07 - Biosecurity in Shrimp Farming
07 - Biosecurity in Shrimp Farming
Introduction
T
he most important diseases of cultured penaeid shrimp, in terms of economic impact,
in Asia, the Indo-Pacific, and the Americas have infectious etiologies. Among the infec-
tious diseases of cultured shrimp, certain virus-caused diseases stand out as the most
significant. The pandemics due to the penaeid viruses WSSV (White spot) and TSV (Taura
Syndrome), and to a lesser extent to IHHNV (Infectious Hypodermal and Hematopoietic
Necrosis virus) and YHV (Yellow Head), have cost the penaeid shrimp industry billions of
dollars in lost crops, jobs, and export revenue (Table 1). The social and economic impacts of
the pandemics caused by these pathogens in countries in which shrimp farming constitutes
a significant industry have been profound. In the wake of the viral pandemics the shrimp
culture industry has sought ways to restore the industry's levels of production to the "pre-
virus" years. The application of biosecurity to shrimp farming is central to those efforts.
"Biosecurity" has become a commonly used term in the shrimp culture industries of the
world only in the past few years. However, the concept that it represents is, and has been,
the foundation of nearly all mature and successful food animal producing industries for
decades. Many producers of cattle, swine, poultry, and many species from aquaculture, like
trout, salmon, and catfish, rely on the principles of biosecurity. But what is "biosecurity"?
Neither recent editions of Dorland's Medical Dictionary nor the Webster's New Collegiate
Dictionary define the term. The term may have been born in one of the food animal pro-
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ducing industries where profits and sometimes even the survival of businesses, are depen-
dent on the strict adherence to the concepts of biosecurity.
In the poultry industry, biosecurity has been defined as an essential group of tools for the
prevention, control, and eradication of economically important infectious diseases (Zavala
1999).
In the wake of the epizootics due principally to the shrimp viruses TSV and WSSV that swept
through the main penaeid shrimp growing regions of both Asia and the Americas, the
shrimp farming industry now seems intent to utilize any of the applicable concepts of biose-
curity in its farms.
The poultry industry's definition of biosecurity may be simply summarized as the exclusion
of pathogens from cultured stocks at farms. The application of biosecurity concepts to many
of the existing types of shrimp farming, as they have been applied to poultry for example, is
not something that can be accomplished easily or in the short term. The industry has thou-
sands of hectares of farms and hundreds of hatcheries, few of which were designed to afford
managers with much of an opportunity to totally prevent a particular pathogen from being
introduced and becoming established. Nonetheless, biosecurity is a broad concept and
much can be done to reduce losses due to particular pathogens by modifying existing farms
and their management routines in order to apply biosecure concepts. Furthermore, the
application of biosecurity concepts to shrimp aquaculture, will contribute significantly to
making the industry much more sustainable and environmentally responsible well into the
future. Key to any effort at excluding pathogens are the following principles and tools:
First on the above list is the requirement that adequate disease diagnosis and pathogen
detection methods are available to the industry. Highly sophisticated methods for pathogen
detection in various sorts of samples are of little value to the shrimp farming industry if
those methods are not sufficiently sensitive or accurate, or if adequate methods do exist but
they are not readily available to an industry that could benefit from their application and
use.
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The global penaeid shrimp farming industry is nearly 30 years old and it now produces
about 800,000 metric tons of shrimp annually from its farms. The importance of the indus-
try to the global economy is reflected in those production numbers and by the millions of
persons employed directly or indirectly by the industry. That farmed shrimp are among the
most important foreign exchange earners for many tropical and subtropical coastal nations
further documents the importance of the industry. Yet ironically, most of the penaeid shrimp
farming industry depends on the capture of wild postlarvae or broodstock to provide the
"seed stock" used to stock farms (Argue and Warren 1999). While some application of biose-
curity principles are possible with an industry that uses wild stocks for seed production, con-
sistency in preventing disease and pathogen introduction is problematic. As long as the
industry remains dependent on wild stocks, it cannot expect to be consistently successful in
excluding pathogens of concern. The use of wild broodstock, and especially wild postlarvae,
leaves the farms that rely on this source of seed stock particularly venerable to the intro-
duction of pathogens of concern.
While numerous methods have been incorporated into the operational design and ma-
nagement of shrimp farms previously affected by TSV and WSSV to eradicate them and to
insure that they are not reintroduced, none can be expected to provide much protection
against crop losses in farms that use seed stock derived from wild stock sources. The use of
only domesticated shrimp stocks that have a known history of being free of pathogens of
concern can help to mitigate this risk. However, a specific pathogen-free history comes only
from a long-term captive breeding and disease surveillance program at a facility that has a
fully functional and effective biosecurity plan.
This section of the manual reviews the concepts and principles of biosecurity, with a partic-
ular emphasis on the available diagnostic methodologies for pathogen detection, disease
diagnosis, and for the development and use of domesticated lines of specific pathogen-free
shrimp stocks. Shrimp taxonomy used in this section is according to Holthuis (1950).
