REPORT

Borovets, EUROPEAN
Bulgaria, COMMISSION FOR THE
5-8 September CONTROL OF
2000 FOOT-AND-MOUTH DISEASE

Session of the Research Group

of the Standing Technical
Committee
 AGA: EUFMD/RG/00

REPORT

of the

Session of the Research Group of the Standing Technical Committee

of the

EUROPEAN COMMISSION FOR THE CONTROL OF FOOT-AND-MOUTH

DISEASE

held at

Borovets, Bulgaria
5-8 September 2000

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS

Rome, 2000
 iii

TABLE OF CONTENTS

Page

INTRODUCTION.................................................................................................... 1

Adoption of the Agenda............................................................................................. 2

Item 1 General information........................................................................... 3

Item 2 Epidemiology/pathogenicity/immunology/genetics............................ 3

Item 3 Control of FMD.................................................................................. 6

Item 4 New developments in FMD diagnostics............................................. 9

Item 5 Collaborative Laboratory Study Phase XVI...................................... 12

Item 6 Vaccines............................................................................................ 13

Item 7 European Pharmacopoeia................................................................ 14

Item 8 Expert Elicitation Workshop.............................................................15

Item 9 Closed Session..................................................................................16

1 Follow-up to the activities of the Working Group

on the European Pharmacopoeia
2 EU antigen bank
3 Information on recent and future missions
to Caucase, Greece and Turkey
4 Follow-up to the Workshop on NSP ELISA
Brescia, January 2000
5 Follow-up to the discussion on SVD at
the previous Sessions.
6 Relations between EUFMD and EC :
utilisation of the Trust Fund
7 Circulation of the information to the Group
by the Secretariat, evolution in the roles and
methods of work for the RG
8 Next group to be designated by the 34th session
of EUFMD, activities of the RG
over the coming years
9 Venues for the next Sessions of the RG

Adoption of the Report.........................................................................................18

Closing remarks....................................................................................................18
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LIST OF APPENDICES

Appendix 1
Emergence of a Pandemic Strain of Foot-and-Mouth Disease Virus Serotype O
Nick J. Knowles; Alan R. Samuel; Paul R. Davies; R. Paul Kitching; R.
Venkataramanan; Toru Kanno; Alexei V. Scherbakov; Vladimir V.Drygin; Qi-Zu
Zhao and Qing-Ge Xie

Appendix 2
Pathogenicity of O/JPN/2000 to susceptible animals
Kenichi Sakamoto; Makoto Yamakawa; Toru Kanno and Yosuke Murakami

Appendix 3
Origin of recent outbreaks of foot-and-mouth disease in North Africa,
the Middle East and Europe
N.J. Knowles and P.R. Davies

Appendix 4
FMD situation in Turkey in 2000
Ismet Gurhan

Appendix 5
Minimal aerosol infectious dose of the 01 Lausanne strain of foot-and-mouth disease
virus for pigs
Soren Alexandersen; Ian Brotherhood and Alex I. Donaldson

Appendix 6
Antigenic and genetic characterization of foot-and-mouth disease type O viruses from
the Far East
A.R. Samuel; L.S. Turner and N.J. Knowles

Appendix 7
Genetically diverse isolates of type O foot-and-mouth disease virus exhibit
remarkable amino acid conservation at their neutralizing antigenic sites
A.R. Samuel

Appendix 8
Early pathogenesis of foot-and-mouth disease in pigs: a quantitative time-course study
using a newly developed TaqMan RT-PCR assay
Soren Alexandersen; Martin B. Oleksiewicz and Alex I. Donaldson

Appendix 9
IgA response of cattle to FMDV infection in probang and saliva samples
Amadori M.; Haas B.; Moos A.; and Zerbini I.
Appendix 10
The role of sheep in the epidemiology of foot-and-mouth disease and proposals for
control and eradication in animal populations with a high density of sheep.
Alex I. Donaldson

Appendix 11
Possible introduction of FMD with sheep intestines from FMD countries
Bernd Haas and Matthias Kramer

Appendix 12
Peculiarities of FMD buffer zone functioning in the CIS countries
V.M. Avilov; V.M. Zakharov; A.A. Gusev; S.A. Doudnikov; N.A. Yariomenko; A.M.
Rakhmanov; A.K. Karaulov

Appendix 13
Results of seromonitoring studies in FMD buffer zone of the CIS countries
T.A. Fomina; V.M. Zakharov; A.A. Gusev; N.Y. Kamalova; S.R. Kremenchugskaya;
O.A. Schekotova

Appendix 14
Sensitivity of primary cells immortalized by oncogene transfection for the isolation of
foot-and-mouth disease virus
M.P. Ferris; G.M. Hutchings;H.J. Moulsdale; J.B.Clarke

Appendix 15
Interferon and foot-and-mouth disease virus
Reinhard Ahl

Appendix 16
Type-independent detection of foot-and-mouth disease virus by ELISA using
monoclonal antibodies that bind to the animoterminus of capsid protein VP2
Brigitte Freiberg; Bernd Haas; Barbara Hohlich; Armin Saalmuller; Eberhard Pfaff
and Otfried Marquardt

Appendix 17
Serotyping of foot-and-mouth disease virus isolated from western buffer zone with
coagglutination test as an alternative to CFT and ELISA
Gulner Unver and Feray Alkan

Appendix 18
Evaluation of a chromatographic strip test for foot-and-mouth disease virus antigen
detection
Scott M. Reid; Anke Brunig; Nigel P. Ferris; Geoffrey H. Hutchings and Lennart
Akerblom

Appendix 19
Development of antigen capture RT-PCR for foot-and-mouth disease virus antigen
detection
Scott M. Reid; Geoffrey H. Hutchings and Nigel P. Ferris
Appendix 20
RT-PCR probing ELISAs for the diagnosis and typing of foot-and-mouth disease
virus using our newly developed SNAP (Simple and Aqueous Phase) hybridization
Soren Alexandersen; Morag A. Forsythe; Scott M. Reid and Graham J. Belsham

Appendix 21
Evaluation of RT-PCR procedures for the diagnosis of clinical samples of foot-and-
mouth disease virus (serotypes O, A, C and Asia 1) under EU Concerted Action PL
98-4032
Scott M. Reid; Geoffrey H.Hutchings; Nigel P. Ferris

Appendix 22
Diagnosis of persisting infections of foot-and-mouth disease virus in cattle: IgA
ELISA, virus isolation and RT-PCR
P.Moonen; L. Jacobs; H. Costa; A. Crienen and R.S. Schrijver

Appendix 23
Spray-drying of inactivated FMDV antigens for diagnostic use
Amadori, M.

Appendix 24
A solid phase competition ELISA for the detection of antibody to foot-and-mouth
disease virus
N.P. Ferris; A.N.Bulut; T. Rendle; F. Davidson and D.K.J.Mackay

Appendix 25
Animal Production and Health Section of the Joint FAO/IAEA Programme of Nuclear
Techniques in Agriculture (Vienna) Coordinated Research Project on: “ The use of
non-structural proteins of foot-and-mouth disease virus (FMDV) to differentiate
between vaccinated and infected animals
J.R. Crowther

Appendix 26
Detection of antibodies against non-structural proteins of FMDV in Bulgaria
G. Georgiev, E. Veleva, A Dimitrova, Y. Ivanov

Appendix 27
Improved indirect ELISA based on the 3ABC polyprotein for differentiating infection
from vaccination in foot-and-mouth disease
Esther Blanco; Marisa Arias and Jose Manuel Sanchez-Vizcaino

Appendix 28
Indirect ELISA using recombinant VP1 capsid protein for the serodifferentiation of
foot-and-mouth disease virus infected animals
M. Wenger; J.D. Tratschin; M.A. Hofmann

Appendix 29
FAO Collaborative Study Phase XVI: Establishing reference standards for FMD
serology
P.Kitching;T. Rendle and B. Newman

Appendix 30
Comparison of FMDV neutralization tests using three different cell lines: validation
of the new FAO reference sera
P. Moonen; G.K.W. Miedema; F. van Hemert-Kluitenberg; G. Chenard and A.
Dekker

Appendix 31
Longevity of the antibody response in pigs and sheep following a single
administration of high potency emergency FMD vaccines
S.J. Cox and P.V. Barnett

Appendix 32
Enhancement of the immune response induced by the inclusion of saponin in
oil adjuvanted vaccines against foot-and-mouth disease
E. Smitsaart; N. Mattion; J.L. Filippi; B. Robiolo; O. Periolo; J. La Torre and
R.C. Bellinzoni

Appendix 33
Experimental vaccination against FMDV with immunocomplexes
H. Yadin; Dalia Chai; A. Bril; B. Gelman and M. Amadori

Appendix 34
Formaldehyde enhances BEI-inactivation rates of foot-and-mouth disease (FMD)
virus by at least a ten-fold
Simon J. Barteling and Nazeem I. Cassim

Appendix 35
Validating the efficacy of emergency foot-and-mouth disease vaccines in sheep
following virus challenge using the non-structural polyprotein 3ABC MAT-ELISA
Paul Barnett; Morag Forsyth and Sarah Cox

Appendix 36
Experimental FMD infections in pigs: a possible design for a PD50 experiment
Hanny Swam and Aldo Dekker

Appendix 37
The second report of the European Pharmacopoeia Working Group. Present and
future contacts with the European Pharmacopoeia and with EMEA
Kris De Clercq (on behalf of the European Pharmacopoeia Working Group)

Appendix 38
Preliminary results from the EUFMD Expert Elicitation Workshop on risk of
introduction of FMD to Europe
John Ryan and Lisa Gallagher

Appendix 39
List of Participants
Introduction

A Session of the Research Group of the Standing Technical Committee of the

European Commission for the Control of Foot-and-Mouth Disease (EUFMD) was
held at Samokov, Borovets, Bulgaria, from 5 to 8 September 2000.

Dr. Y. Leforban, Secretary of the European Commission for the Control of

Foot-and-Mouth Disease welcomed, on behalf of the EUFMD and the Food and
Agriculture Organization of the United Nations, all the participants and observers to
this annual Session ( Appendix 39).

He thanked the Minister of Agriculture and Forests, Dr. Ventsislav Varbanov,

and the Deputy Minister, Dr. Georgi Kirilov for their presence at the Opening
Ceremony. He also stated that it was a great honour for the Commission that the
Session would be opened by the Minister. Appreciation was also conveyed to the
organizers of the Session, Dr. Ilian Bachvarov, Director General of the National
Veterinary Services and Dr. Yanko Ivanov, Deputy Director for preparing the Session
in such an efficient manner. A further note of gratitude was extended to the staff of
the Veterinary Services, in particular to Mrs. Julia Bouchkova and Dr. Jordan Voinov
of the local Veterinary Services for the local arrangements of the meeting.

Dr. Leforban welcomed the members of the Research Group, the observers,
and in particular the representatives of the Scientific Veterinary Committee of the
EC, the Japanese Delegation and the World Reference Laboratory, UK team led by
Dr. Alex Donaldson, of the World Reference Laboratory in UK.

He reminded the meeting that relations between the EUFMD and Bulgaria
have always been very close. Europe is aware of the quality of the Veterinary
Services in Bulgaria and despite the recent political changes in the country, the
Veterinary Services continue to be very strong and efficient. This is demonstrated by
the rapid control of the last FMD occurrences in 1993 and 1996 which were
eliminated as primary outbreaks.

In concluding, Dr. Leforban drew attention to the fact that research activities
are essential to the development of new tools for diagnosis and control of FMD in
Europe and the sessions of the Research Group are the most important forum for the
exchange of information on FMD.

The floor was given to the Minister of Agriculture and Forests, H.E.
Ventsislav Varbanov who gave a warm welcome to the Session and stressed the fact
that this was the first forum of its kind to be held in Bulgaria. He informed the
meeting of the tradition to protect the country against FMD which dates back to 1962
when a specialized laboratory was established to produce vaccine. As in other
European countries vaccination has been discontinued since 1991 in Bulgaria and a
laboratory has recently been reconstructed with EC PHARE programme support. It is
now equipped with the most modern facilities and professional staff. Gratitude was
expressed to FAO for the constant technical assistance provided for FMD control in
the region.
 2

In concluding, he expressed the hope that the present Session would assist the
Bulgarian experts in FMD control to broaden their experience and knowledge, as one
of the main prerogatives for efficient protection against the disease is the coordination
of efforts of each separate country under the auspices of international organizations
such as FAO, EU and OIE.

Dr. Ilian Bachvarov, Director General of the National Veterinary Services

wished the Session success and expressed his certainty that the papers presented
would contribute to the optimization of the European strategy for control of the
disease. He felt sure that in the next millennium FMD, just as rinderpest, would be a
disease of the past in Bulgaria.

Dr. Kris De Clercq, Chairman of the European Commission for the Control of
FMD, reminded the meeting of the main task of the Research Group which is to give
advice to the FAO European Commission and to the Executive Committee. Support
from the participants was requested.

He informed the meeting that the FMD situation in Iran, Iraq, Turkey, the
Caucasian region and the Balkans was of special concern to the Commission. He
thanked the Secretariat, Dr. Leforban, Dr. John Ryan and the members of the Group,
such as Dr. Amadori, who accepted to carry out missions in these regions on behalf
of the EUFMD. The reliability of the WRL in Pirbright for production of
information was acknowledged.

He regretted that for the first time Ms. Joan Raftery was unable to attend the
meeting and on behalf of all present wished her well.

On closing, Dr. De Clercq thanked the EUFMD and representatives of the

Scientific Committee on Animal Health and Animal Welfare of the Commission of
the European Union for their constant collaboration and support to the Research
Group’s activities.

Professor R. Ahl, of the Scientific Committee on Animal Health and Animal

Welfare of the Commission of the European Union conveyed the wishes of the
Committee to the successful outcome of the meeting. He assured the meeting that the
Committee will continue to observe the disease situation in the region and, whenever
possible, will contribute to the progress in FMD research.

The Session was chaired by Dr. K. De Clercq, Belgium. Members of the

Group present were: Dr. M. Amadori, Italy; Dr. S.J. Barteling, The Netherlands; Dr.
A.I. Donaldson, UK; Dr. M. Danes, Romania; Dr. C. Griot, Switzerland; Dr. I.
Gurhan, Turkey; Dr. B. Haas, Germany; Dr. Y. Ivanov, Bulgaria; Dr. J.M. Sanchez
Vizcaino, Spain and Dr. H. Yadin, Israel.

Dr Per Have, Denmark, had sent his apologies for not being able to participate
due to other commitments.

Adoption of the Agenda

The Chairman proposed that the following agenda should be adopted.

 3

Item 1 General information

Item 2 Epidemiology/pathogenicity/immunology/genetics
Item 3 Control of FMD
Item 4 New developments in FMD diagnostics
Item 5 Phase XVI
Item 6 Vaccines
Item 7 European Pharmacopoeia
Item 8 RA workshop
Item 9 Closed Session

The Agenda was adopted as proposed.

Item 1 General Information

Dr. J.R. Crowther gave a brief description of the activities of The Animal
Production and Health Section of the Joint FAO/IAEA Programme of Nuclear
Techniques in Agriculture (Vienna) which promotes research in developing countries.
The main mechanism is through FAO/IAEA Coordinated Research Projects (CRPs).
These provide funds for mainly applied research by individuals within established
laboratories. The funding usually allows for five years efforts and amounts to between
$5,000 and $10,000 per Research Contract Holder (RCH) per year. Research
Agreement Holders (RAH) are also recruited usually from Institutions with a research
background in the relevant area. These individuals, not their Institutions, receive
payment and provide expertise and laboratory back up for the RCHs. There can be
provision of Technical Contracts to provide reagents to facilitate the research being
made (up to $20,000 per contract). A very important aspect of the CRP is the holding
of regular meetings to plan and discuss research data. These Research Coordination
meetings (RCMs) are fully funded and allow all RCHs and RAHs to attend. The
subject areas for the CRP stem from advice usually through a meeting by experts in
Vienna. Following this, the CRP title and aims are advertised and offers received
from candidates. These are processed and successful applicants receive funding. This
may be held in Vienna for procurement purposes, or a proportion paid as cash.
Reports on progress are expected at the RCM and a final document is produced at the
culmination of the CRP in an Agency Technical Document (TECDOC). Publication
in journals during the CRP is promoted. The developments in such CRPs link with
other funding for countries through the Department of Technical Cooperation; this
will be described.

Item 2 Epidemiology/pathogenicity/immunology/genetics

Mr. N. Knowles presented a paper (Appendix 1) on the emergence of a

pandemic strain of FMDV serotype O. A new pandemic strain of FMDV was recently
identified in India in 1990 and further characterized. In the past 10 years this virus has
spread westwards (as far as Greece and Bulgaria) as well as eastwards and had
reached most of South East Asia by the year 2000. The virus has been isolated from a
wide variety of hosts including camels, buffaloes and deer. The viruses identified so
far all belong to the same genetic lineage as determined by the sequence analysis of
their VP1 genes.
 4

Dr. K. Sakamoto presented results of studies on the virulence of O/Jpn/2000

for susceptible animals (Appendix 2). Japanese Black beef cattle as well as Holsteins
were infected experimentally using this isolate. There were only mild clinical signs in
Japanese Black beef cattle and no clinical signs (and no virus excretion) in the
Holsteins after inoculation of 106TCID50. However, pigs developed typical clinical
signs after experimental inoculation. Virus was detected in all experiments by RT
PCR. The origin of the virus is still unclear but it was speculated by the authors that
straw bedding (and maybe fodder) imported from China could have been the source
of introduction. The vaccine which was supplied to Japan (4 million doses) was not
used.

Mr. N. Knowles also presented a further paper on the origin of recent

outbreaks of FMD in North Africa, the Middle East and Europe (Appendix 3). The
origin of recent outbreaks of FMD in these countries were examined using
phylogenetic analyses. The 1999 outbreaks of Type O in Algeria, Tunisia and
Morocco originated in West Africa. In Iran and Turkey most type A viruses belonged
to either the Iran-96 or Iran-99 lineages, however, a new strain was found in eastern
Iran which was related to viruses present in India. The type Asia 1 outbreaks in
Greece in 2000 were closely related to viruses from Turkey, Iran and India.

Dr. I. Gürhan gave an update on the FMD situation in Turkey (Appendix 4). A
total of 90 outbreaks were recorded from 1.1.00 to 31.8.00. The viruses found were of
serotype Asia1 (46), serotype O (39) and A (4). The last Asia 1 outbreaks occurred in
August. A total of 9 samples were in addition submitted to the WRL and confirmed.
The serotype A is closely related to A Iran 96. The FMD institute has produced
monovalent, bivalent as well as trivalent vaccine. The latter is used at present (A
Aydin 98, O1 Manisa, Asia 1). In the spring vaccination campaign 6.3 million cattle
and 3.2 million small ruminants were vaccinated in the whole of Turkey. The autumn
vaccination campaign is to start on 15 September in Thrace. It will include the Thrace
provinces as well as the Anatolian part of Istanbul and of the Canakale provinces. All
ruminants (including sheep and goats) will be included. A total of 1.5 million doses of
the trivalent vaccine A22 + O1 Manisa + Asia 1 have been supplied by the EU vaccine
bank which arrived on 6 September. In Anatolia only large ruminants will be
vaccinated using the trivalent vaccine A Aydin 98 + O1 Manisa + Asia 1 produced in
Turkey. Some remote areas of the Black Sea region will not be vaccinated since no
outbreaks have been reported for the past couple of years.

In the next paper (Appendix 5) Dr. S. Alexandersen examined the 50%

minimal infectious dose (MID50) of airborne FMD virus for pigs using infected donor
pigs as the source of infection. So far data have been generated for O1 Lausanne
FMDV. The MID50 was estimated to be around 200-400 for subclinical infection
(only antibody reaction) while the estimated MID50 for causing clinical disease was
more than 800 TCID50. Doses around 100 TCID50 did not cause any infection and
doses as low as 10-15 TCID50 or less, known to infect ruminants did not infect pigs
by the aerosol route.

Dr. A.R. Samuel presented a paper on the antigenic and genetic

characterization of FMDV type O virus from the Far East (Appendix 6). FMD type O
viruses isolated from outbreaks in Cambodia, Vietnam and Taiwan, Province of China
in 1997 and 1998 were examined both genetically and antigenetically. Phylogenetic
 5

analysis of the complete capsid-coding region showed that the virus could be divided
into two groups which were distinct from the O1 Manisa and O1 Kaufbeuren
reference strains. Representatives of both groups were found in Vietnam. Antigenic
analysis (LPB-ELISA and Mab profiling) was also able to distinguish the two virus
groups.

Dr. A.R. Samuel also gave a presentation (Appendix 7) on the genetically

diverse isolates of FMDV which exhibit remarkable amino acid conservation at their
neutralizing antigenic sites. Eighteen genetically diverse FMD type O viruses were
examined using a panel of neutralizing MAbs. Comparison of the deduced aminoacid
sequences in the previously defined five antigenic sites revealed aminoacid
substitutions which were conservative in nature. These changes were usually limited
to one or two different aminoacids.

Prof. Alexandersen presented a paper (Appendix 8) on the early pathogenesis

of FMD in pigs: a quantitative time course study using a newly developed Taq Man
RT PCR assay. Selected tissues were collected from pigs infected by contact with
FMDV serotype O1 Lausanne and analyzed using a) real time PCR and b) virus
titration in cell culture as well as histopathology. It was proposed that after contact
exposure virus deposits in the pharynx, spreads to the regional lymph nodes and then
produces an initial viraemia. Following this the majority of virus amplification is
generated by several replication cycles in epithelial cells and further viraemic spread.

Dr. M. Amadori presented a paper (Appendix 9) on the IgA response of cattle

to FMDV infection in probang and saliva samples. Probang and saliva samples from
experimentally challenged cattle using FMDV serotype O1 Manisa were collected up
to 234 days pi and subjected to IgA antibody ELISA. Virus isolation and RT PCR
was carried out on the probang samples. Mucosal IgA antibody detection seems to be
more reliable than virus detection in probang samples since a) virus presence in
probang samples was found to be intermittent and b ) IgA response in saliva of
“carrier” animals was much more constant and was still detectable even when no
virus could be detected.

Conclusions:

1. The pandemic strain of FMDV serotype O is a major threat to Europe since it has
shown to be able to spread very extensively and quickly.

2. Phylogenetic analysis has been shown to be very valuable for the characterization
of the recent outbreaks and the identification of their origin.

3. Disease awareness can be sub-optimal in a country which has not experienced

FMD for a long period of time.

4. Variable pathogenicity of the Japanese 2000 type O FMD isolate has been shown
although the experiment was done with few animals.

5. Relatively high doses of FMDV are required to infect pigs by the aerosol route.
 6

6. Taq Man PCR technique is a valuable tool for quantitative studies on FMD. It
shows promise for the diagnosis of FMDV.

7. Detection of the mucosal IgA response to FMDV is an additional method to detect

carrier cattle and cattle exposed to infectious virus.

8. The FMD situation in Turkey is still of concern since a large number of outbreaks
have been reported throughout the year 2000 in Anatolia. At present 3 FMDV
serotypes (O, A and Asia 1) are circulating in Turkey.

Recommendations:

1. Further analysis of known antigenic sites in terms of sequence should be done as

valuable support to serological testing.

