneumonitis.92
Several Penicillium species have also been considered as causes of food allergy, and this occurs on
intake of dry-ripened foods coated with molds, such as dry-fermented sausages and cheese (Table 35.7).
These food allergies can cause facial angioedema, allergic rhinitis, urticaria, oropharyngeal pruritus, or
anaphylactic shock immediately upon ingestion.101–105
In addition, mold-ripened foods can be colonized by Penicillium species such as P. chrysogenum,
P. nalgiovense, P. dipodomis, P. griseofulvum, and P. flavigenum that are well-known penicillin produc-
ers. Small amounts of penicillin in foods have been proposed as a potential causative factor in chronic
urticarial reactions,106 and it can also provoke allergic reaction in penicillin-sensitive individuals.107,108
Sensitivity to mold allergens in individuals can be studied using skin prick and intradermal testing or
in vitro testing for specific IgE antibodies with fungal extracts.92 However, to assess the potential aller-
genic effect of molds, some laboratorial models have been used.

35.4.2 Animal Models to Study Allergies Caused by Penicillium spp.

Animal models, mainly mice, have been employed to study allergenic potential and other different
aspects related with Penicillium allergies (Table 35.8). The use of these models allows studying the effect
of fungal inhalation in airways and its correlation with lung inflammation or allergic airway responses,
such as rhinitis and asthma. Data on allergenicity of fungi and the organic effects can be obtained by
Penicillium and Talaromyces 569

provoking the responses in animals. The main responses evaluated include eosinophil, ­specific IgE and
IgG antibodies, interferon, and interleukins (ILs) levels; bronchoalveolar and lung lavage fluid produc-
tion; and histopathological lesions. Animal assays have also aided in the characterization of the cell
receptors involved in the immune recognition of fungi.109
In addition to mold species, animal strains, dose, and exposure method are important factors affecting
allergic responses. Animal strains have different ability to respond to fungal allergens depending to their
genetic susceptibility. It has been reported that BALB/c mice exhibited stronger inflammatory response
to Stachybotrys chartarum than other congenic mouse strains, such as C3H/HeJ and C56BL/6.120 In con-
trast, intranasal instillation of viable P. chrysogenum conidia increased serum IgE levels in C57 Black/6
mice but not in BALB/c mice.117
Different exposure and sensitization techniques can be used, including inhalation by aerosol of fungal
spores or extracts in suspension, involuntary aspiration, intranasal instillation, and intratracheal instilla-
tion. The advantages and disadvantages of each type of method with regard to modelling fungal exposure
and allergic sensitization in human have been reviewed by Templeton et al.109 Involuntary aspiration and
intranasal or intratracheal instillation are reproducible and allow for a homogenous suspension of fungi to
be administered, but they are not representative of natural exposure. With aerosol, animals are exposed by
normal breathing, but lung exposure concentration is unknown, and its reproducibility is lower.109
Animal studies have demonstrated the ability of Penicillium spp. to provoke allergic and asthma
response. Most of these studies involve acute or chronic exposure of animals to conidia, spore extracts,
hyphae, subcellular fragments, or fungal proteins from P. chrysogenum.
Female C57 Black/6 mouse model was established to examine the in vivo effect in allergic pro-
cesses of different levels of P. chrysogenum viable and nonviable conidia by nasal instillation.114,116,117
The acute instillation of viable P. chrysogenum conidia induced inflammatory reactions in a dose-
dependent manner, while the instillation of nonviable conidia did not.116 Low levels of P. chrysoge-
num conidia (102 spores) neither provoked lung inflammation nor increased serum immunoglobulins.114
About 18% of the viable conidia instilled intranasally were deposited and retained in the lungs and
could produce substances that induce allergic reactions.116 Viable conidia could remain in the lungs long
enough to produce an unknown protein responsible for the chronic Th2-mediated airway inflammation
by increasing total and specific IgE and IgG1, bronchoalveolar lavage fluid levels of IL-4 and IL-5, and
eosinophilia.117 In contrast, repeated exposures to nonviable conidia of P. chrysogenum induced type
Th1 helper responses in mice, with increases in total serum IgG2a and levels of interferon (IFN)-γ in
bronchoalveolar lavage fluid.117
The allergic and inflammatory response of BALB/c mice to mycelium and spores showed differences
in the threshold dose for allergy induction among species.113 These authors classified molds accord-
ing to their allergenic potential: G1 molds induced low-to-moderate responses requiring higher doses
than house dust mite; G2 molds, including P. chrysogenum, required lower doses to induce a similar
response.113 In this sense, the allergic and inflammatory response of BALB/c mice to intratracheal aspi-
ration of mycelium and spores of P. chrysogenum was more robust than that produced by house dust
mite.111 Therefore, molds must be evaluated individually for allergic or asthmatic potential.
Some experiments have been performed to study the response of animals to different extract or com-
pounds from P. chrysogenum. A dose-dependent allergic asthma-like response was observed after
involuntary aspiration of soluble components of P. chrysogenum by BALB/c mice.110 There are some
differences in the effect between single and multiple exposures to high doses. While single exposure
resulted in edema and cellular damage but not immune responses, multiple exposures showed increased
allergen-triggered immediate respiratory responses.110
C57 Black/6 mice have been used to study the effect of allergen protease extract Pen ch released by
viable P. chrysogenum conidia.114,115 Mice previously sensitized with Pen ch or viable P. chrysogenum
conidia by intraperitoneal injections and exposed to intranasal challenge showed increased allergic air-
way inflammation.114 In these mice, significant increases in serum IgE and IgG1, eosinophilia, and mucus
hyperproduction in bronchoalveolar lavage and lung tissue was recorded.114 Furthermore, intraperitoneal
sensitization and posterior instillation of Pen ch produce a strong allergic inflammatory response charac-
terized by increasing serum IgE and IgG1, eosinophils and neutrophils counts in bronchoalveolar lavage,
as well as mucous production and perivascular inflammation by eosinophils and neutrophils in lungs.115
570 Laboratory Models for Foodborne Infections

Guinea pigs have been used for experimental induction of hypersensitivity pneumonitis by using
aerosol inhalation for 12 weeks of a glycoprotein from P. chrysogenum (formerly P. notatum).119 In
exposed animals, specific serum IgM, IgG, and IgE antibodies and sensitized leukotriene CD4 cells were
detected. Moreover, interstitial infiltrates of macrophages and leukotriene cells, cellular bronchiolitis,
and single nonnecrotizing granulomas were observed in lungs. This response is a typical delayed-type
reaction due to chronic contact with the heterologous glycoprotein of Penicillium.119
Swiss Webster Carworth Farms white (CFW) male mice were used to elucidate the role of bioactive
constituents of spores from P. brevicompactum and P. chrysogenum that mediate allergenic responses.118
Inflammatory and cytotoxic responses to intratracheal instillation of brevianamide A and mycophenolic
acid from P. brevicompactum, and roquefortine C from P. chrysogenum were investigated. High doses
of these three metabolites induced a dose-dependent-like inflammatory response expressed as increased
macrophage, neutrophil, MIP-2, TNF, and IL-6 concentrations in the bronchoalveolar lavage fluid in
exposed mice. The analysis of this fluid revealed some compound-specific toxic responses: brevianamide
A and mycophenolic acid provoked vascular leakage according to albumin concentration, and brevian-
amide A induced cytotoxicity, increasing lactate dehydrogenase (LDH) concentration.118
BALB/c mice have also been used to study the effect of intratracheal inoculation of Pen c 13 protease
from P. citrinum. An increase in airway hyperresponsiveness, inflammatory cell infiltration, mucus over-
production, and collagen deposition in the lung, as well as serum levels of total IgE and Pen c 13-specific
IgE and IgG1 were observed.112 Moreover, the exposure to Pen c 13 provoked changes in lung proteome,
with increase of proteins involved in leukocyte extravasation signalling, oxidative stress response, and
actin cytoskeleton organization, and also decrease of vinculin, a junctional protein between cells.112
Therefore, Pen c 13 exposure causes structure alterations and actin cytoskeletal rearrangements, result-
ing in increased permeability and airway structural changes.112

35.4.3 Cell Cultures to Study Allergies Caused by Penicillium

Several cell cultures have been used to study the effect of allergens from Penicillium spp. The effects of
the Pen ch 13 allergen from P. chrysogenum on respiratory epithelial cells have been demonstrated on
A549 cells, a human alveolar type II epithelium-like cell line; 16HBE14o-, an immortalized bronchial
epithelial cell line; and HBEpC, primary cells derived from normal human bronchi.121 Pen ch 13 induced
in all three cell cultures the secretion of mediators linked to local immune responses and inflammatory
process such as prostaglandin-E2 (PGE2), IL-8, and transforming growth factor (TGF)-β1. Additionally,
Pen ch 13 degraded the protein occludin of cultured 16HBE14o-, contributing to disruption of the tight
junctions, damaging the barrier formed by the airway epithelium.121
Spleen cell culture obtained from BALB/c mice as well as NCI-H441cell line derived from human
lung epithelial adenocarcinoma have been used to study the effect of Pen c 13 from P. citrinum. The in
vitro stimulation of splenocytes with Pen c 13 resulted in an immunologic response by increasing pro-
duction of Th2 cytokines IL-4, IL-5, and IL-13.112
NCI-H441 cell line was used to mimic the effect of Pen c 13 on pulmonary epithelial barrier integ-
rity. This antigen produced disruption of tight junctions integrity and increased permeability, which
can be associated with the loss of pulmonary epithelium integrity observed in Pen c 13-sensitized
BALB/c mice.112

35.5 Conclusions
Given that penicillia only show a limited invasive ability for animal tissue, they are mainly soil sapro-
phytes or plant pathogens, being responsible for mycotoxin production and postharvest rotting of fruits
and vegetables. However, Penicillium and Talaromyces spp. have been described as rare causative agents
of infectious diseases in humans, dogs, and fish, as well as environmental allergens among asthmatic
and atopic individuals. T. marneffei, the only dimorphic species of this group, is capable of growing
as a uninucleate pathogenic yeast at 37°C upon invasion of a human host, leading to localized reac-
tions in persons with normal immunity, while causing fatal systemic mycosis in immunocompromised
Penicillium and Talaromyces 571

patients. Diagnosis of penicilliosis is based on the confirmation by culture methods through specimen
­examination, detection of antibodies produced against various fungal proteins, the detection of fungal
proteins in sera or in urine, or by selective amplification of T. marneffei DNA. Bamboo rats are the only
known nonhuman host of T. marneffei, but these animals show no signs of illness. Therefore, laboratory
models to study the pathogenicity of T. marneffei are extremely limited.
In contrast to pathogenic penicillia, mycotoxins produce toxic effects in various organisms, allow-
ing the use of different bioassays to study the toxicity of such secondary metabolites. Vertebrate ani-
mals have been used to test the toxicity of Penicillium mycotoxins. Tests with microorganisms and
invertebrates are preferred to the use of vertebrates due to ethical objections and high maintenance
costs. Organs and cell cultures have also been used, particularly when obtaining information on the
effect on DNA, cell organelles, or cell membranes. In addition, different culture media have been
tested to study the ability and efficiency of Penicillium spp. to produce mycotoxins, including food
analogues media.
Some Penicillium species produce allergens that pose a respiratory health hazard to susceptible indi-
viduals. Airborne spores from dry-ripened foods may cause food allergies or occupational respiratory
diseases to food industry workers. Sensitivity to penicillia allergens can be studied using common tests
with fungal extracts, but rodents and cell cultures are required to assess organic effects, such as eosino-
phil, specific IgE and IgG antibodies, interferon, and ILs levels; lung lavage fluids production; and his-
topathological lesions. The genetic susceptibility of the different animal strains to allergens makes it
necessary to select the strain exhibiting the adequate response to the mold, according to the exposure
and sensitization techniques used. Different types of respiratory epithelial cell cultures have been used
to study the effect of allergens form Penicillium spp. on secretion of mediators linked to local immune
responses. However, the use of cell cultures is otherwise rather limited.

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 Section V

Foodborne Infections due to Protozoa

36
Acanthamoeba

Dongyou Liu

CONTENTS
36.1 Introduction................................................................................................................................... 579
36.1.1 Classification, Morphology, and Genomics..................................................................... 580
36.1.1.1 Classification..................................................................................................... 580
36.1.1.2 Morphology....................................................................................................... 580
36.1.1.3 Genomics...........................................................................................................581
36.1.2 Life Cycle and Epidemiology........................................................................................... 582
36.1.2.1 Life Cycle.......................................................................................................... 582
36.1.2.2 Epidemiology.................................................................................................... 582
36.1.3 Clinical Features and Pathogenesis.................................................................................. 583
36.1.3.1 Amoebic (Acanthamoebic) Keratitis................................................................. 583
36.1.3.2 Granulomatous Amoebic Encephalitis............................................................. 583
36.1.3.3 Cutaneous Acanthamoebiasis and Sinusitis..................................................... 583
36.1.4 Diagnosis.......................................................................................................................... 584
36.1.5 Treatment and Prevention................................................................................................. 585
36.2 Laboratory Models........................................................................................................................ 585
36.2.1 Animal Models................................................................................................................. 585
36.2.1.1 Mice.................................................................................................................. 585
36.2.1.2 Rats................................................................................................................... 585
36.2.1.3 Chinese Hamsters............................................................................................. 586
36.2.1.4 Rabbits.............................................................................................................. 586
36.2.1.5 Pigs.................................................................................................................... 586
36.2.2 In Vitro Models................................................................................................................. 586
36.3 Conclusion..................................................................................................................................... 586
References............................................................................................................................................... 586

36.1 Introduction
Acanthamoeba is a unicellular organism that was first detected by Castellani from Cryptococcus
­pararoseus cultures. As a free-living bacterivore, Acanthamoeba is present in a diversity of
­environments, including soil, dust, water, air, plants, animals, and humans. However, Acanthamoeba
has the ability to take advantage of temporary weakness (trauma or suppressed immune function) in
human host, causing keratitis, encephalitis, and skin infections, often with severe consequences. In
view of the limited treatment options for Acanthamoeba infections at present, there is a need to improve
our understanding of the pathogenicity of this devastating disease, and to devise novel, highly effective
intervention strategies. Utilization of laboratory models to Acanthamoeba infections will be undoubt-
edly beneficial in this regard.

579
580 Laboratory Models for Foodborne Infections

36.1.1 Classification, Morphology, and Genomics

36.1.1.1 Classification
The genus Acanthamoeba is classified in the family Acanthamoebidae, order Centramoebida, sub-
class Longamoebia, class Discosea, kingdom Amoebozoa, domain Eukaryota, kingdom Animalia.
Beside Acanthamoeba, the family Acanthamoebidae contains two other genera (Comandonia and
Protacanthamoeba). Another former genus (Balamuthia) of the family Acanthamoebidae, order
Centramoebida, has been moved to a newly created family Balamuthiidae, order Centramoebida.
Currently, the genus Acanthamoeba consists of at least 25 named species [i.e., A. astronyxis (T7),
A  byersi (T18), A. castellanii (T4), A. comandoni (T9), A. culbertsoni (T10), A. divionensis (T4), A  griffini
(T3), A. hatchetti (T11), A. healyi (T12), A. jacobsi (T15), A. lenticulata (T3), A. lugdunensis (T4), A. mau-
ritaniensis (T4), A. palestinensis (T2), A. paradivionensis (T4), A. pearcei (T3), A. polyphaga (T4), A.
pustulosa (T2), A. quina (T11), A. rhysodes (T4), A. royreba (T4), A. stevensoni (T11), A. terricola (T4),
A. triangularis (T4), and A. tubiashi (T8)] in addition to many unassigned species/isolates [1–3].
On the basis of their morphological features, members of the genus Acanthamoeba are placed into
three groups (designated Groups I, II, and III). Acanthamoeba Group I consists of five species [e.g.,
A. astronyxis (T7), A. tubiashi (T8), A. comondani (T9)], which produce large trophozoites of 25–35 μm
and cysts (with distinctly stellate endocysts and smooth spherical ectocysts) of >18 μm in size, with cysts
that either have four or less arms or more than six arms. This group is mostly environmental and not
convincingly associated with infections in humans or animals. Acanthamoeba Group II comprises 10
species such as A. castellanii (T4), A. griffinii (T3), A. hatchetti (T11), A. polyphaga, and A. stevensoni
(T11), which produce cysts (with polygonal to stellate endocysts and irregular or wrinkled ectocysts) of
<18 μm in size with rounded arms. This group is responsible for the majority of reported human infec-
tions [e.g., amebic keratitis (AK), granulomatous amebic encephalitis (GAE), cutaneous acanthamoe-
biasis, and sinusitis]. Acanthamoeba Group III comprises species A. culbertsoni (T10), A. healyi (T12),
A. palestinensis (T1), A. pustulosa (T2), and A. lenticulata (T5), which possess cysts of <15 μm in size
(smaller than those of Group II) with three to five points. A. culbertsoni from Group III is a recognized
pathogen causing both keratitis and encephalitis [2].
Sequencing analysis of the nuclear small-subunit (18S) rRNA gene indicates that members of the genus
Acanthamoeba may be distinguished into 18 genotypes (T-type 1–18, or T1–18), with different geno-
types exhibiting 5% or more sequence divergence (Table 36.1) [3,4]. However, some genotypes may form
clusters (i.e., T4/T3/T11, T2/T6, T10/T12/T14, T7/T8/T9/T17, and T13/T16) in this analysis, and careful
examination of the sequence data is essential for correct identification. In addition, as A. divionensis,
A. mauritaniensis, and A. paradivionensis display 18S rRNA gene sequence differences of 0.2%–1.1% to
A. rhysodes and with each other, they may be regarded as synonyms of A. rhysodes. Furthermore, recent
18S rRNA gene sequence analysis suggests that some T16 isolates may form distinct, novel genotypes
(T19 and T20) within the genus Acanthamoeba [3,5]. Although T4 isolates have accounted for a majority
of clinical cases of AK and GAE, isolates belonging to other genotypes (e.g., T1, T2, T3, T5, T6, T10,
T11, T12, T15, and T18) have also been implicated in human infections (Table 36.1) [6–11].

36.1.1.2 Morphology
Acanthamoeba trophozoites (usually <30 μm, rarely >65 μm in size) are amoeboid in shape (or wide
and tongue-shaped), with irregular margins and short and fine pseudopodia (or acanthopodia; Greek
“acanth” means “spikes”). Continuously forming and reabsorbing, acanthopodia (formed by bundles of
actin microfilaments extending as rigid cores) protrude from every area of the body’s surface, contribut-
ing to Acanthamoeba locomotion. However, in A. astronyxis and A. comandoni, the acanthopodia may
be quite long.
Under adverse environmental conditions (e.g., food deprivation, desiccation, and changes in tempera-
ture and pH), Acanthamoeba forms a double-walled wrinkled cyst (of 13–20 μm in size). The outer
wall is made up of proteins and polysaccharides, and the inner wall contains cellulose, with the two
walls being separated by a space forming the so-called ectocyst and endocyst. Cysts are resistant to
Acanthamoeba 581

TABLE 36.1
Correlation of Acanthamoeba T-Types with Clinical Diseases
T-Type Species Clinical Disease
T1 Acanthamoeba sp. Encephalitis
T2 A. palestinensis, A. pustulosa Keratitis, encephalitis, sinusitis
T3 A. griffini, A. pearcei Keratitis
T4 A. castellanii, A. divionensis, A. lugdunensis, A. Keratitis, encephalitis
­mauritaniensis, A. polyphaga, A.quina, A. rhysodes
T5 A. lenticulata Keratitis, encephalitis
T6 Acanthamoeba sp. Keratitis
T7 A. astronyxis Unknown
T8 A. tubiashi Unknown
T9 A. comandoni Unknown
T10 A. culbertsoni, Acanthamoeba sp. Keratitis, encephalitis
T11 A. hatchetti, A. stevensoni Keratitis
T12 A. healyi Encephalitis
T13 Acanthamoeba sp. Unknown
T14 Acanthamoeba sp. Unknown
T15 A. jacobsi Keratitis
T16 Acanthamoeba sp. Unknown
T17 Acanthamoeba sp. Unknown
T18 A. byersi Encephalitis, skin infection
Sources: Crary, M.J. Genetic variability and its relationship to Acanthamoeba pathogenesis. Thesis,
The Ohio State University, 2012; Sente, C., et al., Parasite Vectors, 9(1), 127, 2016.

biocides, chlorination, and antibiotics and survive low temperatures (0°C–2°C). There is evidence
that Acanthamoeba cysts remain viable for more than 24 years after storage in water at 4°C and sus-
tain desiccation for more than 20 years. However, Acanthamoeba cysts are sensitive to treatment with
Freon or methylene oxide or autoclaving. When condition improves [e.g., inoculation on nonnutrient
agar (NNA) containing bacteria], excystment takes place with trophozoites emerging from the cyst. As
Acanthamoeba cyst formation (encystment) and excystation may be induced using nonnutrient media,
Acanthamoeba offers a valuable tool to study cellular differentiation [12].
Electron microscopy examination reveals the presence of a Golgi complex, smooth and rough endo-
plasmic reticula, free ribosomes, digestive vacuoles, mitochondria, and microtubules in Acanthamoeba
trophozoites. Apart from the existence of a trilaminar plasma membrane surrounding the cytoplasmic
contents, characteristic spiny surface projections (i.e., acanthopodia) are observable. Within the cyto-
plasm are one or more prominent contractile vacuoles with osmotic function, and a nucleus (with a large
central nucleolus) that is approximately one-sixth the size of trophozoite. Other notable types of vacuoles
in the cytoplasm include lysosomes, digestive vacuoles, and glycogen-containing vacuoles [2].

36.1.1.3 Genomics
From the sequence data of 14 Acanthamoeba species available in GenBank, it is clear that
Acanthamoeba genomes vary from 42.02 to 120.42 Mb in size, with GC contents ranging from 42.7%
to 59.3% (Table 36.2). Interestingly, A. castellanii, a solitary free-living amoebozoan of T4 ­genotype,
possesses a genome of 42.02 Mb including 15,455 compact intron-rich genes. Besides encoding vari-
ous tyrosine kinase signaling proteins, A. castellanii genome also generates a diverse repertoire of
predicted pattern recognition receptors, many of which show predicted orthologous functions in the
innate immune systems of higher organisms. The size of A. castellanii mitochondrial DNA genome
is 41,591 bp [13].
582 Laboratory Models for Foodborne Infections

TABLE 36.2
Genomic Comparison of Acanthamoeba Species
Species T-Type Genome Size (Mb) GC Content (%)
Acanthamoeba astronyxis T7 83.43 42.9
Acanthamoeba castellanii T4 42.02–45 57.8–58.4
Acanthamoeba culbertsoni T10 55.54 50.4
Acanthamoeba divionensis T4 84.77 42.7
Acanthamoeba healyi T12 75.32 58.7
Acanthamoeba lenticulata T3 66.03 56.9
Acanthamoeba lugdunensis T4 99.42 59.1
Acanthamoeba mauritaniensis T4 106.84 58.7
Acanthamoeba palestinensis T2 103.48 59.1
Acanthamoeba pearcei T3 115.61 59
Acanthamoeba polyphaga T4 120.42 59.3
Acanthamoeba quina T11 83.59 59.2
Acanthamoeba rhysodes T4 75.82 57.9
Acanthamoeba royreba T4 79.54 51.3

36.1.2 Life Cycle and Epidemiology

36.1.2.1 Life Cycle
Acanthamoeba undergoes two stages of development in its life cycle: motile trophozoite and dormant
cyst. Trophozoites (Greek “tropho” means “to nourish”) actively feed on bacteria (e.g., Escherichia
coli K-12, Klebsiella aerogenes), algae, yeast, and other protozoa in the environment through pseudo-
pod formation, phagocytosis, or food cup formation, and are infective to humans. In addition, tropho-
zoites are capable of living axenically on nutrients in liquid through pinocytosis. The locomotion of
trophozoites involves the formation of hyaline pseudopodia (acanthopodia), and trophozoites divide
mitotically (through binary fission) under optimal conditions (food supply, neutral pH, ~30°C, and
50–80 mOsmol). Acanthamoeba forms dormant cyst with a resistant double-layer wall under harsh
conditions, facilitating its long-term survival under extreme temperatures and pH, desiccation, and
chemical exposure [2].
In amebic or acanthamoebic keratitis (AK), which is a more common form of Acanthamoeba
infections, Acanthamoeba enters into the cornea following a corneal trauma. The abrased corneal
epithelium secretes high concentrations of mannose-glycoprotein, to which Acanthamoeba binds
with high affinity. After penetration into the corneal epithelium, Acanthamoeba invades the underly-
ing stroma (a collagenous matrix), in which Acanthamoeba produces a collagenase to dissolve the
collagenous matrix [2].
In GAE, Acanthamoeba enters into the body via an open wound (in the skin or the upper respiratory
tract) or via inhalation of airborne cysts, and spreads hematogenously into the central nervous system
(CNS). Inside the brain, Acanthamoeba generates proteases, which in association with host immune sys-
tems induce proinflammatory responses and cause massive brain swelling and neuronal damage, leading
to death in ~95% of the infected individuals within few days [2].

36.1.2.2 Epidemiology
As a free-living organism, Acanthamoeba is ubiquitously distributed in nature and has been found in
the environment (e.g., soil, dust, air, natural and treated water, seawater, swimming pools, sewage, sedi-
ments, air-conditioning units, domestic tap water, drinking water treatment plants, and bottled water),
health care facilities (e.g., dental treatment units, hospitals and dialysis units, eyewash stations, contact
Acanthamoeba 583

lenses, and lens cases), vegetation, animals (e.g., fish, amphibia, reptiles, and mammals), and humans
(skin, nasal cavities, throat, intestines, brain, lungs, and cornea) [14–16].
In addition, Acanthamoeba may harbor various microbial endosymbionts (e.g., Candidatus Caedibacter
acanthamoebae, Candidatus Odyssella thessalonicensis, Candidatus Paracaedibacter acantham-
oebae, Candidatus Paracaedibacter symbiosus, Comamonas acidovorans, Legionella pneumophila,
Pseudomonas aeruginosa, mimivirus, megavirus, and pandoravirus). Further, a large number of other
bacterial species (e.g., Aeromonas, Bacillus cereus, Bartonella, Burkholderia, Campylobacter jejuni,
Chlamydia pnuemoniae, Coxiella burnetii, Cytophaga, E. coli O157:H7, Flavobacterium, Francisella
tularensis, Helicobacter pylori, Listeria, Mycobacterium, Pasteurella multocida, Prevotella interme-
dia, Porphyromonas gingivalis, Rickettsia, Salmonella Typhimurium, Shigella, Simkania negevensis,
Staphylococcus aureus, Vibrio, and Waddlia chondrophila) have been shown to survive and multiply
within Acanthamoeba. This highlights the potential role of Acanthamoeba in serving as bacterial reser-
voirs for human infections [2,12].

36.1.3 Clinical Features and Pathogenesis

Despite their reputation as free-living organisms, Acanthamoeba spp. are opportunistic pathogens with
the ability to cause AK, GAE, cutaneous acanthamoebiasis, and sinusitis in humans.

36.1.3.1 Amoebic (Acanthamoebic) Keratitis

As a most common form of Acanthamoeba infections, AK is a debilitating, painful, and v­ ision-impairing
disease of cornea that usually occurs in immunocompetent individuals with corneal abrasions (as a result
of contact lens wearing or poor lens hygiene). Characterized by a ring-like opaque infiltrate underly-
ing an epithelial ulcer, with clinical symptoms ranging from redness, tearing, photophobia, lid edema,
corneal ulcers, to blindness, AK may be caused by several Acanthamoeba species, including A. castel-
lanii, A. polyphaga, A. hatchetti, A. culbertsoni, A. rhysodes, A. griffini, A. quina, and A. lugdunen-
sis. Histopathologically, trophozoites are initially present in the corneal epithelium and subsequently
invade the underlying stroma, inducing a ring-like stromal infiltrate (composed of neutrophils and other
inflammatory cells) that leads to extensive damage (e.g., conjunctival hyperemia, corneal inflammation,
episcleritis, and scleritis). Further spread of trophozoites into corneal nerves or the retina may result in
neuritis and necrosis, or chorioretinitis [17,18].

36.1.3.2 Granulomatous Amoebic Encephalitis

As a less common but more severe form of Acanthamoeba infection, GAE is usually associated with
immunodeficiency (e.g., HIV), diabetes, malignancies, malnutrition, systemic lupus erythematosus,
renal failure, cirrhosis, tuberculosis, skin ulcers, Hodgkin’s disease, and other predisposing factors (alco-
holism, drug abuse, steroid treatment, cancer chemotherapy, radiotherapy, and organ transplantation).
Characterized by a chronic protracted slowly progressive CNS infection, GAE shows a range of subacute
symptoms, from headaches, confusion, nausea, vomiting, fever, lethargy, stiff neck, focal neurologi-
cal deficits (such as cranial nerve palsies and coma), seizures, to rapid death (within 1 week to several
months). GAE may also involve the lungs [19–21].

36.1.3.3 Cutaneous Acanthamoebiasis and Sinusitis

Cutaneous acanthamoebiasis and sinusitis may occur in AIDS patients with or without CNS involve-
ment. Characterized by the formation of hard erythematous nodules or skin ulcers, cutaneous acantham-
oebiasis causes high mortalities in infected patients (73% without CNS involvement, and 100% with
CNS involvement).
Postmortem biopsy from patients with GAE reveals severe edema, hemorrhagic necrosis, fibrin
thrombi, and inflammation, with multifocal lesions present in the midbrain, brain stem, corpus callosum,
584 Laboratory Models for Foodborne Infections

and cerebellum. Chronic inflammatory exudate (composed mainly of polymorphonuclear leucocytes

and mononuclear cells) is evident over the cortex. Numerous trophozoites (and microscopic cysts) may
be found in neurological tissues and occasionally other organs (e.g., the liver, kidneys, trachea, adrenals,
and lungs). Histological findings in cutaneous lesions include foci of necrosis surrounded by inflamma-
tory cells, vasculitis, trophozoite, and cyst forms, the latter of which may be mistaken for yeast forms
of Blastomyces dermatitidis, sporangia of Rhinosporidium seeberi, Cryptococcus neoformans, or
Prototheca wickerhamii [2].
A central mechanism of Acanthamoeba pathogenesis relates to its ability to attach to the host cell,
a process mediated chiefly by a 130-kDa mannose-binding protein (MBP) expressed on the surface of
Acanthamoeba. Other adhesins produced by Acanthamoeba include a 28.2-kDa laminin-binding protein,
a 55-kDa laminin-binding protein, and a >207-kDa adhesin. Following initial binding, Acanthamoeba
engages in other activities (e.g., phagocytosis and toxin production) that lead to host cell death [22,23].
Other factors that influence Acanthamoeba pathogenesis include cytolytic enzyme production (e.g.,
serine and cysteine metalloproteinases, phospholipases, glycosidases, elastases, sphingomyelinase,
collagenase, fibrinolytic enzyme, and ecto-ATPases, many of which are secreted only by clinical iso-
lates), temperature tolerance, and immune evasion capability, in addition to various host determinants
(interleukin-β, interleukin-α, interferon-γ, tumor necrosis factor-α, host cell apoptosis) [24,25].

36.1.4 Diagnosis
Traditionally, identification of Acanthamoeba is dependent on morphological characterization through
microscopic examination of clinical specimens [direct wet mounts of cerebrospinal fluid (CSF) or bron-
choalveolar lavage (BAL) fluid cytospin preparations, and stained smears of CSF sediment, brain, cuta-
neous lesion scrape/biopsy, or corneal scrape/biopsy]. The sizes and shape of trophozoites and cysts
as well as distinct nuclear structure [characterized by a prominent nucleolus, contractile vacuole, and
cytoplasmic vacuoles, which may be visualized more readily using trichrome or hematoxylin and eosin
(H&E) stains on fixed preparations after cytocentrifugation] help distinguish Acanthamoeba from host
macrophages and other immune cells. In addition, Acanthamoeba cyst wall turns red after periodic
acid-Schiff staining, and its cyst becomes black after Gomori methenamine silver staining. Calcofluor
white (a chemofluorescent dye with an affinity for the polysaccharide polymers) stains amebic cyst walls
bright apple green, which, with Evans blue counterstaining the background, is useful for identifica-
tion of Acanthamoeba cysts in brain or corneal tissue. Acridine orange staining of corneal scrapings
or CSF represents another simple and reliable method for rapid histological diagnosis of AK or GAE.
Furthermore, transmission electron microscopy of infected tissues, immunofluorescent or immunoper-
oxidase cytochemical staining of cryostat sections, or infected tissues embedded in paraffin offer other
approaches for identification of Acanthamoeba [26].
To enhance its detection and identification, Acanthamoeba is grown on NNA, 1.5% containing a
lawn of E. coli or E. aerogenes, axenically in PYG medium (2% proteose peptone, 0.2% yeast extract,
and 0.1 M glucose), or in Oxoid medium (Cline medium, containing serum and hemin) at 28°C–35°C
for 10 days or more (to allow sufficient time for excystment). Alternatively, Acanthamoeba may be cul-
tured on mammalian cell monolayers [e.g., African green monkey kidney (Vero), human embryonic
lung (HEL), human embryonic kidney (HEK), HeLa, B103 rat neuroblastoma, and L929 fibroblasts].
Acanthamoeba ingestion of bacteria or cells produces clear plaques after a week. Microscopic examina-
tion of Acanthamoeba culture isolates provides valuable confirmation [2].
With molecular techniques moving from laboratory bench to clinical setting, a variety of nucleic acid
amplification procedures have been developed for Acanthamoeba identification. These methods exploit
variations of complete or partial nuclear 18S rRNA gene, complete mitochondrial 16S rRNA, complete
mitochondrial genome, and randomly amplified polymorphic DNA patterns. In particular, analysis of
nuclear 18S ribosomal RNA (18S rRNA) gene sequence has made fast, reliable, and repeatable identifica-
tion of Acanthamoeba feasible. By setting a cutoff of <5% sequence divergences, the nuclear 18S ribo-
somal RNA-based method has enabled separation of Acanthamoeba isolates into 18 genotypes (T-types
1–18 or T1–18). Typically, primers CRN5 (5′-TGGTTGATCCTGCCAGTAG-3′) and SSU2-TRUN
Acanthamoeba 585

(5′-TGATCCCTCCGCAGGTTCAC-3′), or primers JDP1 (5′-GGCCCAGATCGTTTACCGTGAA-3′)

and JDP2 (5′-TCTCACAAGCTGCTAGGGGAGTCA-3′), are used to amplify nearly complete Rns
sequence, or a partial Rns region (ASA.S1) that contains a diagnostic fragment with respect to genotype,
respectively. The amplification products are then sequenced by using primers JDP1/JDP2 and internal
primers 892 (5′-CCAAGAATTTCACCTCTGAC-3′) and 892C (5′-GTCAGAGGTGAAATTCTTGG-3′).
The resulting sequences are aligned with other Acanthamoeba sequences in Rns database [6].

36.1.5 Treatment and Prevention

Due to the resilient nature of Acanthamoeba cysts, current treatment of AK involves topical applications
of a cationic antiseptic agent (e.g., polyhexamethylene biguanide 0.02% or chlorhexidine 0.02%) with or
without a diamidine (e.g., propamidine 0.1% or hexamidine 0.1%) for several weeks. A combination of
0.1% propamidine isethionate (Brolene) topically with 0.15% dibromopropamidine is also effective if
treatment is initiated early in infection. For pain relief, topical cyclopegic solutions and oral nonsteroidal
medications may be administered. Penetrating keratoplasty may help restore visual acuity but is unnec-
essary if AK patients are treated within 6 weeks of presentation [27–31].
For disseminated Acanthamoeba infection such as GAE, which evolves rapidly, it is important to
initiate treatment early. Trimethoprim–sulfamethoxazole therapy together with ketoconazole and
rifampin has proven effective for treating two immunocompetent pediatric patients with CNS involve-
ment. A 4-week course of IV pentamidine isethionate, topical chlorhexidine gluconate, and 2% ketocon-
azole cream has also been successfully applied for treatment of disseminated Acanthamoeba infection
in a renal t­ ransplant patient. For CNS infection, 5-fluorocytosine rather than pentamidine is recom-
mended because the latter demonstrates nephrotoxicity and fails to cross the blood–brain barrier. In
addition, 40 mg of 5-fluorocytosine per kg for 2 weeks appears to be useful for treating cutaneous acan-
thamoebiasis (e.g., AIDS patient with cutaneous and sinus lesions). A combination of lipid formulation
of amphotericin B and voriconazole may also be applied for granulomatous dermatitis secondary to
Acanthamoeba infection [32].
For effective prevention of AK, decontamination of contact lens and lens cases with 3% solution
of hydrogen peroxide is valuable. Other precautions include (1) wearing and replacing contact lenses
according to the schedule, (2) removing contact lenses before any activity involving contact with water
(showering, using a hot tub, or swimming), (3) washing hands with soap and water and drying before
handling contact lenses, (4) cleaning contact lenses according to instructions, (5) rubbing and rinsing
storage cases with sterile contact lens solution (not tap water), and (6) replacing storage cases at least
once every 3 months.

36.2 Laboratory Models
36.2.1 Animal Models
36.2.1.1 Mice
Mice represent a model of choice for CNS infection, as mice infected via intranasal inoculation of
Acanthamoeba develop symptoms of head tilt, circling, twirling, limb paresis, and convulsive seizures,
along with amebic rhinitis and pneumonitis. In the capillaries of the lungs and brains, trophozoites and
cysts are present. In addition, mice may also be used for modeling AK, with neutrophil being the pre-
dominant cell type during early stage of AK pathogenesis [33].

36.2.1.2 Rats
Wistar rats are a preferred model for AK because of a low death rate and larger corneas for inoculation.
Intrastromal injection of Acanthamoeba or coinjection with bacteria (e.g., Corynebacterium) provides
an efficient way to initiate AK [34–36].
586 Laboratory Models for Foodborne Infections

36.2.1.3 Chinese Hamsters
Application of “contact lenses” containing Acanthamoeba to the abraded corneal surfaces of Chinese
hamsters for at least 5–7 days leads to acute and self-limiting corneal infection, with clinical features
of neutrophil infiltration, epithelial ulceration, edema, corneal opacity, and neovascularization [37–40].

36.2.1.4 Rabbits
Microinjection of 1 × 104/100 μL Acanthamoeba healyi trophozoites between the corneal epithelium
and Bowman’s layer, anterior to the corneal stroma of New Zealand white rabbits, enabled successful
establishment of AK, which displayed an efficient immune response with less severe pathology and was
strikingly similar to AK in humans [41,42].

36.2.1.5 Pigs
Due to the anatomical similarities of the pig eye to the human eye, pigs offer a useful model for ocular
infections such as AK. However, spontaneous resolution of AK in pigs occurs in 8–10 weeks, in contrast
to the prolonged infection in humans [43].

36.2.2 In Vitro Models

In vitro osmotolerance (0.5 M mannitol) and temperature tolerance (30°C) assays offer a valuable way
to assess Acanthamoeba pathogenicity [44]. Human epithelial cells (e.g., corneal epithelial cells), stro-
mal keratocytes, and stromal cell homogenates may be used for in vitro assessment of AK, with clear-
ing of cell monolayers (CPE) being an indication of Acanthamoeba pathogenicity. In vitro experiment
with human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier,
indicates that Acanthamoeba abolishes the HBMEC transendothelial electrical resistance by degrading
occludin and zonula occludens-1 tight junction proteins, leading to increased permeability.

36.3 Conclusion
The genus Acanthamoeba consists of more than 25 species of free-living protozoa, which are charac-
terized by the generation of pseudopodia (acanthopodia) for locomotion during trophozoite stage and
the formation of a double-walled wrinkled cyst under adverse environmental conditions. While being
seemingly harmless creatures in the environment, certain Acanthamoeba species are surprisingly dan-
gerous, with the ability to take advantage of the temporary weakness in human host for its own gain,
and cause painful, sight-threatening AK in contact lens wearers and deadly GAE and skin infections in
immunocompromised individuals. In addition, Acanthamoeba has the potential to serve as reservoirs
for a number of human bacterial pathogen due to the fact that many bacteria have been shown to survive
and multiply within the parasite. Following the development of molecular test targeting nuclear 18S
rRNA gene, rapid and reliable identification of Acanthamoeba is possible [45]. However, considering the
current lack of options for treating and controlling Acanthamoeba infections, especially GAE, further
research using laboratory models to uncover the molecular details of Acanthamoeba pathogenesis is
critical.

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2006;22(4):175–80.
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2001;43(6):391–5.
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37
Cryptosporidium

Dongyou Liu

CONTENTS
37.1 Introduction.................................................................................................................................... 589
37.1.1 Classification, Morphology, and Genome Structure........................................................ 589
37.1.1.1 Classification..................................................................................................... 589
37.1.1.2 Morphology....................................................................................................... 590
37.1.1.3 Genome Structure............................................................................................. 590
37.1.2 Life Cycle and Epidemiology............................................................................................591
37.1.3 Clinical Features and Pathogenesis.................................................................................. 592
37.1.4 Diagnosis.......................................................................................................................... 593
37.1.5 Treatment and Prevention................................................................................................. 593
37.2 Laboratory Models........................................................................................................................ 594
37.2.1 Animal Models................................................................................................................. 594
37.2.2 In Vitro Models................................................................................................................. 595
37.3 Conclusion..................................................................................................................................... 595
References............................................................................................................................................... 595

37.1 Introduction
The genus Cryptosporidium comprises nearly 30 known species of apicomplexan protozoans that infect
birds, reptiles, fish, and mammals, including humans. Of these, C. parvum and C. hominis along with
a few other species are responsible for producing watery diarrhea (intestinal cryptosporidiosis) with or
without a persistent cough (respiratory cryptosporidiosis) in both immunocompetent and immunodefi-
cient humans.
Although Cryptosporidium was first identified from the stomach of mice in 1907, its role in human
infection was only established in 1976. Since then, cryptosporidiosis has been recognized as a major
cause of chronic diarrhea in patients with AIDS, as a cause of zoonotic and waterborne outbreaks of
diarrhea, and as a cause of diarrhea and unexplained cough in immunocompetent children.

37.1.1 Classification, Morphology, and Genome Structure

37.1.1.1 Classification
Taxonomically, the genus Cryptosporidium belongs to the family Cryptosporidiidae, suborder
Eimeriorina, order Eucoccidiorida, subclass Coccidiasina, class Conoidasida, phylum Apicomplexa,
domain Eukaryota, kingdom Animalia.
The suborder Eimeriorina includes 12 established families (Aggregatidae, Atoxoplasmatidae,
Barrouxiidae, Calyptosporidae, Caryotrophidae, Cryptosporidiidae, Eimeriidae, Elleipsisomatidae,
Lankesterellidae, Sarcocystidae, Selenococcidiidae, and Spirocystidae).
Being the only member of the family Cryptosporidiidae, the genus Cryptosporidium currently
consists of about 30 recognized species, i.e., Cryptosporidium andersoni, Cryptosporidium baileyi,

589
590 Laboratory Models for Foodborne Infections

Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium cuniculus, Cryptosporidium erina-

cei, Cryptosporidium fayeri, Cryptosporidium felis, Cryptosporidium fragile, Cryptosporidium galli,
Cryptosporidium hominis, Cryptosporidium huwi, Cryptosporidium macropodum, Cryptosporidium
meleagridis, Cryptosporidium molnari, Cryptosporidium muris, Cryptosporidium parvum,
Cryptosporidium proliferans, Cryptosporidium rubeyi, Cryptosporidium ryanae, Cryptosporidium sau-
rophilum, Cryptosporidium serpentis, Cryptosporidium suis, Cryptosporidium tyzzeri, Cryptosporidium
ubiquitum, Cryptosporidium varanii, Cryptosporidium viatorum, Cryptosporidium wrairi, and
Cryptosporidium xiaoi [1–4].
Due the fact that members of the genus Sarcocystis in the family Sarcocystidae produce oocysts with
thin walls that often rupture to release sporocysts, each of which possesses four sporozoites similar to
Cryptosporidium oocysts, a number of Cryptosporidium species were erroneously assigned to the genus
Sarcocystis in the early days. However, subsequent ultrastructural examinations revealed the presence
of a unique attachment organelle as a defining feature for the genus Cryptosporidium (indeed, the name
Cryptosporidium refers to the absence of sporocysts within the oocyst) and the family Cryptosporidiidae.
In addition, biological and molecular studies indicated that Cryptosporidium species are more closely
related to gregarine parasites of the subclass Gregarinasina, class Conoidasida rather than to Coccidians
(e.g., Isospora, Cyclospora, and Sarcocystis), since Cryptosporidium species undergo gregarine-like
gamont stages and have the ability to complete their life cycles in the absence of host cells [1,2,5,6].
Cryptosporidium species are capable of infecting many vertebrates, ranging from mammals (includ-
ing man) and birds to reptiles and fish [7]. Within the genus Cryptosporidium, species of medical and
veterinary importance consist of C. andersoni (cattle), C. baileyi (chicken and turkey), C. bovis (cattle),
C. canis (dogs), C. felis (cats), C. galli (birds), C. hominis (humans), C. meleagridis (birds and humans),
C. molnari (fish), C. muris (rodents, other mammals, and humans), C. parvum (cattle, sheep, goats, and
humans), and C. suis (pigs and humans). It is noteworthy that C. parvum and C. hominis are respon-
sible for a majority (90%) of human clinical cases of cryptosporidiosis, while C. canis, C. felis, C. suis,
C. meleagridis, C. muris, and C. andersoni as well as chipmunk genotype I (W17) and skunk genotype
may be occasionally involved in human illnesses (Table 37.1) [1,2,8].
Based on their organ specificity (localization of endogenous development in the host), Cryptosporidium
species and genotypes may be distinguished into two major groups: the larger intestinal group (which has
affinity for the intestine, as well as the lungs and bursa of Fabricius), and the smaller gastric group (which
has affinity for the glands of the glandular stomach). Within the gastric group, two species (C. muris and
C. andersoni) are specific for mammals. Interestingly, C. muris strain TS03 from a naturally infected
East African mole rat (Tachyoryctes splendens) in Kenya was recently renamed as Cryptosporidium
proliferans n. sp. Oocysts of C. proliferans [of 7.7 × 5.3 μm (range 6.8–8.8 × 4.8–6.2 μm) in size, with a
length/width ratio of 1.48] are morphologically distinct from C. parvum, C. muris HZ206, C. andersoni,
C. suis, and C. xiaoi. Phylogenetic analyses on the basis of small subunit rRNA, Cryptosporidium oocyst
wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, heat shock protein 70,
actin, heat shock protein 90 (MS2), MS1, MS3, and M16 gene sequences further highlighted the genetic
differences of C. proliferans from C. muris and other Cryptosporidium species [9,10].

37.1.1.2 Morphology
Cryptosporidium oocysts are round to oval in shape and measure about 4–6 μm in diameter (Table 37.1).
Under a phase-contrast microscope, Cryptosporidium oocysts are highly refractile in wet smear and con-
tain prominent dark granules (black dot) or small vacuoles, in contrast to yeast, which are not refractile
and contain no granules. Further, Cryptosporidium oocysts do not stain with Lugol’s iodine solution, but
yeast do. Excystment of Cryptosporidium oocysts release four sporozoites each.

37.1.1.3 Genome Structure
The genome sequences of C. parvum IOWA, C. hominis TU502, and C. muris were released on CryptoDB
in 2003 and 2011, respectively [11–13]. Composed of eight chromosomes each, these genomes are of
9.1–92 Mb in size, with 26%–30% GC content and 94%–97% nucleotide identity. Altogether, about 4000
Cryptosporidium 591

TABLE 37.1
Morphological and Biological Characteristics of Human-Infecting Cryptosporidium Species
Main
Former Oocyst Dimension Oocyst Length/ Susceptible Predilection
Designation (μm) Width Ratio Hosts Site
Cryptosporidium Bovine genotype, 5.0 × 4.5 (4.5–5.4 × 1.16 or 1.15 Cattle (especially Small intestine
parvum senso genotype 2 4.2–5.0) or 5.2 × 4.6 (1.04–1.22) preweaned
stricto (4.8–5.6 × 4.2–4.8) calves), sheep,
goats, deer, and
humans
Cryptosporidium Human genotype, 5.2 × 4.9 (4–6 × 4–5) 1.1 (1.0–1.1) Humans, dugong, Small intestine
hominis genotype 1, and lamb
genotype H
Cryptosporidium Dog genotype 4.95 × 4.71 1.05 (1.04–1.06) Dogs, coyotes, Small intestine
canis (3.68–5.88 × foxes, and
3.68–5.88) humans
Cryptosporidium 5 × 4.5 (6.0–5.0 × 1.19 Cats Small intestine
felis 5.0–4.5)
Cryptosporidium 4.6 × 4.2 (4.9–4.4 × 1.1 Pigs and humans Small and large
suis 4.0–4.3) intestine
Cryptosporidium 5.2 × 4.6 (4.5–6.0 × 1.13 (1.00–1.33) Chicken and Small intestine
meleagridis 4.2–5.3) turkey
Cryptosporidium 7.4 × 5.6 (7–8 × 1.3 (1.1–1.5) or 1.4 Rodents, other Stomach
muris 5–6.5) or 8.4 × 6.3 mammals, and
(7.5–10 × 5.5–7) humans
Cryptosporidium C. muris-like 7.4 × 5.5 (6.0–8.1 × 1.35 Cattle, bactrian Abomasum
andersoni 5.0–6.5) camels, sheep,
rodents, and
humans

protein-encoding genes are present. Cryptosporidium genomes appear to be unusual among eukaryotes
in having a degenerate “mitosome” (located in the posterior end of sporozoites) instead of a mitochon-
drion, with accompanied loss of many mitochondrial proteins, including those required for the TCA
cycle, oxidative phosphorylation, and fatty acid oxidation as well as de novo biosynthesis of amino acids,
nucleotides, and sugars. These gene losses render Cryptosporidium species heavily reliant on scavenging
nutrients from the host rather than de novo biosynthesis [12–15]. In addition, sequencing analyses of C.
hominis IbA10G2 (the most virulent subtype responsible for all outbreaks in Europe and Australia) and
IaA28R4 (a dominant outbreak subtype in the United States) uncovered major differences in the 5′ and
3′ ends of chromosome 6 and the putative virulence determinant gp60 region, suggesting that genetic
recombination plays a potential role in the emergence of hypertransmissible C. hominis subtypes [16,17].

37.1.2 Life Cycle and Epidemiology

As an obligate intracellular monoxenous parasite with the ability to carry out its entire biological cycle
in a single host, Cryptosporidium produces oocysts (each containing four naked sporozoites) for trans-
mission to humans via various routes (man-to-man, animal-to-man, contaminated water, food, or air).
Inside the stomach, sporozoites exit through a suture along one side of the oocyst wall, pass through the
microvillus brush border, enter into epithelial cells of the jejunum and duodenum, and complete their
biological cycle with both asexual and sexual phases [1,2].
During asexual reproduction, sporozoites multiply several times; and in subsequent sexual reproduc-
tion, sporozoites form oocysts (via trophozoites and merozoites) at the luminal apex of the enterocytes.
The newly formed oocysts gather in an extracytoplasmatic parasitophorous vacuole whose membrane
is made from the enteric epithelium. It is notable that while about 80% of oocysts have a thicker, strong
wall and are shed in the feces and become immediately infective, the remainder 20% of oocysts have a
592 Laboratory Models for Foodborne Infections

thin, ­incomplete wall, rupture in the intestine, and release four infectious sporozoites, resulting in a new
cycle of infection (endogenous autoinfection). The autoinfection allows Cryptosporidium to sporulate
and persist within the same host indefinitely, accounting for the severe chronic forms of cryptosporidi-
osis in immunocompromised persons in the absence of exogenous reinfection. In respiratory cryptospo-
ridiosis, as seen in immune-deficient patients, the infective oocysts spread via nasal secretions into the
­environment [1,2].
Cryptosporidium oocysts are resistant to environmental adversaries and are highly infectious; as few
as 10 C. hominis oocysts are able to produce disease in healthy adults. Although fecal–oral transmission
represents the main route of infection, transmission via coughing and fomites is also possible [1,2].
Most (90%) of the human infections are caused by C. hominis and C. parvum, with C. hominis being
found almost exclusively in humans, while C. parvum is readily transmitted between humans and ani-
mals as well as between humans. Other Cryptosporidium species (C. meleagridis, C. muris, C. felis,
C. canis, C. suis, and C. andersoni) may also be involved in human infections [1,2].
As a most common cause of severe diarrhea in humans, cryptosporidiosis poses a serious public health
risk worldwide. In industrialized nations, cryptosporidiosis is often associated with recreational water
use, animals on petting farms, and day care centers. In developing countries (e.g., sub-Saharan Africa,
Asia, and Latin America), cryptosporidiosis may result from contaminated food or drinking water, or
contaminated recreational water (as in swimming pools) and occurs mostly in children younger than
5 years (with peak occurrence in children younger than 2 years) [1,2].
The population groups with an elevated risk of being exposed to Cryptosporidium include: (1) people
who swim regularly in pools with insufficient sanitation (as some Cryptosporidium strains are chlorine-
resistant); (2) child-care workers; (3) parents of infected children; (4) people caring for others with cryp-
tosporidiosis; (5) backpackers, hikers, and campers who drink unfiltered, untreated water; (6) people,
including swimmers, who swallow water from contaminated sources; (7) people handling infected cat-
tle; and (8) people exposed to human feces [1,2].

37.1.3 Clinical Features and Pathogenesis

Cryptosporidium spp. (notably C. parvum and C. hominis) are apicomplexan parasites that commonly
infect the microvillus border of the gastrointestinal epithelium of humans as well as other vertebrate
hosts, typically producing moderate-to-severe watery diarrhea, abdominal pain, vomiting, nausea, fever,
anorexia, dehydration, and weight loss. While immunocompetent individuals may experience transient
gastroenteritis lasting up to 2 weeks and recover without treatment, young children (especially those
<2 years of age) and individuals with immune deficiencies (e.g., HIV/AIDS), renal failure, inflamma-
tory bowel disease, or undergoing immunosuppressive therapy may develop intractable diarrhea, growth
stunting, and wasting, with fatal consequence [1,2].
Patients with respiratory cryptosporidiosis show inflammation of the nasal mucosa, sinuses, larynx,
and trachea, accompanied by nasal discharge and voice change (upper respiratory cryptosporidiosis), or
productive cough, dyspnea, fever, and hypoxemia (lower respiratory cryptosporidiosis). However, diar-
rhea is absent and fecal examination for oocysts is negative [18].
Occasionally, Cryptosporidium parasites may settle in the hepatobiliary system, pancreas, and urinary
bladder, particularly in patients with dysfunctional immune systems.
In intestinal cryptosporidiosis, Cryptosporidium spp. are predominately located in the epithelial cells
of the jejunum and ileum as well as other parts of the gastrointestinal tract (including biliary organ)
in immunocompromised patients. The parasite stays within a parasitophorous vacuole that protrudes
out of the host cytoplasm into the intestinal lumen. This provides a unique intracellular but extracy-
toplasmic niche for the parasite, and segregates it from direct interaction with other cell types. Host
responses to Cryptosporidium spp. and disease outcomes are therefore dependent on the functionality
of mucosal epithelial cells, including expression of pathogen pattern recognition receptors (PRRs) [e.g.,
the Toll-like receptors (TLRs), which recognize microbes on the cell surface and in endosomes; and
nucleotide binding and oligomerization domain-like receptors (NLRs), which sense microbial molecules
in the cytosol], production of antimicrobial peptides (e.g., β-defensins and LL-37), release of inflamma-
tory cytokines/chemokines [e.g., interleukin-8 (IL-8)], production of epithelial-cell-derived exosomes,
Cryptosporidium 593

and feedback regulation of immune homoeostasis [19,20]. In immunocompetent individuals, mucosal

­epithelial cells are capable of initiating adequate immune responses that contribute to the elimination
of invading Cryptosporidium parasites within a relatively short period [21]. In immunocompromised
individuals and young children, immune responses of mucosal epithelial cells are insufficient to over-
come the parasites, which affect host enteric nervous system (ENS) (e.g., efferent and afferent neurons,
as well as i­ nterneurons) and disturb water and electrolyte balance in the gastrointestinal tract, leading to
malabsorption and hypersecretion [22].

37.1.4 Diagnosis
Traditional method for diagnosing human intestinal cryptosporidiosis is based on microscopic detection
of Cryptosporidium oocysts in stool samples with acid-fast, auramine, or indirect immunofluorescence
stains. Use of fluorescence microscopy (e.g., light-emitting diode light sources) together with fluorescent
stains (e.g., auramine-rhodamine, which makes oocysts appear yellow-orange) provides a more sensitive
detection than the modified acid-fast stain [e.g., modified Ziehl–Neelsen (hot) or Kinyoun (cold), which
demonstrates the acid resistance of oocysts]. Further, application of formalin-ether or formalin-ethyl
acetate sedimentation concentration procedures, or Sheather’s flotation-concentration method (using
a sucrose gradient) improves the sensitivity of microscopic detection. Nonetheless, Cryptosporidium
oocysts (of 4–6 µm in diameter) must be differentiated from other partially acid-fast organisms (e.g.,
Cyclospora cayetanensis) and other similar looking yeast or fungal spores [23,24].
Similarly, human respiratory cryptosporidiosis may be confirmed by microscopic detection of
Cryptosporidium organisms in respiratory secretions [e.g., sputum, tracheal aspiration, bronchoalveolar
lavage (BAL)], bronchial and lung biopsy, or autopsy specimens. Examination of respiratory secretions
using acid-fast, auramine or indirect immunofluorescence stains may show thick-walled Cryptosporidium
oocysts and invasive forms of the parasite (sporozoites and merozoites). Sporozoites and merozoites may
be also detected from BAL fluid specimens using Giemsa stain. Hematoxylin–eosin staining of tis-
sue specimens may uncover sporozoites, merozoites, and oocysts lining the mucosal epithelium at the
luminal surface of the trachea, bronchi, and bronchioles, as well as within bronchial mucous glands.
Bronchial and lung biopsy specimens may yield intracellular and extracellular cryptosporidia, including
sporozoites, merozoites, or oocysts on the bronchoepithelial surface [25].
Serological assays are important for detection of specific antibodies in both symptomatic and
asymptomatic infections. Although antibodies to Cp23 correlate with distant infection, those to Cp17
(also called gp15) suggest recent infection, and those to P2 are associated with repeated infection. In
addition, several serological assays [e.g., direct immunofluorescence assay (DFA), enzyme immune
assays (e.g., EIA, ELISA), and immunochromatographic assays] allow detection of oocyst-specific
antigens [26].
In recent years, nucleic amplification techniques [e.g., polymerase chain reaction (PCR), PCR-
restriction fragment length polymorphism (RFLP) analysis] are increasingly used for sensitive detection
and subtyping of Cryptosporidium species [27–30]. This involves disruption of oocysts by bead-beating,
freeze-thaw, boiling, or chemical lysis for nucleic acid extraction and use of oligonucleotide primers
from selective gene targets [e.g., 18S rRNA, actin, oocyst wall protein (COWP), thrombospondin-related
adhesive protein 1 (TRAP-C1), tubulin, HSP70, gp60, and gp900 (also known as polythreonine protein or
poly-T) in addition to noncoding internal transcribed spacer 1 and microsatellites] for a­ mplification, and
detection of amplified products on various platforms. Application of molecular procedures has enabled
the differentiation of C. andersoni from C. muris, the discrimination of C. canis from C. ­parvum, and
the establishment of C. hominis as a species distinct from C. parvum [1,2].

37.1.5 Treatment and Prevention

Being the only drug approved by the FDA for the treatment of intestinal cryptosporidiosis in immuno-
competent hosts, nitazoxanide (NTZ, marketed under the name Alinia, which is a thiazolide drug with
broad antiparasitic activities) shows efficacy of 56%–96%. Supplemental zinc may improve symptoms,
particularly in recurrent or persistent infections or in patients at risk for zinc deficiency. Nonetheless,
594 Laboratory Models for Foodborne Infections

the relatively high cost of nitazoxanide limits its clinical adoption. Further, nitazoxanide is ineffective
against respiratory cryptosporidiosis and has proven unsuccessful in eradicating the parasite from the
guts of chronically infected children with HIV [31].
In individuals coinfected with HIV, antiretroviral therapy (ART) is useful for controlling chronic
diarrhea and wasting due to cryptosporidiosis. Essentially, ART helps restore immune function of
human patients, leading to parasitological clearance after treatment for several months. Protease
inhibitors, including indinavir, have also been shown to directly interfere with Cryptosporidium
development. Alternatively, paromomycin may be considered for AIDS patients with respiratory cryp-
tosporidiosis [31].
Prevention of human cryptosporidiosis should aim to interrupt fecal–oral transmission of
Cryptosporidium oocysts through provision of clean water and sanitation, maintenance of scrupulous
hygiene in communal settings such as day care centers, implementation of food safety practice, and
development of effective vaccines [32].
Use of filtration technologies (e.g., slow sand filters, diatomaceous earth filter, membranes, bag- and
cartridge-filter products) is useful for the removal of Cryptosporidium in water supply. Ultrafiltration and
UV irradiation are also efficient. However, high-rate filtration and chlorine disinfection are largely inef-
fective in the reduction of Cryptosporidium oocysts from water supply. For wastewater treatment, stabili-
zation ponds and constructed wetlands are valuable for the elimination of Cryptosporidium oocysts [33].

37.2 Laboratory Models
Given the ability of Cryptosporidium to perpetuate in human hosts, use of animal models and in vitro
cultivation system is vital for characterization of Cryptosporidium life cycle, elucidation of host immune
mechanisms, evaluation of potential drugs, and development of vaccines [34–36].

37.2.1 Animal Models
A number of laboratory animals are susceptible to Cryptosporidium infection, although numerous
parameters (e.g., isolate employed, dosage, oocyst age, oocyst storage conditions, chemical pretreat-
ments, and host genetics) may have a bearing on the outcome.
Out of the 19 different strains of adult mice examined, C. parvum has been shown to produce the
highest levels of infection in the beige mouse (C57BL/6J-bgJ). In addition, similar acute patterns of
C. parvum infection in C57BL/6 wild-type and T- and B cell-deficient Rag2–/– newborn mice were also
observed. Using dexamethasone-treated or untreated adult severe combined immunodeficiency (SCID)
mice, it was shown that C. parvum strains (of both animal and human origins) are capable of inducing
intraepithelial neoplasia and invasive adenocarcinoma in the stomach, ileocecal region, and intrahepatic
biliary tree [37]. Further, use of the interleukin-12 (IL-12) knockout mouse model, which mimics acute
human cryptosporidiosis, facilitated the identification of a compound (P131) with significant antipara-
sitic activity [36]. Use of various animal models has helped to confirm that C. hominis at high doses may
also infect calves, lambs, and piglets.
C. muris oocysts obtained from the gastric glands of wild rat stomach have been successfully transmit-
ted to uninfected rats, mice, guinea pigs, rabbits, dogs, and cats. Indeed, C. muris seems to be infective to
hamsters, squirrels, Siberian chipmunks, wood mice (Apodemus sylvaticus), bank voles (Clethrionomys
glareolus), Dolichotis patagonum, rock hyrax, bactrian camels, mountain goats, cynomolgus monkeys,
and humans. Interestingly, C. muris demonstrates notable differences in prepatent and patent periods
in two laboratory rodents—BALB/c mice and the southern multimammate rat (Mastomys coucha).
Specifically, C. muris infection progressed more rapidly in BALB/c mice (with a prepatent period of
7.5–10 days) than M. coucha (with prepatent period of 18–21 days) [38].
In contrast to C. muris, C. andersoni (previously C. muris-like) is not infective to outbred, inbred, neo-
natal, or immunocompetent mice (including common field mice, BALB/c mice, and SCID mice) as well
as common voles, bank voles, desert gerbils, guinea pigs, rats, rabbits, or goats. However, C. andersoni
has the capacity to infect Mongolian gerbils.
Cryptosporidium 595

37.2.2 In Vitro Models

C. parvum and C. hominis are capable of replicating in murine macrophages, and C. parvum has been
shown to grow in the human ileocecal epithelial cell line (HCT-8) [39]. C. hominis was found to prolifer-
ate in cell-free culture [40]. A novel method for prolonged in vitro cultivation of primary human intes-
tinal epithelial cells (PECs) from small intestinal crypts enabled the detailed study of Cryptosporidium
infection [41]. In addition, a reproducible and quantitative C. parvum infection model based on a non-
carcinomatous, human small intestinal epithelial cell type, named FHs 74 Int, was developed [42]. An
optimal protocol for transfection of C. parvum sporozoites in tissue culture was also described.
Using the hollow fiber technology that mimics the gut by delivering nutrients and oxygen from the
basal layer upward while allowing separate redox and nutrient control of the lumen for parasite develop-
ment, Cryptosporidium oocyst production was maintained for more than 6 months, yielding approxi-
mately 1 × 108 oocysts/mL/day [43].
A chicken embryo model supports the development of all the endogenous life cycle stages of C. baileyi,
and provides a new and effective in vitro cultivation system for studies on antigens, virulence, i­ nfectivity,
metabolites, and sensitivity of drugs against the parasite [44]. In a separate report, chick embryo tracheal
organ (TOC) was used to cultivate oocysts or sporozoites of C. baileyi [45].

37.3 Conclusion
Of nearly 30 recognized species within the genus Cryptosporidium, C. parvum and C. hominis are
important waterborne and foodborne pathogens of humans. These parasites cause villus atrophy, crypt
hyperplasia, infiltration of the lamina propria, chloride hypersecretion, glucose malabsorption, and
reduced barrier function, leading to diarrhea, wasting, stunt growth, and explained cough. Considering
its high infectivity (as few as 10 oocysts are sufficient to establish infection in immunocompetent per-
sons) and strong resistance to chlorine disinfection, large-scale outbreaks of human cryptosporidiosis
attributable to contaminated drinking or recreational water have been reported. In order to develop
improved control measures, it is essential to uncover the molecular mechanisms of Cryptosporidium
infection. Application of laboratory models will be fundamental to achieving this goal.

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17. Hadfield SJ, et al. Generation of whole genome sequences of new Cryptosporidium hominis and
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18. Sponseller JK, Griffiths JK, Tzipori S. The evolution of respiratory cryptosporidiosis: evidence for
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38
Cystoisospora belli

Chaturong Putaporntip and Somchai Jongwutiwes

CONTENTS
38.1 Introduction................................................................................................................................... 599
38.1.1 Classification and Phylogeny............................................................................................ 600
38.1.2 Biology, Morphology, and Life Cycle.............................................................................. 600
38.1.3 Epidemiology.................................................................................................................... 603
38.1.4 Pathology and Clinical Aspects....................................................................................... 604
38.1.5 Laboratory Diagnosis....................................................................................................... 605
38.1.5.1 Stool Examination............................................................................................ 605
38.1.5.2 Examination of Duodenal Content................................................................... 606
38.1.5.3 Histopathological Diagnosis............................................................................. 606
38.1.5.4 Molecular Diagnosis......................................................................................... 607
38.1.6 Treatment and Prevention................................................................................................. 607
38.2 Laboratory Models........................................................................................................................ 608
38.2.1 Animal Models................................................................................................................. 608
38.2.2 In Vitro Growth in Mammalian Cells.............................................................................. 608
38.2.2.1 Preparation of Infective Oocysts...................................................................... 608
38.2.2.2 Inoculation of Sporozoites into Cell Lines........................................................610
38.2.2.3 Inoculation of Sporozoites into Human and Murine Macrophages..................610
38.3 Conclusion and Future Perspectives..............................................................................................611
References................................................................................................................................................612

38.1 Introduction
Cystoisospora belli, formerly known as Isospora belli, is an important enteric coccidian protozoa that
is responsible for diarrheal illness in humans. Belonging to phylum Apicomplexa, coccidians parasit-
ize diverse species of hosts ranging from higher invertebrates to vertebrates. First observed as globu-
lar and ovoidal structures in the bile of a cow by Leeuwenhoek in 1674 [1], coccidian protozoa of the
apicomplexan parasites are known to cause diseases in humans. These include the genera Plasmodium
and Babesia infecting mainly erythrocytes, Toxoplasma and Sarcocystis inhabiting various tissues, and
enteric coccidia in the genera Cystoisospora, Cyclospora, and Cryptosporidium, mainly involving intes-
tinal epithelial cells. All enteric coccidian protozoa produce gastrointestinal symptoms indistinguishable
from one another; therefore, laboratory investigation is crucial to make definite diagnosis that is man-
datory for a proper management. Although a number of species in the genus Cystoisospora are known
to infect mammalian hosts, C. belli is the only known pathogenic species to humans, causing cystoiso-
sporiasis. The description of characteristic oocysts of C. belli in stools was initially observed among
military personnel stationed in Egypt, the Middle East, and eastern Mediterranean countries during
1914 and 1921 [2]. Nowadays, it is well recognized that cystoisosporiasis has a cosmopolitan distribu-
tion. Although oocysts of Isospora natalensis were reportedly found in human fecal samples, several
studies have failed to reaffirm the presence of these oocysts as a causative agent of diarrheal illness in

599
600 Laboratory Models for Foodborne Infections

humans since its first report in 1953, raising the possibility that it may not be a valid pathogenic species in
humans [3]. Most of the enteric coccidian parasites of humans are considered to be opportunistic patho-
gens except the genus Sarcocystis. Thus, the prevalence of these coccidian infections seems to increase
as the number of immunocompromised patients increases, especially after AIDS became pandemic. In
general, severity of illness caused by C. belli seems to be more aggressive in patients with compromised
immunity than those with normal immune status. However, chronic emaciating infections have been
reported in immunocompetent patients, suggesting that host immunity per se may not determine the
severity of cystoisosporiasis [4,5]. Meanwhile, difference in intrinsic virulence of this organism has not
yet been explored.

38.1.1 Classification and Phylogeny

Members of the phylum Apicomplexa are intracellular organisms that share common structural features
by possessing apical complex at certain stages of their life cycles. Apical complex is subcellular organ-
elles consisting of polar ring, rhoptries, micronemes, conoid, dense granules, and subpellicular microtu-
bules that can be identified under a transmission electron microscope. Of these, micronemes, rhoptries,
and dense granules are secretory organelles involved in ligand–receptor recognition and host cell inva-
sion. Rhoptries are club-shaped or teardrop membrane-bound organelles with a connecting duct to the
anterior end of the cell. Micronemes are elliptical-shaped vesicles located in close proximity to polar
ring. Dense granules are secretory vesicles found clustering around the anterior portion of the organ-
ism. Apical organelles exist only at the invasive stages of apicomplexan protozoa and become gradually
disintegrated after host cell invasion. Parasitic protozoa in the phylum Apicomplexa inhabit tissues,
blood, or lymphoid cells of the host, and most of them, including genus Cystoisospora, belong to the
class Sporozoea. Previous taxonomic classification based on biology and characters of protozoa placed
the formerly known mammalian Isospora species, currently classified as genus Cystoisospora, in sub-
class Coccidia, suborder Eimeriina, and family Eimeriidae whose members include genera Eimeria and
Cyclospora. These coccidian parasites require single hosts to complete both sexual and asexual repro-
ductions, termed monoxenous life cycle. Although the mature oocysts of mammalian Cystoisospora
contain two sporocysts (diplosporocystic) and each with four sporozoites (tetrasporozoic) akin to some
members of protozoa in the family Eimeriidae, the absence of a polar Stieda body in each sporocyst and
recent phylogenetic inference from the small subunit ribosomal RNA sequences of several members of
apicomplexan protozoa have repositioned members of mammal host species of Cystoisospora within
tissue cyst-forming coccidia that belong to the family Sarcocystidae [6]. The members in this fam-
ily include genera Toxoplasma, Neospora, Hammondia, and Sarcocystis as a monophyletic group. On
the other hand, avian host species Isospora robini and Isospora gryphoni are clustered within another
monophyletic group with genera Caryospora, Eimeria, and Cyclospora, all of which are non-tissue-cyst-
forming coccidia in the family Eimeriidae (Figure 38.1) [7–9]. The presence of tissue cyst in the genus
Cystoisospora favors phylogenetic rather than phonetic classification [10–12]. The genus Cystoisospora
was created by Frenkel in 1977 based on the characteristics of oocysts and the presence of tissue cysts
in paratenic hosts that are distinct from those of the genus Isospora [13]. Meanwhile, Isospora hominis
described over seven decades ago was misclassified, which de facto belongs to the genus Sarcocystis
based on the life cycle and structures of the developmental stages. Other species of Cystoisospora are
parasitic in wild and domestic animals, for example, C. arctopitheci in nonhuman primates, C. canis
and C. ohioensis in dogs, C. rivolta and C. felis in cats, and C. suis in pigs [14]. More than 300 species of
Cystoisospora have been proposed and found to infect a wide range of animals that includes amphibians,
reptiles, birds, and mammals. However, some of these species may require further validation.

38.1.2 Biology, Morphology, and Life Cycle

Developmental stages of Cystoisospora comprise asexual multiplication, sexual reproduction, and spo-
rogony. The life cycle of C. belli completes within the human host without known animal reservoirs.
Humans get infected by ingestion of food or water contaminated with oocysts (Figure 38.2). The infective
Cystoisospora belli 601

Cystoisospora ohioensis GU292304

Cystoisospora belli U94787
Cystoisospora suis U97523

Eimeriidae Sarcocystidae
Cystoisospora felis L76471
Neospora caninum U03069
Hammondia hammondi AF096498
Toxoplasma gondii M97703
Sarcocystis neurona U07812
Caryospora bigenetica AF060975
Isospora robini AF080612
Isospora gryphoni AF080613
Cyclospora cayetanensis U40261
Eimeria tenella U67121

0.02

FIGURE 38.1  Phylogeny of Cystoisospora and other apicomplexan protozoa inferred by using the Maximum Likelihood
method based on the small subunit ribosomal RNA sequences. Tree was constructed by using the MEGA6 program [15].
Scale indicates the number of base substitutions per site. GenBank accession numbers are listed after species.

? ?
Monozoic tissue cyst

Caryospora-like oocyst Mature oocyst Sporozoites

with sporozoites with sporozoites Merozoites

Immature oocyst Mature oocyst

with sporocysts

Sporogony in
environment Endodyogeny/
schizogony
Microgametes
Immature oocyst
with sporoblasts

Fertilization

Caryospora-like Oocyst in stool

oocyst with sporoblast
Macrogamete
Gametogony

FIGURE 38.2  Life cycle of Cystoisospora belli depicting asexual multiplication, sexual development, and sporogony.
(Illustration by Urassaya Pattanawong.)

stage of C. belli is characterized by an oval or rugby-shaped oocyst with a wide range of size variation
in terms of the maximum length and width, both within and between isolates. The average length mea-
sured from more than 700 oocysts is 28.3 μm (range = 17–37). A remarkable narrowing with a neck-like
appearance may be observed at one end of some oocysts, whereas most oocysts are nearly symmetric
at both ends. The maximum width of oocysts averages 13.5 μm (range = 8–21). The mean shape index
or the ratio of length to width of oocysts is 2.1 (range = 1.3–3.3) [9]. The oocyst wall is bilayer, thin,
602 Laboratory Models for Foodborne Infections

smooth, and transparent for all stages of development. The inner wall is membranous, and the outer
wall is rigid and relatively impermeable to external fluids [16]. When freshly passed in feces, the oocyst
remains immature, containing a spherical or slightly elongated, oval-shaped sporoblast that develops
within another rigid cyst wall called sporocyst. Occasionally, some freshly passed oocysts in stool may
contain two sporocysts, each of which possesses one sporoblast. The sporocyst of C. belli lacks a pro-
teinaceous plug, known as a Stieda body, a structure existing in the genus Isospora. The dimension of
sporocyst is 12–14 μm × 7–9 μm.
Sporogony, the process of producing infective sporozoites of Cystoisospora, usually occurs outside the
host (Figure 38.2). The maturation period of oocysts usually completes within 24 h to 10 days [9]. The
mature oocyst possesses two sporocysts, each with four crescentic or banana-shaped sporozoites cluster-
ing together with a clump of granular mass or residual bodies surrounded by sporocyst wall (Figure 38.3).
Detailed investigation of ex vivo development has shown that the percentage of oocyst maturation varies
between isolates. In vitro studies reveal that only 27%–30% of oocysts excreted from stools of patients
become fully sporulated at ambient temperature. However, sporulation process requires optimum condi-
tions that could be variable among studies. Importantly, despite the frequently identified characteristic
mature oocysts containing two sporocysts, about 5% of the oocysts that undergo the maturation pro-
cess possess eight sporozoites in a single sporocyst akin to oocysts of Caryospora, a member of fam-
ily Eimeriidae [9,17]. The formation of Caryospora-like oocysts requires 5–14 days after excretion in
feces [9]. Both types of mature oocysts contain a number of granular residual bodies clumping together.
Environmental factors such as moisture, temperature, and oxygen composition in the atmosphere have
been suggested to influence the maturation of Cystoisospora oocysts. It is noteworthy that incubation
of C. rivolta oocysts at 50°C for 5 min enhances development of Caryospora-like oocysts, and they are
infective to mice and cats [18]. It is therefore likely that Caryospora-like oocysts of C. belli could also
be infective to humans.
Sporozoites are released from the oocysts and become free in the lumen of small intestine, espe-
cially around distal duodenum and jejunum. Thereafter, sporozoites invade small intestinal epithelial
cells where asexual multiplication ensues. All endogenous stages of Cystoisospora reside within para-
sitophorous vacuoles in the cytoplasm of host cells. Sporozoites of mammalian Cystoisospora species,
for example, C. suis, C. rivolta, and C. felis, initially undergo asexual multiplication by endodyogeny,
a cellular division process in which two daughter cells are generated from a mother cell so that these
daughter cells are surrounded by the mother cell membrane [19–21]. Repeated endodyogeny probably
occurs and generates at least three structurally distinct types of merozoites in C. suis, whereas schizo-
gonic development with repeated nuclear division within a single cytoplasmic mass possibly occurs in
epithelium of upper small intestine [22,23]. However, the number of generations of merozoites remains
unknown. Eventually, some merozoites develop into sexual stages consisting of female and male game-
tocytes. Although detailed knowledge on asexual development of C. belli remains to be elucidated,
it could resemble these porcine and feline species of Cystoisospora. In vitro studies have shown that
endodyogenic development of C. belli occurs after inoculation of sporozoites into cell cultures resulting
in the formation of merozoites, while histopathological studies demonstrated multinucleate schizonts
in infected enterocytes [11–13,24–27]. Fully developed merozoites disintegrate host cells, invade other
epithelial cells, and undergo further asexual multiplication cycles (Figure 38.4).

(A) (B) (C) (D) (E)

FIGURE 38.3  Oocysts of Cystoisospora belli. (A) Oocyst from freshly passed stool containing one sporocyst. (B) and
(C) Developing oocysts with two sporoblasts. (D) Mature oocyst with two sporocysts, each with four sporozoites.
(E) Caryospora-like oocyst with eight sporozoites in one sporocyst. Scale = 10 μm.
Cystoisospora belli 603

(A) (B) (C)

FIGURE 38.4  Upper intestinal biopsy showing (A) shortening of villi, (B) and (C) endogenous stages of Isospora belli in
epithelium cells. Single and broken arrows indicate merozoites/schizonts and oocysts, respectively.

Extraintestinal monozoic tissue cysts of C. belli have been found in sections of lymph nodes, spleen,
and liver at autopsy of AIDS patients, suggesting that these stages may represent hypnozoites or dormant
forms that could be responsible for relapse of symptoms [11,12,28–30]. Ultrastructural studies reveal
that the extraintestinal monozoic tissue cyst is surrounded by a parasitophorous vacuole membrane and
characterized by oval or ellipsoidal body measuring about 11 μm × 8 μm. A conspicuous cyst wall is
observed with a thickness varying from 0.7 to 4 μm, enclosing a crescent-shaped zoite. Occasionally,
the surface of monozoic tissue cyst possesses grooves or small projections. The zoite inside monozoic
tissue cyst resembles the sporozoite stage and contains a single rather than multiple nuclei. The anterior
part of these zoites possesses polysaccharide-like and lipid-like granules, and apical organelles consist-
ing of rhoptries, micronemes, dense bodies, and a conoid. Each zoite is surrounded by a parasitopho-
rous vacuole and granular materials forming the cyst wall. Monozoic tissue cysts enlarge over time
without cellular division. The monozoic tissue cysts of C. canis in mouse, a paratenic host, are infective
to dogs similar to ingestion of oocysts [31]. Although the origin of this developmental process remains
elusive, abundant numbers of tissue cysts found at autopsy favor that they probably originated from
merozoites rather than sporozoites from the oocyst stage [28–30]. It is noteworthy that extraintestinal
stages and monozoic tissue cysts have also been found in C. felis after oral inoculation of the oocysts
into kittens [10,32,33].
Sexual reproduction or gametogony usually occurs after initiation of the asexual cycle for about
1 week. Female gamont develops intracellularly without nuclear division and becomes macrogamete,
whereas male gamont undergoes multiple nuclear and cytoplasmic divisions, resulting in the production
of a number of microgametes. Microgametes disrupt host cells and enter into other epithelial cells where
macrogametes are located. After fertilization, zygote is formed within the epithelial cell and sequentially
develops into an oocyst. Intraepithelial oocysts containing granular bodies are excreted in feces upon
rupture of host cell. The oocysts are resistant to the hostile environment, including common disinfec-
tants, and remain viable in cool and moist condition for months [14].