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When the epizootiology of shrimp diseases is considered in terms of their historic and cur-
rent distribution, some trends become quickly apparent. Tables 1-2 list the principal di-
seases of culture penaeid shrimp in the Western and Eastern Hemisphere. Some of the most
important diseases (and their etiological agents) were once limited in distribution to either
the Western or Eastern Hemisphere. However, the international movement of live (for aqua-
culture) and dead (commodity shrimp for commerce) has led to the transfer and establish-
ment of certain pathogens from one hemisphere to the other.
WSSV was moved from Asia to the Americas by this route and TSV was moved in the oppo-
site direction. Perhaps these transfers and introductions could have been prevented if the
industries and governments of the exporting and importing countries had known of the risks
posed by their actions and if the appropriate disease diagnostic and pathogen detection
methods had been readily available when the most damaging transfers were being made.
Unfortunately, since its beginnings, growth and development of the penaeid aquaculture
industry has had as one of its characteristics a "gold rush" mentality. The industry has too
often moved forward on development of new farming regions, transfer of live or dead
shrimp stocks, etc., well before the risks or environmental consequences were considered,
let alone assessed. Many of the most significant shrimp pathogens were moved from the
regions where they initially appeared to new regions even before the "new" pathogen had
been recognized, named, proven to cause the disease, and before reliable diagnostic meth-
ods were developed. The diseases due to the shrimp viruses IHHNV, TSV and WSSV all were
transferred with live shrimp stocks from country to country and from one continent to
another before their etiology was understood.
While biosecurity has as its goal the exclusion of known pathogens for which epizootiolo-
gical data is available and for which there are adequate diagnostic and detection methods,
the application of biosecure practices can also reduce the likelihood of the introduction of
an unknown or poorly understood pathogen. Nonetheless, before the principles of biose-
curity can be applied to a particular shrimp farming region or to an individual facility, it is
necessary to identify which pathogens are to be targeted for exclusion. The listed
pathogens in a biosecurity program must be excludable. Figure 1 illustrates graphi-
cally the interaction between host, environment and pathogen. Therefore, the epizootiolo-
gy of a pathogen (i.e. its hosts, biology, and methods of transmission) must be understood
to permit managers to understand how the pathogen is transmitted and how to prevent its
entry and spread. Adequate diagnostic methods for the disease and detection methods for
its agent are also essential tools in biosecurity. Adequate diagnostic methods means reliable,
sensitive and readily available.
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Figure 1. Pathogens are generally found in any organism's environment, often without ill
effects. The presence of pathogens may result in disease due to a variety of factors related
to the host and its environment. Only when a pathogen is excludable can disease be pre-
vented.
Shrimp have as part of their natural microbial flora and in their aquatic environment, a large
and diverse population of microorganisms, some of which are facultative pathogens ready
to strike when the shrimp become compromised by any number of stressors. Certain Vibrio
species provide a good example of organisms that live in the shrimp's environment, often
as part of the normal microflora inhabiting the surface of their cuticle or colonizing areas of
the gut or hepatopancreas. Some Vibrio species can become deadly pathogens in "stressed"
shrimp.
"Stress" in shrimp is a poorly defined condition that is difficult to measure, and it has more
causes than are even known. Its causes can range from exposure to environmental
extremes to inadequate nutrition. Most penaeid shrimp have the best culture performance
(i.e. growth and food conversion efficiency) at water temperatures near their upper tole-
rance limit for a particular life stage of the species. Farms and management practices must
be tailored to operating near the temperature tolerance limit, and be prepared to take cor-
rective measures when "stress" and disease result from water temperatures becoming too
high for too long. Hence, the farm siting, culture system design, the quality of feed used,
stocking density, the farm's routine management practices, and other factors can have a
profound effect on the amount of "stress" to which farmed shrimp stocks are subjected. A
key feature of biosecurity is that farm design, feeds and feeding, and the quality of mana-
gement are essential components to successful shrimp farming (i.e. broodstock facility,
hatchery, or growout farm).
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The International Office of Epizootics (OIE) is the administrative arm of the World Animal
Health Organization. OIE maintains at its web site (www.oie.int) and regularly publishes the
International Aquatic Animal Health Code and Diagnostic Manual. The OIE currently has
nine crustacean diseases (eight of which are penaeid virus diseases) on its list of pathogens
which pose a threat to international commerce, fisheries, and crustacean aquaculture
(especially shrimp). Tables 5-7 list the OIE notifiable and listed shrimp viruses and the avai-
lable diagnostic and detection methods for each. Before a disease may be included on the
OIE lists of notifiable and listed diseases, several criteria must be met:
Pathogen lists, such as the OIE list, are useful models for setting up a biosecurity program
that is based on exclusion of a list of specific pathogens and the diagnostic methods for sur-
veillance and diagnosis.