2. The minimal aerosol infective doses of FMDV for pigs (using pig adapted strains)
should be determined to improve models to predict airborne spread.

3. Quantitative studies in livestock species of the pathogenesis of FMDV should be

performed to formulate equations to describe the kinetics of infection.

4. Virus specific IgA levels in saliva and nasal swabs in cattle and sheep should be
considered as additional parameters for the detection of carrier animals and
animals exposed to infectious virus.

5. Continued submission to the WRL of additional outbreak samples from various

parts of Turkey and from each of the detected serotypes should be encouraged.

Item 3 Control of FMD

In the first presentation under this item (Appendix 10) Dr.A.I. Donaldson
reviewed the role of sheep in the epidemiology of FMD and highlighted the main
features of virus transmission related to that species. FMD in sheep is often very mild
or subclinical and so their infectivity status may not be clear until they transmit the
virus to indicator hosts, most commonly cattle. Examples were provided of the
numerous occasions when live sheep have been the source of infection in the
transboundary spread of FMD. Infected sheep excrete most FMD virus in the early
acute stages of infection which usually lasts for 4 to 5 days and so this is when they
are most likely to transmit the virus. An important feature of FMD in sheep is that the
infection may be self-limiting. This is supported by evidence from both the field and
preliminary experimental investigations.

It was concluded that there may be certain circumstances when the self
limiting infection of sheep can permit different control strategies to be applied.

A series of recommended actions and strategies were presented (Table 1,

Appendix 11) which aim to achieve particular control and eradication objectives
under different circumstances in which sheep are a major component of the livestock
population.
 7

Dr. B. Haas presented data (Appendix 11) demonstrating that very large
quantities of intestines for sausage casings are imported into Europe from countries
which are endemically infected with FMD virus. These products include small and
large intestines, bladders and stomach from sheep and pigs. Dr Haas pointed out that
neither the OIE International Animal Health Code nor Council Directive 92/118 EEC
nor Decision 94/187 require any treatment of intestines that would destroy FMD
virus. Since work by Bohm and Krebs in 1974 showed that cleaned and salted
intestines from FMD infected sheep still contained FMD virus, it was concluded that
the importation of those products constitutes a risk. It was recommended that studies
are initiated to determine the feasibility of measures to reduce the risk associated with
trade in these products and that this operation be submitted to the OIE Code
Commission for consideration.

The next paper (Appendix 12) was presented jointly by Dr. V. Avilov and Dr.
S. Doudnikov. They described the FMD situation during the last ten years in the
Russian Federation and in the ex-USSR Transcaucasian countries (Armenia,
Azerbaijan and Georgia) and in the Central Asian Region (Kazakhstan, Kyrgystan,
Tajikistan, Turkmenistan and Uzbekistan). In the Russian Federation, outbreaks
occurred in 1990, 1993, 1995 and 2000. All were eradicated as primary foci by
stamping out combined with ring vaccination of all susceptible animals in an area
within a radius of up to 30 km.

Since the break-up of the USSR there has been a decline in the level of FMD
surveillance and disease control activities. There has been a resulting increase in the
number of FMD outbreaks seen in the Transcaucasian and Central Asian regions. The
Transcaucasian region is endemic for FMD. Georgia has had only one year (1994)
during the last ten years without outbreaks, Armenia was FMD-free for only three
years during that period. In Azerbaijan the last officially reported outbreak was in
1996 but there are unconfirmed reports of outbreaks since then. The surveillance data
of the Central Asian region is incomplete and unreliable; this area is probably
endemically infected.

A paper was given by Dr. T.A. Fomina (Appendix 13) who reported the
results of a serological survey for antibodies to the 3 ABC non-structural polypeptide
carried out in the spring-autumn of 1999-2000 in Georgia, Armenia, Azerbaijan and
the Volga region of the Russian Federation. In surveys conducted in 1999 and 2000
in the three Transcaucasian Republics samples from both cattle and sheep gave
positive reactions. However, samples collected in three regions of Kalmykia and
Krasnodar Territory in the Russian Federation were negative for antibodies to non-
structural proteins.

Conclusions:

1. There may be certain circumstances when the self-limiting infection of FMDV in

sheep can permit different control strategies to be applied.

2. Intestines, bladders and stomachs from sheep and pigs for sausage casings
imported into Europe from countries which are endemically infected with FMD
virus constitutes a risk.
 8

3. The deterioration of FMD control procedures in Transcaucasia and Central Asia

has increased the risk of the introduction of FMDV particularly for the Russian
Federation and possibly also for Europe.

4. Russia is currently free from FMD but due to economic pressures the size of its
national buffer zone in northern Caucasia may be reduced.

5. The FMD situation in Kazakhstan, which shares a long border with Russia, has
deteriorated in recent years. This part of Russia’s southern border is not protected
by a buffer zone and so there is a major risk that animal trade could transport virus
northwards through this gap in Russia’s defences.

6. Transcaucasian countries have insufficient resources, infrastructure, legislation

and laboratory support to implement effective disease control measures when
outbreaks of FMD are reported.

7. There is a shortage of vaccine for use in Transcaucasian countries and the locally
produced vaccine (from Armenia and Georgia) is not fully quality controlled.

8. The results of the tests for non-structural antibodies indicate that FMD virus was
circulating in the three Transcaucasian Republics during 1999 and 2000.

Recommendations:

1. Actions and strategies which aim to achieve particular control and eradication
objectives under different circumstances in which sheep are a major component of
the livestock population should be implemented.

2. Studies should be initiated to determine the feasibility of measures to reduce the

risk associated with trade in intestines for sausage casings and these measures
should be submitted to the OIE Code Commission for consideration.

3. From a strategical point of view, and considering the endemic situation of FMDV
in Transcaucasian countries, priority should be given to the strengthening of the
Russian buffer zones and to the improvement of surveillance and control
programmes in the Transcaucasian region.

4. The national buffer zones in Russia (northern Caucasian region and the extreme
east region) should be maintained and surveillance continued, including the
application of the 3ABC ELISA to detect any possible entry of virus.

5. The ARRIAH, Russia, sends to the WRL, Pirbright, representative samples of the
FMD virus isolates which it receives from the ex-USSR countries.

6. The FMD vaccine used in the Transcaucasian region should comply with the OIE
manual requirements.
 9

7. Cattle in the Transcausian region control programme should be vaccinated within

a short period of time in the early spring before they are permitted to move to
highland pastures.

Item 4 New developments in FMD diagnostics

Antigen detection and virus isolation

Mr.S. Reid presented a paper by Ferris et al. (Appendix 14) on the sensitivity
for the isolation of FMDV of primary cells (calf thyroid, calf kidney and piglet
kidney) immortalised by oncogene transfection. Sixty-five immortalised cell lines
were stable upon repeated cell culture passages and many supported the growth of
FMDV (and SVD, in the case of PK cell lines) but none had either the degree of
sensitivity or specificity for all FMD virus serotypes exhibited by primary calf thyroid
cells and IB-RS-2 cell cultures which are routinely employed for vesicular virus
diagnosis.

Prof. R. Ahl gave a talk on the influence of interferon on the replication of

virus in cells, (Appendix 15) which may lead to problems in the isolation of FMDV in
primary or secondary cells. The possibility that interferon might be one factor
responsible for the observed differences in the virulence and pathogenicity of FMDV
was discussed.

Dr. O. Marquardt presented a paper (Appendix 16) on the type independent

detection of FMDV by ELISA. Three MAbs raised against ASIA1, SAT1 and A22
Iraq, respectively, recognized virus of all seven serotypes. Pepscan investigations
revealed that these MAbs bind to the VP2 protein. The possible diagnostic value of
these antibodies was discussed.

Dr. I. Gürhan presented the comparative study of Ünver and Alkan on the
detection of FMDV in field specimens by coagglutination test (COAT), CFT and
ELISA (Appendix 17). COAT was considered as an alternative test for the primary
diagnosis of FMD in regional laboratories with limited equipment, including the
detection of carrier animals. It was suggested that confirmatory tests be performed on
samples positive in the COAT, but negative with CFT and/or ELISA.

PCR-based testing

Mr. S. Reid presented the results of an evaluation of a pen-side test for FMDV
antigen detection using ClearviewTM chromatographic strip-test technology at the
WRL, Pirbright (Appendix 18). The strip-test rapidly and specifically detected FMD
viral antigen in nasal swabs and probang samples both from naturally or
experimentally infected animals as well as in epithelial suspensions of clinical
material and cell culture supernatants. The sensitivity of the devices compared to
antigen detection ELISA was discussed. These results indicated that such strip-test
devices could potentially achieve a more immediate diagnosis at the site of a
suspected FMD outbreak and allow control procedures to be effected more rapidly.
Such pen-side diagnosis would be particularly relevant to FMD control programmes
in endemic regions.
 10

Mr. S. Reid presented a paper describing the principle of an antigen capture

RT-PCR method for the detection of FMDV as well as preliminary results (Appendix
19). The protocols investigated were specific for FMD virus and their sensitivity was
the same as that of the RT-PCR currently used at the WRL. The results obtained
suggests that the entire reaction could be carried out on a 96-well microtitre plate.
These systems could increase the sensitivity of the RT-PCR for FMD diagnosis and
would have the additional advantage of being objectively analysed by an ELISA plate
reader linked to a computer.

Miss. M. Forsyth described a RT-PCR / ELISA detection system (Appendix

20) which had been developed to target a relatively conserved region within the RNA
genome of all seven serotypes of FMDV. Using a Simple and Aqueous Phase
(SNAP) hybridisation step with labelled oligonucleotides, the assay was highly
sensitive, specific and easy to perform. Rapid preliminary genotyping of FMDV was
made possible using multiple hybridisation oligonucleotides and targeting a highly
variable region of the FMDV genome. The method could be modified for diagnosis
and strain differentiation in any system amenable to PCR amplification.

A collaborative study initiated from the first meeting (Tübingen, Germany,

1999) of the European Union Concerted Action on FMD diagnosis (CT 98-4032) was
presented by Mr. S. Reid (Appendix 21). Each participating laboratory had been
supplied with FMDV positive clinical material and supernatant fluids to evaluate the
sensitivity and specificity of locally employed RT-PCR procedures. Only one
laboratory reported results in which FMDV was detected in 36 of the 40 epithelial
suspensions and in 39 of the 40 cell culture supernatant fluids by a diagnostic RT-
PCR. FMDV was detected in all of the cell culture supernatants by a universal
primer/probe set for FMD using a quantitative PCR machine. The results will be
compared with those obtained by other laboratories.

A paper on the diagnosis of persisting FMD infections in cattle by ELISA,

including an IgA ELISA, virus isolation and RT-PCR was presented by Dr. P.
Moonen (Appendix 22). For that purpose, animals were infected with FMDV Atur
14/98 and samples taken at regular intervals during an eight months period after
infection. The RT-PCR method employed was more sensitive in detecting virus in OP
fluids than virus isolation. However, with the method used here, it was not possible to
detect IgA in OP fluid. The possible implications of RT-PCR positive results not
confirmed by any other test were discussed.

Serology

Dr. M. Amadori presented a paper (Appendix 23) dealing with the spray-
drying of inactivated FMD virus as a possible convenient procedure for the
production of large batches of antigen for the antibody tests.

Mr. S. Reid presented the paper by Ferris et al (Appendix 24) which

demonstrated an improved competition ELISA for the detection of antibodies against
selected serotypes of FMDV. The ELISA used purified virus and a competition step
involving the test serum and guinea pig antiserum. An improved blocking buffer
increased the specificity of the ELISA.
 11

Dr. J. Crowther reviewed a Coordinated Research Programme (CRP) on the

use of non-structural proteins of FMDV to differentiate vaccinated from infected
animals (Appendix 25). Fifteen contract holders from South East Asia, South America
and Africa are included and are examining three ELISA systems from Pirbright/
Brescia, Lindholm and United Biomedical Incorporation, USA. Preliminary data were
reviewed indicating performance of the tests. Dr. Crowther stressed the importance of
the harmonisation of tests involving NS proteins and stated that this is a major
element in the approach of the IAEA.

Dr. G. Georgiev presented Bulgaria’s results from 3ABC ELISAs (Appendix

26). Stored serum samples from previous outbreaks in Bulgaria, as well as current
field sera, were studied. The 3ABC ELISA has been found to be highly useful for
Bulgaria and should be used for the continuous surveillance for FMDV antibodies in
the region.

Dr. J.M. Sanchez-Vizcaino (Appendix 27) reported on the use of an SDS gel
purified 3ABC antigen in an indirect ELISA. This purified antigen increased the
sensitivity and specificity of the test. Successful results were obtained using sheep,
cattle and pig sera. Dr. Sanchez-Vizcaino also indicated that all ELISA systems
involving NS proteins should be harmonised and he is willing to cooperate fully in
any future studies.

Dr. M. Wenger presented a paper on an ELISA using recombinant VP1 to

measure antibodies in cattle (Appendix 28). Preliminary ELISA results showed some
cross reactivity between FMDV serotypes O and A at the antigen dilutions used.
Further work should be done in the near future to resolve this problem.

Conclusions:

1. The combination of immunocapture PCR with PCR-ELISA, when fully developed

and validated for FMDV, would increase the capacity for sample analysis.

2. Chromatographic strips utilising MAbs that bind all types of FMDV could provide
the possibility of a pen-side preliminary diagnosis leading to more effective
control but only in certain situations, i.e. in endemic areas.

3. Tests for antibody against non-structural proteins of FMD will have an increasing
role in disease surveillance and control.

4. There is a need to harmonise all tests which are designed to differentiate infected
from vaccinated animals.

Recommendations:

1. To continue validation of chromatographic strip tests.

2. To prepare standard guidelines for the use and application of the strip-tests.
 12

3. To continue work on the immortalisation of primary cells.

4. To continue the evaluation of ‘in-house’ diagnostic RT-PCR using the reference

samples supplied by WRL, Pirbright.

5. To develop robust diagnostic RT-PCRs that allow a high throughput of samples.

6. Data showing analytical and diagnostic sensitivity and specificity of all tests for
the detection of antibodies to non-structural proteins should be collated.

7. The use of ELISAs based on non-structural FMD proteins should be supported

and reference sera identified for internal and external quality assessments of test
performance.

8. The antigenic variation of 3ABC should be investigated.

Item 5 Collaborative Laboratory Study Phase XVI

Dr. P. Kitching presented the results of the FAO Phase XVI Collaborative
Laboratory Study (Appendix 29). Of the 30 laboratories which received reagents, 24
returned results (one of these was submitted late and did not appear in the tables
presented). With a few exceptions, there was good agreement between the
laboratories on the classification of the test sera. Results obtained using a solid phase
blocking ELISA indicated that this test format was more sensitive and specific than
that of the liquid phase blocking ELISA.

Dr. P. Moonen (Appendix 30) argued for a raising of the titre used as a cut-off
value, on the basis that the low cut-off titre generally used results in an unacceptably
high number of false positive results.

Conclusions:

1. The reference sera provided for Phase XVI would be accepted as standards for
import/export testing.

2. Improvements to the currently used LPB ELISA for FMDV antibodies should
be made to reduce the degree of non-specific positive results and cross-
reactivity.

3. Higher cut-off titres can be used by national laboratories in response to

particular requirements within their own countries, such as post-outbreak
surveillance.

Recommendations:

1. The FAO Phase XVII Collaborative Laboratory Study should supply

candidate reference sera for the O PanAsia, A Iran ‘ 96 and Asia1. Some of
 13

this sera should be post-infection which could be tested for the presence of
antibodies to non-structural proteins.

2. A comparison should be made between low titre serum produced naturally and
that produced artificially by diluting a high-positive serum with negative
serum.

3. The reference sera (O1 Manisa, A22 Iraq and C1 Oberbayern) used in phase
XVI should be recommended to OIE as the international reference standards.

4. Protocols for the solid phase competition ELISA and solid phase blocking
ELISA should be circulated to national laboratories of member countries.

Item 6 Vaccines

Dr. K. De Clercq, Chairman of the meeting, announced at the beginning of the

Session that Dr. S. Barteling will step down as a member of the RG at the end of this
mandate. He thanked Dr. Barteling for his considerable number of contributions
during the thirty years he attended the RG meetings as active observer and member.
“Dr. Barteling’s contributions, especially in the field of vaccine production and
developments will surely be remembered,“ he stated. He also thanked him for all the
missions he had carried out on behalf of EUFMD.

Dr. A. Samuel presented the paper by Cox and Barnett (Appendix 31) dealing
with the longevity of the antibody response in pigs and sheep after injection of a
single dose of a potent emergency FMD vaccine. It was shown that double oil
emulsion vaccines formulated with Montanide ISA 206 induced a rapid sero-
conversion in both pigs and sheep. Furthermore protective Ab-titres were maintained
for at least 4.7 months in pigs and 6 months in sheep. The need for such a long-lasting
immunity for emergency vaccination was discussed.

Dr. E. Smitsaart presented a paper describing the results of the incorporation

of saponin in water-in-oil vaccines (Appendix 32). Antibody levels were about 10
times higher if saponin was included. The vaccine also induced high antibody levels
in pigs and goats. Calves with high levels of residual maternal antibodies, also
showed consistent antibody titres after vaccination if saponin was included in the
vaccine. No adverse effects were observed in the vaccinated animals.

Dr. H. Yadin dealt with experimental immunization of cattle with FMD

vaccine mixed with anti-serum against the FMD-type of the vaccine (Appendix 33).
In agreement with other test models, one particular Ag/Ab ratio induced a high
antibody response up to the end of the test at 45 d.p.v. The control vaccine and the
other vaccine - antibody combinations induced lower antibody levels.

Dr. S. Barteling dealt with the combined inactivation of FMD virus by BEI
and formaldehyde (Appendix 34). Inactivation rates of 5 batches of SAT 1, 2, and 3
virus were increased by at least a ten-fold without a detectable loss of 146 S antigen.
A vaccine formulated from these inactivated antigens induced a strong antibody
response in cattle. It was proposed that the cross-linking activity of formaldehyde
could contribute to a superior stability of both antigens and vaccines.
 14

Dr. A. Samuel presented the paper by Barnett et al. (Appendix 35), which
dealt with the use of the 3ABC MAT-ELISA to evaluate the efficacy of emergency
vaccines in sheep. There was a positive correlation between (i) the occurrence of
clinical signs and/or virus isolation from OP fluid, and (ii) the antibody response to
3ABC after airborne challenge infection from donor pigs. The importance of these
results was stressed in view of the inconsistent outcome of the experimental infections
of sheep. On the contrary, there was a weak correlation between the above parameters
and the virus-specific IgA response in OP fluid.

Dr. H. Swam presented an experimental design for evaluating PD50 in pigs

(Appendix 36). Instead of testing 3 diminishing doses in 3 groups of 5 animals she
tested 5 diminishing doses in 3 pigs each. The groups were kept separate and pigs
were removed upon the appearance of clinical signs. This was done in order to reduce
the possibility of secondary FMDV infection.

The need for a potency test for FMDV vaccine designed for pigs was discussed at
length.

Conclusions:

1. High potency vaccines suitable for emergency vaccination have been used in
sheep and pigs and probably confer immunity for up to 6 months.

2. The addition of saponin to a water-in-oil vaccine was shown to increase the level
of humoral antibody response.

3. The combined use of formaldehyde and BEI was shown to shorten the time
needed for complete inactivation.

4. The use of Ab tests for NSPs can substantially contribute to an objective

evaluation of vaccine efficacy in sheep.

Recommendations:

1. Research should continue to investigate the novel vaccine preparations presented

at the meeting.

2. The feasibility of developing a practical PD50 test in pigs should be further

considered. However, it should be restricted to particular cases.

Item 7 European Pharmacopoeia (E.P.)

Dr. K. De Clercq reviewed the main contents of the 2nd report of the
European Pharmacopoeia Working Group as included in the proceedings of the
Maisons-Alfort meeting of the EUFMD Research Group (Appendix 37). He also
reported on the contacts made with the Secretariat of the EP and EMEA. It was
stressed that the report generated much interest and that it will be included in the
agenda of the EP Expert Group meeting which will be held on 3 October 2000. The
 15

particular features of FMD vaccines were also taken into account by a representative
of EMEA, which intends to examine alternatives for the existing licensing procedures
for certain vaccines in Europe (e.g. Rabies, CSF, FMD).

Recommendation:

1. Contacts with the EP and EMEA should be continued and finalized to obtain a
substantial inclusion of the contents of the report proposed by the EP Working
Group.

Item 8 Expert Elicitation Workshop

Prior to the Session of the group, Dr. J. Ryan and Ms. L. Gallagher, Veterinary
Laboratories Agency, Weybridge, UK conducted an expert elicitation workshop on
the Risk of Introduction of FMD into Europe. Six members of the Research Group
and 12 other experts took part (Appendix 38).

The workshop sought to answer 3 key questions by eliciting the opinions of

the experts:

a) what groups of countries in Europe were most likely to experience outbreaks of

FMD in the next 5 years?
b) what groups of external countries posed the greatest risk to Europe?
c) what routes of introduction posed the greatest risk to Europe?

In addition, the experts were asked to predict the minimum, most likely, and
maximum number of outbreaks that Europe would experience over the next 5 years.

The workshop utilised a modified Delphi technique, where the experts

completed two identical questionnaires. The questioning method used was direct
elicitation of probabilities of the introduction of FMD to Europe. After presentation of
the results of the first questionnaire, the experts could discuss these results before
completing the second questionnaire.

European countries were placed in 5 groups and non-European countries with

endemic or sporadic FMD were placed in 8 groups. The questionnaire began by
asking which European groups of countries were most at risk from FMD introduction.
Then for each of those European groups, the experts were asked to assume that an
outbreak had occurred in that group and give the probabilities (in %) that the outbreak
could have come from each of the external source groups. Then for each source
group-European group pair, the expert was asked to assume that an outbreak in the
European group had been traced to the source group and then was asked to assign
probabilities to a list of routes of introduction. The maximum number of routes
considered was 15.

The preliminary results were presented to the meeting and displayed good
convergence between the experts’ opinion.
 16

Conclusions:

1. It was concluded that the results accurately reflected the experts’ opinions on the
risk of introduction of FMD to Europe

2. The exercise was very useful for both participants and organisers and EUFMD
and it provided comprehensive expert opinions on the risk of introduction of FMD
to Europe.

3. The exercise demonstrated that the method used is a valid means for eliciting
expert opinion in a cost effective manner.

Recommendations:

1. EUFMD and the Research Group continue to examine methods of analysing the
risk of introduction of FMD to Europe and continue its collaborative
efforts/activities with specialized European institutes

2. Future elicitations involve the gathering in advance of relevant data especially in

relation to trade prices, volumes and movements of commodities.

3. Future elicitations include experts from the livestock and meat industries and
veterinary experts involved from Veterinary Services, in the control of trade in
livestock and all animal products.

Item 9 Closed Session

The members of the Group and the Representative of the EC Scientific Committee
met in a closed meeting on 8 September. Dr Kris De Clercq, Belgium, chaired the
meeting. Members of the Group present were: Dr. M. Amadori, Italy; Dr. S.J.
Barteling, The Netherlands; Dr. A.I. Donaldson, WRL,UK; Dr. M. Danes, Romania;
Dr. C. Griot, Switzerland; Dr. I. Gurhan, Turkey; Dr. B. Haas, Germany; Dr. Y.
Ivanov, Bulgaria; and Dr. H. Yadin, Israel.