38.1.3 Epidemiology
Transmission of C. belli is solely anthroponotic because humans are the only known natural hosts.
Before 1935, the prevalence of human cystoisosporiasis seemed to be have been low as only 200 cases
were reported [34]. However, in certain circumstances when living condition is overcrowded or sani-
tation is below the standard level, cases of C. belli infections could be encountered. The majority of
infected cases were identified mainly among military or related personnel during 1914–1921 [2]. Until
1961, after awareness of the infection along with the competency of microscopic detection, more than
800 cases were detected in various localities in South America [34]. Before the pandemic of HIV infec-
tions, sporadic or endemic cystoisosporiasis has been reported from several countries both in tropical
and temperate zones. The infection rates of C. belli among immunocompetent individuals are usually
604 Laboratory Models for Foodborne Infections

<2%, while a remarkable higher rate was observed among those with underlying immunodeficiency.
The prevalence of cystoisosporiasis in AIDS patients in Zaire, Haiti, Venezuela, Brazil, and Thailand
is 19%, 15%, 14%, 9.9%, and 7.6%, respectively [9,35–38]. Nevertheless, endemicity, public sanitation,
preexisting anticoccidial drug treatment, and other factors may also affect such prevalence. An 8-year
surveillance of intestinal infections among AIDS patients in Los Angeles County revealed the presence
of C.  belli in 1% of pathogen-positive stool samples [39]. Interestingly, most of the C. belli-positive
samples were from foreign-born patients, especially those from Central America. Travel-related infec-
tions may be a contributing factor.

38.1.4 Pathology and Clinical Aspects

Infection with C. belli is usually confined to mucosal epithelial cells of upper small intestine.
Endogenous stages vary in size and structure depending on their growth and development. Both
asexual multiplication and sporogonic developmental cycles may persist if untreated or probably due
to the failure of gut immune response to control the progression of infection. No obvious patho-
logical change is observed in the early phase of infection except for some leucocyte infiltration in
mucosa and lamina propria, most of which are plasma cells, lymphocytes, and eosinophils [11,12]. In
chronic cystoisosporiasis, pathological features include shortening or blunting of villi or even atrophy,
hypertrophied crypts, and lamina propria infiltrated with eosinophils, lymphocytes, and neutrophils
[4,40,41]. In some cases, histopathology of cystoisosporiasis may masquerade eosinophilic enteri-
tis [5]. However, administration of corticosteroid cannot alleviate the symptoms or even worsen its
clinical course. Postmortem examinations reveal that some cystoisosporiasis cases who had underly-
ing AIDS exhibit extraintestinal involvement of lymphoid tissues, causing enlargement of mesenteric
lymph nodes, liver, and spleen [11,12,28,29].
A spectrum of clinical manifestations of cystoisosporiasis has been noted. Although both immu-
nocompetent and immunocompromised patients are susceptible to C. belli infections, higher prev-
alence of cystoisosporiasis afflicts the latter, especially in AIDS patients. Incubation period takes
about 1 week after ingestion of infective oocysts [42,43]. Asymptomatic infection with spontaneous
clearance of oocysts is rarely observed. Symptomatic infections with C. belli in immunocompetent
hosts present with watery diarrhea, abdominal discomfort, colicky abdominal pain, low-grade fever,
malaise, anorexia, nausea, vomiting, headache, and dehydration. Experimental infections in human
volunteers show that symptoms precede patency of oocysts in stool for a few days. The duration of
symptoms continues for about 1 day, whereas oocysts are discharged in stool for more than 1 month
[42]. Diarrheal episode in some patients can be severe with up to 20 stools per day [44]. Diarrheal
symptoms usually become chronic if left undiagnosed. Chronic infection may turn to malnutrition,
steatorrhea, and cachexia. The excretion of oocysts may last for months or years in chronic cystoiso-
sporiasis. A few reports have shown chronic diarrheal symptoms lasting for over a decade in immu-
nocompetent patients [4,5,41].
Cystoisosporiasis in immunosuppressed patients, such as those who receive immunosuppressive drugs,
prolonged corticosteroid treatment, and more commonly in AIDS patients, is generally more severe than
those with normal immune status in terms of duration of symptoms and volume of stools. The diarrheal
symptoms caused by C. belli seem to be a secretory process that may lead to massive fluid loss, resulting
in dehydration and electrolyte imbalance. Fever is frequently observed in dehydrated cases. Coinfections
with other enteric pathogens can be detected, especially in AIDS patients [9,36,37,45].
Recurrent symptomatic cystoisosporiasis occurs in both immunocompetent and immunodeficient
individuals. In studies of cystoisosporiasis in Haitian AIDS patients, relapse occurs in almost half of
the cases [36,45]. Extraintestinal cystoisosporiasis identified in lymphoid tissues at postmortem in AIDS
patients has suggested that these stages could reinvade mucosal epithelial cells and are responsible for
clinical relapse [11,28,29]. Although relapse is believed to be caused by reactivation of these dormant
forms or monozoic tissue cysts, its pathogenesis remains to be elucidated. An unusual case of endome-
trial cystoisosporiasis has been reported in an otherwise immunologically uncompromised host present-
ing with granulomatous endometritis. Numerous intracytoplasmic cysts resembling C. belli oocysts, but
not unicellular zoite, were found in the endometrial layer of the uterus [46].
Cystoisospora belli 605

TABLE 38.1
Comparative Characteristics of Cystoisospora belli, Cyclospora cayetanensis, Cryptosporidium spp., and
Sarcocystis hominis
Feature C. belli C. cayetanensis Cryptosporidium spp. S. hominis
First report in humans 1914 1977–1978 1974 1925
Location in enterocytes of Intracytoplasmic in Intracytoplasmic in Intracellular, Lamina propria
small intestine apical supranuclear apical extracytoplasmic at
region supranuclear luminal surface
region
Zoonotic potential No Unknown Yes Yes
Complete life cycle within Yes Yes Yes No
humans
Average size of oocyst 13.5 × 28.3 μm 8–10 μm 4.5 to >6 μm 13.5 × 19.5 μm
(sporocyst: 9.3 ×
14.7 μm)
Acid-fast staining of Acid-fast Variably acid-fact Acid-fast Not acid-fast (due
oocysts to thick
sporocyst wall)
Number of sporocysts in 2 (1 for Caryospora-like 2 No sporocyst 2
mature oocyst oocyst)
Number of sporozoites per 8 4 4 8
sporulated oocyst
Sporulation outside host Yes Yes No No
Autofluorescence of Yes Yes No Unknown. Oocyst
oocyst wall usually disrupted
when passed in
stool
Number of oocysts in Low Low to moderate Low to very high Low
stools
Infectivity of oocysts in No No Yes Yes
freshly passed stool
Resistance of oocysts to Yes Yes Yes Unknown
chlorine
Source: Herwaldt, B.L., Clin. Infect. Dis., 31, 1040, 2000.

Laboratory test frequently shows eosinophilia in the majority of infected cases, but a remarkable
a­ lteration of other hematological profiles is not observed. Patients with normal immune status tend
to have a higher level of eosinophils than those with suppressed immune system [9]. Charcot–Leyden
­crystals are frequently observed in stools of cystoisosporiasis patients, indicating active proliferation and
degradation of eosinophils in gut tissues. Because endogenous stages of C. belli neither cause apparent
ulceration of intestinal epithelial cells nor induce intense inflammatory response, a remarkable number
of erythrocyte or leucocyte is not found in stool. Identification of characteristic oocysts in stool allows
definite diagnosis in routine laboratory practice. The efficiency of detection depends on various factors
such as the microscopist’s experience, shedding pattern of oocysts that may be fluctuating during the
course of infection, as well as the choice of diagnostic techniques. Some differential features of coccid-
ian protozoa that shed their oocysts in host stool samples are listed in Table 38.1 [47].

38.1.5 Laboratory Diagnosis
38.1.5.1 Stool Examination
Definite diagnosis of cystoisosporiasis relies on identification of characteristic oocysts in stool or intesti-
nal content. Collection of stool samples containing or suspicious of having oocysts of C. belli essentially
follows standard guidelines for parasitological diagnosis. Although immediate examination of stool
606 Laboratory Models for Foodborne Infections

specimen is not a crucial issue as those for amoebic trophozoites, it is recommended to examine the
stool samples within 24 h after passage. If examination of stool sample is not possible within 1 day, stool
should be kept at less than 10°C, but not frozen, in order to minimize bacterial overgrowth and decay
of fecal materials [48]. Although coccidian oocysts can be preserved in 5% or 10% formalin solution
for microscopy-based diagnostic purpose, preservation of coccidian oocysts in ethanol also gives good
results for subsequent acid-fast stain. Structures of C. belli and Cryptosporidium oocysts kept in 80%
ethanol at ambient temperature for more than 2 years are also well preserved for acid-fast stains [9,49].
Microscopic examination of stool samples should be done by both direct smear and concentration
methods. For fresh stool samples containing mucus, direct smear sampling from the mucus portion
may yield better discovery than the watery part of stool as coccidian oocysts tend to stick to mucoid
substance. Meanwhile, C. belli oocysts usually do not excrete in large amount; therefore, they are
often diluted in voluminous diarrhea precluding efficient diagnosis by direct smear method. Oocysts of
C. belli can be concentrated without morphological alteration by formalin-ethylacetate (or ether) sedi-
mentation procedure to increase diagnostic yield [50]. Despite superiority of formalin-ethylacetate sedi-
mentation to simple smear method, simple sedimentation by centrifugation of previously sieved stool
samples may yield higher diagnostic probability than conventional formalin-ethylacetate sedimentation,
because some oocysts are lost to the fatty layer in the latter method [51]. It is noteworthy that oocysts of
C. belli can be overlooked in unstained stool samples because of their transparency when adjustment of
microscope does not make good contrast. Alternatively, various acid-fast staining methods such as modi-
fied Kinyoun acid-fast and modified Ziehl–Neelsen stains that intentionally deployed for detection of
Cryptosporidium spp. and Cyclospora oocysts are also useful for diagnosing C. belli oocysts. Specimens
can be versatile ranging from fresh stool, sediment from formalin-ethylacetate sedimentation, ethanol-
preserved stool sample, duodenal fluid, and other body fluids [52].
Oocysts of C. belli, Cyclospora cayetanensis, and Cryptosporidium spp. become autofluorescent
bright green under violet excitation (405 nm), bluish violet autofluorescent under ultraviolet excitation
(365 nm), and green autofluorescent under blue-violet light excitation (436 nm) [53]. Faint reddish fluo-
rescence is also observed under green light (546 nm). Characteristic size, shape, and structure of C. belli
oocysts under autofluorescence microscopy differ from those of C. cayetanensis and Cryptosporidium
spp., and thus, the method is useful for diagnosis. Autofluorescence is proven to be more sensitive than
bright-field microscopy in detecting Cystoisospora oocysts [54,55].

38.1.5.2 Examination of Duodenal Content

Duodenal content can be obtained from various procedures such as duodenal capsule technique or duo-
denal aspirate during endoscopic examination of upper gastrointestinal mucosa [56]. Like Giardia intes-
tinalis and Strongyloides stercoralis, aspiration of duodenal content for direct examination of C. belli
can be an adjunctive source other than stool samples that should be submitted to parasitology laboratory
without preservative. Examination using wet smear method should be done soon to visualize motility
of parasites such as falling leaf pattern of G. intestinalis trophozoites and rapid sine-curve movement
of S. stercoralis larvae. If the sample cannot be examined within a few hours after collection, keeping
at <10°C is recommended. Preservation of the content with 5%–10% formalin solution is adequate for
parasitological detection but not for molecular analysis. Oocysts of C. belli can be seen by direct smear
of duodenal content or the acid-fast staining procedure. Mucus in the intestinal tract attracts a number of
oocysts so that the sampling of content containing mucus material may help in increasing the recovery
rate. In case of voluminous content, examination of sediment after centrifugation at 500 × g for 10 min
should be done. If the volume of the content is small and precludes several preparative diagnostic meth-
ods, some authors suggest processing the sample for staining procedure or molecular diagnosis, instead
of preparing for direct wet smear examination [56].

38.1.5.3 Histopathological Diagnosis
Biopsy material from upper small intestine during endoscopic examination provides a useful diagnostic
sample for C. belli as well as other pathogens that parasitize these regions of the gastrointestinal tract
Cystoisospora belli 607

such as G. intestinalis, Cryptosporidium spp., and C. cayetanensis. Biopsy material should never be
allowed to dry but should be fixed with 10% formalin solution, embedded in paraffin or equivalent mate-
rials, and stained with hematoxylin-eosin. Biopsy materials are also good sample sources for molecular
detection provided that they should be kept frozen at less than –20°C without preservatives. The endog-
enous stages of C. belli consisting of asexual and sexual development can be seen in the cytoplasmic
mass of enterocytes or at the lamina propria of upper small intestine, although involvement of biliary
ducts and large intestine has been reported [11,12,28]. Duodenal mucosal architecture of patients with
chronic cystoisosporiasis usually presents with blunting of villi and hypertrophied crypts. Inflammatory
responses in upper intestinal tissues of both cystoisosporiasis and cyclosporiasis are similar, showing
mixed infiltration of polymorphonuclear cells, plasma cells, lymphocytes, and eosinophils, although
the eosinophilic infiltration seems to be more prominent in cystoisosporiasis [40,57]. The presence of
characteristic immature oocysts of C. belli in the intestinal epithelium cells assists in definite diagnosis.
Meanwhile, all endogenous stages of Cryptosporidium spp. are located at the intracellular rim at the
luminal surface of mucosal epithelium, and the diameters of all stages do not exceed 6 μm, making them
easily differentiable from C. belli [58,59]. Extraintestinal stages of C. belli, existing predominantly in
mesenteric lymph nodes, liver, and spleen of some AIDS patients, are mostly crescent-shaped, centrally
located, and contain only one zoite in the tissue cyst. The presence of longitudinal grooves or projections
of the zoite surface may represent simultaneous invasion of two zoites in the same host cell [60]. Neither
multinucleate nor sexual stages exist in the tissue cyst of cystoisosporiasis [30]. Although stool exami-
nation often gives more sensitive results than histopathology, the sampling areas of biopsy are limited
and may not include the affected regions. However, a large study involving 118 adult AIDS patients who
suffered from chronic diarrhea identified C. belli from duodenal biopsy samples in two patients who had
negative results in stool examinations [28].

38.1.5.4 Molecular Diagnosis
Amplification of a specific region in the small subunit ribosomal RNA gene (18S rDNA) of C. belli
by nested polymerase chain reaction (PCR) has been successfully applied to duodenal biopsy and bile
and stool samples without cross-reactivity to other enteric protozoa [8,61]. PCR targeting the 5.8S
ribosomal RNA gene and the internal transcribed spacer II (ITS2) region is proven to be more sensi-
tive than microscopy in diagnosing C. belli [62]. Likewise, quantitative PCR assay with melting curve
analysis using primers derived from the 18S rDNA can differentiate human enteric coccidian proto-
zoa [63]. Diagnosis of C. belli infection by PCR involves the isolation of C. belli DNA from clinical
specimens that can be stool, duodenal fluid, tissue sample, or other potential sources of infections. The
source of C. belli DNA is usually from oocyst stage in stool, while tissue sample contains any stages
of endogenous development. Freshly collected stool specimen, frozen stool sample, or stool preserved
in 80% ethanol can be used for extraction of C. belli DNA. Ethanol in preserved stool samples should
be removed before disruption of oocysts. Duodenal fluid or tissue samples can be kept at −20°C, and
ethanol should not be used for preservation of these samples. Because the oocyst wall of coccidian
protozoa is solid and resistant to diverse environmental conditions, it is recommended that the oocyst
wall should be ruptured so that internal contents, either sporoblasts or sporozoites, are exposed to DNA
extraction solution. Examples of simple procedures to disrupt oocyst wall of C. belli are mechanical
disruption and freeze-thaw method.

38.1.6 Treatment and Prevention

The standard therapy of cystoisosporiasis is a combination of trimethoprim (160 mg) and sulfamethox-
azole (800 mg) given orally 4 times a day for 10 days [36,45]. Usually, the patients respond well to
treatment with symptomatic and parasitological clearance within a few days after initiation of therapy.
Pyrimethamine (50−75 mg/day) is an alternative drug for those who are intolerant or allergic to anti-sulfa
agents. The pharmacological effects of these compounds involve inhibition of parasite folate biosynthe-
sis pathway. Sulfamethoxazole is absorbed well after oral administration and excreted relatively slowly
with a serum half-life of 11 h [64]. Like other sulfonamides, the drug acts by competitive inhibition
608 Laboratory Models for Foodborne Infections

of dihydropteroate synthase, resulting in interruption of 7,8 hydropteroate synthesis from para-amino

benzoic acid and pteridine in the folate biosynthesis pathway [6,64–66]. The drug is usually marketed
in fixed-dose combinations with trimethoprim, which is a potent competitive inhibitor of dihydrofolate
reductase that converts dihydrofolate to tetrahydrofolate in the folate pathway; therefore, these combi-
nations exert synergistic antimicrobial effect [67]. Although resistance to these combinations has been
observed in several pathogenic bacteria, no clear evidence is reported for treatment of cystoisosporiasis.
However, a high rate of relapse has been observed in cystoisosporiasis, which requires maintenance
therapy with trimethoprim–sulfamethoxazole given twice daily for 3 weeks [36,45]. Untoward effects
of these compounds include gastrointestinal disturbance, folate deficiency in those who have low folate
reserve in tissues, and various forms of dermatological adverse drug reactions. In some obstinate cases,
long-term administration with alternative drugs such as pyrimethamine, a dihydrofolate reductase inhib-
itor, can alleviate or confer long-term remission of symptoms [5].
There is no known animal reservoir of C. belli, and transmission is likely to be foodborne or water-
borne; therefore, taking food safety precautions and drinking properly treated water should prevent
acquiring C. belli infection as well as other enteric pathogens. Good personal hygiene and appropriate
sanitary disposal of infected stools can interrupt transmission of this enteric coccidian.

38.2 Laboratory Models
38.2.1 Animal Models
A number of animals have been tested for the susceptibility of infection with orally administered
oocysts of C. belli. These include kittens, puppies, guinea pigs, mice, rats, rabbits, and rhesus macaques
[14,68,69]. However, no consistent evidence of infection has been demonstrated in these studies.

38.2.2  In Vitro Growth in Mammalian Cells

38.2.2.1 Preparation of Infective Oocysts
The fundamental step for in vitro growth study of C. belli in mammalian cells is to obtain viable spo-
rozoites that are suitable for inoculation into cell culture system. The lack of known reservoir hosts
of C. belli and no available standard frozen strain of this protozoa make it necessary to isolate viable
oocysts from infected individuals. After obtaining fresh stool samples from infected human subjects
prior to treatment, add equal or more volume of 2.5% potassium dichromate solution to the samples, and
stir until complete mixing occurs. Stools containing a lot of fecal debris or substances should be filtered
through cheesecloth before adding dichromate solution to stool filtrate. Potassium dichromate confers
antimicrobial activity but does not affect viability of coccidian oocysts while it also provides additional
oxygen source during the process of oocyst sporulation. During storage of stool samples, it is recom-
mended not to fill the tube or collection container all the way to the top, but leave a layer of air between
the top of the feces-dichromate mixture and the cap of collection tube or container to allow the oocysts
expose to some atmospheric oxygen. Sporulation is better achieved by spreading fecal-dichromate mix-
ture into a Petri dish. Complete sporulation of C. belli oocysts usually requires 2 days to 1 week by keep-
ing samples at room temperature (20°C–25°C) [70]. After complete sporulation, potassium dichromate
is removed, and sporulated oocysts are purified. The following method for purification of viable C. belli
oocysts has been described by Oliveira-Silva et al. [24].

1. Removal of 2.5% potassium dichromate from sporulated oocyst suspension is done by adding
more than one volume of phosphate buffer saline solution (PBS, pH 7.2) containing 2% Tween
20 and is centrifuged at 1500 × g for 10 min at 4°C.
2. After discarding supernatant, PBS-Tween 20 solution and diethylether (2:1 v/v) are added to the
pellet for removal of lipid portion of the sample, and centrifugation is repeated under the same
condition. The supernatant is discarded.
Cystoisospora belli 609

3. Further purification of oocysts is done by discontinuous gradient flotation. To obtain better

purification, it requires prior knowledge of the approximate specific gravity of the oocysts.
However, the sucrose gradient of 1.05 and 1.15 g/mL reportedly offers acceptable results. For
gradient preparation, sucrose solutions are allowed to run down steadily inside the 15 mL tube.
More dense sucrose solution must be added to the tube before layering less dense sucrose solu-
tion. The aqueous suspension containing oocysts is then layered on top of the gradient solu-
tion and centrifuged at 1500 × g for 20 min. The oocysts will form a thin layer at the interface
between the two sucrose gradient layers that also separates the oocysts from both heavier and
lighter contaminants.
4. The layer containing oocysts is carefully pipetted to a new tube, and PBS is added for washing
by centrifugation at 1500 × g for 10 min. The washing step is repeated twice.
5. After the purification step, the oocysts are subject to further cleaning by adding 1% sodium
hypochlorite solution and allowed to stand for 10 min at 4°C.
6. Subsequent washing of the pellets is done thrice by adding normal saline solution and cen-
trifuged at 1500 × g for 10 min. Optionally, a small aliquot of the pellet containing purified
oocysts can be sampled for enumeration of oocysts using Neubauer chamber.
7. The purified oocysts are diluted with PBS (pH 7.2) containing 1.5% sodium taurocholate and
0.5% trypsin. Excystation is done by incubation at 37°C for 30 min.
8. To neutralize trypsin, Eagle’s minimum essential medium (MEM) supplemented with 10%
fetal bovine serum is added to the excysted sporozoites and is centrifuged at 1500 × g for
10 min.
9. The number of sporozoites is estimated using the Neubauer chamber.
10. The viability of sporozoites can be assessed by observation of their movement. Alternatively,
trypan blue exclusion test is more feasible to determine the viability of sporozoites by adding
1 volume of 0.4% solution of trypan blue in PBS (pH 7.2) to 10 volumes of cell suspension. After
loading the sample to Neubauer chamber, it is immediately examined under light microscope.
The number of blue staining cells should be counted, and total number of cells is examined
to estimate percentage of viable sporozoites. For each inoculation, the number of sporozoites
should be about 104 sporozoites per well or 106 sporozoites per 12.5 cm2 culture flask.

Alternatively, the following similar protocol has been described by Siripanth et al. for purification of
infective oocysts of C. belli [25].

1. Fecal samples containing oocysts of C. belli that have been sporulated in 2.5% potassium
dichromate solution are filtered through a few layers of gauze or cheesecloth. Subsequently,
oocysts are purified from the remaining fecal materials by sugar flotation. Preparation of sugar
solution is done by dissolving 454 g of table sugar in 355 mL water using gentle or indirect
heat. To keep the viability of oocysts, the originally recommended preservatives such as form-
aldehyde and phenol are not added in the sugar solution. The specific gravity of sugar solution
should be about 1.27. After adding filtered stool sample to a 15 mL centrifuge tube, the tube
is filled with sugar solution until the level reaches below the top of the tube. A lid or cap is
attached if the tube has screw cap, and it is then centrifuged at 280 × g for 5 min. A centrifuge
that has swinging cup should be used. Slowly sugar solution is added by carefully inserting tip
of pipette below the surface to create a slightly inverted meniscus. Oocysts at the surface of the
solution can be subsequently harvested [71].
2. After obtaining the oocysts from the flotation procedure, the sample is washed thrice by adding
distilled water and is centrifuged at 1000 × g for 10 min.
3. 2% sodium hypochlorite solution is added to the sediment containing oocysts and is left on ice
for 10 min.
4. The sample with PBS is washed by centrifugation at 1000 × g for 10 min. This step is repeated
twice.
610 Laboratory Models for Foodborne Infections

5. The sediment is resuspended in Eagle’s MEM medium containing 80 μg/mL gentamicin and
kept at 4°C until use.
6. A Teflon-coated tissue grinder is used to break oocyst wall for releasing sporocysts.
7. The sporocysts are resuspended in sterile 3% sodium taurocholate solution containing 1% tryp-
sin in MEM medium. It is then incubated at 37°C in a CO2 incubator for 2 h.
8. Sporozoites are harvested by centrifugation at 1000 × g for 10 min and washed twice with PBS.
9. The number of oocysts is estimated using Neubauer chamber.
10. Sporozoite viability is checked by using trypan blue exclusion test.

38.2.2.2 Inoculation of Sporozoites into Cell Lines

Mammalian cells reportedly supported in vitro multiplication of C. belli sporozoites are human ileoce-
cal adenocarcinoma (Hct-8), human larynx carcinoma (Hep-2), human lung carcinoma (A-549), African
monkey kidney (VERO cells), bovine epithelial kidney (BEK), and rhesus monkey kidney cells (MK2).
These cell lines are maintained per standard cell culture protocol using appropriate culture media.
Oliveira-Silva, Resende, and colleagues report that VERO cells can support the growth of C. belli better
than Hct-8 and A-549 cells in which the parasites invade these cell lines between 4 and 12 h post inocula-
tion and undergo asexual multiplication after 24 h as demonstrated by the presence of paired merozoites
inside the cytoplasm of these cells [24,27]. Meanwhile, Siripanth et al. observed complete schizogonic
development in Hct-8 cells after 5 days of sporozoite inoculation into the cell culture. After the second
passage of the culture, sporogonic development and formation of microgametocytes reportedly occurred
within 5 days [25]. Furthermore, it is also noted that Hep-2 cells could support the growth of C. belli
as the parasites developed to early schizogony, whereas monozoic cysts could be observed in both BEK
and VERO cells. However, complete developmental cycle with the formation of oocysts could not be
achieved [25].

38.2.2.2.1 Preparation of Cell Culture

Usually, standard cell lines are available from cell banks as frozen stabilates kept in liquid nitrogen.
Initiation of cell culture essentially follows the standard procedure as follows:

1. The frozen cells are thawed by placing the vial immediately in a water bath preset at 37°C.
2. Before opening, the vial is cleaned thoroughly with 70% ethanol.
3. The content is transferred from the vial to a 15 mL tube containing appropriate culture medium.
4. It is centrifuged at 200 × g for 5 min and supernatant is discarded.
5. The remaining pellets containing cells are transferred to a new culture flask preadded with
appropriate cell culture medium. It is mixed by gently pipetting.
6. It is incubated at 37°C. The culture flask is examined using inverted microscope.

38.2.2.3 Inoculation of Sporozoites into Human and Murine Macrophages

Resende et al. succeeded in growing C. belli sporozoites using human and murine macrophages as host
cells [26]. Sporozoites reportedly invaded host cells after 15–17 h post inoculation and multiplied by
endodyogeny within 24 h. Procedures for preparation of human and murine macrophages have been
described by Resende et al. as follows [26].

38.2.2.3.1 Preparation of Human Macrophages

1. Fresh venous blood sample treated with an anticoagulant (e.g., heparin, citrate) is prepared.
2. One volume of Ficoll-Hypaque gradient (v/v) solution is added to a sterile tube.
3. One volume of the blood sample is carefully and slowly layered over the density gradient
medium and centrifuged at 400 × g for 20 min at room temperature in a swinging-bucket rotor
without brake.
Cystoisospora belli 611

4. After centrifugation, four layers appear consisting of plasma at the top, followed by a thin layer
of peripheral blood mononuclear cells (PBMCs), Ficoll-Hypaque layer, and erythrocytes mixed
with granulocytes at the bottom. PBMCs are collected either by gently removing the upper
plasma layer until the PBMCs can be harvested or by inserting the pipette tip directly through
the upper plasma layer to collect cells at the interface. The PBMCs are kept in a new tube.
5. The harvested cells are washed with RPMI-1640 supplemented with 20% fetal calf serum and
centrifuged at 200 × g for 20 min.
6. After discarding the supernatant, the cell pellet is resuspended with RPMI-1640 supplemented
with 20% fetal calf serum and kept in culture flask for 20 days or until complete differentiation
to macrophages with a density of 3 × 106 cells per 12.5 cm2 culture flask.

38.2.2.3.2 Preparation of Murine Macrophages

1. 10 mL of 0.9% NaCl solution containing heparin sodium 10 units/mL is aseptically injected
into peritoneal cavity of an albino mouse. The mouse abdomen is massaged for 30 s.
2. Fluid from mouse peritoneal cavity is aspirated using sterile syringe, and the content is trans-
ferred to a sterile 15 mL tube. It is then centrifuged at 720 × g for 10 min.
3. A small volume of pellet is used for counting the macrophages using a Neubauer chamber, and
viability of cells can be simultaneously evaluated by trypan blue exclusion test. Unstained cells
indicate viability.
4. RPMI-1640 supplemented with 20% fetal calf serum is added to resuspend macrophages. It is
kept in a 12.5 cm2 culture flask. A density of 3 × 106 cells/flask or 3 × 105 cells/well is appropri-
ate for sporozoite inoculation.

About 105–106 sporozoites are inoculated to the cell culture flask when host cells reach appropriate den-
sity. The culture flask is incubated at 37°C in a CO2 incubator. Growth and development of sporozoites
should be monitored every day under inverted microscope until no further development can be observed.
Infected cells can be examined by histopathological and ultrastructural studies.

38.3 Conclusion and Future Perspectives

Taxonomic repositioning of the formerly known Isospora belli to Cystoisospora belli has been based
on biological and phylogenetical characters of this diplosporocystic, tetrasporozoic oocyst-producing
coccidian protozoa recognized as a human pathogen almost a century ago. The genus Isospora, whose
sporocyst contains Stieda body, undergoes monoxenous life cycle development without any demon-
strable monozoic tissue cysts; therefore, no intermediate host exists. The avian parasites in the genus
Isospora, for example, I. robini and I. gryphoni, are phylogenetically closely related with the genera
Eimeria and Cyclospora (Figure 38.1). On the other hand, the genus Cystoisospora includes the for-
merly known mammalian Isospora species and I. belli, whose sporocyst lacks Stieda body and con-
tains monozoic tissue cysts. A number of optional intermediate or paratenic hosts harboring monozoic
tissue cysts have been reported in canine and feline Cystoisospora [60]. Monozoic tissue cysts that are
the dormant forms in these intermediate hosts can serve as an infective stage for transmission. The
efficacy of monozoic tissue cysts of C. felis, C. rivolta, and C. canis from murine paratenic hosts to
produce intestinal infections in their respective feline and canine definitive hosts was comparable to
that produced by infective oocysts [31,72]. Despite the presence of monozoic tissue cysts in cystoiso-
sporiasis in AIDS patients, it remains unknown whether these tissue cysts generally occur during
infections in immunocompetent persons. Meanwhile, optional intermediate or paratenic hosts have
not been clearly demonstrated in C. belli, although attempts to infect a calf and chickens with human-
derived oocysts failed to generate monozoic tissue cysts in these animals [29]. The presence of mono-
zoic tissue cysts observed in BEK and VERO cells after inoculation of C. belli sporozoites may not
directly indicate that bovine and simian hosts are paratenic hosts if host cell entry by Cystoisospora
resembles an active invasion process of Toxoplasma gondii, rendering the parasites to infect a variety
612 Laboratory Models for Foodborne Infections

of host cells. Undoubtedly, identification of natural cryptic optional intermediate or paratenic hosts for
C. belli has significant biological and medical implications.
Developmental cycle of C. belli consists of both asexual and sexual reproductions in a single host.
Freshly excreted oocysts from infected human stools are not readily infective but require some time in
environment to reach maturation, producing eight infective, crescentic-shaped sporozoites per oocyst.
The oocysts are environmentally resistant and can retain viability for months at low temperature.
Invasion of upper small intestinal enterocytes by sporozoites initiates asexual developmental cycle where
endodyogeny and possibly schizogony ensue. However, extraintestinal unicellular zoites or monozoic tis-
sue cysts, found mostly in lymphoid tissues of some AIDS patients, are suggestive of dormant stage that
could be responsible for relapse in almost half of symptomatic isosporiasis cases. The high rate of relapse
in C. belli infection suggests the presence of resistant cryptic stage that is not responsive to treatment. It
remains to be explored whether monozoic tissue cysts of C. belli directly contribute to relapses and what
reactivates their development. Although the majority of multiple relapses in cystoisosporiasis occurred
in humans with compromised immunity, some immunocompetent individuals reportedly suffered from
recurrent relapsing symptoms. It seems that the interplay between host immunity and parasite virulent
factors determines clinical course of C. belli infection. However, the lack of natural reservoir host for
C. belli has hindered experimental investigation to elucidate this issue. Therefore, modern molecular
biology techniques will be important alternative strategies to unravel several unknown aspects of patho-
genesis of human cystoisosporiasis. However, establishment of complete developmental cycle of this
coccidian parasite in cell culture system is mandatory to shed light on this neglected but important
human enteric coccidian pathogen.

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1. Manwell, R.D. Coccidia. In Manwell, R.D. (ed.), Introduction to Protozoology, 1st ed. St. Martin’s
Press, New York, pp. 515–538, 1961.
2. Wenyon, C.M. Coccidiosis of cats and dogs and the status of the Isospora of man. Ann. Trop. Med.
Parasitol. 17, 231, 1923.
3. Elsdon-Dew, R. Isospora natalensis (sp. Nov.) in man. J. Trop. Med. Hyg. 56, 149, 1953.
4. Ravenel, J.M., Suggs, J.L., and Legerton, C.W. Human coccidiosis. Recurrent diarrhea of 26 years dura-
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39
Entamoeba histolytica

Mineko Shibayama, Nidia León-Sicairos, Jesús Serrano-Luna, and Mireya de la Garza

CONTENTS
39.1 Introduction....................................................................................................................................617
39.1.1 Human Amoebiasis...........................................................................................................617
39.1.2 Life Cycle of E. histolytica................................................................................................618
39.1.3 Isolation and Culture Conditions.......................................................................................618
39.1.4 Virulence Factors..............................................................................................................618
39.2 Animal Models of Amoebiasis......................................................................................................619
39.2.1 Intestinal Amoebiasis........................................................................................................619
39.2.1.1 Rats..................................................................................................................619
39.2.1.2 G  uinea Pigs and Hamsters.............................................................................. 622
39.2.1.3 R  abbits............................................................................................................ 622
39.2.1.4 G  erbils............................................................................................................. 622
39.2.1.5 P  igs.................................................................................................................. 622
39.2.1.6 M  ice Models................................................................................................... 623
39.2.1.7 C3H/HeJ Mice................................................................................................ 623
39.2.1.8 C  57BL/6 IL-10-Deficient Mouse.................................................................... 623
39.2.1.9 SCID-HU-INT Mouse.................................................................................... 623
39.2.1.10 Human Colonic Explants Model.................................................................... 623
39.2.2 Amoebic Liver Abscess.................................................................................................... 624
39.2.3 Animal Models for Amoebiasis Treatment...................................................................... 626
39.2.4 Animal Models for Amoebic Vaccine Research.............................................................. 627
39.3 Conclusions and Future Directions............................................................................................... 628
Acknowledgments................................................................................................................................... 629
References............................................................................................................................................... 629

39.1 Introduction
39.1.1 Human Amoebiasis
Amoebiasis is a parasitic infection that is caused by an extracellular enteric protozoan named Entamoeba
histolytica. This parasite is able to coexist with the human intestine through a tolerogenic/hyporespon-
sive immune reaction, and the intestinal barrier is not broken. Through an unknown mechanism, the
host switches perception of the parasites from commensal to invaders and mounts an acute inflammatory
response that leads to colonic lesions of diverse magnitude. The symptoms of intestinal amoebiasis (IA)
are abdominal pain, dysentery, and ulcerative colitis with mucous and blood, which can, in some cases,
evolve into appendicitis and amoeboma, which is an acute manifestation of the disease. Rarely, the
parasites can spread through portal circulation to the liver and produce the main extraintestinal infec-
tion, amoebic liver abscess (ALA). E. histolytica can also reach the lungs and the brain, primarily in

617
618 Laboratory Models for Foodborne Infections

immunocompromised patients. Both IA and ALA can be fatal if left untreated. Amoebiasis is the third
leading cause of death due to parasites after malaria and schistosomiasis. This disease presents a high
index of morbidity and mortality, mainly in developing countries with poor hygiene conditions, where
it can be endemic. It can also affect travelers who visit those countries. According to the World Health
Organization (WHO) [1], 500 million people are infected with amoebae worldwide; 10% of them have
virulent E. histolytica, which leads to 40,000–100,000 deaths annually. Epidemiological studies dem-
onstrated the presence of antibodies (Abs) against E. histolytica in the populations of Mexico, Brazil,
Venezuela, Bangladesh, South Africa, and Egypt, showing the high prevalence of this parasite [2]. The
closely related nonpathogenic species E. dispar and E. moshkovskii are morphologically identical to
E. histolytica in standard stool microscopy analysis and are the cause of asymptomatic amoebiasis [3–8].
Studies of colon microbiota have been valuable in understanding the interplay among amoeba, the host,
and disease status [9–11]. Other host factors also influence the pathogenicity of amoeba, such as diet,
sex, and age [12].

39.1.2 Life Cycle of E. histolytica

The E. histolytica life cycle does not include a vector and consists of two main stages—trophozoite
and cyst. The cycle begins with the ingestion of mature quadrinucleate cysts (resistant forms, infective
phase), which tolerate the acidic pH of the stomach and excyst in the terminal ileum, where they produce
eight uninucleate trophozoites per cyst. Trophozoites, which are also called amoebae (motile forms,
invasive phase), travel to and colonize the large intestine. In response to unknown stimuli, the amoebae
undergo morphological and biochemical changes that lead to the formation of cysts, which are elimi-
nated in the feces and transmitted to other hosts via the ingestion of contaminated water or foods, thus
reinitiating the cycle [3,4,13].

39.1.3 Isolation and Culture Conditions

The excystation of E. histolytica cysts was first carried out in a medium with bacteria, but further
amoebae could be cultured axenically [14]. Currently, amoebae are cultured in vitro in BI-S-33
(TYI-S-33) medium, an axenic medium designed by Diamond et al. [15], which contains peptone,
glucose, ferric citrate, and ascorbic acid and cysteine as reducing compounds; the medium is also
supplemented with bovine serum and vitamins. The most frequently used strain in research is
HM-1:IMSS, which was isolated in 1971 by De la Torre et al. from feces of a patient suffering from
IA [16]. Because this strain has been grown in culture for years, it is less virulent, and in the case
of in vivo studies, amoebae are periodically passed through a susceptible animal, mainly hamster
(see Section 39.2.2).

39.1.4 Virulence Factors
E. histolytica is an active protist parasite that can adhere to the human epithelial mucosa and extracel-
lular matrix proteins, phagocytose erythrocytes and cell debris, secrete toxins and proteolytic enzymes,
cause apoptosis in target cells, and scavenge iron and other nutrients from the host [17–21]. Amoebiasis
is a multifactorial disease because host pathogen recognition receptors (PRRs), immune cells, amoeba
virulence factors, and microenvironment all contribute to the damage of the intestinal mucosa [4,22].
The contact between amoebae and target cells appears to be the first step in cell lysis and phagocytosis.
Intestinal flask-shaped ulcers, which are a hallmark of amoebic colitis, are characterized by severe dam-
age to enteric cells and migration to the lamina propria and blood vessels; an inflammatory reaction is
also present [20,23,24].
A wide variety of molecules have been described as E. histolytica virulence factors that act in con-
cert and favor amoebae invasion. For example, disruption of the colonic mucosa through amoebic
enzymes leads to direct contact between the parasites and the epithelium mainly through the amoebic
Gal/GalNAc lectin (Gal-lectin), which activates the inflammasome. The IL-1β released during invasion
Entamoeba histolytica 619

produces a severe inflammatory response [25]. Amoebapore polypeptides can lyse bacterial and host
cell membranes and are involved in liver abscess and cell apoptosis [26–29]. Several membrane and
secreted cysteine proteases (CPs) that degrade host proteins have been described in E. histolytica;
the most studied are CP1 and CP5 [30–33]. Currently, more than 50 genes are known to encode CPs,
and their expression depends on conditions such as the amoeba strain and culture conditions [34,35].
Lipophosphopeptidoglycan is a surface molecule that activates NKT cells in a CD1d-dependent manner,
and this interaction limits ALA development [25,36,37].

39.2 Animal Models of Amoebiasis

Presently, there is no animal model that can reproduce the whole life cycle of E. histolytica as seen
in humans. Therefore, the studies of experimental amoebiasis have been performed either with intes-
tinal or hepatic models as separate systems. However, the use of different animal strains that are sus-
ceptible and resistant to amoebiasis has led to a better understanding of the host–parasite relationship
regarding amoeba-secreted products and pathogenicity mechanisms, the role of neutrophils and other
host cells and molecules affecting invasiveness, sequential stages of tissue damage, and immune
response. Animal models have also enabled the testing of new therapeutic targets to treat amoebiasis.
In the case of IA, several strains of susceptible mice have been employed; hamsters and gerbils have
been mainly utilized to reproduce ALA [38–41]. In this chapter, we briefly describe the main models
used to study intestinal and hepatic amoebiasis, including the lesions caused by amoebae and treat-
ments for the disease.

39.2.1 Intestinal Amoebiasis
The first in vivo model of IA was developed by Lesh in 1875 [42]. He produced intestinal lesions in a
dog inoculated with feces from a patient suffering from dysentery. Since then, many studies have been
performed in different animals (dogs, cats, rabbits, hamsters, and monkeys); however, the use of these
models is limited at present due to several factors, including a lack of uniformity, difficulty in handling,
and an insufficient number of animals [41]. IA in animals has yielded inconsistent and poor results,
and in many cases, this difficulty has been simply attributed to their natural immune resistance [41,43].
Moreover, although experimental animals are inoculated with several million trophozoites of a known
specific virulent strain, this inoculum then mixes with poorly characterized intestinal flora consisting
of additional protozoa, bacteria, and other microorganisms. This mixture of intestinal organisms, and
our lack of knowledge of the physicochemical characteristics of the intestinal milieu, are important
factors that have not been considered in the past and are likely the primary causes of the heterogeneity
of results [41].
Differences in susceptibility have been reported by different groups, even when using similar labora-
tory animals under apparently identical conditions. Despite these limitations, interesting information
regarding the early stages of IA, pathology, and host immune response has been obtained [41,44–46].
At the molecular level, various research groups have studied the genes or gene products of E. histolytica
related to those of humans through the evaluation of severe combined immunodeficient (SCID) and
SCID-modified mice, microarray analysis, and examination of several aspects of the mechanisms of the
immune response. In the forthcoming sections, we analyze some of the models used to establish IA [43].
Table 39.1 presents the foremost models used in IA.

39.2.1.1 Rats
Wistar strain rats (mainly weanling), or colon loops of adult Sprague Dawley rats, have been used to
model IA. However, due to the great variability of the macroscopic appearance related to the damage
induced by E. histolytica trophozoites, this model is no longer used [47,48].
TABLE 39.1

620
The Foremost Models Used to Study Intestinal Amoebiasis
Animal Model Method Advantages/Main Findings Limitations Reference
Dogs Inoculation with feces from a patient suffering Intestinal lesions in some animals Results were not reproducible, the animals [42]
from dysentery resolved the infection
Newborn guinea pigs, hamsters Intracecal inoculation axenic cultures Lesions in cecum Results were not reproduced in a larger [49]
number of animals
C3H/HeCr, BALB/c, NZB/BIN, Intracecal inoculation of axenic cultures Susceptibility of mice to infection Only early states of ameobiasis can be [56]
B10.A, DBA/2, and C57BL/6 depended on the genetic charge of the studied
inbred mice strains
Conventionally raised animals Inoculation of axenic or monoxenic cultures Early stages of amoeba invasion inside the Only early states of amoebiasis can be [50]
(hamsters or Guinea pigs) on a closed loop of the cecum mucosal and submucosal layers with a studied
substantial inflammatory reaction
Adult Sprague Dawley rats Inoculation of axenic cultures into in vivo The mucus blanket provided a significant Only early states of amoebiasis can be [47]
colon loops barrier to trophozoite access to intestinal studied
epithelium target
Washed-closed cecal loop model An artificial cecal loop inoculated with It helped reported the possible role of Only early states of amoebiasis can be [24,50]
axenic or monoxenic cultures leukocytes in the development of the studied
amoebic ulcer
Balb/c mice Intracecal inoculation of axenic cultures in Trophozoites did not survive in the Some mice were resistant to disease [54]

Laboratory Models for Foodborne Infections

the cecal loop of animals animals, but they colonize longer in
animals with the cecal loop
Rabbit colon preparations Full-thickness rabbit colon preparations Lesions in colon were demonstrated by Only early events of amoebiasis [44]
mounted in Ussing-type chambers, increased decay rates for potential pathogenesis can be studied
incubated  with monoxenic cultures difference, short-circuit current and
transmural resistance
(Continued)
TABLE 39.1 (Continued)

Entamoeba histolytica
The Foremost Models Used to Study Intestinal Amoebiasis
Animal Model Method Advantages/Main Findings Limitations Reference
Mongolian gerbils Intracecal inoculation of gerbils with Microulcerative mucosal lesions appeared Normal cecal mucosa at 96 h postinfection [51]
monoxenic cultures 24–72 h postinoculation, inflammatory
infiltrates and edema of the lamina
propria
C3H/HeJ mice The cecum was directly inoculated with Amoebae release toxic factors that Only intestinal amoeabiasis developed [125]
monoxenic cultures contributed to the inflammatory disease
SCID mouse-human intestinal Human intestinal xenografts were infected Extensive tissue damage, an early Only early intestinal amoebiasis can be [59]
xenograft model with monoxenic cultures inflammatory response composed studied
primarily of neutrophils. IL-1 and IL-8
were produced
Porcine colonic fragments Trophozoites cocultured with porcine Severe acute ulcerative jejunitis, with Lesions in two of the four animals [52]
colonic fragments large hemorrhagic lesions. Typical
large-sized hepatic abscesses
Human colon model ex vivo Parasite penetration into the mucus Human colonic explants are far less [63]
and mucosa, cell lysis, and an available
inflammatory response

621
622 Laboratory Models for Foodborne Infections

39.2.1.2 Guinea Pigs and Hamsters

Only amoebae cultured in axenic conditions have been able to produce ulcers in intestines of guinea pigs.
Diamond induced lesions in newborn guinea pigs by inoculating intracecally; however, the results could
not be reproduced consistently in a larger number of animals [49]. Anaya-Velazquez et al. described
typical intestinal ulcers in guinea pigs and hamsters by producing an artificial cecal loop inoculated
with axenic or monoxenic E. histolytica trophozoites [50]. With this model, the authors showed the early
stages of amoeba invasion of the mucosal and submucosal layers with a substantial inflammatory reac-
tion. Electron microscopy analysis of the ulcers showed the possible role of leukocytes in the develop-
ment of IA [41]. This model is particularly useful for the study of early intestinal lesions produced by
virulent amoebae, and may also be applied to studies on the immunology of invasive IA [50].

39.2.1.3 Rabbits
To develop a model capable of reproducing the events that occur during the initial interaction of E. his-
tolytica trophozoites with the mucosa of the large intestine, Navarro-García used full-thickness rabbit
colon preparations (0.28 cm2) mounted in Ussing-type chambers. The untreated samples had electro-
physiological properties (potential difference, short-circuit current, and electrical resistance) that were
similar in magnitude and duration to those reported for stripped colonic mucosa. However, in samples
exposed to amoeba lysates for up to 80 min, dose-dependent lesions in the colon were observed and
consisted of (1) increased decay rates for potential difference, short-circuit current, and transmural resis-
tance and (2) mucosal lesions involving vacuolization at the bases and shortening of epithelial cells, loss
of intercellular junctions, destruction of microvilli, and necrosis of interglandular epithelial zones. The
authors discussed the specificity and speed of the electrophysiological effects and their correlation with
the microscopic lesions and suggested that this model will help increase our understanding of the initial
pathogenic events of IA [44].

39.2.1.4 Gerbils
By using monoxenic cultures of E. histolytica trophozoites inoculated in Mongolian gerbils, Shibayama
observed that an increase in mucus production occurred during the first hours of the interaction. Furthermore,
microulcerative mucosal lesions appeared 24–72 h postinoculation. Inflammatory infiltrate and edema of the
lamina propria were associated with necrotic foci. Unfortunately, at 96 h, the cecal mucosa was normal,
amoebae were no longer detected, and the gerbils healed spontaneously [51]. However, overall, it was con-
cluded that gerbils are useful as experimental models for studying the early stages of invasive IA.

39.2.1.5 Pigs
More recently, assays with trophozoites of virulent E. histolytica cocultured with porcine colonic frag-
ments have shown that outbred pigs can be used to reproduce some lesions associated with human IA.
A detailed analysis showed that loops inoculated with virulent amoebae showed severe acute ulcerative
jejunitis 14 days postinoculation, with large hemorrhagic areas associated with the presence of amoebae in
the depth of the mucosa in two of the four animals [52]. Furthermore, typical large hepatic abscesses were
observed in the liver of one animal at 7 days postinfection after inoculation by the portal vein or directly
into the liver parenchyma, which showed that the pig model could be useful in simultaneously studying IA
and ALA. Moreover, human colonic explants have been identified as valuable assets in the study of host–
parasite interactions. However, because human colonic explants are far less available, porcine colonic
explants have been investigated as an alternative to human tissues. Porcine colonic explants cultured with
virulent E. histolytica (HM1:IMSS) or an avirulent strain (Rahman) showed that explants cultured with
virulent trophozoites react similar to their human counterparts, including tissue invasion by amoebae and
the triggering of a typical innate immune response against the parasite. In contrast, explants cultured with
avirulent amoebae were healthy. The authors suggest that this study opens the way for the use of porcine
colonic explants in the study of the complex interactions between the parasite and the host [53].
Entamoeba histolytica 623

39.2.1.6 Mice Models
In the past, mice were not commonly used as a model for amoebiasis, primarily since these rodents were
always considered naturally resistant to E. histolytica infection [54]. Depending on the strain, mice pre-
sented different susceptibilities to developing IA; thus, the authors concluded that a genetic factor could
be involved in mouse susceptibility to intestinal infection. It was also postulated that other factors such
as a cholesterol-rich diet, association with bacteria, and the presence of other protozoa could influence
the results found in the different mice strains [43,54]. A neutropenic BALB/c mouse model was devel-
oped, in which the production of a granulomatous inflammatory reaction in the intestinal wall of mice
inoculated with axenically cultured amoebae was reported for the first time [55]. Assays in other strains
corroborated that the establishment of IA depends on the mouse strain; for example, C57BL/6 mice are
highly resistant, whereas C3H/HeJ mice are relatively susceptible. The amoebic colitis in these mice was
limited to the cecum, and the morphology of the inflammatory infiltrate was similar to that observed
in humans. This model of resistant versus susceptible mice could provide useful insight into the human
variability of parasite clearance when compared with invasive disease [56]. In the next section, we ana-
lyze the mice strains that are susceptible to intestinal amoebiasis.

39.2.1.7 C3H/HeJ Mice
In 2002, it was shown that C3H/HeJ mice, which have a mutation at the lipopolysaccharide response
locus, were 60% infected with amoeba after intracecal inoculation, whereas C57BL/6 or BALB/c mice
were resistant, including mice that were deficient for IL-12, IFN-γ, or inducible NO synthase. Infection
was a chronic cecum inflammation that pathologically mirrored the human disease. This model revealed
important immune factors that influence susceptibility to infection and, for the first time, established the
pathological contribution of the host immune response in amoebiasis [57]. The early steps in IA, such as
parasite adhesion to the mucosa, can be investigated in this model, and chronic infection can be obtained
after mechanical injury of the cecal epithelium.

39.2.1.8 C57BL/6 IL-10-Deficient Mouse

Use of this model showed that nonhematopoietic cells mediate the natural resistance of mice to IA, but
this resistance depends on hematopoietic IL-10 activity [58].

39.2.1.9 SCID-HU-INT Mouse
Infection of the SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease with amoe-
bae resulted in extensive tissue damage, which was associated with an early inflammatory response
composed primarily of neutrophils [59]. In this model, it was evidenced that human intestinal epithelial
cells can produce inflammatory cytokines in response to infection in vivo, and it was established as a
system for studying the interactions between E. histolytica and the human intestine [59,60]. Furthermore,
research on an E. histolytica substrain in which the expression of several CPs was downregulated by an
antisense transcript showed that these enzymes play a major role in the advancement of IA in the SCID-
HU-INT mouse [61]. One of them, EhCP-A5, which is not expressed in E. dispar, has been shown to
degrade the cysteine-rich domains of the MUC2 mucin, which are the major structural component of the
colonic mucus gel in the human digestive tract [61].

39.2.1.10 Human Colonic Explants Model

Recently, human colonic explants have been used to study host–parasite interactions in IA [62]. This
ex vivo model allows identifying the first steps of invasion and the comparison of the reaction to differ-
ent strains in the same colon sample. It is possible to examine both sides of the host–parasite interaction
by determining the kinetics of parasite penetration into the mucus and mucosa, structural changes in the
mucosa, cell lysis and the inflammatory response to the virulent wild-type E. histolytica strain, and to
624 Laboratory Models for Foodborne Infections

compare them with those observed after infection with the nonpathogenic parasite E. dispar [63]. Based
on recent results in work with this model, the Gal-lectin and amoebapores are not required for the inva-
sion of human colon explants, thus suggesting that CP-A5, which is involved in extracellular matrix deg-
radation, is not required for crossing the mucus but rather contributes directly or indirectly to penetrating
the lamina propria and inducing inflammation [64].

39.2.2 Amoebic Liver Abscess

The most common extraintestinal amoebiasis is ALA. Macroscopically, liver lesions are formed by
necrotic tissue, which has a yellowish color and creamy consistency, and are surrounded by congestive
parenchyma and a discrete fibrosis. When the damage is extensive, the necrotic material can be substi-
tuted by liquid necrosis, thus creating a cavitary aspect. Microscopically, trophozoites are localized in the
border of the necrotic areas and close to the apparently normal hepatic tissue. Occasionally, the amoebae
are mixed with the necrotic parenchyma and contain erythrocytes and ingested cell debris. The inflamma-
tory foci in the liver tissue vary but show an important chronic inflammatory reaction in the portal areas.
Reports of early liver lesions caused by E. histolytica in humans are very scarce; when ALA is well
established, there is extensive necrotic tissue. The information on early liver lesions is based on experi-
mental studies using susceptible animals such as hamsters and gerbils that were inoculated by intraportal
or intrahepatic routes with virulent amoebae. The first laboratory animal used successfully to produce
ALA was the hamster [65,66]. This was done by inoculating axenic trophozoites via the portal vein and
producing large amoebic abscesses. From these pioneering studies, data have been obtained regarding
pathogenesis, immunopathology, cell and molecular biology, and genetics, among other topics involved
in the host–parasite interaction.
An excellent work in 1984 [23] described the sequential histopathological study of ALA formation
from very early stages in hamsters inoculated by the intraportal route with E. histolytica trophozoites.
The authors demonstrated the role of the host inflammatory reaction in the liver tissue damage for the
first time. They suggested that the amoebae do not produce ALA by direct lysis of the hepatocytes but
rather the inflammatory cell lysis contributes to liver damage. This finding broke the paradigm that
amoebic liver lesions produced by E. histolytica do not produce inflammation in human ALA. These
findings were later confirmed by transmission electron microscopy [39]. Figure 39.1 shows macroscopic
and histopathological analysis of ALA in hamsters. Table 39.2 lists the susceptibility of different rodents
to IA and ALA.
Another set of rodents highly susceptible to hepatic amoebiasis are the gerbils [67]. Hepatic lesions
are similar to those reported in hamsters, although amoebae show less virulent behavior in gerbils than
they do in hamsters. In gerbils, parasites were in direct contact with the hepatocytes and were associated
with inflammatory cells and the liver parenchyma and showed severe damage in both conditions. The
production of ALA and the possibility of producing intestinal lesions have led some researchers to con-
sider this rodent model of disease as being more similar to human hepatic pathology [41]. Studies of host
immune responses and the effect of several vaccine candidates from purified proteins from E. histolytica
(recombinant) administered orally or parenterally have been reported in gerbils [68].
E. histolytica infects only humans and nonhuman primates; studies in laboratory mice models suggest
the existence of natural resistance to amoebiasis. However, the use of different mice strains has increased
the understanding of the cellular and molecular basis of the mechanisms involved in the host–parasite
­relationship. Genetically modified animals, such as the SCID mice reported by Cieslak et al. [69], ­developed
ALA when the animals were challenged intrahepatically with virulent E. histolytica trophozoites. When the
immunocompetent congenic CB-17 (used as control) was used, only one of seven mice developed an abscess.
To study immune responses, the role of macrophages in the resistance to invasive amoebiasis has been
examined in several species. Mice treated with silica are susceptible to developing ALA, probably due to
impairment in macrophage function. However, the absence of macrophages or their activation does not
reduce the natural resistance of mice to the development of ALA [70].
Data on the role of lymphocytes are contradictory. CBA/nu/nu athymic or Balb/c mice treated with an
anti-lymphocyte Ab showed that these cells are not important in the resistance to E. histolytica infec-
tion. However, C3H/mg mice treated with the same Ab developed hepatic lesions, which suggests that
Entamoeba histolytica 625

(A) (B) h

h
40x
(C) h (D)
h

40x n 20x

(E) ec (F)

n ly

40x 20x

FIGURE 39.1  (a) Amoebic liver abscess. Macroscopic aspect. Whitish lesions covering the entire right lobule (visceral
face); 7 days postintrahepatic inoculation with 1 × 106 trophozoites of Entamoeba histolytica. (b–f) Histopathological
analysis. (b) Three hours postinfection. Trophozoites (arrows), inflammatory cells (arrow-heads), and hepatocytes (h). 40×.
(c) Six hours postinoculation. The inflammatory foci increase in number; amoeba (arrow), inflammatory reaction (arrow-
heads) and hepatocytes (h) 40×. (d) Two days postintrahepatic inoculation. The necrotic areas are evident (n), the amoe-
bae increase in number (arrows), inflammatory cells (arrow-head). Some hepatocytes appear apparently normal (h). 20×.
(e) Granulomatous reaction. Five days postinfection. Necrotic center (n), epithelioid cells (ec), and trophozoites (arrows).
40×. (f) Seven days postinoculation. The necrotic areas increased in the liver. Some amoebae are seen (arrows) intermixed
with the lytic necrosis (ly), inflammation (arrow-heads). 20×.

TABLE 39.2
Amoebiasis: Susceptibility in Rodents
Intestinal Liver
Hamster − +
Gerbil +/− +
Guinea pig +/− −
Mouse +/− −
Rat +/− −

lymphocytes are important in the resistance to hepatic amoebiasis [69,71]. More recently, studies using
Balb/c mice treated with a monoclonal Ab (RB6–8C5) against neutrophils were challenged by intrahe-
patic administration of amoebae. The animals were sacrificed at different times postinoculation, and liv-
ers were analyzed histologically. The results showed that the neutropenic animals developed more severe
ALA than did the controls. The authors concluded that neutrophils are important to natural resistance in
amoebiasis, in contrast to the results found in hamster and gerbil models that develop ALA [72].
626 Laboratory Models for Foodborne Infections

Information about the participation of cytokines, complement, and Abs is still incomplete. Cytokines
such as IFN-γ, TNF-α, and MCSF-1 increase the amoebicidal effect of macrophages (peritoneal and
Kupffer cells) [73]. Proteinases and amoebapores of E. histolytica are also involved in ALA produc-
tion. To actively invade, amoebae must degrade different components of the extracellular matrix, which
occurs via the action of proteinases secreted by the parasite. These enzymes degrade collagen, elastin,
fibrinogen, fibronectin, and laminin [21,30]. Another factor that is known to contribute to tissue inva-
sion is the inflammatory reaction produced by the presence of amoebae in the liver. The expression of
TNF-α, IFN-γ, IL-1β, IL-8, and IL-10 was studied throughout ALA evolution in hamster. The authors
showed that neutrophils and macrophages that infiltrate the liver parenchyma in the acute and chronic
stages of ALA are also responsible for the tissue damage. IL-10 does not regulate the local production of
proinflammatory cytokines. The results showed that the exacerbated inflammatory milieu contributes to
damage and probably supports the survival of E. histolytica [74].
It is important to mention that the nonpathogenic E. dispar strain SAW760 cultured in axenic condi-
tions showed early inflammatory reaction around the amoebae. However, hepatic lesions did not progress
to ALA formation. In other recent studies using different strains of E. dispar in xenic or monoxenic
culture conditions, the production of amoebic liver lesions has been reported [75,76].

39.2.3 Animal Models for Amoebiasis Treatment

As in the case of every drug for human use, it is desirable that antiamoebic compounds act exclusively
against the parasite and do not affect human cells or metabolism, although this is not always possible.
The first study of amoebiasis in rats used to evaluate new antiamoebic compounds was reported by Jones
in 1946 [77]. Since then, numerous investigations have been performed in different animal species to
test the efficacy of antiamoebics because results in animals are more validly extrapolated to the effects
in humans than those performed in cultures in vitro.
Several drugs have been tested for both IA and ALA. Metronidazole (Mtz) has been used for 50 years
for trichomoniasis treatment, and its effect in ALA treatment in hamsters was evaluated [78]. Derivatives
of Mtz obtained through minor changes in the molecule have also been assayed in animal models and, in
some cases, are more effective than Mtz. They also extend the parasiticidal effect of Mtz to other proto-
zoa [79–84]. Treatment of amoebiasis should include luminal and/or extraluminal agents (some of them
are known as cysticides), depending on the site of infection [13,85,86]. Symptomatic amoebiasis requires
a two-drug regimen: a tissue-amoebicidal agent, such as Mtz, and a luminal-acting agent, such as iodo-
quinol, diloxanide furoate, or paromomycin [2,87,88]. The treatment for ALA is generally Mtz; this
azole and several luminal drugs have also been used against IA. Mtz has toxic adverse effects because
it produces nausea, vomiting, and abdominal pain; thus, patients often discontinue the treatment. Mtz
induces DNA breaks and chromosomal aberrations in mouse peripheral lymphocytes, and it could be
carcinogenic [85,86]. In addition, strains resistant to Mtz have been found in E. histolytica cultures [89].
Thus, the need for an effective and safe antiamoebic compound is increasing, and several new drugs
have been tested in animal models against the parasite. Plant crude extracts that have been utilized for
amoebiasis in traditional medicine have been examined against E. histolytica infections; in some cases,
the compounds responsible for the antiamoebic effect have been purified, and their mode of action has
been elucidated [90–93]. Generally, a number of compounds are assessed in in vitro cultures, and after-
ward the compound that presented the best effect is evaluated in in vivo models. In both cases, Mtz is
used as positive control [94,95]. For example, the steroidal alkaloid chonemorphine was identified as an
antiamoebic during the course of a screening program for novel antiparasitic agents from plant sources.
Interestingly, this alkaloid led to a 100% cure of ALA in hamsters and cleared 90% of the intestinal
infection in weanling Wistar rats [96].
A comparative study of experimental cecal amoebiasis to evaluate amoebicides in the mouse, hamster,
and rat was developed in India. The authors found different responses for each animal and proposed
the mouse model as the most useful for the primary screening of antiamoebic compounds [97,98]. In
another study, the cytotoxic effect of amide derivatives of trifluoromethionine (TFM) against E. histo-
lytica was evaluated. Amoebae but not mammals possess l-methionine γ-lyase, an enzyme that hydro-
lyzes TFM and its derivatives, which makes it a good target for amoebicidal activity [99]. Interestingly,
Entamoeba histolytica 627

a high-throughput screening for compounds effective against amoeba identified auranofin, an FDA-
approved drug used therapeutically against rheumatoid arthritis. Auranofin is 10 times more potent than
Mtz, which makes it a promising therapy for amoebiasis [100]. The innate immune response by pro-
tein lactoferrin, an iron-chelating molecule that is responsible for avoiding pathogens to acquire iron in
mucosae and infection sites, has been tested in ALA and IA [101,102]. Remarkably, bovine lactoferrin
was able to cure ALA and synergize with Mtz in biological action. This observation is particularly
important given that a reduced dose of Mtz can be used if combined with lactoferrin, with the same effect
on the parasite, thus diminishing the side effects and toxicity of the drug. In addition, other properties of
lactoferrin such as its anti-inflammatory activity can help resolve the amoebic abscesses [101].

39.2.4 Animal Models for Amoebic Vaccine Research

The development of a vaccine against E. histolytica is imperative and represents a great opportunity to
prevent and even eradicate amoebiasis. In general, vaccines are cost-effective and safe and provide high
protection rates in many infectious diseases. However, to date, no amoebic vaccine has been tested in
clinical trials. Recently, several vaccines have been tested successfully in animal models; researchers
hope to obtain good results [103,104].
An important point in the human colonic-defense mechanism is the production of mucosal
­immunoglobulins (Igs), which have important roles in maintaining intestinal integrity [105]. Secretory
IgA (sIgA) is one of the most abundant Abs produced by plasma cells within the lamina propria,
preventing pathogens from adhering to the intestinal mucosal barrier. There is evidence that ­mucosal
anti-Gal-lectin responses are critical for amoebic colonization and invasion [106]. IgA titers correlate
with protection against amoebiasis, and other studies suggest that the presence of IgG has a ­detrimental
effect [107]. Analysis of asymptomatic carriers of E. histolytica showed that these persons had high
­levels of IFN-γ, which reflects a Th1 response, but that patients with invasive amoebiasis had higher
­levels of IL-4, which resembled Th2-type responses [108]. The uncertainty regarding the role of
­adaptive immunity in providing protection against E. histolytica IA and ALA is important to resolve to
develop a vaccine against E. histolytica.
Clinical studies in Bangladesh suggest that children with amoebic colitis develop mucosal IgA
Abs to the surface E. histolytica Gal-lectin [107]. However, immunity was short-lived, and 20% of
the children had a new case of amoebiasis [109]. In contrast, a group in South Africa with a previous
history of ALA and with mucosal Abs to amoebic lectin was less predisposed to E. histolytica or
E. dispar infection [110]. The data from a Bangladesh study suggest that mucosal immunity against
Gal-lectin can reduce the risk of amoebic reinfection. However, in Vietnam, it was reported that
individuals with a prior history of ALA present the same or higher risk of a second case of ALA
[111]. This is important, because patients with ALA produce high quantities of E. histolytica-specific
serum Abs and develop T cell proliferative responses that recognize amoebic antigens [112]. In addi-
tion, there is evidence that indicates that reinfection rates may be lower in persons with mucosal Abs
against Gal-lectin.
The major requirements of an effective vaccine include immunological memory, which depends on
the production of a strong immune response, identification of a protective antigen, and an appropriate
delivery route. A basic concept for vaccine development is to use a live attenuated strain of a virus or
bacterium to stimulate immunity to prevent subsequent reinfection with virulent strains. This approach
requires that the vaccine strain be immunogenic and stably attenuated, with little risk of reversion.
Although there is no animal model that completely mimics human IA and ALA, various in vivo models
have been developed and contribute to advance the development of a potential vaccine. In guinea pigs,
previous infection with a noninvasive strain of E. histolytica resulted in some protection against sub-
sequent intracecal challenge with a virulent strain of the parasite [113]. It is also possible to genetically
modify E. histolytica to create attenuated strains or to use naturally occurring Entamoeba strains that
show reduced virulence compared with the wild-type, such as E. histolytica Rahman or E. dispar [103].
One approach to attenuation is to target E. histolytica virulence factors. Mirelman et al. were able to
silence the expression of the E. histolytica amoebapore A gene and showed that intraperitoneal challenge
in hamster with these amoebae induced IgG antiamoebic Abs [114].
628 Laboratory Models for Foodborne Infections

Other possible vaccine candidates are antigenic proteins, such as Gal-lectin adhesin, which is
the best characterized E. histolytica protein. This protein is a 260-kDa heterodimer localized on
the amoeba surface [115]. Gal-lectin is an attractive candidate for a vaccine because of its immu-
nogenicity and its importance in the development of the disease. The first experiment to use puri-
fied Gal-lectin showed 86% protection against ALA in the gerbil model [116]. Another potential
protein target is the serine-rich E. histolytica protein (SREHP). Previous studies of this protein have
reported its success as an antigen to protect gerbils against ALA [68]. Intradermal immunization
with SREHP as a maltose-binding protein induced 100% protection against ALA in gerbils [68]. The
E. histolytica 29-kDa molecule (Eh29) is also considered an important antigen for an amoebiasis
vaccine. Eh29 is an alkyl hydroperoxide reductase that is involved in the detoxification of reactive
oxygen species secreted by microflora or immune cells [117]. One study of this protein in the ham-
ster model showed 54% protection against ALA [118]. Another study using Eh-29 conjugated with
cholera toxin B subunit (CTB) conferred protection against intracecal amoebiasis in C3H/heJ mice
(IA), which was associated with anti-Eh29 IgA Abs in the intestine and anti-Eh29 IgG-specific Abs
in the serum [119].
The development of DNA vaccines is recent and involves the induction of DNA of known
sequences of some antigens of interest into a bacterial plasmid. DNA vaccines have been shown
to exert strong humoral and cell-mediated responses and were successful in conferring protection
against parasites. One example of this method is the generation of a codon-optimized DNA vaccine
encoding a portion of the Gal-lectin of E. histolytica. When Balb/c mice were vaccinated intrader-
mally with the DNA plasmid, the vaccine stimulated a Th1-type Gal-lectin-specific cellular immune
response as well as the development of serum Abs that recognized a recombinant portion of the
heavy subunit and inhibited the adherence of trophozoites to target cells in vitro [120]. Another type
of DNA vaccine is the multivalent vaccine. One example of this is the EhCPADH complex, which is
formed by two surface molecules: CP112 (EhCP112) and an adhesin (EhADH112). Compared with
immunization with each plasmid alone (EhCP112) or EhADH112), the coimmunization of hamsters
with the two plasmids induced a significantly greater level of anti-E. histolytica IgG. Interestingly,
protection against liver abscesses was detected only in animals that received the plasmid mixture,
and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112
or pcDNA-Ehadh112 [121].
There are other amoebic targets such as the heparin sulfate-binding protein (HSBP) and the 30-kDa
collagen-binding protein (CBP30) recombinant fused to portions of the Trypanosoma cruzi heat shock
protein of 70 kDa, which have been assayed as vaccine antigens in guinea pigs [122] and in hamsters
[123], respectively. Beyond rodent models, only one study has explored the utility of vaccines in a non-
human primate model of amoebiasis [124]. In this model, Gal-lectin was administered with CTB as an
adjuvant in baboons by colonoscopy into the lumen of small bowel and cecum. This vaccine resulted in
a moderate level of protection against E. histolytica reinfection. However, the nonhuman primate study
represents a significant advance toward an anti-E. histolytica vaccine. It is evident that more studies are
needed in this field. The identification of E. histolytica immunogenic proteins and the correct combina-
tion of doses, boosts, and adjuvants is a priority to obtain long-term immunological memory in experi-
mental animal models to advance beyond the preclinical stage in humans.

39.3 Conclusions and Future Directions

Amoebiasis is the third leading cause of death by parasitic diseases; this problem mainly affects develop-
ing countries. In recent years, knowledge of the aspects involved in amoebiasis, the virulence factors of
E. histolytica, and the human–amoeba interaction have advanced rapidly. However, despite the abundant
data obtained using in vitro and in vivo experimental procedures, there are still many unknown features
related to the mechanisms involved in the invasion of this parasite to the intestine and liver. Drug thera-
pies that only affect the parasite and a vaccine that protects people living in zones where amoebiasis is
endemic are also necessary. Improvements in sanitation and hygiene practices are particularly important
to eliminate amoebiasis.
Entamoeba histolytica 629

Acknowledgments
This work was supported by grants No. 1674231 (Serrano-Luna), 179251 (de la Garza), and 237523
(Shibayama-Salas) from Conacyt, México.

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40
Giardia lamblia

Steven M. Singer, Jenny G. Maloney, and Camila H. Coelho

CONTENTS
40.1 Introduction................................................................................................................................. 635
40.2 History of G. lamblia.................................................................................................................. 635
40.3 Life Cycle of G. lamblia and Diagnosis..................................................................................... 636
40.4 Morphology of G. lamblia.......................................................................................................... 636
40.5 Epidemiology.............................................................................................................................. 639
40.6 Encystation.................................................................................................................................. 640
40.7 Excystation.................................................................................................................................. 640
40.8 Adhesion..................................................................................................................................... 640
40.9 Pathology..................................................................................................................................... 641
40.10 Immunology................................................................................................................................ 643
40.11 Antigenic Variation in G. lamblia.............................................................................................. 645
40.12 Giardia and Arginine................................................................................................................. 645
40.13 Drug Resistance in Giardia........................................................................................................ 646
40.14 Conclusion................................................................................................................................... 647
References............................................................................................................................................... 647

40.1 Introduction
The protozoan parasite Giardia lamblia (Protista, Diplomonadida, Hexamitidae, syn. G. intestinalis,
G. lamblia) is the etiologic agent of giardiasis, a common diarrheal disease worldwide. G. lamblia can
infect humans and many other mammals, with prevalence rates in humans ranging from 2% to 7% in
developed countries to 20%–30% in developing countries and 100% prevalence reported in some popu-
lations.1,2 Human outbreaks of G. lamblia are most often associated with fecal contamination of drinking
water. However, several foodborne outbreaks have been documented.3 Until recently, giardiasis was not
the subject of significant scientific investigation, but after its inclusion in the Neglected Disease Initiative
by WHO in 2004 and its reemergence in developed countries, the landscape has changed significantly.4–6
In a bibliometric review covering all studies on giardiasis published between 1971 and 2010, Escobedo
et al.7 reported that especially in the last decade, there has been an increased number of publications and
clinical research studies involving giardiasis. Comparative studies, clinical trials, and pharmacotherapy
assessment were the main identified research areas. This chapter will discuss several aspects of the biol-
ogy of Giardia and the pathology associated with infection while highlighting the laboratory models
currently used to study this parasite.

40.2 History of G. lamblia
Historically, G. lamblia was so common that it was once considered a commensal organism of the
human intestine. G. lamblia was first discovered in 1681 by Antony van Leeuwenhoek during an exami-
nation of his own stool.5 Almost 200 years later in 1859, the protozoan was given the name Cercomonas

635
636 Laboratory Models for Foodborne Infections

intestinalis by Lambl and then later renamed Giardia lamblia by Stiles in 1915 in honor of Professor
A. Giard of Paris and Dr F. Lambl of Prague, who both described the parasite.8 The first association
between G. lamblia and the disease was found in 1954, when it was demonstrated that the ingestion of
G. lamblia cysts by humans could cause diarrheal disease even after ingestion of as few as 10 cysts.9 It
was not until 1981, 300 years after its first description, that G. lamblia was added to the WHO’s list of
parasitic pathogens.8 Soon after its addition to the WHO’s list of parasitic pathogens, Koch’s postulates
were fulfilled, confirming G. lamblia as a pathogen of humans.10

40.3 Life Cycle of G. lamblia and Diagnosis

The life cycle of G. lamblia includes an infectious cyst form and a noninfectious, rapidly multiplying
trophozoite form. The cyst form is extremely hardy in the environment and can be transmitted to humans
by contaminated food, water, hands, or fomites via the fecal–oral route.11 Once ingested, exposure to
stomach acid and digestive enzymes triggers excystation, and the noninfective trophozoite form takes
residence on the mucosal surface of the small intestine where it multiplies rapidly. As the trophozoites
are passed through the intestines, they transform back into the infectious and environmentally hardy cyst
form and are shed with the feces (Figure 40.1). In the environment, cysts can survive in harsh conditions
and may persist for several months in cold water.11 Cysts can survive water temperatures of <10°C for
2–3 months, and some cysts can survive a single freeze–thaw cycle.11 As water temperature increases,
survival rates decrease; at 21°C, cysts can survive in water for about 1 month. At a water temperature of
54°C, cysts tend to die within 1 min, whereas cysts tend to die immediately in boiling water.11 Infected
individuals can shed up to 10 billion cysts a day, and ingestion of as few as 10 cysts can yield infection.9
Because of the low infectious dose of G. lamblia as well as the environmentally hardy nature of the
infectious cyst, G. lamblia is included on the National Institute of Allergy and Infectious Diseases’ list
of category B pathogens.
Diagnosis of infection in both humans and animals is predominately based on microscopic identifica-
tion of Giardia cysts in fecal samples, although in recent years, ELISA- and PCR-based techniques have
been developed.12,13 The course of disease following G. lamblia infection can be highly variable and
ranges in severity from asymptomatic infections to infections that result in severe diarrhea and painful
abdominal cramping.1,2 These clinical outcomes can have a disproportionate effect on children. Infection
in children has been associated with stunting and impaired development of cognitive function in popu-
lations where giardiasis is prevalent.14,15 Infections are treatable, but current treatment for G. lamblia
involves administration of toxic drugs to which parasite resistance is reported.16

40.4 Morphology of G. lamblia
The two forms of the parasite, the trophozoite and the cyst, are highly specialized for survival
within the host and within the environment, respectively. Therefore, by studying the morphology
of G. lamblia, we can better understand both infection and transmission of the parasite. Most of
what we know about G. lamblia morphology is drawn from studies utilizing electron microscopy
to visualize the surface, cytoskeletal, and intracellular components of the parasite. More gross fea-
tures of the parasite can easily be visualized using light microscopy. These studies rely on in vitro
culture techniques, which allow for the production of both trophozoites and cysts outside of a host.
The trophozoites of G. lamblia can be grown in an axenic medium originally created for cultivation
of Entamoeba ­histolytica.17 Axenization of new strains can be somewhat problematic. Protocols for
excystation of cysts in vitro (Sections 40.6 and 40.7) can be used to initiate cultures, although a more
common procedure is to collect trophozoites from the small intestines of infected animals.18 Addition
of antibiotics to culture media then helps propagation of newly axenized strains.19 Cyst production is
more complicated, but several protocols exist (Table 40.1).
Giardia lamblia 637

Contamination of water, food, or

hands/fomites with infective cysts.

Trophozoites are also

passed in stool, but
they do not survive in
the environment.

1
i = Infective stage i
d
d = Diagnostic stage d Cyst

3 4 5
Cyst Trophozoites

FIGURE 40.1  Life cycle of Giardia. Transition from noninfectious, rapidly multiplying trophozoites to infectious, envi-
ronmentally hardy cysts. (Courtesy of US CDC.)

TABLE 40.1
Methods Used for Encystation of Giardia lamblia In Vitro
Pre-encystation Medium Encystation Medium Timing References
TYI-S33 without bile TYI-S33 + 10 mM lactic acid, pH 7.8 72 h pre-encystation 52,54
and 10 mg/mL porcine bile 48 h encystation
None TYI-S33 + 12.5 mg/mL bovine bile, no 72 h encystation 55
lactic acid
None TYI-S33 with delipidated serum 72 h encystation 57
None TYI-S33, pH 7.8 + 10 mg/mL bovine bile 96 h encystation 165
638 Laboratory Models for Foodborne Infections

(B)

500 nm
(A) VD AF

VD

VLF
VF

CF

PF (C)
N

500 nm
Ax

FIGURE 40.2  Electron micrographs of Giardia lamblia trophozoites. (A) Scanning electron micrograph of the ven-
tral surface showing the ventral disc (VD), anterior (AF), ventral (VF), caudal (CF), and posterior (PF) flagellar pairs.
(B and C) Transmission electron micrographs showing the ventrolateral flange (VLF), ventral disc (VD), flagellar
axonemes (Ax), and nuclei (N).