Current Diagnostic Methods
Modern penaeid shrimp diagnostic and research laboratories are based on traditional me-
thods of disease diagnosis and pathogen detection that have been adapted from me-
thods used in fish, veterinary and human diagnostic laboratories. In penaeid shrimp patho-
logy, diagnosticians rely heavily on case history, gross signs and behavior, morphological
pathology (direct bright-field or phase contrast light microscopy and electron microscopy)
and classical microbiology (bacteriology and mycology) (Table 3, Figure 2).
Paradoxically, important techniques involving tissue and cell culture and hematology and
clinical chemistry, which are virtual cornerstones of vertebrate biomedical research, diag-
nostics, and pathology, have either not been successfully applied as routine diagnostic tools
in penaeid shrimp pathology (in the case of cell and tissue culture), or have not provided
routinely practical diagnostic data (in the case of hematology and clinical chemistry). In
marked contrast, methods based on pathogen detection using antibody-based methods
(employing polyclonal and monoclonal antibodies) and, especially, molecular methods
(using gene probes and Polymerase Chain Reaction-PCR) have been found to provide accu-
rate and standardizable methods for disease diagnosis and pathogen detection to the
penaeid shrimp culture industries, especially for certain penaeid viruses (Lightner 1996,
1999a, 1999b; Tables 6 & 7).
Classical Methods
Methods for the detection of pathogens and the diagnosis of diseases that are currently in
use by shrimp pathologists and by diagnostic labs have been reviewed many times in the
past decade (Baticados 1988; Baticados et al. 1990; Liu 1989; Johnson 1990, 1995; Brock 1991,
1992; Brock and Lightner 1990a, 1990b; Brock and LeaMaster 1992; Brock and Main 1994;
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Biosecurity in shrimp farming
Fulks and Main 1992; Lightner 1988, 1992, 1993a, 1993b, 1996; Lightner and Redman 1991,
1992, 1998; Lightner et al. 1992a, 1992b, 1994; Limsuwan 1993). Diagnosticians working with
penaeid shrimp continue to rely heavily on the "classical" diagnostic methods (Table 3).
Among the most important of these are gross and clinical signs, with the most commonly
applied laboratory tests being direct examination and microscopy using the light micro-
scope, classical microbiology with isolation and culture of the agent, and routine histology
and histochemistry (Bell and Lightner 1988; Lightner 1996). Virtually every functional shrimp
pathology/diagnostic laboratory today is equipped to do direct light microscopic methods
and routine procedures in histology and bacteriology.
Other "classic" diagnostic techniques that are important, but are used less frequently,
include techniques such as bioassay and enhancement, which are used for the detection of
subclinical or carrier-state infections by certain pathogens (Lightner et al. 1983b; Overstreet
et al. 1988; Brock and Lightner 1990a; Lightner 1996; Lu et al. 1995a). Methods used by shrimp
pathologists more as research tools, but occasionally for diagnostic purposes, are transmis-
sion and scanning electron microscopy (Momoyama et al. 1995; Johnson and Cassout 1995),
and antibody-based tests with immune sera prepared in mammals (Lightner 1996).
Hematology and Clinical Chemistry
It is interesting, and perhaps somewhat of a paradox, that hematology and clinical chem-
istry, two of the principal diagnostic tools of human and veterinary medicine, are so seldom
used as a diagnostic tool in penaeid shrimp pathology. However, while there have been a
few studies in which changes in hemolymph parameters, such as hemocyte count, clotting
time, glucose, non-protein nitrogen, ammonia, SGOT (Serum Glutamic Oxaloacetate
Transaminase), alkaline phosphatase, total serum protein, etc., were shown to result from
infectious disease in shrimp and lobsters (Stewart et al. 1969; Stewart and Rabin 1970; Hose
et al. 1984; Stewart 1993), almost none of these test have been adapted to routine diagnostic
use. Only hemolymph clotting time and changes in total hemocyte count seem to be used
by shrimp disease diagnosticians (Lightner 1996).
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Tissue Culture
Another paradox of shrimp pathology is the virtual absence of cell and tissue culture me-
thods as diagnostic tools. Cell and tissue culture form one of the diagnostic cornerstones of
plant, veterinary, and human pathology and biomedical research. Tissue culture methods
are central to the diagnosis of most of the viruses of finfish (Thoesen 1994). Even for insects
(which, like crustaceans are arthropods) there are numerous cell lines, some of which have
been available for decades (Vago 1971; Maramorosch and Mitsuhashi 1982). A number of
research groups have developed and improved upon the methods for primary cells from
penaeid shrimp (Chen et al. 1986; Itami et al. 1989; Rosenthal and Diamant 1990; Ellender et
al. 1992; Luedeman and Lightner 1992). Some researchers have used primary cultures of
shrimp cells to attempt to grow in vitro certain shrimp viruses like MBV, YHV, and WSSV
(Chen and Kou 1989; Lu et al. 1995b; Tapay et al. 1996a; Crane and Benzie 1999). While
advances in penaeid shrimp primary cell culture are encouraging, shrimp tissue culture
remains in the research and development phase as a diagnostic tool (Crane and Benzie
1999).