Pf. R. Ahl, ex-member of the Research Group, represented the EC Scientific

Veterinary Committee. EUFMD Secretariat: Yves Leforban, John Ryan
The agenda had been circulated prior to the meeting. The following items were
examined by the meeting:

1 Follow-up to the activities of the working group on the European

Pharmacopoeia

Members of the group agreed that:

- if the new layout risks slowing down the process of changing the EP
Monograph, it was proposed that the actual layout be kept and the focus be
put on the main changes.
- a member of the EP Committee should be invited to the next meeting of the
EUFMD Working Group ( if any)
 17

-lobbying prior to the EP Committee’s meeting in October was not considered

to be appropriate
- the Secretary would send the report of the Working Group to the Director of
OIE.

2 EU antigen bank

Members of the Group expressed their concern:

- regarding the supply from the EU antigen bank to Turkey for vaccination in
Thrace of a trivalent vaccine which includes the A22 strain as this strain does
not protect correctly against new type A currently circulating in Turkey and in
the Middle East. However, it was explained that EU had no possibility to
supply A Iran 96 from its antigen bank at the time of the decision. Therefore,
considering the emergency situation created by the Asia 1 type threatening the
region – and already introduced in Greece – the Commission decided to
provide a very potent trivalent vaccine formulated from the antigens stored in
its bank.
- on the depletion of the EU bank following the supply of FMD vaccine of O
type to Japan, Korea and Turkey.

This was discussed and it was agreed that this concern should be addressed to
the CVO’s of the Executive Committee who could probably refer it to EC.

3 Information on the recent and future missions to Caucase, Greece and

Turkey

The Secretariat and the members of the Group who participated in EUFMD
missions reported on their recent mission.

- Dr M. Amadori and Dr Y. Leforban reported on their mission to

Transcaucasian countries from 24 June to 9 July 2000.

- The Secretary reported on his mission to Greece ( joint EC/EUFMD) mission

from 24 to 28 July 2000. He informed the Group that the information regularly
updated relating to the situation in Greece is available on the web site of the
Ministry of Agriculture of Greece.

- He also informed the Group that an EC/EUFMD mission will visit Thrace
during the first week of October to assess progress in the vaccination
campaign with the trivalent vaccine provided from the EU antigen bank.
Concerning the vaccination in Thrace, the Group recommended that :
vaccination start from the border

*clinical examination be carried out prior to vaccinating

*vaccination be organised over a short period of time ( one month)
*the recommendations of the EC/ EUFMD mission which visited
Thrace in 1998 during the emergency campaign against type A be
taken into account.
 18

- Dr J. Ryan reported on the FAO/TCP project in progress between Iran and

Turkey for strengthening surveillance and control of new FMDV strains.

4 Follow up to the Workshop on NSP ELISA in Brescia in January 2000

Need for an additional workshop for other National FMD labs.

The Chairman reported on the Workshop and asked the members of the
Group whether additional workshops should be organised by EUFMD for
other countries. Dr Danes, Romania, felt that the need existed in his country
and he has already contacted IZSLE, Brescia to train his staff.
It was suggested that if another technical meeting on the subject is organised
by the Balkan countries, other countries in the region, including Romania be
associated.

5 Follow up to the discussion on SVD at the previous Sessions.

The Chairman informed the Group that he will participate in the meeting of
the OIE Regional Commission for Europe to be held in Israel at the end of the
month and will present the replies of the CVO’s to the questionnaire on SVD
which he had circulated.

6 Relations between EUFMD and EC : utilisation of the Trust Fund

The Secretary informed the Group about the ongoing discussion between the
EUFMD/FAO and EC for the signature of a new four-year agreement on the
utilisation of the Trust Fund.

7 Circulation of the information to the Group by the Secretariat; evolution in

the roles and methods of work for the RG

The Secretary informed the meeting of the initiative of the Secretariat

regarding the circulation of information. He stated that he sometimes had
difficulty in identifying the information to be circulated to the Group knowing
that members of the Group have already access to Promed and to OIE
information.

The Group was interested in receiving all epidemiological information on

FMD and the Secretary promised that he would make every effort to ensure
that it would be circulated.

He also stated that he was open to any proposal of the Group for updating the
methods of work of the RG including the organisation of electronic
conferences if necessary

8 Next Group to be designated by the 34th Session of EUFMD together with

the Activities of the RG over the coming years

The Chairman recalled the role of the RG which is to give scientific advice to
the Executive Committee. He also stressed the need for the members to
actively participate in the activities of the Commission by presenting
 19

contributions to the meetings and by participating in the EUFMD missions

when requested.

9 Venues for the next Sessions of the RG

2001 Denmark ( proposed on 2nd or 3rd week of September, week 37 or 38

2002 Turkey
2003 Switzerland
Proposals from Brescia and Madrid for the next sessions.

Adoption of the Report

The draft report of the meeting was discussed by the Session and accepted
with some amendments.

Closing remarks

The Chairman informed the Group that he would be reporting the outcome of
the meeting to the 65th Session of the Executive Committee scheduled to take place in
Germany on 16 and 17 November 2000.

The Chairman expressed the appreciation of the Group to the National

Veterinary Services, Bulgaria, in particular to Dr Yanko Ivanov, Deputy Director
General and to the staff who had arranged the venue for the meeting and who had so
efficiently and generously contributed to its success. He extended thanks to the
members and observers for their contributions

Before closing the meeting the Chairman thanked the secretariat for their
efforts in providing the working papers and coordinating the preparations for the
Session.
 20
Appendix 1

Emergence of a Pandemic Strain of Foot-and-Mouth Disease Virus

Serotype O
N.J. Knowles1, A.R. Samuel1, P.R.Davies1, R.P. Kitching1, R. Venkataramanan2,
T. Kanno3, A.V. Scherbakov4, V.V. Drygin4, Q.-Z. Zhao5 and Q.-G. Xie5

1) Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom;
2) Central Laboratory, All India Coordinated Research Project on Foot-and-Mouth Disease, Indian Veterinary Research
Institute, Mukteswar-Kumaon, Nainital, India; 3) National Institute of Animal Health, Department of Exotic Disease, 6-
20-1 Josuihoncho, Kodaira, Tokyo 187-0022, Japan; 4) All-Russian Research Institute for Animal Health, 600900
Jur'evets, Vladimir, Russian Federation; 5) Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural
Sciences, No. 11 Xujiaping Yanchangbu, Lanzhou, Gansu Province, 730046, People’s Republic of China.
Abstract
A pandemic strain of foot-and-mouth disease virus (FMDV) serotype O, which we have
named PanAsia, has recently been identified. It was first identified in northern India in 1990 and
has spread westward into Saudi Arabia during 1994 and then throughout the Middle East and into
Europe (Turkish Thrace, Bulgaria and Greece) in 1996. In 1993 it was found in Nepal and later in
Bangladesh (1996) and Bhutan (1998). During 1999 it spread to mainland China and then into
Taiwan Province of China. In late 1999 and 2000 it reached most of south-east Asia. Most
recently it has been introduced into the Republic of Korea, Japan, the Primorsky Territory of
Russia and Mongolia (places free of FMD since 1934, 1908, 1964 and 1973, respectively). The
virus has been isolated from a wide variety of host species (cattle, water buffalo, pigs, sheep,
goats, camels, deer and antelope). The viruses causing these outbreaks of FMD all belong to a
single genetic lineage and the nucleotide sequences of their VP1 genes differ by no more than 5%
despite having been isolated over an 11 year period. The rapid spread of a pandemic strain such
as this clearly demonstrates the ability of newly emerging FMD viruses to infiltrate a wide
geographic area and to cause epidemics in countries which have been free from the disease for
many years.

Introduction
Foot-and-mouth disease (FMD) is endemic throughout most of southern Asia, South-East Asia
and the Far East. However, until recently some countries had been free of the disease for a
number of years, e.g. Japan (1908), Taiwan Province of China (POC; 1929), Republic of Korea
(1934). The FMD status of some Asia countries is unknown and only Indonesia has remained
disease-free in recent years. Understanding the epidemiology of FMD is essential for the
formulation of the most effective control strategies and determining the origin of outbreaks is an
important element of epidemiological investigations. This is best done by the analysis of
nucleotide sequence data.
Nucleotide sequencing was first used for the study of the epidemiology of FMD by Beck and
Strohmaier (1987) who investigated the origin of outbreaks of types O and A in Europe over a 20
year period. Since then a number of epidemiological studies have been published for serotype O
(Knowles et al., 1988; Marquardt and Adam ,1990; Samuel et al., 1990, 1993, 1997, 1999; Krebs
et al., 1991; Armstrong et al., 1992; Marquardt and Krebs, 1992; Saiz et al., 1993; Stram et al.,
1995; Singh et al., 1996; Pattnaik et al., 1998).
At the OIE/FAO World Reference Laboratory for FMD (IAH-Pirbright) we have an on-going
molecular epidemiology programme aimed at elucidating the origins of FMD outbreaks and
 21
tracing the movement of virus strains around the world. These studies are supported by many
collaborations with FMD laboratories throughout the world. We present here a study of a new
FMD strain which emerged in India and has spread throughout the whole of Asia.

Materials and methods

Viruses
The origins of the FMDV isolates studied are listed in Table 1.

Oligonucleotide primers
Primers for reverse transcription, PCR and manual sequencing were purchased from
Cruachem, UK. Oligonucleotide primers for sequencing (Cy5 amidite labelled) were purchased
from Pharmacia Biotech. The location and sequences of the primers used are shown in Table 2.

Extraction of viral RNA and reverse transcription

Total RNA was extracted from 500Φl of 10% epithelial suspensions or cell culture
supernatants using Qiagen RNeasyJ kits according to the manufacturer=s protocol. This RNA
was used as a template for reverse transcription (RT) using primer NK61 and the method
described by Knowles and Samuel (1995).

Polymerase chain reaction amplification

A 1301bp PCR product was amplified using primers ARS4 and NK61 using the method
described by Knowles and Samuel (1995). PCR products were analysed on a 2% agarose-TBE
gel containing ethidium bromide at concentration of 0.5Φg/ml. DNA weight markers were run
alongside the samples to facilitate product identification. Post PCR purification was carried out
using Wizard PrepsJ (Promega, UK) according to the manufacturer=s protocol.

Cycle sequencing
DNA sequencing kits (fmolJ, Promega, UK) were used according to the manufacturer=s
protocol with some amendments (see Samuel et al., 1998). Table 2 shows the various primers
used. PCR amplicons were sequenced either manually using 32P-labelled primers as described by
Knowles and Samuel (1995) or using an ALFexpress II automated sequencer (Pharmacia
Biotech, Sweden) as per the manufacturer=s instructions. In the latter case, the manufacturer=s
software, ALF Manager V3.01, was used to process the data.

Computer analysis
Nucleotide sequences were aligned and analyzed on an IBM compatible personal computer
using programs written by one of the authors (NJK). All pairwise comparisons were performed
by giving each base substitution equal statistical weight (ambiguities were ignored). A
phylogenetic tree was constructed using the UPGMA method as implemented in the computer
program NEIGHBOR and dendrograms plotted using the program DRAWGRAM both from the
PHYLIP 3.5c phylogeny package (Felsenstein, 1993). The UPGMA method constructs a tree by
successive (agglomerative) clustering using an average-linkage method of clustering.

Results and discussion

During the course of routine epidemiological sequencing in the WRLFMD over 100 FMDV
 22
isolates are examined each year. These data are supplemented by sequences provided by other
FMD laboratories around the world. Fig. 1 shows a phylogenetic tree constructed using some
representative virus sequences of serotype O from the WRLFMD database. Six of the seven
newly defined topotypes (Samuel and Knowles, 2000) are represented. All the viruses belonging
to the PanAsia lineage share >96% nucleotide identity and fall within the Middle East-South Asia
(ME-SA) topotype. The complete VP1 sequences were determined for a number of recent
isolates and a phylogenetic comparison made (Fig 2). This confirmed the relationships found
using partial VP1 data.
In 1994 we first noted the arrival of a new FMDV type O lineage in Saudi Arabia (Samuel et
al., 1997). It was probably introduced into Saudi Arabia with the live trade in sheep and goats.
Saudi Arabia imports over 6 million live animals annually mainly to support the requirements of
the Muslim festivals. During 1994 the virus spread within Saudi Arabia and then moved into
neighbouring countries so that by 1996 it had reached Turkey. From Turkey it crossed the Evros
river, probably with illegally moved sheep, into Greece, where it caused 39 reported outbreaks.
The same virus also caused an outbreak in Bulgaria in 1996. By 1998 the new strain had reached
most countries in the Middle East (Table 3; Fig. 3). In the years preceding the arrival of the new
virus many genetic lineages of the Middle East-South Asia topotype (Samuel and Knowles,
2000) were observed to be co-circulating in the region, however, the new strain appeared to have
supplanted all these other lineages. However, in 1998 a isolate from eastern Turkey
(O/TUR/6/98) was found to be related to a virus lineage which was circulating in that region in
1994 and which had caused outbreaks in Greece at that time (Fig. 2).
Retrospective examination of viruses from India indicated that this strain was present in the
north of that country as early as 1990. Between 1991 and 1994, the new lineage appeared to
spread to other parts of India and also to Nepal. In 1995 a single case was found in Malaysia,
however, the new lineage was not found in subsequent years. In 1996 the new virus was seen in
Bangladesh and in 1998 in Bhutan. In July 1998 the People=s Republic of China reported an
outbreak of FMD type O in Yunnan Province and in May 1999 further outbreaks were reported in
Tibet, Hainan and Fujian Provinces. Sequencing of viruses from the outbreaks in Tibet
(O/CHA/1/99, O/CHA/2/99 and O/CHA/3/99) and Hainan (O/CHA/4/99) showed that they
belonged to the new lineage (Fig. 1). In June 1999, FMD virus was isolated from subclinically
infected or carrier cattle in Kinmen Prefecture of Taiwan Province of China (POC) during routine
surveillance; Kinmen Island lies about 1 kilometre off the coast of mainland China (Fujian
Province). Sequence analysis of this isolate (O/TAW/2/99) also showed this to belong to the new
lineage (Fig. 1). Later that month FMD virus was detected in Tainan prefecture on the main
island of Taiwan, again in cattle showing no signs of disease. In January 2000, the first clinical
cases in cattle were found in Taiwan POC (Yunlin and Chiayii prefectures) and in February 2000,
approximately 71 young goats in Kaoshiung and Changhwa prefectures died suddenly due to
FMD infection, although no disease was seen in adult goats which had been vaccinated. This new
virus was clearly spreading throughout Asia and we decided to name it the PanAsia strain.
Towards the end of 1999 it became clear that the PanAsian virus was moving into South East
Asia, where a different FMDV type O topotype (named SEA) normally exists (Samuel and
Knowles, 2000). By April 2000 all mainland South East Asian countries, with the exception of
Myanmar, had outbreaks due to the new strain. In March 2000, FMD type O appeared in South
Korea and Japan and sequence analysis revealed the PanAsian strain to be responsible (Fig. 1). In
April 2000, a very severe outbreak of FMD type O in occurred in pigs in the Ussuriysk district of
eastern Russia. Of 625 pigs affected nearly 37% died from the disease. Sequencing of the VP1
 23
gene showed that the PanAsia strain was responsible for this outbreak (Fig. 1). At the end of
April 2000, an outbreak of FMD type O was reported in Ulaanbadrakh soum county, Dornogovi
province, Mongolia. In this outbreak sheep, goats, cattle and camels were affected. Again,
sequence analysis of the VP1 gene showed the virus to be of the PanAsia lineage (Fig. 1).
The extent of this spread is unique for one strain of FMD virus, and its presence in most recent
samples from the Middle East indicates that it has out-competed the other strains and topotypes
of FMD virus previously established in these areas. Its appearance in Taiwan POC (FMD-free
from 1929-1997) and Japan, South Korea and the Ussuriysk district of eastern Russia previously
free of the disease since 1908, 1934 and 1964, respectively, shows that this strain is capable of
spreading to countries where strict measures are undertaken to exclude importation of animal
diseases. Whether this fitness to survive is related to its ability to cause more severe clinical
disease, as has been indicated from reports from the Middle East, or an increased ability to
transmit between susceptible species (the PanAsia strain has been isolated from cattle, water
buffalo, sheep, goats, pigs, camels, deer and antelope) is not clear. However, its spread across
most of Asia and into Europe demonstrates how a newly evolved virus with a competitive
advantage can become established, in spite of controls at international borders to prevent the
spread of animal disease. The presence of FMD in a previously free country can seriously
interfere with the local and export trade in susceptible animals and their products. The
appearance in Taiwan Province of China in 1997 of a pig adapted strain of FMD virus has cost
the country over 1.5 billion dollars annually in lost export markets, apart from the expenditure on
the control programme. A large outbreak of FMD in northern Europe or USA would result in
losses exceeding 100 billion dollars. It is not surprising that many nations view with anxiety the
threat of economic terrorism based on the use of rapidly spreading biological agents targeted at
farm animal. The emergence of this competitively advantaged strain of FMD virus, and its spread
within the territory bounded by Greece in the West and Japan in the east, provides an example of
the economic damage that can result.

References

Armstrong, R.M., Samuel, A.R., Knowles, N.J. and Uluturk, S. (1992). Genetic studies on foot-
and-mouth disease viruses isolated from samples collected in Turkey. Report of the Session of
the Research Group of the Standing Technical Committee of the European Commission for
the Control of Foot-and-Mouth Disease, Berne, Switzerland. Rome: FAO. Pp. 64-69.
Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Department of
Genetics, University of Washington, Seattle.
Forss, S., Strebel, K., Beck, E. and Schaller, H. (1984). Nucleotide sequence and genome
organization of foot-and-mouth disease virus. Nuclic Acids Research 12: 6587-6601.
Knowles, N.J. and Samuel, A.R. (1995). Polymerase chain reaction amplification and cycle
sequencing of the 1D (VP1) gene of foot-and-mouth disease viruses. Report of the session of
the Research Group of the Standing Technical Committee of the European Commission for
the Control of Foot-and-mouth Disease held jointly with the FMD Sub-group of the Scientific
Veterinary Committee of the Commission of the European Community, Mödling, Vienna,
Austria, 19-22 September, 1994. Rome: FAO. Appendix 8: 45-53.
Knowles, N.J., Marquardt, O. and Samuel, A.R. (1988). Antigenic and molecular
characterization of isolates from recent outbreaks of foot-and-mouth disease virus in the
Federal Republic of Germany. Report of the Session of the Research Group of the Standing
 24
Technical Committee of the European Commission for the Control of Foot-and-Mouth
Disease., Prague, Czechoslovakia. Rome: FAO. Appendix 24: 149-154.
Krebs, O., Berger, H-G., Niedbalski, W. and Marquardt, O. (1991). Foot-and-mouth disease virus
O1 Lombardy is biochemically related to O2 isolates. Virus genes. 5: 255-266.
Marquardt, O. and Adam., K-H. (1990). FMDV subtyping by sequencing VP1 genes. Advances
in Veterinary Virology: Proceedings of the 1st Congress of the European Society for
Veterinary Virology, Liege, 1989. Veterinary Microbiology 23: 175-183.
Marquardt, O. and Krebs, O. (1992). Outbreaks of foot-and-mouth disease near Hannover in
1987 and 1989: evidence for two strains of virus. Tierarztliche Umschau 47: 137-140.
Pattnaik, B., Venkataramanan, R., Tosh, C., Sanyal, A., Hemadri, D.,. Samuel, A.R., Knowles,
N.J. and Kitching, R.P. (1998). Genetic heterogeneity of Indian field isolates of foot-and-
mouth disease virus serotype O as revealed by partial sequencing of 1D gene. Virus Research.
55: 115-127.
Saiz, J., Sobrino, F. and Dopazo, J. (1993). Molecular epidemiology of foot-and-mouth disease
virus type O. Journal of General Virology 74: 2281-2285.
Samuel, A.R. (1997). Genetic and antigenic studies on foot-and-mouth disease virus type O. PhD
thesis. University of Hertfordshire, UK.
Samuel A.R. and Knowles N.J. (2000). Foot-and-mouth disease type O viruses exhibit
genetically and geographically distinct evolutionary lineages (topotypes). Manuscript
submitted to Journal of General Virology.
Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1990). Preliminary molecular analysis of foot-
and-mouth disease virus type O in the Middle East. Report of the Session of the Research
Group of the Standing Technical Committee of the European Commission for the Control of
Foot-and-Mouth Disease, Lindholm, Denmark. Rome: FAO. Appendix 24: 133-138.
Samuel, A.R., Ansell, D.M., Rendle, R.T., Armstrong, R.M., Davidson, F.L., Knowles, N.J. and
Kitching, R.P. (1993). Genetic and antigenic studies of foot-and-mouth disease virus type O
from Bulgaria in 1991. Revue scientifique et technique de l'Office International des Epizooties
12: 839-848.
Samuel, A.R., Knowles, N.J., Kitching, R.P. and Hafez, S.M. (1997). Molecular analysis of type
O foot-and-mouth disease viruses isolated in Saudi Arabia between 1983 and 1995.
Epidemiology and Infection 119: 381-389.
Samuel, A.R., Knowles, N.J. and Mackay, D.K.J. (1999). Genetic analysis of type O viruses
responsible for epidemics of foot-and-mouth disease in North Africa. Epidemiology and
Infection 122: 529-538.
Singh, M., Mohan, B.M. and Suryanarayana, V.V.S.(1996). Serological and molecular analysis
of serotype O foot-and-mouth disease virus isolated from disease outbreaks in India during
1987-91. Virus Research 43: 45-55.
Stram, Y., Chai, D., Fawzy, H., Molad, T., Meiri, N., Van-Ham, M., EL-Kilani, S., Fahamy, F.,
Moussa, A. and Yadin, H. (1995). Molecular epidemiology of foot-and-mouth disease (FMD)
in Israel in 1994 and in other Middle-Eastern countries in the years 1992-1994. Archives of
Virology 140: 1791-1797.
 25
Table 1. The designation and origin of FMD type O viruses studied.
Virus designation Geographic origin Date Species
collected
O/ALG/1/99 Souidania, Algeria 1999 bovine
O/1691/ARM/96* Armenia 07/1996 bovine
O/BAR/2/97 Bahrain 1997 nk
O/BAR/8/98 Bahrain 1998 bovine
O/BHU/1/98 Serbithang, Bhutan 08/02/1998 porcine
O/CAM/1/98 Kâmpóng Speu,Cambodia 05/01/1998 porcine
O/CAM/6/99 Kâmpóng Thum, Cambodia 28/01/1999 bovine
O/CAM/2/2000 Angkor Chum, Siem Reap, Cambodia 27/01/2000 bovine
O/CAM/4/2000 Angkor Chum, Siem Reap, Cambodia 29/01/2000 bovine
O/CHA/1/99H Tibet, China 05/2000 bovine
O/CHA/2/99H Tibet, China 05/2000 bovine
O/CHA/3/99H Tibet, China 05/2000 bovine
O/CHA/4/99H Hainan, China 05/2000 bovine
O/CIV/8/99 Côte d=Ivoire 1999 bovine
O/ETH/8/94 Highland area of east Ethiopia 02/02/1994 bovine
O/1696/GRG/97* Georgia 1997 nk
O1/Kaufbeuren/FRG/66 Kaufbeuren, Germany 1966 bovine
O/GNA/10/99 Kawkan/Madina, Guinea 08/05/1999 bovine
O/HKN/1/99 Mong Tseng Tsuen, Yuen Long, Hong Kong 05/01/1998 porcine
O/HKN/10/99 Hong Kong 19/03/1999 porcine
O/IND/6/90I Punjab, India 1990 bovine
O/IND/138/90I Uttar Pradesh, India 1990 bovine
O/IND/17/96I West Bengal, India 1995 bovine
O/IND/304/98I Meghalaya, India 18/05/1998 porcine
O/IND/143/99I Uttar Pradesh, India 1999 buffalo
O/IRN/15/97 Iran 18/02/1997 ovine/caprine
O/IRN/9/99 Iran 1999 nk
O/IRN/24/99 Iran 1999 nk
O/IRN/16/2000 Alostan, Sardasht, West Azerbaijan, Iran 28/06/2000 ovine
O/IRQ/26/2000 Iraq 09/04/2000 bovine
O/ISR/3/99 Upper Galilee, Israel 08/02/1999 bovine
O1/Lombardy/ITL/46 Lombardy, Italy 1946 nk
O2/Brescia/ITL/47 Brescia, Italy 1947 nk
O/JPN/A/2000 Miyazaki, Japan 07/04/2000 bovine
O/JPN/B/2000 Miyazaki, Japan 04/2000 bovine
O/JOR/3/96 Jordan 1996 nk
O/KUW/4/97 Sutybia, Giahra, Kuwait 09/03/1997 bovine
O/LAO/2/2000 Laos 26/01/2000 nk
O/LEB/1/98 Lebanon 1998 bovine
O/MAY/2/2000 Kilang Papan, Batu Arang, Selangor, Malaysia 02/02/2000 bovine
O/MOG/2000* Mongolia 04/2000 nk
 26
O/PAK/1/98 Pakistan 1998 bovine
O/PHI/5/99 Lloilo, Philippines 1999 porcine
O/1734/RUS/2000* Ussuriysk, Russia 04/2000 porcine
O/SAU/2/95 Al-Kharj, Saudi Arabia 07/01/1995 deer
O/SAU/2/97 Riyadh, Saudi Arabia 05/1997 bovine
O/SAU/38/98 Al-Kharj, Saudi Arabia 1998 bovine
O/SAU/7/99 Riyadh, Saudi Arabia 01/09/1999 bovine
O/SKR/1/2000 Papyung, P=aju City, Kyunggi, South Korea 26/03/2000 bovine
O/TAW/81/97 Ilaw, Taiwan 17/04/1997 porcine
O/TAW/2/99 Kinmen, Taiwan 06/1999 bovine
O/TAW/4/99 Penghu Island, Taiwan 02/1999 porcine
O1/Manisa/TUR/69 Manisa, Turkey 04/1969 bovine
O/TUR/6/98 Ovaköy, Balikesir, Turkey 13/02/1998 bovine
O/UAE/7/97 Al Ain, United Arab Emirates 05/1997 bovine
O/VIT/2/97 Vietnam 1997 bovine
O/VIT/3/97 Vietnam 1997 porcine
O/VIT/2/99 Cao Bang, Vietnam 01/05/1999 porcine
O/VIT/17/99 Quang Ninh, Vietnam 01/07/1999 bovine
O/YEM/15/98 Yemen 27/10/1998 nk
All virus designations are WRLFMD reference numbers/names except:
*, ARRIAH reference number
H, LVRI reference number
I, IVRI reference number
nk, not known
 27