Trophozoites of G. lamblia have a distinctive pear shape and exhibit bilateral symmetry. Each cell is
12–15 μm in length and 5–9 μm in width with a flattened ventral surface, a rounded dorsal surface, and
four pairs of flagella: anterior, posterior, caudal, and ventral (Figure 40.2). A major component of the
G. lamblia cytoskeleton is the ventral disc. It is a concave structure that covers the ventral surface of
the trophozoite and is thought to play a major role in attachment of the parasite to the intestinal mucosa.
Another prominent feature of the trophozoite cytoskeleton is the median body, which serves as the
microtubule-organizing center of the cell. Differences in appearance of the median body contribute to
distinction among the species of Giardia. Other morphological features of the trophozoite include the
lateral crest and ventrolateral flange, which are structures with unknown function, although believed to
be participating in adhesion.20–22
Like other eukaryotic cells, Giardia also contain numerous cytoplasmic organelles. In the trophozo-
ites, mitosomes, peripheral vesicles, and ribosome granules can be visualized.23 Interestingly, while a
Golgi complex is easily visualized during the process of encystation, vegetatively growing trophozoites
contain only vesicular structures suggestive of a Golgi complex.24–26 Unlike most other eukaryotic cells,
trophozoites have two nuclei that are localized anteriorly and are symmetric across the longitudinal axis
of the cell. During encystation, the nuclei replicate to produce mature cysts with four nuclei. Following
excystation, each cyst gives rise to two trophozoites.
The transformation of trophozoites of G. lamblia into cysts occurs in response to physiological
stimuli, leading to a process characterized by decreased cell metabolic rate, internalization of fla-
gella, cytoplasmic condensation, peripheral vesicle formation, and production of a cystic membrane.22,27
Encystation is fundamental for survival, transmission, and pathogenesis of G. lamblia. It is consid-
ered an important virulence factor because it allows G. lamblia to survive outside the host and to be
transmitted.23,28
Cysts are ellipsoid or oval and have a size of 7–12 μm. They have a refractive cyst wall, which
is 0.3–0.5 μm thick and is composed of an outer layer and an inner filamentous membranous layer.
Median bodies and structural elements of flagella, the axonemes, and four nuclei in mature and infec-
tious cysts are found in the cytoplasm.29,30 The cyst wall is composed mainly of N-acetylgalactosamine
(GalNAc) homopolymers.31 The precursor for the GalNAc homopolymer is UDP-GalNAc, an amino
sugar phosphate, which is produced from glucose. Enzymes required for the synthesis of UDP-GalNAc
include glucosamine-6-phosphate isomerase, phosphoacetyl-glucosamine mutase, UDP-GlcNAc
49-epimerase, and glucosamine-6-phosphate N-acetylase.32 During encystation, transcript levels and
Giardia lamblia 639

enzyme activity for some of these enzymes increase significantly.33 The external portion of the cyst
wall is covered by a network of filaments, 7–20 nm in diameter, composed primarily of GalNAc homo-
polymer, along with cyst wall proteins. Considering that this is the stable life cycle stage, the cysts
have a metabolic rate of only 10%–20% relative to trophozoites.34 Some cysts are resistant to chlorina-
tion and radiation. Boiling is the most efficient way to eliminate cysts; freezing requires several days
to reduce cyst viability.35–37

40.5 Epidemiology
Within the genus Giardia, there are currently six recognized species:38 G. agilis (a parasite of amphib-
ians), G. ardeae and G. psittaci (both of which are found in birds), G. microti (found in voles and
muskrats), G. muris (found in rodents), and G. lamblia (which infects a wide range of mammalian spe-
cies including humans). The aforementioned taxonomic divisions are based on morphological differ-
ences that have been historically used to define the six Giardia species.38 More recently, G. lamblia,
the only species known to infect humans, has been divided into assemblages A–H based on molecular
evidence.5 Assemblages A and B are known to infect a wide range of mammals including humans,
but assemblages C–G appear to have some degree of host specificity.39 Assemblages C and D are most
commonly found in dogs, assemblage E in hoofed animals, assemblage F in cats, and assemblage G in
rats.40 The most recent addition, assemblage H, was identified in marine animals.39 The classification
of these assemblages is supported by studies using both enzyme electrophoretic data and phylogenetic
analyses of molecular sequence data.5
There are striking genetic differences between the two assemblages, assemblage A and assem-
blage  B, associated with human infections. Whole genome sequencing of two strains of G. lamblia
isolated from humans, WB (assemblage A strain) and GS (assemblage B strain), revealed that these
two assemblages share only 77% nucleotide identity and 78% amino acid identity in protein-coding
regions.41 Because these two assemblages are so genetically different, it is tempting to speculate that
they should contribute to different geographical distributions or clinical outcomes in humans. However,
data to support these geographical and clinical differences are often conflicting. Reports from Asia,
Europe, Africa, and the Americas all demonstrate that each assemblage is found in pockets through-
out these areas.42 Symptomatic differences attributable to assemblage follow a similar trend. While
assemblage A is found to account for more symptomatic infection in some studies, assemblage B is
found to have a greater association with symptoms in others, with some studies reporting no associa-
tion between patient symptoms and assemblage causing the infection.42 In a laboratory infection model
using adult mice, it was demonstrated that while the GS (assemblage B) strain caused measurable
disaccharidase deficiency, infections with the WB (assemblage A) strain did not.43 Similarly, another
assemblage B strain, H3, caused chronic infections that contributed to enhanced weight loss in mice on
a protein-deficient diet, while the assemblage A strain, WB, did not.44 The use of antibiotics to allow
the WB strain to infect mice in the former paper43 and the use of H3 cysts rather than WB trophozoites
to initiate infections in the latter paper44 require caution in interpreting these results. Nevertheless, they
suggest that mouse models may be useful for identifying strain- and assemblage-specific differences
in virulence. Better cohort studies in humans could also help to distinguish the potential differences in
pathogenicity of these two assemblages.
Because G. lamblia infects such a diverse range of mammals, it is conceivable that zoonotic trans-
mission could occur. However, the zoonotic risk of G. lamblia infection is unclear. Some studies have
found an increased risk for human infection in households that harbor domestic pets.45,46 Molecular
evidence has linked human infections to assemblages found only in cattle, cats, and dogs, indicating that
cross-species transmission can occur.47–50 A large-scale European study found that only 1% of human
G.  lamblia infection could be attributed to assemblages C, D, E, and F, indicating that the zoonotic
potential of G. lamblia is low for these assemblages.51 Interestingly, this same study demonstrated that
while assemblage B infections are mostly restricted to humans, assemblage A appears quite frequently
in companion animals, wildlife, and livestock. So, while humans may serve as the main source of assem-
blage B, assemblage A could have a greater zoonotic potential.
640 Laboratory Models for Foodborne Infections

40.6 Encystation
Encystation is the process of differentiation of the trophozoite into the resistant cyst, which occurs due to
external stimuli. During encystation, the main transformations that occur are internalization of flagella
creating an oval shape, formation of encystation-specific vesicles (ESVs), the appearance of a refractory
cyst wall, and replication of the nuclei. The cell also has diminished adherence capacity owing to frag-
mentation of the ventral disc.22 Much of the work on G. lamblia encystation has been done by in vitro
manipulation of axenically cultured trophozoites. By altering the culture conditions of trophozoites, it is
possible to mimic some of the signals a cell might encounter in the host small intestine that are neces-
sary to drive encystation (Table 40.1). In vitro studies have shown that encystation begins when important
modifications of the environment occur such as shifts in pH, concentrations of bile salts, availability of
lipids, and production of lactic acid by intestinal bacteria.52 In a suckling mouse model of G. lamblia
infection, cysts were found in the mid to lower portion of the jejunum, while later the majority of cysts
were in the large intestine and cecum, suggesting that bile concentration may be an important component
of the encystation process.53 It is thought that bile stimulates encystation by sequestering cholesterol.
Common methods used to achieve encystation are based on parasite growth in conventional culture
media without bile, and after 2–3 days, the parasites are switched to a culture media containing a high
concentration of porcine bile with lactic acid and a pH of 7.8.52,54,55 Using these methods, trophozoites
can be induced to transform to viable cysts. However, the ability of in vitro-derived cysts to excyst, either
in animals or in culture, is often poor.
G. lamblia has little capacity for synthesizing lipid molecules de novo and instead depends on envi-
ronmental sources. It has been hypothesized that most of the lipid uptake by the cells is then used for
energy production and biosynthesis of organelles.56 Because lipid metabolism is central to the process
of encystation, these pathways are of interest as a potential drug target.52,57 Recently, attention has been
focused on glycosylceramide pathways in encystation.58 Conversely, specific lipid products from the host
have also been shown to kill trophozoites.59,60
The role of ESVs in the encystation process and their relationship with the normal functions of the Golgi
complex have been the subject of significant interest. Stefanic et al.61 used two-dimensional electrophore-
sis to analyze Golgi-like vesicles isolated from encysting G. lamblia. These vesicles are highly dynamic
spaces that act as hosts of the cyst wall materials and increase only during the induction of encystation. The
authors have shown that the vesicles do not have sorting functions characteristic of mature Golgi, but do
retain protein quality control functions. They also suggested that the ESVs can be involved in anterograde
and retrograde trafficking with the endoplasmic reticulum, similar to the function of the Golgi.61

40.7 Excystation
In vivo, excystation starts following ingestion by the host when the cysts are exposed to the acidic pH
of the stomach. Mobile parasites emerge from within the cyst wall in the host small intestine, but at this
stage, the ventral disc is not fully reorganized. Soon after emergence, the cell divides rapidly generating
two trophozoites capable of adhering to intestinal cells by the ventral disc, which is now fully reorga-
nized.62 Parasite viability requires that cysts produce trophozoites that can emerge from the cyst wall,
perform cytokinesis, and adhere to the intestinal epithelium of a newly infected host. Several protocols
for excystation have been published (e.g.,54,63–65), but the success of the excystation process may vary
depending on whether the source of the cysts is feces or in vitro generation.

40.8 Adhesion
The ability of the parasite to adhere to the intestinal epithelium is an essential factor in its ability to
reproduce and cause disease. In vivo, Giardia trophozoites can adhere to the surface of the small intes-
tine and in vitro to surfaces like glass and polymers.66 Understanding the mechanisms that mediate
Giardia lamblia 641

parasite adhesion can explain how Giardia cells can attach to many surfaces as well as the intestinal
wall of many host animals.67 Several approaches have been developed to evaluate Giardia cell adhesion.
These include adhesion of trophozoites to different glass surfaces, scanning electron microscopy to ana-
lyze attachment, and a microarray-based approach.67–69
Many studies have been published in an attempt to elucidate the adhesion mechanisms of Giardia
cells. The structure responsible for the attachment is the ventral disc, a spiral array of microtubules
associated with microribbons and found just below the plasma membrane in the anterior and ventral por-
tion of the trophozoite.70 Several auxiliary proteins are found associated with the ventral disc including
β-giardin, γ-giardin, δ-giardin, and SALP-1. The periphery of the disc has contractile proteins including
actin, myosin, and tropomyosin, which may serve as the biochemical basis for contraction of the disc, a
fundamental process in parasite adhesion. Hagen et al. used a proteomic approach to detect more than
100 protein candidates associated with the ventral disc as well as the first proteins localized to the lateral
flange.71 Because adhesion is central to parasite viability and these structures are unique to the parasite,
strategies targeting these molecules could be developed as novel therapeutics.

40.9 Pathology
Studies of the pathology associated with giardiasis often use animal models that aim to recapitulate
human infection conditions. Several animal models for studying G. lamblia exist including mouse, ger-
bil, hamster, rat, and recently, nematodes (Table 40.2). Infection with G. muris is a common model of
human giardiasis because the host specificity of this species in mice and rats can be useful for simpli-
fying infection protocols. Both human assemblages of G. lamblia are commonly used in experimental
giardiasis, which can be useful for understanding assemblage-level differences in the human host. In
vitro models are also used to study G. lamblia pathology by exposing human intestinal cell lines to
live parasites or their byproducts. There is a bias in the literature for mouse infections performed with

TABLE 40.2
Common Models Used for Studies of Giardiasis
Model Advantages Disadvantages
Axenic culture of • Rapid • Limited applicability in vivo
trophozoites • Inexpensive
Coculture of Giardia with • More rapid than in vivo • Limited applicability in vivo
epithelial cell lines • Can test hypotheses without
using animals
• Easy to analyze interactions
Adult mouse or rat • Robust infections • Limited applicability to human infection
infection with G. muris • Natural infection cycle • G. muris cannot be cultured axenically—cyst
stocks may have fecal contamination
Neonatal mouse infection • Can use human relevant strains • Limited immunity in neonatal mice
with G. lamblia • Can study more complex
host–parasite interactions
Adult mouse infection • Abundant reagents for analysis • Not all findings translate to human infections
with G. lamblia of immunity
• Many molecular tools available • Infections have little overt pathologya
Gerbil infection • Highly susceptible to infection • Fewer reagents and mutant strains available
and development of symptoms
Hamster infection • Highly susceptible to infection • Fewer reagents and mutant strains available
and development of symptoms
C. elegans • Fewer ethical considerations • Limited applicability to human infection
compared to vertebrates
a This is actually similar to most human infections, however.
642 Laboratory Models for Foodborne Infections

assemblage B strains (particularly GS) and for in vitro experiments performed with assemblage A strains
(e.g., Portland-1 and WB).
There are several pathological mechanisms thought to contribute to the diarrheal disease caused by
G. lamblia infection. These include epithelial barrier breakdown, defects in the epithelial brush border,
and increased intestinal motility.1,2 Changes in epithelial barrier function have been linked to both tight
junction proteins and epithelial apoptosis in epithelial cell lines and in human infections.72–74 Parasite
strain could be important in determining the severity of this pathology. When the human epithelial
cell line HCT-8 was exposed to different assemblages of G. lamblia, the assemblage was seen to influ-
ence both epithelial barrier integrity and apoptosis.73 Similarly, Chin et al. found that some strains of
G. lamblia were superior to others in inducing apoptosis in the SCBN cell line (reported at the time to be
a human small intestinal cell, but later genotyped as being of canine origin).75 Hardin et al. first showed
increased macromolecular uptake in Giardia-infected gerbils.76 Zhou et al. showed that transepithelial
electrical resistance of mucosa isolated from infected mice was reduced compared to mucosa from non-
infected animals, and Chen et al. showed epithelial barrier breakdown and translocation of commensal
bacteria in infected mice.77,78 Reductions in transepithelial electrical resistance were first shown in vitro
using cell lines exposed to Giardia, and these changes were further associated with alterations in tight
junction proteins.79 Parasite factors responsible for changes to the intestinal epithelium have not been
identified at the molecular level, although new structures related to adhesion and potential toxins have
been suggested.68,80,81
Giardiasis is also associated with reduced disaccharidase activity in humans, and this has been associ-
ated with shortening of the intestinal microvilli in animal models. This enzyme deficiency can contrib-
ute to diarrhea by creating a gradient that drives water out of the tissue and into the intestinal lumen.
In a mouse infection model using the murine-specific species G. muris, infection and CD8+ T cell
responses were shown to contribute to microvillous shortening and a deficiency in disaccharidases.82,83
This same deficiency in small intestinal disaccharidases was confirmed in mice using human strains of
G. lamblia.43 In the latter study, parasite assemblage was again shown to be an important factor contrib-
uting to intestinal pathology, as disaccharidase deficiency was observed in assemblage-B-infected mice
but not assemblage-A-infected mice.43
G. lamblia infection has been linked with other changes in bowel function in humans and animal
models. Humans infected with Giardia are prone to postinfectious irritable bowel syndrome.1 Gerbils
infected with G. lamblia had increased intestinal transit and hypercontractility of intestinal smooth
muscle.84 Studies using G. muris and G. lamblia infections in adult mice showed that increased
intestinal transit required adaptive immune responses as well as the neuronal isoform of nitric oxide
synthase.85,86 These studies also showed that increased transit helped facilitate elimination of the para-
site. Follow-up studies using the adult mouse model of G. lamblia infection found that altered motility
is due at least in part to increased contractility of intestinal smooth muscle and that the gut hormone
cholecystokinin (CCK) contributes to this phenotype.87 CCK normally acts to induce contraction of
the gall bladder and release of bile into the digestive tract in response to ingestion of fats. Giardia
requires bile for growth and, thus, appears to have evolved a mechanism to induce the host to provide
it with an essential nutrient.
Infection with G. lamblia is also associated with long-term health consequences. These diseases can
manifest well after infection clearance and have a range of severity. G. lamblia has been linked to several
metabolic outcomes including lowered cognitive function, lower weight and height, failure to thrive, and
nutritional deficiencies.1 Furthermore, acute G. lamblia disease severity is associated with nutritional
deficiency in animals as both mice and gerbils on nutrient-limited diets presented with worsened intesti-
nal pathology as compared to non-nutrient-limited controls.44,88 These studies indicate that the relation-
ship between G. lamblia infection and disease could be complex and bidirectional. The long-term health
consequences of G. lamblia infection represent an intriguing area of investigation as these associations
can be strong but mechanisms are lacking. For example, one study of humans with giardiasis found that
one-third of infected individuals presented with extraintestinal manifestations of infections including
symptoms of the eye, skin, joints, and urinary tract.89 G. lamblia has also been linked with postinfec-
tion development of allergies, muscular complications, chronic fatigue syndrome, and irritable bowel
syndrome,1 but good animal models of these long-term sequelae have not been developed.
Giardia lamblia 643

40.10 Immunology
G. lamblia infection leads to pathological alterations of the host’s intestinal epithelia, yet intestinal
inflammation is not often seen during infection.90 This could indicate that the host immune response
is protective against inflammatory effects. Furthermore, several studies demonstrate the host immune
response having a role in driving pathology in the intestine during infection.43,83 Thus, it is important to
understand which elements of the host immune response contribute to pathology versus those which aid
in clearance and protection during G. lamblia infection.
To successfully colonize the small intestine, G. lamblia must contend with the hosts’ natural barriers
to infection. Both intestinal mucus and epithelial cell turnover could serve as obstacles for trophozoite
attachment, and in vitro studies have shown that mucin can inhibit the ability of trophozoites to attach to
a substrate.91 Recent work has also demonstrated that the host microbiome can play an important role in
successful colonization. Infections of mice from different suppliers with G. lamblia showed that intes-
tinal microbiota conferred protection against infection and that this protective effect could be transmit-
ted through cohousing of mice.92 This same study demonstrated that antibiotic treatment could ablate the
protective effect of the host microbiome, demonstrating that intestinal bacteria composition plays a role in
infection susceptibility.92 Whether the influence of the host microbiome on G. lamblia infection is direct or
a result of bacteria-driven immune stimulation remains unclear. Lactobacillus johnsonii culture superna-
tants have been shown in vitro to have a cytostatic effect on G. lamblia.93 This same bacterium was shown
to inhibit G. lamblia infection of gerbils.94 In a different study, mouse infection with Enterococcus faecium
led to immunological stimulations that decreased G. lamblia colonization success in infected mice, demon-
strating that bacteria could alter host immunity to prevent establishment of G. lamblia infection.95
The innate immune system serves as an early line of defense against pathogens as it does not require
the specificity of adaptive immunity to exert its protective effects. The importance of innate immunity
in the control of G. lamblia infection is not well characterized. However, several studies have shown that
innate immune effectors do have a role in G. lamblia infection. Antimicrobial peptides like defensins
and lactoferrin have been shown to kill G. lamblia in vitro.96,97 Another potent antimicrobial, nitric
oxide (NO), has also been shown to have inhibitory effects on G. lamblia growth and survival in an
in vitro model using human intestinal epithelia cells.98 Studies in G. lamblia-infected mice have shown
that while NO produced by inducible NO synthase (NOS2) may not be effective in controlling infection
alone, it may have a redundant role with MMP-7, a protease required for cleavage of defensins.85,99 These
studies demonstrated that while a loss of either NOS2 or MMP-7 did not affect infection clearance, mice
lacking both MMP-7 and NOS2 had a defect in parasite clearance.
Interestingly, it has also been shown that G. lamblia can inhibit innate cellular immune responses.
G. lamblia extracts are able to inhibit the production of proinflammatory cytokines by murine
bone-marrow-derived dendritic cells stimulated with LPS and other innate immune stimuli.100 At the
same time, production of the anti-inflammatory cytokine IL-10 was increased. The importance of
dendritic cells in stimulating anti-Giardia immunity in vivo was shown using cell transfer systems.101
Giardia infection can also modulate macrophage responses in the intestinal lamina propria. We recently
showed that adult mice infected with G. lamblia have an increase in the proportion of macrophages in
the small intestine and that these cells express both NOS2 and arginase (ARG1).102 In addition, these
macrophages express more IL-10 and less TNF following infection, consistent with an anti-inflammatory
phenotype (JM and SMS, unpublished observation). Giardia has also been reported to actively block
neutrophil recruitment by degrading the chemokine IL-8, which was induced in epithelial cells follow-
ing exposure to the parasite in vitro.103 Combined analysis using in vitro and animal models continue to
elucidate interactions between the host and parasite.
Another important role of the innate immune response is to begin to mobilize the adaptive immune
response. Innate immune recognition of a pathogen drives the release of chemical signals that recruit and
activate other immune cells. In vitro studies have shown that several intestinal chemokines are secreted
in response to G. lamblia infection. CCL2, CCL20, and CXCL1-3 are all secreted when Caco-2 cells, a
colon cell line, are stimulated with G. lamblia.104 The in vivo importance of these chemokines remains
to be explored.
644 Laboratory Models for Foodborne Infections

Communication between the parasite and host is bidirectional, and parasites alter their behavior in
response to intestinal epithelial cells. Ringqvist et al. cocultured G. lamblia trophozoites with the human
adenocarcinoma line, CaCo2, and used proteomics to identify three parasite proteins (arginine deimi-
nase, ornithine carbamoyl transferase, and enolase) in culture medium.105 All three proteins were pre-
viously known as antigens recognized by giardiasis patient sera. The authors showed that G. lamblia
contact with epithelial cells triggers release of metabolic enzymes, which would facilitate effective para-
site colonization of the human small intestine.105
Adaptive immunity has been shown to be essential for control of Giardia infection, with both antibod-
ies and T cells having important roles. Studies using a mouse model of G. muris infection have dem-
onstrated that B cells and specifically IgA produced by these cells are essential for parasite control and
elimination.106–109 B cells appear to be less important for resolving G. lamblia infection. In a study using
B-cell- and T-cell-deficient mice, it was demonstrated that while T cells were required for elimination of
G. lamblia infection in mice, B cells were not.110 However, in a study on the role of IL-6 in G. lamblia
infection, IL-6-deficient mice had a defect in parasite clearance until 60 days after infection when para-
site clearance correlated with the production of antibodies that were reactive to a diverse population of
parasites.111 These studies indicate that while host antibody may not be required for G. lamblia clear-
ance, it could play an important role in parasite population control throughout the course of infection.
These results are consistent with studies on the role of antibody in human infection where patients with
antibody deficiency are at only a slightly increased risk for G. lamblia infection.112 Unlike B cells, T cells
seem to be required for the clearance of G. lamblia infection in mice. In fact, CD4 T cells have been
shown to be essential for parasite clearance as mice with specific deficiency in CD4 T cells are unable
to control infections.43,110,113
The role of several T cell cytokines has been analyzed in animal models. IFN-γ has been shown to
be important for parasite clearance during mouse infection with either G. muris or G. lamblia.110,114,115
IFN-γ production and T cell proliferation were also observed in ex vivo stimulations of human lympho-
cytes with G. lamblia, supporting a role for this cytokine in human infections.116 TNF-α has also been
shown to be important in early control of G. lamblia infection as mice with defects in TNF-α production
demonstrated increased parasite burden during infection.77 Recent studies on the inflammatory cytokine
IL-17 have demonstrated that this cytokine is important for clearance of infection in mouse models of
both G. muris and G. lamblia.117,118 The role of IL-6 in Giardia infection has been analyzed in IL-6-
deficient mice. G. lamblia infections of IL-6-deficient mice have demonstrated that this cytokine is
required for infection control and parasite clearance.111,119 As mentioned earlier, these mice did not have
a defect in antibody production, but instead, defects in dendritic cell function may be responsible for the
observed phenotype.101
Intestinal mast cells also have important functions in control of Giardia infection. Mouse infection
models using G. muris and G. lamblia have demonstrated that mast cell deficiencies lead to deficiencies
in infection clearance.120,121 Mast cell hyperplasia has also been observed in Mongolian gerbils infected
with G. muris.76 Furthermore, G. lamblia-infected mast cell-deficient mice produce less IL-6 mRNA
and IgA than their wild-type counterparts.121 Mast-cells-also appear to contribute to intestinal smooth
muscle contractions during G. lamblia infection in mice and could further support a protective role for
these cells in infection.87
One important goal of immunological studies of G. lamblia is the development of a human vaccine.
Currently, no human Giardia vaccine exists; however, a veterinary vaccine, GiardiaVax, has been used
in a variety of domestic mammals. Studies in humans have found that previous Giardia infection could
convey some level of protection.122,123 This protection indicates that immunological memory to Giardia is
possible in humans and supports the development of a human vaccine. Recently, several studies in mice
have been done with the goal of better understanding how to develop a human vaccine. A reinfection
model in mice with G. lamblia was able to recapitulate the protection observed in humans, indicating
that mouse models could serve as a useful tool for human vaccine development.124 In a mouse model of
G. lamblia infection, a vaccine against parasite cyst wall protein was able to reduce cyst shedding.125–127
In another mouse vaccine study, vaccination against a G. lamblia protein, α1-giardin, was able to confer
protection against infection.128 This protein is highly conserved between both human assemblages of
G. lamblia, indicating it could provide coverage against multiple strains of the parasite.128 The ability
Giardia lamblia 645

of Giardia to undergo antigenic variation of the variant-specific surface proteins (VSPs) (discussed in
Section 40.11) may be a major impediment to the design of a successful vaccine. Rivero et al. used para-
sites engineered to express multiple VSPs simultaneously as a vaccine in gerbils and achieved significant
levels of protection.129 Combinations of approaches may be necessary to generate an effective vaccine
for humans and/or animals.

40.11 Antigenic Variation in G. lamblia

Many pathogenic bacteria and protozoans develop strategies to evade the host immune response and
maintain chronic infections. One of them, present in microorganisms like Neisseria, Candida, and
Trypanosoma, is the frequent interchange of antigens on the surface of the cell. This phenomenon is
known as antigenic variation. There are three important criteria defining this phenomenon: Only one
antigen can be expressed at the surface at a time; there must be a family of homologous genes for encod-
ing the variant proteins that will be expressed; and there must be a mechanism for altering the pattern of
the expression of this antigen in the cell.130
Antigenic variation in Giardia was first observed in vitro as an alteration in the pattern of expression
of highly abundant surface molecules (later termed VSPs) during the in vitro growth of the parasite.131,132
This process was then verified during infections in humans and animals.133–135 According to Nash et al.,
switching among VSPs occurs every 6–16 generations, depending on the strain.136 The expression of
VSPs in trophozoite cells is controlled through post-transcriptional gene silencing, involving a large
number of miRNAs.137,138
Similar to the role of antigenic variation in other pathogens, reports indicate that by altering VSPs on
the cell surface during infection, the parasite can avoid the immune response and prolong infection.135,139
Additional roles for VSPs have also been postulated. More than 200 VSP genes have been described,23
and proteomics studies showed that the VSP expression profile can differ considerably by strain and gen-
otype.140 This variation may allow for repeated infections with giardia, irrespective of antigenic switch-
ing that occurs during a single infection. Earlier, Nash et al. showed that VSPs differed in sensitivity to
host proteases and might prefer a growth advantage depending on their environment.141 This was vali-
dated in vivo when parasite clones expressing different VSPs were shown to exhibit growth preferences
in immunodeficient mice and gerbils.142 Astiazarán-Garcia suggested that VSPs, based on their ability
to bind zinc, could inhibit the function of zinc- or metal-requiring intestinal enzymes, or compete with
the host for zinc and contribute to zinc malnutrition.143 Recently, proteomic comparison of virulent and
avirulent strains of Giardia suggested that differences in the VSP repertoire might contribute to the dif-
ferent levels of virulence observed in animals.144

40.12  G iardia and Arginine

The relationship between Giardia and arginine within the host is of interest because the parasite con-
sumes arginine as an energy source, which could have important implications for immune function and
parasite survival during infection. Giardia consumes arginine through a three-step enzymatic pathway
that involves arginine deiminase (ADI), ornithine carbamoyltransferase (OCT), and carbamate kinase
(CK) with ATP generated as an end product.145 This pathway also serves as a means by which Giardia
could reduce arginine availability in the host and limit its conversion to nitric oxide (NO). In fact, argi-
nine is the sole amino acid substrate for NO production by the enzyme nitric oxide synthase (NOS),
making it a limiting factor for NO production.146
The production of NO from arginine is an important element of the innate immune response as NO is
toxic to many pathogens. In vitro infection models using human intestinal epithelial cells and activated
mouse macrophages have demonstrated that NO has the ability to inhibit trophozoite growth and is
potentially lethal to the parasite and may block cyst formation.98,147 Human intestinal epithelial cells have
increased NOS expression following coculture with G. lamblia in vitro, and increased NOS expression
is also observed in small intestinal tissue from G. lamblia-infected mice.85,102,148 The importance of host
646 Laboratory Models for Foodborne Infections

NO production in vivo is unclear. Recent studies indicate that host-produced NO may be redundant with
α-defensins as only a loss of the expression of enzymes required for the production of both influenced
infection outcomes in mice.85,99 Furthermore, it is likely that G. lamblia influences arginine availability
within the host indirectly. Stimulation of a mouse macrophage cell line with parasite lysate leads to
increased expression of arginase 1 (ARG1), a host-produced arginine-consuming enzyme.102 This same
study found that small intestinal tissue from infected mice also showed increased ARG1 expression
compared to uninfected controls.102
The consumption of arginine through parasite ADI is advantageous for the G. lamblia in potentially
two ways. First, it serves as a means of energy production through conversion of arginine to ATP, and
second, it provides a means by which Giardia might evade the host immune response by limiting NO
production. Limiting arginine availability in the gut could also affect the host immune system in other
ways. For example, it is possible that T cell function is altered as arginine is an important modulator of
T cell proliferation. Arginine depletion could result in the limitation of T cell proliferation and may also
cause an impairment of CD3ζ chain turnover of the T cell receptor after antigen stimulation, inhibiting
signaling through the T cell receptor complex.149 Both of these scenarios would result in limiting the
T cell response during Giardia infection, providing an additional benefit for the parasite through argi-
nine depletion. Thus, giardial ADI or host ARG1 may directly interfere with the host’s ability to gener-
ate a strong, protective cellular immune response against the parasite while limiting the innate immune
response by inhibiting NO production.

40.13 Drug Resistance in Giardia

Current giardiasis treatments cause a variety of side effects, treatment failures are common, and a con-
siderable incidence of drug resistance occurs. Many drugs have been tested for a therapeutic approach
against G. lamblia. Albendazole, quinacrine, metronidazole, nitazoxanide, and isoflavones are impor-
tant drugs used in some studies involving drug resistance in giardiasis.19,150–161 Uzlikova and Nohynkova
showed by flow cytometry analysis that sublethal drug concentrations of metronidazole affect the
replication phase of the cell cycle of Giardia.162 Cells incubated with lethal drug concentrations lose
the ability to adhere to a surface after few hours of incubation. A recent study of drug resistance in
Giardia19 identified the minimum lethal concentrations of 28 drugs, and compared the results with met-
ronidazole, the drug most used to treat giardiasis. Fumagillin, carbadox, and tioxidazole were able to
eliminate metronidazole-resistant G. lamblia isolates. In the same studies, the authors evaluated the
dose-dependent efficacy of fumagillin in a mouse model, and showed that the effective dose of fumagil-
lin was much lower than that for metronidazole.
Nitroheterocyclic drugs like metronidazole are the first line of therapy for giardiasis. These com-
pounds are redox-active and are believed to cause damage to protein and DNA after being activated by
oxidoreductase enzymes in metabolically active cells. In a recent study, Ansell et al. reviewed the molec-
ular phenotype of nitroheterocyclic-resistant G. lamblia isolated from patients. The authors believe that
resistance mechanisms are related to enzymes commonly associated with drug resistance, like pyruvate
ferredoxin, oxidoreductases, and nitroreductases. These authors also highlighted new approaches using
systems biology and advanced bioinformatics to evaluate mechanisms of nitroheterocyclic resistance in
Giardia.163
Using proteomic and transcriptomic analysis, researchers have identified and characterized dif-
ferentially expressed genes in albendazole-resistant clones of G. lamblia. The expression of proteins
and their corresponding mRNA-resistant clones were analyzed at different concentrations, and these
were compared with albendazole-sensitive clones using two-dimensional electrophoresis and mass
spectrometry. The authors identified eight differentially expressed proteins in albendazole-resistant
clones that are involved in the following systems: cytoskeleton, antioxidant metabolism, and energy
metabolism. In addition, it was suggested that resistance to albendazole in G. lamblia is involved
in response to gene regulation and may have an important role in maintaining cellular stability and
­oxidative stress.164
Giardia lamblia 647

40.14 Conclusion
The relationship between G. lamblia and its host is complex and multifactorial with host immunity,
intestinal microbial composition, nutritional status, and parasite strain all having potential roles in host
susceptibility and infection severity. As a disease of the developing world, many of these aspects of
infection remain understudied and poorly understood. Studies that aim to illuminate these relationships
will provide information that could lead to new strategies for combating parasitic disease. These types
of investigations could yield not only better treatments but also better control of a disease with a global
distribution that infects millions of humans and other animals.

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41
Toxoplasma: Animal and In Vitro
Models on Toxoplasmosis

Renato Augusto DaMatta, Andrea Cristina Vetö Arnholdt, and

Farlen José Bebber Miranda

CONTENTS
41.1 Introduction................................................................................................................................... 655
41.1.1 The Disease...................................................................................................................... 655
41.1.2 Biology of the Parasite..................................................................................................... 656
41.2 Laboratory Models to Study Toxoplasma gondii......................................................................... 658
41.2.1 In Vitro Models................................................................................................................. 658
41.2.2 Animal Models................................................................................................................. 660
41.2.2.1 Detection of the Parasite in Experimental Infected Animals...........................661
41.2.2.2 Rodent............................................................................................................... 662
41.2.2.3 Pigs.................................................................................................................... 665
41.2.2.4 Cat..................................................................................................................... 666
41.2.2.5 Chicken............................................................................................................. 667
41.2.2.6 Fish.................................................................................................................... 668
41.3 Concluding Remarks..................................................................................................................... 668
Acknowledgments................................................................................................................................... 669
References............................................................................................................................................... 669

41.1 Introduction
41.1.1 The Disease
Toxoplasmosis is a disease that infects nearly all warm-blooded vertebrates.1,2 It is estimated that about
a third of the world’s human population is serologically positive for this infection.2–4 Transmission of
the parasite has been historically implicated by the ingestion of undercooked or raw meat containing
the parasite in tissue cysts.5 However, in the last two decades, accumulated evidence point also to the
parasite infection by oocysts ingestion through contaminated water, soil, or food—mainly vegetables
and fruits.5,6 Food consumption is culturally based; thus, the venue of infection may vary greatly in dif-
ferent human populations, explaining the variance in different geographical locations.3 Toxoplasmosis
outbreaks through contaminated water in human populations have been reported in detail7–10 showing
how relevant environmental contamination can be.
Most often, toxoplasmosis is asymptomatic in immunocompetent individuals.2–4,11 However, depend-
ing on the Toxoplasma gondii strain,2 the genetic constitution of the host, parasite load, and the mode
of transmission,12,13 infection can cause severe clinical manifestations including ocular toxoplasmosis,14
cerebral toxoplasmosis,15 and congenital toxoplasmosis.1 When acquired by healthy individuals, toxo-
plasmosis most often causes lymphadenopathy, usually of the head and neck, but pneumonia, retino-
choroiditis, myocarditis, neurological disorders, and severe ocular infection have also been reported in

655
656 Laboratory Models for Foodborne Infections

severe cases especially in French Guiana16 and in Brazil.17 Congenital toxoplasmosis can cause mental
retardation, encephalitis, neonatal mortality, and abortion of the fetuses.2,17,18 In immunocompromised
patients, the reactivation of the parasite into fast replicating forms in the central nervous system (CNS)
causes cerebral toxoplasmosis.5 In addition, T. gondii infection has been implicated in schizophrenia and
other neurological disorders.15,19 Domestic animals also develop toxoplasmosis upon infection, causing
relevant economic losses, and wild animals are also infected by T. gondii.1,20 Due to its severe mani-
festations, worldwide distribution, and diverse host infection, toxoplasmosis is a relevant public health
problem.21,22

41.1.2 Biology of the Parasite

The etiological agent of toxoplasmosis is Toxoplasma gondii, an obligate intracellular protozoan parasite
of the Apicomplexa phylum.13,23 This parasite presents three infection forms. Tachyzoites (Figure 41.1)
replicate fast, rupturing host cells, causing lytic cycle during the acute phase of the disease.13,24
Bradyzoites are slow-multiplying forms found within tissue cysts (Figure 41.2) mainly located in the
CNS and skeletal muscles.13 Sporozoites are found in sporulated oocysts shed by the definitive host.13 In
healthy intermediate and definitive hosts, tachyzoites convert to bradyzoites, and the infection becomes
latent. In vitro treatment of infected cells with stress factors, such as proinflammatory cytokines, NO,
serum from infected laboratory animals, drugs, low nutrient, alkaline or acid pH, and extreme tem-
perature cause conversion of tachyzoites into bradyzoites.13,25–27 It was first thought that the immune
host response was responsible for inducing this conversion.13,25,26,28 However, spontaneous conversion of
tachyzoites into bradyzoites in cultures of cells from the CNS29,30 and in skeletal muscle cells31 suggests
that this phenomenon may be orchestrated by the host cell type.31,32 This highlights the possible tissue
tropism found in infected hosts.13,33
The parasite life cycle is complex and involves different host species (Figure 41.3). The definitive
hosts are members of the Felidae family.34,35 After ingestion of the parasite, the infection of the Felidae
intestinal epithelium cells (enterocytes) results first in schizogony followed by the sexual phase of the
cycle.35,36 Infection may occur by feline oral ingestion of bradyzoites (in tissue cysts) or sporozoites (in
sporulated oocysts).35,36 These forms infect enterocytes, and some are converted into tachyzoites in the
lamina propria cells,35 but different generations of merozoites,37 with five distinct forms (named from
A to E), are found in these cells before the appearance of gametes.35 Fertilization generates a diploid
zygote that becomes the unsporulated oocyst shed in the feline’s feces.35 The released oocyst sporulates

(A) (B)

(C) (D)

FIGURE 41.1  Tachyzoites infecting ostrich macrophages. Crescent shape of the parasite can be noted after infection
(2 h—A), replication (24 h—B; 48 h—C) and egress (48 h—D). During egress, host cells are usually destroyed (D). Bar: 20 μm.
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 657

FIGURE 41.2  Toxoplasma gondii cyst (arrow) found in the brain of mice inoculated with heart tissue of an infected pig.
Bar: 50 μm.

Felids Shed unsporulated

definitive oocysts
hosts

Sporulated
oocysts contaminate
the environment
Carnivorism

Homeothermic vertebrates Congenital

intermediate hosts infection

Carnivorism

FIGURE 41.3  Schematic representation of the Toxoplasma gondii life cycle. In the enterocytes of the members of the
Felidae family, the parasite differentiates in gametocytes that generate a zygote that is shed in the feces as unsporulated
oocysts. After 2–5 days in the environment, oocysts sporulate to form two esporocysts, each with four sporozoites that
contaminate the environment. Hosts get infected by the ingestion of oocysts. Oocysts are digested liberating sporozoites
that first infect enterocytes differentiating in tachyzoites that later infect lamina propria cells and multiply rapidly. These
forms disseminate through the organism by the circulatory system. Tachyzoites convert into bradyzoites forming tissue
cysts. Tachyzoites can migrate to the placenta in pregnant hosts causing congenital infection. Carnivorism among the hosts
disseminates the parasite through the ingestion of bradyzoites in tissue cysts. As with oocysts, tissue cysts are digested
liberating bradyzoites that are resistant to this process. These forms infect host enterocytes and differentiate in tachyzoites
similar to sporozoites.

by meiosis and mitosis in the environment and disseminates. Infection of intermediate hosts occurs by
ingestion of oocysts through the consumption of contaminated water or food, or by the ingestion of tis-
sue cysts by carnivorism. In these hosts, the infective forms, sporozoites or bradyzoites, pass through the
enterocytes and convert into tachyzoites38 that infect and multiply in cells of the lamina propria. At this
stage, tachyzoites disseminate through the organism and then convert into bradyzoites, forming tissue
cysts. During their life cycle, tachyzoites may pass from the blood to the placenta and infect the fetus
causing congenital toxoplasmosis (Figure 41.3).
658 Laboratory Models for Foodborne Infections

Sexual reproduction of the parasite in Felidae provides genetic variability that results in different
recombinant strains.24,39,40 Isolated strains from North America and Europe have an unusual population
structure with basically three clonal lineages (types I, II, and III).41 However, recombinant strains are
found in different parts of the world and have diverse virulence.20,39,42 Studies on isolates from Brazil,
Colombia, and French Guiana have shown that most T. gondii strains are recombinant, suggesting greater
sexual recombination of this parasite in South America,16,39,43 thus explaining the wider spectra of clini-
cal manifestations.16,17,43 This indicates that the parasite population structure varies in different locations
around the world, being composed by distinct genotypes with important implications to infection, clini-
cal manifestations, and development of distinct diagnosis and treatment strategies.

41.2 Laboratory Models to Study Toxoplasma gondii

The first description of T. gondii was actually made independently by two groups using two different
laboratory experimental animals: rabbits in Brazil,44 and the gundis (Ctenodactylus gundi), a rodent, in
Tunisia.45,46 Since then, many different laboratory models have been used to study different aspects of
this parasite. Some of them will be described here.

41.2.1  In Vitro Models

In vitro studies are crucial to understand the biology of cells, including intracellular parasitism. All fields
studying cells have adopted cell culture techniques47 resulting in most of what we know, T. gondii biol-
ogy included. The first laboratory procedure to obtain tachyzoites of T. gondii for experimental infec-
tion was to pass parasites into mice by the inoculation of macerated tissue, usually of the brain.48 Later,
parasites were maintained by peritoneal wash of infected mice and used for further experimentation.49,50
At that moment, some key aspects of the biology of the parasite were studied: its intracellular nature,48
drug treatment,49–51 and transmission.52–54 Today, tachyzoites are mostly maintained by culture passage
in cell monolayers. However, the use of animals is still necessary to obtain bradyzoites, merozoites, or
sporozoites, since in vitro system does not generate these forms yet.
The first reproducible culture system for T. gondii was obtained by Guimarães and Meyer.55–57
Tachyzoites can be maintained in cell culture allowing relevant studies on the biology of the parasite.24,46
The infective forms of these parasites are haploid, facilitating transient and stable transfections.58 These
characteristics transformed this parasite in an Apicomplexan and intracellular parasitism model.24
Furthermore, host cells can also be genetically changed,59 allowing a precise questioning about the inter-
play between parasite and host cells. Thus, numerous in vitro laboratory models are used nowadays and
can be created in the future to come.
Aided by these models, relevant information was obtained on the host cell–T. gondii interaction (motil-
ity, invasion, replication, and egress), infective form conversion (tachyzoites to bradyzoites and vice versa),
gene expression, diagnostic methods, screening of new therapeutic compounds, etc. An active field of
study is how T. gondii escapes the cell’s autonomous immunity and also the immune system response as a
whole. One of the first evasion mechanisms described for T. gondii involves the structure in which this par-
asite resides in the host cell: the parasitophorous vacuole (PV). Using transmission electron microscopy, it
was found that live parasites reside in the PV that does not fuse with lysosomes.60 However, parasites that
are dead or opsonized with antibodies do fuse, and the parasites are destroyed by lysosomal digestion.61,62
Further work has been performed in this cell compartment revealing interesting parasite adaptations.63,64
These early findings described the PV harboring T. gondii as “invisible” to the endolysosomal system,
allowing its persistence and multiplication in the host cell. This demonstrates a cellular escape mecha-
nism, clearly showing that the evasion concept could explain T. gondii’s success as a parasite.
Another classical evasion mechanism of T. gondii is its capacity to resist killing by reactive oxygen species
(ROS) produced by host cells, especially macrophages. By the end of the 1970s, it was clear that ROS could
be produced not only by neutrophils, but also by macrophages and monocytes with clear negative correla-
tion to bacterial growth. It was found that in cell-free systems, T. gondii was 100-fold more resistant to H2O2
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 659

than Trypanosoma cruzi.65 This ROS resistance was correlated with high catalase activity of T. gondii,65
which was later confirmed by the activity of superoxide dismutase showing the robust antioxidant arsenal
of this parasite.66,67 As a result, production of ROS is not detected in the T. gondii macrophage contact sites,
explaining parasite survival, persistence, and multiplication in host cells that are professional ROS produc-
ers.68,69 All this work was done with mammalian macrophages, but the similar result was obtained when
chicken macrophages were examined,70 suggesting that the same mechanism operates in birds. Although
this parasite can evade from host cell ROS production in vitro, it was later demonstrated that these radicals
are implicated in the in vivo control of T. gondii71 suggesting that this system still needs further work.
More recently, it has been demonstrated that IRGs (immunity-related GTPases) are involved in the growth
control of T. gondii in mice.72–75 This is significant because most of the data from different aspects of T. gon-
dii biology, including IRGs, point to rodents as the most relevant intermediate host for the parasite, in a clear
example of coevolution of host cell resistance mechanisms and T. gondii virulence factors.73,75 Interferon-
gamma (IFN-γ) induces IRGs in rodent host cells of myeloid and nonmyeloid origin. If an IFN-γ-activated
cell is infected by T. gondii, the GTPases hierarchically and cooperatively accumulate in the PV membrane,
changing their form until disruption, allowing T. gondii to end up in the cytosol where it is killed.73,76
However, some T. gondii strains can escape the IRG system.40,75 Virulence of T. gondii strains to mice is
directly related to its repertory of rhoptry proteins kinases and pseudokinases against the IRGs microbi-
cidal system.40 Rhoptry is a secretory organelle of T. gondii that releases its protein contents into the host
cell during invasion. However, there is high polymorphism of these proteins between strains.77,78 Virulent
strains of T. gondii, such as type I, have rhoptry proteins that efficiently phosphorylate IRGs components,
impairing their association to the PV, resulting in parasite survival and virulence capacity.40,79 Less-virulent
strains have fewer or no effective rhoptry proteins against IRGs, being destroyed by this system. In virulent
strains, these proteins form complexes that inactivate IRGs.80 These complexes also involve proteins from
another secretory organelle of the parasite, the dense granules.81 The composition of these secretory protein
complexes and how they deactivate IRGs is currently an important area of T. gondii research.
In the last decade, it has also been shown that T. gondii hijacks cells from the immune system, allow-
ing the parasite to cross host barriers such as gut epithelial barrier, blood–brain, and blood–retina barri-
ers in a mechanism known as Trojan horse.82–84 Some of the molecular events underlying this mechanism
have been described by our group. In vitro infection of unstimulated murine macrophages leads to down-
regulation of integrin alphaL, alpha4, and alpha5 immediately after infection, allowing cells to de-attach
from extracellular matrix components.82 On the other hand, T. gondii infection of human or murine
macrophages impairs upregulation of CD86, CD80, CD40, and CD1a, while maintaining their migra-
tory ability.85 T. gondii seems to regulate the migratory machinery similar to what metastatic cells do
as demonstrated by increased expression of host metalloproteinases MT1-MMP and ADAM-10 upon
infection of murine macrophages, in parallel with increased secretion of active MMP-9.84 Active MMP-9
is secreted in association with its cell membrane acceptor CD44, and with uPAR and TIMP-1.86 In fact,
secretion of this multiprotein complex is partially dependent on the extracellular plasminogen urokinase
pathway (uPA/uPAR)86 and ERK.84 In vivo expression of MMP-9 is also increased in the ilea and lungs
of orally infected C57Bl/6 mice (Pimentel, P. M., et al., unpublished data). Adoptive transfer experiments
of MMP knockdown cells infected with T. gondii have been developed in order to access its contribution
to in vivo dissemination of host cells carrying the parasite.
Nitric oxide (NO) produced by inducible NO synthase (iNOS or NOS2) expressed in classically acti-
vated macrophages is another microbicidal molecule of vertebrates87,88 involved in T. gondii control.
Infection of iNOS knockout mice or mice treated with iNOS inhibitors (aminoguanidine) have shown
that NO is important to control T. gondii growth, especially during the chronic phase of the disease.89–91
However, NO may also induce host death in the acute phase, being one of the responsible events of
the classical histopathology found in the ileum.91,92 Macrophages in culture classically activated by
IFN-γ and lipopolysaccharide express iNOS and produce high amounts of NO that control T. gondii
growth.90,93,94 After gut infection, T. gondii disseminates throughout the host, and immune response
initiated locally becomes systemic and, depending on the strain, controls parasite growth leading to the
latent infection. During this phase, tachyzoite will come across activated macrophages producing high
amounts of NO. How do these parasites cope with these encounters? We have been working on this
question by studying T. gondii infection of activated macrophages of mice and chickens in vitro. It was
660 Laboratory Models for Foodborne Infections

shown that tachyzoites of T. gondii inhibit NO production after infection of activated macrophages.70,95–99
NO production inhibition is caused by iNOS degradation.70,96,97 Interestingly enough, this degradation
occurs right at the beginning (2 h) of the infection70,97,99 and is caused by the proteasome pathway.99 It was
determined that an autocrine secretion of TGF-β1 caused by the parasite infection was responsible for
iNOS degradation.97 Similar to amastigotes of Leishmania amazonensis,100 T. gondii tachyzoites expose
phosphatidylserine (PS) without cell death, which, when blocked by annexin-V, abolishes the inhibition
of NO production caused by the infection.97 This indicates that tachyzoites of T. gondii may, indeed, use
what is known as “apoptotic mimicry,” a concept established for L. amazonensis that basically states
that the parasites that expose PS mimic the anti-inflammatory response induced by apoptotic cells.101 We
then separated the PS-positive and -negative tachyzoite subpopulations using annexin-V conjugated with
magnetic beads and found that only the PS-positive subpopulation was able to inhibit NO production in
infected macrophages.98 In addition, mice infected with positive or negative PS subpopulations died faster
than the ones infected with both populations. Mice infected with the positive PS subpopulation died of high
parasite burden, while the ones infected with the negative PS subpopulation died of uncontrolled inflam-
matory response.98 These results indicate that both subpopulations are necessary for a balanced response
of the host, allowing survival and parasite transmission. Recently, we have found that four strains of T.
gondii with distinct virulence exposed PS and inhibited iNOS expression immediately after the infection
of activated macrophages (Damasceno-Sá, J. C. et al., unpublished data). Thus, it seems that apoptotic
mimicry is a general escape mechanism to NO microbicidal system that is present in mammals and birds.
Tachyzoite conversion to bradyzoite and formation of tissue cysts may be a naturally occurring phe-
nomenon (not necessarily induced by the immune response) in the course of T. gondii’s life cycle and
occurs mainly in the CNS and in skeletal and cardiac muscles, defining the latent stage of T. gondii infec-
tion.13,31,32,102 Thus, there is great interest in the understanding of this conversion and the need of a well-
established model in order to test drugs, because most treatments are not effective against the bradyzoite
stage. An interesting in vitro model involves the infection of primary cultures of embryonic-derived
skeletal muscle cells by T. gondii.103 The most impressive result concerns the spontaneous capacity of
some T. gondii strains to convert tachyzoites into bradyzoites, forming in vitro tissue cyst-like struc-
tures in these muscular cells.31,32 The infection of these cells with bradyzoites, instead of tachyzoites,
accelerates the formation of cyst-like structures.102 Skeletal muscle cells become microbicidal against T.
gondii after activation with IFN-γ, controlling parasite growth by NO production and IRGs, indicating
that these cells may also help to control T. gondii growth in mice.104 This model has also been used to
show the induction of lipid bodies by T. gondii infection in these cells and its association to the PV.105
The PV association to lipid bodies was also seen in macrophages infected with T. gondii in a lipid body
model developed by our group.106 Embryonic-derived skeletal muscle cells should be further explored as
a model of in vitro infection to study other aspects of the biology of this parasite.
In vitro models lead to important findings on T. gondii. The lytic cycle and different aspects of the parasite
biology are well studied using tachyzoites to infect distinct cell types in culture. Tachyzoite is the most easily
obtained T. gondii form, which explains why this is, by far, the most used model. Infection of culture cells
with bradyzoites has been used to study the formation of tissue cyst-like structures in vitro (see paragraph
above). Bradyzoites are obtained from mice but may be replaced by an efficient in vitro system as earlier
stated. More recently, spontaneous cystogenesis was demonstrated in an established cell line of renal feline
kidney cells infected with bradyzoites, suggesting that cell culture system may eventually replace infected
animals for studies with cysts and bradyzoites.107 The enteroepithelial part of the life cycle of T. gondii in
Felidae is less well known because there is no established cell culture model, with most findings deriving
from cat experiments.35 However, primary cultures of cat intestinal cells have been infected with bradyzoites
resulting in syncytial parasite structures suggesting the in vitro reproduction of schizogony.108 These results
indicate that it is a matter of time for the different parts of T. gondii cell cycle to be obtained in vitro.

41.2.2 Animal Models
The use of animal models to study toxoplasmosis is extremely important. T. gondii infects a plethora
of warm-blooded animals; thus, most animal infections are unique and important to understand the
parasite cell cycle and the coevolution of host mechanisms that control the parasite and of virulence
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 661

strategies for the evasion of these mechanisms. Owing to ethical reasons, the use of animals is always
controversial and must follow standard and strict protocols. In addition, some host vertebrates are
difficult and expensive to maintain, including wild, and large or medium-sized domestic animals.
Many different hosts have been used to study infections by T. gondii, but the mouse is by far the most
common (Table 41.1). A PubMed search using common host names (chosen by the authors) and the
terms “toxoplasma” and later “experimental” or “infection” or “epidemiology” (Table 41.1) showed
the highest number of articles for the mouse followed by the rat, cat, pig, chicken, Calomys, and fish.
The term “infection” had the highest number of articles, and since infection encompasses “epidemiol-
ogy,” this term was also included in the survey to better understand the numbers of published articles.
This reveals how important the cat and the pig are in the study of T. gondii epidemiology (Table 41.1).
Here, we will refer to some relevant findings of the models mentioned in Table 41.1.

41.2.2.1  D etection of the Parasite in Experimental Infected Animals

Initially, T. gondii infection induces a humoral response that is characterized by the production of
IgM, and later IgG, which gives protection to subsequent infections.109 The most used serological
tests in experimental and epidemiological studies are the ELISA (enzyme-linked immunosorbent
assay), DT (dye test), IHA (indirect hemagglutination antibody), IFA (indirect fluorescent antibody),
LAT (latex agglutination test), and MAT (modified agglutination test). Histopathological diagnosis
is highly sensitive when cysts are observed in tissues stained with H&E. However, due to the sparse
distribution of cysts in organs, particularly in medium-sized animals, a greater number of slides need
to be analyzed.
Inoculation of mice with pepsin-digested tissue from possible infected animals (bioassay) has good
specificity, although the sensitivity may be diminished by the amount of the processed material and para-
sitemia. Inoculation of leukocytes and plasma in the peritoneum of mice is a relatively simple method,
confirming the infection by the detection of tachyzoites in the peritoneal cavity, brain cysts, or serology
of mice.110 Another interesting method to detect T. gondii is to seed culture cells with the blood leukocyte
and detect the parasite by direct visualization due to growth.111
Polymerase chain reaction (PCR) is a method used as a diagnostic tool for the presence of parasites in
various organs. In addition, nested PCR is preferable due to greater specificity. However, real-time PCR
can quantify parasite DNA in tissue samples.111,112
The pig as a medium-sized animal model has great potential for use in experimental T. gondii infec-
tions filling the gap left by the murine model that is not able to respond adequately due to physiological
limitations, such as the IRGs microbicidal system that is inexistent in humans. In our opinion, this study
model can answer important issues of great interest and refinement.

TABLE 41.1
Number of Published Articles in a PubMed Search (September 2015)
Crossing the Animal Models of the First Column with “Toxoplasma”
and One of the Terms on the Second, Third, or Fourth Column
Animal Host Experimental Infection Epidemiology
Mousea 520 2358 442
Rats 81 263 76
Cat 70 746 437
Pigs 53 315 270
Chicken 14 90 90
Calomys 8 14 1
Fish 3 28 34
a The use of plural or singular for the animal host denomination was based on
the higher number of articles obtained on the “experimental” column.
662 Laboratory Models for Foodborne Infections

41.2.2.2 Rodent
Rodents are abundantly present in different parts of the world.113 Species of this order are usually infected
with T. gondii, although depending on the location, the prevalence varies and is probably related to the
lack of seropositivity in congenital infected animals of this order.114,115 These animals are probably the
most common intermediate hosts of T. gondii,73,75 with small- or medium-sized Felidae being the fur-
thermost natural definitive hosts.116,117 In addition, the mouse is the most used laboratory animal, and
numerous articles describe experimental infections with T. gondii. As a result, most of what is known
about T. gondii biology comes from laboratory models involving rodents, especially mice.

41.2.2.2.1 Mice
Infection of mice has been crucial for the understanding of many aspects of T. gondii. One of the greatest
advantages of mice as animal models for parasitic infection is the availability of a vast number of mice
strains with different genetic backgrounds, deficiencies, and the capacity to genetically manipulate these
animals.118 This has allowed the discovery of important aspects of T. gondii biology. Some relevant find-
ings concerning characteristics of the T. gondii infection will be described in the following paragraphs.
IFN-γ is crucial to control T. gondii infection.119 The first evidence that this cytokine was important
came from in vitro studies showing that the presence of this cytokine increases the microbicidal potential
of different cell types after infection.26,90,93,94,104 Studies using mice treated with the IFN-γ120 and anti-
bodies that neutralize this cytokine121 proved that IFN-γ is essential to control T. gondii growth in vivo.
The importance of IFN-γ in controlling T. gondii infection was also shown in knockout mice that died
rapidly after infection.122
The interleukin (IL)-12/IFN-γ axis was first shown in mice with severe combined immunodeficiency
(SCID) with the demonstration that IL-12 was mainly produced by dendritic cells (DCs) upon IL-1β
stimulation, resulting in the induction of IFN-γ production by NK cells in this model.123,124 The impor-
tance of IFN-γ and IL-12 and their signaling pathway was demonstrated by the infection of mice lacking
the transcription factor interferon consensus sequence binding protein (ICSBP) with an avirulent strain
of T. gondii that lead mice to rapid death after 14 days of infection.125 Also, the regulatory role of IL-10
was demonstrated in knockout mice that succumbed to infection after 2 weeks due to elevated circu-
lating IFN-γ and IL-12 and intense liver pathology.126 The importance of TLRs in immune response
to T. gondii was revealed by the use of knockout mice to MyD88, a crucial adaptor molecule for TLR
signaling transduction.127,128
Another interesting use of the mous model for T. gondii infection came from studies showing that oral
infection of these animals causes death by a typical pathology of the ileum that involves tumor necrosis
factor-α (TNF-α), IFN-γ, and NO production.92 This is relevant because oral infection is the natural route
of T. gondii infection. Depending on the strain of the parasite, the genetic background of the mice, and the
parasite burden, the ilea pathology varies. When the C57BL/6 mice receive 100 cysts of the ME-49 strain
of T. gondii by the oral route, a potent Th1 response is induced leading to the destruction of the ileum
by intense necrosis. The typical ileum villus morphology is lost due to the high inflammatory response
(Figure 41.4). Eventually, the ileum ruptures, and the mice die by sepsis. The infection of C57BL/6 mice
with the ME-49 strain of T. gondii has been proposed as a model for intestinal bowel disease.129 In addi-
tion, this pathology is not exclusive to mice, since it has also been described in different hosts.130
Although TNF-α and IFN-γ are involved in the ileum pathology, another crucial player is the gut flora.131
Oral infection causes an increase in Gram-negative bacteria, antibiotic treatment reduces IFN-γ levels and
NO production in the small intestine, and gnotobiotic mice do not develop the ileum pathology.131 Thus,
the presence of LPS from the gut flora and the increase in IFN-γ, and possibly of TNF-α, caused by the
immune response against the parasite, induces a Th1 response that ends up destroying the ileum. In this
model, IL-22 induced MMP-2 expression in an IL-17-independent manner, and infection of MMP-2 KO
mice showed decreased immunopathology scores of the gut.132 However, oral infection of MIF KO mice
led to reduced pathogenesis in the ileum, with decreased amounts of IL-12, IFN-γ, and TNF-α and also
MMP-9, whereas no modification in MMP-2 expression was observed.133 Our group has demonstrated
that oral infection of C57BL/6 with the ME-49 T. gondii strain induced high amounts of MMP-9 but no
MMP-2 in the ilea and lungs after 7 days of infection (Pimentel, P. M., et al., unpublished data).
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 663

(A) (C)

(B) (D)

FIGURE 41.4  General aspects of the ilea of C57BL/6 mice infected orally with tissue cysts of Toxoplasma gondii of the
ME-49 strain. (A) Noninfected ileum with apparent normal villi. (B) Higher magnification of the box image “A.” (C) Ileum
from infected mice with necrotic areas disrupting the villi architecture (arrows). (D) Higher magnification of the box of
image “C”; parasites can be seen in the inset. Bars = 200 μm and 10 μm in inset.

Mice models have recently been used to further explore the role of the microbiota and IL-17 in the
gut upon T. gondii infection. Some models show that IL-17 is involved in the immunopathogenesis
at the gut mucosal barrier.134,135 However, it has not been clearly identified whether IL-17 is produced
by Th17 or ROR-γ innate lymphoid cells or both. Elimination of Paneth cells by IFN-γ produced by
Th1 cells induces decreased amounts of antimicrobial peptides, leading to expansion of bacteria of the
Enterobacteriaceae family, amplifying immunopathology at the gut after infection.136 On the other hand,
mice models have also identified mechanisms of containment of spreading of microflora during infec-
tion. Translocation into the luminal space of inflammatory macrophages and neutrophils is observed
early in T. gondii infection, where Fpr1 (N-formyl peptide receptor)-expressing neutrophils promote
encapsulation of γ-proteobacteria,137 leading to bacterial death probably by NETosis.138
Finally, it would be interesting to see in the coming years the exploration of diverse mice models
combined with infection with transgenic parasites designed to verify the cross-talk between T. gondii
virulence factors and host immune response.

41.2.2.2.2 Rats
Infection of rats with T. gondii has been performed since the 1950s. Most studies on rats are per-
formed with two species, Rattus norvegicus and R. rattus, with the latter being the most used owing
to the different strains available.139 Rats may have an important epidemiological role for toxoplasmosis,
since they can be a source of infection to different hosts, especially pigs and possibly the domestic
cats.114,140 Epidemiological studies involving rats are complex, since T. gondii has been isolated by bio-
assay (in mice) from tissue of serologically negative rodents140 including from experimental congenital
infections.114 In addition, various cases have been reported where inoculation with different strains of
T. gondii resulted in different serology findings, the antibodies titers varied greatly with the type of
664 Laboratory Models for Foodborne Infections

test used, and negative serology was not uncommon.140 Thus, epidemiological studies of rats for T. gondii
based on serology must be analyzed with great care.
Most T. gondii strains have been isolated by bioassay with mice using pepsin-digested tissue from dis-
tinct hosts. This standard procedure to isolate T. gondii strains directly classifies the isolates as virulent or
avirulent to mice. Naturally, these strains have been used to experimentally infect other hosts, such as rats.
Rats have a natural resistance to T. gondii that has been known for a long time.56,140–142 Strains that are vir-
ulent to mice may not necessarily cause the disease in other host species. Immunocompetent rats infected
with tachyzoites or tissue cysts of different strains of T. gondii do not develop clinical manifestations being
resistant to the parasite,140 but infection with oocysts of the VAG strain induced clinical manifestations
and, depending on the dosage, killed the rats.143 Because immunocompetent humans are normally resis-
tant to T. gondii infection, the rat T. gondii infection model is considered by different authors to be a closer
animal model for human toxoplasmosis. The rat T. gondii infection model has been used as a congenital
transmission model showing that the parasite first colonizes the placenta and later the fetus.140 It was
found that the transmission to the fetus may be low,140 but higher transmission has also been reported,144,145
although the lactation route is not relevant.144 Differences in the transmission to the fetus in the distinct
congenital rat T. gondii models may be related to the strain of rat and to the strain of parasite used.
One of the most interesting outcomes with the rat T. gondii infection model is the heterogeneous resis-
tance of the different strains of rats to this parasite. After experimental infection, no parasite cysts are
found in the brain of the LEW strain in contrast to the Fisher strain.146 The lack of parasite cysts in the brain
of the LEW rats was further confirmed, and no detectable humoral response was found when compared
to the T. gondii susceptible Fisher and Brown Norway strains where a clear IgG response was observed.147
With the aid of bone marrow chimeras between these rats strains, the resistance of the LEW rat was linked
to cells derived from the myeloid lineage.146 Using congenic lines of the LEW and Brown Norway rats, the
resistance of the LEW strain was determined to be on chromosome 10 in a locus named Toxo1.148 In addi-
tion, T. gondii grew in infected fibroblasts of all congenic rat strains of this study but not in macrophages
from rats with the LEW Toxo1 locus, indicating that these cells are the main immune component related to
the resistance of this rat strain,148 and that possible genetic polymorphisms of the genes at this locus exist
in the rat strains. Although the locus responsible for LEW resistance was found, the gene or genes were not
characterized at that time, and the killing mechanism was not determined, but it was not NO-dependent.148
By using classical and molecular genetic tools with the rat strains that are susceptible or resistant for
T. gondii and distinct studies on infected macrophages, two research groups have recently suggested the
identity of a gene in the Toxo1 locus related to the capacity of the resistant rat macrophage to kill the par-
asite.149,150 These groups found that the NOD-like receptor 1 (Nlrp1), which is part of the inflammasome,
is essential for the resistant rat against the parasite. After bone marrow macrophage infection, this intra-
cellular receptor senses T. gondii and activates the inflammasome, host cells die, the parasite is killed,
and the tissue becomes inflamed. Inflammasomes are high-molecular-weight cytosolic multimeric pro-
tein complexes, which, upon assemblage, activate caspase-1 leading to a lytic cell death program (pyrop-
tosis) and also to the secretion of IL-1β and IL-18, caused by the cleavage of their precursors, inducing
a tissue proinflammatory response that controls infections.151 It was found that Nlrp1 sequence varies
among rat strains and that a variant from the resistant rat strains was responsible for macrophage pyrop-
tosis upon infection and parasite killing; on the other hand, two other variants from T. gondii susceptible
rat strains allowed macrophage survival and parasite growth.150 Activation of caspase-1 of infected mac-
rophages depends on the Nlrp1 variants, with the ones from resistant rats releasing higher levels of IL-18
and IL-1β. Knockdown and over expression of the Nlrp1 variant from the resistant rat in macrophages
establish the importance of this receptor in pyroptosis initiated by the infection and subsequent killing of
T. gondii.150 Genetic and infection studies using the different susceptible or resistant T. gondii rat strains
and their congenics also predicted the Nlrp1 gene to confer the rat macrophages resistance to T. gondii.149
In vitro infection caused parasite and peritoneal macrophage death only of resistant rat strains. The death
of the infected macrophages of the resistant rats was neither apoptotic nor autophagic. Higher production
of ROS and cleavage of caspase-1 were shown on infected macrophages of the resistant rat strain, clearly
correlating T. gondii death with host cells death by inflammasome activation.149 The use of pharmaco-
logical inhibitors of caspase-1 cleavage abrogated parasite killing in the resistance rat macrophages, but
the growth was not totally restored, indicating that the control of T. gondii in resistance rat macrophages
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 665

involves more mechanisms.149 The identification of the Nlrp1 gene in the Toxo1 locus of the rat and its
possible relation to resistance rat to T. gondii infection is an example of how different host models are
important to better understand parasite biology.
Inflammasome is also important in T. gondii control in mice.152,153 In addition, human Nlrp1 has been
implicated in human congenital toxoplasmosis154,155 and monocyte capacity to control T. gondii growth.153
In addition, GRA15 from a specific T. gondii strain was responsible for triggering IL-1β release from
infected human macrophages.156 Thus, inflammasome activation by sensing different strains of the para-
sites in distinct hosts resulting in broad killing potential seems to be a widespread strategy to control
T. gondii growth that varies within host species. The killing of T. gondii by the inflammasome is rapid
and may explain the low serology results seen in rodent epidemiological surveys. If this microbicidal
mechanism is also operating in humans and varies depending on gene polymorphism, there may be low
estimations of T. gondii infection in the human population as well.

41.2.2.2.3 Calomys callosus
The genus Calomys (Waterhouse, 1937) (Rodentia, Sigmodontinae) comprises small rodents that are
endemic in South America, originally being observed from northern Argentina to Peru, including Brazil,
Bolivia, and Paraguay. Currently, 10 species are described by considering morphology and karyotype.157
Calomys callosus mainly inhabits the central region of Brazil and has the capacity to live in different
biomes, giving it a great adaptive capacity.158
Such a rodent has been successfully domesticated and presents advantages as an animal model being
well adapted to the animal-housing environment.159 They are very prolific throughout the year and are
easy to handle. Common infections of rats, mice, and guinea pigs (Cavia aperea) do not affect this
rodent, which is a robust animal.159
High genetic diversity has been observed in isolates of T. gondii from South America.17 Recombinant
strains isolated in Brazil were more pathogenic to mice than clonal isolates from North America and
Europe.160 Because C. callosus has been found in environments with more pathogenic recombinant
strains of T. gondii, this model becomes quite interesting for the study of the parasite–host relationship.161
C. callosus is highly susceptible to infection with the RH strain of T. gondii,162 which proliferates in
large numbers in the newly implanted trophoblasts in pregnant females,163 and especially in the labyrin-
thine zone of the placenta.164 The same applies if the females are in the acute phase of the infection with
the ME-49 strain. However, females with chronic infection with the ME-49 strain are, at least tempo-
rarily, protected against RH infection with no transplacental infection.165 In contrast, the same chronic
infection does not protect against transplacental migration when reinfection is performed with Brazilian
recombinant strains.166 Thus, this is a good model to study neonatal infection.167
Few studies have been done with blood leukocytes in this model, thus demonstrating great poten-
tial for further research. Intraperitoneal infection with the RH strain of T. gondii induces migration of
neutrophils, monocytes, and mast cells after 24 h. In addition, basophils with distinct morphology were
identified after 48 h in the ileum submucosa. This cellular process coincided with the presence of dead
extracellular parasites. In lymphoid tissues, T. gondii was seen in mast cells, with or without the PV.168 In
intraconjunctival infection with the RH parasites, mast cell influx was detected within 5 and 24 h, with
correlation to the inflammatory process.169
Pregnant Calomys were orally infected with the ME-49 strain resulting in ocular injury and the pres-
ence of cysts in 40% of the fetuses. Ocular lesions were found in 25% of the females and 75% of males,
while 25% of each group had binocular lesions.170 These results indicate that the Calomys model is
promising for studying ocular and congenital toxoplasmosis.

41.2.2.3 Pigs
Knowledge obtained with laboratory animals (especially mice) needs to be validated in medium-sized ani-
mal models before clinical human trials.171 Because pigs and humans have similar anatomy, genetics, and
physiology, this animal is an interesting human infectious disease model172 and has been used as a biomedical
model for preclinical experimentation.173 However, few functional experiments have been carried out in these
animals, including T. gondii infection. Thus, there is great potential concerning this experimental model.
666 Laboratory Models for Foodborne Infections

Besides being an interesting animal model for T. gondii infection, pigs are important in the epidemiol-
ogy of this parasite, since the ingestion of tissue cysts from undercooked pig meat is a relevant source for
T. gondii human infection.174 Most pigs are infected by ingesting oocysts,175 but due to their omnivorous
behavior, the ingestion of birds and rodents with T. gondii cysts is important for the cycle.1,175 Pig infec-
tion has a direct correlation with the environment and animal management.176 Low management, access
of other animals to where food and water is offered to pigs, and low control of rodent access are directly
related to the prevalence of T. gondii infection in pig-breeding farms, especially the noncertified ones.177
In pigs, cysts can be found in organs such as the brain, lungs, liver, and spleen, with the possible occur-
rence of retinopathy.178 In addition, these animals do not usually develop severe pathological clinical
manifestations when infected with T. gondii if the immune system is not compromised, similar to what
occurs in humans with competent immune system.110
Although there is potential use of the pig model for T. gondii infection involving the reproductive
system, only a few groups have been working on this issue. The fact that these animals develop congeni-
tal infection strengthens the advantages of their experimental use. In naturally infected pregnant pigs,
there may be damage to the placenta,179 encephalitis, liver necrosis, and pneumonia in fetuses.180 Similar
changes may occur in human fetuses as a result of parasitemia and placental migration, particularly if the
mother has contact with the parasite for the first time during pregnancy.181 In addition, infective tachyzo-
ites of T. gondii can be found in porcine semen for up to 49 days postinfection, demonstrating a potential
for sexual transmission in this species.182 Surprisingly, the hypothesis of congenital toxoplasmosis as a
consequence of infection through human semen has also been recently proposed.183
An aspect evaluated via experimental infection with T. gondii in swine relates to its presence in blood.
Biological evidence in infected mice demonstrates the viability of this parasite in the pig blood, which
can be observed in neutrophils and monocytes, as well as outside these cells.110 In fact, the parasitemia
is persistent in pigs, and an accurate observation can detect tachyzoites for up to 63 days postinfection in
pigs that show no clinical symptoms.110
Some studies have demonstrated important clinical and hematological alterations in pigs infected with
T.  gondii. However, such studies typically use highly infective parasite inoculum or very young ani-
mals,184 factors that possibly enhance the pathology.110 In our study, pigs with developed immune system
were infected with parasite dosages close to a natural infection with the ME-49 strain, and typical clini-
cal manifestations of acute toxoplasmosis were observed, such as general apathy, low fever, and, rarely,
lymphadenopathy. Diarrhea was not observed, although the possibility exists and may depend on the
strain and the infecting dose.184 When orally infected, an increase in the number of band neutrophil fol-
lowed by a tendency to monocytopenia was observed. Under these experimental infection conditions, no
anemia or thrombocytopenia were observed, although liver injury was found as seen by increased serum
alanine aminotransferase readings.110 In addition, a persistent parasitemia without exacerbation of pathol-
ogy was found, which is similar to human toxoplasmosis infections caused by some Brazilian strains.185
This strengthens pig as an animal model to study human toxoplasmosis in immunocompetent patients.

41.2.2.4 Cat
Felids were only discovered as definitive hosts of T. gondii in the last half of the 1960s and in the begin-
ning of the 1970s. Excellent reviews report how the life cycle of T. gondii was discovered.35,186 With the
advent of the serological tests, it was realized that T. gondii infection was disseminated around the globe
in different hosts, including humans. This led to the assumption that transmission through carnivorism
alone could not explain the broad prevalence in the world and how herbivores were being infected; in
addition, transmission by invertebrate vectors was unsuccessful.35,186 Ferguson186 reported that Hutchison
was intrigued by these questions and hypothesized that T. gondii could be fecal–orally transmitted by
domestic animals. By using basic parasitology techniques, cats were fed with brain from infected mice;
the parasite material was purified by flotation from cat feces, stored in water, and then fed to mice that later
became ill with toxoplasmosis.187 This work opened the field to others; the T. gondii oocyst was discov-
ered, the sexual cycle of T. gondii in cats revealed, and the whole life cycle of this parasite solved.34,188–191
The discovery of the T. gondii life cycle explained the broad dissemination of this parasite with rel-
evant importance to the epidemiology of the disease. Oocyst shedding contaminated the environment,
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 667

ended up infecting herbivores, and was also responsible for human toxoplasma outbreaks by water con-
tamination.192 This led to proper management in farms and reduced the contamination of these ani-
mals.35 In addition, more cats release faster and higher number of oocysts if fed with the bradyzoite form
when compared to the tachyzoites or oocysts.193 This indicates that the natural life cycle of the parasite is
indeed by the transmission to the definitive host by carnivorism of small intermediate hosts (like mice)
containing tissue cysts.
Naturally, the cat became an animal model to better understand the sexual cycle of T. gondii, for oocysts
obtainment for further infection of other hosts, and to answer how the parasite behaves in the definitive
host. Experimental manipulation of the cat with corticosteroids indicated that the immune response is
important to stop oocysts shedding and to control parasite growth in the organs of these animals.194 Young
cats (no older than 12 months) shed more oocysts and also present more parasites in their tissue.195
Simultaneous infection of cats with two distinct strains of T. gondii has been done showing that clas-
sical genetic crosses do occur in this parasite.196 These crosses have been used to create linkage maps of
this parasite,197 identifying 11 chromosomes.198 Later, this technique was used to study virulent factors of
the parasites, and a few have been identified.40 Infected cats have also been used to genetically character-
ize the merozoites forms that multiply in cat enterocytes before gametogenesis. A complete distinct set
of genes was identified in these forms when compared to tachyzoites.37 Thus, the cat is fundamental for
the development of T. gondii biology.

41.2.2.5 Chicken
Chickens are less used in experimental trials compared to the murine model. However, in some specific
cases, chickens are an extremely suitable model for experimental studies.199 The ease in handling, low
maintenance cost, and the prolific characteristics of chickens favor their use in experimentations. In
addition, chickens are the world’s largest population of domestic animals with great economic impor-
tance and, therefore, there is interest in studying them.
Chickens may be 100% infected with T. gondii when raised in backyards, whereas those raised in
free-range organic rearing can present prevalence as high as 50%.200 Because of their feeding habits,
chickens are characterized for being indicators of soil contamination by T. gondii oocysts. Moreover,
these animals may be as important in the biological cycle as mice, due to their greater longevity.200 In
chickens, tissue cysts are more common in muscles than in the brain,1 strengthening the importance of
this animal as a source of human infection.200
Chickens do not normally develop a severe clinical disease in natural infections200 and may not develop
the disease when inoculated with the virulent T. gondii strains for mice.201 However, diarrhea, emacia-
tion, blindness, and sudden death have been reported in chickens naturally infected with T. gondii, with
isolation of the parasite in the heart, liver, brain, and lungs. Interestingly enough, the parasite isolates
were avirulent for mice.202,203
Prior to the discovery of T. gondii life cycle and the importance of oral transmission, studies empha-
sized the parenteral infection route, which was not accompanied by clinical manifestations.202–204 After
this period, oral infections have shown variable results with chickens depending on the strain used.
Chickens inoculated with oocysts of the ME-49 strain (Type II) did not develop any clinical symptoms,
whereas chickens infected with the GT1 strain (Type I) became ill, resulting in 20% (one of five animals)
death.205 In addition, oral infection with oocysts of the E strain caused mild fever only in highly infective
doses.206 Although the relative acute phase of the T. gondii infection in chickens does not result in clinical
manifestations, the chronicity of the disease may cause physiological changes and possible production
losses.207 Chicken infection with the M-7741 strain caused diarrhea and inflammatory cell infiltration in
the intestinal wall, with a strong reduction of the density of myenteric neurons. The chronic phase was
correlated with neuronal death and atrophy of the intestinal wall, causing gastrointestinal disorders.207
The relatively mild acute phase followed by a chronic phase with potential epidemiological impor-
tance indicates that the experimental infection of chickens with T. gondii is indeed an interesting model,
particularly with respect to immunological studies. Therefore, interaction with immune cells from pri-
mary cultures, or with the established cell lines such as the HD11 of the chicken macrophages,70,208 may
generate interesting results.
668 Laboratory Models for Foodborne Infections

41.2.2.6 Fish
T. gondii is known to infect nucleated cells of warm-blooded animals, with mice being the most common
and important intermediate host.73,75 However, some studies have indicated that fish and shellfish may
be involved in the natural infection of different hosts by T. gondii. In China, parasitic DNA was identi-
fied in a small number of crayfish Procambarus clarkii, fish Hypophthalmichthys molitrix, and shrimp
Macrobrachium nipponense.209 The authors indicated that the consumption of raw meat of these animals
may be considered a health threat related to T. gondii infection.209 In Brazil, it was shown that oysters
(Crassostrea rhizophorae) may retain oocysts of T. gondii as these animals filter water, and the parasite
DNA was found in a low percentage of individuals.210 There is a need for more studies on the participa-
tion of these animals as carriers of T. gondii.210 However, the importance of these cold-blooded animals
as an effective intermediate host has not been clearly demonstrated, since the presence of DNA in the
sample does not mean viable parasites. It is necessary to differentiate the participation of a potential host
or parasite carrier from a mechanical vector.
Grizzly bears (Ursus arctos) in Alaska showed a T. gondii serology prevalence up to 37% depending
on the area studied.211 Although this animal feeds on fish, no established relationship with these cold-
blooded animals was established, because toxoplasmosis is also a waterborne disease.6 Thus, one cannot
exclude the infection of these animals through contaminated water.
Experimental studies have demonstrated the inability of T. gondii to persist in tissues of goldfish main-
tained at 37°C.212 However, in appropriate conditions, especially temperature, this can be used as a new
experimental infection model for T. gondii in the zebrafish (Danio rerio).213 Proliferation of T. gondii of
the ME-49 and VEG strains was observed in the cardiac myocytes, endothelium, lumen of peripheral
vessels, liver, spleen, brain, pancreas, ovaries, skeletal musculature, and the eye of this animal infected
after acclimation at 37°C. Two weeks after infection, cystic structures were observed in the brain, sug-
gesting that this model may also be fit for chronic studies. The authors report that the only limitation to
the study of toxoplasmosis in this fish was temperature. This is a promising model because this fish can
be kept at temperatures of 37°C, used in high-throughput screenings for new compound, and be geneti-
cally modified.213

41.3 Concluding Remarks
Toxoplasmosis is a disease with worldwide distribution affecting about 2 × 109 people. First considered a
foodborne disease, it is now also recognized as a waterborne disease. It has been suggested that cultural
customs dictate the way of transmission, especially in regard to the consumption of raw or undercooked
meat. This disease can cause different clinical manifestations, with serious implications to immuno-
compromised patients. The disease also affects a large number of wild and domestic warm-blooded
animals causing great economic losses. The etiological agent of this disease is the obligate intracellular
protozoan Toxoplasma gondii, which infects a vast number of hosts and has an elaborate life cycle that
helps to explain its broad dissemination. With the establishment of T. gondii cell culture, the biological
characteristics of the parasite have been delineated through the infection of primary cells, cell lines, and
transfected cells with distinct strains and mutant parasites. Because of its broad host range, most animal
models are suited to investigate this disease.
With a complex life cycle involving various hosts and a diversity of genotypes made up of multiple
strains, T. gondii is a peculiar parasite. Thus, it is imperative to use different models to better understand
the many distinct steps of infection leading to acute and chronic stages of this disease. In vitro models
are in constant development and yield interesting outcomes, but the obtained results must be confirmed
via in vivo models. To date, no animal model that mimics the human infection by T. gondii exists;
however, recent advances on secreted virulence factors by distinct T. gondii strains and specific antimi-
crobial mechanisms of host cells from different host animals that are strain-dependent have increased
our knowledge and shown how adapted this parasite is. Further research is clearly required to fully elu-
cidate the clinical variations of toxoplasmosis and to unravel if specific microbicidal mechanisms oper-
ate in humans and what parasite genes code for virulence factors that help its evasion of host immune
Toxoplasma: Animal and In Vitro Models on Toxoplasmosis 669

functions. In addition, its genetic diversity also demands that diagnosis and treatment of toxoplasmosis
need to be geographically specific. There is no doubt that the models described here and others to come
will contribute to improved understanding of this important parasite and the disease it causes.