Toxicology and Analysis
Non-infectious diseases of penaeid shrimp are common in cultured shrimp (Lightner 1993b).
Many are due to environmental extremes of temperature, salinity, pH, and other factors.
Others are due to nutritional imbalances and deficiencies, and still others are due to toxi-
cants (Lightner 1993a, 1993b). Toxicity syndromes may be due to the shrimp's own metabo-
lites like ammonia and nitrite, or to toxic metabolites from such sources as moldy feed
ingredients or feeds. Industrial and agricultural toxicants (certain heavy metals, some pesti-
cides, and toxic chemicals) also occasionally cause disease and losses in cultured shrimp
(Baticados et al. 1990; Brock 1992; Flegel et al. 1992; Lightner 1993b).
Shrimp affected by some non-infectious disease syndromes present unique gross signs and
lesions that can provide a definitive diagnosis. However, most require confirmation of the
causative agent by laboratory analysis (Brock 1992; Lightner 1993b; Brock and Main 1994).
Examples of some toxic diseases that can be diagnosed solely from histological demonstra-
tion of pathognomonic lesions include hemocytic enteritis caused by blue-green algae tox-
icity, and presumably by other potent endotoxin producing organisms, in which prominent
inflammatory lesions of the midgut are present (Lightner 1996), and a toxicity syndrome due
to the agricultural fungicide benomyl in which unique lesions occur in the hepatopancreas
(Lightner et al. 1996). While aflatoxicosis and certain forms of black gill and shell disease also
display unique histological lesions, these diseases provide examples of toxicity syndromes
in which demonstration of the suspected toxicant in the appropriate sample(s) (i.e., water,
sediment, feed, shrimp, etc.) with the appropriate analytical method is necessary to provide
a definitive diagnosis. Likewise, nutritional diseases, such as cramped muscle syndrome,
soft shell, carotenoid deficiency, and the ascorbic acid deficiency syndrome, present unique
gross signs and histopathology (Baticados et al. 1990; Lightner 1988, 1993a, 1993b), but for
most nutritional disorders, confirmation of the provisional diagnosis depends upon other
information obtained from case history and analytical results.
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Antibody-based Methods
Several antibody-based methods have been developed for use in shrimp disease diagnosis
(Tables 6-8). Monoclonal antibodies (MAbs) have been developed for detection of several
species of Vibrio, the causative agents of vibriosis in shrimp. Song et al. (1992) developed an
enzyme-linked immunoassay (ELISA) test based on MAbs to Vibrio vulnificus and V. harveyi.
Chen et al. (1992) also developed a number of MAbs to a number of Vibrio spp., including
species (V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. harveyi) that are com-
monly reported as causative agents of vibriosis in shrimp. However, none of these MAbs
have been made available to the public.
Polyclonal (PAb) and monoclonal antibodies have also been developed as diagnostic
reagents for shrimp viruses (Tables 6 and 7). Sano et al. (1984) developed a fluorescent PAb
test for BMN, the agent of baculoviral midgut gland necrosis in P. japonicus, Lewis (1986)
reported the application of an ELISA-based PAb test for BP (Baculovirus penaei) of American
penaeids. More recently, PAbs have been developed and applied to the detection of other
shrimp viruses. Tapay et al. (1996b) reported on the application of PAbs for the detection of
rhabdovirus (RPS) of penaeid shrimp (Nalda et al. 1992; Tapay et al. 1996b), and for YHV and
WSSV (Lu et al. 1996; Nalda and Loh 2000) (Table 4).
Monoclonal antibodies (MAbs) have been successfully developed and a few of these have
become available to the shrimp culture industry through commercial sources. Poulos et al.
(1994a) developed MAbs to IHHNV, but reported problems with specifity. Specificity pro-
blems may have been related to the IgM nature of the MAbs developed to IHHNV. While the
IgM MAbs developed reacted specificially with purified IHHNV or its capsid proteins in
Western blots, they reacted nonspecifically with components in normal shrimp tissue,
resulting in false positive reactions with uninfected shrimp tissue samples in ELISA-based
assays (Lightner et al. 1992c; Poulos et al. 1994a). More recently, IgG class MAbs to TSV and
WSSV have been developed (Poulos et al. 1999; Poulos et al. submitted) which do not have
the specificity problem. MAbs to TSV and WSSV are commercially available from
DiagXotics, Inc. (Wilton, CT, USA).
Although the development of antibody-based tests for the more important shrimp
pathogens has lagged behind the development of molecular detection and diagnostic meth-
ods, it is very likely that the use of tests based on polyclonal and monoclonal antibodies will
become much more common in shrimp diagnostic laboratories in the next few years.
Because of their speed, versatility, relatively low cost, simplicity, and reasonably good sen-
sitivity, monoclonal antibody based tests are potentially very useful as a routine diagnostic
tests even in the most modestly equipped diagnostic laboratories (Mailhe et al. 1992;
Reddington and Lightner 1994).