Table 2. Oligonucleotide primers used for RT-PCR and cycle sequencing of foot-and-mouth disease viruses.
Location on the
FMDV genome
Primer Primer sequence (5' 6 3') Sense Gene Position* Use
ARS4 ACCAACCTCCTTGATGTGGCT + 1C 2349-2369 PCR
NK61 GACATGTCCTCCTGCATCTG - 2B 3630-3649 RT & PCR
NK72 GAAGGGCCCAGGGTTGGACTC - 2A/2B 3558-3578 sequencing
1D296F ACAACACCACCAACCCAAC + 1D 3181-3199 sequencing
1D628R GTTGGGTTGGTGGTGTTGT - 1D 3181-3199 sequencing
* on the genome of O1/Kaufbeuren/FRG/66 (Forss et al., 1984; EMBL/GenBank accession no. X00871)
 29
Table 3. Occurrence of the PanAsia lineage of FMDV serotype O between 1990 and 2000.
Year
Country 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000
India x ?
Nepal ? ? ?
Saudi Arabia x x x x
Malaysia x x x x
Yemen x ?
Lebanon x x
Israel x x x
Bangladesh ?
Kuwait x x
Turkey x x x x x x ?
Bulgaria x x
Greece x
Armenia
Georgia
Bahrain x x x x x
United Arab Emirates x ?
Iran x x x
Bhutan x x
Jordan x x x x
Syria x x
China (Tibet)
China (Hainan Province)
Taiwan Province of China x x
Qatar
Iraq
Thailand x x x x
Vietnam x
Laos x x
Cambodia x x x x
Japan
South Korea
Russia x
Mongolia
Afghanistan x
Azerbaijan
Hong Kong (China) x x x x x x x x x x x
Indonesia
Kazakhstan
Kyrgyzstan x
Myanmar x x
North Korea
Oman x
Pakistan x x x
Philippines x x x x x x ?
Sri Lanka x x ?
Tajikistan x
Turkmensitan
Uzbekistan x
 30
solid boxes, PanAsia virus isolated
x, type O virus(es) examined, but PanAsia lineage not found
blank boxes, not examined (possibly because no samples received or no outbreaks reported)
?, sequencing in progress
 O/ALG/1/99
WA O/CIV/8/99
O/GNA/10/99
10 O/1691/ARM/96
O/1696/GRG/97
O/IRN/15/97
O/IND/6/90
O/IND/138/90
O/IND/17/96
O/SAU/2/95
O/JOR/3/96
O/SAU/2/97
O/BAR/2/97
O/KUW/4/97
O/UAE/7/97
O/LEB/1/98
O/BAR/8/98
O/CHA/1/99
O/CHA/3/99
O/MOG/2000
O/CHA/2/99
O/CHA/4/99
O/TAW/2/99
O/MAY/2/2000
O/SKR/1/2000
PanAsia O/LAO/2/2000
O/VIT/17/99
O/CAM/2/2000
19.2% O/CAM/4/2000
O/1734/RUS/2000
O/JPN/A/2000
O/JPN/B/2000
O/BHU/1/98
O/IND/304/98
O/IND/143/99
O/IRN/16/2000
O/IRQ/26/2000
O/SAU/38/98
O/ISR/3/99
ME-SA O/YEM/15/98
O/SAU/7/99
O/IRN/24/99
O/PAK/1/98
O1/Manisa/TUR/69

SEA
O/ETH/8/94
O/CAM/1/98
O/VIT/2/97
Euro-SA O1/Kaufbeuren/FRG/66
O/HKN/1/99
O/HKN/10/99
O/VIT/2/99
Cathay O/TAW/81/97
O/VIT/3/97
O/PHI/5/99

20 18 16 14 12 10 8 6 4 2 0
Percentage nucleotide difference (nt 469-639 of VP1)

Fig. 1. Phylogenetic (UPGMA) tree showing the genetic relationships between the FMD type O viruses studied. The China/Hong Kong (Cathay),
Europea/South America (Euro-SA), Middle East/South Asia (ME-SA), South-east Asia (SEA) and West Africa (WA) topotypes are indicated.
 11
Additionally the PanAsia lineage of the ME-SA topotype is also shown.
 12
WA O/ALG/1/99
O/BHU/1/98
O/CAM/2/2000
O/Tibet/CHA/1/99
O/Tibet/CHA/2/99
PanAsia O/Tibet/CHA/3/99
O/TAW/2/99
O/Hainan/CHA/4/99
O/IRN/9/99
O/SKR/1/2000
O/JPN/A/2000
ME-SA O/MAY/2/2000
O1/Manisa/TUR/69
O/TUR/6/98
20.3% O/CAM/6/99
SEA O/VIT/2/97
O/HKN/1/99
O/TAW/81/97
Cathay O/TAW/4/99
O/VIT/3/97
O1/Kaufbeuren/FRG/66
Euro-SA O1/Lombardy/ITL/46
O2/Brescia/ITL/47

20 18 16 14 12 10 8 6 4 2 0
Percentage nucleotide difference (complete VP1)

Fig. 2. Phylogenetic tree showing the relationships between the complete VP1-coding sequences of some of the FMD type O viruses studied.
 13

2000
97 2000 2000
96
96 96
96-99
98-99 2000
95-99 99-2000 97-99
98-99 99 2000
95-98 93-96 98
97-99
94-99 97-99 96 99 99
90-99 99
99-2000 99
95-98 99-2000
99-2000

2000
95

Fig. 3. Conjectured spread of the PanAsian lineage of the Middle East-South Asia (ME-SA) topotype of FMDV-O.
 32

Appendix 2

PATHOGENESIS OF O/JPN/2000 TO SUSCEPTIBLE ANIMALS

Kenichi Sakamoto, Makoto Yamakawa, Toru Kanno and Yosuke Murakami*

Department of Exotic Disease, National Institute of Animal Health, Japan

Department of Virology, National Institute of Animal Health, Japan*

Introduction

For more than 90 years FMD has been absent in Japan. On the end of March 2000, an
outbreak of FMD was suspected in Miyazaki prefecture, southern part of Japan.
Coincidentally FMD outbreaks occurred in South Korea, Russia and Mongolia.
After first outbreak of FMD in Japan, epidemiological surveillance has been
implemented, other 3 farms were suspected by ELISA for antibody detection, and
confirmed by RT-PCR from probang samples and/or virus isolation. Two cases in three
were in Miyazaki and one in Hokkaido prefecture Northern part of Japan.
FMD is recognized as the disease that is a highly contagious and affected animal
shows clinical signs of vesicular and lameness. Regarding to Japanese outbreak of FMD,
the clinical signs were reported only in the first outbreak. Those were atypical
symptoms without any vesicular on foot nor in mouth and nose. The farm was breeding
ten Japanese Black cattle. They developed pyrexia, erosion of mouth and nose without
any clear vesicular.
The epithelial tissue was collected and subjected to antigen detection ELISA, CF test
and virus isolation with all negatives. The specific RT-PCR products were observed and
it was found that the cattle produced high titer of antibody against FMDV by ELISA.
The virus was isolated from the probang sample of the third case by primary bovine
kidney cell.
The sequence of VP1 gene has been determined and was analyzed by Pirbright
Institute. It showed close relationship with those of Asian strains and the virus was
named as O/JPN/2000.
In this paper we demonstrate the virulence of O/JPN/2000 virus to holstain and
Japanese black cattle and pig.

Materials and Methods

 33

Cells and virus: BHK, IBRS-2, bovine kidney primary cell (BK) and bovine thyloid
primary cell (BTh) were prepared and 10% homogenized samples and probang samples
were inoculated to the cells for virus isolation. The isolated virus was cultured four times
into BK cells and subjected to animal experiments.

Animal: Two major plans were designed for animal experiments. One was for inoculation
of the virus to Holstain and pig with its cohabitation animal. The other was to Japanese
black cattle with cohabitation of Japanese black and pig. The titers of the inoculated
virus were 106TCID50 and 106.5TCID50 respectively. The virus was inoculated
subcutaneously or intracutaneously to tongue of cattle and to foot pat of pig.
Following the schedules, serum, plasma, nasal swab samples were collected from
holstain and pig of the first experiment, and serum, plasma, nasal swab, feces and
probing samples from Japanese black cattle and cohabitated pigs in the second
experiment.

Serological tests: ELISA for antibody detection and virus neutralization tests was
performed with using the sera from the animal. This ELISA was established by
Pirbright Institute as a world standard method. The detail protocol of this liquid-phase
blocking enzyme-linked immunosorbent assay was described in OIE Manual of
Standards for Diagnostics Tests and Vaccines.

RT-PCR: A specific primer set was designed in 3D region of FMDV gene (Kanno et al.).
The specificity of this primer set was formally compared with those of 1D-2A primer set
(Forsyth et al.) and IRES primer set (Doel et al.) at FMD center in Thailand.

Virus isolation: Virus was isolated form the plasma and probing samples of animal
experiment with IBRS-2 cells.

Results

Clinical signs of animal

106TCID50 of O/JPN/2000 of virus was inoculated to holstain cattle. One was put in
together with uninfected holstain one day after virus inoculation. Both cattle did not
showed any clinical signs.
A pig was inoculated same dose of virus into one of its foot pat and two other pig
 34

which were not inoculated virus were in close contact with the inoculated pig one day
after virus inoculation. Three pigs all developed clinical signs such as fever, vesicular on
their feet and lameness.
Two JB cattle were inoculated 106.5TCID50 of the virus. Untreated JB cattle was put
in together with one of the virus inoculated cattle. Two pigs were kept together with
another virus injected cattle.
All three Japanese black, both virus infected and cohabitated cattle showed clinical
signs of erosion and ulcer in their tongue, gums and nasal cavity. But clear vesicular was
not observed in the period of experiments. The cohabitated pigs did not develop any
clinical signs.

Serological test by liquid-phase of ELISA for antibody detection

The virus inoculated animal and the pig which was put in together with a virus
inoculated pig had high titer of antibody. The specific antibody was produced between 4
and 6 days after virus inoculation. The antibody titer of the cohabitated holstain was
between 45 and 128 through 3 weeks of the experiment period. The pigs with Japanese
black cattle did not produce any antibody to the virus.

Virus neutralization test (NT)

The movement of antibody for virus neutralization developed the same tendency with
that of ELISA. The NT titers of holstain and pig which was put in close contact with
infected animal, were less than 4.

Excretion of virus
Excretion of the virus was examined by RT-PCR with nasal swab and feces sample.
Although the virus was not excreted from nasal route of infected and contact holstain,
JB cattle and pigs scattered from same route. The virus was not detected from any feces
samples.

Evidence of virus existence in blood

In order to prove virus existence in blood, plasma samples were subjected to RT-PCR and
virus isolation. The specific PCR product was observed from the samples of 5 and 6 days
after virus inoculation of infected holstain, but not from any samples of contact one. The
product was also detected from 2 to 4 dpi samples of infected pig and 3 to 6 dpi samples
of contact pigs. Regarding to JB cattle, it was amplified from 1 dpi samples of virus
injected cattle and from the sample of 5 days after cohabitation. From the plasma
 35

samples of pigs which were put in close contact, no PCR product was detected.

Discussion

FMD in Japan is atypical case, because of its sensitivity to susceptible animal,

clinical signs and the speed of transmission. From animal experiments with inoculation
of O/JPN/2000 strain of FMDV to cattle and pig, the pathogenesis of this virus was
revealed.
To holstain cattle it affected, but affected cattle did not show any clinical signs even
the virus was injected to the tongue and it had high titer of antibody. The cattle did not
spread the virus from the nasal route. On the other hand, Japanese black cattle showed
clinical signs such as erosion, ulcer in its mouth and nose without vesicular but no lesion
on foot. Such legion were limited and focused on its place. It is considered that virulency
of this virus is rather weak even to JB cattle. It is reported that O/Taiwan/1999 is silent
to their native cattle, Yellow cattle, but infected holstain developed clinical signs with
vesicular. In Korea, the isolated virus, O/SKR/2000, affected holstain with developing
vesicular and its vesicular is said to be small. The virulency of Panasian topotype is
considerably mild to the cattle.
In virus injected and contact pigs, typical clinical signs of FMD such as vesicular on
their feet and lameness were observed.
The results of virus excretion from virus injected and cohabitat animal demonstrated
that incubation period of this virus to JB cattle and pig were estimated 2 days.
The diagnosis of this kind of atypical FMD is troublesome because ELISA for antigen
detection, CF test, virus isolation with epithelial tissue sample result in all negatives.
Although PCR method is only reliable method for diagnosis, this method always stand
beside of contamination reaction on account of its high sensitivity. As far as holstain
cattle is concerned, it does not develop any clinical signs. In order to diagnose them,
serological tests such as ELISA and virus neutralization test have to be carried out.
The presence of this type of FMD will place new obstacles on worldwide trade of
animal and animal products. Importing countries does not have any efficient measure to
keep free from this disease. In Japan the first outbreak of FMD was suspected by a local
private veterinarian. If he miss clinical diagnosis and did not report to the local
veterinary officer, the disease was still remaining in Japan.
36
 37

Antibody movement of Infected Cattle and Contac Cattle

Cattle: Holstain
Detection Method : ELISA for Antibody detection Pirbright Neutralisation Test

Days after VI lnfected Cattle Contact Cattle

ELISA NT ELISA NT
0 32 <4 32 <4
1 <32 <4 *<32 <4
2 <32 <4 45 <4
3 <32 4 32 <4
4 <32 4 64 <4
5 <32 16 90 <4
6 45 32 64 <4
8 181 128 90 <4
10 724 512 128 4
12 1448 512 90 <4
15 2048 1024 45 <4
18 1448 1024 45 <4
21 1448 512 45 <4

Note : No Clinical Signs in infected and contact cattle

* Start in Contact

Antibody movement of lnfected Pig and Contac Pigs

Detection Method ELISA for Antibody detection Pirbright

Days after VI Infected Pig Contact Pigl Pig2

0 <32 <32 <32
1 <32 <32 <32
2 * ! <32 <32 <32
3 <32 *<32 <32
 38

4 362 <32 *<32

5 512 45 <32
6 724 512 <32
8 1448 1448 (Path. St.)
10 724 1448
12 1024 724
15 724 724
18 724 724
21 512 724

Notes : Clinical Signs from *

! Virus Isolated from Plasma

Antibody movement of lnfected Cattle and Contac JB

Animal:Japanese Black Meat Cattle (JB)
Samp les: Serum

Detection Method
ELISA for Antibody detection Pirbright Virus Neutralisation Iest(NT)

Days after VI lnfected Cattle Contact Cattle

ELISA NT ELISA NT
0 45 <4
i <32 <4 ! 45 <4
2 45 4 45 <4
3 32 4 45 <4
4 362 512 45 <4
5 181 256 90 4
6 724 512 45 8
7 1448 256 45 4
9 724 256 90 32
 39

il 512 128 724 512

13 2048 512 1448 256

Note:Clinical signs in both infected and contact cattle

! Start in Contact

Antibody movement of lnfected Cattle and Contact Pigs

Animal:Japanese Black Meat Cattle (JB), LW pig
Samp les: Serum
Detection Method : ELISA for Antibody detection Pirbright

Days after VI lnfected Cattle Contact pigl Contact pig2

ELISA NT ELISA NT ELISA NT
0 45 <4
1 45 4 !<32 4 !<32 <4
2 45 8 <32 4 <32 <4
3 45 8 <32 <4 <32 4
4 724 512 45 <4 45 <4
5 1448 256 <32 <4 <32 <4
6 1448 512 <32 <4 <32 <4
7 2048 128 <32 4 <32 4
9 1024 256 <32 4 <32 <4
il 1448 512 <32 4 <32 <4
13 2896 64 <32 4 <32 4
15 4096 32 <32 <4 <32 <4

Notes ! :Start in Contact

No Clinical Signs in pigs

Virus RNA Detection from Plasma of Infected and Close Contact Animal
Animal: Holstain
 40

Detection Method : RT-PCT

lnfected Contact
Days after VI Cattle Pig Cattle Pigl Pig2
0 — — — — —

1 — *!+ — — —

2 — + — + +
3 — + — *+ +
4 + — — + *+
5 + — — — —

6 + — — — —

8 — NT NT NT NT
10 — NT NT NT NT
12 — NT NT NT NT

Notes
No Clinical Signs in Cattle
*:Clinical Signs from *
! :Virus lsolated from Plasma with IB—RS—2 cell
NT: Not tested

Virus RNA Detection from Plasma of Infected and Close Contact Animal
Animal:Japanese Black Meat Cattle (JB), LW pig
Detect ion Method RT-PCR

Infected Cattle Contact Animal

Days after VI 1 2 Cattle3 Pigl Pig2
0 — — — — —

1 + + — — —

2 + + — — — -

3 + + — — —

4 + — — — —
 41

5 — — — — —

6 — — + — —

7 — — + — —

9 — — — — —

il — — — — —

13 — — — — —

Notes No Clinical Signs in Pigs

Virus Excretion from Nasal Route of Infected and Close Contact Animal
Cattle: Holstain
Detect ion Method RT-PCT

Pig Days after virus Inoculation Infected Cattle Infected

0 - -
1 - +
2 - *! ++
3 - ++++
4 - ++++
5 - ++++
6 - ++
8 - +
10 - +
12 - -

Notes : No Clinical Signs in Cattle

* :Clinical Signs from *
! :Virus lsolated from Plasma
Contact animal showed same behavior
 42

Virus Excretion from Nasal Route of Infected and Close Contact JB

Animal:Japanese Black Meat Cattle (JB)
Samples:Nasal swab(NS) in MEM pH7.4
Detection Method RT-PCT

Days after VI I noculated Cattle Close contact JB

1 2
3

0 - -
1 - + ! -
2 + + -
3 + + -
4 + *+ -
5 *- - -
6 - - -
7 - - +
9 - - -
11 - - -
13 - - -

Notes
! :Start of Cohabitation
*:Clinical Signs
 Appendix 3

Origin of recent outbreaks of foot-and-mouth disease in North Africa,

the Middle East and Europe

N.J. Knowles and P.R. Davies

World Reference Laboratory for Foot-and-Mouth Disease, Institute for Animal Health, Pirbright
Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom.

Foot-and-mouth disease continues to be a threat to Europe despite its eradication from

that continent by the end of the 1980’s. Since then sporadic outbreaks have occurred in
1991 (type O in Bulgaria), 1993 (type O in Bulgaria and Italy), 1994 (type O in Greece),
1996 (type O in Bulgaria and Greece and type A in Albania and the former Yugoslav
Republic of Macedonia) and most recently in 2000 (type Asia 1 in Greece). Previous
studies, using nucleotide sequence data, have shown that the type O outbreaks were
introduced from the Middle East (Samuel et al., 1993; Knowles and Samuel, 1995) and
the type A outbreaks came from the Indian sub-continent, possibly via Saudi Arabia
(Knowles and Samuel, 1998). In the World Reference Laboratory for Foot-and-Mouth
Disease (WRLFMD) we continue to monitor the movement of FMD virus strains around
the world using phylogenetic analyses. The areas surrounding Europe present the greatest
threat and we present here a brief report on the genetic characterization of FMD viruses
isolated from recent outbreaks of type O in North Africa, type A in the Middle East and
type Asia 1 in the Middle East and Greece.

Foot-and-mouth disease type O in North Africa

In February 1999 FMD type O was identified in Algeria. By March 1999, 158 premises
and 139 of the country’s 1,541 districts were affected. All cases were in cattle. In late
February 1999 disease was found in cattle in the Oujda municipality of Morocco and in
early March 1999 disease was detected in Tunisia, both in cattle and sheep. Full details
may be found on the OIE Disease Information website (http://www.oie.int/info/A_info.htm).