Acknowledgments
The authors would like to thank Andrèa Carvalho César for proofing the manuscript. This study was
supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
(FAPERJ), and Fundação de Coordenação de Pessoal de Nível Superior (CAPES).

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 Section VI

Foodborne Infections
due to Helminths
42
Anisakis

Mauricio Afonso Vericimo, Gerlinde Teixeira, Israel Figueiredo Jr., Janaina

Ribeiro, Maria Augusta Moulin Fantezia, and Sergio Carmona São Clemente

CONTENTS
42.1 Introduction................................................................................................................................... 680
42.2 Taxonomy, Life Cycle, and World Distribution of Anisakis Species............................................ 680
42.3 Allergen Nomenclature................................................................................................................. 683
42.4 Pathogenesis, Immunological Response, and Clinical Signs and Symptoms.............................. 683
42.5 Laboratorial Diagnosis.................................................................................................................. 684
42.6 Experimental Models ................................................................................................................... 684
42.6.1 General Considerations.................................................................................................... 684
42.6.2 Guinea Pigs....................................................................................................................... 685
42.6.2.1 Intradermic Route............................................................................................. 685
42.6.2.2 Intraperitoneal Route........................................................................................ 685
42.6.2.3 Intragastric Route.............................................................................................. 685
42.6.3 Pigs................................................................................................................................... 685
42.6.3.1 Oral Route......................................................................................................... 685
42.6.4 Rabbit................................................................................................................................ 686
42.6.4.1 Intragastric Route.............................................................................................. 686
42.6.4.2 Subcutaneous Route.......................................................................................... 686
42.6.4.3 Intramuscular Route......................................................................................... 687
42.6.5 Rats................................................................................................................................... 687
42.6.5.1 Intraperitoneal Larval Implant......................................................................... 687
42.6.5.2 Intragastric Infection........................................................................................ 687
42.6.5.3 In Vivo L3–L4 Transformation Model in Rats................................................. 688
42.6.6 Mice.................................................................................................................................. 688
42.6.6.1 Intraperitoneal Larva Implant........................................................................... 689
42.6.6.2 Intraperitoneal Immunization........................................................................... 689
42.6.6.3 Intragastric Infection........................................................................................ 689
42.6.6.4 Epicutaneous Immunization............................................................................. 690
42.6.6.5 Subcutaneous Immunization............................................................................ 690
42.6.6.6 Intranasal Immunization................................................................................... 690
42.6.6.7 Nematode Molecules as Immunoregulators......................................................691
42.6.7 Fish....................................................................................................................................691
42.6.7.1 Oral Infection.....................................................................................................691
42.6.7.2 Intraperitoneal................................................................................................... 692
42.6.8 In Vitro Cultivation........................................................................................................... 692
42.6.8.1 Culture Media................................................................................................... 693
42.6.8.2 L3–L4 Transformation Model.......................................................................... 693
42.6.9 Larvicidal Models............................................................................................................ 694
42.7 C  onclusion..................................................................................................................................... 694
Acknowledgments................................................................................................................................... 695
References............................................................................................................................................... 695

679
680 Laboratory Models for Foodborne Infections

42.1 Introduction
Parasites from the marine environment have historically been overlooked as a risk for human disease
and are thus not in the main stream of basic or clinical investigation, although they can infect humans,
thereby causing anthropozoonosis, and are therefore a public health risk. Within the marine worms
with clinical importance are those pertaining to the Anisakidae family (Anisakis, Pseudoterranova, and
Contracaecum) and Raphidascarididae family (Hysterothylacium) causing anisakidosis.1 Anisakids are
nematodes whose definitive hosts are marine mammals; intermediate hosts are crustaceans (L2), fish,
and cephalopods (L3), and they have a worldwide distribution. Humans become accidental hosts after
ingestion of raw or undercooked infected seafood.2
There is an estimate of 20,000 human cases of anisakidosis, with an annual registration of 2000
new cases. The highest incidence with approximately 90% of all reported cases occurs in Japan,3 prob-
ably owing to the routine habit of eating raw fish in dishes like sushi and sashimi.4 Other countries
that habitually consume raw or undercooked seafood also record an expressive number of cases of the
disease. This is the case of European countries, mainly in the coastal areas of Germany, Netherlands,
and Scandinavian countries that consume salted, pickled, and smoked herring, or Spain, where typical
appetizers are ceviche (fresh seafood marinated in lemon juice) and Boquerón’s en vinegar (pickled
anchovies).1 In the Americas, there has been an increase in the number of reported anisakidosis cases,
probably because of the popularization of oriental cuisine and the consumption of dishes like Lomi-lomi
salmon and ceviche.1,4,5 The improvement of diagnostic methods is probably another explanation for the
increase in the report of new cases.
Since the first descriptions of human cases, the number of researchers who investigate actual and
potential human marine infections has increased, and several animal models have been developed in
order to understand the host–parasite relationship associated with the sensitization of individuals who
accidentally ingest anisakid larvae. In this chapter, we will contextualize several in vitro and in vivo
experimental models that are employed to reproduce and understand the natural history of human dis-
ease and explore the molecular and biological aspects of these parasites.

42.2 Taxonomy, Life Cycle, and World Distribution of Anisakis Species

The taxonomic classification of anisakids6 consists of:
⇨ Kingdom: Animalia
  ⇨ Phylum: Nematoda
   ⇨ Class: Rabdititia (=Secernentea)
    ⇨ Subclass: Rabditia (=Phasmidea)
     ⇨ Order: Ascarida
      ⇨ Superfamily: Ascaridoidea
       ⇨ Family: Anisakidae
        ⇨ Genus:   Anisakis
              Pseudoterranova
              Contracaecum
       ⇨ Family: Raphidascarididae
        ⇨ Genus:    Hysterothylacium
Within the Ascaridoidea superfamily, the Anisakidae family is considered the largest and includes spe-
cies that can parasitize fish, reptiles, mammals, and fish-eating birds. The representatives of this fam-
ily are dependent on the aquatic environment for the development of their biological cycle and usually
involve invertebrates and fish as intermediate or paratenic hosts.
Among the Anisakidae family, the species of the genus Anisakis have low specificity for the definitive
host, but, in general, live in the stomach of cetaceans such as whales, dolphins, and porpoises. Those
Anisakis 681

of the Pseudoterranova genus are more specific having the pinnipeds (seals, walruses, and sea lions) as
definitive hosts. The species belonging to the Contracaecum genus have as definitive hosts fish-eating
birds and pinnipeds, and unlike the other genera, Contracaecum larvae can parasitize both marine and
freshwater fish. Finally, the definite hosts of the species belonging to the Hysterothylacium genus of the
Raphidascarididae family are pinnipeds, fish, and shellfish.7–10 Because the biological cycles of the four
genera are similar, and the aim here is the experimental approach to study these worms, we will only
depict the Anisakis life cycle.
Adult worms release their eggs in the gut of the definite host. Through the feces, the eggs gain access
to the seawater, where they embryonate and form the first larval stage (L1) and progress to the second
stage (L2). The L2 are eaten by small crustaceans such as krill (first intermediate host), where they prog-
ress to the third-stage larvae (L3), the infective stage for the definitive host. Second intermediate hosts
(fish or shellfish) ingest the crustaceans, which in turn are eaten by bigger fish, transferring L3 through
the food chain and resulting in their accumulation in the larger fish until eaten by sea mammals, which
are their definite hosts.11,12 Once L3 have been eaten by their definite hosts, they progress to the fourth
larval stage (L4) and finally become adults13,14 (Figure 42.1).
When fish are captured, soon after their death, L3 migrate to the viscera, peritoneal cavity, and mus-
cles. The degree of migration depends on environmental conditions, the parasite, and fish species. When
humans consume raw or undercooked infected fish or shellfish, they may become accidental hosts. As
the parasites are not adapted to humans, they do not reach sexual maturity although they may cause mild
irritation to anaphylactic shock.5,15,16
At least 200 fish and 25 cephalopods species have been described as being infected with anisakid
larvae.17–19 Within all anisakids, species pertaining to the Anisakis genus are considered the most

6—Marine mammals eat

infected fish and squids.
Larvae molt to the adult 1—Marine mammals expel
worm, which produce eggs nonembryonated eggs

2—In contact with water,

eggs become
embryonated and larvae
are formed in the egg

7—Humans become
accidental hosts by
ingesting raw or
undercooked sea food
3—Eggs hatch,
ANISAKIS LIFE CYCLE
and L2 larva
swim freely
in water

5—Infected crustaceans
are consumed by fish and
4—Free swimming
squids. These are eaten
larvae are ingested
by larger fish, and so on,
by small crustacean
forming increasingly larger
and develop to L3
numbers of L3 in the gut

FIGURE 42.1  Life cycle of anisakids.

682 Laboratory Models for Foodborne Infections

pathogenic and cause the largest number of human occurrences.20,21 Although morphologically very
similar, the genus has nine species that have been identified by molecular technologies and have
distinct definitive host distribution worldwide.22–24 As depicted in Figure 42.2, larvae are classified
by their morphology and genetic characteristics in “clades (I and II).” Clade I contains the Anisakis
simplex complex, which includes A. simplex (strict sense), A. pegreffi, and A. simplex (complex); the
other sister species in this clade are A. typica, A. ziphidarum, and A. nascettii. The definitive host of
the Clade I species are mainly distributed in the Atlantic and Pacific oceans. A. simplex (ss) and A.
pegreffi are also found in the Mediterranean, Arctic, and Antarctic Seas.2,12 Anisakis species pertain-
ing to Clade II are classified as A. physeteris complex, which includes A. physeteris, A. brevispiculata,
and A. paggiae species. Although considered to have a cosmopolitan distribution, they are mainly
found in the Atlantic Ocean.2,25
The Pseudoterranova decipiens complex consists of species that include the P. decipiens (sensu
stricto) or P. decipiens B, P. krabbei, P. bulbous, P. azarasi, and P. cattani. They are considered cosmo-
politan and are very abundant in the Atlantic Ocean, occurring from the Arctic to Antarctica.26
The Contracaecum genus has species that are able to parasitize both freshwater and marine organ-
isms. From this genus, the species that most frequently cause anisakidosis pertain to the C. osculatum
complex, which is a set of five members including C. osculatum types A, B, C, D, and E, where C. oscu-
latum sensu stricto corresponds to type C.27 The most frequent geographic distribution of these species
is the Alaskan and Japanese waters, the Baltic Sea, and Antarctic and Atlantic Ocean.7,28
Although the Hysterothylacium genus has a worldwide distribution, it is described as a rarely occur-
ring causative agent of anisakidosis. Apparently, the first human case of H. aduncum was registered in
1996 in Japan.29 Molecular identification of Anisakis and Hysterothylacium larvae from marine fish of
the East China Sea and the Pacific coast of central Japan showed that approximately 10% of the larvae
pertained to the Hysterothylacium genus (H. amoyense—5.0%, H. aduncum—1.6%, H. fabri—3.4%,
and H. spp.—2.9%), while the majority of the remaining Anisakidae nematodes belong to the Anisakis
genus.30 This result correlates well with the clinical finding.
Using classical techniques (morphological taxonomy), human anisakidosis is most frequently described
as being caused by Anisakis and Pseudoterranova genus.29,31–33 Among the Anisakis species, A. simplex
(ss) is reported as responsible for the highest number of human cases. However, after the introduction of
molecular biology in taxonomy, A. pegreffi has been more frequently described as the agent responsible
for anisakiosis in some countries such as Italy.18,34,35

Anisakis Host species

A. physeteris complex

A. physeteris I Physeteridae

A. brevispiculata
Clade 2

II Kogiidae
A. paggiae

A. ziphidarum
Clade 1

III Ziphiidae, Delphinidae

A. nascetti

A. typica IV Delphinidae
A. simplex complex

A. simplex C V Ziphiidae, Delphinidae

A.pegreffi VI Physeteridae, Ziphiidae, Neobalaenidae, Delphinidae

A. simplex (s.s.) VII Phocoenidae, Balaenopteridae, Monodontidae, Delphinidae

FIGURE 42.2  Cladistic distribution of anisakid larvae.

Anisakis 683

Ani s 13
Ani s 14 hemoglobin
Unknown Ani s 3
UniProt - K9USK2
UniProt-?? Tropomyosin
Gonzalez-Fernandez (2015) Ani s 2
Kobayashi et al. UniProt - Q9NAS5
Asturias et al. (2000) Paramyosin
UniProt - L7V1|9
Pérez-Pérez et al. (2000)
Ani s 12
Unknown Ani s 1
UniProt - L7V0K0 Serine protease inhibitor
Kobayashi et al. (2011) (Kunitz type)
UniProt - L7V3Q3
Ani s 11 Moneo et al. (2000)
Unknown
Ani s 4
UniProt - E9RFF3
Cystatin
Kobayashi et al. (2011)
UniProt - Q14QT4
Moneo et al. (2005)

Ani s 10
Unknown Ani s 5
UniProt - D2K835 SXP/RAL-2
Caballero et al. UniProt - A1|KL2
Kobayashi et al. (2007)

Ani s 6
Ani s 9 Ani s 8 Ani s 7 Trypsin inhibitor like cysteine
SXP/RAL-2 SXP/RAL-2 n/a rich domain
UniProt - B2XCP1 UniProt - A7M6S9 UniProt - A9XBJ8 UniProt - A1|KL3
Rodriguez-Perez et al. (2008) Kobayashi et al. (2007) RodrÍguez et al. (2008) Kobayashi et al. (2007)

FIGURE 42.3  Updated Anisakis allergen compiled mainly from data extracted from the Allergome database in combina-
tion with published literature. The colors of the wedges indicate the origin of the antigens: Dark gray—somatic antigens;
medium gray—excretory-secretory antigens; light gray—unknown origin. (http://www.allergen.org/treeview.php.)

42.3 Allergen Nomenclature
The abbreviation of the name of the gender (first three letters) and of the species (first letter) followed by
a number indicating the chronology of the allergen purification was adopted as the systematic nomencla-
ture of allergens, implemented by the Nomenclature Sub-Committee of the World Health Organization
(WHO) and International Union of Immunological Societies. So, the A. simplex allergens are called
“Ani s #” (e.g., Ani S1).36 A. simplex (ss) has 14 allergens characterized by origin and molecular aspects.
The immunoreactivity pattern for these allergens has been studied both with human sera and with
experimental animals. A synthesis of the structural classification of Anisakis allergens is presented in
Figure 42.3 based mainly on the allergen database AllFam,37 which can be accessed on the web at http://
www.meduniwien.ac.at/allergens/allfam. The data was complemented from other published literature.
For example, data from the Conserved Domain Database (CDD) and from the domain of unknown func-
tion (PF; DUF, Pfam) were used.

42.4 Pathogenesis, Immunological Response, and Clinical Signs and Symptoms

In humans, the ingestion of the live anisakid larvae causes distinct clinical forms of illness: gastric,
intestinal and/or ectopic anisakidosis, and/or allergic reactions, which may vary from mild to severe
reactions. Although not a very common finding, gastroallergic anisakiosis (GAA) is a well-established
clinical entity, characterized by acute IgE-mediated urticaria, angioedema, or anaphylaxis shortly after
an A. simplex acute infection. The immunologic response that accompanies this parasite presents a sig-
nificant polyclonal stimulation of different immunoglobulin isotypes comprising a mixed Th1- and Th2-
mediated reaction.38 The ingestion of dead anisakid larvae or proteins derived from the larvae may also
trigger mild to severe allergic reactions. Therefore, anisakid extracts should be included in the standard
sets of allergens used to investigate undefined allergies and anaphylactic reactions.39
The insertion of the cephalic portion of the larva in the mucosal wall and the secretion of proteases
that permit its fixation result in a local inflammatory reaction that leads to the clinical symptoms such as
684 Laboratory Models for Foodborne Infections

epigastric pain, nausea, diarrhea, vomiting, and fever.40 Furthermore, when larvae penetrate the submu-
cosa, it may sensitize the host with its excretory-secretory (ES) products by stimulating the development
of a predominantly Th2 immune response, which favors the production of IgE antibodies, responsible for
allergies.41 Persistence of larvae in the tissue can result in direct damage and, in turn, the development
of a eosinophilic granuloma, characterized by an inflammatory infiltrate of eosinophils and neutrophils
associated with a diffuse interstitial edema and proliferation of connective tissue around the body of the
larva.42–46
There is evidence in the literature that the continued exposure to Anisakis antigens by fish factory
workers, anglers, and their families can sensitize them through inhalation or direct contact.47,48 Farmers
are another group of workers who can become sensitized to Anisakis antigens when in direct contact
with the corresponding allergens, e.g., fish meal.47 Gastrointestinal conditions, asthma, conjunctivitis,
and occupational contact dermatitis have been frequently described in Anisakis-sensitized patients.47,49–53
Signs and symptoms can range from discreet allergic symptoms, urticaria up to angioedema, and fatal
anaphylactic reactions with or without gastrointestinal symptoms.54,55
The human immune response to Anisakis sp. antigens is highly heterogeneous, varying both in quan-
tity and in quality between individuals.56 Studies in patients showed that infection with Anisakis larvae
induces a strong immune response with the production of specific antibodies reaching maximum titers
within the first month of infection.57 Infections with low numbers of larvae and continuous exposure
frequently result in the production of high levels of IgE, whereas the exposure to high numbers of lar-
vae frequently results in the production of IgG.58,59 The analysis of the cytokine profile obtained from
the peripheral blood and intestinal biopsy samples of newly infected patients reinforces the concept that
the Th2 response plays an important role in the immunopathogenesis of anisakiosis.38 Further detail
pertaining to the immune response shall be presented during the experimental section.

42.5 Laboratorial Diagnosis
Initially, specific IgG was used to diagnose anisakiosis; however, as the IgG titers persist elevated for a
relatively long period, it is not a good parameter to differentiate current from previous A. simplex infec-
tions. Another observation is that anisakid allergy is frequently associated with high levels of specific
IgG4.60–62
A good diagnostic tool can be the use of the proportion of specific IgE and IgG4 titers. This strategy
has been used to evaluate allergic diseases caused by a variety of other nematodes even if the nematode
is not observed by a gastroscopy.60,63,64 Thus, the serological diagnosis of a gastroallergic anisakiosis can
be a good alternative.57,60,63,65 Chronic urticaria (CU) associated with anisakiosis is another clinical set-
ting in which IgG4 can be used for diagnosis and follow-up. Unlike patients who continue their exposure
to the fish, those that are subjected to a fish-free diet experience a significant reduction in CU symptoms
accompanied by significant reduction of IgG4 levels.65 The comparison of the levels of IgE, IgG, and
IgG4 to A. simplex in CU and GAA patients showed that the latter presented significantly higher levels
of all tested immunoglobulins.66

42.6 Experimental Models
42.6.1 General Considerations
Even if the conditions that are used in animal experimentation do not exactly match those that occur in
the natural history of disease, this is a widely used method for acquiring knowledge of various diseases
in human and veterinary medicine. Through in vivo experimentation, it is possible to answer specific
questions about the pathophysiology of diseases generating information that can then be extrapolated
to the clinical setting, permitting a better understanding of the disease, leading to better prevention and
better treatment.
Anisakis 685

Since the discovery of the first human anisakiosis cases in the 1960s, many animal species have been
used as a model for this disease. The first studies used rabbits and guinea pigs to understand the migra-
tion trajectory of the larvae to the tissues and granuloma formation. However, to study the allergic reac-
tions induced by Anisakis larvae, most researchers prefer to use rats and mice. We chose to present the
animal models by species and route of infection/sensitization.

42.6.2 Guinea Pigs
42.6.2.1 Intradermic Route
In order to evaluate the in vivo chemotactic effect of A. simplex larvae extract, Tanaka and Torisu67
used guinea pigs as experimental animals. These researchers found that a few hours after intrader-
mal injection of crude larvae extract (CE), a dose-dependent accumulation of eosinophils occurred at
the site of injection. To confirm this effect, these authors carried out in vitro chemotaxis assays using
Boyden chambers.68 Using the same concentration of the extract with which eosinophil chemotaxis was
observed, no chemotactic activity was found for neutrophils, supporting the idea that the CE plays an
important role in the development of eosinophilia in anisakiosis.

42.6.2.2 Intraperitoneal Route
Early in the 1980s, in the attempt to determine the etiologic mechanism of the allergic reactions associ-
ated with anisakiosis, guinea pigs were sensitized by implanting live Anisakis sp. larvae in the perito-
neal cavity.69 The Schultz–Dale70,71 reaction was used to determine the presence of type I reactivity. In
short, intestinal fragments of intraperitoneal-sensitized guinea pigs with live Anisakis larvae responded
intensely when stimulated with Anisakis larvae hemoglobin and with less intensity when stimulated with
CE from other anisakids (Contracaecum and Pseudoterranova), whereas no response was observed
when Toxocara canis or Ascaris suum extracts were used. These results confirm the IgE-mediated etiol-
ogy of the allergic reactions associated with anisakiosis.

42.6.2.3 Intragastric Route
To determine the migratory pattern and viability of live Anisakis larvae, these were delivered to the
gastric cavity. Larvae gained different organs and tissues passing the stomach wall through an active
migration mechanism without a preestablished migratory pattern. Live larvae without any morphologi-
cal changes were recovered up to the fifth day after administration. These were able to reinfect another
guinea pig maintaining the same migration capability. However, as of the sixth day post infection, all
larvae disappeared leaving no hint of its presence in any part of the body.
Guinea pigs experimentally infected with A. simplex have also been used to test drugs.72 For example,
oral treatment with ivermectin or albendazole was tested and found to present high in vivo efficacy
against the larvae present in different organs of the guinea pigs.73

42.6.3 Pigs
42.6.3.1 Oral Route
Anisakis larvae infection in pigs was studied by feeding the animals with fish offal contaminated with L3.
In these studies, researchers observed that the severity of injury was proportional to the number of larvae
ingested. Histological alterations due to larvae interaction with the mucosa included primary mechanical
damage accompanied with bleeding, ulceration of the mucosa and submucosa, and intense cellular infil-
tration with connective tissue proliferation around the larva.74 The histological changes of the stomach
mucosa from experimentally infected pigs with Anisakis sp. and Pseudoterranova sp. larvae involved
intense inflammatory reaction around the larva with the presence of numerous eosinophilic cells.75 That
686 Laboratory Models for Foodborne Infections

is, feeding pigs L3 of C. osculatum results in the same histopathological findings that c­ orresponded to
findings of infections caused by other Anisakis sp. pathogens.76

42.6.4 Rabbit
The histological aspects of intestinal sections of experimentally infected rabbits resemble those of acciden-
tally infected humans, suggesting a similarity of the pathogenesis. Thus, rabbits were successfully intro-
duced as experimental anisakiosis models soon after the publication of the first human anisakiosis cases.77

42.6.4.1 Intragastric Route
In the early 1970s, the experimental determination of the pathogenesis of anisakiosis was performed by
administrating live larvae to the stomach of rabbits and semiquantitatively grading the inflammatory
reaction of the surrounding tissue where larvae penetrated.78 Three days after the oral administration of
40 Anisakis larvae, only a very small number entered the stomach wall, many of which were still alive,
and the degree of the inflammatory reactions of the gastric mucosa surrounding the distinct larvae varied
between mild, moderate, and severe in an individual animal and between individuals.
Necrosis and massive amounts of granulocytes, including eosinophils, were the main findings on day 3
after infection. On day 5, the larval viability declined, and an infiltrate of plasma cells and immunoblasts
was observed along with the granulocytes in the center of the reaction, while fibroblasts were already
present in the periphery. After 7 days, the fibroblast infiltrate became more intense; by 10 days, granula-
tion tissue was observed; and by a month, the necrotic tissue was substituted by new connective tissue
surrounded predominantly by mononuclear cells with moderate amounts of eosinophils.
The serological reactivity in association with the histopathological pattern was also studied in rabbits
infected with 30 live A. simplex larvae through the oral rout. Although most larvae were recovered in the
stomach, some migrated from the gastrointestinal tract and reached extragastric tissues, resulting in the
formation of abscess that contained dead larvae. By 30 days, the reactions progressed to granulomatous
abscesses followed by calcification of the larvae.79
From the serological point of view, IgG peaked by 30 days, coinciding with the granuloma resolution
and calcification of the larva followed by an abrupt decline. Another study that infected rabbits with
10 larvae showed a peak of IgM on the 11th day, whereas IgG peaked approximately a month later.80
Intragastric sensitization of rabbits with Anisakis larvae was also employed to assess the recognition
pattern of somatic and secreted antigens of infective Anisakis larvae comparing possible relationships
with antigens from other nematodes of Ascaroidea family using radioimmunoprecipitation techniques.81
Such as in serum derived from Anisakis infected patients, infected rabbits preferentially respond to
somatic antigens, and the recognition sequence occurs to different components of secreted antigens.
The differences in the recognition of secreted/excreted antigens and somatic components may be due to
the duration of sensitization and the degree of penetration by nematodes in the tissues. Kennedy et al.81
also demonstrated that a 14-kDa component derived from A. simplex cross-reacts with a homologues
component derived from Ascaris suum, Ascaris lumbricoides, and Toxocara canis, species from the
Ascaroidea family.

42.6.4.2 Subcutaneous Route
A chemotactic factor selectively attractive for eosinophils found in the extract from Anisakis larva was
termed eosinophil chemotactic factor of parasites (ECF-P).67 To determine whether the eosinophilic
phlegmonous inflammation typically observed in human anisakiosis could be experimentally repro-
duced, normal and subcutaneously immunized rabbits received intraserosal injection of ECF-P into the
ileum of rabbits. All rabbits developed a significant eosinophilic inflammation at the injection site in a
dose-dependent manner. Although immunized rabbits presented high anti-ECF-P antibody titers while
normal animals had no detectable antibody, there was no significant histological difference between the
lesions observed in either group of rabbits. These results support the argument that, especially in the
Anisakis 687

early phase of primary infection with anisakiosis, ECF-P may contribute to the development of eosino-
philic phlegmonous inflammation without any immunological intervention.67,82

42.6.4.3 Intramuscular Route
One of the experimental protocols involves the intramuscular route to investigate if larval antigens of
A. simplex present molecular similarity to interleukin IL-4. The resulting rabbit anti-mouse IL-4 anti-
bodies were tested against A. simplex ES and CE antigens in ELISA. The anti-IL-4 antibodies showed
a strong cross-reactivity, which was confirmed by western blot analysis. A complementary assay, the
absorption of the anti-IL-4 sera with A. simplex antigen, demonstrated a 70%–80% inhibition of antigen
binding when retested in ELISA. These results support the hypothesis that A. simplex proteins share
several epitopes with IL-4 or conversely that A. simplex larval ES and somatic products present IL-4-
like molecules. This finding implies that the parasite may control and modulate the mucosal Th1-Th2
dichotomy for its own benefit in an attempt to avoid its expelling.83
Currently, experimental Anisakis research has not used rabbits as a model to study allergic reactions
caused by this nematode. However, intramuscular inoculation with Anisakis antigens has been employed
when the aim is to characterize allergens and to produce laboratory reagents.84–88

42.6.5 Rats
Rats have been used extensively to investigate the immune response to Anisakis larvae. Although the
oral route is the natural form of infection, in the experimental scenario, investigators have shown a
limited usefulness of per os administration due to the difficulty in accurately determining the parasite
load, since many larvae are expelled through the anus, hampering the establishment of the relationship
between parasite load and immune response.89 Although the surgical implant may appear to be an inad-
equate route of infection, the argument used to validate this technique and to expect that the antibody
production profile would be the same regardless of the route is that orally administered larvae pass from
the intestinal lumen into the peritoneal cavity after infection.90,91 Another observation that supports this
hypothesis is that extragastrointestinal anisakiosis has also been observed in humans who are infected.92

42.6.5.1 Intraperitoneal Larval Implant

Immunization of rats by intraperitoneal L3 larvae implantation was used to determine the immune
response to SE and CE. In contrast to oral infection in rabbits, intraperitoneal implantation of live larvae
in rats induced a strong response to both SE and CE antigens. After 63 days of implantation, no larva was
found alive; thus, the hypothesis is that the immune response was due to the release of somatic antigens
in the peritoneal cavity.81
ES-specific IgM and IgG titers of rats inoculated with increasing A. simplex L3 load (1, 5, or 20 larvae)
show a positive correlation after the primary inoculum but not to the secondary inoculum. IgM and IgG
titers of animals inoculated with 20 larvae did not further increase. However, after the second inoculation,
those animals that received one or five larvae presented antibody titers comparable to the 20 L3 inoculation.
The primary inoculation induced low ES-specific IgE antibody titers in all groups, and in the second-
ary inoculation, a negative correlation was obtained. In other words, rats receiving one larva developed
higher IgE titers than rats receiving larger inoculums. IgE titers of single larvae-inoculated rats peaked
at 3–5 days after secondary inoculation and disappeared by day 14, which is consistent with the dura-
tion of infection. Thus, monitoring ES-specific IgE may be a useful diagnostic tool for human intestinal
anisakiosis, because in the natural scenario, infections typically course with low larvae loads.93

42.6.5.2 Intragastric Infection
Let us return to the intragastric/intraperitoneal duel. Authors argue that despite the importance of the
live larvae intraperitoneal inoculum studies, the human natural history of gastroallergic anisakiosis is
688 Laboratory Models for Foodborne Infections

given orally; so experiments using this pathway are important.59 Rats were infected with L3 Anisakis by
the oral route twice with an interval of 9 weeks to investigate the kinetics of isotype-specific antibody
expression, and it was found that IgM’s peak with similar titers after primary and reinfection presents
the same antigenic recognition. After reinfection, as expected, IgG1 and IgG2a levels were higher and
showed accelerated kinetics; however, IgG2b level was substantially lower. The biological allergy state
peaked earlier (1 week) than the immunochemical allergy state (2 weeks). Since no meaningful cor-
relation between specific IgE avidity and biological allergy state was found and elevated IgM levels at
reinfection occurred, the hypothesis is that the allergic response induced by oral L3 infection might not
be related to specific IgE avidity.94
A procedure developed recently to deliver live larvae directly to the stomach of mice by an esophageal
catheterization95 was adapted to perform live Anisakis spp. infection in rats.96 The aim of this study was
to understand the histopathological effects of acute (single) and chronic (multiple reinfections—24, 48,
72, and 96 h intervals) Anisakis infection. Live larvae were found anchored to the mucosa at different
locations (whose milieu varied from a very acidic to basic pH gradient), passing through the stomach
wall and in organs out of the gastrointestinal tract. The histopathology showed an acute inflammatory
reaction, with eosinophil predominance and a mild fibrotic reaction. Even though not all larvae were
recovered as previously placed as an obstacle to the oral route, this protocol can be considered a good
experimental model because the histopathological alterations are similar to those described in human
anisakiosis.97
Although there are reported cases of allergic reactions due to the ingestion of cooked and frozen
seafood, there is also evidence that only live larvae trigger the allergic reactions. Consequently, the
debate on the risk of Anisakis-associated hypersensitivity by ingestion of properly cooked and frozen
fish remains. To elucidate this fact, an experimental model was designed to study the antibody produc-
tion kinetics after oral inoculation with live or dead Anisakis L3. The results show that animals produce
specific IgM, IgG, and IgE to ES antigens after primary and secondary inoculation with live L3 but not
after dead L3 (frozen, heated, cut, or homogenized). These results suggest that the ingestion of cooked or
frozen seafood containing Anisakis L3 is safe even for allergic individuals.98

42.6.5.3  In Vivo L3–L4 Transformation Model in Rats

To study the morphological transformations of L3 to L4 Anisakis type I, P. decipiens, Contracaecum
type B and Hysterothylacium L3, recovered after experimental infection in rats, and Anisakis type I L4
derived from humans were examined with the aid of scanning electron microscopy to examine the ante-
rior and posterior extremities and the cuticular structures of the larvae. Rats were sacrificed at different
times after oral administration, and a careful search in the digestive tract, abdominal cavity, muscles,
and viscera was performed. Molting from L3 to L4 was observed as of the third day onward in rats
that received Anisakis type I and P. decipiens. Anisakis larvae penetrated the stomach and the intes-
tinal wall, and a single larva of Pseudoterranova penetrated to muscularis mucosa of the stomach. No
Contracaecum larvae were recovered. Electron microscopy revealed that L4 of Anisakis type I from rat
and man were similar, while the L4 of Anisakis type I and P. decipiens showed ultrastructural differ-
ences, which might be of clinical value for the identification of fragments recovered during endoscopy
in man.99

42.6.6 Mice
Because of the accumulated data in the last decades, especially concerning IgE synthesis of the anti-
body associated with allergic reactions, mice are considered better animal models than other species to
investigate allergic reactions.100–102 In food-associated allergies, it is still unclear what conditions make
certain foods strong IgE inductors. There are reports of more than 170 foods causing food allergies, but
only 8 (peanut, tree nuts, milk, egg, wheat, soy, fish, and shellfish) account for 90% of all food-allergic
reactions.103 It is known that the major reaction to food proteins when ingested in physiological condi-
tions usually is a phenomenon called oral tolerance, while the parenteral administration of the same
Anisakis 689

food proteins in experimental models induces sensitization.104–110 This intriguing dichotomy has inter-
ested immunologists.
It is also known that in natural helminthic infections, IgE and eosinophilia are major hallmarks of
the immunological response as the consequence of a Th2 lymphocyte profile activation by helminths.
Among other interleukins, Th2 cells secrete IL-4 and IL-5; the former promotes immunoglobulin class
switching to IgE, and the latter stimulates eosinophil development and activation. Furthermore, in IgE
experimental models, animals are immunized with the antigenic preparations mixed with adjuvants such
as aluminum hydroxide102 or pertussis toxin111,112 and commonly administered by a parenteral route. In
experimental models where the aim is to develop oral sensitization and food allergy, antigens are associ-
ated to cholera toxin.113 Complete or incomplete Freund’s adjuvant is another commonly used adjuvant,
which is considered a good IgG inducer.114
The humoral and cellular immune responses to live A. simplex larvae observed in mice models share
similarities with those observed in human disease. However, due to the difficulty in introducing live
larva into the gastric cavity of mice, the majority of the experiments have been conducted by immuniz-
ing the animals with CE or with ES antigens of cultivated larvae, thus indicating the relevance of the
investigation of the immune mechanisms that control the allergic responses to live and dead Anisakis
spp. larvae.115–118

42.6.6.1 Intraperitoneal Larva Implant

A. simplex L3 were surgically implanted into the abdominal cavity of mice to investigate histopatho-
logical alterations.119 Necropsy performed at 7, 14, or 21 days post infection evidenced that larvae were
mostly found embedded in the gut mesentery and only rarely invaded the viscera. On day 7, adjacent
to viable parasites, an intense neutrophil aggregation characterizing an acute inflammatory reaction
was observed; by day 14, this reaction evolved to a mature eosinophilic granuloma with large numbers
of fibroblasts and associated collagen. Granulocytes and occasionally multinucleate giant cells were
observed at the still viable host–parasite interface. By day 21, the L3 were dead, invaded by inflamma-
tory cells, and the lesions displayed the predominance of connective tissue. Multinucleate giant cells and
eosinophils adjacent to parasite remnants or scattered within the walls of the granulomata were frequent.
Hematological findings, regardless of the number of implanted worms, showed that on days 7 and 14
mice presented neutrophilia of varying magnitude accompanied with an eosinopenia that began to return
to normal values by day 21. Both hematological and histological findings are consistent with those seen
in human anisakiosis.

42.6.6.2 Intraperitoneal Immunization
To help understand some of the unknown immune interactions between helminth infection and allergy,
mice were intraperitoneally sensitized to develop a hypersensitivity reaction with A. simplex proteins,
followed by an intravenous or oral A. simplex challenge. The sensitized mice presented as of the third
week specific IgE, IgG1, and IgG2a to numerous A. simplex allergens, some of which were similar to
those found in human serum. When challenged with intravenous A. simplex antigens but not after an oral
antigen challenge, anaphylaxis and plasma histamine release were observed. The cellular and molecular
profile showed that A. simplex stimulated splenocytes to release IL-10, IFN-γ, IL-4, IL-13, and IL-5 with
a mixed Th1/Th2 pattern.120 This seems to be a good model to investigate the peculiar allergic reactions
to parasitic proteins.

42.6.6.3 Intragastric Infection
Live Anisakis L3 were orally inoculated in C57BL/10 and BALB/c mice to investigate isotype-specific
immune responses to ES and CE products. The C57BL mouse strains typically produce a Th1-Type
cytokine profile, while BALB/c mice produce a Th2-Type cytokine profile. Both ES and CE antigens
stimulated similar antibody patterns; however, CE stimulated the production of higher antibody levels.
690 Laboratory Models for Foodborne Infections

BALB/c mice produced a faster IgM response than C57BL/10 mice, while the latter produced higher
IgG1 and IgG2b antibodies with practically undetectable IgG2a levels.121 Further anisakiosis studies
using BALB/c mice showed that after multiple immunizations using Freund’s adjuvant, mice showed a
single maximum peak of IL-4 between weeks 8 and 14, whereas animals inoculated with a single larva
per os showed two IL-4 peaks—the first with moderate levels between days 6 and 12 p.i. and the second
maintained from week 3 to 9.122 After Perteguer and Cuellar showed the consequences of natural sensi-
tization,121,122 the authors of this chapter proposed a simplified method to introduce live larvae with an
intragastric tube. This technique results in similar data as those published in the literature.95,118

42.6.6.4 Epicutaneous Immunization
As cited before, contact dermatitis is one of the consequences of antigen exposure to Anisakis proteins
in seafood-processing workers. Therefore, to understand the basic mechanisms in the development of
allergic sensitization through the skin, repeated epicutaneous exposure of Anisakis proteins in wild-type
(WT), IL-4-, IL-4Rα-, IL-13-, and IL-4/IL-13-deficient mice was evaluated by following the systemic
signs and symptoms. Epicutaneous sensitization with Anisakis larval antigens induced the WT local-
ized inflammation, epidermal hyperplasia, production of TH2 cytokines, antigen-specific IgE and IgG1,
and anaphylactic shock after intravenous challenge. IL-13-deficient mice failed to develop epidermal
hyperplasia and inflammation, and in IL-4-, IL-4/IL-13-, and IL-4Rα-deficient mice, anaphylaxis was
reduced. These results suggest that interleukin-13 plays a central role in contact dermatitis development,
whereas IL-4 drives the Th2 profile and resultant anaphylactic reactions.48

42.6.6.5 Subcutaneous Immunization
The subcutaneous route is a technique frequently utilized in immunological studies. The footpad is a
frequently used location, since the draining lymph nodes are easily removed making it possible to study
the local immunological response. Taking the advantages of this strategy, the cellular immune response
to A. simplex L3 antigens was compared in mice that were infected either after being pre-sensitized, with
homologous CE antigen or sham sensitized. As the pre-sensitization simulates a primary immune response
the authors addressed the difference between a primary infection and a simulation of a reinfection. This
footpad sensitization protocol induced an increase in the size and weight of the popliteal lymph nodes
(PLN). A high proportion of systemic CD4+ and TCRαβ+ T cells in both groups was observed. A reduc-
tion in B cells accompanied by a decrease of CD8α+ T cells was observed in pre-sensitized and infected
mice, while those only exposed to infection present the greatest increase in CD8α+ and TCRαβ− T cells.

42.6.6.6 Intranasal Immunization
To examine the immunological mechanisms underlying the development of allergic airway inflamma-
tion, WT and interleukin-4 receptor alpha (IL-4Rα)-deficient mice were sensitized to Anisakis anti-
gens through different routes.123 Live or heat-killed Anisakis larvae were administered intraperitoneally,
while Anisakis extract was administered by the intranasal route. Subsequently, all animals were chal-
lenged intranasally with an Anisakis extract. Allergen-specific antibodies developed only in intra-
peritoneally sensitized mice; however, both routes of sensitization induced IL-4Rα-dependent allergic
airway responses in WT mice in an IL-4/IL-13-dependent pathway. Unexpectedly, infection with live
Anisakis larvae induced airway hyperresponsiveness that was abrogated when IFN-γ was neutralized
in vivo. Thus, infection leads to IL-4/IL-13-independent, IFN-γ-dependent airway hyperresponsive-
ness. Together, these results demonstrate that both infection with larvae and inhalational exposure to
Anisakis proteins are potent routes of allergic sensitization, explaining food- and work-related allergies
in humans, which can involve either IL-4/IL-13 or IFN-γ. Importantly for diagnosis, detectable Anisakis-
specific antibodies may not accompany allergic airway inflammation.
In vitro studies demonstrated that a 24-kDa protein (22U homologous; As22U) derived from A. simplex
larva elicits several Th2-related chemokine gene expression, meaning that it may be one of the impor-
tant allergens for the clinical setting. In order to examine their hypothesis, six intranasal applications of
Anisakis 691

ovalbumin (OVA) or recombinant As22U (rAs22U) and OVA were performed. When compared to the
group that only received OVA, the animals challenged with rAs22U associated to OVA presented severe
airway inflammation, immune cell recruitment in special, eosinophils, increased levels of IL-4, IL-5, and
IL-13 in the Bronchoalveolar lavage fluid (BALF), significantly increased airway hyperresponsiveness,
and significantly higher anti-OVA-­specific IgE and IgG1. After receiving rAs22U, the GRO-α (CXCL1)
gene expression increased immediately while eotaxin (CCL11) and TARC (CCL17) gene expressions
increased significantly at 6 h. Thus, rAs22U may be responsible for a Th2/Th17-mediated airway allergic
inflammation.124 Using the same experimental protocol, two other Anisakis antigens (Ani s 1 Ani s 9)
were tested, eliciting similar results expressing Th2 (IL-4, IL-5, IL-13, e IL-25) and Th17 (IL-6 e IL-17)
cytokines because of the intranasal exposure.125

42.6.6.7 Nematode Molecules as Immunoregulators

In the last decades, a variety of immunoregulatory molecules have been isolated from a number of nema-
todes. The identified biological activities include actions equivalent to cytokines, protease inhibitors,
macrophage migration inhibitory factor-like protein (MIF), proteins as poison expressed sequence tags
(ESTs), and allergen.126–132 The A. simplex macrophage MIF protein obtained from third-stage larvae
of A. simplex was cloned (rAs-MIF) and tested in a murine OVA/Alum-induced asthma model.129 The
rAs-MIF treatment coupled with OVA/alum induced a complete inhibition of eosinophilia, reduced lung
goblet cell hyperplasia, profoundly improved lung hyperactivity, and reduced the quantity of Th2-related
cytokines (IL-4, IL-5, and IL-13) in the BALF and allergen-specific IgG2a in sera. Conversely, the
BALF of the rAs-MIF-treated group contained significantly higher of IL-10 and TGF-β than controls. In
addition, rAs-MIF recruited regulatory T cells (CD4+CD25+Foxp3+) to the spleen and lungs.
These authors evaluated the function of rAs-MIF on a dextran sodium sulphate (DSS)-induced intes-
tinal inflammation. Mice treated with rAs-MIF recovered weight loss and the disease activity index
(DAI) value. The cytokine profile evaluation showed that rAs-MIF-treated mice presented higher lev-
els of splenic and mesenteric lymph nodes (MLN) TGF-β and IL-10 with lower levels of IFN-γ, IL-6,
and IL-13. In addition, Treg were greatly increased in the MLNs of the rAs-MIF-treated mice. In vitro
experiments showed that rAs-MIF stimulated IL-10 production via toll-like receptor 2.133
Further studies on rAs-MIF also showed that TLR2 gene expression was significantly increased fol-
lowing rAs-MIF treatment. To further understand the relation between TLR2 and the amelioration
mechanisms of rAs-MIF, the OVA/Alum allergic airway inflammation protocol was induced with or
without rAs-MIF associated or not to anti-TLR2-specific antibody and comparing WT and TLR2 knock-
out mice. As a result, the amelioration effects of rAs-MIF in allergic airway inflammation model, as pre-
viously described, were diminished to fewer than two of the TLR2 blocking models. The expression of
TLR2 on the surface of lung epithelial cell was significantly elevated by rAs-MIF or Pam3CSK (TLR2-
specific agonist) treatment.134 Pretreatment with α-mTLR2 Ab or Pam3CSK inhibited the elevation of
IL-10 gene expression by rAs-MIF, suggesting that the anti-inflammatory effects of rAs-MIF might be
closely related to TLR2.

42.6.7 Fish
Many marine fish are infected with third-stage larvae of A. simplex (sensu lato). To ensure food safety,
it is important to determine whether these larvae are present in the flesh of commercial fish species.
However, there is little information regarding the tissue specificity of anisakid species. Thus, the ratio-
nale for the use of fish as an experimental model to study Anisakidae nematode is to understand the
infective capacity in commercially relevant fish species, the parasite mechanisms of aggression, and the
host’s immunological response.

42.6.7.1 Oral Infection
Rainbow trout (Oncorhynchus mykiss) and olive flounder (Paralichthys olivaceus) received L3 larvae of two
sibling species of A. simplex per os and were accompanied for 5 weeks. In the rainbow trout, A. simplex s.s.
692 Laboratory Models for Foodborne Infections

predominantly migrated into the body muscle while a small number of freely moving A. pegreffi larvae were
recovered within the body cavity. In the olive flounder, A. simplex s.s. larvae were found both in the body
cavity and the muscle, while A. pegreffi larvae were found only in the body cavity encapsulated in lumps.135
In another set of in vivo investigations, A. simplex was used to experimentally infect rainbow trout
(Oncorhynchus mykiss), Baltic salmon (Salmo salar), and brown trout (Salmo trutta). Of the three spe-
cies, Baltic salmon was the most susceptible, presenting the highest number of successfully established
nematodes, whereas brown and rainbow trout had a higher natural resistance. The preferred A. simplex
larvae microhabitat in the brown trout was the stomach, pyloric caeca, and intestine, while the majority
of larvae found in rainbow trout were located at the pyloric caeca. In the Baltic salmon, the most suscep-
tible fish species, nematodes were dispersed in and on the spleen, head, kidney, liver, swim bladder, and
musculature. CD8+ cells were present while IgM+ -bearing cells were absent in the inflammatory tissue
around the nematodes of all three fish species. MHCII-bearing cells were present in the encapsulated
A. simplex in rainbow and the brown trout, but not in Baltic salmon.136
Yet, another set of recent experiments show that closely related salmonids differ in their susceptibility
toward different anisakid larvae and agree that parasites select different microhabitats in the hosts.137
Orally infected rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), and Atlantic salmon
(Salmo salar) with larval stages of H. aduncum, C. osculatum, or A. simplex were studied to determine
parasite survival and location up to14 days post infection (dpi). Although the most prevalent and numer-
ous nematode in brown trout at 2 dpi was H. aduncum, a large proportion of the worms were already
recovered dead with no tissue penetration. This fish species exhibited the highest natural resistance to
A. simplex. Rainbow trout exhibited the highest susceptibility to C. osculatum larvae at 2, 7, and 14 dpi
with eventual pyloric cecum penetration. A. simplex larvae established a more successful infection in
salmon compared to rainbow trout, although at 2 and 7 dpi this fish showed the highest intensity and
abundance of larvae, but not after 14 days. Although the pyloric ceca was the preferred microhabitat for
Anisakis in both rainbow trout and salmon, larval penetration into muscle and liver was found.

42.6.7.2 Intraperitoneal
Since hydrolytic enzymes play an important role in the nematode host tissue penetration, determination
of what enzymes are present within the ES proteins seems important. Lipase, esterase/lipase, valine and
cysteine arylamidases, naphthol-AS-BI-phosphohydrolase, and α-galactosidase activities were found. To
further elucidate the influence of intraperitoneally injected ES, substances on the immune system of fish-
specific gene expression in spleen and liver of the rainbow trout (Oncorhynchus mykiss) were measured.
The results demonstrate a generalized downregulation of immune-related gene expression, suggesting
a suppressive immunomodulatory role for ES proteins. From the ecological point of view, this makes
biological sense. One can argue that when worm enzymes directly target the host’s immune molecules, a
decreased immune response with an increased worm survival is the consequence.138

42.6.8  In Vitro Cultivation

The in vitro cultivation of nematodes has been for long a goal of the field of parasitology. These tech-
niques permit the understanding of parasite behavior, physiology, and metabolism as well as the molecu-
lar nature of the ES products and their relationship with the host. This, in turn, permits more adequate
vaccine production designs, vaccine efficacy testing, antigen production for serological reagents, detec-
tion of drug-resistance, screening of potential therapeutic agents, and conducting epidemiological
studies. However, the complexity of the parasite’s life cycle involving different host species for their
developmental stages frequently makes their cultivation a difficult task. Each parasite requires different
cultivation conditions with specific nutrients, temperature, and incubation conditions.
A search in biological databases indicate that the first papers regarding parasite cultivation, in general,
were published in the 1910s139 and the first Anisakis cultivation papers in the 1970s.140 An important
systematization of the developed technique was performed by Silverman,141 which has been updated in
a diversity of technical books.142,143
Anisakis 693

For many clinically important parasites, in vitro cultivation is an important diagnosis tool. An array
of commercial systems, which have been developed, such as the Harada-Mori culture technique for
larval-stage nematodes, permit rapid diagnosis. In comparison, although in vitro cultivation techniques
are used more often than in vivo techniques, the in vivo techniques are sometimes used for diagnosing
parasitic infections such as trypanosomiasis and toxoplasmosis. Parasite cultivation continues to be a
challenging diagnostic option. Thus, an overview of intricacies of parasitic culture and an update on
popular methods used for cultivating parasites are presented.

42.6.8.1 Culture Media
The first description of Anisakidae nematode cultivation occurred in the early 1960s. P. decipiens larvae
removed from the flesh of fresh fish were immediately transferred to 199 culture media enriched with
glucose, beef embryo extract, beef liver extract, and antibiotics. In this study, the authors obtained larvae
that reached morphological changes consistent with adult worms.144 Subsequently, with adjustments of
the initial conditions, A. marina developed successfully to adult worms. The first larval developmental
changes were observed within 4 days after the release of cuticles in the medium. The development of
gonadal tissue characterizing the preadult stage occurred between 26 and 98 days. The complete matu-
ration characterized by the worm wall thickening and gonadal maturation can be distinguished in vitro.
The first free larvae were observed after 4–8 days at a temperature of 13°C–18°C and after 20–27 days
at a temperature of 5°C–7°C. The larvae are very active, and their mobility has no fixed direction. In
seawater, they can live for 3–4 weeks at temperatures of 13°C–18°C and for 6–7 weeks at 5°C–7°C; tem-
peratures above 20°C led to increased mortality, and a temperature of 34°C was absolutely inadequate,
indicating that the first intermediate host must be cold-blooded.145

42.6.8.2 L3–L4 Transformation Model

Improvements of in vitro Anisakis L3 culture conditions were introduced in 1976; these allowed to
explore the formation of cuticles and ecdysis.140 Different culture media (199, Krebs-Ringer), carbon
dioxide concentration, temperature, and storage conditions were tested. Among the tested conditions,
culture media 199 gave the best results, with the highest number of molts and viability. The carbon
dioxide concentration of 5% in low concentrations is more efficient in the first 40 h of cultivation. Using
fluorescent tracers, it was determined that larvae do not feed (salt and glucose) until their digestive tract
is complete, in other words, when they enter the fourth stage of development (L4).
To simulate the natural conditions of the fish’s body, where the larvae remain for long periods in ana-
biose, and to determine the temporal resistance, Anisakis L3 were collected from herring and kept in
saline solution culture (0.65% NaCl) at about 5°C. The mortality of Anisakis in culture presented three
phases: Phase 1 (months 1–2)—low mortality; Phase 2 (months 3–5)—significant increase in mortality
rate; and Phase 3 (months 6–8)—only the strongest survive larvae. Thus, the larvae kept in saline solu-
tion survived for about 35 weeks.146
CO2 fixation is an important metabolic process for many organisms. In anisakid nematodes, CO2 has
been shown to be required for its development, at least in vitro. Comparing culture conditions, molting to
L4 was reduced to one-third, after a 30-day culture in air, which corresponds to one-third of the survival
of L3 cultivated in air + 5% CO2. Thus, at suitable temperatures, a high pCO2 is vital for the optimum
development of L3 to adult (M3). Regarding the activity of the CO2-fixing enzymes, phosphoenolpyru-
vate carboxykinase (PEPck) activity (305 nmol/min·mg protein) was much higher than that of PEPC
(6.8 nmol/min·mg protein). The activity of these enzymes in the worms cultivated in air + 5% CO2 was
highest during M3 and, in general, was higher than that of those cultivated in air only, especially dur-
ing molting from L3 to L4. The presence of CO2 stimulates the molting from L3 to L4 and prolongs the
survival at least in vitro.147–153
A. simplex L3 larvae tend to prefer fish tissues with high lipid content.154 In vitro tests were carried
out to study the behavior of A. simplex L3 in response to different concentrations of cod liver oil lip-
ids. Larvae were placed into culture dishes containing agar separated into three segments, containing
694 Laboratory Models for Foodborne Infections

0.2%–7% of cod liver oil. The results demonstrate that although L3 move randomly, they do not stop
randomly. The tendency to move out of a certain area was inversely correlated with lipid concentration.
A second observation indicates that the intentional migration range of larvae is short. In conclusion, L3
prefer high fat content and seek it over short distances. These in vitro data agree with previous observa-
tions that A. simplex L3 randomly tend to migrate out of the fish gut into the flesh.155

42.6.9 Larvicidal Models
With the growing number of human anisakiosis cases, an alternative was the search for active larvicidal
compounds. In vitro and in vivo assays were undertaken to evaluate herbs used to season fish based on
epidemiological observations that prevalence of anisakidosis in the Chinese regions where raw fish is
often seasoned with ginger (rhizome of Zingiber officinale) and/or “perilla mint,” “Chinese basil,” or
“wild basil” (common names for Perilla frutescens [Lamiaceae]) is smaller. 156,157
In vitro studies showed that [6]-Shogaol and [6]-gingerol components derived from ginger rhizome
induced an important reduction in larvae mobility and altered both their cuticle and digestive tract.158
Further studies revealed that [10]-gingerol, [10]-shogoal, and other compounds derived from ginger also
has a very effective larvicidal effect.159
In vivo protocols where rats were infected by delivering larvae directly to the stomach through the
use of a gavage were used to evaluate the action of essential oils on Anisakis L3. Simultaneously or 2 h
after infection, each rat received one of five monoterpenes. To determine the localization and viabil-
ity of the larvae and to determine gastrointestinal histopathological changes, rats were sacrificed at
various time points. The order of in vivo larvicidal activity was peril aldehyde > citral > citronellol >
cuminaldehyde > carvacrol. When peril aldehyde, citral, and citronellol were given together with the
nematodes, no hemorrhages were observed, leading to the conclusion that these monoterpenes somehow
inhibit the fixation and/or penetration capacity of the larvae. The time gap of 2 h between the infesta-
tion and the administration of any of the tested compounds is sufficient for the larvae to develop their
pathogenicity in the rats.160
Three sesquiterpenic derivatives (nerolidol, farnesol, and elemolto) were studied to determine their
in vivo larvicidal activity. The order of in vivo larvicidal activity was nerolidol > farnesol > elemolto; the
first two caused the death of all nematodes, which showed cuticle changes and intestinal wall rupture.
Only 20% of infected rats treated with nerolidol or farnesol showed gastric wall lesions in comparison to
86.6% of control animals, suggesting that nerolidol and farnesol are good candidates for further research
as biocidal agents against L(3) larvae of Anisakis type I.161
The histological parameters to evaluate the effect of potentially larvicidal compounds were the analy-
sis of the cuticle and intestinal wall structure. Fixed formalin A. simplex L3 was assessed by optical
microscopy study of transverse thin sections (0.5–1 μm) stained with hematoxylin eosin, Masson’s tri-
chromic dyes, or toluidine blue.
Knowing that essential oils can irritate the mucosa, gut inflammatory reaction was studied after oral
administration of the tested compounds.161 A marker of neutrophilic infiltration is the titration of myelo-
peroxidase activity (MPO), determined by solubilization of myeloperoxidase with hexadecyltrimethyl-
ammonium bromide and measured with a dianisidine-H2O2 assay.162

42.7 Conclusion
A range of laboratory models are available to investigate foodborne infectious diseases, including those
due to Anisakis nematodes. As presented in the short epidemiological and taxonomical review of the
anisakid family along with the review of laboratory models used to study anisakiosis, there are still many
open questions regarding the life cycle, host–pathogen interaction, pathogenesis, and immune response
of the anisakid family larvae. These questions should be addressed because these nematodes are more
frequently contaminating foods due to the diffusion of oriental and Spanish cuisine, making this an
emerging anthropozoonosis of clinical importance.
Anisakis 695

Acknowledgments
We thank Maira Platais for help in the translation process.

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43
Clonorchis sinensis

Bayissa Chala Legissa and Sung-Tae Hong

CONTENTS
43.1 Introduction................................................................................................................................... 703
43.1.1 History.............................................................................................................................. 703
43.1.2 Life Cycle and Morphology............................................................................................. 703
43.1.3 Pathogenesis and Pathology............................................................................................. 705
43.1.4 Clinical Manifestations.................................................................................................... 707
43.1.5 Epidemiology.................................................................................................................... 707
43.1.6 Diagnosis.......................................................................................................................... 708
43.1.7 Chemotherapy and Control Measures.............................................................................. 709
43.2 Laboratory Models for C. sinensis Research................................................................................ 709
43.2.1 Rationale on Models......................................................................................................... 709
43.2.2 Laboratory Animals for Host Susceptibility to C. sinensis............................................. 709
43.2.3 Animal Models for Immune Response and Reinfection...................................................710
43.2.4 Animal Models for Metabolism Study..............................................................................711
43.2.5 Animal Models for Chemotherapy....................................................................................711
43.2.6 Animal Models for Histopathology and Carcinogenesis..................................................711
43.2.7 In Vitro Models..................................................................................................................712
43.3 Conclusion......................................................................................................................................713
References................................................................................................................................................713

43.1 Introduction
43.1.1 History
Clonorchis sinensis was first discovered by James McConnell at the autopsy of a Chinese male in
1874. Following a detailed description of the fluke, Spencer Cobbold suggested that the species be
named Distoma sinense in 1875. Afterward, its nomenclature was changed to Opisthorchis sinensis
by Blanchard in 1895 and finally renamed as Clonorchis sinensis by Arthur Looss in 1907. Commonly
called the “Chinese or oriental liver fluke,” C. sinensis belongs to the family Opisthorchiidae. Similar to
those in the families Dicrocoeliidae and Fasciolidae, members of the family Opisthorchiidae infect the
biliary tree of mammals including humans. Currently, C. sinensis is recognized as a major parasite of
the human intrahepatic bile duct causing clonorchiasis [1].

43.1.2 Life Cycle and Morphology

The adult flukes of C. sinensis are leaf-shaped and measure 15–20 mm long and 3–5 mm wide
(Figures 43.1 and 43.2). The human and mammalian reservoir hosts (dogs, pigs, cats, and rats) acquire
the infection by ingestion of raw or undercooked fish containing infectious metacercariae. Inside these
mammals, the metacercariae are liberated from the fish flesh by pepsin digestion in the stomach and the

703
704 Laboratory Models for Foodborne Infections

FIGURE 43.1  Adult worm of C. sinensis, acetocarmine stained.

FIGURE 43.2  Gross view of recovered adult worms of Clonorchis sinensis from a man after chemotherapy in Korea. The
uterus of worms that are red in color is full of eggs.

metacercariae excyst in the duodenum after trypsin digestion [2]. The excysted juvenile flukes migrate
into the common bile duct through the ampulla of Vater to reach the intrahepatic biliary duct. One
experimental study using rabbits observed that the juvenile flukes moved to the intrahepatic bile duct
as early as 10–20 min after they excysted and entered into the duodenum [3]. The flukes develop into
adults within 4 weeks and begin to produce eggs, which mix in feces and pass through the intestine [1].
Clonorchis sinensis 705

FIGURE 43.3  Gross view of the snail host, Parafossarulus manchouricus.

After their discharge by mammalian hosts, the eggs are ingested by freshwater snail, which is the
intermediate hosts, in the rivers or lakes. The first intermediate snail hosts are Parafossarulus or
Bithynia species (Figure 43.3). In the snail host, the miracidium hatches out in the rectum and pen-
etrates the wall to reach the perirectal tissue. In the perirectal tissue, the miracidium undergoes meta-
morphoses into a sporocyst, which in turn gives rise to rediae and then cercariae. One miracidium may
amplify to 1000–1500 cercariae, which emerge into the water [4]. The emerging cercariae freely swim
and penetrate the skin, gills, fins, or muscles of freshwater fish within 24–48 h. In the fish, the cercariae
encyst to become metacercariae. Numerous species of small-sized freshwater fish such as Pseudorasbora
parva, Pungtungia herzi, Pseudogobio esocinus, Acheilognathus intermedia, Odontobutis interrupta,
and others serve as the second intermediate host of C. sinensis, harboring the metacercariae [5,6].
A study investigating freshwater fish in their infection status with metacercariae of C. sinensis in Korea
reported that the number of metacercariae per fish was found to be 48 (1–1142) in P. parva and 60
(1–412) in P. herzi (Figure 43.4), implying that the two species are able to be the index fish for the estima-
tion of C. sinensis transmission in a certain locality [5]. When humans or mammals consume infected
raw freshwater fish or poorly cooked ones (including smoked or pickled fish), the metacercariae infect
the host and develop into adults.

43.1.3 Pathogenesis and Pathology

Excysted C. sinensis metacercariae migrate into the end of the bile ducts of their host and use the
bile ducts as their nest [1]. Pathogenesis of liver-fluke-mediated tissue damage may be directly via
mechanical and chemical irritation and also by immune-mediated response during their reforming
process. Mechanical injury caused by oral suckers of the parasite during feeding activities and motility
stimulates the biliary epithelial cells. Simultaneously, chemical and immunological irritation by the
excretory–secretory products induces cell proliferation and inflammation in addition to the mechanical
stimulation [7].
The infected bile duct is widened as a sac-like nest over 5–7 mm in diameter and becomes tortuous
(Figure 43.5). The endothelium of the dilated ducts undergoes several changes. The extent of ductal
changes is proportional to the number of worms present, with some infections running into the thou-
sands; more than 6000 adult worms have been recovered from a single patient [8]. In an experimental
study using Sprague Dawley rats infected by C. sinensis, the bile duct endothelial cells proliferated more
than 20-fold at the base of the mucosal layer [9]. The bile duct mucosal layer revealed severe hyperplasia.
At the base of the hyperplastic tissue, many mucin-secreting cells appear as a metaplasia. Some dysplas-
tic cells are also noticed among the hyperplastic cells. Eosinophilic inflammation is commonly found in
the mucosa and along the bile ducts. Fibroblasts proliferate, and collagen fibers are deposited surround-
ing the duct [10]. The pathological changes were observed 1 year after chemotherapy in experimentally
infected rabbits [11].
706 Laboratory Models for Foodborne Infections

FIGURE 43.4  Second intermediate host. (A) Pseudorasbora parva. (B) Pungtungia herzi.

FIGURE 43.5  Histopathology of the Clonorchis sinensis-infected intrahepatic bile duct in an experimentally infected
cat. HE stained, original magnification, ×40. The duct is enlarged with adenomatous hyperplasia and periductal fibrosis.
Clonorchis sinensis 707

43.1.4 Clinical Manifestations
Clonorchiasis is rather well adapted to human hosts, and most of the light infected cases show no clinical
findings. When present, clinical features of clonorchiasis are often nonspecific and related to inflamma-
tion and intermittent obstruction of the biliary ducts. Adult worms cause obstruction and blockage of
the bile flow, which may induce epigastric discomfort or pain, swelling, and jaundice. In severe cases
with heavy worm burdens, abdominal pain, nausea, and diarrhea can occur. However, in long-standing
and untreated infections, chronic inflammation of the biliary system can lead to a neoplastic change as
cholangiocarcinoma (CCA) [1].

43.1.5 Epidemiology
Globally, clonorchiasis is a common foodborne zoonosis distributed mainly in East Asia including
China, Korea, Vietnam, the Philippines, and the far eastern part of Russia [1,12]. It may also occur
in other regions where there are immigrants from endemic areas [13]. It is estimated that more than
200 × 106 people are at risk of infection, 15–20 × 106 people are infected, and 1.5–2 × 106 show symp-
toms or complications [1].
Several studies in Korea reported that the infection status of C. sinensis has shown only a little decrease
despite wide use of praziquantel, an effective anthelmintic, for 30 years [14,15]. Cho et al. [14] reported
that the prevalence of C. sinensis was 11.1% among 24,075 inhabitants in the riverside areas of southern
Korea. Recently, several small endemic foci were reported in southern rural communities where eating
raw fish is common [15].
The Korea Centers for Disease Control and Prevention (KCDC) performed the eighth nationwide
survey on prevalence of intestinal parasitic infections in Korea and reported that the overall egg-positive
rate of C. sinensis was 1.9% in 2012 compared to 4.6% in 1971, 1.8% in 1976, 2.6% in 1981, 2.7% in
1986, 2.2% in 1992, 1.4% in 1997, and 2.4% in 2004 [16]. The egg-positive rates, which are regarded as
average prevalence of clonorchiasis in the general population in the surveyed year, have been decreasing
slowly. Hong and Fang [1] explained that the slow decrease is due mainly to frequent treatment failure
or reinfection after treatment. Still, about 1 × 106 residents who live along the river beds are infected in
southern endemic areas in Korea [1,16].
In a study conducted during 2000–2004 in Korea, C. sinensis prevalence (2.1% in Chuncheon, 7.8% in
Chungju, and 31.3% in Haman) was significantly correlated with CCA incidence rates (0.3, 1.8, and 5.5 per
100,000 persons, respectively) [17]. Furthermore, earlier case–control study in Busan, Korea, including 41
patients with CCA and 203 cases of hepatocellular carcinoma (HCC) revealed that CCA was significantly
correlated with C. sinensis eggs in stool, past history of liver flukes, hepatitis history, and a history of heavy
consumption of alcoholic beverages [18]. Shin et al. [19] described that the odds ratio of CCA due to C. sinensis
infection was 4.7, and about 10% of CCA cases in Korea were due to this organism. The overall odds ratio of
CCA is 4.47 for clonorchiasis, and about 5000 CCA cases are estimated annually in the world [20].
In Guangdong, China, where fish farming is common, the prevalence of human clonorchiasis is about
14%, and it may reach 80% in rural endemic areas [8]. A recent analysis of data from three parasitic
disease surveys conducted in Hengxian County, China, in the last 22 years showed substantial increases
in the patterns of clonorchiasis prevalence and infection intensity and decrease in trends of prevalence
of soil-transmitting helminths [21]. Another recent study in Guangdong, China, reported that incidence
rates of clonorchiasis showed a direct increasing trend by years with temperature change [22].
The distribution of clonorchiasis is determined not only by the distribution of snail intermediate hosts
but also by the fish-consuming custom of residents in the endemic community. Fishing and eating of raw
fish is an old popular tradition that has remained habitual and constant for thousands of years in endemic
areas. The eating habit has resulted in persistent transmission of the liver fluke to humans [23,24]. There
are several well-known risk factors that are associated with the transmission of clonorchiasis such as
poor educational level of local residents, lack of sanitation, habit of eating raw or undercooked freshwater
fish, development of freshwater aquaculture, and lack of systematic control activities in many endemic
areas. In particular, freshwater aquaculture has rapidly expanded, with a resulting increase in fish con-
tamination that has resulted from a lack of quarantine measures for fish products [25].
708 Laboratory Models for Foodborne Infections

Regarding accumulated infection, infection rate and burden as measured by egg-positive rate and
number of eggs per gram of feces (EPGs) have been recorded as increasing with age in men [23,26]. The
reason for its increase among adult men reflects behavioral patterns of fishing as an occupation or recre-
ation. Since the lifespan of C. sinensis is estimated to be around 30 years, the infection occurs repeatedly
through the life of humans [23]. That finding suggests that host immunity is not so efficient to prevent
reinfection or superinfection in humans.

43.1.6 Diagnosis
Diagnosis of clonorchiasis is essential in the control programs as well as for treatment. Early diagnosis
and treatment is important to prevent serious complications of clonorchiasis in humans. Detection of
eggs or adult worms in feces, bile, or duodenal fluids is a definite diagnosis. However, since detection of
worms from a human body needs invasive procedures, the gold standard diagnosis of C. sinensis infec-
tion is microscopic identification of eggs in feces. The Kato-Katz (KK) method is reliable for diagnosis
of clonorchiasis by egg detection [27], but the formalin-ether concentration technique is more sensitive
than the KK method in cases of mild infection [28]. Furthermore, Choi et al. [28] reported that is good
correlation between the EPGs determined by KK method and by direct smear. Since the KK method is
far from straightforward for differential diagnosis between eggs of C. sinensis and heterophyid flukes,
well-trained technicians are required for mixed infections [6]. Morphological identification of these eggs
by fecal examination is very tricky and requires much more experience (Figure 43.6).
Methods other than fecal examination include serology tests, detection of DNA, and image diagnosis.
There have been many serological methods used for diagnosis of C. sinensis infection, but ELISA is
more commonly used [29]. The ELISA diagnosis using excretory-secretory antigen (ESA) and crude
antigen (CA) of C. sinensis showed sensitivities of 92.5% and 88.2% and specificities of 93.1% and
87.8%, respectively [30]. The data implied that ESA was a better serodiagnostic antigen than CA for
ELISA. ELISA detects specific IgG antibodies in serum, and the antibodies may last over 2 years after
treatment, which may make it less specific.
Clonorchiasis has been diagnosed using ultrasound scanning, computerized tomography (CT), and
magnetic resonance imaging (MRI) [31,32]. Choi and Hong [31] reported that radiological images
employing CT or MRI of the liver were correlated with worm burdens (EPG counts) frequency and
also severity. The images of the liver and bile ducts can be used as a good practical alternative diag-
nostic method of clonorchiasis. Although most of the positive images are acceptable for diagnosis,
their sensitivity and specificity are lower than those of the fecal examination. This is mainly because
those images recognize the tissue changes caused by the worms but not the worms itself. The images
of cholangiocarcinoma associated with clonorchiasis show both the tumor with obstruction images

FIGURE 43.6  Eggs of C. sinensis by the Kato-Katz smear. Original magnification, ×400.
Clonorchis sinensis 709

and diffuse dilatation of the peripheral intrahepatic bile ducts [31]. The remaining ductal dilatation
with thick wall after chemotherapy further reduces the image specificity [11]. In endemic areas, it
is recommended to make diagnosis of clonorchiasis by any feasible method and to treat positives
more actively.

43.1.7 Chemotherapy and Control Measures

Praziquantel exhibits satisfactory efficacy and represents the first-line drug of choice for clonorchiasis [4].
The recommended treatment regimen by WHO is 25 mg/kg three times daily for two consecutive days [13].
In heavily endemic areas of China, using mass chemotherapy with praziquantel, it was reported that four
selective annual treatments for egg-positive subjects reduced the egg-positive rates from 54.9% in 2001
to 15.0% in 2004 or from 73.2% in 2001 to 12.3% in 2004 [26]. In general, thorough cooking of fish,
proper diagnosis, health education, and praziquantel treatment are major requirements for its control.
Unfortunately, control of snail intermediate hosts by molluscicides was not considered feasible because
of widespread distribution of snails, low prevalence of fluke infection among snails even in endemic
communities, hazards to surrounding ecology, and economic considerations [26].
As clonorchiasis is one of the important foodborne neglected tropical diseases (NTDs) in Asia, it
is included as a foodborne zoonosis in control programs of NTDs by the World Health Organization
[33]. Currently, fish farming and neighboring environmental ponds that are routinely contaminated by
untreated sewage have resulted in the establishment of infection in fish populations at large. This phe-
nomenon along with the involvement of animal reservoir hosts will make control of the liver fluke infec-
tion even more challenging.

43.2 Laboratory Models for C. sinensis Research

Much of our knowledge and understanding on pathogenic mechanism of foodborne pathogens have been
obtained by animal experiments or in vitro cultures. Animals are used as basic research models of hel-
minths to study their life cycle in the laboratory as well as to study pathology, immunology, diagnosis,
or treatment. In vitro cultivation of helminths or cell culture models, especially the secondary cell line,
is a valuable tool that may help us in determining its virulence potential. The studies on C. sinensis have
developed several appropriate animal models for specific aspects of the parasite, such as host suscepti-
bility for infection and reinfection, host immune response, diagnosis, chemotherapy, pathogenesis, and
carcinogenesis.

43.2.1 Rationale on Models
Animal research has provided valuable information about many physiological processes that are relevant
to humans and human pathogens. Rodents (e.g., mice, rats) and rabbits are the most commonly used
laboratory animal models for biomedical studies. Of course, several other mammals have been used. The
animal model for C. sinensis research may depend on host–parasite relationship, feasibility of animal
quarters, research purpose, and cost.

43.2.2 Laboratory Animals for Host Susceptibility to C. sinensis

Most mammals have been known to be susceptible to C. sinensis infection. Sohn et al. [34] investigated
worm recovery rates of six laboratory mammals after infection and reinfection with C. sinensis metacer-
cariae. The recovery rates of the mammals were from 17.6% in mice to 68.0% in hamsters (Table 43.1).
The recovery rates suggest that rats and hamsters are highly susceptible; guinea pigs, rabbits, and dogs
are moderately susceptible but mice are not. Rats, mice, guinea pigs, and rabbits were recognized to be
resistant to reinfection after chemotherapy [34]. Another study reported that rats were found susceptible
to C. sinensis infection with an 83.3% recovery rate [35].
710 Laboratory Models for Foodborne Infections

TABLE 43.1
Recovery Rates of C. sinensis in Mammals by Experimental Infection
Recovery Rates (%)
Mammals No. of Heads Mean ± SD
Rats 10 63.9 ± 20.4
Mice 11 17.6 ± 13.0
Hamsters  9 68.0 ± 15.0
Guinea pigs 10 34.7 ± 13.7
Rabbits  7 35.0 ± 7.6
Dogs  3 41.6 ± 14.5
Source: Sohn, W.M., et al., Korean J. Parasitol., 44, 163–166, 2006.

Mice are the most commonly used laboratory animal model in biomedical research, but they are
not susceptible to C. sinensis infection [34,36]. Many strains of mice have been developed for diverse
research purposes, and the susceptibility was different in the strains [36]. ICR, Balb/c, C57BL/6, DDY,
CBA/N, and C3H/HeN mouse strains were not susceptible to C. sinensis infection and showed very low
recovery rates below 10%. Only the FVB strain showed 17.6% recovery rate, which is little higher than
other strains [34]. Since the FVB strain is relatively susceptible compared to other strains, this strain has
been used for in vivo studies of C. sinensis as a mouse model [37,38]. The FVB mice show cystic dilata-
tion of the infected bile duct, which is a unique pathological change [38].
On the other hand, there have been reports that rats are resistant (to some degree) to reinfection
by C. sinensis. The significant increase in the levels of bile IgA antibodies and serum IgE antibodies
in resistant reinfected rats might play a role in the development of resistance to reinfection by C. sinensis [39].
Rabbits, dogs, and cats are susceptible hosts for C. sinensis infection. Rabbits are commonly used to
maintain adult worms in the laboratory. Dogs, cats, and pigs are not used commonly for experimental
infection of C. sinensis, but their natural infection has been reported to be common in China [26,40].
They are good reservoir hosts of C. sinensis in nature.

43.2.3 Animal Models for Immune Response and Reinfection

C. sinensis stimulates the host cells and tissues in the bile duct by both mechanical means and releas-
ing excretory-secretory proteins (ESPs) [41]. The ESPs contain mostly enzymes, but various other
soluble immunogenic components have been identified and have been found to be good serodiagnostic
antigens by ELISA [30]. A recent study using mice reported that T2 ribonuclease in trematode ESPs
has been identified as a potent regulator of dendritic cells (DCs) [42]. A T2 ribonuclease in ESPs of
C. sinensis, named CsRNASET2, could modulate maturation of DCs and might play an important
role in C. sinensis-associated immune regulation in mice. In another recent mouse model study, it has
been reported that CA from C. sinensis plays a role in the anti-inflammatory function of dendritic DCs
by inducing IL-10 and TGF-β through activation of extracellular signal-regulated kinase, which is a
mitogen-activated protein kinase [43].
Previous studies demonstrated that C. sinensis-derived CAs suppress development of allergic
responses. Jeong et al. [44] reported that C. sinensis venom allergen-like (CsVAL) peptide treatment
inhibited activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which were
involved in degranulation of IgE-sensitized mast cells. Furthermore, immunization with CsVAL
suppressed development of skin inflammation, and this was demonstrated by assessing ear thickness
and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity
in vivo mouse model.
In an attempt to evaluate immunological responses and to develop an appropriate model, C57BL/6
and CBA/N mice were experimentally infected with C. sinensis [45]. The result of the study showed
high proliferation of splenocytes, increased IL-17, and more severe gross and histopathological
Clonorchis sinensis 711

changes in CBA/N mice as compared to C57BL/6 mice. Based on these results, it has been suggested
that CBA/N could be an appropriate mouse model for studying early immune responses in liver fluke
infection [45].
A few experimental studies on reinfection or superinfection of C. sinensis used mostly rats because
they are susceptible to primary infection but resistant to reinfection [34,39,46]. Meanwhile, hamsters,
dogs, and humans are susceptible to primary and also reinfection or superinfection, and the infection
is accumulated in those hosts. The resistance of the rats was mainly modulated by the local immune
response in the infected tissue [39].
The findings of laboratory studies suggest that C. sinensis infection in mammals may suppress host
immunity, which makes the host susceptible. The host immunity provokes the host resistant to reinfec-
tion or superinfection in rats but not in humans. Humans are repeatedly reinfected.

43.2.4 Animal Models for Metabolism Study

Another remarkable issue in host–parasitic interaction is the energy-acquiring mechanism of the parasite
from its host. In this perspective, glycolytic enzymes are crucial molecules for trematode survival and
have been targeted for drug development. It has been reported that hexokinase of C. sinensis (CsHK),
the first key regulatory enzyme of the glycolytic pathway, was significantly inhibited compared to that
of the corresponding negative control by praziquantel and anti-rCsHK serum [47]. In addition, the study
pinpointed that there were differences in both mRNA and protein levels of CsHK among the life stages
of the adult worm, metacercaria, excysted metacercaria, and eggs of C. sinensis, suggesting different
energy requirements during different development stages. Another recent report showed that subcuta-
neous immunization with recombinant C. sinensis hexokinase (rCsHK) decreased worm burden and
EPGs in challenged Sprague Dawley rats, implying that CsHK is a potential vaccine candidate and is a
promising drug target for clonorchiasis [48]. Those studies used rats because they are susceptible and
easy to handle.

43.2.5 Animal Models for Chemotherapy

One study evaluated cure of C. sinensis infection and recovery of the bile duct pathology after pra-
ziquantel medication with a dose of 50 mg/kg × 2/day × 2 days in rabbits [11]. The rabbit is an optimal
model of C. sinensis infection because the liver of rabbits is big enough and its pathology is similar
to that of humans.
On the other hand, there have been several reports on in vivo study to evaluate other chemothera-
peutic agents than praziquantel to treat clonorchiasis. Those studies investigated cure rates, reduction
of worm burdens, and morphological damage of C. sinensis adults using rats [49,50]. The new agents
were artemether, artesunate, OZ78, and tribendimidine, and they were screened in various doses in rats
or rabbits. Of them, tribendimidine was found promising and was used to a human trial. One clinical
trial in China reported a cure rate of 44% with a 400 mg single dose of tribendimidine and of 58% after
therapy for 3 days [51].
The chemotherapy study using experimental animals should evaluate cure rates or worm reduction
rates after medication. Rats or rabbits are commonly used for this purpose because they are susceptible
and because C. sinensis can grow well in the host. It should be noted that doses of antihelmintics vary
between rodents and humans.