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Methods for improving shrimp farming in Central America
Molecular methods (gene probes and DNA amplification using the polymerase chain reac-
tion - PCR) have recently been applied to the diagnosis of certain infectious diseases of
penaeid shrimp. Development and application of the first gene probe to the diagnosis of the
shrimp virus IHHNV (Table 4) was reported only 8 years ago (Lightner et al. 1992c; Mari et al.
1993a). The first generation of IHHNV gene probes was developed by extracting ssDNA* from
IHHNV purified from infected L. vannamei and L. stylirostris and cloned into E. coli-DH5
cells (Mari et al. 1993a). From the resultant libraries of cloned fragments of IHHNV DNA, five
clones with DNA inserts of 2.0 Kbp or larger were selected for further development. From
these clones, the first DNA probes to a shrimp virus were developed (Mari et al. 1993a).
When labeled with (what was once traditional) radioactive tags, the use of gene probes was
an option for only the best equipped diagnostic and research laboratories. However, the
application of non-radioactive labeling methods has made gene probe technology readily
available to shrimp research and diagnostic laboratories. The first non-radioactive gene
probes for shrimp pathology were developed employing the non-radioactive Genius TMI Kit
(Boehringer Mannheim, Inc.), which contains digoxigenin-11-dUTP (DIG) as the DNA label
and uses an ELISA-based system for final detection (Lightner et al. 1992c; Mari et al. 1993a).
This led to the development of the non-radioactive DIG-labeled gene probes for IHHNV and
to their commercial application in diagnostic kits marketed under the product name
"ShrimProbesTM" by DiagXotics (Wilton, CT, U.S.A.).
Since the first gene probe to the shrimp parvovirus IHHNV was developed in 1992 (Lightner
et al. 1992c; Mari et al. 1993a), the technology has been applied to the development of addi-
tional gene probes to other shrimp viruses, a rickettsia-like bacterium and a microsporidian
(Table 8). At the present time, DIG-labelled gene probes have been developed and are avail-
able for the parvoviruses IHHNV and HPV, the picornavirus TSV, for the shrimp baculovirus-
es BP, MBV, for WSSV, and YHV, a rod-shaped ssRNA virus (Tables 5-8) (Lightner et al. 1992c,
1994; 2000; Bruce et al. 1993; Mari et al. 1993b, 1995; Poulos et al. 1994b; Nunan and Lightner
1997; Lightner 1996; Wang et al. 1995; Flegel et al. 1996). Using essentially the same technol-
ogy, additional DIG-labeled gene probes have been developed for the causative agent of
necrotizing hepatopancreatitis, an intracellular, rickettsial-like bacterium (Krol et al. 1991;
Frelier et al. 1992, 1993, 1994; Lightner et al. 1992d; Lightner 1996; Loy and Frelier 1996), and
for the microsporidian Agmasoma sp., which parasitizes P. monodon and P. mer-
guiensis in southeast Asia (Pasharawipas and Flegel 1994; Pasharawipas et al. 1994). Many of
these probes are commercially available as DIG-labeled probes or in kit form in the
ShrimProbeTM line (Table 8) from DiagXotics, Inc. (Wilton, CT, U.S.A.).
DIG-labeled gene probes may be applied to shrimp diagnostics and pathogen detection in
several ways. The protocol for the GeniusTM System (Boehringer Mannheim Inc., GeniusTM
System User's Guide for Membrane Hybridization) was adapted for "dot blot" hybridization
assays using homogenized tissue samples "blotted" and fixed onto membranes prepared
from nitrocellulose or positively charged nylon. The method has been applied successfully
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Biosecurity in shrimp farming
to the detection of the penaeid shrimp viruses IHHNV and WSSV (Lightner 1996), the NHP
bacterium (Loy and Frelier 1996; Lightner 1996), and in the detection of the microsporidian
Agmasoma sp. (Pasharawipas and Flegel 1994).
In situ hybridization using protocols adapted from the GeniusTM System developed by
Boehringer Mannheim (Nonradioactive In Situ Hybridization Application Manual) may be
used to detect viral and other genomic sequences with specific complementary DNA probes.
The use of non-radioactive, DIG-labeled gene probes has been shown to provide a highly
specific diagnostic method, since any non-specific tissue effects (which may result in a false
positive diagnosis in a dot blot assay with homogenized tissue samples) can be readily dis-
tinguished from specific histological lesions that have reacted with the labeled probe
(Lightner 1996). In situ hybridization methods have been developed for the shrimp viruses
IHHNV, HPV, MBV, BP, the WSSV group, YHV, and TSV (Tables 6 and 7), for rickettsial-like bac-
terium NHP, and for the microsporidian Agmasoma sp. (Flegel et al. 1996; Lightner 1996).