The VP1 genes of representative virus isolates from all three countries were amplified by
PCR and the products partially sequenced. Comparisons with sequences held on the
WRLFMD database showed the North African viruses to have close relationships with
viruses isolated in 1999 from Côte d’Ivoire and Guinea (Fig. 1). A more distant
relationship was found with a virus originating in Ghana in 1993 suggesting that this
lineage is endemic in West Africa. Zebus smuggled into the south of Algeria in February
were intercepted in the Sahara, south of El Bayadh and Bechar wilayas and in the south
of El Oued wilaya, and were destroyed. These animals did not present clinical signs of
FMD, but this indicates a possible route of entry for the disease.

Previous epidemics of FMD type O affecting North Africa have been shown to originate
from the Middle East (Samuel et al., 1999) as indicated in Fig. 2a.
Foot-and-mouth disease type A in the Middle East

FMD type A is much less widespread than type O in the Middle East. However, previous
studies have shown that new type A viruses may initially be recognized in Iran and
subsequently spread westwards towards Europe (N.J. Knowles and A.R. Samuel,
unpublished data). Monitoring of FMD type A viruses by nucleotide sequence analysis
has shown three distinct genetic lineages to be present in Iran (Fig. 3). Figure 4 shows the
Iranian and Turkish provinces from which we have received type A viruses between 1998
and 2000. The distribution of the three topotypes is indicted by shading. It is not clear
where these virus ultimately originate since we receive few samples from Afghanistan
and Pakistan. A more complete analysis of Indian type A strains is in progress in
conjunction with the Indian Veterinary Research Institute at Mukteswar.

Foot-and-mouth disease type Asia 1 in the Middle East and Greece

In July 2000 FMD type Asia 1 was confirmed on six farms in the Evros region close to
the border with Turkish Thrace. In early August 2000, three more farms were confirmed
as being infected, two at Ferres (Evros region) and one at Potamia (Xanthi region). The
latter outbreak was approximately 100 km from the Evros outbreaks.

Prior to these outbreaks in Greece, FMD Asia 1 had been detected in Iran (1999) and
Turkey (1999-2000). Phylogenetic analysis of partial VP1 sequences show that viruses
from all three countries were closely related (Fig. 5). They were also related to FMD Asia
1 viruses present in Pakistan (1998), India (1995) and Saudi Arabia (1994). Fig. 6 shows
the conjectured spread of this recent Asia 1 strain. It was probably present in India prior
to 1995 and spread to Saudi Arabia in 1994. However, the route of spread of Asia 1 to
Greece in 2000 probably involved animals movements through Pakistan, Iran and
Turkey.

Previous outbreaks of Asia 1 have spread from the India subcontinent via Iran to Turkey
(Firoozi Bandpay et al., 1974), however, the last outbreak of FMD Asia 1 in Greece in
1984 (Anon., 1984) was linked genetically to viruses occurring in Israel and Lebanon
(Ansell et al., 1994).

Conclusions

FMD occurring in countries surrounding Europe may have originated in many parts of
the world. In the past in North Africa FMD type O has originated for either the Middle
East (Samuel et al., 1999) or West Africa (Fig. 1). It has been suggested that an A24-like
found in Algeria and Morocco in 1977 originated from South America, while A5 strains
present in Libya and Tunisia in 1979 and Morocco in 1983 probably came from Europe
(Beck and Strohmaier, 1987; Knowles et al., 1998).

Many viruses occurring in the Middle East originate from the Indian subcontinent and
enter either through the Gulf States or by an overland route via Afghanistan and Iran.
However, FMD viruses may enter the Middle East from Africa and even exotic serotypes
such as SAT 1 and SAT 2 have occasionally caused epidemics.

Routine molecular epidemiological studies are invaluable for monitoring the movement
of FMD viruses and can detect the approach of novel strains before they reach Europe.
References

Anonymous (1984). Greek outbreak in Thrace buffer zone. Bull. Off. Int. Epiz. 96: 20-
23.

Ansell, D.M., Samuel, A.R., Carpenter, W.C. and Knowles, N.J. (1994). Genetic
relationships between foot-and-mouth disease type Asia 1 viruses. Epidemiology and
Infection 112: 213-224.

Beck, E. and Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus

strains by nucleotide sequence determination. Journal of Virology 61: 1621-1629.

Firoozi Bandpay, M.R., Amighi, M., Mastan, M.B. and Maleknejad, P. (1974). The
outbreak of foot-and-mouth disease (Asia 1 type) in 1973 in Iran. Bull. Off. int. Epiz. 81:
681-687.

Knowles, N.J. and Samuel, A.R. (1995). Polymerase chain reaction amplification and
cycle sequencing of the 1D (VP1) gene of foot-and-mouth disease viruses. Report of the
Session of the Research Group of the Standing Technical Committee of the European
Commission for the Control of Foot-and-Mouth Disease held jointly with the FMD Sub-
group of the Scientific Veterinary Committee of the Commission of the European
Community, Mödling, Vienna, Austria, 19-22 September, 1994: Appendix 8: 45-53.

Knowles, N.J. and Samuel, A.R. (1998). Molecular techniques in foot-and-mouth disease
epidemiology. Towards Livestock Disease Diagnosis and Control in the 21st Century:
proceedings of an International Symposium on Diagnosis and Control of Livestock
Diseases jointly organized by the International Atomic Energy Agency and the Food and
Agriculture Organization of the United Nations, held in Vienna, 7-11 April 1997. Vienna:
International Atomic Energy Agency, pp. 185-201.

Knowles, N.J., Ansell, D.M. and Samuel, A.R. (1998). Molecular comparison of recent
foot-and-mouth disease type A viruses from West Africa with historical and reference
virus strains. Report of the session of the Research Group of the European Commission
for the Control of Foot-and-Mouth Disease, Aldershot, United Kingdom, 14-18
September 1998. Rome: FAO, Appendix 4: 41-48.

Samuel, A.R., Ansell, D.M., Rendle, R.T., Armstrong, R.M., Davidson, F.L., Knowles,
N.J. and Kitching, R.P. (1993). Field and laboratory analysis of an outbreak of foot-and-
mouth disease in Bulgaria in 1991. Revue scientifique et technique de l'Office
International des Epizooties 12: 839-848.

Samuel, A.R., Knowles, N.J. and Mackay, D.K.J. (1999). Genetic analysis of type O
viruses responsible for epidemics of foot-and-mouth disease in North Africa.
Epidemiology and Infection 122: 529-538.
 O/ALG/1/99
O/ALG/2/99
O/ALG/3/99
O/MOR/2/99
O/MOR/11/99
O/CIV/8/99
O/GNA/6/99
O/GNA/10/99
West Africa O/TUN/1/99
O/TUN/5/99
O/GHA/5/93
O/GHA/9/93
O/BAR/2/97
O/IRN/5/97
O/KUW/3/97
O/KUW/4/97
O/UAE/7/97
O/IRN/15/97
O/IRN/16/97
O/ISR/1/96
O/SAU/2/95
ALG = Algeria O/SAU/5/95
BAR = Bahrain O/SAU/14/95
O/SAU/20/95
BHU = Bhutan O/SAU/28/95
CIV = Cote d'Ivoire O/YEM/8/95
O/KUW/1/96
ERI = Eritrea O/JOR/3/96
ETH = Ethiopia O/LEB/1/98
O/LEB/3/98
GHA = Ghana O/SAU/2/97
GNA = Guinea O/KUW/1/98
O/BAR/8/98
IRN = Iran O/IRN/20/98
IRQ = Iraq O/IRN/8/99
O/IRN/12/98
ISR = Israel O/IRN/1/99
JOR = Jordan O/BHU/1/98
O/BHU/2/98
LEB = Lebanon O/BHU/6/98
KUW = Kuwait O/ISR/2/99
O/ISR/3/99
MOR = Morocco O/ISR/5/99
PAK = Pakistan O/LEB/9/98
QTR = Qatar O/LEB/12/98
O/LEB/14/98
SAU = Saudi Arabia O/LEB/17/98
TUN = Tunisia O/SAU/36/98
O/SAU/38/98
TUR = Turkey O/SAU/41/98
UAE = United Arab Emirates O/SAU/25/98
O/YEM/3/98
YEM = Yemen O/YEM/6/98
O/SAU/1/99
O/SAU/2/99
O/ISR/A/99
O/YEM/15/98
O/IRQ/2/99
O/IRQ/9/99
O/QTR/3/99
O/IRN/8/94
O/IRN/8/95
O/IRN/13/95
O/IRN/27/95
O/IRN/10/95
ME-SA O1/Manisa/69
O/PAK/1/98
O/ERI/1/96
O/ETH/8/94
Euro-SA O/YEM/4/95
O1/Kaufbeuren/66

18 16 14 12 10 8 6 4 2 0
Percentage nucleotide difference (nt 469-639 of VP1)
Fig. 1. Foot-and-mouth disease type O epidemic in North Africa in 1999

1999
TUNISIA 1989-1992 TUNISIA

& 1994
MOROCCO MOROCCO

ALGERIA LIBYA EGYPT ALGERIA LIBYA EGYPT

WESTERN WESTERN
SAHARA SAHARA

MAURITANIA MALI NIGER MAURITANIA MALI NIGER

ERITREA ERITREA
CHAD CHAD
SENEGAL SUDAN SENEGAL SUDAN

THE GAMBIA DJIBOUTI THE GAMBIA DJIBOUTI

BURKINA BURKINA
GUINEA-BISSAU GUINEA-BISSAU
GUINEA TOGO GUINEA TOGO
NIGERIA ETHIOPIA NIGERIA ETHIOPIA
SIERRA-LEONE COTE SIERRA-LEONE COTE
BENIN CENTRAL BENIN CENTRAL
D'IVOIRE D'IVOIRE
GHANA AFRICAN REPUBLIC GHANA AFRICAN REPUBLIC
LIBERIA LIBERIA
CAMEROON CAMEROON
UGANDA SOMALIA UGANDA SOMALIA
EQUATORIAL EQUATORIAL
GUINEA KENYA GUINEA KENYA
GABON GABON
RWANDA RWANDA
CONGO ZAIRE BURUNDI CONGO ZAIRE BURUNDI

ANGOLA TANZANIA ANGOLA TANZANIA

a) b)
ANGOLA ANGOLA

ZAMBIA MALAWI ZAMBIA MALAWI

MOZAMBIQUE MADAGASCAR MOZAMBIQUE MADAGASCAR

ZIMBABWE ZIMBABWE

NAMIBIA BOTSWANA NAMIBIA BOTSWANA

SWAZILAND SWAZILAND

SOUTH LESOTHO SOUTH LESOTHO

AFRICA AFRICA

Fig. 2. The origin of foot-and-mouth disease type O epidemics in North Africa between 1989 and 1999.
 A/ALB/1/96
A/ALB/4/96
A/MCD/1/96
A/MCD/3/96
A/MCD/5/96
A/MCD/6/96
A/MCD/8/96
A/ALB/3/96
A/IND/300/94
A/SAU/16/95
A/SAU/19/95
A22/IND/17/77 VS
A22/IRQ/24/64
A22/Mahmatli/TUR/65
Indian/Middle Eastern
A/SAU/104/93 (P16)
A/SAU/111/93 (P21)
(A22) topotype
A/IRN/4/2000
A/IRN/7/2000
A/IRN/8/2000
A/IRN/10/2000
A/IRN/1/94
A/IRN/19/95
A/IRN/87
A/SAU/29/93
A/SAU/99/93 (P14)
A/TUR/9/91
A/TUR/1/92
A/TUR/3/92
A/TUR/14/91 Turkish topotype
A/TUR/12/91
A/TUR/3/95
A/TUR/8/96
A/TUR/16/96
A/TUR/11/97
A/IRN/22/99
A/TUR/6/99
A/TUR/4/99 Iran-99 topotype
A/IRN/1/96
A/IRN/7/96
A/IRN/3/96
A/IRN/11/96
A/IRN/17/96
A/IRN/1/97
A/TUR/12/97
A/TUR/1/98
ALB, Albania A/TUR/2/98
A/TUR/14/98
BRA, Brazil A/TUR/21/98
FRA, France Iran-96 topotype
A/TUR/23/98
A/TUR/25/98
HOL, Holland A/TUR/27/98
A/TUR/32/98
IND, India A/TUR/17/98
IRN, Iran A/IRN/1/98
A/IRN/3/98
IRQ, Iraq A/IRN/4/98
A/TUR/4/98
MCD, Macedonia A/IRN/28/99
SAU, Saudi Arabia A/IRQ/17/2000
A/TUR/4/2000
TUR, Turkey A/TUR/1/2000
A/TUR/5/99
A24/Cruzeiro/BRA/55 European/South
A5/Allier/FRA/60
A10/HOL/42 American topotype
20 18 16 14 12 10 8 6 4 2 0
Percentage nucleotide difference (nt 469-639 of VP1)

Fig. 3. Relationships between various FMD type A viruses and recent isolates from Iran, Iraq and Turkey

Kowcaeli Sakarya

Artvin
Tokat Kars
Ankara
Kutahya Erzincan
E. Azerbaijan
Malatya
Aydin
Burdur Gilan
W. Azerbaijan
Markazi
Qom

Qazvin Khorasan

Topotype

Iran-96
Iran-99
Iran-96 & Iran-99 Fars

India / Middle East (A22 -like)

not done

Fig. 4. Foot-and-mouth disease type A topotypes present in Turkey and Iran between 1998 and 2000
 AFG/4/71
BAN/30/79
SAU/2/80
SAU/11/85
TUR/15/73
BAN/13/78
BAN/1/79
BAN/1/87
BHU/1/86
NEP/23/85
WBN/IND/117/85
NEP/19/86 (1985)
IND/9/89 (1988)
NEP/8/87
NEP/36/87
NEP/58/88
NEP/28/90 (1989)
HKN/20/80
YEM/15/79
YEM/2/80 (1979)
IND/8/79
IND/91/79
SAU/48/91
SAU/9/92
AFG, Afghanistan SAU/10/92
BAN, Bangladesh SAU/32/92
IND/10/82
BAR, Bahrain IND/60/79
IND/5/89 (1988)
BHU, Bhutan IND/A/93 (Mandya Karnataka)
GRE, Greece GRE/2/2000
IND, India IRN/58/99
TUR/6/2000
IRN, Iran TUR/8/99
ISR, Israel TUR/3/2000
TUR/10/99
LAO, Laos IND/233/95
IND/26/95
LEB, Lebanon IND/40/95
14.3% NEP, Nepal IND/43/95
SAU/39/94
PAK, Pakistan SAU/40/94
SAU, Saudi Arabia PAK/3/98
IND/51/95
TAI, Thailand PAK/2/98
TUR, Turkey BAR/1/85
GRE/1/84
YEM, Yemen ISR/3/89
LEB/3/83
LEB/1/84
N1214/Armenia/USSR/84
N1181/Azerbaijan/USSR/83
ISR/1/84
IND/63/72 MukVS
PAK/1/85
PAK/4/69
PAK/1/71
PAK/1/74
PAK/2/76 (1975)
PAK/1/54
Bangkok/TAI/60

16 14 12 10 8 6 4 2 0
Percentage nucleotide difference (nt 466-636 of VP1)

Fig. 5. Comparison of recent FMD type Asia 1 viruses from Iran, Turkey and Greece.

Greece Turkey (1999-2000)

(July 2000)
Iran (1999)

Pakistan
(1998)
Saudi Arabia India
(1994) (1995)

Fig. 6. The likely recent spread of FMDV-Asia1

 Appendix 4
FMD Situation in TURKEY in 2000

During the last 8 months 3 serotypes (A, O and Asia 1) of FMDV caused totally 90 outbreaks in
Turkey (Table 1). Type O and Asia 1 was responsible for most of these outbreaks. Type A has
not been reported since 4 months.

Table 1: FMD outbreaks in 2000.

MONTH OUTBREAKS SUSCEPTIBLE INFECTED DEATHS
MONTH Type Total Cattle Sheep Cattle Sheep Cattle Sheep
MONTH A O As Nt. Total Cattle Sheep Cattle Sheep Cattle Sheep
January - 4 - 1 5 789 2575 32 45 - 1
February - 6 - - 6 2213 - 23 - - -
March 2 4 4 - 10 9257 - 174 - 15 -
April 2 10 3 - 15 11378 500 175 80 10 80
May - 6 9 - 15 29288 2900 458 220 34 3
June - 7 16 - 23 26053 1168 1819 76 12 1
July - 2 7 - 9 3870 1153 399 1 1 -
August - - 7 - 7 2115 5100 113 - 2 -
TOTAL 4 39 46 1 90 10972 13396 3193 422 74 85
A: Type A, O: Type O, As: Type Asia 1, NT: Not typed.

In 2000, 9 vesicular epithelium collected from the infected animals in the field were sent to
Pirbright IAH for strain identification. The, genetic analysis carried out by WRL, Pirbright
showed that, the A type FMDV circulating in Turkey is still close to A/98 (Table2).

Table 2: FMDV samples tested in Pirbright IAH.

Item IAH Ref. Şap Enst. Ref. Province Serotype
1 TUR 4/2000 437/2000 Erzincan A
2 TUR 1/2000 101/2000 Malatya A
3 TUR 9/2000 568/2000 Sivas Asia 1
4 TUR 5/2000 441/2000 Niğde O
5 TUR 2/2000 235/2000 Balıkesir O
6 TUR 3/2000 308/2000 Kırıkkale Asia 1
7 TUR 7/2000 515/2000 Diyarbakır O
8 TUR 6/2000 511/2000 Sivas Asia 1
9 TUR 8/2000 544/2000 Konya O
.
The vaccine production at the FMD Institute (SAP Institute) for 2000 is given in Table.3. The
production is still going on at the Institute.

Table.3. Vaccine production in 2000.

Vaccine strain Amount produced (cattle doses)
O Manisa 69 3.432.960
A Aydın 98 (homologue Iran 96) 53.600
Asia 1 74 1.391.040
A Mahmatlı 65 + O Manisa 69 840.960
A Aydın 98 + O Manisa 69 + Asia 1 74 216.000
Total 5.934.560

Thrace Region of Turkey is composed of 5 provinces (Edirne, Kırklareli, Tekirdağ, European

parts of Istanbul and Çanakkale) separated from Anatolia with Bosphorus. FMD susceptible
animal population is about 1.155.243 (388.981 large ruminants and 766.262 small ruminants).
2

This year the cattle and sheep were vaccinated in spring campaign with bivalent (A Mahmatlı 65
and O Manisa 69) FMD vaccine. Whereas, in autumn, trivalent (A, O and Asia 1) vaccine which
will be supplied by EU is going to be applied in the region. The programme is vaccination of not
less than 80% of the animal population in Thrace including the Asian parts of Istanbul and
Canakkale. In Anatolia, the cattle were vaccinated with monovalent vaccine (O Manisa) in
spring. The vaccination figures for the first half of 2000 is shown in Table.4.

Table.4. Vaccination figures for the first round of 2000.

Region Animal Population Vaccination
Region Large rum. Small rum. Large rum Small rum.
Thrace 388.981 766.262 368.110 394.233
Anatolia 10.818.019 36.725.738 5.529.548 2.784.644
TOTAL 11.377.917 39.376.960 6.291.891 3.178.877

A serosurvey is going on to examine the antibody levels of the livestock in the field. In this
work, random selection “directed towards the object” was used for nomination of the 31
provinces. 6 villages per province and 10 cattle from each village were randomly selected. The
tests are already being done at the FMD Institute, Ankara. The results will be evaluated by the
15th of September.

Other activities:
A comparative study has just started for the potency control of the FMD vaccine with on going
cattle challenge test, guinea pig PD50 and serology following vaccination of the cattle in the field.
The objective of that work is to establish the correlation between the results of testing in cattle
and in laboratory animals, and eventually obviating the need at least for the routine cattle testing
of vaccines.

There are two international projects related to FMD going on in Turkey:

- The TCP titled “Regional Technical Co-operation Project between I.R. of Iran and
Republic of TURKEY for Emergency Control of FMD due to the type A virus variant in
the Region” was started at the end of 1999. The objectives of this proposal were to
combat the new strain of type A FMDV in Iran and TURKEY and to strengthen the
overall future control of FMD and of other serious communicable diseases of animals in
the Region.
- The project titled “The Control of FMD in Turkey” is supported by EU. Quality control
of the vaccine produced in TURKEY and used for the vaccination campaigns by
European Commission which was firstly discussed during the EU/FMD Executive
Committee Meeting in Antalya, Turkey in May/1998 is also included in this

Reconstruction of the “Ankara Şap Enstitüsü”: The Diagnosis, Control and Research Department
of the Institute are being recreated. It was planned to complete this work not later then the end of
2000.

In the present situation the vaccination programme in Turkey for the autumn campaign 2000 will
be as follows:
- Thrace, including the Anatolian part of Istanbul and Çanakkale: Vaccination of all
ruminants with 1.5 million doses trivalent vaccine (A22 Mahmatlı, O1 Manisa and Asia 1
types) supplied by EU.
- Anatolia: Vaccination of all large ruminants with trivalent vaccine (A Aydın 98, O1
Manisa and Asia 1). In some area in the Black Sea Region where no FMD outbreak was
occurred for years were excluded in this campaign, whereas, in the case of necessity
strategic vaccination will be applied.
 48

Appendix 5

Minimal aerosol infectious dose for the O1 Lausanne strain of foot-and-mouth disease
virus for pigs.

Soren Alexandersen, Ian Brotherhood and Alex I. Donaldson

Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, GU24 ONF,
UK.

Summary
Foot-and-mouth disease (FMD) can be spread by a variety of mechanisms, including,
under certain epidemiological and climatic circumstances, by the wind. Simulation models
have been developed to predict the risk of airborne spread of FMD during outbreaks and have
played an important part in guiding the deployment of manpower resources during
emergencies to support disease control measures. The minimal infectious dose for different
species of livestock by inhalation is an important determinant of airborne spread of the virus,
however, the minimal dose of airborne virus for pigs is not known. The objective of the study
was to obtain that data in order to enhance the capability of models which can predict the
airborne spread of virus. Under experimental conditions, pigs were exposed individually to
different doses of naturally generated aerosols of FMD virus, strain O1 Lausanne, held
separately, and monitored for signs of infection and disease. The data obtained indicated that
pigs are rather resistant to natural aerosol challenge although doses around 300 TCID50 in
certain cases caused subclinical infection. Doses of 100 TCID50 or less consistently failed to
infect pigs by this route. The results are discussed in relation to the mathematical modeling
and epidemiology of the airborne spread of FMD.