43.2.6 Animal Models for Histopathology and Carcinogenesis

As adult worms commonly parasitize the intrahepatic ducts, they provoke pathological changes in the
biliary tracts, which may lead to further complications. The induced pathological changes of the infected
bile duct start to present different clinical symptoms, although severity of the pathology depends upon
intensity and chronicity of the infection. Both experimental findings and epidemiological evidence have
implied that long-term infections by liver flukes lead to chronic pathological changes, including chol-
angitis, cholecystitis, cholelithiasis, cholangiectasis, adenomatous hyperplasia, hepatomegaly, hepatic
712 Laboratory Models for Foodborne Infections

fibrosis, and CCA [52–54]. In extreme cases, liver enlargement, thickening of the bile ducts, fibrosis, and
some destruction of liver parenchyma are evident. However, unlike Fasciola, Clonorchis does not invade
liver tissues and therefore, does not cause extensive liver necrosis [8].
Several well-documented epidemiological, histopathological, and experimental studies of
C. sinensis have provided convincing evidence of a relationship between this trematode infection and
the tendency for malignant transformation of the biliary epithelium in humans and experimentally
infected animals. The International Agency for Research on Cancer (IARC) of the World Health
Organization recategorized C. sinensis as a Group 1 biological carcinogen with Opisthorchis viver-
rini, as it ­produces human CCA [55,56].
The neoplastic response that is associated with clonorchiasis is CCA, which is a highly malignant
epithelial cancer of the biliary tract [57]. In humans, primary sclerosing cholangitis, hepatolithiasis,
fibropolycystic diseases of the biliary tract, Caroli’s disease, and liver fluke infection have been con-
sidered as predisposing conditions for development of CCA [18,57,58].
Concerning history of experimental animals for induction of CCA, Thamavit et al. [59] first reported the
possible development of CCA in the hamster model by administering Opisthorchis viverrini and admin-
istration of N-nitrosodimethylamine (NDMA; exogenously) in drinking water. Since then, similar exper-
iments were also performed following C. sinensis infection in combination with 2-­acetylaminofluorene
or N-nitroso compounds (NDMA or N-nitrosodihydroxydi-n-propylamine) in hamsters [­ 60–62] and rats
[63]. These studies supported the role of C. sinensis as a promotor instead of an initiator. Experimental
infection with C. sinensis alone in animals has never induced bile duct tumors [62]. During the past two
decades, the primary experimental model for CCA induced by C. sinensis [60–62] as well as O. viverrini
is the Syrian golden hamster [64,65]. The study using rat model on the possible modifying potential of
C. sinensis infection and NDMA suggested that C. sinensis infection might facilitate the proliferation of
NDMA-induced preneoplastic lesions of the liver [63].
One experimental animal study using animals other than hamsters or rats reported that intrahe-
patic cholangiocarcinomas (ICCs) could originate from fully differentiated hepatocytes. Using a
mouse model of hepatocyte fate tracing, they found that activated NOTCH and AKT signaling coop-
erated to convert normal hepatocytes into neoplastic biliary cells that acted as precursors of rapidly
progressing, lethal ICCs [66].

43.2.7  In Vitro Models

One study suggested that it was possible to establish and maintain the complete life cycle of C. sinensis
in the laboratory [67]. It is evident that in vitro maintenance or culturing of C. sinensis in an active
state (for the whole life cycle) is helpful for further characterization of its physiology and important
regulatory molecules. In this regard, it was reported that bile acids and conjugated bile salts favored
the survival of newly excysted juvenile C. sinensis (CsNEJ) under in vitro model. In a similar fash-
ion, the study also indicated that NCTC 109 medium was found to be optimal for the in vitro mainte-
nance of CsNEJs and 1× Locke’s solution to be suitable for analyzing the biological effects of bioactive
compounds [68]. C. sinensis worms were maintained in vitro using 0.85% normal saline (NaCl) or
1× PBS, mostly to collect ESP [69]. Normal saline and PBS lack essential nutrients, and the worms in
those solutions may pass all reserved ESP and eggs within a few days but not produce new ESP [70]. It
has been reported that the RPMI-1640 medium maintained living C. sinensis adults better and longer (up
to 114 days) in vitro than other media. On the other hand, 1× Locke’s solution best supported the worms
alive among inorganic solutions for 57 days, confirming Locke’s solution and RPMI-1640 media could
maintain C. sinensis adult worms much longer than other inorganic solutions or nutrient media [70].
These studies helped define the best conditions for in vitro maintenance of the organism for long dura-
tions in different inorganic solutions and nutrient media under laboratory conditions. In vitro cultivation
in any nutrient media could not foster adult worms from larvae.
It has already been reported that the ESPs of C. sinensis play important roles in host–parasite interac-
tions. Recently, it has been revealed that the protein compositions of ESPs at different secretory times are
variable. Moreover, it was observed, using the MTT assay, that the ESPs were found to inhibit prolifera-
tion of hepatic stellate cells (LX-2) [41].
Clonorchis sinensis 713

For the biliary hyperplasia, several in vitro experiments provided strong evidence that C. sinensis pos-
sessed mitogenic factors in its ESPs and exhibited different levels of cell proliferations. For instance, C.
sinensis ESPs were reported to exhibit antiapoptotic effect in human CCA cells [71]. Likewise, exposure
of human CCA cells (HuCCT1) to C. sinensis ESPs demonstrated increased free radical generation
through activation of NADPH oxidase, inducible nitric oxide synthase (iNOS), and xanthine oxidase,
subsequently leading to nuclear factor-kappa B (NF-κB)-mediated inflammatory processes [72].
There has been a well-established body of knowledge about these highly reactive free radicals that
can damage biologically relevant molecules of cells. Consequently, oxidative stress arising as a result of
an imbalance between free radical production and antioxidant defenses is associated with damage to a
wide range of molecular species including lipids, proteins, and nucleic acids [73,74]. The damage to cells
caused by free radicals, especially the damage to DNA, may trigger the development of neoplasms and
also other health-related disorders.

43.3 Conclusion
C. sinensis, a foodborne trematode infecting intrahepatic bile duct of humans, is considered as one
of the target NTDs by WHO. Since its host specificity is low, most of the laboratory mammals except
for mice are susceptible, and rats or rabbits are commonly used for common experimental infection.
For experimental CCA studies, hamster is the only successful animal model. It is recommended to
develop other animal models such as murine model for experimental studies on immunology and oncol-
ogy of C. sinensis. In vitro cell cultivation is a good option for molecular studies of ESPs and generates
molecular insights into its carcinogenesis.

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52. Choi D, et al. Cholangiocarcinoma and Clonorchis sinensis infection: a case-control study in Korea.
J Hepatol. 2006; 44: 1066–73.
53. Sripa B, et al. Liver fluke induces cholangiocarcinoma. PLoS Med. 2007; 4: e201.
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55. International Agency for Research on Cancer. A Review of Human Carcinogens Part B: Biological
Agents, Vol. 100 B. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon,
France: IARC; 2009.
56. Grosse Y, et al. A review of human carcinogens-part A: pharmaceuticals. Lancet Oncol. 2009; 10: 13–4.
57. Sirica AE. Cholangiocarcinoma: molecular targeting strategies for chemoprevention and therapy.
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cinoma in Syrian golden hamsters administered 0.03% N-2-fluorenylacetamide (FAA). Jpn J Parasitol.
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61. Lee JH, Rim HJ, Bak UB. Effect of Clonorchis sinensis infection and dimethylnitrosamine administration
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cholangiocarcinoma during two-step carcinogenesis. Korean J Parasitol. 1994; 32: 13–8.
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hamster model. Mol Carcinog. 2006; 45: 279–87.
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74. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol. 2001; 54: 176–86.
44
Fasciola and Fasciolosis

Antonio Muro and Jose Rojas-Caraballo

CONTENTS
44.1 Introduction....................................................................................................................................717
44.2 Life Cycle.......................................................................................................................................719
44.3 Genome, Transcriptome, and Proteome........................................................................................719
44.4 Host–Pathogen Interaction............................................................................................................ 720
44.4.1 Immunity...........................................................................................................................721
44.4.2 Evasion Mechanisms........................................................................................................ 722
44.5 Clinical Manifestations................................................................................................................. 722
44.5.1 Acute Phase...................................................................................................................... 722
44.5.2 Chronic Phase................................................................................................................... 722
44.6 Diagnosis....................................................................................................................................... 723
44.6.1 Parasitological Techniques............................................................................................... 723
44.6.2 Immunological Techniques.............................................................................................. 723
44.6.3 Molecular Techniques...................................................................................................... 723
44.7 Treatment...................................................................................................................................... 723
44.8 Experimental Models in Fasciolosis............................................................................................. 724
44.8.1 Fasciolosis in Mice........................................................................................................... 724
44.8.1.1 Experimental Infection..................................................................................... 724
44.8.1.2 Physicopathological Changes........................................................................... 724
44.8.1.3 Microarray Analysis......................................................................................... 726
44.8.2 Fasciolosis in Rats............................................................................................................ 726
44.8.2.1 Experimental Infection..................................................................................... 726
44.8.2.2 Physicopathological Changes........................................................................... 726
44.8.3 Fasciolosis in Rabbits........................................................................................................ 727
44.8.3.1 Experimental Infection...................................................................................... 727
44.8.3.2 Physicopathological Changes............................................................................ 727
44.9 Experimental Assessment of Vaccine Candidates...................................................................... 728
44.10 Conclusion and Future Perspective............................................................................................. 730
References............................................................................................................................................... 730

44.1 Introduction
Classified in the phylum Platyhelminthes, subphylum Neodermata, class Trematoda, subclass Digenea,
order Echinostomida, and family Fasciolidae, the genus Fasciola consists of two liver fluke species that
cause fasciolosis in human and animal hosts, i.e., Fasciola hepatica and Fasciola gigantica. Jean DeBrie
was the first person who referred fasciolosis to “putrefaction from liver of sheep” in 1379. However, no
association between fasciolosis and the causal agent, the trematode F. hepatica, was mentioned at that
time. On the contrary, there was a belief that the disease could be attributed to toxic substances from
plants that were consumed by animals.1 The discovery of F. hepatica and its life cycle in the intermediate

717
718 Laboratory Models for Foodborne Infections

host (Galba truncatula) was the result of many observations made by several researchers worldwide. The
first observation was by Sir Anthony Fitzherbert (1523) and the Italian physicist Fanensi Gabucin (1549),
who described the presence of worms similar to pumpkin seeds in the blood vessels of sheep and goats.
At this time, it was thought that the onset of the disease was spontaneous. In 1688, the Italian physicist
Francesco Redi demonstrated that the disease was not a spontaneous phenomenon, but was caused by
oviposition of adult worms. In 1698, Govert Bidloo, a professor of anatomy, documented the presence
of worms in the bile ducts of sheep, deer, and calves and also described for the first time the presence of
worms in the liver of human beings.
During the second half of the 18th century, the intermediate phases in the life cycle of this parasite
were described. These observations were made by Johann Swammerdam (1758) while he was dissect-
ing a snail (Paludina vivipara), and he noted living organisms that evidently did not belong to the snail.
Otto Müller (1798) found microscopic living organisms similar to tadpoles in backwaters, and he called
them cercariae, but the life cycle of these worms remained a mystery, and nobody thought that more
than one host will be required for completing its life cycle. In 1803, Johan Zeder noted egg hatching
from different trematode species and also the release of a ciliated embryo in water. In 1807, Christian
Nitzsch described for the first time the encystment of cercariae. He noted that after a period of time,
cercariae stick on the surface of aquatic plants, they lose the tail, and then they were covered with a
gelatinous substance. Guido Wagener (1857) noted miracidia penetration into snail and subsequent
development of rediae. The first person who suggested that larval stages of liver worm were developed
in the G. truncatula snail was the German helminthologist David Weinlad (1875) when he noted cer-
cariae inside the snail. He also described for the first time the encystment of the cercariae on aquatic
plants and the subsequent digestion by sheep, and he argued that these cercariae were the juvenile
stages of liver worm. However, how the encysted cercariae reached the liver and bile ducts remained
unknown. In 1914, the Russian helminthologist Dimitry Sinitsin demonstrated, using rabbits as animal
models, that juvenile stages of worms, once released in the small intestine, penetrate the intestinal wall
and migrate to the liver by the peritoneal cavity. By then, it was clearly evident that F. hepatica involves
the snail as an intermediate host in its life cycle, resulting in the so-called liver-putrefaction disease,
now known as fasciolosis.1–5
During the past decades, the epidemiology of human fasciolosis has markedly changed, and the
number of reported cases has increased since 1990, and currently, it is considered as a reemerging
disease worldwide. The geographic distribution of the two parasitic species causing fasciolosis is
quite different. F. hepatica is cosmopolitan, while F. gigantica is mainly distributed in Africa and
Asia, although its presence has also been reported in some regions of Europe and Russia.6 Although
the European origin of F. hepatica is well accepted, the incidence of human fasciolosis has been
reported in 51 countries ­worldwide. It is estimated that more than 1 million people are currently
infected, and there are more than 90 million people living in high-risk areas of acquiring the disease.
The distribution of F. hepatica around the world has been mainly caused by cattle exportation from
Europe to other continents and also because F. hepatica has been able to easily adapt into other
mammalian hosts.
The ability of F. hepatica to successfully adapt into other mammalian hosts has been well
described in the literature. For example, F. hepatica has been well adapted to camelids in Africa
and the South of America, in some marsupials in Australia, and also in black rats, otters, and pigs,7–9
which increases fasciolosis transmission rates. F. hepatica has also been able to adapt to different
intermediate hosts. The main species responsible for F. hepatica transmission is the snail G. trun-
catula, with an important role in the distribution of fasciolosis around the world, as these snails
inhabit ponds or waters that are formed, for example, in the rainy season, and its presence has also
been detected in all continents. The snail responsible for the transmission of F. gigantica (Radix)
lives mainly in standing and deep waters rich in aquatic vegetation, and its presence is limited to
certain geographic areas.
The incidence of human fasciolosis could be underestimated, as not all cases are described in inter-
national reports, and they remain as internal reports, thesis, or publications in local journals, without
international diffusion. Also, fasciolosis is not a mandatory notifiable disease in some countries,10 and in
many instances, they are not diagnosed, as they remain asymptomatic.
Fasciola and Fasciolosis 719

Andean (Bolivia, Perú, Chile, and Ecuador), Caribbean (Cuba), north African (Egypt), western
European (Portugal, France, and Spain), and Caspian Sea (Iran) countries have the highest prevalence
of fasciolosis in the world. In Bolivia, the north plateau is the area most affected with human fasciolo-
sis,11 where the prevalence is higher than 72%, and in some individuals even about 5000 eggs have been
found in per gram of feces.12,13 High prevalence has also been noted in the Puno plateau, Cajamarca, and
Mantaro Valley (Perú).14,15

44.2 Life Cycle
An infected mammalian host releases F. hepatica eggs to the environment through the feces. They are
ovoid, are yellow-brown in color, and measure about 130–145 μm long and 70–90 μm wide. Development
of eggs and hatching are closely related to physical and chemical factors such as relative humidity, tem-
perature, oxygen concentration, and pH. It has been demonstrated that the optimal temperature for egg
development is 23°C–26°C; under these conditions, the eggs are embryonated in 2–3 weeks at pH 7.0. The
precise mechanisms involved in egg hatching still remain unknown. Miracidia (hatched from eggs), which
measure about 130 μm long and have great mobility due to their ciliated tegument, swim quickly to find the
intermediate host (snails). And this happens within 24 h; otherwise, they die. Several factors are involved
in the localization and penetration of miracidia into the snail, the most important being the presence of
stimulatory molecules in the snail mucose, such as glucose, amino acids, and lipids. Miracidia penetrate
into snail, lose cilia, and transform into sporocyst. Sporocysts are transformed into rediae, which in turn
transform into cercariae. It has been estimated that one miracidia can develop up to 600 cercariae, through
parthenogenetic proliferation. The cercariae have a propeller tail for swimming actively, which is subse-
quently lost before its encystment and posterior transformation into metacercariae, which attach to aquatic
plants. A gelatinous substance covers the metacercariae to protect from environmental conditions until
they are ingested by the mammalian host. The infection occurs after digesting metacercariae attached
on the surface of aquatic plants. Approximately 1 h after the ingestion of metacercariae, the excystment
occurs in the small intestine. After that, excysted metacercariae perforate the intestinal wall, migrate to
the peritoneal cavity, perforate the Glisson’s capsule, and penetrate into the hepatic parenchyma, reach-
ing the inside of the liver, causing hemorrhage and fibrosis. In this stage, the parasite attains maximum
growth. Finally, the parasites reach the bile ducts (7 weeks after the infection), and they remain here until
full maturity. Occasionally, juvenile parasites can also be found in rare anatomical locations such as lungs,
pancreas, lymph nodes, and thymus. Eight weeks after the infection, eggs can be found in the bile and later
in the feces, thus completing the entire life cycle. Adult worms of F. hepatica can remain alive in the liver
of the mammalian host even for several years and can also produce approximately 20,000 eggs daily.2–5,16
The main phases of the life cycle of F. hepatica are shown in Figure 44.1.

44.3 Genome, Transcriptome, and Proteome

The availability of the genome sequence of different pathogens, including some helminths, during the last
few years, has allowed a better and deeper understanding concerning relationships between host and patho-
gen as well as the identification of target molecules essential for host invasion, molecules involved in drug
resistance generation, and also molecules with potential as vaccine candidates to prevent the diseases.17–19
Recently, a draft of the F. hepatica genome has been published.20 This genome has a length of
approximately 1.3 Gb, being larger than other trematodes. F. hepatica genome is three times the size of
Schistosoma (363–397 Mb) or two times that of Clonorchis sinensis (547 Mb) and Opisthorchis viverrini
(634 Mb). There is no evidence of genome duplication or repeat expansion to explain the large genome.
It is possible that much of the noncoding gene is involved in regulation.
Through the investigation of the transcriptome of F. hepatica, more than 44,597 open reading frame
(ORF) sequences have been identified using a 454-Roche-based methodology, starting with RNA isolated
from adult worms.21,22 A subsequent annotation and sequence analysis using the Basic Local Alignment
Search Tool (BLAST) in search for both orthologous and paralogous proteins with the related trematodes
720 Laboratory Models for Foodborne Infections

(A) (B)

(C) (D)

FIGURE 44.1  The biology of F. hepatica. (A) Metacercariae. (B) Adolescariae. (C) Adult. (D) Eggs.

Schistosoma mansoni (http://schistodb.net/) and S. japonicum (http://schistodb.net/) was performed

together with a functional profile using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes
and Genomes (KEGG). A great similarity between F. hepatica and S. mansoni proteins was identified,
which was not only at the amino acid sequence level but also at the structural and functional level.
Nevertheless, data concerning the genome and transcriptome from F. hepatica still remain scarce, and
several studies have been performed to describe F. hepatica’s proteome in more detail using different
larval stages of its life cycle. Two-dimensional gel electrophoresis (2D-E) and mass spectrometry analysis
(MS) have been the two techniques used more widely for the characterization of F. hepatica proteome.
A proteomic analysis from the tegument of F. hepatica demonstrated high similarity with the S. mansoni
proteome, resulting in the identification of 229 proteins, classified as follows: cytoskeleton (18), defense
(12), metabolism (12), unknown (11), calcium-binding proteins (12), chaperones (7), proteases and inhibi-
tors (7), mitochondrion (3), secretion (2), and nuclear (1).23 A proteomic study from the surface proteins
using an ultra-high performance liquid chromatography (UPLC)-MS-based technique allowed the iden-
tification of 64 proteins, which are the fatty-acid-binding proteins (FABPs) found in high abundance, and
among them the FABP1 was the most abundant. Glutathione-S-transferase (GST) was also identified as a
high-abundance protein from the surface of the tegument and has been considered as an important vac-
cine candidate against F. hepatica. Juvenile flukes have also been subjected to proteomic characterization,
allowing the identification of a group of proteolytic proteins (cathepsins) (44%) together with proteins
associated with detoxification, energy, and metabolism (16.9%), such as enolase, thioredoxin, and malate
dehydrogenase.24 Cathepsins have also been identified as the most abundant proteins in the products of
excretion-secretion (ES) from F. hepatica, previously obtained by in vitro cell culture. This group of pro-
teins has a molecular weight close to 30 kDa and an isoelectric point between 5.0 and 6.0. Other proteins
such as enolase, GST, thioredoxin, and FABPs were also identified in the ES antigen.25
A special interest concerning cross-immunoprotective antigens between F. hepatica and S. mansoni
has been raised during the last few years. Using adult worms, a proteomics approach allows the identi-
fication of 28 cross-reactive proteins present in both parasites, 10 out of them being highly expressed in
S. mansoni, 9 out of them with high expression in F. hepatica, and the others with a similar expression
pattern. Cross-reactive proteins were classified as FABPS, cathepsins, thioredoxin, and GST.26

44.4 Host–Pathogen Interaction
After ingestion of the metacercariae, the newly excysted juveniles perforate the intestinal wall and
migrate through the hepatic parenchyma to reach the bile ducts. The induced damage to the mammalian
Fasciola and Fasciolosis 721

host during its migration is caused mainly by factors such as mechanical abrasion by its spicules or the
suction caused by their suckers. The hepatic parenchyma suffers a huge destruction with several internal
injuries and bleeding during parasite migration, which are caused not only by the juvenile flukes but also
by the inflammatory and immunological responses that are generated by the mammalian host. Peritonitis
can also be produced as a result of secondary bacterial infections.
Adult worms are located in the bile ducts, but they can also be found in the cystic duct, gall bladder,
Vater blister, and also in the choledochus. Bile ducts are highly dilated with hyperplasia and obstruction
of bile flow.27,28

44.4.1 Immunity
Although one of the biggest challenges in the study and understanding of the immunology of infections
caused by parasites is the vaccine development, the extreme importance of function and regulation of
different types of T-lymphocytes and cytokines they produce help us to gain a better understanding con-
cerning the immunological reactions between the host and the parasite and figure out the mechanisms
evolved by the parasites not only to successfully invade and establish the cells and tissues from the
mammalian host but also to evade the immune response from the host. Helminths are organisms that
can measure from few centimeters to even meters. This is the reason why they do not invade cells but
tissues, and the induced immune response by the mammalian host is also different from the common
immune response generated by other pathogens. Immunomodulation from the host immune response
becomes the most important factor by which helminths successfully survive for long periods of time in
the host. During helminthic infections, the cells of the innate immune system are activated, resulting in
the generation of different cellular phenotypes that promote the differentiation of Th2 cells with secre-
tion of IL-4, IL-5, and IL-13, which stimulate the production of IgG1, IgE, and IgA. At the same time,
the differentiation of Th1 cells that produce high levels of IFN-γ and are supposed to be highly protective
is suppressed.29–31
The chronic infection occurs when the helminths are well established in the mammalian host. At this
time, the Th2 phenotype still remains through the secretion of IL-4, but the T-regulator (Treg) phenotype
is generated with secretion of IL-10 together with other cell types that secrete TGF-β with the expression
of the cytotoxic T-lymphocyte antigen (CTLA).32 Treg cells also play an important role in the suppression
of Th1 cells and also regulate Th2 response that prevent tissue damage in the mammalian host caused
by migration and feeding of parasite.33 It has also been proposed that in helminthic infections, the mam-
malian host suppresses Th1 responses with destructive potential to the parasite and generates a Th2
response instead, which prevents tissue damage.29,34 However, not all the infections caused by helminths
induce the same pattern in the mammalian host.35 Thus, infection caused by Schistosoma spp. induces a
Th1 response at early stages (acute). Only when the helminth has established in the host and egg libera-
tion by adult worms occurs, Th1 response switches to Th2 as a consequence of immunomodulation by
the antigens from the egg parasites.36 A similar behavior has also been observed in the infection caused
by B. malayi using a murine model. Microfilariae induce a Th1 response in the acute phase, and once the
adult worm has been established in the host, a Th2 phenotype differentiation occurs.37
Once F. hepatica enters into the intestinal wall, a complex network of cellular and molecular interac-
tions between the parasite and different cell types from the host, such as dendritic cells, macrophages,
and mastocytes, occurs. The parasite secretes a great number of molecules, which induce both Th2 and
Treg nonprotective immune response in the host, and favors adaptation and survival of the parasite inside
the host. It has been estimated that one adult worm from F. hepatica can remain alive in the host for
2 years and for even 20 years in sheep.1,32
Using a murine model of fasciolosis, it has been reported that the first attack from the immune system
of the host to the parasite occurs during the next 4 h after the infection when the metacercariae have
excysted in the duodenum. It consists of the generation of immunoglobulins that attempt to perform an
effector mechanism, but the parasite releases cysteine proteases that are able to break these immuno-
globulins, thereby preventing its effector action.38–40 It has also been demonstrated that peritoneal mac-
rophages induce the expression of Th2 phenotype cells together with a decrease in the Th1 phenotype
at 24 h after infection. Simultaneously in this early stage of infection, the juvenile parasites also release
722 Laboratory Models for Foodborne Infections

immunomodulatory molecules, which have a direct effect on the function of the innate immune system
such as dendritic cells and macrophages allocated in the intestinal wall and peritoneal cavity.41,42
Once the infection has been well established, there is a secretion of Th2-type cytokines such as IL-4,
IL-5, and IL-13 by the splenocytes; in later stages of infection (3 weeks), there is also generation of Treg
cytokines IL-10 and TGF-β by macrophages and dendritic cells. It has also been demonstrated that in
the peritoneum, most of the T-CD4+ cells secrete only IL-10 without IL-4 or IFN-γ, thus inducing a
suppression of both Th1 and Th2 responses, which are ineffective against the parasite.43 Chronic fas-
ciolosis in cattle has a similar immune response pattern as that of the murine model of fasciolosis, with
predominance of Th2 and suppression of Th1 phenotype. Serological studies have demonstrated high
levels of IgG1 and little or no IgG2a,44–46 and the susceptibility to F. hepatica has been correlated with
an increase in both the IgG1/IgG2a and the IL-4/IFN-γ ratio.47 Owing to such immunoregulation/immu-
nosuppression, the host infected with F. hepatica becomes more susceptible to acquire other infections,
for example, in those infections where a Th1 response is necessary to prevent acquiring a disease. More
specifically, it has been observed that mice infected with F. hepatica and coinfected with Bordetella per-
tussis could not develop a strong Th1 immune response which in turn becomes protective.48–50 However,
not all coinfections with F. hepatica present the same behavior. For example, Toxoplasma gondii induces
a high Th1 response, with high levels of IFN-γ even in the presence of F. hepatica.51

44.4.2 Evasion Mechanisms
F. hepatica has developed several mechanisms to evade the immune response raised by the host. Anatomic
location of adult worms in the bile ducts can be considered as an evasion mechanism as it represents,
immunologically speaking, an inaccessible site. Furthermore, F. hepatica releases eggs and antigens
together with the bile fluid, which also protect them from the immune response of the host. The tegument
of F. hepatica also plays an important role in immune evasion, which is composed by the glycocalyx,
which changes its chemical composition during the phases of migration and maturation of the parasite.
In the adolescariae stage, the tegumental change occurs every 3 h, thus avoiding the contact and attack
of ligated antibody effector cells such as eosinophils and neutrophils. Juvenile flukes are highly resistant
to the action of the complement of the host, and it is supposed to be due to the presence of sialic acid in
surface glycoproteins, which activates the alternative pathway of complement. F. hepatica has mecha-
nisms to prevent the action of nitric oxide, free oxygen radicals, superoxide dismutase (SOD), glutathi-
one peroxidase, and GST. The parasite also secretes molecules that suppress or immunomodulate the
immune response from the host.27,28

44.5 Clinical Manifestations
44.5.1 Acute Phase
The acute or invasive phase from fasciolosis is caused by the migration of the worms through the perito-
neum and the hepatic parenchyma, causing mechanical damage and allergic and toxic reactions within
the first 2–4 months after infection. The more frequent clinical manifestations include fever, abdominal
pain, flatulence, nausea, diarrhea, constipation, urticaria, and respiratory symptoms such as cough, dys-
pnea, and chest pain. A physical exploration of patients with fasciolosis reveals hepatomegaly, ascites,
and jaundice, while analytical data reveal leukocytosis, eosinophilia, anemia, and high activity from
hepatic enzymes.27,28

44.5.2 Chronic Phase
The chronic phase from fasciolosis (also called obstructive phase) could occur within the next
months or years after the infection. Adult worms migrate and establish within the bile ducts, caus-
ing inflammation and hyperplasia of epithelium and also thickening and dilatation of bile ducts and
gallbladder, leading to cholangitis, cholecystitis, and gallbladder obstruction. Analytical data reveal
Fasciola and Fasciolosis 723

leukocytosis, eosinophilia, middle anemia, high activity from hepatic enzymes, hypoalbuminemia,
and hypergammaglobulinemia.27,28

44.6 Diagnosis
The diagnosis of human fasciolosis is difficult, as there are no specific symptoms and also because there
is no egg-parasite detection in the early phases of infection. Two methods are widely used for the diag-
nosis of F. hepatica: parasitological- and immunological-based. There are also other methods based on
the detection of parasite DNA, such as polymerase chain reaction (PCR), which are not commonly used
because of technical difficulties.

44.6.1 Parasitological Techniques
The detection of F. hepatica eggs in feces is performed through sedimentation techniques, and it rep-
resents the most common methodology used for the diagnosis of fasciolosis.27 The eggs released in the
feces have an ovoid morphology, normally with a green-yellow coloration (caused by the direct contact
with bile), and it is 60 μm in width and 130–150 μm in length. Parasitological techniques have several
advantages, as they are not time consuming and are economical. However, these methods cannot be used
in the acute phase of the disease, as eggs are released at 3–4 months after infection. In areas with a high
prevalence of animal fasciolosis, a false-positive detection could occur, which is caused by the ingestion
of bovine-liver-containing eggs from F. hepatica.52

44.6.2 Immunological Techniques
The antibodies produced against the antigens of F. hepatica have high sensitivity, and can be diagnosed
even in the acute phase of the disease. ELISA assay is used, with antigens from F. hepatica, mainly
excretory-secretory antigens,53,54 cathepsin L1,55,56 proteins from the tegument,57 saposin-like proteins,58
leucine aminopeptidase,59 and other recombinant antigens.60 Although there are several antigens for such
detection, cathepsins remain the main source of antigens for the detection of circulating antibodies.61
Monoclonal antibodies have also been used for the detection of antigens in sera samples and also for
the detection of coproantigens. Some examples of monoclonal antibodies currently used are ES78 and
MM3.62,63 A new lateral flow test was constructed with a recombinant cathepsin L1, using protein A and
MM3 monoclonal antibodies as detector reagents.64

44.6.3 Molecular Techniques
Although molecular techniques for the detection of parasitic DNA, such as PCR, have high specificity
and sensibility, because of technical difficulties in areas with high incidence, PCR is not commonly
used for the diagnosis of F. hepatica infection. Also, this is the only technique that identifies the caus-
ative organism both in the intermediate host and in the mammalian one. The molecular techniques
more widely used for the detection of fasciolosis are (1) conventional PCR,65,66 (2) real-time PCR,65,66
(3) LAMP (loop-mediated isothermal amplification),67 and (4) multiplex PCR.68,69

44.7 Treatment
To date, the number of chemotherapeutic agents for the successful treatment of fasciolosis is very scarce,
as most of them are effective only against adult worms and ineffective or partially effective against
immature worms. Triclabendazole (TBZ) is currently the drug of choice for the treatment of fasciolo-
sis, as it is highly active against both mature and immature worms. Furthermore, it is a safe compound
when administered and is well tolerated.70 The recommended dosage is 10 mg/kg/day for 2 consecutive
days, obtaining a curative rate between 92.2% and 93.9%. Adverse effects caused by TBZ are normally
724 Laboratory Models for Foodborne Infections

minimal, the more frequent being abdominal pain and sweating. In some cases, it can also induce
­nausea, vomiting, chills, cough, fever, and itching. In animals, the dose regimen may vary depending on
the infected species; thus, bovines receive a single dose of 12 mg/kg, whereas sheep and goats receive a
single dose of 10 mg/kg. For animals that are used for human consumption, such treatment should not be
applied during the 28 days prior to sacrifice. On the other hand, milk from treated animals should not be
consumed for the next 4 days after treatment.71
Structurally, TBZ belongs to the benzimidazole’s family, and its mechanism of action is associated
with the β-tubulin function, attaching to and hampering biological process associated with microtu-
bules.72 The resistance to TBZ was reported for the first time in Australia in the mid-1990s.73 Since then,
resistance to TBZ has also been reported in European countries such as Ireland, Scotland, Wales, Spain,
and Netherlands.74–76 The alternative treatment for the treatment of fasciolosis caused by TBZ-resistant
strains is based on the administration of artemisinin-derivative compounds.

44.8 Experimental Models in Fasciolosis

F. hepatica naturally affects grazing ruminants, including sheep, cows, lambs, and goats, and also affects
human beings. Currently, fasciolosis is considered as an emerging pathogen because of the high number
of human infections reported. Because of the difficulty in investigating the immunology, pathology, and
cellular and molecular mechanisms associated with fasciolosis in grazing ruminants, several experi-
mental models that mimic fasciolosis have been used for decades in the laboratory. The main species
currently used to study fasciolosis include mice, rats, and rabbits, although several studies have also
been performed in sheep. The use of mice, rats, and rabbits in scientific experimentation has been well
documented.

44.8.1 Fasciolosis in Mice
Mice have been used for a long time to study the immunology of the infection and are also used as an
experimental model to assess vaccine efficacy. Furthermore, the use of natural infection models to study
fasciolosis has been hampered because of technical issues such as cost, space, manipulation, and the
availability of specific reagents for immunological studies. The University of Salamanca’s Research
Center for Tropical Diseases (CIETUS) has been working for the last decade on the development of an
appropriate laboratory model to study the infection caused by F. hepatica, including both BALB/c and
CD1 mice. Thus, a hyperinfection model using lethal doses of F. hepatica metacercariae to study both
the immunopathology induced by the infection and the immunoprotective efficacy of several vaccine
candidates was described.77 The current approaches to study fasciolosis in mice are discussed in the fol-
lowing paragraphs.

44.8.1.1 Experimental Infection
Mice are orally infected with five to seven F. hepatica metacercariae, which are suspended in water
or phosphate buffered solution (PBS). This suspension is then administered to each mouse using an
intragastric gavage technique.78,79 Nonlethal dosage in mice is frequently used to study the dynamics
of the infection as mice remain alive during the whole study.80 Other authors have used a large number
of metacercariae to infect mice, including 10 and 15 metacercariae. In both cases, a human endpoint is
established at 3 weeks after experimental infection.49

44.8.1.2 Physicopathological Changes
It is well known that helminth-associated infections induce a strong Th2 immune response in the mam-
malian host.35 Thus, typically the success of the infection is investigated by measuring the level of anti-
bodies raised against the excretory-secretory antigens. It is worth noting that the hyperinfection in mice
with five to seven metacercariae induces death of animals between days 28 and 34.77,79
Fasciola and Fasciolosis 725

44.8.1.2.1 Measuring Antibody Levels

After experimental infection, mice are weekly bled to investigate the dynamics of antibody production.
Blood is recovered on heparin-containing microtubes, centrifuged, and the plasma is carefully removed
from the surface of the microtube. An ELISA technique is then used to investigate the levels of IgG,
IgG1, and IgG2a, according to previously established protocols.79 Normally, the levels of both IgG and
IgG1 increase as the infection occurs, and little or no IgG2a is detected.49,77

44.8.1.2.2 Hepatic Transaminases
Alterations in hepatic transaminase levels are often used to assess the course of infection, and such mea-
sures can be done either in the serum or in the liver of infected mice. Typically, aspartate transaminase
(AST), alanine transaminase (ALT), and γ-glutamyl transpeptidase are measured.

44.8.1.2.3 Eosinophilia Alteration during Fasciolosis

The infection caused by F. hepatica also induces changes in the number of eosinophils in peripheral
blood. It has been demonstrated that eosinophilia occurs immediately after infection, reaching its maxi-
mum level at 7 days postinfection, where it begins to decrease.81

44.8.1.2.4 Cytokines
To assess cellular immune responses induced by F. hepatica, the most representative Th1- and Th2-
Treg and Th17-associated cytokines are measured. For this purpose, infected mice are humanely
euthanized and necropsied for spleen recovery. Later, splenocytes are recovered, in vitro cultured,
and stimulated to quantify the levels of the aforementioned cytokines. The results clearly demon-
strate that F. hepatica induces a Th2-polarized immune response with high levels of IL-4 and IL-5
and little or no IFN-γ.49

44.8.1.2.5 Hepatic Damage
Measuring the hepatic damage induced after infection also assesses the success of the infection by
F. hepatica. At the time of necropsy, the liver is isolated and macroscopically evaluated considering the
size, consistency, color, and dilatation of the bile ducts and repletion of the vessels, and scored as intense
(+++), mild (++), moderate, (+) or without lesions (−).77,79 There is no consensus in the quantitation of
hepatic damage, and most of the times authors use their own scale and parameters. It is worth noting
that not all of the mouse strains are equally susceptible/resistant to the infection with F. hepatica.49 An
example of liver lesions in healthy and infected mice is shown in Figure 44.2.

(A) (B)

FIGURE 44.2  The hepatic damage induced by the experimental infection of F. hepatica in mouse. (A) Liver of one
uninfected healthy mouse. (B) Liver of one mouse infected and necropsied at 21 days after infection. In this stage, bile
duct enlargement is observed together with scars on the hepatic surface and irregular yellowish white area on the liver and
parenchyma.
726 Laboratory Models for Foodborne Infections

44.8.1.2.6 Worm Burden
Although determining worm burden is very useful to evaluate the success of infection with F. hepatica,
the results of this approach are slightly inappropriate when using mouse as an experimental model, as
they could not harbor too many adult worms in the liver, thereby leading to inconclusive statistical infer-
ence. Furthermore, when using lethal doses, mice die before worms reach the liver and bile ducts.

44.8.1.3 Microarray Analysis
Recently, we have investigated the gene expression profile during F. hepatica infection at 7 and 21 days
after experimental infection using a microarray-based methodology. Differential expression of genes occurs
as the infection progresses. For this purpose, we have isolated the RNA from the liver of infected mice at
7 and 21 days postinfection and investigated the gene expression profile compared to untreated mice. Using
functional profiling, we identified several upregulated key genes involved in the induction of hepatic injury.78

44.8.2 Fasciolosis in Rats
Rats have also been widely used to study not only the immunology of the infection but also the protective
efficacy of several vaccine candidates. Rats are more resistant to the infection, and typically they do not die,
thus allowing a more detailed study during advanced stages of the infection. Moreover, previous reports have
demonstrated the important role of black rats, Rattus rattus, in the epidemiology of fasciolosis, mainly in
the Mediterranean island of Corsica. It has been demonstrated that these rats become naturally infected with
the trematode F. hepatica. Although human infections of fasciolosis have also been reported in Corsica, the
prevalence of the disease remains low.82 Thus, the investigation of the immune response and the assessment
of vaccines in rats have attracted interest, and several studies have been developed since then.

44.8.2.1 Experimental Infection
Rats are orally infected with the metacercariae of F. hepatica using the intragastric gavage technique.
However, there is no consensus regarding the number of metacercariae that are needed for a successful
infection, and this decision depends almost exclusively on each researcher’s criteria and experience.83–85

44.8.2.2 Physicopathological Changes
44.8.2.2.1 Antibody Response
Using rats as laboratory experimental model allows a more compressive study of the dynamics of anti-
body production during infection with F. hepatica for a long period of time. Typically, IgG, IgG1, IgG2a,
IgG2b, and IgG2c are measured during infection in rats by the ELISA technique using sera or plasma
samples. IgG antibodies are detected from 1 week after experimental infection, and an increase occurs
during the following 5 weeks. From 7 to 10 weeks after infection, IgG remains unchanged.86 IgG1
and IgG2a are also detected from the first week after the infection, and this increases during the next
10 weeks, with similar patterns. High levels of IgM are induced by 2 weeks after infection, which slowly
decreases as the infection occurs. However, high levels still remain by 10 weeks after infection. IgE has
a biphasic behavior, with peaks being detected at 5 and 9 weeks after infection.87

44.8.2.2.2 Cytokines
Most of the experiments aimed at the study of the production of cytokines after the experimental infec-
tion with F. hepatica are performed in the spleen cells at different times of infection. An increase in the
percentage of T-CD4 cells after the experimental infection is observed while the level of T-CD8 cells
remains unchanged. It has also been demonstrated that the amount of cells producing IFN-γ remains
unchanged during the first week after the infection, but they suffer a decrease at 2 weeks after the infec-
tion,84 thus demonstrating a downregulation of the Th1 phenotype as the infection moves along. This
behavior has also been reported in mice infections.49 An increase in the level of both IL-4 and IL-10 is
observed at 1 and 2 weeks after the infection.83,84
Fasciola and Fasciolosis 727

44.8.2.2.3 Reactive Oxygen Species

It is well known that the infection of rats with F. hepatica is characterized by the formation of reac-
tive oxygen species (antioxidants). Thus, the study of both enzymatic and nonenzymatic antioxidants
in the serum of infected rats is frequently performed to study the time-course of the infection of
fasciolosis.88

44.8.2.2.4 Worm Burden
Using rats as an experimental model in the study of fasciolosis allows a more accurate investigation of
worm burden than using mice. From third week after infection until the seventh week, flukes are har-
bored in the hepatic parenchyma. Later, they continue migrating and reach the bile ducts at week 11,
where they remain for a long period of time.89

44.8.2.2.5 Hepatic Damage
As in mice, the hepatic damage induced by experimental infection is assessed considering the size,
consistency, color, dilatation of the bile ducts, and repletion of the vessels, and scored as intense (+++),
mild (++), moderate (+), or without lesions (−).77 However, the pathology in the liver has previously been
described from the first week up to 12 weeks after infection.90

44.8.3 Fasciolosis in Rabbits
The use of rabbits as an experimental model to study the immunology of the infection caused by
F.  hepatica has been very limited mainly because immunological assays for this species remain unavail-
able. However, rabbits have been widely used for the production of monoclonal antibodies (as large
quantities of sera could be obtained) and also for the evaluation of the protective efficacy of vaccine
candidates. Currently, there is only one report concerning the cytokine profile in rabbits immunized with
a saposin-like (SAP2) recombinant protein by means of quantification of mRNA by real-time PCR.91
As in rats, rabbits could harbor a large number of adult worms in the liver, making them suitable to
statistically assess worm burden.

44.8.3.1 Experimental Infection
To perform the experimental infection, rabbits are orally infected with the metacercariae of F. hepatica
suspended in either distilled water or PBS. As in the rat model, the number of metacercariae required for
the infection is variable and depends on the researcher’s criteria and experience. Because a large number
of metacercariae are commonly used to infect rabbits, the administration protocol could also be differ-
ent. In some cases, the metacercariae of F. hepatica are contained within a gelatin capsule, thus avoiding
the count of the metacercariae. The number of metacercariae required to infect rabbits is usually in the
range of 25 to 50.91–95

44.8.3.2 Physicopathological Changes
44.8.3.2.1 Antibody Response
After experimental infection, rabbits are weekly bled to investigate the dynamics of antibodies raised
against the parasite. ELISA technique is used to measure antibodies. From the third week after experi-
mental infection, IgG is detectable, reaching a maximum peak at sixth week after infection. From week
6 onward, antibody levels remain unchanged until 10th week.91

44.8.3.2.2 Worm Burden
Rabbits are humanely slaughtered between 12 and 20 weeks after experimental infection, and the flukes
are isolated from bile ducts. Both immature and mature flukes are isolated from the liver. For this pur-
pose, livers are cut, soaked in water or PBS at 37°C, squeezed, and passed through 300-μm mesh sieve,
according to previously described methodologies.91,92
728 Laboratory Models for Foodborne Infections

44.8.3.2.3 Hepatic Damage
At necropsy, independent, experienced pathologists assess hepatic damage. They macroscopically
­examine the livers and assign a score according to the intensity of the damage. There is no consensus
regarding the scale used in the quantitation of hepatic damage, but researchers have their own scales,
which are applied independently. Typically, hepatic damage is classified as mild, moderate, and intense.96

44.9 Experimental Assessment of Vaccine Candidates

During the last decade, we performed several studies, in which we assessed the immunoprotective
efficacy of different vaccine candidates. The FABPs and cathepsins from F. hepatica have been
considered many times as promissory vaccine candidates.97 The immunoprotective efficacy from
the so-called Fh12, a F. hepatica FABP-derived protein weighing 12 kDa and obtained in its native
form, was assessed in mice and sheep using the ADAD vaccination system. Results in mice showed a
survival rate of 40% compared to the control group, whereas immunized sheep displayed lower fluke
recovery (24.5%), a significant reduction in the number of eggs in the bile (58.1%) and feces (40.3%)
compared to control groups.98 Later, we evaluated the efficacy of the so-called Fh15, another FABP
from F. hepatica having a molecular weight of 15 kDa, in mice and sheep as experimental models.
Here, we demonstrated a survival rate of approximately 50% in mice, with low titers of IgG1. In
sheep, a significant reduction in worm burden was achieved (43%) with less hepatic damage than in
unimmunized control sheep.99
By means of a bioinformatics approach, we have identified two T-cell-containing epitopes derived
from the amino acid sequence from the recombinant Fh15 protein (termed IKMVSSLKTKIT and
VKAVTTLLKA) and produced as recombinant-GST-linked proteins. Its protective efficacy was then
assessed in mice and rabbits, demonstrating that those peptides induced survival rates of 48.2% and
59.1% in mice, respectively. In rabbits, the immunization procedure induced a reduction in worm burden
(46%), showing its potential as vaccine candidates.100
Other studies with the Fh12 protein in mice and sheep were performed by introducing a lipidic
­aminoalcohol as immunomodulator into the ADAD vaccination system. The mice showed survival rates
ranging from 40% to 50%, while vaccination in sheep induced lower fluke recovery (42%), lower adult
worm counts (57%), lower fecal egg count (38%), and less hepatic damage.101 In another study with the
recombinant Fh15 protein, we have demonstrated that the protocol of immunization with the ADAD
vaccination system improves the survival rates in a murine model of mice. The immunization procedure
induced high levels of IgG2a and IFN-γ, survival rates of about 50%, and less hepatic damage.16
More recently, we have used a bioinformatics approach to select vaccine candidates against
F. hepatica. From the F. hepatica available protein sequences in databases, we selected those with
the following criteria: (1) proteins having a signal peptide sequence according to the SignalP4.0
server and (2) proteins with no transmembrane domain according to the TMHMM2.0 server. The
criteria highly fit to select those proteins that seem to be secreted are considered as important vac-
cine candidates. Once selected, the proteins are grouped into common families, and its amino acid
sequences are aligned using the BLAST. Then, we identified peptides containing B and T cell
epitopes within the conserved or semiconserved amino acid sequenced from proteins. The immune
response induced in mice by the immunization with the peptides containing B and T cell epitopes is
well characterized by means of the production of antibodies (IgG, IgG1, IgG2a, IgE, IgM), cytokines
(IFN-γ, TNF-α, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-13), and T cell populations (CD4, CD8, CD27,
CD62L, CD197). Those peptides inducing a high immune response in mice are then selected to
assess their immunoprotective efficacy in mice. We have demonstrated that peptides containing B
and T cell epitopes are highly immunogenic, as high levels of IgG, IgG1, and IgG2 are reached, with
high levels of IFN-γ, IL-4, IL-17, and IL-10 together with increased CD62L T cell populations. Those
peptides also resulted in the induction of immunoprotection, as high levels of survival rates were
achieved.79 The success of the vaccination trials conducted by our group in different experimental
models of fasciolosis is summarized in Table 44.1.
 Fasciola and Fasciolosis
TABLE 44.1
Vaccination Trials Carried Out by Our Group to Assess the Immunoprotective Efficacy of Vaccine Candidates against Experimental Infection with F. hepatica
Immunization Infection Protection Assessment
Experimental
Model Antigen Vaccine Dose (μg) mc Survival Rate (%) Worm Burden Reduction (%) Hepatic Damage Reduction (%) References
Mice B, T epitopes Synthetic 10 5 33–67 ND 22–33 79
Fh15 Recombinant 20 5 11–50 NS 14–44 16
Fh12 Native 20 5 40–50 ND ND 101
Fh15 Recombinant peptides 20 5 12–43 ND ND 100
Fh15 Recombinant 20 5 40–63 ND NS 99
Fh12 Native 20 5 10–42 NS ND 98
Rabbit Fh15 Recombinant peptides 50 20 ND 48–59 NS 100
Fh15 Peptides 50 20 ND 5–46 NS 100
Sheep Fh12 Native 150 100 ND 42–57 25–42 101
Fh15 Recombinant 150 100 ND 24–43 NS 99
Fh12 Native 150 100 ND 24–34 NS 98
mc, metacercariae; ND, not detected; NS, not significant.

729
730 Laboratory Models for Foodborne Infections

44.10 Conclusion and Future Perspective

Fasciolosis remains a neglected tropical disease, with an increasing reemergence in many parts of the
world. It has been estimated that 17 × 106 people are currently infected, with most of the cases being
reported in Southeast Asia, the Middle East, Africa, and some countries in the South of America, and
more than 170 × 106 people are in risk of acquiring the disease. Moreover, fasciolosis caused by F. hepatica
also represents a serious veterinary problem causing economic losses of more than 3 × 109 dollars per year.
Despite the availability of an effective treatment for F. hepatica infection, issues related to the
emergence of resistant strains and the toxicity and side effects induced by the current treatment have
decreased its efficacy. Although the immunology and the immune response induced in the infected
mammalian host have been well described, little is known about the precise molecular mechanisms dur-
ing F. hepatica infection and establishment in the host. Recently, a draft of the genome of F. hepatica has
been published, which represents a valuable source of information to explore issues related to coding and
regulatory genes and its implication in the success of the infection. It is also important to apply new tech-
nologies such as microarray and RNA-seq-based methodologies in experimental models of fasciolosis to
improve the measurement and quantitation of both immunological and pathological key markers and to
gain a better understanding of mechanisms involved during the infection, migration, and establishment
of the parasite in the mammalian host.
Currently, there is no effective vaccine for the prevention of fasciolosis, and for this reason, use of
experimental models to investigate naturally infected hosts is indispensable for vaccine development.
Existing data indicate that experimental models are valuable not only to study the immunology of
the infection, but also to assess the immunoprotective efficacy of several vaccine candidates against
F. hepatica. Compared to the natural models of infection, experimental models have several obvious
advantages, especially in the areas of material availability and technical simplicity.

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45
Haplorchis

Dongyou Liu

CONTENTS
45.1 Introduction................................................................................................................................... 735
45.1.1 Classification and Morphology......................................................................................... 736
45.1.1.1 Classification..................................................................................................... 736
45.1.1.2 Morphology....................................................................................................... 736
45.1.2 Life Cycle and Epidemiology........................................................................................... 737
45.1.3 Clinical Features.............................................................................................................. 737
45.1.4 Diagnosis.......................................................................................................................... 738
45.1.5 Treatment and Prevention................................................................................................. 738
45.2 Laboratory Models........................................................................................................................ 738
45.2.1 Animal Models................................................................................................................. 738
45.2.1.1 Rodents............................................................................................................. 738
45.2.1.2 Dogs.................................................................................................................. 739
45.2.1.3 Foxes................................................................................................................. 739
45.2.1.4 Chicks............................................................................................................... 739
45.2.2 In Vitro Models................................................................................................................. 739
45.3 Conclusion..................................................................................................................................... 739
References............................................................................................................................................... 739

45.1 Introduction
The genus Haplorchis contains several small intestinal flukes that require mollusks (first intermediate
host), fish (second intermediate host), humans, other mammals, or birds (definitive hosts) for complet-
ing life cycles. In comparison to the liver flukes Opisthorchis viverrini and Clonorchis sinensis, which
are well known and extensively investigated (as shown by 1320 and 1166 entries in recent PubMed
searches using the terms Opisthorchis and Clonorchis, respectively), Haplorchis species are relatively
understudied (as shown by 149 entries in PubMed search using the term Haplorchis). Nevertheless,
this does not make Haplorchis a less significant pathogen. In fact, intestinal flukes Haplorchis tai-
chui and Metagonimus yokogawai (see Chapter 46) of the family Heterophyidae are listed by WHO
as foodborne trematodes of medical importance and public health concern in Asia, along with liver
flukes O. viverrini (see Chapter 47) and C. sinensis (see Chapter 43) of the family Opisthorchiidae.
Given the medical prominence of H. taichui, this chapter will focus on the genus Haplorchis, despite
the presence of several other human-infecting genera (e.g., Heterophyes, Centrocestus, Pygidiopsis,
Stellantchasmus, and Procerovum) in the family Heterophyidae.

735
736 Laboratory Models for Foodborne Infections

45.1.1 Classification and Morphology

45.1.1.1 Classification
The family Heterophyidae encompasses a large group of intestinal flukes that are classified in the
superfamily Opisthorchioidea, suborder Opisthorchiata, order Opisthorchiida, subclass Digenea, class
Trematoda, phylum Platyhelminthes, kingdom Animalia.
Of the three families (Opisthorchiidae, Heterophyidae, and Cryptogonimidae) within the superfamily
Opisthorchioidea, the families Opisthorchiidae and Heterophyidae include many human pathogenic
flukes such as O. viverrini, O. felineus, C. sinensis, Haplorchis spp., and Metagonimus spp. Recent
molecular phylogenetic analyses indicated that the families Opisthorchiidae and Heterophyidae share a
paraphyletic relationship, with the former nested within the latter [1].
The family Heterophyidae is composed of at least 30 genera, including Adleriella, Apophalloides,
Apophallus, Ascocotyle, Centrocestus, Cercarioides, Cryptocotyle, Euhaplorchis, Euryhelmis, Galacto­
somum, Haplorchis, Heterophyes, Heterophyopsis, Leighia, Metagonimoides, Metagonimus, Neostictodora,
Phagicola, Phocitrema, Phocitremoides, Pholeter, Pricetrema, Procerovum, Pseudasco­cotyle,
Pseudogalactosoma, Pygidiopsis, Pygidiopsoides, Scaphanocephalus, Stellantchasmas, and Stictodora [2,3].
To date, more than 20 species have been identified in the genus Haplorchis, with 5 implicated in human
infections: Haplorchis pleurolophocerca, H. pumilio (synonym: Monorchotrema taihokui), H. taichui
(synonyms: M. taichui, M. microrchia, H. microrchis), H. vanissimus, and H. yokogawai (synonym:
M. yokogawai) [2].

45.1.1.2 Morphology
Similar to other members of the family Heterophyidae (often referred to as heterophyids), Haplorchis
species are small flukes (of 0.3–1 mm in length and 0.14–0.2 mm in width), with tegument covered by
spines. In addition, they possess a small oral sucker (which is armed with spines) and a ventrogenital
sucker complex (also with spines). Other organs present in Haplorchis species include pharynx, simple
intestinal system, ceca, one testis (compared to two testes seen in some other heterophyids), and vitellaria
(both located in posterior part of the body); however, cirrus and bursa are absent.
Within the Haplorchis genus, H. pleurolophocerca adult worms (of 0.32–0.42 × 0.14–0.17 mm in size) are
characterized by having only one testis and a ventrogenital sucker complex armed with spines. Freshwater
snails Melanoides tuberculata and Cleopatra bulimoides are the first intermediate host (harboring cer-
cariae); freshwater fish Gambusia affinis is the second intermediate host (harboring metacercariae); and
cats are the natural definitive hosts. H. pleurolophocerca infection in humans has been reported in Egypt.
H. pumilio adult worms (of 0.45–0.89 × 0.2–0.4 in size) are characterized by the presence of only
one testis and a ventrogenital sucker complex armed with 27–39 (average 32) gonotyl and chitinous
spines. The freshwater snail Melania reiniana var. hitachiens appears to act as the first intermediate
host; freshwater fish belonging to the families Cyprinidae, Siluridae, and Cobitidae are the second
intermediate host; and dogs and cats serve as the natural definitive hosts. This parasite is present in the
Philippines, Thailand, Laos, Vietnam, South China, Taiwan, Malaysia, India, Sri Lanka, Iraq and Egypt;
and H. pumilio infection in humans was first documented in Thailand in 1983 [4].
H. taichui adult worms (of 0.47–0.64 × 0.18–0.22 mm in size) are characterized by the presence of only
one testis and 14–20 (av. 15) large chitinous, fan-shaped spines (hooklets) on the ventrogenital sucker
complex. Under scanning electron microscope (SEM), the whole body surface of newly excysted juvenile
H. taichui shows numerous transverse rows of scale-like spines and two types of sensory papillae (type I,
ciliated knob-like swellings and type II, round swellings of the tegument) [5]. The first intermediate hosts
are freshwater snails Melania obliquegranosa, Melania juncea, or M. tuberculata [6]; the second inter-
mediate hosts are freshwater fish, including Cyclocheilichthys repasson, Cyprinus auratus, Cyprinus
carpio, Gambusia affinis, Hampala dispar, Labiobarbus leptocheila, Puntius binotatus, Puntius bre-
vis, Puntius gonionotus, Puntius leicanthus, Puntius orphoides, Puntius palata, Pseudorasbora parva,
Rhodeus ocellatus, and Zacco platypus in addition to Raiamas guttatus, Mystacoleucus marginatus,
and Henichoryhnchus siamensis; and the natural definitive hosts are dogs, cats, and birds. This parasite
Haplorchis 737

is found in Taiwan, the Philippines, Bangladesh, India, Sri Lanka, Malaysia, Thailand, Laos, Vietnam,
South China, Iraq, Palestine and Egypt. The first human case of H. taichui infection was reported in the
Philippines [4,7].
H. vanissimus adult worms (of 0.38–0.51 × 0.25–0.51 mm in size) utilize freshwater fish as the second
intermediate host, and pelicans and wild mammals as the definitive hosts. Human infection with H.
vanissimus was described in the Philippines [4].
H. yokogawai adult worms (of 0.47–0.64 × 0.18–0.22 mm in size) are characterized by having
only one testis and numerous (uncountable) minute chitinous spines on the ventrogenital sucker com-
plex. The first intermediate host is freshwater snail (M. tuberculata or Stenomelania newcombi); the
second intermediate host is freshwater fish, belonging to Cyclocheilichthys armatus, Hampala dispar,
Labiobarbus leptocheila, Misgurnus sp., Mugil spp., Onychostoma elongatum, Ophiocephalus striatus,
and Puntius spp.; and the natural definitive hosts are dogs, cats, cattle, and other mammals. The fluke is
known to occur in the Philippines, South China, Malaysia, Indonesia, Thailand, Laos, Vietnam, India,
Australia, and Egypt; and human infection with H. yokogawai was first noted in the Philippines [4].
The complete mitochondrial genome of H. taichui measures 15,130 bp in length and contains 12
protein-coding genes, 2 ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs
(tRNAs). Similar to other trematodes, the atp8 gene is absent, although a single long noncoding region
is present between trnE and trnG in H. taichui mitochondrial genome. On the whole, H. taichui appears
to be more closely related to Opisthorchiidae than other trematode groups [8].

45.1.2 Life Cycle and Epidemiology

Haplorchis eggs (each containing a miracidium) produced by adult worms residing in the intestine of the
definitive hosts (e.g., humans, cats, dogs, foxes, wolves, and pelicans) are discharged into fresh or brack-
ish water, and subsequently taken up by the first intermediate host (snails). In the gut of snails, miracidia
(contained within eggs) evolve via three stages (sporocysts, rediae, and cercariae) to become cercariae,
which upon release by snails penetrate the epithelium of the second intermediate host (fish). Encysting in
the muscles (and sometimes in the head) [9], cercariae develop into metacercariae. Finally, infected fish
(harboring metacercariae) are consumed by humans and other fish-eating mammals and birds, in which
metacercariae burrow between the villi of the small intestine and mature into adult worms [10].
Due to the abundance of intermediate hosts (snails and fish) and the customs of eating raw, incom-
pletely cooked, smoked, or pickled fish, heterophyds such as Haplorchis spp. are prevalent in Asia
(including Korea, Japan, China, Thailand, Vietnam, Laos, the Philippines, Indonesia, and India),
Mediterranean countries, Egypt, Ukraine, Hawaii, and Brazil [9,11–14]. In the past 20 years, human
cases of heterophyd infections have been also reported in Canada and USA. It is estimated that about
50 million people are infected with heterophyds worldwide, and children appear to be more susceptible
to the infections than adults.
Using Kato-Katz fecal smear technique, Sohn et al. [15] confirmed the high prevalence of the
intestinal fluke H. taichui among humans and fish in Luang Prabang Province, Laos. The worm load
for H. taichui averaged 7691 per infected person, and 138 (67%) of 207 freshwater fish (17 species)
purchased in a market in Luang Prabang District harbored H. taichui metacercariae (average 520 meta-
cercariae per fish).

45.1.3 Clinical Features
With an incubation period of between 1 and 15 days, Haplorchis infections in humans are generally
asymptomatic or mild and transient in immunocompetent individuals. However, in heavily infected or
immunocompromised individuals, Haplorchis infestations may cause weakness, discomfort, abdominal
pain, diarrhea, loss of appetite, nausea, and vomiting [16].
In case that Haplorchis eggs released in the intestine by adult worms gain entry into the blood and
lymph vascular systems via mucosa, and migrate to the valves and myocardium, the heart, the brain, or
spinal cord, they may induce cardiac failure, neurological disorders, and occasional fatalities.
738 Laboratory Models for Foodborne Infections

Microscopic examination of the small intestine from patients with H. taichui infection may reveal
mucosal ulceration, mucosal and submucosal hemorrhages, fusion and shortening of villi, chronic
inflammation, and fibrosis of the submucosa [17].

45.1.4 Diagnosis
Diagnosis of Haplorchis infections has traditionally relied on microscopic observation of adult worms
or eggs in feces. This is sometimes problematic as adult worms may not always be present and because
Haplorchis eggs are indistinguishable from those of O. viverrini and C. sinensis [18].
The development of molecular methods based on nucleic acid amplification has offered new oppor-
tunities to improve the identification of Haplorchis adult worms, metacercariae, and eggs. By target-
ing ribosomal RNA gene and mitochondrial cytochrome c oxidase subunit I (mCOI) gene, polymerase
chain reaction (PCR) and its derivatives [e.g., PCR-restriction fragment length polymorphism (RFLP),
multiplex PCR, nested PCR, real-time PCR, and PCR pyrosequencing] have made rapid, sensitive, and
precise determination and phylogenetic analysis of heterophyid trematodes possible [19–23].
Thaenkham et al. [24] showed that using primers (COI-OV-Hap F&R primers) from mitochondrial
cytochrome c oxidase subunit I (COI) gene, two common fish-borne trematodes in Thailand, O. viverrini
and H. taichui, can be rapidly and precisely differentiated. In addition, Thaenkham et al. [25] described a
PCR-RFLP based on 28S ribosomal RNA gene and restriction enzyme MboII digestion for identification
of metacercaria of C. sinensis, O. viverrini, H. taichui, H. pumilio, and H. yogokawai.
Similarly, Sato et al. [19] designed primers from ITS 1 and ITS 2 regions in ribosomal DNA that
enabled amplification of 800, 820, 1250, and 930 bp products (ITS1) and 380, 390, 380, and 530 bp prod-
ucts (ITS2) from the eggs of O. viverrini, C. sinensis, H. pumilio, and H. taichui, respectively, allowing
their effective discrimination.
Furthermore, Wongsawad et al. [26,27] developed species-specific primers for H. taichui (Hapt_F
5′-GGCCAACGCAATCGTCATCC-3′ and Hapt_R1 5′-CTCTCGACCTCCTCTAGAAT-3′, which
yield a 170 bp PCR product) and O. viverrini (OpV-1F: 5′-AATCGGGCTGCATATTGACCGAT-3′ and
OpV-1R: 5′-CGGTGTTGCTTATTTTGCAGACAA-3′, which generate a 319 bp PCR product). Use of
these primers facilitated the specific confirmation of both parasites in fish and snail intermediate hosts.

45.1.5 Treatment and Prevention

Haplorchis infection in humans can be treated with praziquantel (75 mg/kg body weight) in three doses
of 25 mg/kg for 1 day [28]. Alternatively, a single dose of praziquantel (40 mg/kg) and pyrantel pamoate
(10 mg/kg) followed by purge with magnesium sulfate will eliminate adult worms.
Prevention of human Haplorchis infections requires conducting campaigns to educate people about
the dangers of eating raw fish, the proper way to prepare fish (e.g., skinning fish and removal of gills
before cooking), and to avoid eating raw, pickled, and smoked fish from endemic areas.

45.2 Laboratory Models
45.2.1 Animal Models
45.2.1.1 Rodents
Mice (Mus musculus) are a suitable experimental definitive host for adult H. taichui. Sukontason et al.
[29] noted that mice infected orally with 200 active H. taichui metacercaria developed mature adult
worms in a few days, with egg production as early as 3 days postinfection, and increasing daily to about
50–60 eggs. Kay et al. [30] also found mice highly susceptible to H. pumilio infection, with peak adult
worm recovery 7 days after oral inoculation with metacercariae.
Rats may be used as definitive host for H. taichui infection. Saenphet et al. [31] observed that
male rats orally fed with 300 H. taichui metacercariae produced adult worms as early as 3 days
postinfection.
Haplorchis 739

Syrian golden hamster (Mesocricetus auratus) is also capable of supporting growth of Haplorchis
parasites, producing adult worms in the intestine 14 days after being fed metacercariae via a stomach
tube.

45.2.1.2 Dogs
Nissen et al. [32] showed that dogs (3–6 months old) infected with 500 H. pumilio metacercariae yielded
adult flukes without clinical symptoms. The infection lasted for at least 2 months, and up to 2 eggs/g feces
were excreted. The predilection site of the flukes appeared to be the lower part of jejunum (93% of total
worm burden).

45.2.1.3 Foxes
Nissen et al. [33] reported that foxes orally infected with 2000 H. pumilio metacercariae displayed
anorexia at 12 days postinfection (which last for approximately a week), produced between 116 and 2070
adult flukes per animal mostly in the lower part of the jejunum, and excreted H. pumilio eggs (initially
about 16–20 with a maximum of 1443 ± 1176 eggs/g of feces).

45.2.1.4 Chicks
Kumchoo et al. [34] demonstrated the suitability of chicks (Gallus gallus domesticus) as definitive host
for infection with H. taichui, since chick infected orally with 200 H. taichui metacercariae generated
mature adult worms as early as days 3 postinfection. Kay et al. [30] also confirmed that chicks are useful
host for H. pumilio metacercariae infection, although mice appear to be significantly more susceptible
to infection than chicks.

45.2.2  In Vitro Models

Newly excysted metacercariae of H. taichui survived for 12–14 days in a candle jar at 37°C using RPMI
1640 with blood agar, with ovary and testes observed after 3 days of incubation [35].

45.3 Conclusion
The genus Haplorchis consists of more than 20 intestinal trematode species whose complex life cycles
involve two intermediate hosts (snail and fish) and a definitive host (mammals and birds). Of the five
Haplorchis species (H. pleurolophocerca, H. pumilio, H. taichui, H. vagabundi, and H. yokogawai)
implicated in human infections, H. taichui is particularly common and poses a major public health
threat in Asia, parts of Africa, and the Americas. In order to decipher the pathogenic mechanisms and
immunobiology of haplorchiasis, use of laboratory models is crucial. Fortunately, a number of laboratory
animals (e.g., mice, rats, hamsters, dogs, foxes, and chicks) have been shown to support the growth and
maturation of Haplorchis adult worms from metacercaria recovered from fish. Further experimentation
will undoubtedly contribute to an improved understanding of human haplorchiasis and to the develop-
ment of novel intervention strategies against Haplorchis parasites.

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distinct? Parasitol Int. 2012;61(1):90–3.
2. Pearson JC, OwYang CK. New species of Haplorchis from Southeast Asia, together with keys to the
Haplorchis-group of heterophyid trematodes of the region. Southeast Asian J Trop Med Public Health.
1982;13:35–60.
3. ITIS report. Available at http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_
value=57114. Accessed on February 12, 2016.
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4. Chai JY, Shin EH, Lee SH, Rim HJ. Foodborne intestinal flukes in Southeast Asia. Korean J Parasitol.
2009;47(Suppl):S69–102.
5. Sukontason KL, et al. Surface ultrastructure of excysted metacercariae of Haplorchis taichui
(Trematoda: Heterophyidae). Southeast Asian J Trop Med Public Health. 2000;31(4):747–54.
6. Chontananarth T, Wongsawad C. Prevalence of Haplorchis taichui in field-collected snails: a molecular
approach. Korean J Parasitol. 2010;48(4):343–6.
7. Sommerville C. The life-history of Haplorchis pumilio (Looss, 1896) from cultured tilapias. J Fish Dis.
1982;5:233–41.
8. Lee D, et al. Complete mitochondrial genome of Haplorchis taichui and comparative analysis with other
trematodes. Korean J Parasitol. 2013;51(6):719–26.
9. Kumchoo K, Wongsawad C, Chai JY, Vanittanakom P, Rojanapaibul A. High prevalence of Haplorchis
taichui metacercariae in cyprinoid fish from Chiang Mai Province, Thailand. Southeast Asian J Trop Med
Public Health. 2005;36(2):451–5.
10. Skov J, Kania PW, Dalsgaard A, Jorgensen TR, Buchmann K. Life cycle stages of heterophyid trema-
todes in Vietnamese freshwater fishes traced by molecular and morphometric methods. Vet Parasitol.
2009;160:66–75.
11. Nithikathkul C, Wongsawad C. Prevalence of Haplorchis taichui and Haplorchoides sp. meta-
cercariae in freshwater fish from water reservoirs, Chiang Mai, Thailand. Korean J Parasitol.
2008;46(2):109–12.
12. Chai JY, et al. Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a moun-
tainous area of Phongsaly Province, Lao PDR. Korean J Parasitol. 2010;48(4):339–42.
13. Wijit A, Morakote N, Klinchid J. High prevalence of haplorchiasis in Nan and Lampang provinces,
Thailand, proven by adult worm recovery from suspected opisthorchiasis cases. Korean J Parasitol.
2013;51(6):767–9.
14. Krailas D, Veeravechsukij N, Chuanprasit C, Boonmekam D, Namchote S. Prevalence of fish-borne trema-
todes of the family Heterophyidae at Pasak Cholasid Reservoir, Thailand. Acta Trop. 2016;156:79–86.
15. Sohn WM, et al. Prevalence of Haplorchis taichui among humans and fish in Luang Prabang
Province, Lao PDR. Acta Trop. 2014;136:74–80.
16. Watthanakulpanich D, et al. Haplorchis taichui as a possible etiologic agent of irritable bowel
syndrome-like symptoms. Korean J Parasitol. 2010;48:225–9.
17. Sukontason K, Unpunyo P, Sukontason KL, Piangjai S. Evidence of Haplorchis taichui infection as
pathogenic parasite: three case reports. Scand J Infect Dis. 2005;37:388–90.
18. Sato M, Sanguankiat S, Pubampen S, Kusolsuk T, Maipanich W, Waikagul J. Egg laying capacity of
Haplorchis taichui (Digenea: Heterophyidae) in humans. Korean J Parasitol. 2009;47:315–8.
19. Sato M, Thaenkham U, Dekumyoy P, Waikagul J. Discrimination of O. viverrini, C. sinensis,
H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta
Trop. 2009;109:81–3.
20. Sato M, et al. Copro-DNA diagnosis of Opisthorchis viverrini and Haplorchis taichui infection in
an endemic area of Lao PDR. Southeast Asian J Trop Med Public Health. 2010;41:28–35.
21. Dung DT, Hop NT, Thaenkham U, Waikagul J. Genetic differences among Vietnamese Haplorchis
taichui populations using the COI genetic marker. J Helminthol. 2013;87(1):66–70.
22. Chontananarth T, Wongsawad C, Chomdej S, Krailas D, Chai JY. Molecular phylogeny of trema-
todes in Family Heterophyidae based on mitochondrial cytochrome c oxidase subunit I (mCOI). Asian
Pac J Trop Med. 2014;7(6):446–50.
23. Tantrawatpan C, et al. Development of a PCR assay and pyrosequencing for identification of impor-
tant human fish-borne trematodes and its potential use for detection in fecal specimens. Parasites
Vectors. 2014;7:88.
24. Thaenkham U, Visetsuk K, Dung Do T, Waikagul J. Discrimination of Opisthorchis viverrini from
Haplorchis taichui using COI sequence marker. Acta Trop. 2007;103:26–32.
25. Thaenkham U, et al. Rapid and simple identification of human pathogenic heterophyid intestinal
fluke metacercariae by PCR-RFLP. Parasitol Int. 2011;60:503–6.
26. Wongsawad C, Wongsawad P, Chai JY, Anuntalabhochai S. Development of a HAT-RAPD marker
for the detection of minute intestinal fluke infection. Exp Parasitol. 2009;123(2):158–61.
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27. Wongsawad C, Wongsawad P. Opisthorchis viverrini and Haplorchis taichui: development of a

multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-
RAPD. Exp Parasitol. 2012;132:237–42.
28. Belizario VY Jr., de Leon WU, Bersabe MJ, Purnomo, Baird JK, Bangs MJ. A focus of human
infection by Haplorchis taichui (Trematoda: Heterophyidae) in the southern Philippines. J Parasitol.
2004;90(5):1165–9.
29. Sukontason K, Sukontason KL, Boonsriwong N, Chaithong U, Piangjai S, Choochote
W. Development of Haplorchis taichui (Trematoda: Heterophyidae) in Mus musculus mice. Southeast
Asian J Trop Med Public Health. 2001;32(Suppl 2):43–7.
30. Kay H, et al. Optimization of an experimental model for the recovery of adult Haplorchis pumilio
(Heterophyidae: Digenea). J Parasitol. 2009;95:629–33.
31. Saenphet S, Wongsawad C, Saenphet K, Rojanapaibul A, Vanittanakom P, Chai JY. Haplorchis
taichui: worm recovery rate and immune responses in infected rats (Rattus norvegicus). Exp Parasitol.
2008;120:175–9.
32. Nissen S, Nguyen LA, Dalsgaard A, Thamsborg SM, Johansen MV. Experimental infection with the
small intestinal trematode, Haplorchis pumilio, in young dogs. Vet Parasitol. 2013;191(1–2):138–42.
33. Nissen S, Thamsborg SM, Kania PW, Leifsson PS, Dalsgaard A, Johansen MV. Population dynam-
ics and host reactions in young foxes following experimental infection with the minute intestinal fluke,
Haplorchis pumilio. Parasites Vectors. 2013;6:4.
34. Kumchoo K, Wongsawad C, Chai JY, Vanittanakom P, Rojanapaibul A. Recovery and growth of
Haplorchis taichui (Trematoda: Heterophyidae) in chicks. Southeast Asian J Trop Med Public Health.
2003;34(4):718–22.
35. Chaithong U, Sukontason K, Boonsriwong N, Sukontason KL, Piangjai S. In vitro development
of Haplorchis taichui (Trematoda: Heterophyidae). Southeast Asian J Trop Med Public Health.
2001;32(Suppl 2):31–5.
46
Metagonimus

Jong-Yil Chai

CONTENTS
46.1 Introduction................................................................................................................................... 744
46.1.1 Classification and Morphology......................................................................................... 744
46.1.1.1 Metagonimus yokogawai Katsurada, 1912....................................................... 744
46.1.1.2 Metagonimus takahashii Suzuki, 1930.............................................................745
46.1.1.3 Metagonimus miyatai Saito, Chai, Kim, Lee, and Rim, 1997..........................745
46.1.1.4 Metagonimus minutus Katsuta, 1932................................................................ 746
46.1.1.5 Metagonimus katsuradai Izumi, 1935.............................................................. 746
46.1.1.6 Metagonimus otsurui Saito and Shimizu, 1968............................................... 746
46.1.1.7 Metagonimus hakubaensis Shimazu, 1999...................................................... 746
46.1.2 Biology and Life Cycle......................................................................................................747
46.1.3 Epidemiology and Distribution........................................................................................ 748
46.1.4 Pathogenesis and Clinical Features...................................................................................749
46.1.5 Diagnosis.......................................................................................................................... 750
46.1.6 Treatment and Control.......................................................................................................751
46.2 Laboratory Models.........................................................................................................................751
46.2.1 General Considerations.....................................................................................................751
46.2.2 Animal Models Used for Studies on Morphology and Taxonomy...................................752
46.2.2.1 Dogs...................................................................................................................752
46.2.2.2 Cats....................................................................................................................752
46.2.2.3 Hamsters............................................................................................................752
46.2.2.4 Mice and Rats....................................................................................................753
46.2.2.5 Rabbits, Guinea Pigs, and Other Mammals......................................................753
46.2.2.6 Ducks and Chickens...........................................................................................753
46.2.3 Animal Models Used for Studies on Worm Development and Host Susceptibility.........753
46.2.3.1 Dogs...................................................................................................................753
46.2.3.2 Cats................................................................................................................... 754
46.2.3.3 Hamsters........................................................................................................... 754
46.2.3.4 Mice and Rats................................................................................................... 754
46.2.3.5 Rabbits, Guinea Pigs, and Other Mammals......................................................755
46.2.3.6 Ducks and Chickens...........................................................................................755
46.2.4 Animal Models Used for Studies on Pathogenesis and Pathology...................................756
46.2.4.1 Dogs, Cats, Rats, and Mice................................................................................756
46.2.5 Animal Models Used for Studies on Immunity and Host–Parasite Interaction...............756
46.2.5.1 Dogs, Cats, Rats, Mice, and Hamsters..............................................................756
46.2.6 Animal Models Used for Studies on Chemotherapy and Control....................................758
46.2.6.1 Dogs and Rats....................................................................................................758

743
744 Laboratory Models for Foodborne Infections

46.2.7 I n Vitro Culture and Others...............................................................................................758

46.2.7.1 I n Vitro Culture..................................................................................................758
46.2.7.2 E  xperimental Infection of Fish..........................................................................758
46.3 Conclusion and Future Perspectives.............................................................................................758
References................................................................................................................................................759

46.1 Introduction
Twelve genera (29 species) of the family Heterophyidae are so far known to infect humans around the
world: Apophallus, Ascocotyle, Centrocestus, Cryptocotyle, Haplorchis, Heterophyes, Heterophyopsis,
Metagonimus, Procerovum, Pygidiopsis, Stellantchasmus, and Stictodora.1 Among them, Haplorchis,
Heterophyes, and Metagonimus are the three most important genera in public health significance. The
genus Metagonimus is distinct morphologically from Haplorchis and Heterophyes and also differs in its
life cycle and geographical distribution.1,2
The genus Metagonimus was established by F. Katsurada in 1912 with M. yokogawai as the type
species.3,4 Later, six more species of Metagonimus have been described in the literature: M. takahashii
Suzuki, 19305; M. minutus Katsuta, 19326; M. katsuradai Izumi, 19357; M. otsurui Saito and Shimizu,
19688; M. miyatai Saito et al., 19979; and M. hakubaensis Shimazu, 1999.10 From public health points
of view, M. yokogawai, M. takahashii, and M. miyatai are the three major species frequently causing
human infections.2,11 M. minutus was also listed as a human-infecting species12; however, no literature
background is available to verify it. With regard to M. katsuradai, an experimental human infection was
reported7; however, no natural human infections have been documented yet. M. otsurui was originally
described from experimental hamsters infected with metacercariae from freshwater fishes,8 and later,
natural infection of the Japanese water shrew was discovered13 but never from humans. M. hakubaensis
was first found from experimental rats that were fed the metacercariae in lampreys in Japan,10 but human
infection is yet unknown.
Human Metagonimus spp. infections have been found exclusively in the Far East, including the
Republic of Korea (=Korea), China, Japan, and the Far Eastern Russia.1,2,11 However, the life cycle of
M. yokogawai was also detected in Taiwan and eastern Europe. The potential for human infections with
other species of Metagonimus remains to be elucidated.
The pathogenicity of Metagonimus spp. to humans and animals has been reported to be generally mild.1
The habitat of the adult flukes in immunocompetent mice was confined to the mucosal layer, mainly
in  the intervillous space and crypt of the small intestine, causing mucosal inflammations, but did not
extend beyond the submucosa and underneath.14 However, in immunosuppressed mice, the adult flukes
could invade into a deeper layer of the submucosa where mesenteric vessels are available.15 This suggests
a potential deep invasion of Metagonimus flukes to elicit significant pathogenesis and pathology in the
human host. To verify this suggestion, experiments with proper laboratory animal models are strongly
needed. In this chapter, laboratory models so far used to study the life cycle, host–parasite relationships,
pathogenesis, immunity, and other related aspects of Metagonimus infection are briefly reviewed.

46.1.1 Classification and Morphology

The characteristic morphological features of Metagonimus spp. include a minute body (0.5–1.5 mm in
length), a small laterally deviated ventral sucker with no ventrogenital apparatus and no genital sucker,
a medially located ovary, and two testes located almost side by side or a little oblique near the posterior
extremity.2,3,16 A brief history of the discovery, taxonomic debates if any, and morphological characteris-
tics of each Metagonimus species are given as follows.