The polymerase chain reaction (PCR) has had numerous recent applications to pathogen
detection and shrimp pathology research (Tables 6 and 7). In PCR, small, otherwise unde-
tectable, amounts of DNA can be amplified to produce detectable quantities of the target
DNA. This is accomplished by using specific oligonucleotide primers designed for the target
DNA sequence. The resultant PCR product may then be compared to a known standard
using gel electrophoresis, by reaction with a specific DNA probe of PCR products blotted
directly onto a membrane or to the PCR products in Southern transfers. In some applica-
tions PCR products themselves may be labeled with DIG and used as specific DNA probes
(Innis et al. 1990; Perkin Elmer 1992).
When DNA sequence information is known for specific nucleic acid sequences (of penaeid
shrimp viruses, bacteria, etc.) primers can be synthesized to target specific nucleotide
sequences. The unique target sequences may belong to a virus, a bacterium, or to any nucle-
ic acid sequence. Various computer programs exist which aid in selection of optimal
primers, provided target DNA sequence information is available (Innis et al. 1990; Perkin
Elmer 1992).
PCR has been applied to research and pathogen detection for most of the shrimp viruses of
concern to modern day shrimp culture (Tables 6 and 7) (Wang et al. 1996; Nunan et al. 2000;
Lightner 1999b). Other applications of PCR to shrimp pathology research and pathogen
detection include reports of the application of PCR to the detection of bacterial pathogens
such as the NHP bacterium (Loy et al. 1996) and Vibrio penaeicida (Genmoto et al. 1996).
There is a growing need to standardize and validate the DNA-based diagnostic methods and
the laboratories that use them (Walker and Subasinghe 1999). Standardization of DNA-based
diagnostic methods is almost inherent in the nature of the tests. That is, a specific DNA
probe, or a specific set of primers, that is used to demonstrate the presence or absence of a
unique DNA or RNA sequence does not vary from batch to batch. Hence, with proper con-
trols, these DNA-based methods are readily standardized (Reddington and Lightner 1994).
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Methods for improving shrimp farming in Central America
However, despite the growing dependence of the shrimp culture industry on DNA-based
diagnostic methods, none of the tests that are available from commercial sources nor from
the literature have been validated using controlled field tests. Likewise, there are no formal
accreditation or certification programs yet in place to assure that test results from techni-
cians and laboratories running the tests are indeed accurate and properly controlled
(Lightner and Redman 1998; Lotz and Lightner 1999; Lightner 1999b).
A variety of strategies have been attempted for the control of viral diseases in penaeid
shrimp aquaculture. These strategies range from the use of improved culture practices (i.e.
where sources of virus contamination are reduced or eliminated, sanitation practices are
improved, stocking densities are reduced, etc.) to stocking "specific pathogen-free" (SPF) or
"specific pathogen resistant" (SPR) species or stocks. Most recently, there have been some
studies made on the use of vaccines and immunostimulants for the prevention of viral dis-
eases in shrimp.
Improved husbandry practices have been successfully employed for the control of BP, and
for nearly a decade, this virus has seldom been reported as a serious constraint to success-
ful shrimp culture. This was accomplished because BP's infection cycle can be interrupted
with routine hatchery management practices. BP is a gut-infecting baculovirus which is
transmitted from shrimp to shrimp exclusively per os 1 (Johnson and Lightner, 1988;
Overstreet et al., 1988; Overstreet, 1994). Hence, with BP, as well as with MBV and BMN (all
gut-infecting baculoviruses of penaeid shrimp), infection from parent to off-spring in the
hatchery has been prevented by eliminating fecal contamination of spawned eggs by virus-
contaminated feces from spawning adults and by the use of adequate sanitation practices
(Momoyama, 1988, 1989a, 1989b, 1989c, 1989d).
A number of innovative methods have been developed for reducing or eliminating fecal
(containing baculovirus) contamination of spawned eggs. The simplest of these has been the
1
Per os = by mouth
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Biosecurity in shrimp farming
use of hatching vessels in which embryonating shrimp eggs are rinsed with clean seawater,
and in which hatched nauplii are passively rinsed by continuously flowing clean seawater
and separated from contaminants and "diseased" siblings by collection using the normal
phototaxic response of "healthy nauplii". Chemical rinses of spawned eggs and collected
nauplii with disinfectants like chlorine, ozone, iodophores, and formalin are also common-
ly used in shrimp hatcheries in the Americas to help prevent BP, as well as vibriosis and other
diseases. The use of routine sanitation and disinfection procedures for hatchery equipment
and tanks after each use are also commonly used to prevent the occurrence of viral infec-
tions due to BP, or to limit their tank to tank spread when they do occur.
Some hatcheries individually spawned their gravid females, collected any fecal strands, and
scanned these using simple bright field microscopy for BP occlusion bodies. In like manner,
some hatcheries sacrificed broodstock females after spawning, excised the HP, and exa-
mined it for the presence of BP occlusion bodies using direct microscopy of tissue squash
preparations. To prevent BP infections and disease from occurring in the larval rearing tank
systems, the spawns from females, found to be BP-positive from examination of their feces
or excised HP, were discarded.