INTRODUCTION

FMD is most commonly spread by the movement of infected animals, by contaminated

animal products, or by mechanical means. An additional mechanism is the spread of FMD by
the wind. This occurs infrequently as it requires particular climatic and epidemiological
conditions 1;7.
The determination and expression of the biological determinants of the airborne spread of
FMD such as virus excretion, virus survival and infectious doses in quantitative terms and the
marrying of those with the physical determinants of airborne particle diffusion has resulted in
the development of mathematical models which can predict the risk of airborne spread of
FMD 4-8;11;12;16;19;20. A parameter which has not been quantified, however, is the minimal
infectious dose (MID) of airborne FMD virus for pigs. Estimates have been made using
artificially generated aerosols of virus 23 but these may not be valid as it is now recognized
that the pathogenesis of FMD in animals exposed to artificially generated aerosols is markedly
different 5;6. Furthermore, the mouse assay system used by Terpstra is less sensitive for FMD
virus than is the bovine thyroid cell culture system 21 and so there may have been an under-
estimation of the doses.
The objective of the present investigation was to establish the infectious dose of airborne
FMD virus delivered to experimental pigs as a natural aerosol and to use the values obtained
 49

as input data for the atmospheric prediction model Rimpuff to improve and refine its
capability 16.

MATERIALS AND METHODS

Animals
Five separate experiments were performed. Two or three inoculated “donor” pigs were
used in each experiment as a source of natural aerosols of FMD virus. Five or ten “recipient”
pigs, i.e. animals exposed to airborne FMD virus, were used in each experiment.
The donor pigs were inoculated intradermally or intradermally and subcutaneously in the
heel bulbs 2 with approximately 105-7 BTY TCID50 of virus..
Clinical examination of the donor pigs for signs of FMD was carried out at least once and
sometimes twice per day. Rectal temperatures were recorded daily. For experiments 1 and 5
the donor pigs were selected at 3 days post inoculation (dpi), in experiments 2 and 3 they were
selected at 2 dpi and for experiment 4 a combination was used of two donor pigs at 3 dpi and
one donor pig at 2 dpi. Donor pigs were killed soon after they had been removed from the
aerosol production chamber.
Recipient pigs were housed singly in cubicles constructed within biosecure isolation
rooms.
Each recipient pig was examined daily for signs of FMD over a three week period. Any
animal which developed clinical signs of FMD was killed immediately, otherwise they were
killed at the end of the experiments.
Samples of epithelial tissue were collected from any animal which developed lesions and
tested by ELISA 10;13;18 to confirm the presence of FMD antigen.

Virus
The O1 Lausanne Sw/65 strain of FMD virus was used. Two different stock viruses were
used, one which had been passed in cattle and IB-RS-2 cells (for Experiments 1 and 5), the
other passed 3 times in pigs (for Experiments 2-4).

Exposure of pigs to natural aerosols of FMD virus

The procedures used were similar to those described previously for recipient cattle and
sheep experiments and using donor pigs to produce a natural aerosol containing FMDV 6;11.
Some aspects were changed to adapt the method for use with pigs as recipients and to take
advantage of recent technological advances.
At 3 dpi (Experiments 1, 5 and partly 4) or 2 dpi (Experiments 2, 3 and partly 4), either
two (Experiment 1) or three (Experiments 2 to 5) donor pigs were selected and placed in a
610 litre aerosol production chamber located in the corridor immediately outside the isolation
room. The chamber was closed, disinfected and connected to an “exposure tunnel” of wide-
bore ducting. The end of the tunnel was secured just beneath the filter housing of an extractor
air vent.
Before exposure each recipient pig was placed on its back on a cradle, a blood sample
taken and the animal was then sedated by injection with Propofol (Rapinovet 10 mg/ml,
Schering-Plough Animal Health, Welwyn Garden City, UK) into the anterior vena cava at a
minimal dose. A mask connected to the exposure tunnel was fitted and the pig was allowed to
breathe air drawn from the tunnel. The air flow in the tunnel had been adjusted previously to
 50

between 0.25 and 0.5 m/sec. Recipient pigs were exposed individually for periods ranging
from 2 to 10 min.
The humidity in the tunnel was raised above ambient in Experiments 3 to 5 by introducing
a fine mist of water vapor into the chamber. The relative humidity was monitored by an
electronic humidity meter.
Three Experiments (1 to 3), using a series of 10 pigs in each, and two Experiments (4 and
5) using a series of 5 pigs in each were performed giving a total of 40 recipient pigs.
Blood samples were collected from the pigs in Experiments 1 at 14 and 22 dpe, and from
Experiments 2 to 5 at 7, 10, 14 and 21 dpe. Nasal swabs were collected from the pigs in
Experiment 1 at 14 and 22 dpe and from the pigs in Experiments 2 to 5 at 7 dpe.

Air sampling methods

Air samples were collected from the corridor after the donor pigs had been placed in the
aerosol production chamber and after the last recipient pig had been exposed to airborne virus.
Sampling was done with an all-glass cyclone sampler 11.
After the exposure of each recipient pig, an air sample was collected from the exposure
tunnel using a Porton all-glass impinger or a 3-stage liquid impinger.

Measurement and recording of respiratory function

The respiratory function (rate and volume) of each recipient pig was measured with an
ultrasonic phase-shift respiratory flowmeter (BRDL Flowmetrics, Birmingsham) 3;24 located
between the tunnel and the exposure mask. The analog signal from the instrument was
converted to digital and recorded on a laptop computer. The signals were recorded every 80
millisec. The instrument and ancillary equipment were tested beforehand to ensure the
accuracy and linearity of the method. The millivolt readings recorded were converted directly
to air flow and registered in millilitres/sec. The volume of respirated air was calculated as the
average of calculated inspiration and expiration in order to minimize any fluctuations caused
by leaks or uneven flow. The values for experiment 1 are shown in Table 1. The dose
inhaled by each pig was determined by multiplying the calculated volume of respiration
during exposure by the concentration of virus per litre of air.

Assay for virus

The infectivity in the collection fluid from air samplers, in blood samples and nasal swabs
were assayed by inoculation of monolayer cultures of primary bovine thyroid (BTY) cells in
roller tubes 21. The specificity of the cytopathic effect observed in cell cultures was confirmed
by antigen ELISA 10;13;18.

Assay for antibodies

Serum samples were tested by an enzyme-linked immunosorbent assay (ELISA) for the
presence of antibodies to FMD virus 9;14.

RT-PCR

Blood and nasal swab samples were tested for the presence of FMD viral RNA by the reverse
transcription polymerase chain reaction (RT-PCR) method described in 17.
 51

RESULTS
Airborne virus recovery, respiratory function and exposure doses
The amount of virus in air samples, the concentration of virus in the air, the volumes of air
inhaled by the pigs and the total dose to which each pig in experiment 1 was exposed are
shown in Table 1. Similar results for experiments 2-5 are described in the text below. The
average dose a pig received in each experiment was calculated by: (I) excluding all the pigs
which did not receive a measurable dose of virus (negative air sample); (II) adding the
measurable amounts of virus received by the other pigs in the experiment; and (III) dividing
that sum by the number of those pigs. Therefore, the number of pigs in each experiment on
which calculations were based consisted only of the number of pigs which received detectable
amounts of virus.

Air sampling of corridor air

Samples of air from the corridor collected before the exposure of recipient pigs i.e. just
after the donor pigs were placed in the aerosol production chamber were negative for virus in
Experiments 1 and 4 but positive in Experiments 2, 3 and 5, however, the quantity of virus
was very low compared to that to which recipient pigs were exposed. All of the air samples
collected from the corridor after the exposure of recipient pigs were negative.

Clinical signs, viraemia and seroconversion

The only recipient pig to develop clinical signs of FMD was No. UA9 in Experiment 1. It
was killed the next day when the clinical signs were severe. Pig UA6 showed decreased
activity and reduced appetite at 3 to 4 dpe. However, at 5 dpe its appearance and appetite
were normal. Examination of pig UA9 at post mortem (5 dpe) revealed vesicular lesions on
all four feet, the gingival mucosa, the tongue and snout. Serum and epithelial tissue samples
collected at 5 dpe contained 102.2 TCID50/ml and 105 TCID50/g, respectively. A serum
sample from pig UA9 tested for antibody to FMD virus had a ELISA titre of 1: 64 at 5 dpe.
None of the other 8 recipient pigs in Experiment 1 or any of those in Experiments 2 to 5
developed clinical signs of disease. Blood and nasal swabs taken at 14 dpe (Experiment 1)
and at 7 dpe (Experiments 2 to 5) were negative for FMD virus by cell culture and by RT-
PCR. Nasal swabs taken at 22 dpe (Experiment 1) were negative for FMD virus by cell
culture. However, serum samples taken at 14 and 22 dpe (Experiment 1) showed that 6 pigs,
including pig UA6, of the 9 remaining recipient pigs had antibodies to FMD virus (Table 2).
Interestingly, the antibody titre for pig UA6 at 22 dpe had increased, while the other pigs had
the same or a lower titre than at 14 dpe, indicating that they had experienced an infection of
very short duration.
Antibodies were not detected in any of the recipient pigs in Experiments 2 to 4. In
Experiment 5, a single pig had an ELISA titre of 1:22 at 14 dpe and 1:45 on day 21
suggesting that this animal had a transient infection.

The results can be summarized as follows:

Experiment 1: 9 pigs received an average dose of 293 TCID50. One pig developed clinical
FMD, six were subclinically infected and two remained normal.
Experiment 2: 7 pigs receiving an average dose of 75 TCID50. None were infected.
Experiment 3: 3 pigs receiving an average dose of 53 TCID50. None were infected.
Experiment 4: 5 pigs receiving an average dose of 250 TCID50. None were infected.
Experiment 5: 5 pigs receiving an average dose of 330 TCID50. One pig was subclinically
infected.
 52

The data in Experiments 1 and 5 and Experiments 2 to 4 were analyzed as groups

together. Thus of the 14 pigs in Experiments 1 and 5 which received an average dose of 306
TCID50 from donor pigs infected with stock virus 1, one pig became clinically infected and 7
were subclinically infected. Thus the MID50 for subclinical infection is around 260 TCID50
per pig calculated after Kärber (as described in 15). We are currently testing the two ELISA
antibody borderline samples (1:45) in neutralization tests. If these animals are excluded from
the positive animals, then the calculated MID50 (subclinical) will be increased to 360
TCID50.
The average dose of 306 TCID50 also caused clinical disease in one animal, indicating a
50% disease dose of around 820 TCID50. However, since only one animal was included in
this estimate the standard error of the estimate may be large.
In Experiments 2 to 4 a total of 15 pigs received doses from 53 TCID50 to 75 TCID50 (10
pigs) and 250 TCID50 (5 pigs) from donor pigs infected with stock 2 virus, however, none of
them became infected. It should be noted, that the relatively high average dose of 250
TCID50 in Experiment 4 was mainly due to a single sample collected after the 5 recipient pigs
in the experiment had been exposed to virus. Therefore, the true average exposure dose in
Experiment 4 may more likely be around 100 TCID50.

DISCUSSION

The objective of the studies was to estimate the MID and MID50 of FMDV which can
infect pigs by natural aerosol. While only one pig, out of 40 pigs exposed, developed severe
clinical disease, a total of 7 were subclinically infected i.e., had evidence of active infection
with FMDV. Including the sub-clinically infected pigs in the calculations, an MID50 around
300 TCID50 was obtained. In fact, virus plumes causing virus exposure doses down to
around 100 TCID50 may constitute a significant risk for aerosol transmission in pigs.
However, doses as low as 10-15 TCID50, or less, known to consistently cause infection in
ruminants are unlikely to be sufficient for aerosol transmission to pigs under natural
conditions.
From a theoretical perspective 15;22, one third of a MID50 (i.e. around 85-100 TCID50 for
the aerosol route) could infect a small number of pigs which could then amplify the virus and
cause an outbreak. Nevertheless, none of the 15 pigs exposed to 53 to around 100 (250?)
TCID50s became infected, perhaps suggesting a threshold level below which infection does
not occur, or more likely, are below the detection limits of the diagnostic assays used.
However, since we cannot exclude the possibility of relatively low doses (around 100
TCID50s) infecting pigs, albeit probably at an incidence lower than 10%, this could have the
potential to create an outbreak.
It is not possible to compare the results in the present study with those reported previously
by Terpstra23 because he used a mouse assay system which is less sensitive than BTY cells
and the pigs he exposed were killed before they had the opportunity to develop an antibody
response. However, our finding that relative high doses of aerosol virus are needed to infect
pigs fits well with certain experimental studies and with observations from the field.
The new MID50 values for natural aerosol infection of pigs, sheep and cattle (Fig. 1) will
be included in the Rimpuff model in order to improve its capability to forecast the airborne
spread of FMDV.

ACKNOWLEDGMENTS
 53

We thank Geoffrey Hutchings, Teli Rendle, Nigel Ferris, Scott Reid and Morag Forsyth for
their excellent technical assistance. Natasha Smith and Martin Broomfield are thanked for
their assistance with the handling and management of experimental animals. The research
was supported in part by the UK Ministry of Agriculture, Fisheries and Food and the Danish
Ministry of Food, Agriculture, and Fisheries.

REFERENCES

1. Anonymous. Report of the Committee of Inquiry on Foot-and-Mouth Disease (1968). Part 1,

56-57. 1969. London, Ministry of Agriculture, Fisheries & Food, Her Majesty's Stationery
Office.
Ref Type: Report

2. Burrows, R. 1966. The infectivity assay of foot-and-mouth disease virus in pigs. J Hyg
(Lond) 64:419-29.

3. Butler, P. J., A. J. Woakes, K. Smale, C. A. Roberts, C. J. Hillidge, D. H. Snow, and D. J.

Marlin. 1993. Respiratory and cardiovascular adjustments during exercise of increasing
intensity and during recovery in thoroughbred racehorses. J. Exp. Biol. 179:159-180.

4. Donaldson, A. I. 1972. The influence of relative humidity on the aerosol stability of different
strains of foot-and-mouth disease virus suspended in saliva. J Gen Virol 15:25-33.

5. Donaldson, A. I. and N. P. Ferris. 1980. Sites of release of airborne foot-and-mouth disease

virus from infected pigs. Res. Vet. Sci. 29:315-319.

6. Donaldson, A. I., C. F. Gibson, R. Oliver, C. Hamblin, and R. P. Kitching. 1987.

Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with O1 and SAT
2 strains. Res Vet Sci 43:339-46.

7. Donaldson, A. I., J. Gloster, L. D. Harvey, and D. H. Deans. 1982. Use of prediction

models to forecast and analyse airborne spread during the foot-and-mouth disease outbreaks
in Brittany, Jersey and the Isle of Wight in 1981. Vet. Rec. 110:53-57.

8. Donaldson, A. I., K. A. Herniman, J. Parker, and R. F. Sellers. 1970. Further

investigations on the airborne excretion of foot-and-mouth disease virus. J Hyg (Lond)
68:557-64.

9. Ferris, N. P. 1987. Development and use of Elisa in the control of foot-and-mouth disease.
IAEA-Proceedings 348:65-77.

10. Ferris, N. P. and M. Dawson. 1988. Routine application of enzyme-linked immunosorbent

assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine
vesicular diseases. Vet. Microbiol. 16:201-209.

11. Gibson, C. F. and A. I. Donaldson . 1986. Exposure of sheep to natural aerosols of foot-and-
mouth disease virus. Res Vet Sci 41:45-9.

12. Gloster, J., R. F. Sellers, and A. I. Donaldson. 1982. Long distance transport of foot-and-
mouth disease virus over the sea. Vet Rec 110:47-52.
 54

13. Hamblin, C., R. M. Armstrong, and R. S. Hedger. 1984. A rapid enzyme-linked

immunosorbent assay for the detection of foot-and- mouth disease virus in epithelial tissues.
Vet. Microbiol. 9:435-443.

14. Hamblin, C., I. T. Barnett, and R. S. Hedger. 1986. A new enzyme-linked immunosorbent
assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I.
Development and method of ELISA. J. Immunol. Methods 93:115-121.

15. Lennette, E. H. 1964. Diagnostic procedures for viral and rickettsial diseases, p. 48-51.
American Public Health Association, Inc., Broadway, New York.

16. Mackay, D. K. J., R. Lattuda, P. J. Verrier, R. S. Morris, and A. I. Donaldson. 1998.

Epiman (EU) A computerized decision support system for use within the European Union.
Appendix 24170-175.

17. Reid, S. M., M. A. Forsyth, G. H. Hutchings, and N. P. Ferris. 1998. Comparison of

reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and
virus isolation for the routine diagnosis of foot-and-mouth disease. J Virol Methods 70:213-7.

18. Roeder, P. L. and P. M. Blanc Smith. 1987. Detection and typing of foot-and-mouth disease
virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for
primary diagnosis. Res. Vet. Sci. 43:225-232.

19. Sellers, R. F. 1971. Quantitative aspects of the spread of foot and mouth disease. Vet. Bull.
41:431-439.

20. Sellers, R. F. and J. Parker. 1969. Airborne excretion of foot-and-mouth disease virus. J
Hyg (Lond) 67:671-7.

21. Snowdon, W. A. 1966. Growth of foot-and mouth disease virus in monolayer cultures of calf
thyroid cells. Nature 210:1079-1080.

22. Sutmoller, P. and D. J. Vose. 1997. Contamination of animal products: the minimum
pathogen dose required to initiate infection. Rev Sci Tech 16:30-2.

23. Terpstra, C. 1972. Pathogenesis of foot-and-mouth disease in experimentally infected pigs.

Bull Off Int Epizoot 77:859-74.

24. Young, I. S., R. Alexander, A. J. Woakes, P. J. Butler, and L. Anderson. 1992. The
synchronization of ventilation and locomotion in horses (Equus caballus). J. Exp. Biol.
166:19-31.
 55

TABLE 1. Doses of virus to which the pigs in Experiment 1 were exposed.

Group Pig No. Titre Flow rate Titre in air Air inspired Dose inspired
(TCID50/ml) litre/min TCID50/litre litre total total

UA 6 0.8 11.9 2.65 158 420 TCID50

UA 7 1.0 13.6 3.67 103 378
A UA 8 0.8 12.1 2.60 120 312
UA 9 0.6 11.0 1.80 120 216
UA 10 0.6 12.1 1.65 126 208

UA 11 0.8 11.9 2.65 150 400

UA 12 0.4 13.6 0.92 34* 30
B UA 13 0.4 11.0 1.14 114 130
UA 14 - 12.1 - 85 ND
UA 15 1.0 12.0 4.16 129 537

The average dose of detectable virus received by 9 pigs was 293 TCID50. The range was from non-detectable(1 pig)to 537. All the pigs were
exposed for 10 min, series A at an air-flow of 0.5m/sec and series B at 0.25m/sec.
ND: Not determined.
* Poor connection.
 56

TABLE 2.
ANTIBODY TESTING BY LIQUID PHASE BLOCKING ELISA

Experiment I
Antibody data on serum from plain tubes. Blood samples taken on PED 14 and on PED18.
Neg means negative, i.e., <16. Normally a titre of 45 is cut off, however on these samples 32
can be considered weakly positive.

Antibody titre
Prebleed PED14 PED22

Pig
UA6 neg 90 181

UA7 neg 181 90

UA8 neg neg neg

UA9 neg (Killed on PED 5 Titre then 64)

UA10 neg neg neg

UA11 neg 128 128

UA12 neg 181 32

UA13 neg 45 neg

UA14 neg neg neg

UA15 neg 90 neg

 57

Pigs

1.00 260 half dose

log relation
0.90

0.80 800 half dose

log relation
0.70
Cattle half dose at 5
0.60

0.50
Sheep half dose at 7
0.40

0.30 Pig oral

0.20

0.10 Cattle oral

0.00
0.1 1 10 100 1000 10000 10000 10000 1e+07

FIGURE LEGENDS

Fig. 1. Graph showing probability of infection on the Y axis and the specific dose needed on
the X axis (log scale). The 50% infectious dose for aerosol infection of cattle, sheep and pigs
(at slightly different estimates) as well as the doses required to infect cattle and pigs by the
oral route are depicted. The pig aerosol dose estimates are from the current study, the cattle
and sheep aerosol values are re-calculated from the original data and the dose needed for oral
infection of cattle and pigs are from the literature.
 58
Appendix 6

Antigenic and genetic characterisation of foot-and-mouth disease type O

viruses from the Far East

A.R. Samuel, L.S. Turner and N.J. Knowles

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright

Woking, Surrey GU24 0NF, United Kingdom

Introduction
Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease of cloven-hoofed
animals. It has occurred in most parts of the world and is endemic in sub-Saharan Africa, southern
Asia, the Middle East and parts of South America. North and Central America, the Caribbean,
Australia and New Zealand are free from the disease. Western Europe and North Africa although
normally free from disease have sporadic outbreaks.
FMD is endemic in many parts of the Far East, including Myanmar, Thailand, Laos, Cambodia and
Vietnam. Malaysia was free until recently when in 1990 outbreaks of Asia 1 occurred near to the
border with Thailand. During the period 1992-1998 outbreaks have been caused by types O, A and
Asia 1.
In March 1997 Taiwan province of China reported three outbreaks of FMD to the Office
International des Epizooties. Subsequently, the OIE/FAO World Reference Laboratory for FMD
(WRLFMD) at Pirbright, confirmed that FMDV serotype O had been isolated from samples that had
been submitted. Molecular analysis of the samples identified them as belonging to a group of viruses
which were distinct to Hong Kong and the Phillippines and which were almost certainly related to
strains presumed to be present in China. Despite zoo-sanitary measures and the slaughter of diseased
and in-contact animals the disease rapidly spread to most parts of the island. This was the first reported
case of FMDV in Taiwan province of China since the 1930's.
Relatively little is known about the antigenic characteristics of viruses of serotype O from the Far
East. This paper examines the relationship between recent isolates of type O FMDV from the Far East
using the results of deduced amino acid sequences from nucleotide sequence data, liquid phase
blocking enzyme linked immunosorbent assays (LPB-ELISA) and antigenic profiling ELISA data
using panels of MAbs elicited against distinct genetic FMD viruses of serotype O
(O1/Kaufbeuren/FRG/66 and O1/Manisa/Turkey/69).

Materials and methods

Viruses
The origin of the FMD type O viruses studied is listed in Table 1.

Strain differentiation liquid phase blocking ELISA (LPB-ELISA)

The LPBE-ELISA was performed according to the WRL laboratory protocol and is as described in
Kitching et al. (1988). Briefly the assay measures the residual antigen remaining after overnight
reaction between dilutions of reference antiserum and a pre-titrated mass of the homologous antigen
and the test antigen. The serum titre obtained has been shown to correlate with that obtained using the
virus neutralization test (Hamblin et al., 1987). Relationship values were calculated as described by
Brooksby (1968) and interpreted according to the criteria described in Samuel et al. (1990).

Antigenic profiling ELISA using MAbs

Antigenic profiling with MAbs and analysis of the data was carried out according to the method
 59
described by Samuel et al. (1991). The MAbs used are a panel elicited against O1 European FMD
viruses and which define the five neutralizing sites (see Kitson et al., 1990; Crowther et al., 1993); a
panel elicited against O1/Manisa/Turkey/69 (see Samuel et al., 1998) and three bovine monoclonal
antibodies (Barnett et al., 1998)
For evaluation of these results, MAb binding has been divided into three percentage ranges. Values
of 65- >100% were considered as equal to that of the homologous virus; 30 - 65% was considered as
having a reduced affinity and less than 30% were considered to give no reaction.

Oligonucleotide primers
Oligonucleotide primers with a Cy5 amidite fluorescent dye for use with the ALFexpressJ
automated sequencer were purchased from Pharmacia Biotech. The sequences of primers used in this
study are detailed in Table 2.