46.1.1.1  M
 etagonimus yokogawai Katsurada, 1912
(syn. Loxotrema ovatum Kobayashi, 1912; Metagonimus ovatus Yokogawa, 1913; Loossia romanica
Ciurea, 1915; Loossia parva Ciurea, 1915; Loossia dobrogensis Ciurea, 1915)16
Metagonimus 745

This species was originally reported from an experimental dog fed with metacercariae from the sweet-
fish (Plecoglossus altivelis) in 1912 from Taiwan and named as Heterophyes yokogawai.3,30 However, the
adult worm morphology was significantly different from Heterophyes, and subsequently it was renamed
as M. yokogawai in the same year.3,4 It is the most highly prevalent of all Metagonimus spp. and has a
most wide geographical distribution, including the Republic of Korea, Japan, China, Taiwan, Russia,
Ukraine, India, Romania, Serbia, Bulgaria, Israel, Egypt, the Balkan States, and Spain.2,3,12,17,18–22 Some
old literature on M. yokogawai were actually referring to M. takahashii or M. miyatai, and caution is
required when reviewing M. yokogawai sensu stricto.3
The characteristic morphologies of M. yokogawai include the presence of two testes, which are closely
adjacent to each other near the posterior end of the body.2,16 However, in M. miyatai and M. takahashii,
the two testes are more or less separated from each other (see below).2,3,23 Another differential character
of M. yokogawai is the distribution of vitelline follicles; they extend in lateral fields from the level of the
ovary down to the posterior end of the posterior testis, but not beyond the posterior testis.16 By contrast,
in M. takahashii, vitelline follicles are abundant from the level of the ovary down to the posterior testis
and usually extend beyond the posterior testis level.9,23 In M. miyatai, vitelline follicles distribute from
the level of the ovary down to the anterior level of the posterior testis; there are no vitellaria distribution
beyond the posterior testis.9,23 In addition, M. yokogawai has the uterine tubule, which does not overlap
or cross over the middle portion of the anterior testis, whereas M. takahashii and M. miyatai have the
uterine tubules that overlap the whole anterior testis.9,23
The adult flukes of M. yokogawai are slightly smaller (0.80–1.32 × 0.42–0.54 mm) than those of
M. takahashii (0.86–1.19 × 0.44–0.57 mm) and M. miyatai (1.00–1.30 × 0.46–0.63 mm).3,9 The eggs
of M. yokogawai are also smaller (0.026–0.030 × 0.015–0.018 mm) than those of M. takahashii
(0.030–0.036 × 0.017–0.020 mm) and M. miyatai (0.029–0.032 × 0.017–0.020 mm), although there are
some overlaps in the range.3,23 The adult flukes of M. minutus differ from those of M.  yokogawai,
M. takahashii, and M. miyatai by having a smaller body (0.46 × 0.28 mm) and smaller egg size (0.023 ×
0.013 mm).6,23 The adult flukes of M. katsuradai, M. otsurui, and M. hakubaensis differ from those of
M. yokogawai, M.  takahashii, M. miyatai, and M. minutus in that the formers have a larger ventral
sucker than the oral sucker.7,10,23

46.1.1.2  M etagonimus takahashii Suzuki, 1930

(syn. Metagonimus yokogawai var. takahashii Asada, 1934; Metagonimus yokogawai Koga type Koga,
1938)16
This species was first reported by S. Takahashi in 1929 from the small intestine of mice and dogs
fed with metacercariae encysted in various freshwater fish species in Japan.24 It was at that time called
a larger egg-type M. yokogawai because the worm was morphologically very similar to M. yokogawai,
except that its eggs were larger than those of M. yokogawai.24 However, in 1930, M. takahashii was
reported as a new species, accepting the larger egg size as a specific character.5 However, its validity was
questioned because the difference from M. yokogawai was minor, and thus, the name was compromised
as M. yokogawai var. takahashii.25 Later, however, the taxonomic validity of M. takahashii was strongly
supported by differential morphological characters of larval and adult stages and also by the different
host specificities of the two species at experimental infection with the cercariae.26,27
M. takahashii morphologically differs from M. yokogawai and M. miyatai in the position of the two
testes, the distribution of vitelline follicles, and the size of their eggs.23 The so-called Koga type of
Metagonimus encysting in the dace Tribolodon hakonensis28 is regarded a synonym of M. takahashii.3,16,29
The adults of M. takahashii differ from those of M. katsuradai, M. otsurui, and M. hakubaensis in that
the latter three species have a smaller ventral sucker than the oral sucker.10,23 M. minutus has a smaller
body and egg size compared to M. takahashii.10,23

46.1.1.3  M etagonimus miyatai Saito, Chai, Kim, Lee, and Rim, 1997
This species was first described by F. Katsurada9,30 (only by a figure drawing) together with
M. yokogawai in 1912 in Taiwan and then by I. Miyata in 1941 in Japan.31 However, its specific status
746 Laboratory Models for Foodborne Infections

was not acknowledged, and no special name was given until 1984 when S. Saito began to call it as the
“Metagonimus Miyata type.”9 It was in 1997 when the specific level of this fluke became acknowledged
and described as a new species, M. miyatai.9 The new species description was based on adult flukes col-
lected from dogs and hamsters experimentally fed with the metacercariae from the sweetfish, dace, com-
mon fat-minnow Morocco steindachneri, pale chub Zacco platypus, and dark chub Zacco temmincki in
Korea and Japan.3,9 Human infections have been reported from Korea and Japan.9,23
The specific morphological characters of M. miyatai include the two markedly separated testes from
each other, the posterior one being just touching the terminal end of the worm, its vitelline follicles
never distributing beyond the posterior testis, and the egg size, which is intermediate between those
of M. yokogawai and M. takahashi.2,9,16 M. miyatai is also genetically distinct from M. yokogawai and
M. takahashii.32–34 M. miyatai differs from M. minutus in its larger body and egg size.6,23 It also dif-
fers from M. katsuradai, M. otsurui, and M. hakubaensis in that the latter three species have a smaller
ventral sucker compared with the oral sucker, whereas in M. miyatai, the ventral sucker is larger than
its oral sucker.10,23

46.1.1.4  M
 etagonimus minutus Katsuta, 1932
This species was originally described in Taiwan in 1932 based on adult flukes from cats and mice experi-
mentally fed with the metacercariae in the brackish water mullet.6,16 It has body and egg sizes smaller
than those of M. yokogawai, M. takahashii, and M. miyatai.2,3 The body size of M. minutus is similar to
that of M. katsuradai, but its egg size is smaller than that of M. katsuradai.3,6,7 The relative size of the
oral and ventral suckers is also a characteristic feature; the oral sucker is bigger than the ventral sucker
in M. katsuradai, and the oral sucker is smaller than the ventral sucker in M. minutus.3,6,7 M. minutus is
listed as a human-infecting species; however, no relevant background literature is available.12,16

46.1.1.5  M etagonimus katsuradai Izumi, 1935

This species was first described in Japan based on adult flukes from experimental rats, mice, rabbits, dogs,
and cats fed with the metacercariae from freshwater fish, including Pseudorasbora parva, Zacco platy-
pus, and Tanakia lanceolata.7,16 The possibility of human infection was experimentally proved (by the
author himself and family).7 M. katsuradai differs morphologically from M. yokogawai, M. takahashii,
M. miyatai, and M. minutus in having a smaller ventral sucker than the oral sucker.2,7,16 It also differs
from M. otsurui in the position of the seminal receptacle—on the left side of the ovary in M. katsuradai
and on the right side of the ovary in M. otsurui.8 M. katsuradai is distinct from M. hakubaensis in that
the former has long ceca that enter the posttesticular region, whereas the latter has short ceca ending at
the mid-level of the posterior testis.10

46.1.1.6  M etagonimus otsurui Saito and Shimizu, 1968

This species was described in Japan in 1968 based on adult flukes from golden hamsters experimentally
fed with the metacercariae in freshwater fishes, including Tridentiger obscurus, Chaenogobius castanea,
and C. urotaenia in a lake in Ibaragi Prefecture.8 Later, this species was reported again from another
fish species, Tridentiger brevispinis, in Aomori Prefecture, Japan.35 M. otsurui has a smaller ventral
sucker compared to its oral sucker, which is similar to M. katsuradai and M. hakubaensis but differs
from M. yokogawai, M. takahashii, and M. minutus, which have a smaller oral sucker than their ventral
sucker.8 M. otsurui differs from M. katsuradai in that the seminal vesicle is at the left side of the ovary
in the latter, whereas it is at the right side of the ovary in the former.8 M. otsurui has long ceca, whereas
M. hakubaensis has short ceca.8,10

46.1.1.7  M
 etagonimus hakubaensis Shimazu, 1999
This species was described in Japan in 1999 based on adult flukes from experimental rats fed with the
metacercariae in a lamprey, Lethenteron reissneri, a fish species collected in Hakuba Village, Nagano
Metagonimus 747

Prefecture.10 M. hakubaensis differs from M. yokogawai, M. takahashii, M. miyatai, and M. minutus in

that the former has a larger oral sucker than its ventral sucker, whereas the later four species have a larger
ventral sucker than their oral sucker.8,10 The ceca of M. hakubaensis are short, but the ceca of M. otsurui
and M. katsuradai are long.8

46.1.2 Biology and Life Cycle

Three types of hosts are required to complete the life cycle of Metagonimus spp. The first one is the
snail intermediate host, in particular, freshwater or brackish water snails of the genus Semisulcospira or
other related genera.2 The second host is fish, including a wide variety of freshwater and brackish water
species.1,2 The definitive hosts include humans and animals, including mammals and birds.2
Semisulcospira coreana or Semisulcospira libertina has been reported to be the molluscan inter-
mediate host of M. yokogawai,36 and S. libertina, S. coreana, or Koreanomelania nodifila have been
recognized as the snail hosts for M. takahashii.26,36 The snail host for M. miyatai includes S. libertina,
S. dolorosa, or S. globus.37,38 M. katsuradai takes S. libertina39 and Juga tegulata40 as the molluscan
host in Japan and Russia, respectively. The snail host for M. hakubaensis is S. dolorosa,10 and that for
M. otsurui is S. libertina and S. reiniana.13 However, the molluscan host of M. minutus has not been
reported. Metagonimus spp. produce cercariae of the ophthalmo-pleuro-lophocercous type, which are
characterized by a pair of eyespots and a long slender tail without bifurcation but covered with membra-
nous fin-like structure.16 When they are shed in water, they swim freely and infect freshwater fish, i.e.,
the second intermediate host.4
The major fish host for M. yokogawai is the sweetfish, P. altivelis, in Korea and Japan.1–4 The dace
Tribolodon hokonensis or T. taczanowskii and perch Lateolabrax japonicus have also been reported to
be infected with M. yokogawai metacercariae.2,4,16,41 In comparison, the fish hosts for M. takahashii were
reported to be the crussian carp Carassius carassius, carp Cyprinus carpio, dace T. taczanowskii, and
perch L. japonicus.1,2,4 The metacercariae of M. miyatai were detected in Zacco platypus, Z. temminckii,
P. altivelis, T. hakonensis, T. taczanowskii, Opsariichthys bidens, Morocco steindachneri, and Phoxinus
lagowskii steindachneri.9,37 The fish host for M. minutus is the brackish water fish, M. cephalus,7 and that
of M. katsuradai are Tanakia lanceolata (formerly Acheilognathus lanceolata), T. limbata, T. rhombea,
T. moriokae, Pseudorasbora parva, Z. platypus, and Gnathopogon sp.3,4,7 With regard to M. otsurui, the
second host includes the freshwater fish, including Tridentiger obscurus, T. brevispinis, Chaenogobius
castanea, Chaenogobius urotaenia, and Rhinogobius flumineus.8,13,35 The fish host for M. hakubaensis
is a lamprey, Lethenteron reissneri.10 The cercariae of Metagonimus spp. attach to the skin or scale of
the fish host and penetrate into the dermis and then the muscles leaving their tail outside the body of the
fish.4 The metacercariae usually encyst in the fish muscle but rarely under the scale or in the fin.4,16,35
The metacercariae can persist in the fish muscle for at least 2.5 years and can be alive throughout the life
span of the fish host.3,4,16
The definitive hosts of Metagonimus spp. are fish-eating birds and mammals. With regard to
M. yokogawai, dogs,42 rats,43 cats,44 foxes,45 and kites (bird)45 have been known to be natural definitive
hosts. However, the significance of each animal host as the source of human infections has not been prop-
erly verified.2,4,16 Mice, rats, cats, dogs, gerbils, hamsters, and ducks are experimental definitive hosts
for M. yokogawai.2,46,47 The usefulness of these animal hosts as laboratory models for Metagonimus
spp. infection will be discussed later in this chapter. As for M. takahashii, pelicans, kites, and other
species of birds,48 and mice, rats, dogs, cats, and other mammals24,44,49–51 were reported to be the natural
and experimental definitive hosts. Natural definitive hosts for M. miyatai include the dog, red fox, rac-
coon dog, and black-eared kite.4,9 Mice, rats, hamsters, and dogs are experimental definitive hosts for
M. miyatai.9,29,37,38,52,53 For M. minutus, vertebrate animals (detailed animal species is unknown) were
reported to be natural definitive hosts,54 and rats and mice were used as experimental definitive hosts.6
With regard to M. katsuradai, dogs are the only reported natural definitive hosts,3 whereas its experimen-
tal definitive hosts include mice, white mice, rats, rabbits, puppies, kittens,7 and golden hamsters.54 As
for M. otsurui, the natural definitive host is Japanese water shrews,13 and the experimental hosts include
golden hamsters and rats.13,35 M. hakubaensis was experimentally infected to the hamster, rat, mouse,
dog, cat, chicken, and quail,10,55 and Japanese water shrews were proved to be a natural definitive host.56
748 Laboratory Models for Foodborne Infections

46.1.3 Epidemiology and Distribution

The principal mode of human M. yokogawai infection is ingestion of raw or improperly cooked flesh of
the fish host, notably the sweetfish (P. altivelis) and the dace (T. taczanowskii), in Far Eastern countries.16
Human infections are widely scattered along riverside areas, where local people traditionally eat these
raw fish.4,16
In the Republic of Korea, almost all large and small rivers and streams in eastern and southern
coastal areas are endemic foci of M. yokogawai.1,2,17 The Sumjin, Tamjin and Boseong Rivers, Geoje
Island, and Osip Stream (Gangwon-do) are hyperendemic areas with 20%–70% egg-positive rates of
the riparian residents.2,17,57,58 This high prevalence is persisting in many of these areas, although in
some areas the intensity of infection is slightly reduced.59 The national prevalence of heterophyid eggs
(mostly M. yokogawai) in 2012 was 0.26%; thus, the number of infected people in the Republic of Korea
is about 130,000.60
With regard to the infection status of sweetfish, a survey undertaken in 2011 revealed that 88 (60.7%) of
145 sweetfish from 10 streams of eastern and southern coastal areas were found infected, with the average
number of metacercariae per fish of 61.61 Four (9.8%) of 41 stray cats, a natural definitive host, purchased
from a market in Seoul had M. yokogawai infection,44 whereas 78 (17.8%) of 438 cats from a market in
Busan were found infected with M. yokogawai (under the name Metagonimus sp.),62 and 61 (30.0%) of 188
stray cats caught near the five large rivers in Korea were positive for Metagonimus spp. worms.63 Among
170 wild rats captured from various localities in Korea, 4 (2.4%) were infected with M. yokogawai.43
Interestingly, M. yokogawai eggs were detected from Korean mummies of the 17th century, Joseon
Dynasty.64,65 It indicates that the life cycle of M. yokogawai was actively maintained at about 400 years
ago in Korea.64 The area where the mummy was found64 is now a well-known high-endemic area of
M. yokogawai infection.4,17
In Japan, a lot of epidemiological studies have been performed since 1912 when M. yokogawai was
first described.3 It was thought that the infection was distributed nationwide, except in Hokkaido.3 Later,
however, its presence in Hokkaido was also confirmed.45 Until the 1960s, the reported prevalence in
Japanese people had been 0.5%–35.1% depending on the locality.3 However, in some areas, for example,
along the Takatsu River, Shimane Prefecture, the prevalence in residents was high up to 71.9% in 1965,66
and in Akita Prefecture, two areas surveyed in 1972 revealed 8.5% and 45.9% egg prevalence among the
residents, respectively.67 In addition, an epidemiological survey was performed around the Hamamatsu
Lake, Shizuoka Prefecture, during 1982–1988, in which a 13.2% egg-positive rate among 4524 lakeside
people examined was found infected.68 Thereafter, it seems that the prevalence has been decreasing
steadily in Japan.4
In 1966–1967, 11 prefectures, including Gifu, Kyoto, Hyogo, Okayama, Shimane, Yamaguti, Ehime,
Kochi, Miyazaki, Kumamoto, and Kagoshima, were reported as high-endemic areas of metagonimiasis
with heavy metacercarial burdens in the fish host (>1000 metacercariae per sweetfish).69 Recently, in
2006, the prevalence of metacercariae ranging from 0% to 100% was observed in sweetfish caught from
18 small rivers in Shizuoka Prefecture, with variable numbers of metacercariae detected per fish.70
In Taiwan, where M. yokogawai was originally described as a new species from the sweetfish
P. altivelis,3 surprisingly few investigations have been conducted on adult fluke infections in humans
and animals.4 Studies on larval infections are also scarce.46,47 Freshwater fish species, including
Z. platypus, Varicorhinus barbatulus, Distoechodon turmirostris, Hemibarbus labeo, Sinibrama mac-
rops, Opsariichthys pachycephalus, and Acrosscheilus formosanus, were added as the fish intermediate
hosts in Taiwan.46,47
In mainland China, little information is available on human M. yokogawai infection, although it
was briefly mentioned that the infection exists in Guangdong, Anhui, Hubei, and Zhejiang Province.12
However, it is of interest to note that eight human infections were diagnosed by genetic analysis of fecal
eggs in Guangxi Province in 2012.71 In Heilongjiang Province, 6.2% of 178 farm dogs were found to har-
bor the adult flukes (2–7850 specimens per dog).72 In addition, in Yangshuo County of Guangxi Province,
18 of 31 fish species examined, including Z. platypus and Hemibarbus maculatus, were infected with
M. yokogawai metacercariae.73 Meanwhile, in Yunnan Province, the cercariae of M. yokogawai were
detected from S. libertina snails.4,74
Metagonimus 749

In Russia, human M. yokogawai infection is highly endemic in the Amur and Ussuri valleys of the
Khabarovsk Territory.12 The prevalence among the ethnic minority was reported between 20% and
70%.12 In the north of Sakhalin Island, the infection rate was 1.5% among Russians and 10% among eth-
nic minorities, and sporadic cases were also reported in the Amur district and the Primorye Territory.12,16
The population at risk is estimated to be about 859,000, which is 14.7% of the total population in these
territories.12
In eastern Europe, its existence in fish hosts and wild animals has been reported in Ukraine,21
Serbia,18,22 Bulgaria,19,75 and Czech Republic,20 although no human infections have been confirmed so far.
Regarding M. takahashii infection, in the Republic of Korea, the adult flukes were first recovered from
rabbits experimentally fed with the metacercariae in 1960.76 Thereafter, the presence of human infec-
tions was first demonstrated from riparian people along the Hongcheon River, Gangwon-do, in 1988.77
Subsequently, in 1993, an endemic focus was discovered from Umsong-gun, Chunchungnam-do, along
the upper reaches of the Namhan River.23 The riparian people along this river were mixed-infected with
M. miyatai, with an egg-positive rate of 9.7% for both species.23 A year later, in 1994, M. takahashii adult
flukes (8–402 in number) were recovered in six residents living in Kochang-gun, Gyeongsangnam-do.78
The major sources of human infections were the crucian carp C. carassius.26 The perch, L. japonicus,
was also identified as the fish host in Gyeongsangnam-do.79
In Japan, several articles were published on M. takahashii after this species was first recorded in 1930.3,4
However, before the studies of S. Saito in the 1970s–1990s,13,35,36 its precise epidemiological character-
istics, including the prevalence and geographical distribution of human infections, have not been clearly
defined. However, it is worthwhile to refer to Takahashi,24 who originally discovered M.  takahashii;
in 1929 in Okayama City, 43 (0.64%) of 6680 residents were infected with M. takahashii, whereas 54
(0.81%) were infected with M. yokogawai.3 Later, in 1957 in Fuchu City, Hiroshima Prefecture, 11 (4.8%)
of 231 residents examined were infected with M. takahashii, whereas 81 (35.1%) were infected with
M.  yokogawai.3 Quite recently, in 2003, Semisulcospira snails collected around the Lake Biwa were
found infected with the cercariae of M. takahashii, together with those of M. yokogawai, M. hakubaensis,
M. otsurui, and M. katsuradai.80
In the Republic of Korea, before M. miyatai was designated as a distinct species, its human infec-
tions were noticed by detecting eggs in the feces, which were slightly larger in size than those of
M. yokogawai.52 Later, adult flukes (under the name Metagonimus Miyata type) were recovered from
32  riverside people living along the Namhan River in Umsong-gun (those people were concurrently
infected with M. takahashii) and Yongwol-gun (infected only with M. miyatai).23 In Yongwol-gun, the
egg-positive rate of M. miyatai among 77 riparian residents was 48.1%.23 Adult flukes were also recov-
ered from residents along the Hantan River, Chorwon-gun81 and Talchon River, Chungwon-gun.82
In Japan, epidemiological studies, particularly on human infections, are scarce. Saito et al.9 enlisted
dogs, foxes, raccoon dogs, and black-eared kites for animal definitive hosts in Shimane, Kochi, and
Yamagata Prefectures. In Hiroi River basin, Nagano Prefecture, cercariae and rediae were found in
Semisulcospira snails, and metacercarial infection was detected in P. lagowski steindachneri fish.37 In
Shizuoka Prefecture, many small rivers were found to have M. miyatai metacercariae in fish.70
The geographical distribution of M. katsuradai is confined to Japan.3,7 In Hyogo Prefecture, the larval
infection rate of Semisulcospira snails was 2.0% for M. yokogawai, 8.6% for M. takahashii, and 1.9% for
M. katsuradai in 1939.3 In Kobe City, dogs were reported as a natural definitive host.3 Around the Lake
Biwa, cercariae of M. katsuradai (having six oral spines) were discovered in 2003.80 Natural human
infections have never been documented.4

46.1.4 Pathogenesis and Clinical Features

In laboratory animal models using mice, rats, cats, and dogs, adult M. yokogawai flukes inhabited the
middle part of the small intestine; they invaded the crypt of Lieberkühn by day 2–3 postinfection and
localized between villi in later stages.2,14,15,83,84 The most prominent histopathological findings were
villous atrophy and crypt hyperplasia, accompanied by inflammatory reactions in the stroma.14 The
infected intestinal mucosa showed blunting and fusion of the villi, edema of the villus tip, congestion,
goblet cell hyperplasia, mastocytosis, and inflammatory cell infiltrations in the stroma, and markedly
750 Laboratory Models for Foodborne Infections

decreased villus/crypt height ratio.2,14,15,84 Almost the same intestinal histopathology seems to occur in
human M. yokogawai infection.2,16,85
In immunocompetent hosts, the location of M. yokogawai worms is confined to the intestinal mucosa,
not beyond the submucosa or muscularis mucosa.14 However, immunosuppression could allow a deeper
invasion of worms into the submucosa or underneath.15 Immunosuppression can also enhance survival
of the worms and prolong their life spans.15,86 Poor absorption of intestinal secretions from the secretory
crypt cells seems to induce watery diarrhea in humans or animal hosts.2,16 In M. miyatai infection in
mice, the intestinal histopathology was similar to that in M. yokogawai infection, although the degree of
mucosal damage was less severe than in M. yokogawai infection.87
A big question regarding the pathogenicity of M. yokogawai is whether this fluke can cause extrain-
testinal infections in humans and animal hosts.16 It was reported in 1940 in the Philippines that several
species of heterophyid flukes, including Stellantchasmus falcatus, Haplorchis spp., and Procerovum
spp., caused erratic parasitism in the heart, brain, and spinal cord in humans, which was often fatal.2,17,88
Such erratic parasitism may have occurred in immunocompromised patients.2,16 In M. yokogawai-
infected human patients, no direct evidence of extraintestinal infections has been reported.16 However,
it is worth noting that intracerebral hemorrhage and diabetes mellitus occurred in a metagonimiasis
patient, and the intracerebral hemorrhage may be an acute complication, whereas diabetes mellitus may
be a chronic sign.89
The clinical symptoms in human metagonimiasis, unless complicated by other diseases and there
are no signs of immunosuppression, include mild to moderate degrees of abdominal pain, diarrhea,
lethargy, anorexia, and weight loss.2,16 However, the severity of symptoms may vary depending on vari-
ous host-side factors.4,16 One factor is the intensity of infection—the number of worms infected in each
patient; heavier infection results in more severe illness.2,16,17 The second factor is the immune status of
the host.16,17 In immunocompromised patients, M. yokogawai infection can possibly cause severe clinical
manifestations, including erratic parasitism in vital organs.4,16A severe clinical case of M. yokogawai
infection complicated with multiple intracerebral hemorrhages and diabetes mellitus may have been
immunosuppressed.89 A third factor is the history of previous infections that might confer some degree
of acquired immunity in individual patients.16 New visitors to an endemic area are likely to suffer from
a severe illness from a primary infection.2,16 On the other hand, individuals living in an endemic area for
a long time, who inevitably are infected repeatedly, may be protected from severe pathology and symp-
toms caused by M. yokogawai worms.4,16

46.1.5 Diagnosis
Human Metagonimus infection can be diagnosed by detecting eggs in fecal samples.16 The direct
smear, cellophane thick smear, and Kato-Katz thick smear techniques can be applied in field con-
ditions, whereas in the laboratory equipped with centrifuges, concentration techniques, for exam-
ple, formalin-ether sedimentation technique and brine (or zinc sulfate) floatation techniques, can be
performed.4,16
However, morphological differentiation of eggs in fecal samples may be problematic in areas hav-
ing coprevalence of Clonorchis sinensis and/or heterophyid fluke (Metagonimus spp., Heterophyes
spp., Pygidiopsis summa, and Haplorchis spp.) infections.4 Eggs of M. yokogawai can be differed
from eggs of other heterophyids by their length of 26.9–31.6 µm, elliptical shape with length/width
ratio of 1.5–2.1, clean shell surface (without muskmelon patterns), less prominent operculum, lack
of shoulder rims, and dark yellow or brown color.90,91 Eggs of M. takahashii and M. miyatai are
very similar to those of M. yokogawai, except their sizes being larger in the former and smaller in
M. yokogawai.23,90,91 In patients infected with less than 100 worms, the probability of detecting eggs
in one Kato-Katz smear is almost zero,4,16 assuming that the daily number of eggs produced per
M. yokogawai worm in the human host is 14–64.92 In such cases, serological tests, including ELISA,
are helpful.16
Molecular techniques can be applied to detect Metagonimus spp. infections in the feces71 or food
materials including fish.93
Metagonimus 751

46.1.6 Treatment and Control

Praziquantel in a single oral dose of 10–20 mg/kg is the drug of choice for treating human metagonimia-
sis.16 Praziquantel is safe without particular adverse reactions at this dose even in children and pregnant
women.94 Control measures for metagonimiasis can include control of the snail host, control of the
fish host, and mass chemotherapy of residents in endemic areas.4 However, snail control and fish con-
trol are not feasible.4 Mass chemotherapy can temporarily reduce the prevalence and infection intensity
(individual worm load), but reinfection continues to occur in the area unless the consumption of raw or
improperly cooked fish is stopped.4 Therefore, prevention is the best way. Health education not to eat
raw fish will help prevention of the disease.4 The infectivity of M. yokogawai metacercariae in fish can
be controlled by gamma-irradiation at 200 Gy.95 However, this method is not feasible in the field due to
various reasons such as the necessity of an irradiator, high cost, and low preference of irradiated fish by
the consumers.95

46.2 Laboratory Models
46.2.1 General Considerations
Metagonimus spp. are zoonotic, and various animals are known to serve as natural definitive hosts.
In endemic areas of metagonimiasis due to M. yokogawai or M. miyatai, for example, humans are
the principal host and the most important epidemiological element. The infected humans may suffer
from mild to moderate degrees of abdominal discomfort, diarrhea, indigestion, and weight loss.1,2,4,17
However, the mechanisms of diarrhea and other clinical symptoms as well as host immune responses
against Metagonimus spp. infection are poorly understood. Therefore, for proper understanding of
human metagonimiasis, laboratory models, in particular, experimental metagonimiasis in laboratory
animals (Table 46.1), or in vitro culture models, are indispensable. For this purpose, mammalian ani-
mals like mice, rats, dogs, and hamsters have been most commonly used, and rarely cats, rabbits,
chickens, and ducks were used. The study subjects using laboratory animals have been the morphol-
ogy, taxonomy, growth and sexual development of worms, their longevity and fecundity, comparative
susceptibility of different animal species, pathogenesis and pathology, host–parasite interactions, host
immune responses, chemotherapy, and control.

TABLE 46.1
Species of Metagonimus Reported in the Literature and Laboratory Animals Used
Laboratory Animals
Species Human Infection Source of Infection Models
Metagonimus yokogawai Natural Sweetfish, dace, perch Dog, cat, hamster, rat,
mouse, rabbit, guinea pig,
gerbil, mink, raccoon dog,
fox, duck, chicken
(unsuccessful)
Metagonimus takahashii Natural Crussian carp, common carp, Dog, cat, hamster, rat,
dace, perch mouse, rabbit
Metagonimus miyatai Natural Sweetfish, dace, minnow, pale Dog, hamster, rat, mouse,
chub, dark chub duck (unsuccessful)
Metagonimus minutus Uncertaina Mullet Cat, mouse
Metagonimus katsuradai Experimental Carp, pale chub, Acheilognathus Dog, cat, hamster, rat,
lanceolata mouse, rabbit, duck
Metagonimus otsurui None Freshwater fish Dog, hamster, chicken
(unsuccessful)
Metagonimus hakubaensis None Freshwater fish Dog, hamster, rat
a Enlisted among the species infecting humans,12 but without literature background.
752 Laboratory Models for Foodborne Infections

46.2.2 Animal Models Used for Studies on Morphology and Taxonomy

46.2.2.1 Dogs
When M. yokogawai was first described as a new species in 1912, dogs were used as the principal
laboratory host.30,96,97 Thereafter, a lot of researchers used dogs for experimental M. yokogawai infec-
tion.24,41,98–101 Muto98 studied the morphology of M. yokogawai using dogs as the laboratory host, and
Saito101 compared the morphological and biological characteristics of M. yokogawai, M. yokogawai
Miyata type (now M. miyatai) and Koga type (now considered synonym of M. takahashii), and
M. takahashii. Kagei and Kihata99 studied the morphology as well as development and egg-laying capac-
ity of M. yokogawai in various animals including dogs. Ahn41 morphologically confirmed M. yokogawai
adults recovered from dogs fed with the flesh of the perch from Gurye-gun (Seomjin-gang River), Korea.
Ahn et al.100 studied the egg-laying capacity of M. yokogawai.
For studies on M. miyatai, dogs were also popularly used.9,50,53 Ahn50 used a dog to obtain adult
flukes of M. miyatai (as Metagonimus Miyata type) after feeding the metacercariae from Z. platypus in
Korea. Saito et al.9 obtained adult flukes of M. miyatai from dogs experimentally fed with freshwater
fish including Z. temmincki and Morocco steindachneri in Japan and established M. miyatai as a new
species. To study M. takahashii, Takahashi24 used dogs to compare the morphology of adult flukes and
susceptibility of different laboratory animals. Surface ultrastructure of M. takahashii was also studied
using worms recovered from experimental dogs.102 Dogs were further used to obtain adult flukes and
establish M. katsuradai,7 M. otsurui,8 and M. hakubaensis55 as new species.
The use of dogs as a laboratory definitive host for Metagonimus spp. includes several merits.24,41,98–101
One is the high recovery rate of worms with a long-time establishment in the small intestine of dogs.
They are highly susceptible to Metagonimus infection with larger worm size and longer-time survival
compared to mice and rats. Another merit is full growth and maturation of worms as comparable as
those that develop and mature in the human host. A third is the relatively large body size of dogs as an
alternative host that can mimic the human body. A fourth may include easier laboratory management
compared to cats.

46.2.2.2 Cats
Cats have been used rarely as a laboratory host for Metagonimus spp.7,97 Kobayashi97 infected cats
and dogs with the metacercariae of M. yokogawai isolated from sweetfish muscle to obtain the adult
flukes. Kim et al.79 infected a cat with metacercariae from perches and obtained M. takahashii adult
flukes. Katsuta6 also infected cats with metacercariae of M. minutus and obtained adult flukes. Izumi7
used cats and several other mammalian animals to recover the adult flukes of M. katsuradai. Cats
were also used for pathology103,104 and immunology studies105,106 in Metagonimus spp. infection (see
46.2.4 and 46.2.5).

46.2.2.3 Hamsters
Hamsters were occasionally used as an experimental definitive host for Metagonimus spp. Yokogawa
and Sano107 infected hamsters, mice, rats (cotton rats and Wistar rats), and guinea pigs with the metacer-
cariae of M. yokogawai to obtain the adult flukes and to observe the development of worms in different
hosts. Kagei and Kihata99 also studied the morphology, recovery, and the life span of M. yokogawai in
hamsters. Miyamoto and Kutsumi108 infected hamsters and dogs to recover M. yokogawai adult flukes. A
similar study was performed recently by Li et al.46 in Taiwan. Hamsters were also used to differentiate
morphological characters of M. yokogawai from those of M. miyatai9,29,37,70,109 and M. takahashii.26,29,109
Moreover, unique morphological features of M. katuradai54, M. otsurui,8,13 and M. hakubaensis55 were
also studied using hamsters.
The merits of using hamsters for an experimental definitive host include their high susceptibility to
Metagonimus spp. infection with high worm recovery and longer worm survival.46,107 Suitability as a
laboratory animal and easy handling, compared with dogs and cats, are also among the merits.
Metagonimus 753

46.2.2.4 Mice and Rats

Takahashi24 used mice and rats to study the morphology and life cycle of M. yokogawai in comparison to
M. takahashii. Koga,28 Gushima,110 Komiya et al.,111 and Yokogawa and Sano107 also used mice and rats
to infect M. yokogawai, although the susceptibility of these animals was less satisfactory compared with
animals like dogs, cats, and hamsters. Chun112 infected mice, rabbits, and guinea pigs to obtain the adult
flukes of M. yokogawai, and Hong and Seo113 observed worm development in mice. Kagei and Kihata99
compared the developmental status of M. yokogawai in mice, hamsters, and dogs.
For studies on M. miyatai and/or M. takahashii, mice or rats were used by Kim,52 Kim et al.,38 Ahn,50
Ahn and Ryang,53,77 and Lee et al.114 In addition, Chai et al.29 used rats and hamsters to obtain the adult
flukes of M. yokogawai, M. miyatai (as Metagonimus Miyata type), and M. takahashii (as Metagonimus
Koga type). Mice and cats were used to study the morphology of M. minutus,6 and mice, rats, rabbits,
cats, dogs, and/or ducks were used to infect the metacercariae of M. katsuradai.7,39 For the new species
description of M. hakubaensis,10 rats were used as the experimental host.
Rats were also used to study the surface ultrastructure of the adult flukes of M. yokogawai,115
M.  miyatai,116 and M. takahashii.117 The surface ultrastructure was similar to each other, but a few
unique findings were found, for example, the presence of type III sensory papillae in M. yokogawai but
not in two other species.115–117 Molecular and genetic differentiation and taxonomic studies were per-
formed using adult flukes of M. yokogawai, M. miyatai, and M. takahashii recovered from experimental
rats.32,34,118 These three species were genetically unique and considered to be varied from each other.
The use of mice and rats as an experimental definitive host of Metagonimus spp. was in consideration
of fairly good morphology of the recovered adult flukes and easy handling in the laboratory.

46.2.2.5 Rabbits, Guinea Pigs, and Other Mammals

Rabbits were not uncommonly used to obtain the adult flukes of Metagonimus spp., M. yokogawai,24,112,119
M. takahashii,119 and M. katsuradai.7 Moreover, guinea pigs107,112 and gerbils46 were also used in studies
of M. yokogawai for the same purpose. Other mammalian animals used for studies on M. yokogawai
included American minks, raccoon dogs, and red foxes.45

46.2.2.6 Ducks and Chickens

Studies on the morphology of Metagonimus spp. using avian hosts have been scarce. As for M. yokogawai,
a recent study in Taiwan47 reported that domestic ducks can host this fluke within 14 days after infection
but were considered not susceptible to this fluke infection. Similarly, M. miyatai metacercariae were
experimentally fed to ducks, but no adult flukes could be recovered.53 However, Kurokawa39 succeeded
in obtaining two adult flukes at 7 days after experimental infection of a duck with M. katsuradai meta-
cercariae. On the other hand, Saito and Shimizu8 failed to obtain adult flukes of M. otsurui from two
chickens experimentally fed with the metacercariae.

46.2.3 Animal Models Used for Studies on Worm Development

and Host Susceptibility
46.2.3.1 Dogs
Dogs were most popularly used as a laboratory definitive host to complete the development and sexual
maturity of M. yokogawai24,41,98–100,108 and other Metagonimus spp.7,8,24,28,38,39,50,53 Yokogawa96 reported
that M. yokogawai attained its full development in dogs by day 5 (120 h) after infection with the meta-
cercariae. Muto98 found eggs of M. yokogawai discharged in the feces of dogs experimentally infected
with the metacercariae from day 9 after infection. Kagei and Kihata99 reported that the development and
egg-laying capacity of M. yokogawai were better in dogs compared with mice. They observed eggs in the
uterus of worms recovered from dogs as early as day 3 after infection and eggs in the feces of dogs from
day 6 after infection. Ahn et al.100 reported that the daily egg output (EPDPW; eggs per day per worm)
754 Laboratory Models for Foodborne Infections

of M. yokogawai was 35–45 eggs at day 7 postinfection in the dog host. Miyamoto and Kutsumi108
confirmed that the dace Tribolodon spp. is a second intermediate host of M. yokogawai by recovery of
adult flukes from experimentally infected dogs from day 8 to 129 postinfection. Kang et al.84 infected
dogs with the metacercariae of M. yokogawai and obtained the worm recovery rate of about 20% even at
week 6 postinfection; a repeated infection led to displacement of worms from the duodenum and jejunum
down to the ileum.
With regard to M. miyatai, dogs were used for studies on the worm development and/or fecundity.9,38,50,53
Ahn and Ryang53 reported that the worm development of M. miyatai and susceptibility was better in dogs
than in rats; the worm maturity was the best at day 35 after infection in dogs. In M. takahashii, few stud-
ies have been performed using dogs as the definitive host. Takahashi24 observed better development of
M. takahashii worms in dogs compared to those developed in mice. Similarly, Koga28 studied the satis-
factory worm development of M. takahashii (under the name, large egg-type Metagonimus) in dogs and
cats. Dogs were further used to study the worm development and host susceptibility of M. katsuradai,7,39
M. otsurui,8 and M. hakubaensis.55 Kudo et al.55 reported that dogs and hamsters were more susceptible
to M. hakubaensis infection than cats, rats, mice, chickens, and quails.
Regardless of the species of Metagonimus, dogs were found to be a highly susceptible host to experi-
mental Metagonimus infection. The worms grow quickly (within 3–6 days) in the small intestine of dogs
compared with other laboratory hosts including mice and rats, and survive remarkably a longer time.
Their fecundity in dogs was fairly good.

46.2.3.2 Cats
Kobayashi97 first used cats to infect the metacercariae of M. yokogawai isolated from the sweetfish.
Thereafter, only a few researchers73,103–106 used cats to study M. yokogawai. These studies were mainly
to recover the adult flukes or to observe the intestinal pathology and host immune responses103–106 rather
than to observe the life cycle, development, and sexual maturity of worms. Sohn et al.73 infected cats and
dogs to recover adult M. yokogawai after an experimental infection with the metacercariae in freshwater
fish from China.
Studies using cats were also scarce for other Metagonimus spp. As for M. takahashii, Kim et al.79
infected a cat with the metacercariae in the perch Lateolabrax japonicus from Korea and obtained
adult flukes 21 days later. Similarly, Katsuta6 obtained M. minutus adult flukes from cats experimentally
infected with the metacercariae in Taiwan, and Izumi7 recovered adult flukes of M. katsuradai from
experimental cats.

46.2.3.3 Hamsters
Hamsters were popularly used to study the development and longevity of Metagonimus spp. worms.
Yokogawa and Sano107 reported that hamsters were a highly suitable animal host for M. yokogawai infec-
tion compared with mice, rats, and guinea pigs. However, Kagei and Kihata99 reported that although some
M. yokogawai worms survived up to a year in dogs, no worms were retained in the intestine of hamsters
after 5 months. Miyamoto and Kutsumi108 reported similar findings; in dogs, a considerable number of
worms survived at day 129 postinfection, but in hamsters, only a few worms survived at day 29 postinfec-
tion. However, Li et al.46 obtained a high worm recovery rate (75.3%) of M. yokogawai from hamsters at day
14 postinfection. Compared with hamsters or dogs, mice were not a suitable host for M. yokogawai infec-
tion.99,107 Therefore, it can be said that the suitability of laboratory animals to infection with M. yokogawai
is the highest for dogs, followed by hamsters, rats, mice, and other rodent animals. Hamsters were also used
for studies on the development and host susceptibility of M. miyatai37 and M. otsurui.13

46.2.3.4 Mice and Rats

Mice and rats have been popularly used as laboratory animal models for Metagonimus spp. infection,
although the susceptibility of these animals is lower compared with bigger animals like dogs. Komiya
et al.111 reported that M. yokogawai developed well in the intestine of mice by day 6 after infection and
Metagonimus 755

survived until day 26. Yokogawa and Sano107 compared the recovery and growth of M. yokogawai in
mice, Wistar rats, cotton rats, hamsters, and guinea pigs. They reported that the worms obtained full
maturation by week 1 after infection in all of these animals except guinea pigs, which showed no worms
even at 1 week. Hamsters and cotton rats retained more worms until week 6 after infection, whereas
mice and Wistar rats retained smaller number of worms.107 Hong and Seo113 observed the full growth
and development of M. yokogawai in mice by day 7 postinfection, although the rate of growth varied
individually. Kagei and Kihata99 reported that M. yokogawai developed well in mice, hamsters, and
dogs; however, the worm survived shorter than 1 month in mice, whereas it survived until 4 months in
hamsters and up to a year in dogs. On the other hand, Li et al.46 obtained a fairly high worm recovery
rate (70.0%) of M. yokogawai from ICR mice at day 14 postinfection, comparable with that (75.3%)
obtained in hamsters. Chai et al.86 infected M. yokogawai to various strains of mice (CBH, A, C57BL,
DBA, and KK) and observed the susceptibility of each mouse strain judged by the worm develop-
ment and worm recovery. KK mice (diabetic mice) showed the highest worm recovery at day 7 after
infection.86
Mice and rats were also used for studies on the development and maturation of M. miyatai and/or
M. takahashii.29,38,50,51–53,77 Kim et al.38 observed the development and site distribution of M. miyatai
worms (under the name Metagonimus Miyata type) in the intestine of mice. Ahn and Ryang53 reported
that M. miyatai (under the name Metagonimus Miyata type) continuously increased the body size in
rats until day 30 postinfection when the number of uterine eggs also peaked. Some retarded worms
were recovered until day 55 postinfection in rats.53
Guk et al.51 studied on the comparative growth and development of M. yokogawai, M. miyatai, and
M. takahashii in different strains of mice (BALB/c, ddY, C57BL/6J, C3H/HeN, and A/J). They found
that ddY mice were the most suitable host for M. yokogawai with the highest worm recovery, that
BALB/c and ddY were equally suitable for M. miyatai infection, and that BALB/c, ddY, and C3H/HeN
mice were equally suitable for M. takahashii infection.51 Generally, mice were fairly susceptible to M.
yokogawai infection with high worm recovery, good worm dimension, and high worm fecundity but
less susceptible to M. miyatai and M. takahashii infection.51
Mice were also used to study the growth and development of M. minutus; the sexual maturation was
completed by day 7 postinfection.6 By comparison, the development of M. katsuradai was completed
earlier in mice, by day 5 postinfection; moreover, eggs were seen in the feces of mice from day 4 (90 h)
postinfection.7 Fully mature specimens of M. hakubaensis were recovered in experimentally infected
rats at days 7–22 postinfection.10

46.2.3.5 Rabbits, Guinea Pigs, and Other Mammals

Rabbits were seldom used to observe the growth and development of Metagonimus spp. However, sexu-
ally mature worms of M. yokogawai were obtained from rabbits fed with the flesh of the sweetfish after
days 7–12 postinfection,112 although the susceptibility of rabbits to Metagonimus spp. infection is esti-
mated to be fairly low. Successful development of M. takahashii76 and M. katsuradai7 was also observed
in rabbits.
Guinea pigs107,112 and gerbils46 were used to infect Metagonimus spp., but the results were generally
not satisfactory. Some eggs of M. yokogawai were detected in the feces of guinea pigs from day 10
to 35 postinfection; however, no adult flukes were recovered at day 45 postinfection.112 Gerbils were
infected with the metacercariae of M. yokogawai, and a 6.0% worm recovery rate was obtained at day
14 postinfection.46

46.2.3.6 Ducks and Chickens

Ducks were used to observe the infectivity and development of M. yokogawai.47 About 20% of worms
attained their sexual development by day 7 after infection; however, the worms were quickly expelled
thereafter.47 However, in ducks experimentally infected with M. miyatai, a small number of worms were
recovered at day 2 postinfection, and no worms were recovered at day 10 postinfection.53 Kurokawa39
756 Laboratory Models for Foodborne Infections

succeeded in recovery of two adult flukes at day 7 after infection of a duck with M. katsuradai meta-
cercariae. Chicks were used for infection with M. yokogawai73 and M. otsurui,8 but no worms were
recovered.

46.2.4 Animal Models Used for Studies on Pathogenesis and Pathology

46.2.4.1 Dogs, Cats, Rats, and Mice
The pathogenesis and pathology of metagonimiasis have been studied mainly in dogs,28,84 cats,28,103,104
rats,14,83,120,121 and mice,15 exclusively in M. yokogawai infection. Koga28 was the first who described the
pathogenesis and intestinal pathology of dogs and cats experimentally infected with M. yokogawai meta-
cercariae. He28 reported transient diarrhea in light infections and severe catarrh sometimes with bloody
mucous diarrhea in heavy infections in these animals. However, worms were confined to the mucosa of
the small intestine and never invaded into the deeper layers such as the submucosa and underneath.28
Chai14 observed the clinical course and intestinal pathology of experimental rats infected with the meta-
cercariae; diarrhea occurred from day 6 after infection and continued until sacrificed at week 4 after
infection. The gross intestinal pathology included marked dilatation of the intestinal loop, foamy watery
content in the lumen, and sometimes mucosal hyperemia.14 Such watery content in metagonimiasis is
considered to be due to poor absorption of intestinal secretion from crypt cells.103 Microscopically, the
worms were located in the intervillous space or on the top of the villi, and the worm location was con-
fined to the mucosal layer; the pathological changes could be categorized largely into two kinds: villous
atrophy and crypt hyperplasia.14 Villous atrophy included shortening of villi, blunting and edema at the
tip of the villi, villous adhesion, and inflammation with edema and congestion of the villous stroma.14
Mast cell hyperplasia is another prominent feature in the intestine of M. yokogawai-infected rats;
it peaked at week 3 postinfection.120 It was further observed in experimental rats that the number of
intraepithelial lymphocytes within the epithelial layer of the small intestinal villi increased rapidly at day
5 postinfection but decreased to a lower level during days 10–24 postinfection and then normalized.83
Moreover, the position of the intraepithelial lymphocytes changed significantly at day 5 postinfection
toward the apical side of the villi; from this time, active worm expulsion seemed to occur.83 When rats
were coinfected with M. yokogawai and another kind of intestinal fluke Neodiplostomum seoulense
(under the name Fibricola seoulensis), the intestinal lesions became severer than in rats infected with
only one fluke species.121 In cats,103 the intestinal pathology due to M. yokogawai infection was similar
to that reported in rats,14 and some of the pathological changes continued until week 10 postinfection. As
an early phase mechanism of inducing intestinal lesions observed in dogs, uncompensated enterocytes
deficit due to marked destruction of the villi and upper parts of the crypt or villous–crypt junction was
proposed.104 In dogs, similar intestinal pathology was observed until week 6 after infection.84 There is
one paper reporting the intestinal pathology of metagonimiasis in a human patient; M. yokogawai worms
were found free in the jejunal lumen or impacted in the intervillous spaces, and massive lymphoplasma-
cytic and eosinophilic infiltration in the stroma, erosion of nearby enterocytes, goblet cell depletion, and
occasional villous edema were observed.85
The location of worms was strictly confined to the mucosal layer (intervillous space, crypt, or on the
top of villi) in laboratory animals including dogs, cats, and rats.14,28,84,103 However, in immunosuppressed
mice, the location of worms became deeper into the submucosa and muscular layer of the intestinal
wall.15 This strongly suggests that the invasiveness of Metagonimus worms may be related to the host
immunity, protecting them from deeper invasion.

46.2.5 Animal Models Used for Studies on Immunity and Host–Parasite Interaction

46.2.5.1 Dogs, Cats, Rats, Mice, and Hamsters
Immunological characteristics including acquired resistance, antigenicity of worms, and host–
parasite relationships have been studied using dogs,84,122,123 cats,105,106 rats,83,110,120,124 mice,86,87,110,125–127
and hamsters70 mostly in M. yokogawai and rarely in M. miyatai infection. Gushima110 was the first
to study immune responses of rats and mice after experimental infection with M. yokogawai in 1939.
Metagonimus 757

He performed repeated infections of rats (2–3 times) and mice (2–6 times) with M. yokogawai at 4- to
30-day intervals and reported the following results: Acquired resistance was obtained in the reinfected
animals, and the resistance became stronger in accordance with the frequency of reinfection and the
lapse of the days after reinfection. Worm development was retarded in reinfected animals, particularly in
genital organs, and animals possessing immature worms got reinfected more easily, whereas those pos-
sessing fully developed worms got reinfected with a markedly lower incidence rate.110 He also notified a
tendency of changing parasitic locations from the upper part of the small intestine to the lower part of the
intestine.110 He further notified that the resistance could be acquired by an injection of emulsified worms
after refrigeration, drying, and powdering.110 The shifting of the parasitic locations from the upper part
of the small intestine to the lower part of the intestine was also observed in 1983 in dogs reinfected with
M. yokogawai.84
Chai et al.86 reported that the susceptibility of mice to M. yokogawai infection was highly dependent
on the immune status of the host. In immunocompetent ICR mice experimentally infected with M. yok-
ogawai, most of the worms were expelled spontaneously before day 7 after infection.86 However, ICR
mice immunosuppressed with prednisolone injection allowed a longer survival (until day 21 postinfec-
tion) of a greater number of worms than in immunocompetent control mice.86 Eosinophils were sug-
gested to have an important role to induce such protective immunity against M. yokogawai infection.86,126
Other effector mechanisms conferring protective immune responses of the host may include intestinal
intraepithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), mucosal mast cells (MMC), and
goblet cells.1,2,4,11 The kinetic of intestinal IEL was studied by Chai et al.83; the IEL number was mark-
edly increased at day 5 postinfection, which decreased thereafter until day 24 postinfection and then
normalized. The day 5 postinfection was well corresponded with the time of active worm expulsion
from the host intestine.86 Moreover, the location of the IEL was moved to the apical portion of the villi
from the original basal or intermediate portion around the day 5 after infection.83 Mucosal mastocytosis
was observed throughout the infection period, from week 1 to 4, in rats,120 and, from week 1 to 3, in
mice,125 experimentally infected with M. yokogawai.120 Thus, MMC seemed also a significant factor
for inducing immunopathological effects and damages on the mucosal regions and finally, expulsion
of worms. Increased mucosal permeability was also suggested to be a factor related to the expulsion of
worms from mice.125
Antigenicity of body portions of M. yokogawai was studied using adult worms recovered from experi-
mental cats.105,106 Tegumental cells and tegumental syncytium as well as the intestinal content of the
worms were suggested to be the major antigens of M. yokogawai.105,106 However, a purified 100-kDa anti-
gen reacting to the tegumental and subtegumental layers of M. yokogawai appeared to be not a species-
specific antigen but a common antigen crossly recognizing even the liver fluke (Clonorchis sinensis),
lung fluke (Paragonimus westermani), and another intestinal fluke species (Gymnophalloides seoi).124
The antigenicity of M. yokogawai was generally stronger than that of M. miyatai as assessed by the lower
levels of the villus/crypt height ratio and the lowered expression patterns of the proliferating cell nuclear
antigen in the former.87 In addition, the parasitic location of M. yokogawai was generally at the upper
level of the small intestine in experimental hamsters, whereas that of M. miyatai was generally at the
middle level of the small intestine.70
An interesting observation of the worm posture within the ecological niche of M. yokogawai in the
dog’s small intestine was that the worms, during the early stage of infection (around day 3 postinfec-
tion), invade the slit of the Lieberkhün’s crypts by making their anterolateral bodies as one or more
reversible tube-like structures.122 Thereafter, until week 10 of observation, worms with such protruded
anterior bodies decreased continuously in number when most of the worms were floating on the top of
the villi.122 Another point of interest in host–parasite interactions includes the mechanisms of diarrhea
in metagonimiasis. It has been suggested that diarrhea is caused by excessive water content in the small
intestine of M. yokogawai-infected hosts.14,103,127 The excessive water content is considered to be due
to poor absorption of intestinal secretions resulting from mucosal inflammation in the affected small
intestine.103 The poor absorption of intestinal secretions is likely resulting from the reduced absorptive
surface due to the decreased villous height; however, this may be a reversible phenomenon.123 It was also
reported that the activities of brush border membrane-bound enzymes decreased in the small intestine of
M. yokogawai-infected mice and participated in the generation of diarrhea and malabsorption.127
758 Laboratory Models for Foodborne Infections

46.2.6 Animal Models Used for Studies on Chemotherapy and Control

46.2.6.1 Dogs and Rats
In vivo experimental studies have never been performed to study on the effects of anthelmintic drugs
against Metagonimus spp. infection. Instead, there were a few in vitro studies to evaluate the effects of
anthelmintics such as praziquantel,128 bithionol, and menichlopholan.129 M. yokogawai adult flukes were
harvested from experimentally infected rats fed with the metacercariae and exposed to 0 (control), 1, 10,
and 100 µg praziquantel/mL in vitro.128 The tegumental surface of the drug-treated M. yokogawai showed
vacuolization and bleb formation followed by worm contraction, which led to death of the worms.128
Similarly, M. takahashii adult flukes recovered from experimental dogs and rats lost their motility and
tegumental damage after in vitro exposure to bithionol and menichlopholan.129 For the purpose of con-
trolling metagonimiasis, Chai et al.95 tried irradiation of sweetfish infected with M. yokogawai meta-
cercariae by 200 Gy, which was highly effective to control infectivity as well as development of the
metacercariae in rats.

46.2.7  In Vitro Culture and Others

46.2.7.1  In Vitro Culture
Yasuraoka and Kojima130 tried in vitro cultivation of M. yokogawai metacercariae using a culture
medium containing chick embryo extract, human serum, and NCTC 109 medium. The trial appeared
to be successful as fully mature flukes possessing 70–90 eggs in their uteri could be obtained by this
in vitro cultivation.130 The maximum worm size was reached at 16 days after beginning of the culture,
and the maximum worm survival was 35 days by in vitro culture.130

46.2.7.2 Experimental Infection of Fish

Saito27 experimentally infected sweetfish (P. altivelis) and goldfish (Carassius auratus) with the cer-
cariae of both M. yokogawai and M. takahashii and found that the sweetfish serves as the second inter-
mediate host of only M. yokogawai and the goldfish serves as that of only M. takahashii. This host (fish)
specificity of M. yokogawai and M. takahashii has been approved by other studies.76,101

46.3 Conclusion and Future Perspectives

Metagonimus spp. are important foodborne parasites and are mainly distributed in the Far Eastern
countries, including Korea, China, Taiwan, Japan, and Russia. The major etiological agents of human
metagonimiasis are M. yokogawai, M. takahashii, and M. miyatai. However, four other species, namely
M.  minutus, M. katsuradai, M. otsurui, and M. hakubaensis, are potentially infective to humans.
Laboratory models for studies on Metagonimus spp. infection include mammalian animals (e.g., dogs,
cats, hamsters, rats, and mice) and avian animals (e.g., ducks). The topics of research using these labora-
tory models encompass morphology and taxonomy, worm development and host susceptibility, patho-
genesis and pathology, immunity and host–parasite interaction, and chemotherapy and control, among
others. In vitro cultures using culture medium have also been employed for studies on development and
maturation of worms.
The adult flukes parasitize the small intestine of humans and also animal hosts. They can cause muco-
sal pathology that consists of villous atrophy, crypt hyperplasia, and mucosal inflammations, but the
symptoms in human metagonimiasis are generally mild unless heavily infected. However, in immuno-
compromised patients, complications such as erratic parasitism in the heart, brain, and spinal cord, or
intracerebral hemorrhage, may occur, as reported in other heterophyid fluke infections. The possibility
of erratic parasitism in metagonimiasis should be ruled out. For this reason, new laboratory models are
urgently needed, in particular, larger animal models including monkeys and pigs that may allow passage
of eggs and/or worms into the mesenteric venule, resulting in erratic parasitism.
Metagonimus 759

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and diabetes mellitus, J. Nippon Med. Sch., 75, 32–35, 2008.
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47
Opisthorchis viverrini

Thidarut Boonmars

CONTENTS
47.1 Introduction................................................................................................................................... 765
47.2 Morphological Characteristics of O. viverrini.............................................................................. 765
47.3 Animal Models for O. viverrini.................................................................................................... 766
47.3.1 Establishment of O. viverrini Infection in Animal Models..............................................767
47.3.2 Gross Pathology of Hamster and Gerbil Liver..................................................................767
47.3.3 Pathology of Liver Fluke Infection in Hamster and Gerbil..............................................767
47.3.4 The Level of White Blood Cells in the Liver Fluke Infection in Hamsters..................... 768
47.3.5 Opisthorchiasis-Associated Cholangiocarcinoma............................................................ 769
47.4 Conclusion..................................................................................................................................... 770
References............................................................................................................................................... 771

47.1 Introduction
Opisthorchis viverrini is a trematode (commonly called liver fluke) that is classified in the family
Opisthorchiidae, order Opisthorchiata, subclass Digenea, class Trematoda, phylum Platyhelminthes,
kingdom Animalia (Figure 47.1). With a high prevalence in Southeast Asia, particularly Thailand, Lao
PDR, Cambodia, and Vietnam,1 this parasite lives in the hepatobiliary tract of humans, causing hepato-
biliary diseases such as cholangitis, lithiasis, gallstone, hepatitis, and also cholangiocarcinoma. Humans
normally acquire infection with this liver fluke by ingestion of uncooked cyprinoid fish that contains or
is contaminated by the infective stage (namely “metacercaria”) of O. viverrini. After ingestion, the cyst
wall of metacercaria is digested by gastric juice and duodenum juice, and the excysted juvenile migrates
to the biliary tract and grows to adult in the hepatobiliary tract where it dwells (Figure 47.2). This infec-
tion (often called “opisthorchiasis” or “opisthorchiosis”) induces many changes in hepatobiliary tract,
such as dilatation of the bile ducts with hyperplasia, desquamation, proliferation of the bile duct epithe-
lial cells, glandular formation, and fibrous connective tissue infiltration of the walls, and other complica-
tion of hepatobiliary diseases (e.g., cholangitis, cystitis, cholecystitis including cholangiocarcinoma).2–4
Besides O. viverrini, which mainly occurs in Thailand, Lao PDR, Vietnam, and Cambodia,1 Clonorchis
sinensis and Opisthorchis felineus within the family Opisthorchiidae also cause liver fluke infections in
humans, dogs, and cats. While C. sinensis infection appears in China, Korea, Japan, and Vietnam,5
O. felineus infection is present in Russia and European countries.6

47.2 Morphological Characteristics of O. viverrini

The adult worm of O. viverrini is transparent and flat; its anterior part is small and slender; and its size
is about 5.4–10.2 mm × 0.8–1.9 mm. Oral sucker and ventral sucker are located at the anterior part of
the body. Two testes (deep lobes) are tandemly located at the posterior part. The excretory bladder is
S-shaped and located between two testes. Vitelline glands are located at both sides of the trunk with

765
766 Laboratory Models for Foodborne Infections

Oral sucker

Ventral sucker

Vitelline gland

Ovary

Testes

FIGURE 47.1  Adult worm of O. viverrini.

1.Ingestion of metacercaria
Adult

2.Metacercaria is
excysted and then
4.Cercaria invade to junvenile migrate to
cyprinod fish and bile ducts and gallbladder
develop to
metacercaria

Cercaria Radia Sporocyst Miracidium Egg

3.Snail eat egg and develop many stage to cercaria

FIGURE 47.2  The life cycle of O. viverrini.

columns (Figure 47.1). In vivo study showed that O. viverrini in different hosts show varied shape and
size.7 Rat and mouse are resistant to O. viverrini, whereas hamster and gerbil are susceptible to this
parasite. In general, worm recovery from hamster is higher than that from the gerbil, and the recovered
worm from gerbil is larger than that from hamster. The worm size correlates to the size of the reproduc-
tive organs (testes, uterus, and vitelline gland), eggs per worm, and worms per gram of feces as shown
in Figure 47.3 and Table 47.1.8

47.3 Animal Models for O. viverrini

A study of liver fluke infection in animals showed that many animals, including dogs, rabbits, hamsters,
gerbils, and mice, display varied susceptibility to O. viverrini infection and pathological changes.7–9
One hundred percent worm recovery was found in hamster and gerbil, and the highest number of worm
recovery was observed from the hamster. Severe pathology was found in gerbil, as evidenced by cirrho-
sis and abdominal edema.9 Therefore, hamsters are the appropriate experimental animals for studying
O. viverrini and cholangiocarcinoma.
Opisthorchis viverrini 767

(A) (B)

FIGURE 47.3  The representative adult of O. viverrini from hamster (A) and gerbil (B) at 1.5 month.

TABLE 47.1
The O. viverrini Infection in Animal Models
Animal Number of Size of Adult Worm
Model Metacercariae (Length × Height) in mm % Infection References
Hamster 50 1.43 ± 0.08 × 4.4 ± 0.3 28 ± 9.8 7
Gerbil 50 1.81 ± 0.06 × 5 ± 0.12 60.5 ± 14.44
Mouse 50 — 0 8
Rat 50 — 0

47.3.1 Establishment of O. viverrini Infection in Animal Models

Normally, O. viverrini metacercaria from infected freshwater cyprinoid fish is needed for animal infec-
tion. Typically, fish is blended into a small size and digested with the pepsin-HCl solution at 37°C for
1 h. The digested solution is filtered through a sieve and then washed with normal saline multiple times
until the sediment is easy to observe under stereomicroscope, and then 50 O. viverrini metcercariae are
gastric-intubated (Figure 47.4).

47.3.2 Gross Pathology of Hamster and Gerbil Liver

Based on the uninfected control, the color of the infected liver appears reddish-brown, similar to unin-
fected control in the early stages of infection. In a chronic infection, the liver color is pale, and the
infected bile duct is much larger than the uninfected one. Moreover, bile duct size and thickness increase
with the time of infection. Normally, the color of the bile ducts outside is colorless, but the infected bile
duct and gallbladder are creamy. As mentioned earlier, host differences have an impact on parasite devel-
opment and pathology.8,10,11 Liver flukes recovered from infected gerbils are larger than those recovered
from infected hamster, correlating with the parasite reproductive development and the liver pathology.
Large parasites cause blockage of the bile duct, leading to duct enlargement. The inflammation at the
liver tissue quickly leads to cirrhosis (Figure 47.5). The bile flow of the bile duct is interrupted and easy
to break, resulting in egg granulomas in the liver tissue (Figure 47.6).

47.3.3 Pathology of Liver Fluke Infection in Hamster and Gerbil

Pathology within the liver is correlated to the gross pathology. The liver of uninfected hamster does not
contain inflammatory cell aggregation surrounding the hepatic bile ducts, which are different from those
of the infected hamster. The peak of inflammatory cell aggregation surrounding the hepatic bile ducts
is observed during 14–30 days postinfection and then gradually decreases at 2, 3, and 6 months postin-
fection. During 14–30 days postinfection, neutrophils and eosinophils in the liver tissue are increased.
During chronic infection, monocytes are increased. Moreover, the fibrosis surrounding the hepatic
768 Laboratory Models for Foodborne Infections

Cyprinoid fishes Fishes digestion OV-Metacercariae collection Hamster were orally infected
50 OV-metacercariae

FIGURE 47.4  Metacercariae preparation and animal infection. OV, O. viverrini.

(A) (B) (C)

(D) (E) (F)

FIGURE 47.5  Representative gross liver pathology of the infected hamster (A–C) and gerbil (D–F) at 30 days (A,D),
60 days (B,E), and 90 days (C,F). Gall bladder (G); common bile duct (arrow). Note: Liver obstruction and cirrhosis could
be observed at all time points.

(A) (B) (C)

(D) (E) (F)

FIGURE 47.6  Representative liver pathology of the infected hamster (A–C) and gerbil (D–F) at 30 days (A,D), 60 days
(B,E), and 90 days (C,F). Parasite (P). Note: Fibrosis could be observed in infected gerbil at all time points.

bile ducts appears and thickens in line with the duration of the infection, which corresponds to gross
­pathological bile ducts (Figure 47.7).

47.3.4 The Level of White Blood Cells in the Liver Fluke Infection in Hamsters
The levels of white blood cells in infected hamster in the first period (<2 months postinfection) are signif-
icantly increased in comparison with that in the uninfected hamster (Table 47.2). Among various types
of white blood cells, lymphocytes are higher in all phases of the infection. Neutrophils significantly
increase at 6 months postinfection. Eosinophils peak at the first period of ~1 month and decrease at
Opisthorchis viverrini 769

FIGURE 47.7  Egg granulomas.

TABLE 47.2
Blood Cell Counts in Uninfected/Infected Hamster
Estimated White Blood Cell Count (Months Postinfection)
1 2 3 6
Experimental
Group Mean ± SEM Mean ± SEM Mean ± SEM Mean ± SEM
Total white blood Normal 2040.0 ± 160.0 2012.5 ± 173.7 1800 ± 115.5 1266.7 ± 176.4
cell count OV 2600.0 ± 739.4 2960.0 ± 865.8 2850.0 ± 895.8 2250.0 ± 639.7
Neutrophil Normal 32.0 ± 3.3 26.7 ± 2.1 25.7 ± 5.4 23.9 ± 2.0
OV 42.2 ± 4.7 20.9 ± 1.8 31.5 ± 6.7 33.5 ± 2.8*
Monocyte Normal 2.8 ± 1.0 4.5 ± 1.0 3.8 ± 1.9 0.0 ± 0.0
OV 17.3 ± 1.6* 21.0 ± 3.5* 14.0 ± 2.4* 0.5 ± 0.5
Eosinophil Normal 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
OV 3.8 ± 1.6* 1.2 ± 0.5* 1.0 ± 0.6 0.0 ± 0.0
Lymphocyte Normal 65.2 ± 4.0 68.8 ± 1.6 72.0 ± 4.0 76.1 ± 2.0
OV 35.0 ± 5.0* 56.9 ± 4.4* 54.0 ± 5.1* 66.0 ± 2.9*
*p = 0.05. OV, O. viverrini.

2 months postinfection. Monocytes are significantly higher during the first 1–3 months and s­ ignificantly
decrease at 6 months postinfection.

47.3.5 Opisthorchiasis-Associated Cholangiocarcinoma
Cholangiocarcinoma (CCA) is defined as a carcinoma arising from bile duct epithelium within the
liver, from the large hilar duct and the extrahepatic duct. CCA may arise anywhere in the biliary tree,
but the most frequent sites are the ampulla of Vater, lower end of the common bile duct, hepatic duct, and
the junction of hepatic duct to the common bile duct. CCA is usually small, extending for 1–2 cm along
the duct, thickening the of the duct.12 CCA is a relatively rare cancer in the western world, with the inci-
dence of 1–2 cases per 100,000 people13 but rising worldwide over the past several decades. Recent epi-
demiological studies revealed that the incidence of CCA has increased in the United States, the United
Kingdom, and Australia.14,15 The prevalence of CCA is higher in Thailand, Loa PDR, and Cambodia
770 Laboratory Models for Foodborne Infections

where opisthorchiasis is endemic.3,16 CCA is a crucial public health problem in the Northeastern part of
Thailand. The incidence of CCA is approximately >50% of all liver cancer in Thailand and ~90% of all
liver cancer in the northeastern part of Thailand.17 This incidence rate is the highest in the world.18 It is
well known that both liver flukes O. viverrini and C. sinensis have been classified as class I carcinogens
by the International Agency for Research on Cancer owing to their association with CCA and hepato-
carcinoma in humans.3,19,20
The risk factors for CCA in northeastern part of Thailand was a clear association with past or pres-
ent infection with O. viverrini, as indicated by raised serum antibodies, and at least two-thirds of cases
can be attributed to this cause, and males may be at a higher risk than females.21,22 CCA patients who
have been infected with a liver fluke develop this cancer, and patients who had been infected and treated
for opisthorchiasis have increased risk of developing CCA than the noninfected population.23 Several
experiments and clinical reports revealed the correlation between O. viverrini and CCA development,
including clinical studies3,19,20,21,24 and experimental models.25–28
The mechanism of O. viverrini-associated CCA relates to chronic inflammation resulting in a
combination of mechanical damage, parasite secretions, and immunopathology.28 The primary
pathological change in epithelial desquamation may cause mechanical irritation by the liver fluke
and/or produce metabolic products from the parasite. The mechanical damage is caused by oral
sucker and ventral sucker of fluke during activities of feeding and migrating in the bile duct. The
fluke eggs are trapped in the lumen of the bile duct, leading to ulcerates, granulomatous inflamma-
tion, and cholelithiasis.29 The excretory-­secretory (ES) products released by O. viverrini cause cell
proliferation.30,31 After infection for 2  weeks, the bile duct epithelium hyperplasia and periductal
inflammatory cells, predominantly eosinophils and lymphocytes, are observed. Furthermore, immu-
nopathological ­processes lead to the long-standing hepatobiliary damage. Host immune response can
induce the inflammatory cell infiltrations surrounding the hepatic bile ducts for killing the parasite
by reactive oxygen species (ROS) and reactive nitrogen species (RNS).32 ROS not only destroy para-
site, but also destroy host tissue and DNA leading to DNA fragmentation, which is well known during
apoptosis. Moreover, RNS, which are produced during inflammation, may play a role in initiation
and subsequent modulation stage of CCA development through DNA mutation leading to cancer
development.33,34 In O. viverrini-associated CCA, Sripa and Kaewkes found O. viverrini antigens
located in epithelium bile ducts in hamster model, and Thuwajit et al.30,31 reported that the ES prod-
ucts of O. viverrini increased NIH3T3 cell proliferations, which may enhance bile duct proliferation
and account for nitrative and oxidative DNA damage in hamsters infected with O. viverrini.34 The
development of cholangiocarcinogenesis involves many changes of cancer-relevant genes. Several
studies on the molecular mechanisms of O. viverrini-associated CCA have highlighted the potential
influences of KRAS and TP53 mutations in a large number of biological processes, including hamster
CCA,35 interleukin 6 (IL-6),36 transforming growth factor-β (TGF-β), IL-8, tumor necrosis factor-α
(TNF-α) and platelet-derived growth factor (PDGF),37 proteomic profiling of CCA,38 Rb1 and related
genes in CCA,39 comparative protein expression profiles (MUC5AC, Akt2, CK8, annexin II, and
VEGF A) of hilar and peripheral hepatic CCA,40 MMP-7 and MMP-9 in CCA,41 p38delta/MAPK13
as a diagnostic marker for CCA,42 and c-ski and related genes in CCA.43 However, infection with O.
viverrini alone may not produce CCA, which requires a combination of factors such as O. viverrini
infection, nitrosamine, host immune response, telomerase, alcohol, and hepatobiliary disease.

47.4 Conclusion
Opisthorchis viverrini is a liver fluke living in the hepatobiliary tract that causes opisthorchiasis
in human and animal reservoir host. The highest prevalence of this disease was found in Southeast
Asia, especially in Thailand, Cambodia, and Lao PDR. Chronic infection with O. viverrini enhances
the hepatobiliary diseases including CCA, which is a rare cancer but a highly fatal disease in the
world. However, the highest prevalence of CCA is in northeastern Thailand, which correlates to
the O. viverrini infection and other combination factors. To date, one effective anthelmintic drug
Opisthorchis viverrini 771

is praziquantel, but the CCA remains high. Therefore, use of animal models for the investigation
of O. viverrini pathogenesis is important. Syrian hamster is the best animal model for the study of
O. viverrini infection and CCA.

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J Cancer Prev 5, 118–25 (2004).
18. Vatanasapt, V. et al. Cancer incidence in Thailand, 1988–1991. Cancer Epidemiol Biomarkers Prev 4,
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19. de Martel, C., Plummer, M. & Franceschi, S. Cholangiocarcinoma: descriptive epidemiology and risk
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23. Haswell-Elkins, M.R., Satarug, S. & Elkins, D.B. Opisthorchis viverrini infection in northeast Thailand
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31. Thuwajit, C. et al. Gene expression profiling defined pathways correlated with fibroblast cell ­proliferation
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32. Coussens, L.M. & Werb, Z. Inflammation and cancer. Nature 420, 860–7 (2002).
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35. Tangkawattana, S., Kaewkes, S., Pairojkul, C., Tangkawattana, P. & Sripa, B. Mutations of KRAS and
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in cholangiocarcinoma patients: evaluation of diagnostic accuracy. BMC Gastroenterol 9, 30 (2009).
42. Tan, F.L. et al. p38delta/MAPK13 as a diagnostic marker for cholangiocarcinoma and its involvement
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48
Paragonimus

Dongyou Liu

CONTENTS
48.1 Introduction................................................................................................................................... 773
48.1.1 Classification and Morphology..........................................................................................774
48.1.1.1 Classification......................................................................................................774
48.1.1.2 Morphology........................................................................................................774
48.1.2 Life Cycle and Epidemiology............................................................................................774
48.1.3 Clinical Features...............................................................................................................775
48.1.3.1 Acute Paragonimiasis (Early-Stage Disease)....................................................775
48.1.3.2 Chronic Pleuropulmonary Paragonimiasis (Late-Stage Disease).................... 776
48.1.3.3 Ectopic Paragonimiasis..................................................................................... 776
48.1.4 Diagnosis.......................................................................................................................... 776
48.1.5 Treatment and Prevention................................................................................................. 777
48.2 Laboratory Models........................................................................................................................ 777
48.2.1 Animal Models................................................................................................................. 777
48.2.1.1 Rodents............................................................................................................. 778
48.2.1.2 Cats................................................................................................................... 778
48.2.1.3 Dogs.................................................................................................................. 778
48.2.2 In Vitro Cultures............................................................................................................... 778
48.3 Conclusion..................................................................................................................................... 779
References............................................................................................................................................... 779

48.1 Introduction
The genus Paragonimus covers a large group of flatworms that are associated with parenchymal and/or
pleural lung infections (thus collectively known as lung flukes). Although Naterer made initial obser-
vation of lung flukes in 1828, it was Coenraad Kerbert who first described Paragonimus ­westermani
(originally named Distoma westermani) infection in a Bengal tiger ­suffering from ­pneumonia at the
Amsterdam zoological gardens in 1878. In the subsequent year, B. S. Ringer ­identified P. wester-
mani in the lungs of a human patient who died of an aortic aneurysm, thus confirming the first case
of human paragonimiasis. Following the establishment of the genus Paragonimus (“para”—Greek
word meaning “on the side of” and “gonimos”—Greek word meaning “gonads or genitalia”) by
Max Braun in 1899, in which P. westermani was the type species, much work has been done toward
the identification of many other species in this genus, including the independent, near-simultaneous
discovery of the only North American Paragonimus species (P. kellicotti) by Henry Ward and D. S.
Kellicott in 1894 [1,2].

773
774 Laboratory Models for Foodborne Infections

48.1.1 Classification and Morphology

48.1.1.1 Classification
Taxonomically, the genus Paragonimus belongs to the family Paragonimidae, suborder Troglotremata,
order Plagiorchiida, class Trematodea, phylum Platyhelminthes (i.e., flatworms), kingdom Animalia.
Currently, the genus Paragonimus comprises more than 50 species, including Paragonimus africanus,
Paragonimus bangkokensis, Paragonimus caliensis, Paragonimus compactus, Paragonimus ecuador-
iensis, Paragonimus harinasutai, Paragonimus heterotremus, Paragonimus hokuoensis, Paragonimus
hueitugensis, Paragonimus iloktsuenensis, Paragonimus kellicotti, Paragonimus macrorchis,
Paragonimus mexicanus, Paragonimus miyazakii, Paragonimus ohirai, Paragonimus paishuihoensis,
Paragonimus peruvianus, Paragonimus proliferus, Paragonimus pseudoheterotremus, Paragonimus
pulmonalis, Paragonimus sadoensis, Paragonimus siamensis, Paragonimus skrjabini, Paragonimus
uterobilateralis, Paragonimus vietnamensis, and Paragonimus westermani, in addition to a number of
unassigned species [3–6]. Of these, predominant human-infecting Paragonimus species (and their geo-
graphic distribution) are P. westermani (Asia, India, Philippines, and New Guinea), P. miyazakii (Japan),
P. skrjabini (China, Southeast Asia), P. heterotremus (Thailand, China, Southeast Asia), P. hueitungensis
(China), P. uterobilateralis (West Africa), P. africanus (West Africa), P. kellicotti (North America), and
P. mexicanus (Central America, South America) [1,2,7–16].

48.1.1.2 Morphology
Paragonimus adult worm has an oval- shaped body (of 7.5–12 mm in length, 4–6 mm in width, and 3.5–5 mm
in thickness), composed of smooth muscle that is protected by a thick tegument, which is in turn covered
with variably scattered spines. Both the oral terminal sucker (0.19 mm in diameter) and ventral sucker
(or acetabulum, 0.12 mm in diameter) are round and muscular. The lobed ovary is located behind the
acetabulum and anterior to the two branched testes. Between the ovary and the acetabulum are the semi-
nal receptacle, the tightly coiled uterus, and the thick-walled terminal part (metraterm). The digestive
system comprises a truncated pharynx and esophagus that bifurcates early into paired ceca [17].
Paragonimus eggs (80–120 × 45–70 μm) are ovoid, thick-shelled (measuring 2–4 μm in thickness),
golden brown, and operculate (showing a shoulder- or ridge-like structure). The eggs are produced by
self-fertilization (hermaphrodites), and more often by cross-fertilization in lung or pleura-encysted pairs.
Following release into bronchioles, the eggs are coughed up along with sputum, and then either expecto-
rated or swallowed for later discharge in feces.
Morphological features useful for the identification of Paragonimus adult worm include size and
shape, the patterns of lobation of the ovary and testes, and the appearance of the tegumental spines.
For example, the P. kellicotti group has tegumental spines arranged in groups, whereas the P. africanus
group shows individual spines. Microscopic observation of the thick shell-walled, unembryonated, oper-
culated eggs within sputum or feces is a clear indication of paragonimiasis, since other helminthic para-
sites that produce operculate eggs (e.g., trematodes Clonorchis sinensis, Opisthorchis species, Fasciola
hepatica, and Fasciolopsis buski, as well as cestode Diphyllobothrium latum) do not cause pulmonary
infections. In addition, the morphological features of Paragonimus adult, cercariae, metacercariae, and
metecercarial cyst may also be exploited for differentiation from other trematodes [17].

48.1.2 Life Cycle and Epidemiology

Paragonimus species exhibit a circuitous life cycle that involves a mammal as definitive host (in which
the adult resides and undergoes sexual reproduction) and aquatic snails and crustaceans as specific and
sequential intermediate hosts.
The life cycle begins when fertilized, operculate eggs produced by sexually competent adult worms
are discharged in sputum or feces from mammalian definitive host, enter fresh or brackish water, and
hatch (embryonate) into ciliated miracidia. The miracidia then invade the soft parts of freshwater snails
(the first intermediate host), and undergo two asexual reproductive cycles (including the formation of
sac-like sporocysts in the hemocoel of the snail and two generations of rediae in the lymphatic system
Paragonimus 775

of the snail) to become infective cercariae (with anterior stylets and short tails). The infective cercariae
released by snails into freshwater (or infected snails eaten by freshwater crustaceans) penetrate the gills
and other soft tissue sites on the exoskeletons of crustaceans (the second intermediate host) and develop
into metacercariae [18].
Following consumption of raw, undercooked, or alcohol-pickled freshwater crustaceans (crabs or cray-
fish or undercooked infected tissues from a paratenic/nonpermissive mammalian host such as rats, pigs,
and possibly water birds), the permissive mammalian definitive host acquires the metacercariae, which
move from the intestine into the abdomen and the lungs [19]. In the lungs, the metacercariae mature into
adult worm, encyst (being spherical to ovoid in shape and up to 2 cm in diameter, each containing two or
more adult flukes or a diploid or triploid variant as in the exceptional case of P. westermani), and cross-
fertilize each other. With the eventual rupture of the cyst in the lungs, the eggs are released and coughed
up or swallowed and excreted in the feces for the next cycle of infection.
The first molluscan intermediate hosts of Paragonimus species include 54 species of freshwa-
ter snails from the families Pleuroceridae and Thiaridae of the superfamilies Cerithioidea and
Rissooidea; the second crustacean intermediate hosts consist of freshwater crabs or crayfish (belong-
ing to 53 species of 21 genera); and the definitive hosts are omnivorous and carnivorous crustacean-
eating mammals such as dogs, cats, tigers, and humans [20]. In the case of P. kellicotti, besides
domestic dogs and cats, wild animals [including skunks (Melphitis mephitis), red foxes (Vulpes
vulpes), coyotes (Canis latrans), mink (Mustela vison), and bobcats (Felis rufus)] also function as
competent definitive hosts.
Reflecting the natural distribution of permissive definitive and intermediate hosts that support infec-
tion and the customs of consuming raw or undercooked seafood or wild boar or using raw seafood
preparation as folk medicine, Paragonimus species have an endemic presence in East and South Asia
(e.g., China, Korea, Japan, Laos, Thailand, Vietnam, Malaysia, and the Philippines), sub-Saharan Africa
(e.g., Cameroon), and in the Americas (from Peru to Canada). It is of interest to note that P. westermani
and other Asian species have been traditionally referred to as Asian lung flukes, and P. kellicotti and
Latin American species have been called American lung flukes [11,21]. Worldwide, about 293 million
people are at risk of Paragonimus infection, and nearly 20 million people (of both sexes and all age) are
being infected [2].