In the Western Hemisphere, SPF stocks of L. stylirostris and L. vannamei have been deve-
loped and these are being cultured successfully in some locations (Wyban, 1992; Wyban et
al., 1992; Carr et al., 1994; Pruder et al., 1995; Lightner, 1996b). The International Council for
Exploration of the Seas (ICES) Guidelines (Sindermann, 1990), were followed for the deve-
lopment of these stocks. The determination of which specific pathogens the selected stocks
were to be free of was based on a working list of specific, excludable pathogens (Wyban,
1992; Lotz et al., 1995). The most current working list for the U.S. Marine Shrimp Farming
Consortium includes eight viruses (WSSV, YHV, TSV, IHHNV, HPV, BP, MBV, and BMN), certain
classes of parasitic protozoa (microsporidians, haplospordians, and gregarines), and
helminth parasites (cestodes, trematodes, and nematodes). In the spirit of the ICES
Guidelines, each "SPF candidate population" of wild or cultured shrimp stocks of interest
were identified (Table 3).
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Methods for improving shrimp farming in Central America
The process of developing an SPF strain begins with taking samples of the available stock
and these are tested using appropriate diagnostic and pathogen detection methods for the
specific pathogens of concern. If none were found, a founder population (F0) of the "candi-
date SPF" stock was acquired and reared in primary quarantine. During primary quarantine,
the F0 stock was monitored for signs of disease, sampled, and tested periodically for specif-
ic pathogens. If any pathogens of concern were detected, the stock was destroyed. Those
stocks that tested negative for pathogens of concern through primary quarantine (which ran
from 30 days to as much as 1 year for some stocks) were moved to a separate secondary
quarantine facility for maturation, selection, mating, and production of a second (F1) gen-
eration. The F1 stocks were maintained in quarantine for further testing for specific
pathogens of concern. Those that tested negative were designated as SPF and used to pro-
duce domesticated lines of SPF and "high health" (Wyban et al., 1992; Pruder et al. 1995). SPF
and high health stocks of L. vannamei were used successfully in U.S. shrimp farms in 1993
and 1994, and resulted in nearly double the production per crop that had been previously
obtained at the same farms in previous years when the farms cultured non selected lines of
L. vannamei, which in previous crops, had been persistently affected by "runt deformity syn-
drome" (RDS) due to chronic infection by IHHNV (Pruder et al., 1995; Lightner, 1996a, 1996b)
(Figure 2).
136
Biosecurity in shrimp farming
Figure 2.
Independent quaran-
tine procedure for
isolating and devel-
oping SPF stock.
Another interesting application of disease management through avoidance and the use of
SPF stocks began in 1995 in Belize. The shrimp culture industry in this Central American
nation is relatively small with less than 10 farms. Belize was seriously impacted by the TSV
panzootic in 1994 (Lightner 1996b; Dixon and Dorado 1997). The shrimp farms in Belize are
geographically isolated from other shrimp farming regions in adjacent countries (because
most are situated on the Pacific coast), and with its location on the Caribbean side of the
continent, it has no natural occurring wild stocks of the penaeid species (L. vannamei and
L. stylirostris) that are likely to serve as reservoir hosts of TSV and IHHNV. Belize was unique-
ly suited to attempt to eradicate TSV and IHHNV. Seven of its farms were depopulated of all
their shrimp stocks in late 1995; then each farm was thoroughly disinfected and dried out to
eradicate potential sources of these viruses. The eradication program included pond disin-
fection (liming pond bottoms with calcium oxide at 5,000 kg/ha or chlorine at 10 ppm resid-
ual for 24-48 hr), pond dry-out and bottom tilling (to a depth of ~10 cm to ensure oxidation
of contaminated pond-bottom detritus), farm implement and building disinfection (with
chlorine or with formalin gas), spraying insecticides (to kill potential reservoir hosts like wild
crabs and shrimps in supply drainage canals), and removal of frozen shrimp from storage
from the country's packing plants (Dixon and Dorado, 1997). For the 1996 and 1997 seasons,
these farms were stocked exclusively with SPF L. vannamei. From 1995 to late 2000, TSV has
not been detected in Belize, and IHHNV has been found only at low prevalence rates. In the
absence of TSV, the per crop average production of L. vannamei in 1996 at one farm was 891
kg/ha (heads on) with a stocking density of 15.5 PL/m2, and survival to harvest has averaged
67%. In comparison, the same farm's production during the TSV panzootic of 1994 was 390
kg/m2 (stocked at 18.3 PLS/m2) with a survival of 36% (Dixon and Dorado, 1997). While the
Belize experiment in TSV and IHHNV eradication may have been successful, duplication of
its accomplishments elsewhere in the Americas may not be feasible. In these regions where
total stock eradication is not feasible, other methods for virus disease management are
being used.