RT-PCR
Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the primer set
L463F and NK61 essentially as described by Knowles and Samuel (1994).

Cycle sequencing
A fmolTM DNA sequencing kit (Promega, UK) which utilises the method described by Murray
(1989) was used according to the manufacturers protocol with the following amendments.
Approximately eighty fmoles of cDNA template was used in the reactions and 1.5 pmoles of one of the
Cy5 amidite labelled primers.
The reactions were heated to 95°C for 2 minutes and subjected to 30 cycles of the following
programme on a thermal heating block (Hybaid Omnigene, Hybaid UK): 95°C for 30 seconds, 42°C
for 30 seconds and 70°C for 1 minute.The reactions were terminated by adding 4μl of Cy5 sequencing
stop solution (Pharmacia Biotech. Sweden) and cooled to 4°C. The reactions were heated to 95°C for 3
minutes prior to loading on an ALFexpressJ DNA Sequencer (Pharmacia Biotech, Sweden). The
software AM V3.01 (Pharmacia Biotech., Sweden) was used to process the data which was then
exported as an ASCII text file and aligned manually and analysed using the EpiSeq v2.0 suite of
computer programs (NJ Knowles, unpublished data).

Results
Nucleotide sequence analysis
Phylogenetic analysis of the comparison of 180 nt at the 3' end of the VP1 gene is shown in Fig. 1.
From the dendrogram it can be seen that all the isolates from Taiwan belong to a distinct, larger group
of viruses from Hong Kong and more recent isolates from the Phillippines. The isolate O/VIT/3/97 also
belongs to this group of viruses. The other strains from Vietnam studied do not form part of this group
but are more closely related to strains from other regions of the Far East which included most of the
isolates from Cambodia, Myanmar and Vietnam. These groupings were confirmed by comparison of
the complete P1 capsid-coding region (Fig. 2).
The capsid coding region of selected isolates (as detailed in Table 1) is shown as a lineup of the
deduced amino acid sequence in Fig. 3.

LPB-ELISA
The results obtained from the LPB-ELISA (see Table 3) show that the isolates from Vietnam fall
into two groups. Firstly, the isolate O/VIT/3/97 more closely resembles the O1/Manisa/TUR/69
reference strain (r=>1.0). The isolates O/VIT/4/97 and O/VIT/7/97 have r values of 0.5 with
O1/Manisa/TUR/69 and more closely resemble other Far Eastern reference strains (O/Thailand/89, and
O/Phillippines/95). The isolate O/VIT/2/97 has an r value of <0.1 with O1/Manisa/TUR/69 and low
reactivity with Phi/95 and O/HKN/6/83 reference strains.
 60
The isolates from Taiwan all have similar high reactivity (r is equal to, or greater than 1.0) with the
O1/Manisa/TUR/69 reference sera and r values (where tested) of 0.2 - 0.4 with the O1/BFS 1860
reference sera. The isolates also had high reactivity with the Phillippines/95 reference sera (r =1.0).
The Cambodian isolates however, gave low r values with the O1/Manisa/TUR/69 (<0.2) reference
sera and values between 0.5 and 1.0 with the more european like O1 reference strains.
Antigenic profiling with monoclonal antibodies
Analysis with the panels of MAbs (O1/Manisa/TUR/69 and European O1 panel) would indicate that
the Cambodian isoaltes are similar to each other, the Taiwan and O/VIT/3/97 isolates are similar to
each other (with minor differences). The O/VIT/2/97 and O/VIT/7/97 isolates have some different
reactivities to each other with individual Mabs (Fig. 4).
All the Cambodian isolates sequenced would appear to be the same as each other in the five
recognised antigenic sites. Serological analysis by LPB-ELISA and MAb analysis with the European
O1 and theO1/Manisa panel would also suggest that antigenically there is little measurable antigenic
differences between them.
The capsid sequence data for the Vietnam isolates show that they fall into main two groups (Figs. 1
and 2). The isolates from cattle O/VIT/2/97 and O/VIT/7/97 are more similar to each other but
different to O/VIT/3/97 which appears to be closer to O/TAW/9/97, both of which were isolated from
pigs. The MAb analysis of O/VIT/3/97 and O/TAW/9/97 with the two MAb panels does not show
large changes in the reactivity of individual MAbs, apart from the anti O1/Lausanne/Swiss/65 MAb
D9. This MAb may be influenced by the substitution at VP1138 GD (glycineaspartic acid), although
in the epitope mapping studies carried out by Kitson et al. (1990) this residue has not previously been
implicated in the binding of MAb D9. The O1/Manisa/Turkey/69 MAb panel shows very small
differences between O/VIT/3/97 and O/TAW/9/97(a slight decrease in reactivity of MAbs 85 and 113
with O/TAW/9/97). The LPB-ELISA results with bovine serum show that >r= = 1.0 for both isolates.
This suggests that the antigenic changes measurable by the MAb analysis do not result in gross
antigenic differences. One other feature worthy of mention is the proline residue at VP274 in both the
O/TAW/9/97 isolate and O/VIT/3/97. Other isolates from the distinct Hong Kong genetic lineage
(topotype) that have been sequenced in this region have been serine. However, amongst other type O
isolates, from Asia and the Middle East, this residue is more commonly proline (Samuel, 1997).
Although O/VIT/2/97 has an alanine (A) at VP271 instead of the threonine (T) of O/VIT/7/97 this
would not really account account for the loss of reactivity of O/VIT/7/97 with MAb SA113. Although
MAb SA113 is directed against the BC loop of VP2 the other isolates still reacting with this MAb also
have the threonine at VP271. Because this is a conformational epitope, other as yet unidentified residues
are obviously influencing the binding of MAb SA113. The differences in amino acids (ND) at residue
VP1197 could account for the lack of reactivity of O/VIT/7/97 with MAb SA177 which reacts with an
epitope at the carboxy terminus of VP1. These changes could account for the differences in the
reactivity of these two isolates with the reference sera in the LPB-ELISA. Certainly changes which
affect site 2 directed MAbs (BC loop VP2) have previously been reported to affect the results obtained
with the LPB-ELISA (Samuel, 1997).
No antigenic differences are evident between the Cambodian isolates with the MAb panels or with
the LPBE ( r = 0.2 with O1/Manisa antiserum). O1/Manisa MAb SA120 reacts with O/CAM/3/98 but
not with O/CAM/1/98 or O/CAM/9/98. The most interesting aspect of these results is the fact that
although the LPB-ELISA results indicate poor reactivity with O1/Manisa serum the O1/Manisa MAbs
react more strongly with these isolates than with the other isolates under test which have high reactivity
with O1/Manisa in the LPB-ELISA. The reasons for this are unclear, but it must be assumed that for
these isolates the O1/Manisa MAbs are reacting with epitopes which are more conserved than the ones
that the antibodies which make up the bovine polyclonal response are elicited against.
These results show that not only is it possible to determine the epidemiological relationships between
strains by molecular epidemiology and the gross antigenic differences between isolates by a technique
such as the LPB-ELISA but also how amino acid substitutions may affect the binding of well
 61
characterised MAbs. Careful study of data such as this should allow a better understanding of the
molecular basis of antigenic variation. However, it is important to consider that although antigenic
differences can be measured by MAbs, polyclonal based serological tests still have an important role to
play. This is because they are a measure of overall antibody response and do not rely solely on
antibodies directed against the neutralizing antigenic sites; which may not be as important as an
indication of cross protective ability as has previously been thought (Dunn et al., 1997).

Acknowledgments
We would also like to thank various people for providing reagents that have been used for this study.
Robin Butcher (formerly of IAH, Pirbright) for heterohybridomas; Dr Emiliana Brocchi (Brescia,
Italy), Dr Barry Baxt (Plum Island, USA), Dr Sandra Farias (Porto Alegre, Brazil) and Sinan Aktas
(IAH, Pirbright) for monoclonal antibodies; and Nigel Ferris (IAH, Pirbright) for rabbit and guinea pig
sera.

References

Brooksby, J.B. (1968). Variants and immunity: definitions for serological investigations. Proceedings of the
International Symposium on Foot-and-Mouth Disease: Variants and Immunity, Lyon, 1967. Symposium Series
in Immunobiological Standardisation 8: 1-10.
Barnett, P.V., Samuel, A.R., Pullen, L., Ansell, D., Butcher, R.N. and Parkhouse, R.M.E. (1998).
Monoclonal antibodies, against O1 serotype foot-and-mouth disease virus, from a natural bovine host,
recognize similar antigenic features to those defined by the mouse. J. gen. Virol. 79: 1687B1697.
Crowther, J.R., Farias, S., Carpenter, W.C. and Samuel, A.R. (1993). Identification of a fifth neutralizable
site on type O foot-and-mouth disease virus following characterization of single and quintuple monoclonal
antibody escape mutants. J. gen. Virol. 74: 1547-1553.
Dunn, C. and Donaldson, A.I. (1997). Natural adaptation to pigs of a Taiwanese isolate of foot-and-mouth
disease virus. Vet. Rec. 141: 174-175.
Hamblin, C., Barnett, I.T.R and Hedger,R.S. (1987). A new enzyme linked immunosorbent assay (ELISA)
for the detection of antibodies against FMDV. I.Development and method of ELISA. J. Immunol. Meth. 93:
115-121.
Kitching, R.P., Rendle, R. and Ferris, N.P. (1988). Rapid correlation between field isolates and vaccine
strains of foot-and-mouth disease virus. Vaccine 6: 403-408.
Kitson, J.D.A., McCahon, D. and Belsham, G.J. (1990). Sequence analysis of monoclonal antibody resistant
mutants of type O foot-and-mouth disease virus: evidence for the involvement of the three surface exposed
capsid proteins in four antigenic sites. Virology 179: 26-34.
Knowles, N.J. and Samuel, A.R. (1994). Polymerase chain reaction amplification and cycle sequencing of the
1D (VP1) gene of foot-and-mouth disease viruses. Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm.
Cont. FMD, Vienna, Austria. Rome: FAO.
Murray, V. (1989). Improved double-stranded DNA sequencing using the linear polymerase chain reaction.
Nucleic Acids Res. 17: 8889.
Samuel, A.R. (1997). Genetic and Antigenic studies on foot-and-mouth disease virus type O. PhD thesis. Univ.
of Hertfordshire, UK.
Samuel, A.R., Ouldridge, E.J., Arrowsmith, A.E.M., Kitching, R.P., and Knowles, N.J. (1990). Antigenic
analysis of serotype O foot-and-mouth disease virus isolates from the Middle East, 1981-1988. Vaccine 8:
390-396.
Samuel, A.R., Knowles, N.J., Samuel G.D. and Crowther, J.R.. (1991). Evaluation of a trapping ELISA for
the differentiation of foot-and-mouth disease virus strains using monoclonal antibodies. Biologicals. 19:
299-310.
Samuel, A.R., Aktas, S. and Knowles, N.J. (1998). Identification of antigenic epitopes on
O1/Manisa/Turkey/69 using monoclonal antibodies. Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm.
Cont. FMD, Pirbright, UK, 14-18 Sept., 1998.
Xie, Q.C., McCahon, D., Crowther, J.R., Belsham, G.J. and McCullough, K.C. (1987). Neutralisation of
foot-and-mouth disease virus can be mediated through any of at least three separate antigenic sites. J. gen.
Virol., 68: 1637-1647.
 62
Table 1. Origin of FMD type O viruses examined.
WRLFMD Date
ref. No. Geographic origin collected Species
O/CAM/1/98 Khley Tramey, Banset, Kg Speu,Cambodia 05/01/98 porcine
O/CAM/3/98 Kg Speu, Cambodia 05/01/98 porcine
O/CAM/6/98 Kg Speu, Cambodia 08/01/98 porcine
O/CAM/9/98 Sakorak, Poehamrarm, Kg Speu, Cambodia 08/01/98 bovine
O/VIT/2/97 Vietnam not known bovine
O/VIT/3/97 Vietnam not known porcine
O/VIT/4/97 Vietnam not known bovine
O/VIT/7/97 Har village, Yrongpa district, Gialai province, Vietnam 31/08/97 bovine
O/TAW/3/97 Ghanghwa Prefecture, Taiwan 03/97 porcine
O/TAW/9/97 Taipei Prefecture, Taiwan 03/97 porcine
O/TAW/10/97 Taipei Prefecture, Taiwan 03/97 porcine
O/TAW/12/97 Taipei Prefecture, Taiwan 03/97 porcine
O/TAW/112/97 Sinchu Prefecture, Taiwan 07/12/97 porcine
O/TAW/114/97 Taichung City, Taiwan 08/12/97 porcine

Table 2. Oligonucleotide primers used for RT-PCR and sequencing.

Primer Sense Gene Primer Seq. (5Ν-3Ν)
NK61 negative 2B GACATGTCCTCCTGCATCTG
L-463F positive L ACCTCCRACGGGTGGTACGC
NK72 negative 2A GAAGGGCCCAGGGTTGGACTC
ARS3 negative VP3 CATGTAACGCGCCTTCGCGTC
ARS4 positive VP3 ACCAACCTCCTTGATGTGGCT
ROD5 positive VP2 TTTTGAGGATGTGAGCGGACC
1D-628F positive VP1 GTTGGGTTGGTGGTGTTGT
1D-564R negative VP1 CARATAACACACACGGGAAAGC
1D-386R negative VP1 GTGTTTGACATGTGCTTTG
1C-329R negative VP3 TTGAACACTTTCCCGTA

Table 3. Relationship (‘r’) values obtained by liquid phase blocking ELISA

with Far East FMDV isolates and reference/vaccine polyclonal sera.
Viruses Antisera
Manisa BFS Campos IND/53/79 Dalton PHI/95 HKN/6/83 TAI/89
1860
O/CAM/1/98 <0.2 0.5 0.5 1.0 0.5 - - -
O/CAM/8/98 <0.2 1.0 0.7 1.0 0.4 - - -
O/CAM/6/98 <0.2 0.5 0.5 0.5 0.7 - - -
O/CAM/9/98 <0.2 0.7 0.7 0.4 0.5 - - -
O/VIT/2/97 <0.1 - - - - 0.2 0.2 0.5
O/VIT/3/97 >1.0 - - - - 0.8 0.6 >1.0
O/VIT/4/97 0.5 - - - - 0.6 0.5 >1.0
O/VIT/7/97 0.5 - - - - 0.6 0.4 1.0
O/TAW/3/97 >1.0 0.3 - 0.5 - - 0.5 -
O/TAW/9/97 1.0 0.2/0.4 - <0.2/1.0 - - 0.3/0.8 -
O/TAW/10/97 >1.0 - - - - - - -
O/TAW/12/97 1.0 <0.2 - 0.5 - - 0.4 -
O/TAW/112/97 >1.0 - - - - 1.0 - -
O/TAW/114/97 >1.0 - - - - 1.0 - -
63
Table 3. Relationship (‘r’) values obtained by liquid phase blocking ELISA
with Far East FMDV isolates and reference/vaccine polyclonal sera.
Viruses Antisera
Manisa BFS Campos IND/53/79 Dalton PHI/9 HKN/6/83 TAI/8
1860 5 9
O/CAM/1/98 <0.2 0.5 0.5 1.0 0.5 - - -
O/CAM/8/98 <0.2 1.0 0.7 1.0 0.4 - - -
O/CAM/6/98 <0.2 0.5 0.5 0.5 0.7 - - -
O/CAM/9/98 <0.2 0.7 0.7 0.4 0.5 - - -
O/VIT/2/97 <0.1 - - - - 0.2 0.2 0.5
O/VIT/3/97 >1.0 - - - - 0.8 0.6 >1.0
O/VIT/4/97 0.5 - - - - 0.6 0.5 >1.0
O/VIT/7/97 0.5 - - - - 0.6 0.4 1.0
O/TAW/3/97 >1.0 0.3 - 0.5 - - 0.5 -
O/TAW/9/97 1.0 0.2/0.4 - <0.2/1.0 - - 0.3/0.8 -
O/TAW/10/97 >1.0 - - - - - - -
O/TAW/12/97 1.0 <0.2 - 0.5 - - 0.4 -
O/TAW/112/97 >1.0 - - - - 1.0 - -
O/TAW/114/97 >1.0 - - - - 1.0 - -
 10 20 30 40 50 410 420 430 440 450
Consensus gagQSsPaTGSqNQSGNTGSIiNNYYMQQYQnsmDtqlgDNAiSGGSNEG YaQYsgTinlhfmftgptdakarymiayappgmeppatpeaaahcihaew
O1/Kauf/66 -------------------------------------------------- -T-----------------------V----------K-------------
O1/Manisa/69 ------------------------------------------T------- -T--*--------------*----------------K-------------
O/CAM/1/98 -----*-------------------------------------------- --------------------------------------------------
O/CAM/3/98 ---------------------*---------------*------------ ----------------------------G---------------------
O/CAM/6/98 -------------------------------------------------- --------------*-----------------------------------
O/VIT/2/97 -------------------------------I**-***------------ -T-----N------R--I-*----------------T--------*----
O/VIT/7/97 -----------*-------------------**V-****----------- -----*--* *
O/VIT/3/97 -R-----T------------------------------------------ -T---------------------*-V----*--*--R--*----------
O/TAW/9/97 *----T----------------------------*------------- -T-----*
O/HKN/1/96 -----R-T----------------------------*I------------ -----*-* *--------

60 70 80 90 100 460 470 480 490 500

Consensus sTDTTSTHTtNTQnNDWfsKLAssAfSGlfGALLaDKKTEETTlLEDrIL dtglnskftfsipylsaadYAYTASdtAEtTNVQGWvClfQITHGKAdGD
O1/Kauf/66 -------------------------------------------------- -------------------------GV-----------------------
O1/Manisa/69 *----------------------*-----*-------------------- ---------*A---------------------------------------
O/CAM/1/98 -------------K-----------------------------*------ --------------------------------------------------
O/CAM/3/98 ----------------------------*--------------------- --------------------------------------------------
O/CAM/6/98 -------------------------------------------------- -------------------------*------------*-----------
O/VIT/2/97 -----------------------------------------------P-- ----------*----------------------------S----------
O/VIT/7/97 -----------------**---*--*--------D--------------- ----------*----*-------------*---------S----------
O/VIT/3/97 ---------N------------NT-------------------------- --------------------------A------------S----------
O/TAW/9/97 ---------N------------NT-------------------------- ****-------V---------*--S-------*--
O/HKN/1/96 ---------N------------NT-------------------------- --------------------------V-----------------------

110 120 130 140 150 510 520 530 540 550
Consensus TTRNGHTTsTTqSSvGVTYGYaTaEDFVSGPnTsGlETRVvQaERFFKTH alvVlASAgKDFeLRLPVDARtqTTsaGeSAdPVTaTVENYGGETQvqRR
O1/Kauf/66 -------------------------------------------------- ---------------------AE------------T----------I---
O1/Manisa/69 ----------------------------------------A--------- ----F---------------------------------------------
O/CAM/1/98 -----------*--F----------------*-*-*-------------- -------------------------AV-----------------------
O/CAM/3/98 -------------------------------------------------- -------------------------AV-----------------------
O/CAM/6/98 -------------------------------------------------- -------------------------AV-----------------------
O/VIT/2/97 --------------*------S-T-------------------------- *-*-----------------------V-*------------------*--
O/VIT/7/97 --------*-----*------*--------------------E------- --*-----------------------V--------------------*--
O/VIT/3/97 -------------------------------------------------- --I---------D------------------A--------------A---
O/TAW/9/97 -------------------------------------------------- -**-----A---D-------------------------------------
O/HKN/1/96 -------------------------------------------------- ------------D-------------------------------------

160 170 180 190 200 560 570 580 590 600
Consensus LFDWVtSDsFGRCHLLELPTDHKGVYGSLTdSYaYMRnGWDVEVTAVGNQ QHTDvaFIlDRFVKVtpkdQiNvLDlMQtPshTlVGALLRtaTYYFaDLE
O1/Kauf/66 -------------------------------------------------- -----S--M--------QN---I-----I-----------AS----S---
O1/Manisa/69 --------P----------------------------------------- -----S------------------------G-------------------
O/CAM/1/98 ------------------------------N------------------- -------------------------------Y------------------
O/CAM/3/98 ------------------------------N------------------- -------------------------------Y------------------
O/CAM/6/98 ------------------------------N------------------- -------------------------------Y------------------
O/VIT/2/97 -----A-------------------------------------------- ----------------*-G------*------------------------
O/VIT/7/97 -------------------------------------------------- ------------------G-T-----------------------------
O/VIT/3/97 --------P----------------------------------------- ----I----------K--E-V-------I-A---------------S---
O/TAW/9/97 --------P------------------------G---I------------ ----I----------K--E-V-------I-A--M------------S---
O/HKN/1/96 -------------------------------------------------- ----I----------K--E-V-------I-A---------------S---

210 220 230 240 250 610 620 630 640 650
Consensus FNGGCLLVaMVPELcsiekRElYQLTLFPHQFINPRTNMTAHIsVPfvGV vAvKHEGnLTWVpNGAPetALDNTTNPTAYHKapLTRLaLPYTAPHrVLA
O1/Kauf/66 --------------Y--Q-------------------------T------ I------D----------K-------------------------------
O1/Manisa/69 -----------------Q-------------------------T------ ------------------A--------------R----------------
O/CAM/1/98 ----------------L-R------------------------------- -----------------V---------------*----------------
O/CAM/3/98 ----------------L-R------------------------------- -----------------V---------------*----------------
O/CAM/6/98 ----------------L-R------------------------------- -----------------V---------------*----------------
O/VIT/2/97 ----------------LKE--K---------------------------- --*---------*--------------------*----------------
O/VIT/7/97 ----------------LKE--K---------------------------- --I------------------------------*----------------
O/VIT/3/97 -----------------S-------------------------T--YL-- L------D------------------------*-----*-------*---
O/TAW/9/97 -----------------S-------------------------T--YL-- L------D------------------------E-----------------
O/HKN/1/96 --------P------*-S-------------------------T--YL-- L------D------------------------E-----------------

260 270 280 290 300 660 670 680 690 700
Consensus NRYDQYKVHKPWTlVVMVVAPLTVNtEGApQIKVYaNIAPTNVHVAGElP TVYNGnckYaqgspaNvRGDLQVLAQKaaRpLPTSFNyGAIKatrVTELL
O1/Kauf/66 ------------------------------------------------F- -----E-R-NRNAVP-L----------V--T-------------------
O1/Manisa/69 -------------------------S----------------------F- ---------GD-TV----------------T-------------------
O/CAM/1/98 -------------------------------------------------- -----D--------T---------------------------*-------
O/CAM/3/98 -----------------------------------V-------------- --------------T---------------------------*-------
O/CAM/6/98 -------------------------------------------------- -----D--------T---------------------------*-------
O/VIT/2/97 -----------------------------Q-------------------- ------------PL-----------------------------*Q-----
O/VIT/7/97 -----------------------------Q-------------------- ------------PL------------------------------Q-----
O/VIT/3/97 -------------------------N------------------------ -----SS--GGT-TN------------TE-T------F------------
O/TAW/9/97 -------------------------N------------------------ -----SS--GDT-TN-------------E-T------F------------
O/HKN/1/96 -------------P-----------N------------------------ -----SS--GDT-TN-------------E-A------F------------

310 320 330 340 350 710 720 730

Consensus SKEGIFPVACSDGYGGLVTTDPKTADPaYGKVfNPPRNmLPGRFTNllDV YRMKRAeTYCpRpllAihPseArhkqkivapqkqll
O1/Kauf/66 ---------------------------V----------Q----------- -------------------T-----------V--T-
O1/Manisa/69 ----------------------------------------------F--- -------------------DQ----------V----
O/CAM/1/98 --------------------------------Y----------------- -------------*--------**-----**--*-*
O/CAM/3/98 --------------------------------Y----------------- -----------------------*----------
O/CAM/6/98 --------------------------------Y----------------- -------------*--------****----*
O/VIT/2/97 --------------------------------Y-------------F--- ----------*-**V----N--****----*
O/VIT/7/97 --------------------------------Y-------------F*-- -------------*-----D--**-*-*
O/VIT/3/97 ---------------------------V----------L----------- ----------------*Q--D-*L**-----
O/TAW/9/97 ---------------------------V----------L----------- ------*----------Q--D--*--R--
O/HKN/1/96 ---------------------------V----------L----------- -----------------Q--T----------*----

360 370 380 390 400

Consensus AEACPTfLhFeGdVPYVTTKTdsdrvLAQFDlSLAaKhMSNTfLagLAQy
O1/Kauf/66 --------R---G------------------M-----Q------------
O1/Manisa/69 --------R---G------------------F------------------
O/CAM/1/98 --------------------------------------------------
O/CAM/3/98 --------------------------------------------------
O/CAM/6/98 --------------------------------------------------
O/VIT/2/97 ------------*----------***---------E--------E----S
O/VIT/7/97 ------L-----G--------****-----------------*--*----
O/VIT/3/97 ----------D---------------------------------------
O/TAW/9/97 ----------D---------------------------------------
O/HKN/1/96 ----------D---------------------------------------

Fig. 3. The deduced amino acid sequences of the capsid region of some of the FMD type O viruses studied.
 O/CAM/1/98
O/CAM/3/98
O/CAM/2/98
O/MYA/9/89
O/MYA/13/89
O/VIT/6/97
O/VIT/7/97
O/VIT/9/97
O1/Manisa/TUR/69
O1/Kaufbeuren/FRG/66
O/HKN/8/85
O/HKN/10/87
O/HKN/4/86
18.2%
O/HKN/15/90
O/PHI/10/94
O/PHI/1/95
O/TAW/9/97
O/TAW/10/97
O/TAW/114/97
O/VIT/3/97
O/HKN/9/93
O/HKN/9/94
O/HKN/1/96

18 16 14 12 10 8 6 4 2 0
Percentage nucleotide difference (nt 475-639 of VP1)

Fig. 1. UPGMA tree showing the genetic relationships between FMD type O viruses in the VP1 gene.