48.1.3 Clinical Features
In comparison with other taxonomically related flukes in the class Trematoda (intestinal fluks, liver
flukes, and blood flukes or schistosomes), lung flukes (formerly oriental lung fluke, pulmonary disto-
matosis, and benign endemic hemoptysis) of the genus Paragonimus demonstrate a unique tropism in
their definitive hosts for encysting within the parenchyma or on the pleural surfaces of the lungs, and
occasionally in ectopic sites such as the brain (cerebral paragonimiasis), mesentery and visceral organs,
abdominal wall, muscles, and subcutaneous tissues (trematode larva migrans). Thus, human paragoni-
miasis may be categorized as acute paragonimiasis (early-stage disease), chronic pleuropulmonary para-
gonimiasis (late-stage disease), and ectopic paragonimiasis (with the parasite in a location other than the
lungs).

48.1.3.1 Acute Paragonimiasis (Early-Stage Disease)

Following the ingestion of the infective metacercariae and subsequent migration to the pleural space,
the acute paragonimiasis may be asymptomatic or subclinical in about 20% of patients, while presenting
with fever, abdominal and chest pain, diarrhea, fatigue, and urticaria in other patients (often with heavy
worm burdens) with the possibility of eosinophilia (as high as 500–80,000 × 109 eosinophils/mm3 com-
pared to the normal reference range of up to 0.45 × 109/mm3).
The differential diagnosis of acute paragonimiasis with fever, abdominal pain, and diarrhea may
include viral and bacterial causes of acute gastroenteritis (e.g., caliciviruses, Salmonella, Shigella,
Vibrio, and Campylobacter species, as well as Giardia parasite). The occurrence of eosinophilia as seen
in acute paragonimiasis may also be attributed to other migrating parasites (e.g., Ascaris) [22].
776 Laboratory Models for Foodborne Infections

48.1.3.2 Chronic Pleuropulmonary Paragonimiasis (Late-Stage Disease)

The chronic pleuropulmonary paragonimiasis reflects host responses to Paragonimus worms that migrate
to their final destination in the pulmonary parenchyma, encyst, and produce fertilized eggs. Depending
on the precise location and number of the parasitic cysts and associated sequelae, cough, blood-tinged
sputum (with hemorrhage into the airspace resulting in hemoptysis, and into the pleural space leading
to hemothorax), dyspnea, pleuritic chest pain, fever, crepitation, weakness, rhonchi, hoarseness, and
breathlessness may be evident (implying bronchiectasis, interstitial pneumonitis, or bronchopneumonia).
The radiological findings based on computed tomography (CT) or magnetic resonance imaging (MRI)
scans in patients with pleuropulmonary paragonimiasis include consolidation, pleural effusions (due to
worm penetration into the pleural cavity and the lung), cystic lesions, linear streaks (due to worm pen-
etration and migration of the lung parenchyma, resulting in burrow tracts of 2–4 × 2–7 mm), nodules (due
to cyst formation), pleural thickening, ring shadow, calcified lesions, and adenopathy [23,24].
For differential diagnosis, chronic pleuropulmonary paragonimiasis showing cough and hemoptysis
in conjunction with cavitary changes (cyst production) may be confused with tuberculosis. In this case,
use of acid-fast stains for respiratory specimens and Paragonimus serological testing are valuable. Other
causes of lung disease that may be misdiagnosed with pleuropulmonary paragonimiasis include bacterial
pneumonia, lung abscess, and echinococcosis. In addition, the presence of eosinophilia and an elevated
IgE level observable in pleuropulmonary paragonimiasis may also be found in other parasitic infec-
tions (e.g., strongyloidiasis, ascariasis, toxocariasis, and ancylostomiasis), fungal infections of the lungs
(e.g., coccidioidomycosis and bronchopulmonary aspergillosis), and noninfectious diseases (e.g., Churg–
Strauss syndrome, which is an autoimmune vasculitis predominantly involving blood ­vessels of the
lungs).

48.1.3.3 Ectopic Paragonimiasis
Besides the pleurae and lungs, Paragonimus parasites may migrate to other locations (e.g., the brain,
skin, breast, adrenal gland, heart, mediastinum, and genital organs), especially during heavy infections.
More common in children than in adults, cerebral paragonimiasis often produces two manifestations:
an expansive, space-occupying lesion in the brain (particularly the cerebral cortex) and a meningitis or
meningoencephalitis due to the migration of the worm, with common clinical signs ranging from head-
ache, vomiting, seizures, personality changes, decline of cognitive function, coma, to death (through
herniation caused by increased intracranial pressure). The next common site for ectopic lesions of para-
gonimiasis is the skin, resulting in multifocal skin lesions (e.g., subcutaneous nodules).
The pathogenic mechanisms of paragonimiasis largely reflect the host’s immune responses to
Paragonimus worms and eggs that enter and migrate from the intestines to the lungs, causing edema,
effusion, and subsequently fibrosis that entraps and restricts the lungs, contributing to the associated
signs and symptoms (e.g., shortness of breath). While nodules most likely contain immature cysts,
the mature cysts harboring the adult worms may gradually become fibrotic and die. The cysts with
dead worms degenerate, form scars that hold residual eggs entrapped in the fibrous tissue, and become
calcified.

48.1.4 Diagnosis
Given its largely nonspecific symptoms, clinical diagnosis of paragonimiasis based on clinical observa-
tions, radiological findings, dietary history, and travel history is insufficient and inconclusive [23,24].
Therefore, several conventional laboratory procedures have been routinely used to confirm the pres-
ence of Paragonimus eggs spp. in sputum (through alkaline decontamination and centrifuge sedimenta-
tion) and/or feces (through formalin–ether concentration). Considering that microscopic examination of
respiratory and/or stool specimens has limited sensitivity (30%–40% for a single sputum specimen and
54%–89% for multiple sputum specimens in comparison to 11%–15% for a single stool specimen and
about 25% for three stool specimens) and relatively low specificity, serological tests (e.g., ELISA for IgG
in serum and/or pleural effusion fluid) are helpful when clinical suspicion for paragonimiasis is high
Paragonimus 777

but Paragonimus eggs are not detected [14,25]. In addition, molecular methods based on nucleic acid
amplification have been applied in recent years to further enhance the sensitivity, specificity, and speed
of laboratory diagnosis of paragonimiasis [26–30].
Among various nucleic acid amplification techniques that have been developed to date, polymerase
chain reaction (PCR) has been a standout due to its unprecedented sensitivity, specificity, speed, and
robustness. In a relatively short period since its inception, PCR (and its variants such as conventional
PCR, random amplified polymorphic DNA-PCR, multiplex PCR, and real-time PCR) has proven to be
highly valuable for improved detection and/or species discrimination of Paragonimus parasites directly
from clinical samples (e.g., lung biopsy specimens, bronchoalveolar lavage fluid, sputum, pleural fluid,
or feces) [31]. The commonly used genetic targets for Paragonimus diagnosis include the internal tran-
scribed spacer (ITS) regions of ribosomal genetic complexes, particularly ITS2, and the mitochondrial
cytochrome c oxidase gene (CO1) [32–36].
Sugiyama et al. [37] established PCR-restriction fragment length polymorphism (RFLP) and spe-
cific PCR with primers from the second internal transcribed spacer (ITS2) region of ribosomal DNA
to discriminate the metacercariae of the lung flukes, P. westermani and P. miyazaki, both of which
are found in the same freshwater crab species and are morphologically similar. In addition, Sugiyama
et al. [38] successfully applied primers from the second internal transcribed spacer (ITS2) region of the
nuclear ribosomal DNA in PCR-RFLP and multiplex PCR for precise differentiation between individual
metacercariae of P. heterotremus and P. westermani that occur in Thailand. Using a similar approach,
Sugiyama et al. [39] developed multiplex PCR and PCR-RFLP (with ScrFI) that enabled differentia-
tion of metacercaria from five Paragoniumus species occurring in Thailand, including P. heterotremus
(ca. 310 and 520 bp), P. westermani (ca. 140 and 520 bp), P. siamensis (520 bp), P. bangkokensis ­(520 bp),
and P. harinasutai (520 bp). Digestion of the 520 bp products with restriction enzyme ScrFI yielded three
bands (ca. 60, 210, and 250 bp) for P. harinasutai, two bands (ca. 250 and 270 bp) for P. bangkokensis,
and an uncut band (520 bp) for P. siamensis. In a more recent study, Tantrawatpan et al. [40] described a
real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) with melting curve analy-
sis for specific and sensitive detection of P. heterotremus eggs in the feces of infected cats, with the
detection limit of 10(–3) ng of P. heterotremus genomic DNA or 10 eggs of P. heterotremus per gram of
cat feces.

48.1.5 Treatment and Prevention

Being able to separate or disrupt the tegument of Paragonimus species, praziquantel (75 mg/kg/day
given in three doses for 2 days) represents the therapeutic agent of choice for pulmonary paragoni-
miasis, with a 71%–75% cure rate after 1 day, and 86%–100% cure rate after 2 days, and a complete
(100%) cure rate after 3 days. The side effects of praziquantel may include dizziness, headache, and
gastrointestinal disturbance. Other medications such as bithionol (at 30–50 mg/kg on alternate days for
10–15 doses), niclofan, and triclabendazole (at 10 mg/kg twice a day) also provide high cure rates for
paragonimiasis [41,42]. However, surgery may be necessary for patients with complicated pleural dis-
ease or cerebral paragonimiasis. Prevention of human paragonimiasis should be centered on thorough
cooking of an infected crustacean (crab or crayfish) to kill all stages of the parasite, and avoiding eating
raw, even if pickled, crustaceans.

48.2 Laboratory Models
48.2.1 Animal Models
A number of laboratory animals have been attempted as experimental models for Paragonimus spe-
cies, including rodents (rats, mice, Syrian hamsters, and Mongolian gerbils), dogs, and cats, with varied
success [43]. While dogs and cats, especially puppies and kittens, are susceptible to most Paragonimus
species prevalent in Asia, rats and mice serve well as paratenic hosts when infected naturally or experi-
mentally with some Paragonimus species.
778 Laboratory Models for Foodborne Infections

48.2.1.1 Rodents
Rats have been found to be susceptible to P. westermani and P. ohirai infection, with metacercaria
d­ eveloping to adult worms that are capable of producing eggs [44,45].
Narain et al. [46] demonstrated that outbred Wistar rats infected with P. heterotremus metacercariae
produced adult worms in the lungs and pleural cavity, with some immature flukes being present in the
skeletal muscles, highlighting the utility of outbred Wistar rats as a model for pulmonary P. heterotremus
infection.
Weina et al. [47] showed that Syrian hamsters infected with 3–16 metacercariae of P. kellicotti
displayed acute pleuritis, reactive mesothelial hyperplasia, subpleural accumulations of reactive and
mature plasma cells, neovascularization, fibrohistiocytic thickening with and without giant cells,
raised fibroconnective tissue lesions, and granulomatous inflammation with hemorrhage within
35 days post infection (dpi). In addition, perivascular plasmacytic (lymphocytic) infiltrate, multifocal
bronchopneumonia, and parenchymal necrotizing suppurative granulomatous inflammation, hemor-
rhagic pneumonia, and diffuse sprinkling of eosinophils, neutrophils, and intraalveolar macrophages
were also observed. These results confirmed the suitability of Syrian hamsters as a small animal
mortality model for P. kellicotti.
Fischer et al. [48] reported that Mongolian gerbils (Meriones unguiculatus) infected intraperitone-
ally or by oral gavage with three to eight metacercariae of P. kellicotti, developed signs of apathy,
weight loss, dehydration as early as 14 dpi, and yielded mature, gravid lung flukes as early as 39 dpi,
with 69% of infected gerbils succumbing to the infection by 49 dpi. Necropsies revealed pulmonary
hemorrhage with necrosis, and flukes as long as 8 mm were recovered from intrathoracic tissues.
Thus, gerbils appear to be a suitable small animal for producing P. kellicotti parasite material and
for studying parasite migration, immunobiology, and pathogenesis. Sanpool et al. [49] also described
that Mongolian gerbils injected intraperitoneally with P. macrorchis metacercariae formed adult
worms in the lungs 45 dpi.

48.2.1.2 Cats
Weina et al. [50] noted that cats infected orally with 2–30 P. kellicotti metacercariae underwent changes
in the lungs of intense eosinophilic pneumonia, granulomatous pneumonitis, squamous epithelial-lined
cyst formation of bronchogenic origin, and partial resolution of the host response. Fan et al. [51] showed
that cats fed with P. pulmonalis metacercariae developed mature adult worms in the lungs and the pleu-
ral cavity. Interestingly, while eggs from worms encased in a cyst were passed in the feces, those from
worms in the pleural cavity were only found on the lung surface and pleural cavity.

48.2.1.3 Dogs
Xue et al. [41,42] observed that dogs infected with 100 P. westermani metacercariae developed adult
worms in the lungs. In early study, Yokogama et al. [44] demonstrated dogs’ susceptibility to pulmonary
P. ohirai infection after animals fed with metacercariae developed adult worms in lungs. Tsubokawa et al.
[45] also noted that dogs infected with P. westermani metacercaria yielded egg-producing adult worms.
Furthermore, Singh et al. [43] found that only puppies appeared suitable for pulmonary P. ­heterotremus
infection whereas rats, mice, guinea pigs, and rabbits were largely refractory to oral infection with
metacercariae.

48.2.2 In Vitro Cultures

Yokogawa et al. [52] reported that excysted metacercariae of P. westermani maintained at 37°C in equal
parts of cat serum and Tyrode’s solution for 3 weeks, then supplemented with chick embryo extract
and cat blood cells, remained alive after 98 days. The average length of metacercaria increased from
0.6 to 2.0–2.5 mm at rest (and up to 4 mm when stretching) along with the development of the male
­reproductive system (especially the testes).
Paragonimus 779

48.3 Conclusion
The genus Paragonimus comprises more than 50 trematode species whose adult worms inhabit the lungs
of various mammalian hosts (so called lung flukes), and whose eggs and larval stages are found in snail
and crustacean hosts. Of the nine human-infecting Paragonimus species, five occur mainly in Asia
(P. westermani, P. miyazaki, P. skrjabini, P. heterotremus, and P. hueitungensis), two in Africa (P. uter-
obilateralis and P. africanus), and two in the Americas (P. kellicotti and P. mexicanus). Humans usually
acquire Paragonimus infection through consumption of raw or undercooked crustaceans or meat from
a paratenic host, with serious consequence. Clearly, continued application of rapid, sensitive, and spe-
cific laboratory diagnostic procedures and prompt implementation of appropriate treatment regimens are
critical for limiting the harmful effects of human paragonimiasis. However, to keep an upper hand over
Paragonimus parasites, it is essential to use appropriate laboratory models that will help e­ lucidate the
molecular mechanisms of host–parasite interactions and pathogenesis, and contribute to the ­development
of innovative control strategies against paragonimiasis.

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49
Taenia

Dongyou Liu

CONTENTS
49.1 Introduction................................................................................................................................... 783
49.1.1 Classification and Morphology......................................................................................... 784
49.1.1.1 Classification..................................................................................................... 784
49.1.1.2 Morphology....................................................................................................... 784
49.1.2 Life Cycle and Epidemiology........................................................................................... 786
49.1.2.1 Life Cycle.......................................................................................................... 786
49.1.2.2 Epidemiology.................................................................................................... 786
49.1.3 Clinical Features.............................................................................................................. 786
49.1.4 Diagnosis.......................................................................................................................... 787
49.1.5 Treatment and Prevention................................................................................................. 787
49.1.5.1 Treatment.......................................................................................................... 787
49.1.5.2 Prevention......................................................................................................... 788
49.2 Laboratory Models........................................................................................................................ 788
49.3 Conclusion..................................................................................................................................... 788
References............................................................................................................................................... 789

49.1 Introduction
The genus Taenia encompasses a large group of parasitic tapeworms that undergo three distinct stages
of development (i.e., adult worm, eggs, and larva/metacestode) during their life cycles and require two
mammalian hosts for transmission. The ribbon-like adult worm (so called tapeworm) resides in the intes-
tines of carnivores or omnivores, causing intestinal taeniasis, whereas the cyst-like larva (metacestode)
occurs in the internal organs of herbivores or omnivores, causing cysticercosis or neurocysticercosis
(representing different forms of taeniasis).
Of the 45 described Taenia species, three [i.e., Taenia solium (pork tapeworm), Taenia saginata (beef
tapeworm), and Taenia asiatica (Asian tapeworm)] are known to infect humans. Indeed, as the only
definitive host for these three species, human infection with the adult worms leads to intestinal parasit-
ism, which is largely asymptomatic. However, the larva (metacestode) of T. solium causes cysticercosis
(cysticercus cellulosae) in pigs and humans, that of T. saginata causes cysticercosis (cysticercus bovis) in
cattle, and that of T. asiatica causes cysticercosis (cysticercus viscerotropica) in pigs (Table 49.1).
Although tapeworms were mentioned in ancient Egyptian works dated back to 2000 BC, as well as
in the History of Animals written by Greek author Aristotle (384–322 BC), DNA analyses point out the
likelihood that ancestors of modern humans in Africa had acquired parasite through consumption of
infected antelope and bovid meats more than 10,000 years ago and subsequently passed it to other ani-
mals such as domestic pigs. A more recent description of cysticercosis was made by Johannes Udalric
Rumler in 1555; however, the connection between tapeworm and cysticercosis was not established until
the middle of the 19th century.

783
784 Laboratory Models for Foodborne Infections

49.1.1 Classification and Morphology

49.1.1.1 Classification
The genus Taenia belongs to the family Taeniidae, order Cyclophyllidea (terrestrial cycles, scolex
with suckers), subclass Eucestoda (segmented, hermaphroditic), class Cestoda (tapeworms), phylum
Platyhelminthes (flatworms), kingdom Animalia.
Members of the family Taeniidae are characterized by their terrestrial life cycle, which includes three
developmental stages (eggs, larva, and adult) and involves a carnivorous/omnivorous definitive host for
adult worm, and a herbivorous/omnivorous intermediate host for larva (metacestode). Of the two well-
recognized genera (Echinococcus and Taenia) within the family Taeniidae, the genus Echinococcus is
monophyletic, with a notable similarity in morphology, developmental processes, and genetic makeup,
and is separated into nine species: E. granulosus sensu stricto (including the former genotypic variants
G1–3), E. felidis (the former “lion strain”), E. equinus (the “horse strain,” genotype G4), E. ortleppi (the
“cattle strain,” genotype G5), E. canadensis, E. multilocularis, E. oligarthra, E. vogeli, and E. shiquicus
[1]. On the other hand, the genus Taenia is a highly diverse, paraphyletic group with 45 species identified
to date, including T. arctos, T. asiatica (synonym T. saginata asiatica), T. brachyacantha, T. crassiceps,
T. dinniki, T. gonyamai, T. hydatigena, T. jaipurensis, T. kotlani, T. krabbei, T. krepkogorski, T. laticol-
lis, T. madoquae, T. martis, T. multiceps, T. mustelae, T. omissa, T. ovis, T. parva, T. pisiformis, T. regis,
T. retracta, T. rileyi, T. saginata (synonym Taeniarhynchus saginata), T. saigoni, T. serialis, T. simbae,
T. solium, T. taeniaeformis, T. twitaelli, and T. ursina [2,3]. Among these, T. solium, T. saginata, and T.
asiatica are infective to humans.
Recent examination of nuclear and mitochondrial genes enables further clarification of the genetic
relationships among the representative members of the Taenia genus. Indeed, the nuclear phylogenetic
trees of 18S ribosomal DNA and concatenated exon regions of protein-coding genes (phosphoenol-
pyruvate carboxykinase and DNA polymerase delta) revealed that both T. mustelae and a clade formed
by T. parva, T. krepkogorski, and T. taeniaeformis are only distantly related to other Taenia species.
Similar topologies were also observed in mitochondrial genomic analyses using 12 complete protein-
coding genes. In addition, a sister relationship between T. mustelae and Echinococcus spp. was noted.
These findings led to the proposals to resurrect the genus Hydatigera for T. parva, T. krepkogorski, and
T. taeniaeformis and to create a new genus, Versteria, for T. mustelae as well as T. brachyacantha [4].

49.1.1.2 Morphology
Taenia adult worms (Taenia is derived from Greek ταίvɩα, tainia meaning ribbon, bandage, or stripe)
are flat, ribbon-like in appearance, and white or yellowish-white in color. and consist of a knob-like
head called scolex, a short neck, and a ribbon-like body (also known as strobila). The scolex (hold-
fast organ) often possesses suckers (acetabula), rostellum, and spiny hooks. It is noteworthy that the
scolex of T. solium is spheroidal, with a rostellum (of 1 mm in diameter), which is armed with two
rows of 22–32 hooks; that of T. saginata is cuboidal (of 2 mm in width) without rostellum or hooks;
and that of T. asiatica is spheroidal, with a cuspidal rostellum (of 0.8 mm in width), which has no
hooks (Table 49.1). The strobila (up to 22 m long depending on the species) is composed of a chain
of segments called proglottids, each of which contains a set of male and female reproductive organs
(thus hermaphroditic). The reproductive organs are made up of tubular unbranched uterus (filled with
eggs), ovary, genital pore, testes, and vitelline gland, with testes and ovary opening into a common
genital pore located on the side. Being an acoelomate animal, the adult worm has no body cavity or
digestive system, and relies entirely on its penetrable tegument to absorb nutrients. As body growth
starts from the neck region, immature proglottids are found near the neck, mature proglottids in the
middle, and gravid (oldest) proglottids at the posterior end. On average, T. solium adult worm has
about 1000 proglottids, each producing 50,000 eggs; T. saginata adult worm has about 1000–2000
proglottids, each producing 100,000 eggs; T. asiatica adult worm has 700–900 proglottids, each
producing 80,000 eggs [5].
Taenia eggs are spherical, of about 40–48 µm in diameter, and surrounded by a thick striated wall,
which encases an embryo (oncosphere). After gravid proglottids detach from the strobila or discharge
Taenia 785

TABLE 49.1
Morphological and Biological Characteristics of Human-Infecting Taenia Species
Taenia solium (Pork Taenia saginata (Beef Taenia asiatica (Asian
Tapeworm) Tapeworm) Tapeworm)
Adult worm Adult worm of 2–8 m in length Adult worm of 4–12 m in length Adult worm of about 3.5 m in
resides in human small (as long as 25 m) resides in length resides in human small
intestine; its scolex has four human small intestine; its intestine; its scolex has four
suckers that surround the scolex has four suckers, and an suckers surrounding the
rostellum, with 22–32 hooks apical pit instead of a rostellum rostellum, without hooks; its
arranged in two rows; its and hooks; its strobila contains strobila contains 700–900
strobila contains 1000 1000–2000 proglottids; its proglottids; its gravid proglottid
proglottids; its gravid gravid proglottid has no has posterior protuberances
proglottid has no posterior posterior protuberances
protuberances
Metacestode Cysticercus cellulosae (5–8 × Cysticercus bovis Cysticercus viscerotropica
(Cysticercus) 3–6 mm) occurs mostly in pig (6–10 × 4–6 mm) occurs inside (2 × 2 mm) occurs in liver and
muscle, as fluid-filled cyst muscle, liver, and lungs of lungs in pigs, wild boars, and
(0.5–1.5 cm in diameter) cattle, as fluid-filled cyst occasionally cattle, as fluid-filled
containing single scolex with containing single scolex cyst containing single scolex
hooks; its external surface without hooks; its external with two rows of rudimentary
lacks wart-like formations surface lacks wart-like hooks; its external surface has
formations (or not prominent if wart-like formations
present)
Genome 122–131 Mb in size, with 169 Mb in size, with 13,161 168 Mb in size, with 13,323
11,902–12,481 coding genes coding genes coding genes
Clinical features Humans infected with adult Humans infected with adult Humans infected with adult
worms show minimal worms are usually worms are largely asymptomatic
symptoms; accidental ingestion asymptomatic, although heavy
of embryonated eggs or infection may lead to weight
proglottids (via autoinfection loss, dizziness, abdominal pain,
or contaminated food) by diarrhea, headaches, nausea,
humans leads to cysticercosis, constipation, chronic
which shows three indigestion, and loss of
morphologically distinct forms appetite; cysticercosis is not
[the ordinary “cellulose” observed in humans
cysticercus, with a fluid-filled
cyst and an invaginated scolex;
the intermediate form with a
scolex; the “racemose” form
(20 cm in length and 60 mL of
fluid) with no evident scolex]
Distribution Cosmopolitan, and highly Prevalent in Africa, Europe, Prevalent in Taiwan, South Korea,
prevalent in Mexico, Latin Southeast Asia, South Asia, Indonesia, the Philippines,
America, West Africa, Russia, and Latin America Thailand, China, Vietnam, Japan,
India, Pakistan, China, and and Nepal
Southeast Asia
Sources: Parija, S.C. and Ponnambath, D.K., Trop. Parasitol., 3, 120–124, 2013; Wang, S. et al., Nature Commun., 7, 12845, 2016.

along with feces, eggs are released. Being highly resistant to desiccation and sewage treatment, these
eggs can survive on pastures for weeks. Upon ingestion by intermediate host, the eggs hatch and develop
into infective cysticerci in selective organs (e.g., muscle, liver, and lungs).
Taenia larvae (metacestodes) such as those of T. solium, T. saginata, and T. asiatica form small,
pearly-white, fluid-filled cysts (cysticerci) (hence the common name bladder worms) within which a
single invaginated protoscolex (infective stage) is located [5]. The larvae of other Taenia spp. may show
varied appearance, with some forming a strobilocercus (containing noninvaginated protoscolex) and oth-
ers forming a large coenurus (containing several invaginated protoscoleces).
786 Laboratory Models for Foodborne Infections

49.1.2 Life Cycle and Epidemiology

49.1.2.1 Life Cycle
Members of the family Taeniidae are unique among the subclass Eucestoda in requiring two obligate
mammalian hosts for their life cycles: a carnivorous/omnivorous definitive host for the adult stage, and a
herbivorous/omnivorous intermediate host for the larval stage (metacestode).
Typically, the life cycle begins when vegetation, feed, or water contaminated with taeniid eggs (or
gravid proglottids) are consumed by a herbivorous/omnivorous intermediate host. Once inside, the
embryonated eggs hatch into motile oncospheres within the duodenum and penetrate the intestinal wall,
enter the bloodstream, and differentiate into metacestodes within 70 days (either cysticercoid, cysticer-
cus, or a hydatid cyst) in selective internal organs (liver, lungs, brains, and muscles, etc.). When raw or
undercooked pork and beef containing the fluid-filled cysticerci are eaten by a carnivorous/omnivorous
final host, the cyst (bladder) is digested away, the inverted scolex evaginates under stimuli from the
digestive enzymes of the host, the scolex then embeds itself into the intestinal wall, and the neck buds off
segments to form the strobila. New eggs may appear in the feces of the definitive host within 6–9 weeks,
starting the next cycle of infection [6].

49.1.2.2 Epidemiology
With humans as the definitive host and pigs as the intermediate host, T. solium is present in many parts
of the world where humans live in close proximity with pigs and eat undercooked pork [7]. In addi-
tion, humans may function as aberrant intermediate host after accidental ingestion of embryonated
eggs, either through autoinfection or consumption of contaminated food, leading to neurocysticercosis,
which demonstrates a predilection for brain tissue and other soft muscle tissues [8]. Geographically,
T. solium infection is particularly common in Mexico, Latin America, West Africa, India, Pakistan,
Southeast Asia, China, Russia, and Slavic countries of Europe. Previous reports indicate that the sero­
prevalence of T. solium in areas of Guatemala, Bolivia, and Peru may be as high as 20% in humans and
37% in pigs.
In the case of T. saginata, humans and cattle act as the definitive and intermediate hosts, respectively.
This parasite is found in places where beef is eaten, and is relatively common in Africa, the Middle East,
the Philippines, Eastern Europe, and Latin America.
T. asiatica utilizes humans as the definitive host, and pigs, wild boar, and possibly cattle as the inter-
mediate host. While the adult worm is found in the small intestine of humans, pea-sized, fluid-filled cyst
(cysticercus) is present in liver, serosa, and lungs of pigs and in the liver of cattle. Geographically, the
parasite is essentially restricted to Taiwan, Korea, Indonesia, Nepal, Thailand, and China, in addition to
Japan, the Philippines, and Vietnam [9,10].

49.1.3 Clinical Features
Infection with T. solium adult worm in humans generally involves one to two worms and is often
asymptomatic, although heavy infection may lead to mild intermittent diarrhea or constipation, ane-
mia, indigestion, inappetite, and emaciation, together with urticaria, anal pruritus, and eosinophilia.
However, accidental consumption of T. solium eggs from contaminated vegetables/water or ingestion
of T. solium eggs or proglottids, which rupture within the host intestines, may result in cysticercosis
or neurocysticercosis [11]. In the case of cysticercosis, solid lumps of 1–2 cm in size may develop
under the skin near the trunk and extremities, become painful and swollen, and then resolve. In the
case of neurocysticercosis, cysts of 5–20 mm in diameter may form in the parenchyma of the brain.
In more severe cases (racemose neurocysticercosis), lesions as large as 6 cm in diameter, lobulated,
and may occur in subarachnoid space and fissures. Clinical presentations may include severe head-
aches, dizziness, epilepsy, seizures, dementia, hypertension, lesions in the brain, blindness, tumor-
like growths, hydrocephalus, paraplegy, meningitis, convulsions, and even death.
Taenia 787

Similarly, human infection with T. saginata adult worm is asymptomatic, but heavy infection causes
dizziness, abdominal pain, diarrhea, headaches, nausea, constipation, chronic indigestion, inappetite,
and weight loss. Other rare clinical signs include ileus, pancreatitis, cholecystitis, and cholangitis.
Further, human infection with T. asiatica adult worm is usually asymptomatic. Nevertheless, in severe
cases, damage and bleeding in the stomach and intestine may be observed.

49.1.4 Diagnosis
Identification of Taenia species (T. saginata, T. asiatica, and T. solium) in the definitive host has tradi-
tionally relied on microscopic detection of eggs or proglottids in feces. However, this approach lacks
desired sensitivity and specificity given that the eggs from different Taenia species are morphologically
indistinguishable [12].
Use of computed tomography (CT) or magnetic resonance imaging (MRI) enables detection of cysti-
cerci in the brain, as in the case of human neurocysticercosis. In addition, application of, X-rays facili-
tates detection of calcified cysticerci in the subcutaneous and muscle tissues.
Serological methods (ELISA and IET) based on adult worm excretion-secretion antigens (ES Ag)
(ES33 and ES38) of T. solium have proven useful for confirmation of T. solium taeniasis, with sensitivity
and specificity of >97% and 91%, respectively [13]. Furthermore, serological detection of coproantigens
in fecal specimens allows genus-specific discrimination. Enzyme-linked immunoelectrotransfer blot
(EITB) detects an immunoblot band of 21.5 kDa from T. asiatica, permitting effective differentiation of
asiatica from other taeniid infections.
Utilization of molecular methods targeting various gene regions of Taenia species has greatly
enhanced the diagnosis of taeniasis and cysticercosis [14–25]. Polymerase chain reaction (PCR) amplifi-
cation of ribosomal 5.8S and HDP2 gene sequences enables differentiation among T. solium, T. saginata,
and T. asiatica [26,27]. Sequencing analysis of the variable regions (V1–V5) within nuclear 18S rRNA
gene reveals pertinent phylogenetic details within the genus Taenia. The mitochondrial gene sequence
(mtDNA) provides a valuable molecular marker for uncovering evolutionary relationships among dis-
tantly related taxa, and also for investigating the phylobiogeography of closely related species. A mul-
tiplex PCR based on the primers Ta4978F, Ts5058F, Tso7421F, and Rev7915 from the mitochondrial
gene sequence has streamlined differential diagnosis, molecular characterization, and epidemiological
surveys of Taenia species [28]. Moreover, use of primers (Ta7216F, Ts7313F, Tso7466F, and Rev7915)
from valine transfer RNA and NADH dehydrogenase subunit two genes in multiplex PCR generates
species-specific products of 706, 629, and 474 bp for T. asiatica, T. saginata, and T. solium in a sensitive,
specific, and speedy manner [29].

49.1.5 Treatment and Prevention

49.1.5.1 Treatment
Praziquantel (at 5–10 mg/kg orally once for adults and children) is frequently used to treat active tae-
niasis. It should be noted that praziquantel is cysticidal and may cause inflammation around dying cysts
in patients with cysticercosis, leading to seizures. Niclosamide (at 2 g orally once for adults, 1.5 g orally
once for children weighing >34 kg, 1 g orally once for children weighing 11–34 kg, and 500 mg orally
once for children under 2 years of age) represents an alternative medication for treating Taenia infec-
tion. Stools from treated patients should be collected for 3 days and examined for tapeworm proglottids
for species verification. Furthermore, stools from patients should be reexamined for Taenia eggs 1 and
3 months after treatment to ensure the complete clearance of infection. Another useful medication is
albendazole, which is an effective and safe drug for treating neurocysticercosis. Due to lower cost, fewer
drug interactions, and higher efficiency for both adult worm and larvae, albendazole and mebendazole
are generally preferred over praziquantel [30]. In cases of intraventricular, racemose, or spinal neuro-
cysticercosis, surgical intervention (e.g., direct excision of ventricular cysts, shunting procedures, and
removal of cysts via endoscopy) may be needed.
788 Laboratory Models for Foodborne Infections

49.1.5.2 Prevention
The key for preventing Taenia infections in humans is to break their transmission cycle through stringent
meat inspection, condemnation of infected carcasses for human consumption, proper cooking (80°C) or
freezing (–10°C for 9 days) of meat, sanitary disposal of feces, prohibiting the use of sewage for fertil-
izing pastures, washing salad vegetables, strict personal hygiene, and improved access to clean water.
To prevent Taenia infections in pigs and cattle, it is important to avoid the exposure of pigs and cattle
to environments contaminated with human feces; to eliminate the use of untreated sewage effluent to
irrigate land that is later used by pigs and cattle for forage and food crops; and to vaccinate pigs with
genetically engineered 45W-4B antigens or S3PVAC consisting of three protective peptides (KETc12,
KETc1, and GK1) against T. solium infection [31,32].

49.2 Laboratory Models
As humans act as the only definitive host for T. solium, T. saginata, and T. asiatica, use of laboratory
models is not only necessary but also important in helping elucidate the pathogenesis, host–parasite
relationship, and other related issues concerning taeniasis and cysticercosis [33,34]. From published
data, it is clear that pigs, dogs, cats, and rabbits, even in immunosuppressed status, are relatively poor
hosts for experimental investigation of taeniid infections, On the other hand, various rodent hosts (rats,
mice, hamsters, gerbils, and chinchillas) support the maintenance, growth, and maturation of taeniid
tapeworms, contributing to our improved understanding of taeniasis and cysticercosis [35,36].
In an early study, Wang et al. [37] showed that immunosuppressed Syrian hamsters are susceptible to
T. solium infection, with adult worm developing in the intestine after oral ingestion of cysticerci contained
in severe combined immunodeficiency (SCID) mouse muscles. Separately, Avila et al. [38] demonstrated
that hamsters, gerbils, and chinchillas are adequate experimental models for investigating the growth and
immune responses of T. solium adult worm infection. Furthermore, chinchillas and, to a lesser extent,
hamsters appear to be more permissive than gerbils, allowing longer-term survival and development of
T. solium parasites. In a more recent report, Verastegui et al. [39] described a useful neurocysticercosis
model based on Holtzman rats. It was noted that rats injected extraparenchymally as well as intraparen-
chymally with activated T. solium oncospheres developed cysticerci in the brain tissue after 4 months; that
the cysticerci were present in the parenchymal, ventricle, or submeningeal brain tissue; and that epilepsy
was observed in 9% of rats with neurocysticercosis. The age of rats appeared to be crucial for the success
of infection, although the sites of injection and T. solium oncosphere dosages were largely irrelevant.
Several reports showed that nonobese diabetic SCID (NOD/Shi-scid) mice of both sexes are suscep-
tible to infection with in vitro hatched oncospheres of human-infecting Taenia species (T. solium, T. saginata,
and T. asiatica), producing cysticerci comparable to those developed in their known intermediate host
animals [40–44]. On the other hand, only female SCID mice of BALB/c, C57BL, or C.B-17 inbred
strains allow the development of cysticerci from oncospheres of human taeniid species [44].
Additionally, normal C3H/HeN female mice were showed to initiate Th1 cells immune response
against T. asiatica during the early stages of oncosphere infection [45]. Similarly, hamsters are also
capable of eliciting cytokine expression at the anchor site of T. solium [46–48].
Chowdhury et al. [49] successfully induced neurocysticercosis in rhesus monkey with 12,000 T. solium
eggs, resulting in development of hyperexcitability, epileptic seizures, muscular tremors, digital cramps at
10 days postinfection, and paralysis of limbs and death days post infection. Necropsy of the infected mon-
key revealed numerous cysticerci in the brain, and histopathological examination uncovered liquefactive
necrosis and formation of irregular cystic cavities lined by atrophied parenchymal septa with remnants of
neuropil of the cerebrum.

49.3 Conclusion
Members of the genus Taenia are parasitic tapeworms that involve two mammalian hosts (a carnivorous/
omnivorous definitive host and a herbivorous/omnivorous intermediate host) during their life cycles.
Taenia 789

Taenia infection usually begins when Taenia eggs and proglottids produced by adult worm that reside in
the small intestine of definitive hosts are discharged in stools, and subsequently ingested by intermediate
host. Inside intermediate host, Taenia eggs hatch to release oncospheres, which then penetrate through
intestinal wall, migrate via blood circulation to various internal organs and muscles, and mature into
cyst-like larvae (metacestodes). Once taken up by definitive host, Taenia larvae (metacestodes) release
protoscoleces, which utilize their hooks to anchor onto the intestinal wall and grow into adult worms that
produce eggs for the next cycle of infection.
Of 45 Taenia species identified to date, three (T. solium, T. saginata, and T. asiatica) cause taeniasis
in humans and cysticercosis in pigs and cattle as well as humans. Given that the adult worms of these
human-infecting Taenia species only occur in humans, and the larvae are present in pigs and cattle,
detailed examination of taeniid pathogenesis and host–parasite relationship have been hampered by
ethical and cost concerns. For this reason, use of laboratory models is critically important in helping
to improve our understanding of taeniasis and cysticercosis, and leading to innovative control measures
against these parasites. Fortunately, of various types of laboratory animals available, rodents (e.g., rats,
mice, hamsters, gerbils, and chinchillas) have been shown to support the maintenance, growth, and
maturation of human taeniid tapeworms (in both taeniasis and cysticercosis/neurocysticercosis forms)
and to elicit competent immune responses against these parasites. It is envisaged that in our ongoing
effects to elucidate the molecular basis of host–parasite interactions and to develop novel, tailor-made
control strategies against Taenia parasites, rodents will continue to play an indispensable role [35,50].

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50
Trichinella

Ljiljana Sofronic-Milosavljevic, Natasa Ilic, and Alisa Gruden-Movsesijan

CONTENTS
50.1 Trichinella and Trichinellosis....................................................................................................... 793
50.2 Mouse............................................................................................................................................ 794
50.2.1 Contribution of MHC-Linked and Background Genes to the Control of
the Response..................................................................................................................... 794
50.2.2 Response of the Host during Intestinal and Muscle Phase of the Infection.................... 795
50.2.3 Applied Studies on Mouse Model for Trichinellosis........................................................ 797
50.3 Rat................................................................................................................................................. 797
50.4 Swine............................................................................................................................................. 798
50.4.1 Applied Studies on Swine Model for Trichinella Infection............................................. 799
50.5 Wild Boar...................................................................................................................................... 800
50.6 Horse............................................................................................................................................. 800
50.7 Other Animal Species as Models for Trichinella Infection.......................................................... 801
50.8 Conclusion..................................................................................................................................... 802
References............................................................................................................................................... 803

50.1  T
 richinella and Trichinellosis
Trichinellosis is a foodborne disease caused by the consumption of raw or undercooked meat of dif-
ferent animal origins, e.g., pork, horse, or game, infected with helminth of the Trichinella genus [1,2].
The genus consists of two clades that inhabit mainly striated muscles of the host. Parasites from the first
clade encapsulate, and those from the second do not [3]. Among the so-far identified 12 taxa of the genus,
the species and genotypes of the first clade parasitize only mammals and include T. spiralis, T. britovi
with its closely related genotype T8, T. nativa and related T6, T. murrelli and related T9, T. nelsoni, and
T. patagoniensis. The second clade comprises three species: T. pseudospiralis, which infects mammals
and birds, and T. papuae and T. zimbabwensis, which parasitize mammals and reptiles, respectively [3].
Although Trichinella has cosmopolitan distribution and infects more than 2500 people annually [4],
its infection results in minimal health burden and low mortality rate in human population [5]. Of all
Trichinella taxa, T. spiralis accounts for most human infections [2].
Trichinella spp. are unique among various helminthes in that all three life cycle stages of the parasite-
infective muscle larvae, adult, and newborn larvae develop in one host. Infection is acquired by con-
sumption of infected raw or undercooked meat. Under the influence of gastric juice, larvae are released
from infected meat in the stomach, molt, and develop into the adult stage inside the enterocytes of small
intestine. After mating, newborn larvae move into the circulation and spread throughout the tissues and
organs, and only those that penetrate striated muscles mature into muscle larvae [6]. This parasite has
ability to transform the infected muscle cell into a new type of cell in the host body, the so-called nurse
cell (NC) [7,8]. From this immunologically privileged place, parasite achieves long-lasting communica-
tion with the host through muscle larvae excretory-secretory products (ES).

793
794 Laboratory Models for Foodborne Infections

Unlike what happens in humans, T. spiralis can reach a high worm burden in animals without induc-
ing clinical symptoms [2]. Although infectivity for laboratory animals differs among various Trichinella
taxa, infection provides valuable in vivo models for basic research.
This chapter reviews the most significant observations gained in biological, pathological, and immu-
nological studies performed on rodent models (inbred mice and rats, or genetically manipulated), which
have the benefit of being easy to handle and relatively cost-effective. It also covers data from some larger
animal models using domestic and wild swine, horses, and other extremely costly animals that host
Trichinella species (including even marine mammals and reptiles).

50.2 Mouse
Mice are the most frequently used mammalian model system due to their close genetic (over 95% of the
mouse genome is identical to human genome) and physiological similarities to humans. Creation of vari-
ous inbred strains by genetic manipulation, as well as knock-out and transgenic animals, enabled studies
of complex phenomenon like susceptibility to the infection and immune responses elicited.
Trichinella is not a picky nematode, as it invades and completes its life cycle in the variety of host
organisms, including mice. Among Trichinella species, the most extensively studied is T. spiralis.
Immune response to primary infection with this intestinal and tissue-dwelling parasite starts with recog-
nition of the antigens and proceeds to the inflammation of the intestinal mucosa, leading to the reduction
of female fecundity, cytopathological damage, and expulsion of adults from intestine during primary
infection, whereas in challenge infection, it induces accelerated expulsion of larvae and adults from the
intestine and reduction of growth and fecundity [9,10]. Differences in the resistance to T. spiralis infec-
tion between various strains have a genetic background. Some strains possess alleles that enable rapid
(strong) response, which means that they expel worms more quickly, reduce female worm fecundity, and
as a consequence have lower muscle larvae burden than slow (weak) responders, which possess another
set of alleles [9,11]. Although Wakelin [9] used time of expulsion as a parameter for determining the
response phenotypes of inbred strains of mice infected with T. spiralis, the number of recovered larvae
from the muscles should also be considered for distinguishing between susceptible and resistant strains,
since it takes into account all variables that affect the response to infection.

50.2.1 Contribution of MHC-Linked and Background Genes to the

Control of the Response
Response to primary T. spiralis infection is governed by more than one gene, located within and out-
side the mouse major histocompatibility complex H-2, judged by the results of crossing experiments
involving various types of responders [12,13]. H-2 genes code for proteins that are directly or indirectly
involved in antigen presentation to T cells, thus controlling lymphocyte responsiveness to infection.
First experiments were conducted using panel of B10-background congenic mice, baring various alleles
at H-2 loci [12]. Mouse strain B10.BR carrying H-2k haplotype was shown to be associated with suscep-
tibility, whereas mouse strain bearing H-2q (B10.Q) and H-2s (B10.S) were both resistant to T. spiralis
infection. Investigations conducted on H-2 recombinant mice revealed that at least two genes, mapping
within this locus, were involved in the resistance to T. spiralis, and they were designated as Ts-1 and
Ts-2 genes [12]. The rapid expulsion responses after challenged infection are also controlled by H-2 and
non-H-2 genes [14].
Another set of experiments explored the role of non-H-2 genes in the rejection process, and for that
reason, mice with identical H-2 genes but different backgrounds were used [13,15]. Wassom et al. [15]
using susceptible H-2k strains, and Wakelin and Donachie [13] using resistant H-2q haplotype, came to
similar conclusion that the time of expulsion is controlled by non-H-2 genes.
For discovering genes involved in the resistance to T. spiralis infection, crosses among strains
with large phenotypic differences (strong responder—NFS, intermediate responder—C3H, and
weak responder—B10.BR) and segregation analyses of crosses, intercrosses, and backcrosses among
Trichinella 795

phenotypic generations were analyzed [16]. The results pointed out that there are at least two dominant
non-H-2 genes that control rejection of adult worms. The presence of both of them generates strong
responding phenotype, while each gene on its own is responsible for intermediate phenotype.

50.2.2 Response of the Host during Intestinal and Muscle Phase of the Infection
Response of the host implies immunological and nonimmunological mechanisms at the level of intestine
and muscles, engaged in the process of defense from T. spiralis invasion. Using genetically defined strains
of mice or mice with targeted deletions in cytokine or cytokine receptor genes, it is possible to identify
particular components and mechanisms of the host response important for the outcome of T. spiralis
infection. Effective immunity in intestinal phase of T. spiralis infection is crucial for the establishment
of parasitism, since it controls elimination of adult worms from the gut, fecundity of female parasites,
and therefore the number of newborn larvae that migrate toward muscle cells. Parasite provokes T-cell-
mediated immune response at intestinal level, which is accelerated upon reinfection [17]. Investigation
encompassing four strains of inbred mice that exhibit a range of responsiveness to T. spiralis infection—
NIH (high), CBA (intermediate), C57BL/10 (intermediate to low), and B10.BR (low)—revealed that
the resistance is correlated with the intensity of intestinal inflammatory responses. NIH and CBA mice
were the most resistant to T. spiralis and have the most intense intestinal inflammatory responses [18].
Intestinal inflammation is absent in congenitally athymic mice infected with T. spiralis [19], indicating
an important role of T cells, especially CD4+ T cells [20] in host-protective immunity. T. spiralis induce
inflammatory responses by distorting the gut epithelial cell layer. This is a signal that generates release
of Th1 cytokines such as IFN-γ within 2 days [21], followed by predominant Th2 response that occurs
between days 2 and 8 in all the aforementioned strains [18].
Th1 type of response leads to susceptibility to worm infection. Mice deficient in cytokine IFN-γ expel
T. spiralis more rapidly than wild-type mice [22]. Th1 type responses are under control of proinflam-
matory cytokine IL-12, produced by dendritic cells and macrophages. Transgenic mice overexpressing
IL-12 show delayed worm expulsion, accompanied with increased IFN-γ and decreased IL-4 and IL-13
production [23].
Resistance to T. spiralis infection is mediated by Th2 type of response [24], primarily IL-4, IL-9, and
IL-13 cytokines [17]. The presence of type-2 cytokines causes eosinophilia, intestinal mastocytosis,
and elevated IgE production. Primary infection of C57BL/6 IL-4 knock-out (KO) mice with T. spiralis
pointed out the importance of IL-4 in the expulsion of parasites [25], and treatment with anti-IL-4
receptor monoclonal antibody caused prolonged adult infections and higher muscle larvae burdens [26].
IL-4 deficiency also leads to impaired Th2 antibody response, both IgE and IgG1. However, BALB/c
IL-4 KO mice showed no difference in the time of expulsion or in enteropathy from wild-type mice.
Moreover, expulsion was delayed in wild-type C57BL/6 mice compared to BALB/c mice with no sig-
nificant differences in Th2 responses, which indicates that the function of cytokines depends on genetic
background [27]. Studies with IL-4-receptor-α deficient mice proved that expression of this receptor on
bone-marrow- and non-bone-marrow-derived cells has a crucial role in worm expulsion and thereby in
host protection [28]. Another important cytokine in Th2 response—IL-13—which shares the same IL-4
receptor α chain (IL-4Rα), stimulates B cell proliferation and antibody class switching to IgE, eosino-
philia, and mastocytosis [29,30]. Binding of either of these cytokines to this receptor results in the phos-
phorylation and activation of signal transducer and activator of transcription factor 6 (Stat6), which leads
to eosinophilia, mastocytosis, and expulsion of T. spiralis [22,31]. Degranulation of mast cells that occur
in response to T. spiralis antigens increases the permeability of intestinal epithelial cells, thus promoting
parasite expulsion [32]. Signaling via Stat6 also induces intestinal smooth muscle cell hypercontractility,
another mechanism necessary for efficient worm expulsion [33]. In T. spiralis infection, intestinal muscle
hypercontractility is under control of Th2 responses [34]. Hypercontractility does not occur in athymic
mice and in animals with major histocompatibility class II or CD4+ cell deficiencies, implicating T cell
control [35,36]. A shift to Th1 response via an overexpression of IL-12 significantly inhibits T. spiralis-
induced muscle hypercontractility and delays worm expulsion [23].
Th2 cytokines IL-3 and IL-5 are also produced during T. spiralis infection [24]; however, treat-
ment with anti-IL-3 and IL-5 antibodies, or using IL-3 KO mice, revealed that these cytokines are not
796 Laboratory Models for Foodborne Infections

significant for host protection. Since IL-5 controls eosinophilia provoked by T. spiralis [37], it could be
concluded that eosinophils are not essential for the expulsion of the parasites and thus do not affect host
protection, although they exert larval cytotoxicity in T. spiralis infection [38]. However, experiments
with IL-5-deficient mice revealed that IL-5 and eosinophils are involved in rapid expulsion during chal-
lenge (secondary) T. spiralis infections [39,40]. Another role for IL-5 and eosinophils was suggested in
a model of T. spiralis infection in mouse strains that constitutively overexpress IL-5. It appears that high
levels of IL-5 and eosinophilia may be advantageous for the parasite, since the number of muscle larvae
is much higher in transgenic mice than in wild-type [41]. This was confirmed by the work of Huang
et al. [42], who pointed out immunoregulatory role of eosinophils in the early muscle phase of the infec-
tion. They have found that eosinophils produce IL-10 necessary for the expansion of IL-10-producing
DCs and CD4+ T cells, which then inhibit inducible nitric oxide synthase (iNOS) expression and the
production of NO, thus protecting intracellular larvae.
Using IL-9 transgenic mice that constitutively overexpress this cytokine, the role of IL-9 in resistance
to T. spiralis was revealed for the first time [43]. Enhanced level of IL-9 in vivo caused high levels of par-
asite-specific IgG1, marked intestinal mastocytosis, and accelerated worm expulsion. IL-9 might medi-
ate effective expulsion of T. spiralis by enhancing the release of mouse mast cell protease-1 (mMCPT-1).
The involvement of IL-9 in mucosal immunity, i.e., recruitment and/or survival of mast cells, was sug-
gested [44]. These data imply that IL-9 is a specific effector molecule involved in host defense against
T. spiralis infection.
Mast cells appear to be involved in the expulsion in nematode infections [45]. Mice in which mast cell
depletion was induced either by genetic manipulation [46] or by application of specific antibodies [47]
exhibit a significant delay in expulsion of T. spiralis. Mast cells contribute to intestinal inflammation
through the production of proinflammatory cytokines, including tumor necrosis factor, proteinases,
and other inflammatory mediators such as histamine, leukotrienes, and prostaglandins [48]. It appears
that mastocytosis in the infected mucosa may represent an immunopathological rather than a protective
response. Mice deficient in TNF receptor, infected with T. spiralis, exhibit minimal pathology in the
gut and the absence of intestinal mastocytosis, and yet they are able to expel adult worms. TNF may
mediate pathological effects in gastrointestinal helminth infections via the production of iNOS [49].
iNOS-deficient mice infected with T. spiralis show reduced pathogenic but not protective response to
T. spiralis [50].
When newborn larvae reach striated muscle tissue, they invade muscle cells and rearrange them to
form a completely new entity, the NC [7]. NC is also composed of satellite cells that undergo cell divi-
sion and join the invaded muscle cell [8,51,52]. Tissue damage provoked initially by parasite penetration
accounts also to the presence of inflammatory cells, which produce high levels of reactive oxygen species
and other free radicals, after activation [53]. These actions result in an inflammatory response, which
causes transient myositis.
Inflammatory response to various encapsulated and nonencapsulated Trichinella species differs [54].
T. pseudospiralis, a nonencapsulated species, induce a lower inflammatory reaction around the parasites
than encapsulated species (T. spiralis and T. britovi). There is also difference in the level of inflammation
between encapsulated species; that is, T. spiralis is accompanied by the higher inflammatory response,
compared to that induced by T. britovi [55].
The immunological response to T. spiralis muscle invasion in mice is mixed, although mainly Th2
type, characterized by the production of IL-5, IL-10, IL-13, and IFN-γ [56] and by the presence of
parasite-specific IgG1 and IgE during the chronic infection [57]. Eighty percent of IgG1 specific for
larval antigens recognize a single shared epitope, highly immunogenic sugar, tyvelose [58]. Although
chronic, lifelong infection in mice is characterized by persistent B cell response, local inflammatory
response to the T. spiralis–NC complex is limited, suggesting the existence of suppressive parasite
and/or host factors.
The cellular infiltrate surrounding the NC is composed of macrophages, able to invade the cytoplasm
of the NC [57]. Among other cells (CD4+ T cells, CD8+ T cells, Treg, B cells), eosinophils enter sites
of infection immediately after tissue invasion by T. spiralis larvae, and by producing IL-10, control
the activation of proinflammatory macrophages and neutrophils that otherwise kill parasite larvae by
releasing NO [42]. Inflammatory infiltrates fail to form around NCs in T-lymphocyte-deficient mice,
Trichinella 797

identifying these cells as the coordinators of the cellular response to muscle infection [59]. In IL-10
KO mice, the extent of the inflammatory infiltrate around the NC was markedly increased compared
to control mice during the acute phase of the infection, although the cellular composition remained the
same [57,60]. The other source of IL-10 that participates in reduction of inflammation is CD4+ CD25-T
effector cells [56]. Another cytokine with immunosuppressive effect on T cells is TGF-β, produced by
regulatory T cells (Treg) [61], which acts together with IL-10 to control local inflammation. When IL-10
KO mice were treated with anti-TGF-β, they develop a strong inflammation around the NC–parasite
complex with a worm burden significantly reduced compared to control mice [56]. Control of inflam-
mation in the chronic phase of the infection is IL-10-independent and could be ascribed to strong Th2
response [57].

50.2.3 Applied Studies on Mouse Model for Trichinellosis

Mouse models of Trichinella infection represent useful tool either for investigation of efficacy of anti-
helminthic drugs [62–64] or for study of mechanisms involved in immunomodulation of inflammatory
diseases or tumors by parasite (reviewed in Ref. [65]).
To control trichinellosis, transmission from animal hosts (usually pigs) to humans should be pre-
vented, and this could be achieved by successful prophylactic vaccination. However, this goal has not yet
been reached. Most of the studies concerning development of vaccines against Trichinella infection were
conducted on a mouse model using at first whole extracts or ES products of the parasite. These vaccines
were more efficient (50%–80% of protection) than those composed of isolated parasite antigens, as seen
by the reduction in larval or adult worm burden [66,67]. Implementation of more refined vaccines using
recombinant or synthetic proteins or peptides, with different immunogenic properties, or third genera-
tion of DNA vaccines made of plasmids containing sequences coding for foreign proteins, resulted in
variable protective effect (reviewed in Ref. [68]).

50.3 Rat
While carnivores are considered as particular and permissive species for Trichinella infections, rats are
considered as selective hosts in which only a restricted number of species other than T. spiralis reproduce
through either natural [69,70] or experimental infection [71]. As experimental model, rats are used for the
investigation of Trichinella life cycle, host–parasite relationship, and immune response provoked by this
parasite as well as for investigation of parasite’s potential to manipulate the immune response of the host
and to suppress some severe diseases.
Since they are characterized as selective host species for Trichinella infection, rats have been used as
a model for investigation of Trichinella species-specific defense. These experiments were performed by
comparing T. spiralis and T. nativa infections in Wistar rats [71,72]. In T. spiralis-infected rats, muscle
larvae burden and in vitro newborn larvae production were many fold higher compared to T. nativa-
infected rats, which indicates that the infectivity of T. nativa is lower compared to T. spiralis. No differ-
ences between two Trichinella species were observed in intestine histology, serological response, and
weight dynamics of infected rats. This helped understanding that defense against different Trichinella
species not only is solely enteral, but also happens in the parenteral phase of the parasite’s life cycle.
Investigations of the influence of host genetics on the susceptibility to primary T. spiralis infection
on rat model system gave valuable contribution to understanding of parasitological and parasite-induced
immunological differences between strains. Rat strain variation in response to T. spiralis infection
examined on five inbred strains (AO, LOU, PVG, WKA/H, and LEWIS) was evaluated through differ-
ent rate of worm elimination in each strain [73]. LOU and LEWIS rats were the most efficient in expel-
ling T. spiralis. In these two rat strains, the lowest level of serum IgE was found, while the intestinal IgE
response was the most pronounced. Variations in rat response to T. spiralis were amended by investiga-
tion of interstrain differences with respect to muscle larvae burden between AO and DA rats [74], and it
was shown that DA rats express higher susceptibility in comparison with AO rats.
798 Laboratory Models for Foodborne Infections

Immune responses of rats to Trichinella infection during the intestinal phase involve both Th1 and
Th2 cell subsets [75]. During the muscle stage of infection, with the emphasis on rat strain differences,
T. spiralis provoked the increased level of anti-inflammatory cytokines (IL-4 and IL-10), indicating a
shift toward Th2 and regulatory response, without the restriction of Th1 type of response in DA rats,
while the response in AO rats remained in favor of Th1 type [74]. In vivo application of dendritic cells
(key cells in the initiation and regulation of immune response) primed with T. spiralis ES products
creates an immune status that resembles the one observed during Trichinella infection [76].
Research on effector mechanisms involved in protection during the intestinal phase of the T. spiralis
infection indicated that the rapid worm expulsion is IgE-mediated and T-cell-mediated [10]. Extensive
investigation of the relationship between intestinal immune responses and worm expulsion, which occur
upon the infection with T. spiralis, performed on several rat strains (BN strain—mast cell and IgE high
responders; F344—low responders on the level of mast cells and IgE activation; DA strain—intermedi-
ate responders comparing to BN and F344; Ws/Ws strain—mast cell deficient rats), revealed that mast
cells in addition to IgE could contribute to the worm expulsion following T. spiralis infection in rats [77].
Studies of host–parasite relationships in Trichinella-infected animals with respect to different media-
tors involved in infected muscle cell transformation also used rat model system. Potential differences in
signals derived or provoked by different Trichinella species (encapsulated and nonencapsulated) were
studied regarding the production of NO, a known cell-to-cell mediator. It was found that host macro-
phages differ in NO production between encapsulated and nonencapsulated Trichinella species [78].
Macrophages stimulated with T. pseudospiralis muscle larvae antigens produced increased levels of NO
only at the highest antigen concentrations, while T. spiralis antigens induced increased production of
NO even when applied in much lower concentrations compared to doses of nonencapsulated Trichinella
antigens.
Investigation of the dynamics of antibody response was performed on Wistar rats using different doses
of T. spiralis muscle larvae. It was demonstrated that rats survive the high infection doses (up to 16,000
muscle larvae) and that low doses (10 muscle larvae) can be infective and can cause detectable levels of
specific antibodies [79]. Time of seroconversion and antibody titers showed to be dose-dependent in rats.
Significant contribution to the body of evidence considering biochemical parameters of the host
and their changes during the infection with Trichinella was given by the study of lipid status, levels of
enzyme with antiatherogenic properties (paraoxonase-1), and oxidative stress in rats infected with this
parasite [80]. It was demonstrated that during the infection, serum activity of cardioprotective enzyme
paraoxanase-1 was significantly reduced accompanied with the increased levels of triglycerides and LDL
cholesterol and decreased levels of HDL.
Very important information regarding the potential of Trichinella infection or its products to manipu-
late the host immune response and to suppress some severe diseases like experimental autoimmune
encephalomyelitis (EAE), as experimental model of multiple sclerosis, were obtained in experiments
performed on DA rat model system. It was demonstrated that chronic infection with this parasite as well
as the application of Trichinella ES products, or dendritic cells stimulated with these products, has a
strong impact on the course of EAE, i.e., on the reduction of the disease severity by shifting the immune
response toward Th2 and regulatory type [81,82].

50.4 Swine
Although human Trichinella infections can be a consequence of the consumption of infected meat origi-
nating from different animal species including feral animals, T. spiralis-infected pork meat still remains
the main source of the infection [2,83].
Owing to the model of experimentally infected pigs, predilection sites for T. spiralis and other
Trichinella species were discovered in these animals [84,85]. It was established that all Trichinella
spp. accumulate preferentially in diaphragm, tongue, and masseter of infected pigs. Investigation of
infectivity of the Trichinella genotypes to pigs showed that T. spiralis is highly infective and T. britovi
and T. nelsoni are moderately infective but as persistent as T. spiralis, while T. nativa, T. murrelli,
T. pseudospiralis, and Trichinella (T6) are low infective and nonpersistent [86].
Trichinella 799

Several methods such as trichinelloscopy, artificial digestion, immunofluorescence assay (IFA), and
ELISA were compared for establishing reliable test for detection of experimental porcine trichinellosis.
It was concluded that protection of public health from Trichinella in pork and prevention of the infec-
tion spreading can be ensured only by application of parasitological methods and, in particular, artificial
digestion [87,88]. This was due to the finding that digestion detects infection as early as the larvae enter
and encapsulate in muscle tissue i.e., 17–21 days post infection (p.i.), while serological methods cannot
detect infection before seroconversion occurs, i.e., mean times were 5–6 weeks p.i. [88]. Although serol-
ogy cannot be used as a procedure to certify the safety of pork meat, it could be a useful method for
surveillance and epidemiological studies of swine Trichinella infection [89–91].
Trichinella-specific antibodies in swine could be detected by 1.5 years p.i. [92], and specific anti-
bodies raised by the infection with any of 12 different taxa of the genus Trichinella could be reliably
detected by one common antigen since all taxa share similar antigenic pattern [2,86,90,93]. The inves-
tigations performed on experimentally infected pigs provided significant impact on the development
of serological tests, and defining their performances and the identification of highly immunogenic
antigens enabled the improvement of diagnostic tests. Diagnostic tools for antibody detection in swine
evolved from application of the whole larvae in IFA, via total larvae extract and up to ES or its com-
ponents that belong to TSL-1 in ELISA and western blot [87,94,95]. The specificity of anti-Trichinella
antibodies relies on recognition of antigens from TSL-1 group, which consists of protein and unique
sugar component (tyvelose) [96]. Experimentally infected pigs are currently used for development of
international biological standards or reference materials in an aim to validate in-house tests or com-
mercial kits and to improve the interlaboratory comparability for the serological detection of anti-
Trichinella IgG in pigs [97].
Trichinella larvae recovered from experimentally infected pigs were used during development and
evaluation of specificity and sensitivity of polymerase chain reaction methods for the identification of
species or genotypes of Trichinella single muscle larvae [98,99].
Besides the use of the aforementioned outbred swine, few studies that used SLA-inbred miniature
swine proved to be an important experimental model for mapping of genetic resistance to Trichinella
infection. The influence of genes within the MHC (SLA) versus background (non-MHC) genes on the
initiation, maturation, and effector arm of host antiparasite defense mechanisms was investigated. Swine
selectively bred so that they are homozygous at their SLA complex for three SLA haplotypes, aa, cc, or
dd, were used to determine whether those genes regulate swine immune response to T. spiralis [100]. The
obtained results indicated that swine with SLAc/c haplotype have lower worm burden correlating with
the earlier development of humoral antibody response. This and work of other authors implied that this
“resistance” to infection relies not only on SLA locus (expression of one allele from SLAa haplotype) but
also on other non-SLA-encoded genes [101].
Some extension of this work performed on outbred swine indicated that besides the role of SLA
class II molecules, SLA class I phenotype also influences mechanisms that mediate the presentation of
Trichinella antigens and consequently the host immune response. Swine that expressed particular SLA I
phenotype (2–12–3 positive) found to be more resistant to infection (with lower larvae burden), compared
to negative animals [102].

50.4.1 Applied Studies on Swine Model for Trichinella Infection

Experimental infection in swine was used for evaluation of some other aspects of Trichinella infection
model. One of them is the effect of concomitant infection of T. spiralis with two other helminthes Ascaris
suum and Metastrongylus apri. Data obtained in this study indicated that T. spiralis had a synergistic
interaction with M. apri (higher establishment of larvae in the lungs), and an antagonistic interaction
in concurrent infection with A. suum (reduced numbers of migrating larvae and juvenile worms) [103].
Another model combined T. spiralis and T. gondii and was used for evaluation and validation of bead-
based assay for simultaneous detection of anti-T. spiralis and anti-T. gondii-specific antibodies in swine
serum samples [104]. Experimentally infected swine were also the source of sera used for identifying
several immunodominant T. spiralis adult antigens, which may present promising candidates for devel-
opment of an early diagnostic tool [105].
800 Laboratory Models for Foodborne Infections

Among the trials that aimed to develop vaccine against establishment of Trichinella infection in
swine, Marti et al. [106] showed that vaccine based on newborn larvae (NBL) antigen was considerably
more protective than the one based on muscle larvae stichosomal fractions or ES proteins [107,108].
Experimentally infected swine are currently in use for determination of specific epitopes on NBL [109],
adult and muscle larvae antigens [110], targeted by host antibodies, which could potentially be used for
new approach to vaccine development as well as for early diagnosis of Trichinella infection.

50.5 Wild Boar
Wild boar infections with both domestic and sylvatic Trichinella spp. occur quite frequently in Europe,
North America, Asia, and Africa, and infected meat of this animal species presents an important risk fac-
tor for human Trichinella infection [111,112]. First experimental Trichinella infection was performed to
determine infectivity, muscle larvae distribution, and ability of investigated species to generate immune
response in wild boars [113]. This study revealed that T. spiralis is highly infective for wild boars;
T. britovi, T. nelsoni, and T. pseudospiralis are moderately infective, while T. nativa, T. murrelli, and
Trichinella T6 are not well adapted for this host species. Predilection sites, independently of Trichinella
genotype and infective dose, are diaphragm and tongue [113,114]. Antibody response increased rapidly
from week 3 to 5 p.i. for all investigated Trichinella genotypes except for T. pseudospiralis where it
increased gradually. For encapsulated species of Trichinella, high antibody level remained until the end
of examined period (10 weeks), whereas for nonencapsulated species, antibody levels declined during the
postinfection period. Data concerning wild boars correlate with those observed in domestic pig model
system [86].
A long-lasting experiment was designed using wild boars to investigate the freezing tolerance of
Trichinella-encapsulated muscle larvae T. spiralis and T. britovi [115]. It was demonstrated that muscle
larvae of both Trichinella species could be inactivated after 1 week on −21 ± 2°C. In contrast to this find-
ing, Gari-Toussaint et al. [116] reported six cases of human trichinellosis originating from the consump-
tion of T. britovi-infected wild boar meat that had been frozen for 1 week at −35°C. It has been suggested
that wild animals’ muscles contain substances that protect Trichinella muscle larvae from freezing [117].
Hence, although the study on experimentally infected wild boars provided some new data on Trichinella
freezing tolerance, freezing method is not recommended as a control measure to inactivate this parasite
in game meat [89].

50.6 Horse
Infected horse meat has been recognized as a source of human trichinellosis since 1975 when the out-
breaks were reported for the first time in Italy and France [118,119]. Because horse has been considered
as an atypical host for Trichinella spp., many aspects of the biology and epidemiology of Trichinella
infection in the horse were poorly investigated and understood during the previous century. Still, being
recognized as a serious food safety risk for consumers of fresh horse meat, the infection was started to be
investigated in experimental horse model from the 1970s. Those very early studies were aimed to detect
muscle larvae and specific antibodies presence during the course of the infection, but latter investigations
included the route of infection, tissue distribution of muscle larvae, parasite life span, and assessment
of reliability of parasitological and serological diagnostic methods for the detection of infected animals.
The first report on distribution of Trichinella muscle larvae (L1) in individual muscles was published
by Pampiglione et al. [120] referring to the examination performed on 6-month-old horses infected
with 10,000 L1 in which the highest muscle larvae burden was detected in tongue, masseter, and dia-
phragm. In this millennium, all three aforementioned muscles were confirmed to be predilection sites
for Trichinella muscle larvae [114]. The study included 30 horses experimentally infected with three
Trichinella genotypes: T. spiralis, T. britovi, and T. pseudospiralis. Limited study on three ponies, [121]
Trichinella 801

as well as studies of other authors [92,122,123], corroborated that the tongue is the main predilection
site. Results obtained in experimentally infected horses were in accordance with those observed in natu-
rally infected animals [124], and they supported the contention that food safety inspection of the horse
meat should target the tongue as a tissue most promising to yield positive results in horses infected with
Trichinella.
Several studies have been undertaken to describe the infection in this atypical host [123,125–127].
Horses mostly do not develop the disease during Trichinella infection, except the rare transient muscular
disturbance observed between 2 and 6 weeks p.i. in case of animals infected with a high dose (50,000)
of L1 larvae. Moreover, hematological parameters were not significantly changed, except for the level of
eosinophils, which was slightly increased (up to 15%) independently of infective larvae dose. Levels of
enzymes that indicate the muscle damage (lactate-dehydrogenase, aldolase, and creatine phosphokinase)
were increased from 2 to 17 weeks p.i. [125].
Experimental infection of horses with Trichinella spp. was used for the examination of kinetics of
antibody production and dependence of antibody response on different Trichinella spp. that cause the
infection in horses. Detection of specific, anti-Trichinella antibodies depend on Trichinella species
(T. spiralis induced higher level of anti-Trichinella IgG than T. murrelli due to its antigenicity), the infec-
tive dose applied for horse infection, as well as on the antigen used for antibody detection [124]. A num-
ber of studies demonstrated that in high-dose infection (more than 20,000 L1), anti-Trichinella-specific
IgG appears between 2 and 5 weeks p.i. and disappears between 16 and 40 weeks p.i. [120,125,128],
judging by indirect IFA and ELISA tests. Low doses of infective larvae (1100 L1) also induce antibody
production detectable by ELISA for up to 18 weeks p.i. [129]. The short period of time (in average up to
26 weeks p.i.) during which anti-Trichinella antibodies in infected horses could be detected by ELISA
makes this kind of test unreliable for detection of Trichinella infection in horses [119,125,130]. On the
other hand, other serological methods were proven to be effective in specific antibody detection for a
longer period (IFA of paraffin sections of L1 and western blot analysis of ES and tyvelose-BSA, at least
8 months) [129]. It was supposed that for diagnosis of horse trichinellosis, additional tests including
antigen detection should be performed. One such test was a dot-blot test for the detection of Trichinella
ES muscle larvae antigens in circulation, which allowed the detection between weeks 4 and 32 p.i. in the
sera of all experimentally infected horses [131].
The most extensive and long-term experimental study was performed on 35 horses infected with 1000,
5000, or 10,000 T. spiralis muscle larvae in which the course of infection was followed for 1 year [92].
It was shown that the persistence of infective muscle larvae is at least 1 year, but serological response
could be detected only by 26 weeks p.i. (ELISA with ES). This study offered the conclusion that parasite
recovery methods are the only suitable detection assays for both meat inspection and epidemiological
studies of Trichinella infection in horses.
The question considering the enigma of early disappearance of detectable antibody response in horses
versus the existence of live muscle larvae raised by this research could partly find the answer in data
showing the presence of seven IgG subclasses in horse and five other isotypes [132,133] and the presump-
tion that anti-horse IgG used in previous studies may be inappropriate for detecting subclass-specific
anti-Trichinella reactivity in the horse [92].
Since horses are herbivores and nevertheless, natural infections with Trichinella occur from time to
time, an intriguing question considering the source of infection emerged. Eating behavior of these ani-
mals studied by intentional feeding of horse with meat products [134] revealed that 31% of horses readily
consumed it and that this feeding practice could be a source of Trichinella infection.

50.7 Other Animal Species as Models for Trichinella Infection

In order to investigate the susceptibility to Trichinella infection, define predilection sites as well as freeze
tolerance for muscle larvae and determine serological response parameters in wildlife. Some animal
species that are, conditionally speaking, unusual for experimental purposes were used as animal models
in such surveys.
802 Laboratory Models for Foodborne Infections

Studies comparing the muscle distribution of different Trichinella genotypes were performed, among
other species, on foxes. In these animals, where all Trichinella species were established in high numbers,
the encapsulating species were found primarily in the tongue, extremities, and diaphragm, whereas the
nonencapsulating species were found primarily in the diaphragm [114]. Foxes were one of the experi-
mental models used for studies of Trichinella larvae freeze tolerance. Since T. nativa is the most frequent
Trichinella species in arctic wildlife and well adapted to the cold climate, it was useful for testing the
tolerance on repeated freezing and thawing. Results obtained in experimentally infected foxes showed
that T. nativa isolates originating from carnivores from higher northern latitudes expressed highest toler-
ance to freezing and that temperature fluctuation around freezing point did not influence larval infectiv-
ity [135]. Foxes were also experimentally infected with T. nativa for the purpose of serological survey,
which showed that western blot is a more reliable method compared to ELISA for antibody detection
in this animal species [136]. Potential of Trichinella to cross the placental barrier was investigated on
experimentally infected foxes, and it was found that, unlike some other animal species (ferrets, guinea
pigs, and mice), vertical transmission, measured as recovery of muscle larvae in the offspring, does not
happen in foxes [137].
Since marine mammals may acquire Trichinella infection in nature by scavenging even small amounts
of infected tissue left by hunters or predators, the important data considering infection in this group of
animals were obtained on seals experimentally infected with T. nativa in order to determine the suscepti-
bility of seals to this parasite infection and freeze tolerance of larvae in the seal meat [138]. Seals showed
high susceptibility to Trichinella infection and measurable antibody response unrelated to the infection
dose. Experiments investigating freeze tolerance of muscle larvae in the meat indicated that there was
correlation between resistance to freezing and the age of host–parasite tissue complex; that is, freeze
tolerance increases with time post infection. The results obtained in this study were complemented with
research in which the meat of seals experimentally infected with T. nativa was used for the preparation of
traditional northern foods in an aim to assess the infectivity of larvae present in those foods [139]. Data
indicated significant food safety risk associated with the food containing infected seal meat.
One of the most unusual experimental models for trichinellosis is caiman. Notification of Trichinella
zimbabwensis (nonencapsulated species) infection in farmed crocodiles in Zimbabwe due to the feeding
practice [140] raised a question of susceptibility of reptiles to infection with other Trichinella spp. The
experimental infection of caimans with different Trichinella isolates showed that these animals can be
infected with nonencapsulated T. pseudospiralis, which is, from evolutionary point of view, likely to
infect reptiles since it can be infective for both birds and mammals, while the infection with encapsu-
lated species was impossible [141].