137
Methods for improving shrimp farming in Central America
Successful application of the ICES Guidelines and the SPF concept requires that specific
pathogens are excludable. In situations where specific pathogens may not be excludable, the
development and use of SPR stocks may be the only alternative. The fact that IHHNV and
TSV have become widely distributed in the Americas indicates that either government or
industry supported pathogen exclusion mechanisms and regulations must be implemented
and enforced to achieve the goals of using SPF shrimp stocks, or, alternatively, that SPR
stocks be developed and used.
One alternative approach to developing SPF domesticated shrimp stocks, is to select and
breed survivors of "specific pathogen-infected" (by pathogens like IHHNV, TSV, or WSSV)
stocks to develop "specific pathogen-resistant" or SPR stock. Following this scheme, French
researchers successfully developed a stock of IHHNV resistant P. stylirostris in French
Polynesia (Weppe, 1992; Lightner, 1996b). This stock, designated as SPR-43, was developed
by the French Research Institute for Exploration of the Seas (IFREMER) by breeding gener-
ations of IHHN survivors at the IFREMER stations in Tahiti and New Caledonia. After sever-
al generations, survival and culture performance of the stock improved. The stock was
found to carry IHHNV at low rates of prevalence and severity of infection. When experi-
mentally challenged with IHHNV, the SPR-43 stock was found to be resistant to IHHN disease
(Weppe et al., 1992).
A second line of SPR L. stylirostris was developed in Venezuela. Its development followed
the same strategy that was used by the IFREMER in Tahiti and New Caledonia. The founder
stock was introduced to Venezuela from Panama. The founder may have been infected with
IHHNV when it was introduced, or it became infected from imported stocks of L. vannamei
after being imported into Venezuela. Generations of IHHN survivors were selected and
reared until that stock began to perform as well as did the normally IHHNV resistant stocks
of L. vannamei at the same farm. When the TSV panzootic swept through the Americas, the
Venezuelan stock of L. stylirostris was found to be TSV resistant. This stock possesses resis-
tance to IHHNV and TSV, and it was marketed as Super ShrimpTM in the Americas (Lightner
and Redman 1998). Beginning in 1997 in some regions of Mexico, the SPR stocks of Super
ShrimpTM (L. stylirostris) replaced L. vannamei stocks, which at that time made up >90% of
the shrimp farmed in Mexico (Rosenberry 1996). However, the use of Super Shrimp declined
in 1999-2000 after the WSSV epizootic reached Mexico and after an apparently new strain of
TSV emerged that was pathogenic to Super Shrimp.
138
Biosecurity in shrimp farming
selected stocks (Carr et al. 1997; Lotz and Lightner 1999). Natural selection for TSV resistance
appears to be occurring in wild stocks as well. In regions like Ecuador and Honduras where
TSV has been enzootic for several years, the practice of direct stocking of farms with wild
PLs is providing steadily improving survival rates, even though shrimp displaying classic
signs of TSV infection are commonplace. The same selective process for IHHNV resistance
seems to be occurring in some wild stocks of L. stylirostris (Lightner, 1996b).
Initial efforts to select and breed TSV resistant, domesticated strains of L. vannamei have
resulted in improvements in harvest survivals of 20 to 40% (Lightner, 1996b). Some selected
lines of L. vannamei are highly resistant to TSV, giving >90% survival in laboratory challenge
studies (White et al. 1999; Wyban 2000) and improved survival at farms in regions where the
virus is enzootic (Wyban 2000). Some breeding programs using L. vannamei have reared
survivors of WSSV epizootics and from them produced selected lines of F-1 progeny, some
of which have shown improved survival rates compared to unselected stocks at farms
affected by WSSV (Faria et al. 2000). The use of selective breeding for resistance to selected
shrimp pathogens may provide the shrimp farming industry with improved, SPR stocks.
139
Methods for improving shrimp farming in Central America
Dry and till ponds after the harvest. Better results are obtained if entire farm is de-
populated, dried, limed, and tilled.
Some farms use a pesticide (Sevin or Dylox) to kill all crabs resident in the ponds
and seawater supply system. Other farms found this to be impractical or unneces
sary.
Treat all "new" seawater by coarse filtration and store in reservoir for 1 week or
longer.
Fill ponds with seawater passed through a 150-200 mesh filter bag being certain that
no tears or leaks occur that would permit unfiltered water to enter pond.
Reduce water exchange and use only water filtered through 150-200 mesh screen to
fill ponds.
Stock with PLs only from WSSV-free sources (confirmed by testing of broodstock
and PLs by PCR) or domesticated SPF stock.
Use probiotic products to improve pond bottom condition and improve nutrient
cycling.
140
Biosecurity in shrimp farming
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Table 6
Diagnostic and pathogen detection methods for the OIE notifiable and listed viral diseases of
penaeid shrimp (modified from Lightner 1996, 2000; Lightner and Redman 1998).
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Methods:
BF = bright field LM of tissue impression smears, wet?mounts, stained whole mounts;
LM = light microscopy;
PH = phase microscopy
DF = dark-field microscopy
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165