50.8 Conclusion
Animal models for Trichinella and trichinellosis are a tool that allows detailed investigation of host–
parasite relationships. The host’s susceptibility to different Trichinella genotypes indicates the impact of
genetic background of the host and the parasite on the parasitism establishment. The vast range of exper-
imental animal models reveals insight into parasite muscle distribution and predilection sites, which
form the basis of meat sample properties for accurate parasitological detection and diagnosis. Studies
using experimental Trichinella infection yield valuable information on the course of innate and adaptive
immune response. Knowledge about dynamic of specific antibody synthesis provides a guide for design-
ing and implementing specific and sensitive serological tests in surveillance, monitoring, and control
programs. The rodent models highlight the importance of antigen-presenting cells to drive T-helper
type 2 and T-regulatory responses in Trichinella infection that are responsible for parasite long-lasting
survival and creation of tolerogenic environment in the host body. Animal models have been, and will
continue to be, vital in helping elucidate the mechanisms that underline the host–parasite coevolution,
which is, among others, responsible for the training of host immune systems for accurate discrimination
between foreign and self-antigens.
Trichinella 803

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117. Dick TA. Infectivity of isolates of Trichinella and the ability of an arctic isolate to survive freezing
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Index

A E4 proteins, 19
intermediate genes and, 19
ABC transport, 357 L4 genes, 19–20
A2B toxin, 133 pathogenesis, 21–22
Acanthamoeba, 579 structural proteins
animal models for, 585–586 capsid core proteins, 17–18
classification, 580–582 hexon, 16
clinical features and pathogenesis, 583–584 hexon-associated proteins, 16
amebic (acanthamoebic) keratitis, 583 minor coat protein pIIIa, 16
cutaneous acanthamoebiasis and sinusitis, 583 penton base and fiber proteins, 17
granulomatous amoebic encephalitis, 583 pVI, 16–17
diagnosis, 584–585 pVIII and pIX, 17
epidemiology, 582–583 virions, TEM of, 15
genomics, 580–582 Adherent invasive E. coli. See AIEC
life cycle, 582–583 Adhesion, 640, 641
morphology, 580–582 assays, 252
pathogenesis, 583 Adolescariae, 720
precautions, 585 AdV infections, 14
treatment and prevention, 585 cytokine response induced by, 22–23
in vitro models, 586 gastroenteric, 23
Acanthamoebic keratitis, 582 Aerobic endospore-forming rods, 132
Acanthamoebidae, 580, 582 Aeromonas, 2, 244
Acute liver failure animal models for
HEV-induced, 47 C. elegans, 243
Acute lung infection mouse models rodents, 242–243
of P. aeruginosa infection, 377 Tetrahymena spp., 243
Acute-on-chronic liver failure (ACLF), 48 zebrafish, 243
Acute poststreptococcal glomerulonephritis bacteremia, 241
(APSGN), 3, 228 biliary tract infections, 241
Acute rheumatic fever (ARF), 228 biology of, 240
Adaptive immune response classification of, 238
to astrovirus infection, 34 diagnosis of, 241–242
to HEV infections, 47 epidemiology of, 240
to urinary tract infections, 365 extraintestinal infections, 240–241
Adenovirus (AdV), 13 gastroenteritis, 240
cell entry, 20 genomics of, 239
cell lines as laboratory models for, 14 history of, 237
classification of, 14 isolates, 241–242
clinical diseasess caused by, 13 morphology of, 238–239
conserved features, 15–16 risk factors for, 240
DNA packaging and virion assembly, 21 treatment and prevention of, 242
DNA replication, 20–21 in vitro models for, 243
enteric. See Enteric AdVs Aflatoxins (AFs), 456–457
genome organization, 15–16 A-hemolytic species, 224
genus-common proteins, 16 A. hydrophila, 241
host–pathogen interaction, 22 AIEC, 323
life cycle of, 21 Airborne GAS epidemics, 227
as model systems, 23 A/J mice, 145
morphology of, 14–15 Alanine aminotransferase (ALT) levels
nonstructural proteins HEV infection and, 44–45
E1A proteins, 18 Albendazole, 516
E1B gene products, 18 Alcmaeon of Croton, 4
E2 gene products, 18–19 Alfalfa seedling infection model, 277
E3 proteins, 19 Alkaline peptone water (APW), 241

809
810 Index

Allergens, 566–570 colostrum-deprived piglets, 294

Alternaria, 441 ferret model, 294–295
Alternaria alternata, 441 Galleria mellonella, 295
Alternaria infectoria, 441 mouse models, 295–298
Alternaria mycotoxins three-day-old chick model, 292–294
foodborne diseases due to, 442–443 of Campylobacter jejuni colonization, 290–292
laboratory models for studying. See Foodborne for C. difficile infection, 164
Alternaria analysis, laboratory models for for C. perfringens enterotoxins
production of. See Mycotoxins of Alternaria species, calves and lambs, 164
production of goats, 164
quantification techniques, 444–445 nonhuman primates, 164
Alternaria spp. piglets, 163–164
colonization of ripening ears, 441 rabbit intestinal loops, 163
impact on foods, 441 rats and mice, 162–163
toxic secondary metabolites produced by, 442 enterococcal infections, 180
toxigenic strains, investigation and analysis of, for Enterocytozoon bieneusi, 515
443–445 for foodborne Alternaria, 446, 447
Alternaria tenuissima, 441 for Haplorchis infections
Ames Salmonella test, 449–450 rodents, 738
Aminoglycosides, 179, 255 importance in research, 345
Amoebapore polypeptides, 619 of L. monocytogenes infections, 187–189
Amoebiasis gerbils, 188–189
animal models for, 619–628 guinea pigs, 188
C57BL/6 IL-10-deficient mouse, 623 mice and rats, 187–188
C3H/HeJ mice, 623 nonhuman primates, 189
gerbils, 622 oral transmission, 190–191
guinea pigs and hamsters, 622 rabbits, sheep, and goats, 187
human colonic explants, 623 for O. viverrini infection, 766
mice models, 623 CCA, 769–770
pigs, 622 freshwater cyprinoid fish, 767
rabbits, 622 hamster and gerbil, 767
rats, 619 white blood cells levels, hamster, 768–769
SCID-HU-INT mouse, 623 of pathogenic E. coli
susceptibility in rodents, 625 C. elegans, 326
Amoebic (acanthamoebic) keratitis, 583 pigs, 326
Amoebic liver abscess, 624, 625 rabbits, 325
Amoebic vaccine research, animal models for, 627 rodents, 325
Ampicillin dextrin agar (ADA), 241 for Penicillium species allergenicity, 568
Amylosin, 134 of Salmonella infection
Animal cell lines calves, 398
for Fusarium mycotoxins, 539–546 C. elegans, 398
for mycotoxins of Aspergillus species, 467, 479–487 chick model, 398
Animal infection models of prion diseases, 122–123 guinea pigs, 398
Animal models mice, 397–398
for Acanthamoeba, 585–586 nonhuman primates, 398
Chinese hamsters, 586 rabbits, 398
mice, 585 zebrafish, 398
pigs, 586 for shigellosis
rabbits, 586 C. elegans, 405
rats, 585 chicken, 405
for Aeromonas infections guinea pigs, 402–403
C. elegans, 243 human cell lines, 405–406
rodents, 242–243 mice, 403–404
Tetrahymena spp., 243 pigs and piglets, 404
zebrafish, 243 rabbits, 404
for amoebiasis, 619–628 rhesus monkeys, 405
for BoNT intoxication zebrafish, 405
mouse, 160–162 types of, 8
rats, 162 Animal species for HEV cell-culture models, 57–58
of Campylobacter jejuni Animal viruses as HNoV surrogates
cellular infection models, 292 bovine noroviruses, 86
Index 811

feline calicivirus (FCV-F9), 77–80 Aspergilloses, 455

murine norovirus (MNV-1), 80–84 Aspergillus, 455
norovirus-like particles, 86–87 Aspergillus fumigatus, 455
porcine sapovirus, 85–86 Aspergillus mycotoxicoses. See Mycotoxins of
rabbit caliciviruses, 86 Aspergillus species
Tulane virus (TV), 84–85 Aspergillus species
Anisakidae nematode cultivation, 692 economic benefit of, 456
Anisakids, 680 fungal diseases caused by, 456
larvae mycotoxins. See Mycotoxins of Aspergillus
cladistic distribution of, 682 species
illness caused by, 683 Astroviridae, 29
life cycle of, 681 Astrovirus capsid protein, exposure to, 33
taxonomic classification, 680 Astroviruses
Anisakiosis classification of, 29
eosinophilia, 685 diversity of, 29
experimental models for, 684 future perspectives, 35
fish, 691 genome, 31
general considerations, 684 immunity to, 34–35
guinea pigs, 685 infection transmission routes, 30–31
mice, 688–690 life cycle, 31–32
pigs, 685–687 pathogenesis, 32–34
rats, 687–688 species-specific, 29
laboratorial diagnosis, 684 virion, model of, 31
Anisakis, 680, 683 Astrovirus-induced changes in apical expression of
allergen nomenclature, 683 digestive enzymes and exchangers, 33
antigens, 691 Astrovirus-infected turkeys, lymphocytes from, 34
cultivation, 692 Athymic nude mice, 8
L3, 693, 694 Autoinducers, 357
laboratorial diagnosis, 684 Avastrovirus (AAstV), 29
migration, 681 A. veronii, 241
Anthrax, 132 Aviadenovirus, 14
Anthrax LF, 145–146 Avian HEV, 46
Anthrax toxin receptor 1 (TEM8), 133 chickens as model system for, 60–61
Anthrax toxin receptor 2 (CMG2), 133 Azospirillum brasilense, 564
Anthropozoonosis, 680
Antibiotic-associated diarrhea (AAD), 156 B
Antibody production, 725, 726, 727
Anti-HAstV antibodies, 34 BA. See Bongkrekic acid
Anti-HAstV-specific T cells, 34 Bacillus anthracis
Anti-HEV antibodies, 55 PXO1 and PXO2, 132
Anti-HEV IgG, 47 zoonotic pathogen, 132
Anti-HEV IgM, 47 Bacillus cereus
Antimicrobial resistance, B. fragilis, 253 diversified along evolution, 132
Antimicrobials, 253 species, 132
Anti-OVA-specific IgE, 691 virulence traits, 133–134
Antiretroviral therapy (ART), 594 Bacillus cytotoxicus, 132
against HEV, 48 Bacillus megaterium, extracellular factors of, 140
Apicomplexa, 600 Bacillus mycoides, 132
phylum., 656 Bacillus pseudomycoides, 132
Apodemus sylvaticus, 594 Bacillus spp., 131
Apoptotic mimicry, 660 culture media for, 132
Arabidopsis thaliana of P. aeruginosa infection, 382 ex vivo models of infection, 140–141
Arginase, 646 toxin production regulation, 138–139
Arginine deiminase, 645 virulence factors of, 133–134, 140, 142
ART. See Antiretroviral therapy Asian house shrew model for, 144
Artemia salina, 449, 562 eukaryotic cell lines for. See Eukaryotic cell lines
Arthroconidiation, 556 mice model for, 144–146
Artisanal cheeses, 201 monkeys model for, 144
Ascaridoidea, 680 rats model for, 146
Ascomycota, 555 zebrafish model for, 144
Asian house shrew in vivo studies on cereulide, 144 in vivo models of infection, 140–141
812 Index

Bacillus subtilis, 564 -infected Caco-2 cells, 139

Bacillus thuringiensis, 132, 563 -secreted proteins, toxic activity of, 146
Bacillus weihenstephanensis, 132 sensu lato, virulence of, 133
Bacteremia, 145, 178, 185 sphingomyelinase, 142
Bacteria–enterocyte interaction, 133 toxins of
Bacterial biofilms, 357, 359–361 cereolysin O, 138
Bacteriophage MS2, 87–88 cereulide, 138
Bacteroidales species CytK, 137
β-lactamase production in, 255 emetic, 138
resistance genes in, 255 HBL and NHE, 135, 137
resistance to antibiotics, 254 hemolytic action of, 137
Bacteroides, 247 virulence factors by, 142
taxonomic and ecological aspects of, 249 B. fragilis
Bacteroides fragilis, phenotypic characteristics of, 250 antimicrobial resistance, 253
Bacteroides species, 247, 257 B. fragilis enterotoxin (BFT), 251, 252, 256
biology of, 249 biology of, 249
diagnosis of, 252–253 capsule of, 250
laboratory models, 256 enzymes in, 250–251
susceptibility to antimicrobials, 253–256 host immunologic defense response, 250
aminoglycosides, 255 LPS in, 251
β-lactam agents, 253 occurrence in stool samples, 249
carbapenems, 253 pathogenesis, 252
chloramphenicol, 255 virulence factors, 249
lincosamides, 255 vs. B. vulgatus, 250
macrolides, 255 B. gladioli pv. cocovenenans
multidrug resistance, 256 cellular characteristics of, 278
nitroimidazoles, 255 intoxication, 279
PBP, 253, 255 laboratory infection models, 278–279
plasmids, 256 pathogenic effects of, 278
porins, 255 taxonomical classification of, 278
quinolones, 255–256 toxins, 278–279
tetracycline, 255 B-hemolytic species, 224
variability to antimicrobial susceptibility, 249 Bile duct, infected, 705, 710–711, 767
virulence factors, 249, 252 Bile salts–Irgasan–brilliant green (BIBG), 241–242
Bacteroides vulgatus, 252 Bioassays, 212
Balb/cJ mice, 145 Biofilm-based P. mirabilis, 360
BALF. See Bronchoalveolar lavage fluid Biofilm formation assay, 361
Baltic salmon, 692 Biofilm formation, Cronobacter spp., 312–313
B. anthracis Bioinformatics, 646
infection model, 145 Biovars, 260
pathogenesis of, 133 Black death. See Plague
PI-PLC from, 138 B-lactam agents, 253
routes of infection, 133 B-lactam antibiotics, 253
spores adhered to epithelial cells, 140 B. licheniformis virulence factors, 134
toxin plasmid pXO1, 138 Boar sperm motility inhibition bioassay, 141
toxins murine models, 145 Bombyx mori, 143
virulence factors of, 133 for foodborne Candida infections, 503, 504
Bayesian analysis of genetic population structure model of P. aeruginosa infection, 382
(BAPS) analysis, 177 Bongkrekic acid, 278–279
B. cereus Botulinum neurotoxin (BoNT), 155, 156
adhesion to animal models for
Caco-2 cells, 139 human muscles, 162
cultured human enterocytes, 139 mouse bioassays, 160–162
epithelial cells of bacteria, 140 rat, 162
cultures, crude cell-free spent culture cell culture models for, 165–166
supernatants of, 143 in food samples, investigation of, 157–159
effect in GI tract, rat model for, 146 SMW inhibitors to, 161–162
gastrointestinal infection Bovine noroviruses as HNoV surrogate, 86
murine model of, 146 Bovine spongiform encephalopathy (BSE),
studies in rhesus monkeys, 144 ELISA for, 117
Index 813

B. pumilus virulence factors, 134 C

Bradyzoites, 656, 657, 660
Brain endothelial cells, Cronobacter interaction Caco-2 cell line for foodborne Candida infections,
with, 311–312 504–505
Bronchoalveolar lavage fluid, 691 Caco-2 cells, 9, 186, 313, 447
Brucella, 259 C. sakazakii infection of, 308
bactericidal activity against, 263 cytotoxicity, 166
characteristics of, 260 Caenorhabditis, 345
clinical disease in humans, 263 Caenorhabditis elegans, 141–142, 232, 275, 343
invasion and cellular pathogenesis, 261–263 as Bcc model, 277
microbiological characteristics of, 261 characteristic features of, 346
Brucella infections C. sakazakii interaction with, 313
in humans, 261, 263 discovery and exploration of, 345
immunity against, 263 for foodborne Candida infections, 503, 504
laboratory model of, 265 innate immune system of, 347
Brucella species K. pneumoniae inside intestine of, 347
associated with foodborne infections, CFU of, 348, 349
260–261 microscopic images of, 348
as pathogens, 259–260 K. pneumoniae pathogenicity to, 347
virulent strains of, 260 as model for bacterial infection, 417–418
Brucellosis, 259 as model for host–pathogen interaction, 346, 418
foodborne. See Foodborne brucellosis as model for Vibrio spp. infection, 418–421
long-term protection against, 263 of P. aeruginosa infection, 380–381
prevalence of, 259 p38 MAPK pathway in. See P38 MAPK
transmission of, 259 pathway
BSE, cell lines for, 121 Cag pathogenicity island (cagPAI), 332
B. subtilis and B. mojavensis virulence Caiman models for Trichinella infection, 802
factors, 134 Calcium depletion experiments, 139
B. thuringiensis, 132 Calf model of Salmonella infection, 398
virulence factors by, 142, 143 Calomys callosus, 665
Burkholderia, 271, 279 Campylobacter contamination, 290
Burkholderia cepacia complex (Bcc), 271 Campylobacteriosis, 290
animal models for pathogenicity, 276–278 Campylobacter jejuni, 289
alfalfa seedling infection model, 277 cell invasion by, 289
C. elegans, 277 colonization of animal models
duckweed, 277 acquisition of nutrients, 292
insects, 277 cellular infection models, 292
maceration of onion tissue, 277 colostrum-deprived piglets, 294
rat agar bead model, 276 ferret model, 294–295
zebrafish embryos, 278 Galleria mellonella, 295
bacteria of, 275 intestine, 289, 291
Bcc infections, 275, 276 mouse models, 295–298
organisms, 275–276 stressors, 291–292
from unpasteurized milk, 275–276 three-day-old chick model, 292–294
Burkholderia gladioli, 271 gastroenteritis due to. See Gastroenteritis
pathovars, 271 long-term complications, 289–290
taxonomic description of, 271 standard route of infection, 290
Burkholderia mallei, 271 survival mechanisms, 290
as food contaminants, 272 Campylobacter pyloridis, 331
glanders due to, 272 Candida albicans, 497
laboratory infection models of, 272–273 Candida anamorphs, 498
Burkholderia pseudomallei, 271 Candida infections
animal model for melioidosis, 274–275 causative agents of, 497
melioidosis due to, 273–274 foodborne. See Foodborne Candida infections
transmitted to humans, 273 variety of, 497
virulence mechanisms, 275 Candida species, 497
Burn-wound sepsis model of P. aeruginosa positive and negative effects on food
infection, 378 products, 497
B. weihenstephanensis, virulence factors Cannomys badidus, 558
by, 142 Capsid core proteins, 17–18
814 Index

Capsid protein, 52–53 “Cereus group,” 132

encapsidation of viral genome by, 51 CF. See Cystic fibrosis
ORF2 codes for, 51 CF mouse model of P. aeruginosa infection, 374, 377
Carbamate kinase, 645 Cheddar cheeses, 201
Carbapenem resistance, 249 Cheese, M. bovis and MAP in, 200–201
Carbapenems, 253 Chemical inactivation methods
Carnivorism, 657 for HNoV, 82
Cavia aperea, 665 Chemokines, 643
C. botulinum neurotoxin Chicken model
mouse bioassays for, 160–162 for Haplorchis infections, 739
oral poisoning with, 161 of shigellosis, 405
C. difficile enterotoxins Chickens as HEV animal model, 60–61
human colonic epithelial cell lines for, 166 Chick model of Salmonella infection, 398
C. difficile infection Chimpanzees
animal models for, 164 HEV and HCV infection in, 46
C. difficile toxins for HEV infection studies, 58–59
inflammatory cytokine release, 166 Chinchilla otitis media model, 232
proliferation and infection, 157 Chinese hamster cell line, 448
Cecal epithelium, 623 “Chinese or oriental liver fluke,” 703
C. elegans CHL cells, 448
IECs, 180 Chloramphenicol, 255
of pathogenic E. coli, 326 Chlorine dioxide (ClO2) gas, FCV-F9 as HNoV
of Salmonella infection, 398 surrogate for, 79
of shigellosis, 405 Cholangiocarcinoma (CCA), 769–770
C. elegans model Cholecystokinin, 642
for Aeromonas infections, 243 Cholera, 413–414
for Y. enterocolitica infections, 431 Choleraesuis. See Salmonella enterica
Cell culture models, 165–166 Cholera toxin B subunit (CTB), 628
L. monocytogenes infections, 186 Chronic atrophic gastritis, 336
for staphylococcal foodborne poisoning, 215–216 Chronic urticaria, 684
Cell-free supernatants (CFS), 85 Cis-regulatory genomic regions HEV genome, 52
Cell lines Citrinin, 458–459
for botulinum neurotoxin, 165–166 CJD
for foodborne Alternaria, 446–449 diagnostic laboratories, 120
for Penicillium mycotoxins, 566 epidemiological surveillance, 120
for prion infections, 121–122 prion-infection, cell-culture systems for, 121
for rotavirus infection, 106 Clonorchiasis
used in rotavirus research, 106 chemotherapy and control measures, 709
Cell-mediated host immune response, 512–513 clinical manifestations of, 707
Cell models diagnosis of, 708–709
for Y. enterocolitica infections, 431 epidemiology of, 707–708
host defense mechanisms, modulation of, Clonorchis sinensis, 703, 735
432–433 laboratory models for
mammalian cells, adhesion and invasion of, 432 chemotherapy, 711
Cell monolayers (CPE), 586 histopathology and carcinogenesis, 711–712
binding to extraintestinal tissues, 163 host susceptibility, 709–710
expression, 166 immune response and reinfection, 710–711
intestinal and systemic effects of, 163 metabolism study, 711
in vivo effects of, 163 rationale for, 709
Cell resistance mechanisms, 659 in vitro models, 712–713
Cellular immune responses induced by F. hepatica, 725 life cycle and morphology of, 703–705
Cellular infection models for C. jejuni infection, 292 metacercariae, 705
Cellular stability, 646 Clonorchis sinensis-infected intrahepatic
Central nervous system, 656 bile duct, 706
Cercariae, 718, 719 Clostridium botulinum, 155
Cercomonas intestinalis, 636 foodborne botulism associated with, 156
Cereolysin O (or Hemolysin I), 138 in food samples, investigation of, 157–159
Cereulide, 138, 140 neurotoxins. See Botulinum neurotoxin (BoNT)
spermatozoa bioassay for, 141 Clostridium intoxication, animal models for
synthesis, regulation of, 138 mouse bioassays, 160–162
in vivo studies on, 144 rat, 162
Index 815

Clostridium neurotoxins and enterotoxins pathogenesis of, 306

foodborne outbreaks due to, 156–157 selective media for detecting, 305–306
Clostridium perfringens, 155 serum tolerance of, 310
Clostridium species, 155 source of, 306
Coagulase-negative (CNS) staphylococci, 209 Crude larvae extract, 685
identification of, 211 Cryptococcus pararoseus, 579
Coagulase-positive (CPS) staphylococci, 209 Cryptosporidiosis, 593
identification of, 211 Cryptosporidium, 589
CO2 fixation, 693 animal models for, 594
Collagen-binding protein, 628 biological characteristics, 591
Colostrum-deprived piglets for C. jejuni infection, 294 classification, 589–590
Colpidium campylum, 562 clinical features and pathogenesis, 592
Complement activation pathways inhibition by astrovirus diagnosis, 593
infection, 34 genome structure, 590
Computer-assisted sperm analysis systems, 141 life cycle and epidemiology, 591
Confocal laser scanning microscopy (CLSM), 361 morphological characteristics, 590–591
Congenital toxoplasmosis, 656, 657 oocysts, 593
Conserved Domain Database, 683 treatment and prevention, 593
Contracaecum, 681 in vitro models for, 595
Contracaecum osculatum sensu stricto, 682 Cryptosporidium andersoni, 589
Correlation of Acanthamoeba T-types, with clinical Cryptosporidium baileyi, 589
diseases, 581 Cry toxin, 142
CPB mouse lethality, 163 Cutaneous acanthamoebiasis and sinusitis, 583
CPE. See Cell monolayers CWD prion infectivity, 120
C. perfringens Cyclopiazonic acid, 458
foodborne illness caused by, 157 Cyclops fuscus, 562
foodborne investigation cell culture assays, 166 Cyclospora cayetanensis, 605, 606
isolates, Caco-2 cell cytotoxicity by, 166 Cynomolgus macaques for HEV infection
toxins in food samples, investigation of, 159–160 studies, 58–59
toxins produced by, 156 Cynomolgus monkeys for human rotavirus
type A food poisoning, 157 research, 99
type C disease, pathogenesis of, 163–164 Cysteine proteases, 619
C. perfringens enterotoxins, animal models for Cystic fibrosis
calves and lambs, 164 mouse models of chronic lung infection, 374, 377
goats, 164 pathogenesis of, 374
nonhuman primates, 164 Cystic fibrosis transmembrane conductance regulator
piglets, 163–164 (CFTR), 133
rabbit intestinal loops, 163 Cystoisospora belli, 599
rats and mice, 162–163 animal models for, 608
Crassostrea rhizophorae, 668 biology of, 600
CRISPR 2 analysis, 179 classification and phylogeny, 600
Cronobacter muytjensii comparative characteristics of, 605
interaction with dendritic cells, 308–309 extraintestinal stages of, 607
interaction with epithelial cells, 307–308 life cycle of, 600–603, 601
molecular events induced by, 307 oocysts of, 602
OmpA of, 309 phylogeny of, 601
Cronobacter muytjensii-induced necrotizing in vitro growth in mammalian cells, 608
enterocolitis inoculation of sporozoites, 610
prevention by Lactobacillus, 309–310 preparation of infective oocysts, 608–610
rat model of, 306–307 Cystoisosporiasis
Cronobacter sakazakii, 305 epidemiology, 603
genotypic and phenotypic characteristics, 306 laboratory diagnosis, 605
interaction with Caenorhabditis elegans, 313 examination of duodenal content, 606
motility and biofilm formation, 312–313 histopathological diagnosis, 606
OmpA of, 309 molecular diagnosis, 607
Cronobacter species, 305 stool examination, 605
composition of, 305 pathology and clinical aspects, 604
infection, neutrophils and macrophages role in, 309 treatment and prevention, 607
infections in neonatal intensive care units, 305 Cyst production, 636
interaction with brain endothelial cells, 311–312 Cytokine response induced by AdV infections, 22–23
molecular-based detection techniques for, 306 Cytokines, 643, 726
816 Index

Cytoplasm, 320 E. faecalis strains

Cytoplasmic membrane, 320 MLST analysis of, 177
Cytoskeleton, 646 rabbit model of endocarditis, 180
Cytotoxicity, 135 E. faecium, cgMLST scheme for, 179
Cytotoxin K (CytK), 137 E. faecium isolates, BAPS analysis of ST of, 177
Egg granulomas, 769
D Egg yolk agar (EYA), 158
EHEC, 322
DAEC, 323 E. hirae, G. mellonella infection with, 180
Danio rerio for Bacillus spp. virulence, 144 EIEC, 323
DBP (genus-common protein), 16 ELISA. See Enzyme-linked immunosorbent assay
3D-cell-culture system, 55 Embryonic stem (ES) cells, 8
Debaryomyces hansenii, 498 Emetic assays, 214
Dendritic cells, 662 Emetic reflex, 144
C. muytjensii interaction with, 308–309 Emetic toxin, 138
Diarrhea, 517 Emm gene, sequencing analysis of, 225–226
Diarrhea Encystation, 638
astrovirus infections, 32, 34 Encystation-specific vesicles, 640
rotavirus, 96 Endocarditis, 178
Diarrheagenic Escherichia coli pathotypes, rabbit model of, 180
319–320 Endotoxins, 2
Dictyostelium discoideum as P. aeruginosa infection Entamoeba, 627
model, 381 Entamoeba histolytica, 617, 636
Diffusely adhering E. coli. See DAEC isolation and culture conditions, 618
Digestive enzymes and exchangers, apical life cycle of, 618
expression of, 33 virulence factors, 618
Digit abduction score (DAS) assay, 162 Enteric AdVs, 13–14
Direct immunofluorescence assay, 593 Enteric fever serovars, 394
Disaccharidase activity, 642 Enteroaggregative E. coli. See EAEC
DNA-binding protein (DBP), 16, 20 Enterobacter cloacae. See Cronobacter
DNA microarray, 158 Enterobacteriaceae, 392
DNA polymerase (pol), 16, 20 Enterobacter sakazakii. See Cronobacter
DNA replication of HAdVs, 20–21 Enterococcal endocarditis, 178
Dogs for Haplorchis infections, 739 “Enterococcal group,” 175
Dolichotis patagonum, 594 Enterococcal infections, 177
Drechmeria coniospora, 418 animal models of, 180
Drosophila melanogaster clinical features of, 178
for foodborne Candida infections, 503–504 MDR enterococcal infections, risk factors for,
as P. aeruginosa infection model, 379–380 177–178
Drug resistance, 646 pathogenesis of, 178
Duckweed for Bcc infections, 277 source of, 177
treatment and prevention, 179
E in vitro models of, 181
Enterococcal meningitis, 178
EAEC, 322–323 Enterococcal virulence factors, 180
Early (E) genes of AdV, 15 Enterococci, transmission of, 177
E1A proteins, 18 Enterococcus, 175
binding with cellular factors, 18 biochemical characteristics, 176
E1B gene products, 18 biology and epidemiology, 177–178
E1B proteins, 22 classification, 175–176
E2 gene products, 18–19 colonizers of GI tract, 177
E3 proteins, 19 genomics, 177
E4 proteins, 19 identification of species, 178–179
Ebselen, 162 morphological characteristics, 176
E-cadherin, 190 Enterococcus faecium, 643
E. coli O157:H7, 2 Enterocytozoon bieneusi, 511
Ectopic anisakidosis, 683 biochemistry and physiology of, 512
Edema factor (EF), 133 detection techniques
murine models of, 145 animal models, 515
EDIM infection, 98 immunofluorescence, 515
E. faecalis isolates vancomycin resistance, 179 light microscopy, 514
Index 817

nucleic-acid-based detection, 515 cytotoxic effect on, 135, 137

transmission electron microscopy, 514 direct interactions with, 139–140
in vitro culture, 515 Hep-2, 138
from water samples, 516 NHEB and HBLL2, 137
epidemiology of, 513 Ped-2E9 cell line, 134
in food, 513 U937 cells, 137
host range, 513 Vero cells, 137
immunology of, 512–513 European brown hare syndrome virus (EBHSV), 86
intestinal microsporidiosis, 514 Excretory-secretory, 684
life cycle of, 511–512 Exotoxins, 2
structure of, 511–512 foodborne bacterial. See Foodborne bacterial
transmission routes of, 513–514 exotoxins
treatment of, 516 Experimental disease models, 8
Enterocytozoon bieneusi infection Extraintestinal infections, 240–241
clinical symptoms and pathology of, 514 Ex vivo models of infection, 140–141
treatment of, 516 Eye, model organisms recapitulating aspects of, 374
Enterohemorrhagic E. coli. See EHEC
Enteroid models for rotavirus infection, 106 F
Enteroinvasive E. coli. See EIEC
Enteropathogenic bacteria, 143 F-actin network, 136
Enteropathogenic E. coli. See EPEC Facultative intracellular pathogen, 186
Enterotoxigenic B. fragilis (ETBF), 251–252, 256 Fasciola, 717
Enterotoxigenic E. coli. See ETEC life cycle of, 718
Enterotoxigenicity, 209 Fasciola gigantica, 717
Enterotoxin, 251 geographic distribution of, 718
Enterotoxin-producing staphylococci, 216 transmission of, 718
foodborne intoxications by, 209–210 Fasciola hepatica, 717
SEs produced by, 211 adaptability into mammalian hosts, 718
Environmental scanning electron microscopy genome of, 719–720
(ESEM), 361 geographic distribution of, 718
Enzyme-linked immunosorbent assay (ELISA), 557 host–pathogen interaction, 720
BoNT detection, 158–159 evasion mechanisms, 722
for BSE, 117 immunity, 721–722
for PrPsc detection, 118 life cycle of, 719, 720
Eosinophil chemotactic factor of parasites, 686 proteome of, 720
Eosinophilia alteration, 725 transcriptome of, 719–720
Eosinophils, 685 transmission of, 718
EPEC, 322 vaccine candidates, 728–729
Epidemic anthrax, 132 Fasciola hepatica infections
Epidemiological tracking, 396 clinical manifestations of, 722–723
Epidermal growth factor receptor (EGF-R), 22 diagnosis of, 723
Epithelial cells, C. muytjensii interaction with, 307–308 immunology of, 721–722
Epithelial organotypic models for foodborne Candida in mice, 724–726
infections, 505–506 in rabbits, 727–728
ES. See Excretory-secretory in rat, 726–727
Escherichia coli, 317, 327, 564 treatment of, 723–724
biology of, 321 Fasciolosis, 717–718, 730
classification of, 318 clinical manifestations of, 722–723
epidemiology of, 321 diagnosis of, 723
genomics, 320 epidemiology of, 718
morphology of, 318, 320 immunology of, 721–722
pathogenic. See Pathogenic E. coli incidence of, 718–719
pathotypes, diarrheagenic, 319–320 in mice, 724–726
Escherichia species, 317 parasitic species causing, 718
ETEC, 321–322 in rabbits, 727–728
Etodolac, 337 in rat, 726–727
Eukaryotic cell lines treatment of, 723–724
B. anthracis culture, 134 Feline calicivirus (FCV-F9)
B. cereus culture supernatants, 134, 135 chemical effects on, 78
binding of toxins to, 137 heat treatment evaluation on, 78
Caco-2 cells, 134, 137, 138 as HNoV surrogate, 77–80
818 Index

Feline calicivirus (FCV-F9) (cont.) gastrointestinal models, 502

ClO2 gas effects, 79 intestinal cell lines, 505
high-intensity ultrasound (HIU), 79 mouse, 502–503
HPP treatments, 79 piglets, 503
natural oils and extracts, 79–80 zebrafish, 503
viruses on lettuce, 79 Foodborne diseases, 1, 200
infection with, 80 causes of, 3
persistence/survival study of, 80 by CPEs, 157
surface inactivation methods using, 78 infections, 344
Ferrets struggle against, 1
as HEV animal model, 60 symptoms of, 3
model for C. jejuni infection, 294–295 Foodborne GAS epidemics, 227
Fimbriae, 320 Foodborne HEV infections, 44
Fimbrial expression, 356 Foodborne illness. See Foodborne diseases
Fish Foodborne microbes
intraperitoneal, 692 impact on human health, 2
oral infection, 691–692 Foodborne pathogens
5ʹNCR of HEV genome, 52 definition, 1–2
Flagella, 320 high-impact, 2
Flagella-mediated motility, 356–357 Fox models
Fluorescent in situ hybridization (FISH)-based for Haplorchis infections, 739
techniques, 515 for Trichinella infection, 802
Foodborne Alternaria analysis, laboratory models Fumagillin, 516
for, 445 Fumonisins, 458, 524–525
animal models, 446, 447 Fusaria, 523–524
cell line systems, 446 Fusariosis, 523
Caco-2 cells, 447 Fusarium solani species complex (FSSC), 524
Chinese hamster cell line, 448 Fusarium species, 523–524
CHL cells, 448 human infections caused by, 524
HT-29 cell line, 447–448 mycotoxins of. See Mycotoxins of Fusarium species
human breast adenocarcinoma cell line, 449
human colon carcinoma cells, 448–449 G
human vulva carcinoma cell line, 449
Ishikawa, 449 Gal-lectin, 627
liver hepatocellular carcinoma cell line, 449 Galleria mellonella, 142
MLC cells, 448 for C. jejuni infection, 295
mouse hepatoma Hepa-1c1c7 cell line, 449 for foodborne Candida infections, 503, 504
murine macrophage cell line, 448 for glanders infection, 273
rat hepatoma H4IIE cells, 448 infection with E. hirae, 180
in vitro tests, 446, 449–450 as P. aeruginosa infection model, 381
Foodborne bacterial exotoxins GalNAc homopolymer, 638
botulinum neurotoxin (BoNT), 2 Gametogenesis, 667
heat-stable enterotoxins, 2 GAS. See Group A Streptococcus
lipopolysaccharide (LPS), 2 Gastric carcinogenesis
pore-forming toxins (PFTs), 2 chemoprevention of, 337
superantigens, 2 exacerbating factors for, 336–337
Foodborne botulism, 156, 161 H. pylori infection role in
Foodborne brucellosis hyperplastic lesions and, 334–335
inactivation in food products, 264 intestinal metaplasia, 336
laboratory model of, 265 modifying factors for, 333–334
from meat or fish products, 264–265 intestinalization of, 336
transmission by milk, 264 oxidative stress and, 337
Foodborne Candida infections, 498 prevention of, 336
investigation of, 498–501 Gastric markers, 336
laboratory models for analyzing, 501–506 Gastric microorganisms, discovery of, 331–332
Bombyx mori, 503, 504 Gastroallergic anisakiosis, 683
Caco-2 cell line, 504–505 Gastroenteritis, 23, 85, 415–416
Caenorhabditis elegans, 503, 504 AdV infection, 22
Drosophila melanogaster, 503–504 Bacillus species and, 132
epithelial organotypic models, 505–506 due to AdVs, 13
Galleria mellonella, 503, 504 pathogen, 289
Index 819

Gastrointestinal anthrax, murine model of, 145 Group A Streptococcus (GAS) infections
Gastrointestinal infections, 240 aminal models for
pathognomonic signs of, 144 C. elegans, 232
Gastrointestinal injuries associated with SEs, 216 chinchilla otitis media model, 232
Gastrointestinal models for foodborne Candida insects, 232
infections, 502 nonhuman primate, 232
Gastrointestinal virus surrogate, bacteriophage MS2 as, rodents, 230–232
87–88 zebrafish, 232
Genbank information, 201 clinical features and pathogenesis, 227–229
Gene polymorphism, 665 APSGN, 228
Gene regulation, 646 ARF, 228
Genomic rearrangements, 203 GAS pharyngitis, 227
Genotypes, 668 impetigo, 228
Genotype 3 strain (3c), 45 PANDAS, 228–229
Genotype 3 virus culture systems, 55 scarlet fever, 228
Genotype 4 virus culture systems, 56 complications of, 225
Gerbils, 188–189, 643 diagnosis, 229
Germfree pig model. See Gnotobiotic (Gn) pig model foodborne or airborne, 227
Giardia intestinalis, 606 of skin, 228
Giardia lamblia, 635 treatment and prevention, 229
antigenic variation in, 645 in vitro models for, 232–233
biology of, 635 Group B Streptococcus (GBS), 225
drug resistance in, 646 Group C Streptococci, 225
encystation methods, 637 Group D streptococci, 175, 225
epidemiology, 639 Group G streptococci, 225
history of, 635 Group H streptococci, 225
immunology, 643 Guinea pig models
life cycle of, 636, 637 for Brucella infections, 265
models used for studies of, 641 for listeriosis, 188
morphology of, 636 of Salmonella infection, 398
trophozoites, electron micrographs of, 638 of shigellosis, 402–403
Giardia muris, 641 Gut-derived sepsis mouse models of P. aeruginosa
Giardiasis, 644, 646 infection, 377–378
diagnosis, 636 Gut epithelium, 186
drug resistance in, 646 Gut microbiota and Gn pig’s response to
epidemiology, 639 HRV, 105
nitroheterocyclic resistance in, 646 Gut, model organisms recapitulating aspects of, 374
pathology, 641
GiardiaVax, 644 H
Glanders, 271, 272
laboratory infection models of, 272–273 Hamster models
Glycopeptide, 179 for Acanthamoeba, 586
Gnotobiotic (Gn) pig model for HRV research of C. difficile infection, 164
advancements made using, 102 of glanders infection, 272
features of, 103–104 for melioidosis, 274
HGM and non-HGM-transplanted, 105 for studying prion infections, 122–123
history of, 97, 99 Haplorchis infections
HRV vaccines and therapeutics, 104 animal models for
probiotics and commensal microbiota role in, chicks, 739
104–105 dogs, 739
protective immunity mechanisms, 103 foxes, 739
Wa HRV infection, 104 rodents, 738–739
Gnotobiotic pigs, 86 clinical features of, 737–738
Gram-negative Fe(III) acquisition systems, 357 diagnosis of, 738
Granulomatous amoebic encephalitis, 583 treatment and prevention of, 738
Group A rotaviruses, 96 in vitro models of, 739
Group A Streptococcus (GAS). See also S. pyogenes Haplorchis species, 735
immune dysfunction, 3 classification of, 736
local diseases, 3 genome of, 737
localized inflammatory lesions, 3 life cycle and epidemiology of, 737
pharyngitis, 227 morphology of, 736–737
820 Index

HAstV infections, 32–34 genotype 4 virus culture system, 56

biphasic age distribution of, 32 genotype 3 virus culture systems, 55
gastrointestinal disease, 32 HEV strain propagation, 56
HBMEC. See Human brain microvascular endothelial Kernow C-1 virus strain, 55
cells primary hepatocytes, 56
Hecolin, 48 HEV genome
Hektoen enteric (HE) agar, 402 cis-regulatory genomic regions, 52
Helicase of HEV, 50 components of, 49–50
Helicobacter pylori, 331 ORFs, 49–50
eradication, prophylactic effect of, 336 proteins encoded by
gastric microenvironment of, 332 capsid protein, 51
genome of, 332 helicase, 50
immunohistochemical view of, 332 hypervariable region, 51
infection macro domains, 51
in childhood, 333 methyltransferase, 50
and gastric carcinogenesis, 333–334 nonstructural proteins, 50
hyperplastic lesions, 334–335 ORF3 protein, 51–52
intestinalization of gastric cancer, 336 PCP, 50
intestinal metaplasia, 336 RdRp, 51
and NETs, 334 schematic diagram of, 50
pathogenesis of, 332–333 HEV genotype 1, 56
synergistic effects of, 336–337 HEV genotype 3, 56
transmission routes, 333 HEV genotype 4
Hemolymph, enterococcal virulence factors in, 180 culture system, 56
Hemolysin (HpmA), 358 HEV genotypes
Hemolysin BL (HBL), 135, 137 characteristics of, 48–49
activity, 143 diagnostic antigens from, 47
production, regulation of, 139 HEV IgM-positive patients, viral RNA in, 47
Hemolysin III, 138 HEV infection disease course in nonhuman primates,
Hemolysin IV, 137 58–59
Hemolytic uremic syndrome (HUS), 318 HEV infections
Hemorrhagic septicemia, 241 adaptive immune response to, 47
Heparin sulfate-binding protein, 628 antiviral therapy for, 48
Hepatic damage, 725, 727, 728 clinical manifestations of, 46
Hepatic transaminases, 725 diagnosis, 47–48
Hepatitis E virus. See HEV due to undercooked pork, 44
Hepeviridae, 42 genotype 1 infections, 48
Heterotopic proliferative glands (HPGs), 335 genotype 3 infections, 48
HEV, 41 in humans, 44–46
epidemics of, 42 incidence of, 42
genotypes, 49 innate immune response to, 46–47
life cycle, 52–53 severity of, 45
peak shedding of, 46 shellfish consumption, 44
phylogenetic tree, 42–43 symptoms of, 44
sporadic cases of, 42 HEV ORF2 protein expression, 47, 48
thermal stability of, 55 HEV replication, 54
tools for studying cell-culture system for, 55
HEV cell-culture models. See HEV cell-culture in PLC/PRF/5 and A549 cells, 56
models HEV RNA testing, 47
infectious cDNA clones, 53–54 HEV strain propagation, 56
transmission of, 43–44 HEV strains
HEV animal models genetic diversity among, 42
chickens, 60–61 virulence of, 45
ferrets, 60 HEV vaccine, 48
nonhuman primates, 58–59 Hexon, 16
pigs, 59 Hexon-associated proteins, 16
rabbits, 60 High hydrostatic pressure effects TV inactivation, 84
rodents, 59–60 Highly activated antiretroviral therapy (HAART),
HEV cell-culture models, 54 512–513
animal species for, 57–58 High pathogenicity island (HPI), 357
3D-cell-culture system, 55 High-pressure processing, 79
Index 821

High-salt diet, 336–337 Human gut microbiota (HGM), 99

High temperature, short time (HTST) pasteurization of Human ileocecal epithelial cell line, 595
milk, 200 Human intestinal epithelial cell line, 166
HlyII, 137 Humanized mice, 8
HLyII production, regulation of, 138 Human norovirus (HNoV)
HNoVs challenges facing cultivation of, 76
inactivation in seeded oysters, 77 genome, 75
source of live infectious, 77 genotype, 76
HNoV surrogates, 76 incubation period, 76
animal viruses as outbreaks, 76
bovine noroviruses, 86 surrogates, 76
feline calicivirus (FCV-F9), 77–80 Human rotavirus (HRV) research, animal models
murine norovirus (MNV-1), 80–84 for, 100
norovirus-like particles, 86–87 Gn calf model, 97
porcine sapovirus, 85–86 gnotobiotic (Gn) pig model, 97, 99
rabbit caliciviruses, 86 mouse model, 97, 98
Tulane virus (TV), 84–85 nonhuman primates, 97, 99
“Hog-cholera bacillus,” 391 rabbits and rats, 97
Homologous animals, 8 Human sapoviruses, 85
Homozygous nude mice, 8 Human umbilical vein endothelial cells (HUVECs),
Horse models of Trichinella infection, 800–801 311–312
Host defense mechanisms, modulation of, 432–433 Human vulva carcinoma cell line, 449
Host innate immune response to HEV infections, 46–47 Hydrolytic enzymes, 692
Host iron compounds, 357 Hypercontractility, 642
Host–pathogen interactions, 143 Hyperinfection model, 724
adenovirus, 22 Hyperpotassemia, 162–163
Proteus mirabilis, 362–363 Hypervariable region (HVR), 51
studies, C. elegans as model for, 346, 418 Hypophthalmichthys molitrix, 668
Host range restriction, 101 Hysterothylacium, 681
Host susceptibility, laboratory models for, 709–710 Hysterothylacium aduncum, 682
Housekeeping genes, 177
HRV infections, 96 I
HRV vaccine-induced immune responses
HGM and non-HGM-transplanted Gn pigs, 105 IA. See Intestinal amoebiasis
HRV vaccines and therapeutics, 104 ICell neurons, 165
HT-29 cell line, 447–448 IEC-6 cells, 309–310
Human AdV-type 40 C. muytjensii interaction with, 307
genome structure of, 16 IFN-α-2a treatment, 48
Human amoebiasis, 617–619 IgM+ -bearing cells, 692
Human astroviruses (HAstVs), 29 IHC-based analyses, 119
immunity to, 34–35 Ileum submucosa, 665
serotypes, 31 Immune dysfunction, foodborne pathogens causing, 3
Human blood, model organisms recapitulating Immune response
aspects of, 374 to astrovirus infection, 34–35
Human botulism, categories of, 156–157 against M. bovis, 200
Human brain microvascular endothelial cells to urinary tract infections, 364–365
(HBMECs), 586 Immunization for UTIs, 364
C. muytjensii entry into, 311–312 Immunocompromised intranasal mouse lung
Human breast adenocarcinoma cell line, 449 infections, 276
Human cell lines Immunocompromised patients
for Fusarium mycotoxins, 539–546 AdV infections, 23
for mycotoxins of Aspergillus species, 467, 479–487 astrovirus infection, 32
of shigellosis, 405–406 chronic HEV infections in, 45
Human colon carcinoma cells, 448–449 Immunofluorescence, 515
Human colonic epithelial cell lines, 166 Immunoglobulins, 627
Human-derived cell systems, 165 Immunological techniques, 723
Human E-cadherin, 188 Immunological tools, 212
Human embryonic kidney (HEK) cells for enteric Immunopathogenesis, 684
AdVs, 14 Immunoregulatory molecules, 691
Human fasciolosis. See Fasciolosis Impetigo, 228
Human feeding studies, 77 Inactivation methods on PEV, 86
822 Index

Incubation period in macaques, 46 Invasion assays, 252

Inducible nitric oxide synthase (iNOS), 34–35, 307 Invertebrate models for mycotoxins of Aspergillus
Industrial enzymes, 456 species, 459–460
Infant botulism (IB), 156–157 Invertebrates
Infantile gastroenteritis, 99 insects
Infection models Bombyx mori, 143
of glanders, 272–273 Galleria mellonella, 142
of melioidosis, 274–275 nematodes, 141–142
Infectious cDNA clones In vitro animal models, 3, 7
of HEV, 53–54 cell lines, 4–5
Kernow C-1 virus strain, 55 In vitro chemotaxis, 685
Infectious endocarditis (IE), 177, 178 In vitro culture for Enterocytozoon bieneusi, 515
Infective oocysts, preparation of, 608–610 In vitro culture models, advantages of, 9
Inflammatory cytokine, 644 In vitro cytotoxicity, 166
Inflammatory reaction, 683 In vitro-derived cysts, 640
Inh metalloproteases, 143 In vitro models
Innate immune response for Aeromonas infections, 243
to astrovirus infection, 34 for Clonorchis sinensis, 712–713
to HEV infections, 46–47 cultured eukaryotic cells, 134–138
mammal vs. C. elegans, 347 enterococcal infections, 181
triggered by rotavirus infection, 101 of Salmonella infection, 398–399
Innate immunity to urinary tract infections, 365 In vivo animal models, 3, 4, 5–7
Interchain disulfide reduction, 162 mice, 8
Interferon consensus sequence binding protein, 662 nonhuman primates (NHP), 5
Interferons (IFNs), 22 In vivo experimentation, 684
HEV interference with, 46 In vivo L3–L4 transformation model, 688
Interleukin, 662 In vivo models of infection, 140–141
Intermediate (I) genes of AdV, 15–16 Ionizing radiation, 84
nonstructural proteins encoded by, 19 Iron acquisition systems, 357
Internalins, 186 Ishikawa, 449
Internal transcribed spacer, 607 Isolator pig model. See Gnotobiotic (Gn) pig model
Intestinal amoebiasis, 617 Isomorphic animals, 8
models used to study, 620 Isospora belli, 599
Intestinal amoebiasis, 619 Isospora gryphoni, 600
Intestinal anthrax, outbreak of, 132 Isospora hominis, 600
Intestinal Bacteroidales, diagnosis of, 252–253 Isospora natalensis, 599
Intestinal cell lines for foodborne Candida Isospora robini, 600
infections, 505
Intestinal colonization, 105 K
Intestinal commensal microbiota, 105
Intestinal epithelial antiviral host defense, 101 Kanagawa phenomenon (KP), 416
Intestinal epithelial cells (IECs), 180 Keratitis models of P. aeruginosa infection, 378–379
Intestinal epithelial innate immune response, age- Kernow C-1 virus strain, 55
dependent mechanisms of, 101 Klebsiella pneumoniae infection, 344
Intestinal epithelium, 145 agent for foodborne diseases, 343–344
Intestinal function changes astrovirus infection, animal models for
32–34 C. elegans, 346–351
Intestinal histology changes with astrovirus Galleria mellonella, 346
infection, 32 murine models, 345–346
Intestinal immunity, 180 diseases caused by, 344
Intestinalization of gastric cancer, 336 multidrug resistance, 344
Intestinal markers, 336 pathogenicity of, 347
Intestinal metaplasia, 336 p38 MAPK pathway role against, 349–350
Intestinal microbiota, diagnosis of, 252–253 Toll receptor role against, 350
Intestinal microsporidiosis, 511, 514 virulence gene upregulation during, 351
Intestinal stem cells, 186 Kluyveromyces marxianus, 498
Intestinal villus enterocytes, 102 Knock-in mice, 8
Intra-abdominal infections, 178 Knockout mice, 8
Intragastric infection, 687 for HRV research, 98
Intranasal immunization, 364 rotavirus protective immunity, 103
Intraperitoneal larval implant, 687 Kojic acid, 564
Index 823

L LPS, 306, 365

in B. fragilis, 251
Laboratory models of Brucella, 261, 262
characteristics of, 4–9 Lung, model organisms recapitulating aspects
considerations for selecting, 9 of, 374
milestones in use of, 4 Lutjanus campechanus, 556
rationales for using, 3–4 Lymphadenopathy, 556
in vitro animal models. See In vitro animal Lymphocytes, 624
models from astrovirus-infected turkeys, 34
in vivo animal models. See In vivo animal
models
Lactate dehydrogenase, 570 M
Lactobacillus bulgaricus, NEC prevention by, Maceration of onion tissue, 277
309–310 Macrobrachium nipponense, 668
Lactobacillus johnsonii, 643 Macro domains, 51
Lactobacillus rhamnosus GG (LGG) Macrolides, 255
Gn pig model colonized with, 104–105 Macrophage migration inhibitory factor-like
mechanisms reducing HRV pathogenesis, 105 protein, 691
Lamina propria, 618, 622, 624, 627, 643 Macrophages, 659
Larval stage, 681 role in Cronobacter infection, 309
Late (L) genes of AdV, 16 Major histocompatibility complex class I proteins
nonstructural proteins encoded by, 19–20 (MHC-I) CD8+ T cells, 22
LDH. See Lactate dehydrogenase Malta fever
Leishmania amazonensis, 660 prevalence of, 259
Leprosy, 197 transmission of, 259
Lettuce, FCV-F9 on, 79 Mamastrovirus (MAstV), 29
Light microscopy, 514, 636 Mammalian cell entry (Mce) gene, 200
Lincosamides, 255 Mammalian cells, adhesion and invasion of, 432
Lipase, 692 Mammalian HEV genotypes, transmission of, 46
Listeria, 189 MAPK pathway, 133
Listeria monocytogenes, 185 Marmosets, 272
Listeriosis, 187 Mass spectrometry method, 212
Liver damage due to immune response, 47 Mastadenovirus, 14
Liver fluke. See Opisthorchis viverrini Maternal antibodies, 104
Liver-fluke-mediated tissue damage, pathogenesis and M. avium subsp. paratuberculosis (MAP), 198
pathology of, 705–706 in cheese, 200
Liver hepatocellular carcinoma cell line, 449 genomes from, 201–202
Liver injury, HEV infection, 44 Mce gene in, 200
L4-100-kDa protein, 19–20 mycobactin and, 199–200
L. monocytogenes virulence of, 199–200
facultative intracellular pathogen, 186 M. bovis
genome, 186 in cheese, 200–201
InlA m-expressing, 190 immune response against, 200
mouse-adapted strains of, 190 infection, 201
oral transmission, 190–191 survival of, 201
virulence potential of, 189 transmission of, 197
L. monocytogenes infections M. bovis AF2122/97
animal models of, 187–189 genomes, 201–202
gerbils, 188–189 M. bovis strains
guinea pigs, 188 virulence factors of, 199
mice and rats, 187–188 M. caprae, 198
nonhuman primates, 189 Mce genes, 200
rabbits, sheep, and goats, 187 M cells, 186
cell culture models, 186 MDR enterococcal infections, risk factors for,
gastrointestinal symptoms, 185 177–178
incidence of, 185 Meiosis, 657
public health concern with, 185–186 Melioidosis, 271
L. monocytogenes isolate, virulence of, 189 animal model for, 274–275
Local diseases, 3 antibiotic treatment for, 273–274
Localized inflammatory lesions, 3 incidence of, 273
Loop-mediated isothermal amplification (LAMP), 158 transmission routes, 273
824 Index

Meningitis neuroblastoma rat glioma hybrid cell line, 165

causes of, 310 oropharyngeal colonization model, 231
Cronobacter interaction with brain endothelial cells, Mice models, 192
311–312 for Acanthamoeba, 585
neonatal rat model of, 310–311 of Brucella infections, 265
occurrence of, 310 of C. difficile infection, 164
Mesenteric lymph nodes, 691 of C. jejuni infection, 295–298
Metacercariae, 719, 720 for foodborne Candida infections, 502–503
Metacestodes, 785 H. pylori-associated stomach carcinogenesis in, 334
Metachronous cancer development, 337 for human disease research, 8
Metagonimiasis, 758 inbred, 8
animal models for for listeriosis, 187–188
chemotherapy and control, 758 for mycotoxins of Aspergillus species, 460–479
immunity and host–parasite interaction, of prion infections, 122–123
756–757 of Salmonella infection, 397–398
pathogenesis and pathology, 756 of shigellosis, 403–404
Metagonimus, 744 for Trichinella infection, 797
Metagonimus spp. of Trichinella infection, 797
biology and life cycle of, 747 for Y. enterocolitica infections, 428–430
classification and morphology of Mice models for HRV research, 97, 98
Metagonimus hakubaensis, 746–747 age-dependent mechanisms, 101
Metagonimus katsuradai, 746 knockout mouse models, 101
Metagonimus minutus, 746 rotavirus protective immunity, 102–103
Metagonimus miyatai, 745–746 type I and II IFNs, 101
Metagonimus otsurui, 746 vaccine development, 103
Metagonimus takahashii, 745 Microarray analysis, 726
Metagonimus yokogawai, 744–745 Microbes growth, maintenance media, 2
morphology and taxonomy, laboratory models for Microbial pathogens, 2
cats, 752 Microbiome, 643
dogs, 752 Microsporidia, 511
ducks and chickens, 753 molecular diagnostic tests for, 515
general considerations, 751 Microsporidian species, 511
gerbils, 753 Microsporidiosis, 517
guinea pigs, 753 Migration inhibitory factor-like protein, 691
hamsters, 752 Minor coat proteins, 16–17
mice and rats, 753 Miracidia, 719
pathogenicity of, 744 MLC cells, 448
worm development and host susceptibility, animal M. nematophilum, 418
models of MNV infection, 80
cats, 754 Moina macrocopa, 562
dogs, 753–754 Molecular techniques, 723
ducks and chickens, 755–756 Mongolian gerbil models, H. pylori-associated stomach
gerbils, 755 carcinogenesis in
guinea pigs, 755 high-salt diet, 336–337
hamsters, 754 hyperplastic lesions, 334–335
mice and rats, 754–755 intestinal metaplasia, 336
Metagonimus spp. infections, 744 modifying factors for, 333–334
diagnosis of, 750 Mouse neutralization test (MNT), 160
treatment and control of, 751 M. tuberculosis, 200
in vitro culture, 758 H37Rv genomes, 201–202
Metagonimus yokogawai infection life cycle, 198
epidemiology and distribution of, 748–749 Mce gene in, 200
pathogenesis and clinical features of, 749–750 Mucins, 332
Metalloproteinase ZapA, 358 Mucosal vaccines, 364
Methyl-accepting chemotaxis protein (MCP), 313 Multidrug resistance, 177, 256
Methyltransferase, 50 Multilocus sequence typing (MLST), 177, 179, 226
Metronidazole, 626 MUMi indexes, 202
Mice Murine footpad model, 231
with conditional gene modifications, 8 Murine impetigo model, 230
hepatoma Hepa-1c1c7 cell line, 449 Murine implanted chamber model, 231
host–pathogen interactions modeling using, 144–146 Murine macrophage cell line, 448
Index 825

Murine model of gastrointestinal anthrax, 145 trichothecenes, 525

Murine nasal-associated lymphoid tissue (NALT), 231 zearalenone (ZEA), 524
Murine norovirus (MNV-1) Mycotoxins of Penicillium spp. and Talaromyces spp.,
as HNoV surrogate, 80–83 558–566
for inactivation methods, 81–84 cell cultures in toxicity bioassays, 565
limitations of, 83–84 culture media for mycotoxin production, 565
RNA and ORFs, 80 general description, 558
survival of, 80–81 invertebrate organisms
transmission routes, 80 in toxicity bioassays, 562
Murine pneumonia models cytokine response, 22–23 used in bioassay tests, 562
Murine rotaviruses, 97, 98 microorganisms, in toxicity bioassays, 563
protective immunity mechanisms, 102–103 vertebrate animals
Murine subcutaneous air sac model, 230 in toxicity bioassays, 564
Murine subcutaneous ulcer model, 230 used in several bioassay tests, 565
Murine systemic disease model, 230–231 Myeloperoxidase activity, 694
Murine vaginal colonization model, 231 Myenteric neurons, 667
Mycobacterial Interspersed Repetitive Units
(MIRUs), 203 N
Mycobacterial life cycle, 198
Mycobacterium avium complex (MAC), 198 N2a (cell line), 121
Mycobacterium bovis, 197 Natural plant and fruit extracts as laboratory
Mycobacterium leprae, 197 surrogate for HNoVs, 83
Mycobacterium spp., 197 Necrotizing enterocolitis (NEC)
foodborne illnesses associated with, 200–201 C. muytjensii-induced, rat model of, 307
genomics of, 201–202 etiology of, 306
genomic rearrangements, 203 prevention by Lactobacillus, 309–310
repeat sequences, 203 Negative disease models, 8
pathogenic members, 198 Neglected tropical diseases (NTDs), 709
zoonotic transmission of, 197 Nematodes, 141–142
Mycobacterium tuberculosis complex (MTBC) Nematodes, in vitro cultivation of, 692
foodborne transmission of, 198 Neonatal Gn pigs, diarrhea in, 102
genomes, 201–202 Neonatal-meningitis-causing E. coli. See NMEC
infection, 197 Neonatal rat model of meningitis, 310–311
members of, 198 Neuraminidase, 251
mycobactin and, 199–200 Neuroendocrine tumors (NETs), 334
virulence of, 199–200 Neurogenic cell lines for botulinum neurotoxin, 165
Mycobactin, 199–200 Neuronal cell lines, 122
Mycotoxins of Alternaria species, 2–3, 442–443 Neutrophils role in Cronobacter infection, 309
bioaccessibility of, 450 Niclosamide, 787
EFSA on, 443 Nitric oxide (NO), 307, 645, 659
production of synthase, 645
environmental conditions for, 442–443 Nitroheterocyclic drugs, 646
evaluation of, 442 Nitroimidazoles, 255
investigation and analysis of, 443–445 NK cell levels, 47
Mycotoxins of Aspergillus species, 456 NMEC, 323
aflatoxins, 456–457 “Non-albicans” Candida species, 497
citrinin, 458–459 Nonbacterial gastroenterititis, 76
cyclopiazonic acid, 458 Nonenterococcal streptococci, 175
fumonisins, 458 Nonhemolytic enterotoxin (NHE), 135, 137
laboratory models for production, regulation of, 139
human and animal cell lines, 467, 480–487 Nonhuman primate models
invertebrate models, 459–460 for Brucella infections, 265
mouse models, 460–479 for L. monocytogenes infection, 190
laboratory models for studying, 456 for melioidosis, 274
ochratoxins, 457 of Salmonella infection, 398
patulin, 458 Nonhuman primate pharyngitis model, 232
Mycotoxins of Fusarium species, 523 Nonhuman primates (NHP), 5
fumonisins, 524–525 for CPE intoxication pathogenesis, 164
laboratory models for as HEV animal models, 58–59
human and animal cell lines, 539–546 sepsis model, 232
rodents, 525–539 Nonneuronal cell lines, 122
826 Index

Nonstructural proteins, ORF1, 50 Paragonimiasis

Nontuberculous mycobacteria (NTM), 198 chronic pleuropulmonary, 776
Nontyphoidal Salmonella (NTS) diagnosis of, 776–777
clinical features of, 394 early-stage disease, 775–776
infection with, 394 ectopic, 776
pathogenesis of, 395 treatment and prevention of, 777
serovars, 392, 394 Paragonimus species, 773
Noroviruses (NoVs) classification of, 774
genogroups of, 76 experimental models for
structure, 75 cats, 778
Norovirus-like particles as HNoV surrogate, 86–87 dogs, 778
Norwalk virus (HNoV genogroup I.1), inactivation of, 77 rodents, 778
Nosocomial infection, 96 in vitro cultures, 778
NO synthase, 643 life cycle and epidemiology of, 774–775
Nucleic-acid-based detection, 515 morphology of, 774
Nucleic-acid-based techniques, 445 paragonimiasis. See Paragonimiasis
Null mutations, 359 Paralichthys olivaceus. See Olive flounder
Parasitemia, 666
O Parasitological techniques, 723
Parasitophorous vacuole, 658
Ochratoxins (OTs), 457, 565 Parenteral carbapenem, 180
Oligonucleotide microarray method, 515 Parenteral immunization, 364
Olive flounder, 691 PAS (Per-Arnt-Sim sensory) domains, 313
Oncorhynchus mykiss. See Rainbow trout Passive antibodies, 104
Onion tissue, maceration of, 277 Pathogen-associated molecular patterns
Opisthorchiasis-associated cholangiocarcinoma, (PAMPs), 261, 365
769–770 Pathogenic E. coli
Opisthorchis sinensis, 703 AIEC, 323
Opisthorchis viverrini, 735, 765 animal models of
infection, animal models for, 766 C. elegans, 326
freshwater cyprinoid fish, 767 pigs, 326
hamster and gerbil, 767 rabbits, 325
opisthorchiasis-associated cholangiocarcinoma, rodents, 325
769–770 DAEC, 323
white blood cells levels hamster, 768–769 diagnosis, 324
morphological characteristics of, 765–766 EAEC, 322–323
Oral gavage, 190 EHEC, 322
Oral infection models, 189 EIEC, 323
Oral transmission in experimental animal models, EPEC, 322
190–191 ETEC, 321–322
ORF1 polyprotein NMEC, 323
polyprotein processing of, 50 SCEC, 323
translation, 53 STEAEC, 323
ORF2 protein, 53 treatment and prevention, 324–325
ORF3 protein, 51–52, 53 UPEC, 324
Organoids for rotavirus infection, 106 in vitro models of, 326
Ornithine carbamoyltransferase, 645 Pathogen recognition receptors, 618
Orphan disease models, 8 Pathogens, 556–558
Osteolysis, 556 diagnosis, 557
Otitis media models of P. aeruginosa infection, 379 foodborne, clinical diseases caused by, 3
Outer membrane protein A (OmpA), 308 general description, 556–557
Oxidative stress, 337, 646 molecular epidemiology, 557–558
Ozone treatment, FCV-F9 as HNoV surrogate for, 79 treatment, 558
Pattern recognition receptors (PRRs), 262, 592
P Patulin, 458
PBP, 253, 255
Parabacteroides distasonis PCP, 50
β-lactamase production in, 255 PCR assays for Bacteroides species, 256
resistance genes in, 255 PCR-based methods, 211
Parabacteroides, phenotypic characteristics of, 250 PE (Pro-Glu) and PPE (Pro-Pro-Glu)
Parafossarulus manchouricus, 705 protein, 199
Index 827

Pediatric autoimmune neuropsychiatric disorders Predictive animals, 8

associated with streptococcal infections Pregnancy, HEV infection during, 45
(PANDAS), 228–229 Primary cells, 215–216
Pelvic infections, 178 Primary hepatocytes as culture system, 56
Penicillia allergens, 571 Prion agents, main component of, 117
Penicillin, 249 Prion diseases, 117
Penicillin-binding proteins (PBP), 249 diagnostic methods for, 117–119
Penicillium, 555 Prion infection
animal models to study allergies, 567 animal models for, 122–123
cell cultures to study allergies caused by cell-culture models for, 120–122
Penicillium, 570 cell lines, 121
as environmental allergen, 555 general cell-culture procedures, 120
species causing food allergies, 567 cell-culture procedures for, 120
Penicillium corylophilum, 556 in vitro models for
Penicillium sensu stricto, 555 PMCA, 119–120
Penicillium verrucosum, 563 RT-QuIC assay, 120
Penton base and fiber proteins, 17 Prion protein (PrP), Western blotting for, 117, 118
Peripheral blood mononuclear cells (PBMCs), 216 Probiotic LGG, immune-modulating effects of, 104–105
Peritonitis, 721 Procambarus clarkii, 668
Phagocytic cells, 140 Proinflammatory cytokines, 656
Phosphatidylserine, 660 Prokaryote–eukaryote interactions, 139
Photobacterium phosphoreum, 564 Protein misfolding cyclic amplification (PMCA)
Phylogenetic whole genome tree, 202 modified version of, 120
Physical inactivation methods for HNoV, 82–83 normal brain homogenate, 120
Piglet models for PrPres amplification, 119
for foodborne Candida infections, 503 Proteins encoded by HEV genome
of shigellosis, 404 capsid protein, 51
Pig models helicase, 50
for Acanthamoeba, 586 hypervariable region, 51
of pathogenic E. coli, 326 macro domains, 51
for Trichinella infection, 798–800 methyltransferase, 50
for Y. enterocolitica infections, 430–431 nonstructural proteins, 50
Pigs ORF3 protein, 51–52
as HEV animal model, 59 PCP, 50
infectious cDNA clones, 54 RdRp, 51
PEC in, 86 Proteins kinases, 659
as reservoirs of HEV, domestic, 44 Protein toxins, 135
shigellosis in, 404 HBL and NHE, 135, 137
Pilus genes, genetic analysis of, 226 HlyII, 137
Plague, 427 Proteobactin, 357
Plasmid-borne toxin genes, 159 Proteus, 355
Plasmid profiling of Salmonella infection, 396 Proteus mirabilis, 355, 564
Plasmids, 256 biofilm formation, 357, 359–360
P38 MAPK pathway, 418 evaluation techniques, 361
immune defense against pathogens, 347, 349–350 cell-to-cell communication, 361
immune regulators, 347 exogenous AHL addition to, 357
Pneumonia outbreaks, etiologic agents for, 13 filamentous morphology of, 357
Poly-δ-d-glutamic capsule, 133 flagella-mediated motility, 356–357
Polymerase chain reaction (PCR), 158, 515, 607, 661 food deterioration and spoilage, 356
for C. botulinum BoNT genes, 158 host–pathogen interaction, 362–363
for C. perfringens toxin genes, 159 nosocomial infections caused by, 356
Polymicrobial infections, treatment of, 179 quorum-sensing system, 357, 360
Porcine and human physiology, differences between, 104 urease production, 357
Porcine brucellosis, 261 virulence factors, 356–358
Porcine enteric caliciviruses (PEC), 86 biofilm, 357, 360
Porcine rotavirus infection, 97, 99 fimbrial expression, 356
macroscopic changes induced by, 102 iron acquisition systems, 357
Porcine sapovirus as HNoV surrogate, 85–86 LuxS, 357
Porins, 255 metalloproteinase ZapA, 358
Powdered infant formula, 306 toxins, 358
Praziquantel, 787 wild-type adhesion to Vero cell line, 363
828 Index

Proteus mirabilis infection of Salmonella infection, 398

associated with biofilm formation, 360 of shigellosis, 404
complications in, 355 for Y. enterocolitica infections, 431
vaccines against, 364 Rabbit subdermal abscess model, 180
Proteus toxic agglutinin (Pta), 358 Rainbow trout, 691, 692
Proton pump inhibitors (PPIs), 334 Random transposon mutagenesis, 359
Prototheca wickerhamii, 584 Raphidascarididae, 680
PrPres amplification method, 119–120 Rat autoimmune carditis model, 231
PRRs. See Pathogen recognition receptors Rate models for Trichinella infection, 797–798
Pseudokinases, 659 Rat hepatoma H4IIE cells, 448
Pseudomonas, 373 Rat models
Pseudomonas aeruginosa for Acanthamoeba, 585
infection models, 373, 375–376, 383 for Bacillus spp. infections, 146
acute lung infection mouse models, 377 for Brucella infections, 265
Arabidopsis thaliana, 382 for HEV, 59–60
Bombyx mori, 382 for listeriosis, 187–188
burn-wound sepsis model, 378 for melioidosis, 274
Caenorhabditis elegans, 380–381 Rat oropharyngeal colonization model, 231
CF mouse model, 374, 377 Rattus norvegicus, 663
Dictyostelium discoideum, 381 RdRps, 53
Drosophila melanogaster, 379–380 HEV, 51
Galleria mellonella, 381 Reactive oxygen species, 658, 727
gut-derived sepsis mouse models, 377–378 Real-time quaking-induced conversion (RT-QuIC)
keratitis models, 378–379 test, 120
otitis media models, 379 Recombinant protein-based candidate vaccine for
zebrafish, 379 HEV, 48
opportunistic human pathogen, 373 Repeat sequences in Mycobacterium spp., 203
prevalence of, 373 Restriction fragment length polymorphisms, 557
Pseudomonas syringae, 564 Retinopathy, 666
Pseudorasbora parva, 706 RFLP. See Restriction fragment length
Pseudoterranova, 680, 681, 685 polymorphisms
Pseudoterranova decipiens, 682 Rhesus macaques for melioidosis, 274
Pulsed-field gel electrophoresis (PFGE), 179 Rhesus monkeys
Pungtungia herzi, 706 B. cereus ingested with foods, 144
Putative subgenomic promoter, 52 for HEV infection studies, 58–59
PVI (minor coat protein), 16–17 for human rotavirus research, 99
PVIII and pIX (minor coat protein), 17 of shigellosis, 405
Pyloric gland mucous cell mucin (GMCM), Rhizomys pruinosus, 558
332, 336 Rhizomys sinensis, 558
Pyoderma. See Impetigo Rhombencephalitis, 189
Ribavirin therapy for genotype 1 HEV
Q infections, 48
Rice bran supplementation HRV-specific immune
Quinolones, 255–256 responses, 104
Quorum sensing, 357, 360, 414. See also Bacterial Rodent models
biofilms for Aeromonas infections, 242–243
for Fusarium mycotoxins, 525–539
R for Haplorchis infections, 738–739
for HEV, 59–60
Rabbit caliciviruses as HNoV surrogate, 86 for melioidosis, 274–275
Rabbit hemorrhagic disease virus (RHDV), 86 of pathogenic E. coli, 325
Rabbit ileal loops (RILs), 143 for S. pyogenes strain, 230–232
Rabbit intestinal loop model Rodents, 738–739
CPB along alimentary canal, 163 Rotaviruses
CPE binding to extraintestinal tissues, 163 circulating in humans, 96
Rabbit models diarrhea, 104
for Acanthamoeba, 586 genome, 95
for Brucella infections, 265 groups, 96
for HEV, 60 heterologous, 101
for listeriosis, 60 homologous, 101
of pathogenic E. coli, 325 host range restriction, 101
Index 829

immunity and vaccine development Sarcocystis, 600

in Gn pig models, 103–106 Sarcocystis hominis, 605
in mouse models, 102–103 Sar-55 strain of genotype 1 HEV infectious cDNA clones, 54
pathogenesis, 101–102 Scarlet fever, 228
replication, 101 Scavengers, 1
strains preference for neonates, 101–102 SCEC, 323
transmission routes, 95 Schizogony, 656
vaccination, 96 Sciatic-nerve-injury-induced neuroimmunological
vaccines, 96 changes, 162
heterologous attenuated oral, 101 Seals models for Trichinella infection, 802
Rotavirus fecal shedding, 98 Secondary autoimmune sequelae, 229
Rotavirus infection Secondary cells, 215–216
in adults, 102 Septicemia and meningitis, 310–312
age-dependent pathogenesis of, 101 Serine-rich E. histolytica protein, 628
causes of, 96 Serological typing scheme for Streptococcus sp.,
cell lines and organoid/enteroid models for, 106 225–226
general progression of events in, 102 Serotyping and biotyping, Salmonella
symptoms of, 96 infection, 395
RRNA sequence analysis Serovars, 189, 392–393
enterococcal species, 178–179 Serum tolerance of Cronobacter spp., 310
RT-QuIC assay, 120 Severe combined immunodeficient (SCID), 662
microarray analysis, 619
S mouse-human intestinal xenograft, 623
Sexual reproduction, 658
Salmonella, 391 Shellfish consumption, HEV infections due to, 44
biology of, 393 Shellfish testing positive for HNoV, 77
classification of, 392 Shiga-toxin-producing enteroaggregative E. coli. See
epidemiology of, 393–394 STEAEC
genomics of, 393 Shigella species, 401
morphology of, 392–393 bacteriology of, 402
Salmonella bongori, 392 serotypes of, 402
genome of, 393 transmission of, 402
Salmonella enterica, 391 Shigellosis
serovars in, 392 animal model for assaying
Salmonella infection C. elegans, 405
animal models for chicken, 405
calves, 398 guinea pigs, 402–403
C. elegans, 398 human cell lines, 405–406
chick model, 398 mice, 403–404
guinea pigs, 398 pigs and piglets, 404
mice, 397–398 rabbits, 404
nonhuman primates, 398 rhesus monkeys, 405
rabbits, 398 zebrafish, 405
zebrafish, 398 incidence of, 401
clinical features of, 394 pathogenesis of, 402
diagnosis of transmission, 402
epidemiological tracking, 396 Short-sequence repeats (SSRs), 203
plasmid profiling, 396 Siderophores, 357
serotyping and biotyping, 395 Signature-tagged mutagenesis (STM), 359
virulence and resistance gene profiling, 395 Silkworm model, 232
pathogenesis of, 395 Simulated endocardial vegetations (SEVs), 181
prevention of, 396–397 Sinusitis, 583
treatment of, 396 Sister chromatid exchange (SCE) method, 565
in vitro cell models for, 398–399 SMP-601
Salmonella infections, 392 pharmacodynamics of, 180
Salmonella pathogenicity islands (SPIs), 393 SMW inhibitors to botulinum neurotoxin (BoNT), 161–162
Salmonella typhimurium, 392, 564 SNPs, 199
genome of, 393 Sodium dodecyl sulfate (SDS), 82
Salmonellosis. See Salmonella infection Sodium/hydrogen exchanger-3 (NHE-3) molecule, 34
Salmonicida sensu stricto, 241 Soft tissue infections, 178
Salmo salar. See Baltic salmon Spermatozoa bioassay, 141
830 Index

Spinal motor neurons sensitivity to BoNT/A1, 166 Streptococcus faecium, 175

Splenocyte assays, 216 Streptococcus pyogenes, 3
Spontaneous disease models, 8 Streptococcus species, 224
Sporeforming microorganisms, 131 biology and epidemiology of, 226–227
Sporozoites, 656 classification of, 224–226
Sporozoites, inoculation of, 610 genomics, 226
S. pyogenes, 224 history of, 223–224
biology and epidemiology of, 227 human pathogenic, 227
serotyping scheme, 225 morphology of, 226
S. pyogenes pharyngitis, 227 Streptococcus 16S rRNA sequences, 225
SREHP. See Serine-rich E. histolytica protein Streptolysins, 224
Stachybotrys chartarum, 569 Strongyloides stercoralis, 606
Staphylococcal enterotoxin-like toxins, biological Subclinical mastitis, 276
characteristics of, 210–211 Subgenomic RNA, HEV, 52
Staphylococcal enterotoxins (SEs) Sulfamethoxazole, 607
biological characteristics of, 210–211 Suncus murinus. See Asian house shrew
emetic activity of, 212 Superantigenic activity bioassays, 216
foodborne poisoning by. See Staphylococcal Superantigens, 216
foodborne poisoning Surface mucous cell mucin (SMCM), 332, 336
mechanisms of enteric illness, 211 Swine host of HEV, 46, 59
SEA and SEC, 214 Swine models. See Pig models
SEB and SEA, 211 Systemic diseases, 3
as superantigens, 216
Staphylococcal foodborne poisoning T
animal models for studying, 217
abdominal viscera, 213 Tachyzoite, 660
considerations for selection of, 212 Tachyzoites infecting ostrich macrophages, 656
dogs and cats, 215 Taenia, 783
ferrets, 214 Taenia adult worms
goats, 214–215 epidemiology of, 785
house musk shrew, 214 life cycle of, 785
monkeys, 212–214 morphology of, 784–785
pigs and piglets, 214 Taenia asiatica, 786, 787
rabbits, 215 Taenia infections
rodents, 212 clinical features of, 786–787
cell culture models for, 215–216 diagnosis of, 787
definition of, 209 laboratory models for, 788
diagnosis of, 211–212 prevention of, 788
by enterotoxin-producing staphylococci, 209–210 Taenia saginata, 786, 787
foods involved in, 210 Taenia solium, 786, 787
gastrointestinal injuries associated with, 216 Taenia species, 783, 789
symptoms of, 210 classification of, 784
Staphylococci, 209 Talaromyces, 555
Staphylococcus aureus, 209 Talaromyces helicus, 556
STEAEC, 323 Talaromyces radicus, 556
Sterne strain, 145 Tapeworms, 783
Stomach cancers, 336 Terminal protein precursor (pTP), 16, 20
Stomach carcinogenesis, H. pylori-associated, 333–334 Tetracycline, 255
chemoprevention of, 337 Tetrahymena pyriformis, 562
exacerbating factors for, 336–337 Tetrahymena spp. model
hyperplastic lesions and, 334–335 for Aeromonas infections, 243
intestinalization of, 336 TGF. See Transforming growth factor
intestinal metaplasia, 336 Thermal inactivation kinetics of MNV-1, 83
modifying factors for, 333–334 Thermotolerant lineages, 132
prevention of, 336 Thiabendazole paste containing spores, 145
Strabismus, 162 Thioredoxin Reductase-thioredoxin Redox System, 162
Strep sore throat, 227 Three-day-old chick model for C. jejuni infection,
Streptococcal diseases, human, 224 292–294
Streptococci classification scheme, 175 3ʹNCR of HEV genome, 52
Streptococcus faecalis, 175 Th1 responses, 263
Index 831

Thrombocytopenia, 516, 666 Trimethoprim–sulfamethoxazole, 608

Thrombospondin-related adhesive protein, 593 Trophozoites, 628, 640
TLR3, age-dependent expression of, 101 Trypanosoma, 645
Toll-dependent p38 MAPK pathway, 350–351 Trypanosoma cruzi, 628, 659
Toll-like receptor (TLR) adaptor, 263 Tuberculosis, 200
Toll-like receptors, 592 Tulane virus (TV)
Toll-like receptors (TLRs), 365 as HNoV surrogate, 84–85
Toxic chemicals, 2 infectivity, 85
Toxins, 2, 140 replication and biological studies, 85
types of, 2 survival and dissemination of, 84
Toxoflavin, 278 Tumor necrosis factor (TNF)
Toxoplasma, 655, 661 sensitivity and potential apoptosis, 22
Toxoplasma gondii, 611, 655 viral proteins interacting with, 22
biology of, 656 Type III secretion systems (T3SSs), 416
cyst, 657 Typhoidal Salmonella (TS)
laboratory models to study, 658 pathogenesis of, 395
in vitro models, 658 serovars, 392, 394–395
life cycle of, 657 Typhoid-fever-causing S. Typhi strain, genome
Toxoplasmosis, 655 of, 393
animal models on, 655, 660
clinical manifestations, 655 U
detection in experimental infected animals, 661
cat, 665 U exon, 16
chickens, 667 Ultraviolet (UV) light treatments, 79
mice, 662 Undercooked pork/wild boar meat, HEV
pigs, 665 infections due to, 44
rats, 663 UPEC, 324
rodent, 662 Upper intestinal biopsy, 603
in vitro models on, 655 shortening of villi, 603
Transforming growth factor, 570 stages of Isospora belli in epithelium cells, 603
Transfusion-associated hepatitis E infection, 43 Urease production, 357
Transgenic mice, 8 Urinary tract infections (UTIs), 178, 355
Transmissible spongiform encephalopathies (TSEs), 117 antibiotics for, 364
Transmission electron microscopy, 514 incidence fo, 364
Transposons, 359 protective immune response to, 364–365
TRH toxin, 416 vaccine development for, 364–365
Trichinella, 794 UVC exposure, 85
Trichinella infection, experimental models for
caiman models of, 802 V
fox models of, 802
horse models of, 800–801 Vaccines for HEV, 48
mouse models of, 797 Variable number of tandem repeat (VNTR), 203
rate models of, 797–798 Variant-specific surface proteins, 645
seals models of, 802 Vascular permeability reaction (VPR), 143
swine models of, 798–800 Vero cells, 140
wild boar models of, 800 Vibrio alginolyticus
Trichinella spiralis, 794 adhesion process of, 415
Trichinella spiralis infection extracellular proteases, 415
differences in resistance to, 794 human infections by, 414–415
host response during intestinal and muscle phase of, model organisms for studying, 415
795–797 Vibrio cholerae
immune response to, 794 antibiotic treatment, 414
MHC-linked and background genes controlling cholera caused by, 413–414
response to, 794–795 growth of, 413
Trichinella spp., 794 infection initiation by, 414
Trichinellosis, 794 model organisms for studying, 414
Trichothecenes, 525 virulence of, 414
Triclabendazole (TBZ), 723–724 Vibrio parahaemolyticus
Trifluoromethionine, 626 genes regulating virulent pathway in, 416
Trimethoprim, 607 infections, 415–416
832 Index

Vibrio parahaemolyticus (cont.) Yersinia enterocolitica, 427

model organisms for studying, 416–417 pathogenic, virulence of, 427
pathogenesis of, 416 Yersinia enterocolitica infections
strains, markers of, 416 C. elegans model of, 431
Vibriosis, 413 cell models of, 431
Vibrio spp. infection, C. elegans as model for, 418–421 host defense mechanisms, modulation of,
Viral infections controlled via MHC-I, 22 432–433
Viremia, 44, 46 mammalian cells, adhesion and invasion of, 432
and virulent HRV, 102 mouse model of
Virion assembly AdVs, 21 lifestyle and interaction with host, 428
Virulence and resistance gene profiling of Salmonella virulence of pathogen, 428–430
infection, 395 pig model of, 430–431
Virulence-associated proteins, 178 rabbit model of, 431
Virulence genes, 199 Yersinia outer protein (Yop) virulon, 427
VLPs as HNoV surrogate, 87 Yersinia pestis, 427
VREF infections Yersinia pseudotuberculosis, 427
FDA-approved agents for, 179 Yersinia pseudotuberculosis infections
intervention measures for controlling, 179 C. elegans model of, 431
SMP-601 against, 180 cell models of, 432–433
Ysc apparatus, 427
W
Z
Wa HRV infection, Gn pig model of, 99, 102
Water contamination, HEV risk with, 43 Zearalenone (ZEA), 524
Western blotting for PrPsc detection, 118–119 Zebrafish models
Whole genome sequencing (WGS), 199 for Aeromonas infections, 243
Wild boar models for Trichinella infection, 800 for Bacillus spp. virulence, 144
“Winter vomiting disease,” 76 for Bcc infections, 278
Worm burden, 726, 727 for C. difficile infection, 164
Wound, model organisms recapitulating aspects of, 374 for foodborne Candida infections, 503
of P. aeruginosa infection, 379
X of Salmonella infection, 398
of shigellosis, 405
Xanthothricin. See Toxoflavin for S. pyogenes, 232
Xylose lysine desoxycholate (XLD) agar, 402 Ziehl–Neelsen stains, 606
Zinc metalloproteases, 143
Y Zingiber officinale, 694
Zoonotic infections, 260
Yersinia, 427 in Peruvian human skeletons, 198
Yersinia adhesin A (YadA), 427 Zoonotic tuberculosis, 200, 201