O/VIT/7/97
O/VIT/2/97 O/CAM/3/98
O/CAM/6/98
O/CAM/1/98

O1/Manisa/Turkey69
O/HKN/1/96

O/TAW/9/97
1

O/VIT/3/97

O1/Kaufbeuren/FRG/66

Fig. 2. Neighbor-joining tree showing the genetic relationships between FMD type O viruses in the P1 capsid-coding region.

1
 O/CAM/1/98 O/CAM/198
a) 100
b) 100

80 80

60 60

40 40

20 20

0 0
D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 120 85 127 113 177 98 176 84 107 144

O/CAM/3/98 O/CAM/9/98 O/CAM/3/98 O/CAM/9/98

100 100 100 100

80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 120 85 127 113 177 98 176 84 107 144 120 85 127 113 177 98 176 84 107 144

O/VIT/2/97 O/VIT/7/97 O/VIT/2/97 O/VIT/7/97

100 100 100 100

80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 120 85 127 113 177 98 176 84 107 144 120 85 127 113 177 98 176 84 107 144

O/VIT/3/97 O/TAW/9/97 O/VIT/3/97 O/TAW/9/97

100 100 100 100

80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20

0 0 0 0
D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 D9 C6 C9 C8 14EH9 OC3 C96 C2 MH5 120 85 127 113 177 98 176 84 107 144 120 85 127 113 177 98 176 84 107 144

Fig. 4. MAb profiles seven of the FMD type O viruses studied. a) O1 European MAb panel; b) O1/Manisa/Turkey/69 MAb panel.

2
 67
Appendix 7

Genetically diverse isolates of type O foot-and-mouth disease virus exhibit

remarkable amino acid conservation at their neutralising antigenic sites

A.R. Samuel
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright,
Woking, Surrey, GU24 0NF, United Kingdom.
Summary
Isolates of type O FMDV selected for their genetic diversity have been examined using a panel
of neutralizing monoclonal antibodies and analysis of the deduced amino acid sequence of the
capsid proteins. The amino acid substitutions responsible for antigenic variation, as measured by
the monoclonal antibodies, have been examined and show good correlation to those already
identified as critical for antibody binding. The variation observed at the five defined sites is
conservative, limited, in most cases, to one or two permissible substitutions at the critical
residues.

Introduction
Monoclonal antibodies have been used to map epitopes that neutralize the infectivity of
various strains of foot-and-mouth disease virus (FMDV) belonging to five of the seven serotypes
(O, A, C, SAT1 and SAT2). Serotype O FMDV is probably the most extensively studied and five
discrete neutralizing antigenic sites have been characterised. Kitson et al. (1990) identified four
independent antigenic sites involving the three capsid proteins of the O1/Kaufbeuren/FRG/66
strain. Site 1 consists of linear epitopes located on the the βG-βH loop of VP1 (residues 144-154)
and the carboxy terminus (residue 208) of VP1 which lies close to the base of the βG-βH loop of
the adjacent protomer. The structure of the βG-βH loop of VP1 was difficult to resolve in
crystallography studies due to its highly flexible nature and ability to adopt a number of positions
above the viruses surface (Acharya et al., 1989; Parry et al. (1990). However, treating the crystals
with dithiothreitol (DTT) and examining them in the reduced state allowed resolution of the loop
structure (Logan et al., 1993). Site 3 involving the βB-βC loop (residues 43-45) of VP1 is located
near the five-fold axis of the virus particle. Site 2, at the three-fold axis, involves the βB-βC loop
(residues 70-77) and the adjacent βE-αB loop (residue 131) of VP2. Site 4, also close to the
three-fold axis, consists of residues 56-58 of 1C located at the top of the insertion within βB. The
fifth site identified by Crowther et al. (1993) is a conformational epitope that consists of, in part,
the βG-βH loop of VP1. Crowther et al. (1993) also reported that a mutant isolated by successive
selection using the Mabs defining the five sites was able to escape neutralization by bovine and
guinea pig polyclonal sera elicited against the parent strain (O1/Kaufbeuren/FRG/66). This paper
examines eighteen isolates of type O FMDV belonging to genetically distinct lineages with seven
Mabs whose epitopes are located within these five antigenic sites. Although the sequence of the
capsid coding region has been published for various European O1 strains of FMDV (Forss et al.,
1984; Beck and Strohmaier, 1987) those of viruses which are more geographically/genetically
diverse have not. This study was undertaken to further the understanding of the molecular
 68
basis of antigenic variation of the type O FMD viruses. Field isolates from different genetic
groups (topotypes) have been examined because it is more likely that they will have been
exposed to different environmental pressures e.g. intensive vaccination campaigns, natural
immunity, natural evolution etc. It is hypothesised therefore, that these selective pressures will
have maximised the likelihood of genetic and antigenic evolution. If structural constraints are
driving conservation via the primary protein sequence then it could be anticipated that the most
genetically diverse viruses would also be those that tolerate the most radical substitutions,
manifested by antigenic diversity.
Although the classical serotypes have been shown to have good correlation with genetic
groupings by phylogenetic analysis and, for type C at least, the subtypes within that serotype also
group together genetically (Martinez et al., 1992), the link between sequence homology and
antigenic relationships is not clear. It has been reported that quite distantly related isolates may
have similar antigenic characteristics due to molecular mimicry at important antigenic sites
(Samuel et al., 1988a; Hérnandez et al., 1992). Conversely, very close sequence homology may
belie large antigenic differences, the multiple site mutant described by Crowther et al. (1993)
being a good example of this.

Materials and methods

FMDV isolates
All isolates were passaged once on flasks confluent with IB-RS-2 monolayers and when 100%
cpe was evident the supernatent was harvested, clarified at 4000rpm for 10 minutes and then
either diluted 1:1 with sterile glycerol for serological testing or the vRNA extracted as described
below.

RNA extraction
RNA was extracted from 200 Fl of supernatant collected from an infected 25 cm2 flask of
IB-RS-2 cells using a Qiagen RNeasyJ kit according to the manufacturers protocol. Samples
were stored at -20°C until required.

Reverse transcription of vRNA

The vRNA, extracted as described above, was used as a template for reverse transcription
(RT) by the method described by Knowles and Samuel (1994) and stored at -20°C until required.
The cDNA product was then used as a target for polymerase chain reaction (PCR) amplification.

Polymerase chain reaction amplification

The region of the cDNA corresponding to the capsid coding region of the virus genome was
either amplified as a single fragment by the method described by Knowles and Samuel (1994)
using primers located in the L and 2B genes (L-463F and NK61, respectively) or as two
fragments using the primer sets pMH2/pARS3 and pNK61/pARS4 (1096 bp and 1301 bp,
respectively). PCR products were analysed on a 2% agarose-TBE gel containing ethidium
bromide (EB) at concentration of 0.5 Fg/ml. DNA weight markers were run alongside the
samples to facilitate product identification. Post PCR purification was carried out using Wizard
 69
PrepsJ (Promega, UK) according to the manufacturers protocol (see Samuel et al., 1998).
Cycle sequencing
An fmolJ DNA sequencing kit (Promega, UK) was used according to the manufacturers
protocol with some amendments (see Samuel et al., 1997). Table 1 shows the various primers
used. Sequencing was performed on an ALFexpressJ Automated Sequencer (Pharmacia Biotech,
Sweden). The software AM V3.01 was used to process the data which was then aligned manually
and analysed using the program EpiSeq (N.J. Knowles, unpublished data).

Phylogenetic analysis of sequence data

All pairwise comparisons were performed by giving each base substitution equal statistical
weight (ambiguities were ignored). A binary tree was constructed according to sequence
relatedness across the interval of nucleotides 1570 to 2205 (the VP1 gene) using the unweighted
pair group mean average (UPGMA) method as implemented in the computer program
NEIGHBOR and a dendrogram was plotted using the program DRAWGRAM both from the
PHYLIP 3.5c phylogeny package (Felsenstein, 1993). The UPGMA method constructs a tree by
successive (agglomerative) clustering using an average-linkage method.

Antigenic profiling by ELISA using monoclonal antibodies

The field isolates were profiled by trapping ELISA as previously described by Samuel et al.
(1991). The MAb panel used to profile the isolates consisted of MAbs D9, B2, C6, C9 and C8
produced against O1/Lausanne/Switzerland/65 (Cappuchi et al., 1984); 14EH9 anti-O1/Brugge
(Kitson et al., 1990) and OC3 anti-O1/Caseros (Crowther et al., 1993) (see Table 2).

Results
Antigenic profiling by ELISA
From the results of the antigenic profiling which are shown in Figure 2. It can be seen that
some individual MAbs exhibit remarkable cross reactivity between genetically distant strains e.g.
MAb D9 (site 1), OC3 (site 5). The site 2 MAbs (C6 and C9) show less cross reactivity as does
MAb C8 (site 3) and MAb B2 (site 1).

Sequence analysis
Figure 1 is a UPGMA dendrogram constructed from the complete VP1 nucleotide sequences
of the eighteen selected isolates. The figure shows that the isolates form distinct genetic groups
and that generally isolates from similar geographical regions ie the Far East or Middle East have
greater nucleotide sequence identity than comparisons between strains from different
geographical regions. The percentage nucleotide sequence divergence is >20% for some of the
isolates.

Discussion
Neutralizing antigenic sites 1 and 5
Site 1 consists of linear epitopes on the βG-βH loop of VP1 (a.a140-160). It can be seen from
the sequence alignment in Figure 3. that there is a lot of variation in the protein sequence on the
 70
N-terminal end of the loop although as would be expected viruses which are genetically more
closely related exhibit conservation of the primary protein sequence. All of the isolates studied by
antigenic profiling reacted strongly with the site 1 MAb D9. The residues identified as critical for
binding of this antibody are VP1144, VP1148 and VP1154 (Kitson et al.(1990). Some of the
monoclonal antibody escape mutants selected by Kitson et al.(1990) with Mab D9 also had
additional substitutions at VP1171 and VP1208 although their involvement in resistance to binding
is equivocal. However, from the deduced amino acid sequence data it can be seen that there are
no substitutions with respect to O1/Kaufbeuren/FRG/66 at any of the residues implicated in D9
binding. Significant variation is however evident in adjacent residues towards the N-terminus of
the GH loop but these do not appear to influence MAb D9 binding. MAb B2 (Site 1) shows less
cross reactivity with the field isolates. The critical residue for this MAb is VP1144 and the epitope
is thought to be overlapping, but not identical to D9's. It is probable that the variability in binding
is due to the involvement of residues that are in the variable VP1137-144 region. One other point
worthy of mention is that the isolates O/HKN/1/96 and O/HKN/6/95 do not have a cysteine at
VP1134. This residue has been shown to form a di-sulphide bond with acysteine residue at VP2130
(Achayra et al., 1989) and is thought to provide a stabilising mechanism to the VP1 βG-βH loop
in type O. However, despite a substitution to serine at VP1134 there is no apparent deleterious
effect to the binding of the linear epitope of MAb D9 or the conformational epitope recognised
by Mab OC3 which is perhaps, somewhat suprising.
At the Carboxy terminal end of the 140-160 region, 3' of the RGD triplet (a.a.=s VP1145,
VP1146 and VP1147), the amount of sequence variation is less, presumably to conserve the
structure of the 3,10 α-helix (Logan et al., 1993) which probably gives some structure to the
βG-βH loop so that RGD is presented correctly to the cellular receptor, integrins αV,β3 and α5β1
(Berinstein et al., 1995; Pfaff et al., 1994). However, the five site mutant described by Crowther
et al. (1993) has a run of three consecutive substitutions at residues VP1148, VP1149 and VP1150
which proves that sequence variation is tolerated in this region. Why so little variation is found in
field isolates is unclear. Nearly all the FMDV isolates which have been antigenically profiled
with MAb OC3 (Site 5) react with this antibody, although mutations which abrogate OC3
binding can be readily selected in-vitro. It could have been argued that the mutations are tolerated
in-vitro by the virus using an alternative receptor (see discussion on Site 4 below) and not
involving RGD for binding; in-vivo the substitutions could then possibly be sufficient to disrupt
recognition of the ligand for the primary receptor (RGD) by the cellular integrin. Point mutations
at residues on the carboxy side of the RGD motif have been reported to affect the ability of some
FMDV isolates to replicate (Rieder et al.,1994; Leippert et al., 1997). However, it is possible to
passage the five site mutant in guinea pigs and cattle without reversion of the mutations (C. Dunn
personal communication) and so any hypothesis regarding the lethality of these types of
mutations in field isolates is not supported.

Neutralizing antigenic site 2

Site 2 (BC loop VP2) as defined by MAbs C6 and C9 is a conformational epitope that
involves critical residues at; C9-VP270, VP272, VP275 and VP277 and for C6 VP270, VP271, VP273
and VP2131. In the sequences obtained with the field isolates these residues are invariant. There
 71
74
is, however, variation at residue VP2 . Three residues are evident at this position proline, serine
and in one instance glutamine (see Figure 4). Although this residue has not previously been
implicated as critical for binding of site 2 MAbs; it can be seen from the ELISA data that the
isolates O/UGA/3/72, O/HKN/1/96 and O/HKN/6/95 all bind MAb C6 and like O1/Kaufbeuren
they are all serine at VP274. Variation is apparent in residues adjacent to VP2131 although this
residue is well conserved in the field isolates.

Neutralizing antigenic site 3

Most of the isolates antigenically profiled react with the site 3 MAb (C8) with the exception of
O/HKN/1/96 and O/HKN/6/95. The residues shown by Kitson et al.(1990) to be critical for MAb
C8 are VP143 and VP144. The two isolates from Hong Kong have a threonine to lysine
substitution at VP143 and it is this substitution that causes the lack of binding (see Figure 5).
Barnett et al. (1998) reported that a bovine Mab mapped to a critical a critical residue at VP146 .
It can be seen from the sequence alignment that this residue is in fact more variable than VP143
and VP144 which only exhibit a range of two amino acids at these positions (VP143 - threonine
and lysine; VP144 - proline and histidine). This could be due to selection pressures in the field
due to the immune status of host species, the bovine in particular.

Neutralizing antigenic Site 4

ELISA data for the site 4 directed MAb (14EH9) which has a critical residue at VP358; (Kitson
et al.(1990) shows that lack of reactivity with the MAb for the FMDV isolates O/HKN/1/96,
O/HKN/6/95 and O/LAO/1/82 can be attributed to a glutamic acid to aspartic acid (EÿD)
substitution at VP358 (see Figure 6). The isolate O/IRN/38/95 does not have a substitution at
VP358 but does have a glycine to aspartic acid (GÿD) substitution at VP360.
It is possible that Site 4 is only important for neutralization of type O in-vitro. Recent studies
have shown that heparan sulphate is involved in efficient infectivity in cell culture (Jackson et
al., 1996). Although it has been shown that integrins can utilise synergistic binding sites
(Orlando et al., 1991; Aota et al., 1994) it has been reported by Fry et al. (1999) that FMDV type
O can use heparan sulphate as an alternative receptor in tissue culture adapted strains and that
antigenic variation caused by host defence mechanisms could act as a motor for receptor
switching.
The relatively conservative variation permitted at specific residues is perhaps suprising and
the inability of MAbs to bind to field isolates can, in most instances, be correlated with
substitutions already identified as critical. It could have been expected that over time, mutations
due to errors by the RNA polymerase and selection from the viral quasi-species would have
given rise to the fixation of a wider variety of substitutions on residues exposed on the viruses
surface. However, Green et al. (1998) have reported that if further immune pressure is applied to
antigenic sites 2 and 5 of MARM=s to O1/Kaufbeuren/FRG/66 then the residues critical for
antibody binding merely revert to those residues observed in the wild type. If the limits of
permissible variation within a serotype can be defined and this can be related to data on the
relative importance of individual sites to the host species polyclonal response; then predicting the
effects that changes at the molecular level have on the way the virus is recognised by the host
 72
immune system will be a distinct possibility. Site 2 has already been reported as being
immunodominant in some individual animals (Barnett et al., 1996; Samuel, 1997). Monoclonal
antibody selected mutants with changes in the BC loop of VP2 have also been shown to be
sufficiently different antigenically that the effects are measurable by a polyclonal based assay
such as the LPB-ELISA (Samuel, 1997). The accumulation of this type of data may in the future
facilitate the prediction of which Acocktails@ of FMDV isolates could be used in vaccines, to
afford better protection against antigenic variants that may arise in the field.
 73
Data such as this is useful for authenticating the use of Mabs for diagnosis. However, some
caution is required. Although site 2 has been shown to immunodominant for the response of
individual animals against O1/Kaufbeuren/FRG/66, it should be established whether subtle
structural differences due to changes in the primary protein sequence of isolates from different
genetic groups cause changes in the immunodominance of individual sites or the critical residues
within them. However, Aktas and Samuel (manuscript in press) have found that the critical
binding residues for MAbs produced against the O1/Manisa/Turkey/69 isolate are similar at sites
2 and 5 which would suggest that these sites, at least, are presented to the immune system in a
similar fashion to those of O1/Kaufbeuren/FRG/66.

Acknowledgements
The author would like to thank David Ansell for technical assistance and Nick Knowles for
critical review of the manuscript. This work was supported by the Ministry of Agriculture
Fisheries and Food, UK.

References

Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D. and Brown, F. (1989). The
three-dimensional structure of foot-and-mouth disease virus at 2.9 Å resolution. Nature, 37:
709-716.

Aktas, S. and Samuel, A.R. (2000). Identification of antigenic epitopes on the foot-and-mouth
disease virus isolate O1/Manisa/Turkey/69 using monoclonal antibodies. Rev. sci. tech. Off. int.
Epiz. 19: (3), in press.

Aota, S., I., Nomizu, M. and Yamada, K.M. (1994). The short amino acid sequence
Pro-His-Ser-Arg-Asn in human fibronectin enhances cell adhesive function. J. Biol. Chem. 269:
24756-24761.

Barnett, P.V., Samuel, A.R., Pullen, L., Ansell, D.M. , Butcher R. and Parkhouse, R.M.E. (1998).
Monoclonal antibodies, against O1 serotype foot-and-mouth disease virus, from a natural bovine
host, recognize similar antigenic features to those defined by the mouse. J. Gen. Virol. 79:
1687-1697.

Beck, E., and Strohmaier, K. (1987). Subtyping of European foot-and-mouth disease virus strains
by nucleotide sequence determination. J. Virol. 61: 1621-1629.

Berinstein, A., Roivainen, M., Hovi, T., Mason, P. And Baxt, B. (1995). Antibodies to the
vitronectin receptor (integrin αVβ3) inhibit binding and infection of foot-and-mouth disease virus
to cultured cells. J.Virol. 69: 2664-2666.

Cappuchi, L, Brocchi, E., Simone, F. and De Panina G. F. (1984). Characterization of

 74
monoclonal antibodies produced against foot-and-mouth disease viruses. Report of a session of
the Research Group of the Standing Technical Committee of the European Commission for the
Control of Foot-and-Mouth Disease, Brescia, Italy, 26-28 June 1984 Rome: FAO Appendix 6:
32-39.

Crowther, J.R., Farias, S., Carpenter, W.C. and Samuel, A.R. (1993). Identification of a fifth
neutralizable site on type O foot-and-mouth disease virus following characterization of single
and quintuple monoclonal antibody escape mutants. J. Gen. Virol. 74: 1547-1553.

Felsenstein, J. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author.
Department of Genetics, University of Washington, Seattle, 1993.

Fry, E., Lea, S., Jackson, T., Newman, J., Ellard, F., Blakemore, W., Abu-Ghazaleh, R., Samuel,
A., King, A. and Stuart, D.I. (1999). The structure and function of a foot-and-mouth disease
virus/oligosaccharide receptor complex. EMBO J., 18: 543-554.

Forss, S., Strebel, K., Beck, E. and Schaller, H. (1984). Nucleotide sequence and genome
organisation of foot-and-mouth disease virus. Nucleic Acids Res. 12: 6587-6601

Green, E.G.F., Samuel, A.R., Dunn, C. and Ansell, D.M. and (1998). A multiple mutant derived
from O1/Kaufbeuren/FRG/66 displays a limited antigenic repertoire when subjected to extended
immunological pressure. Report of the session of the Research Group of the European
Commission for the Control of Foot-and-mouth Disease, Aldershot, United Kingdom, 14-18
September 1998 Rome: FAO: Appendix 7: 62-68

Hérnandez, J., Martinéz, M.A., Rocha, E., Domingo, E. and Mateu, M.G. (1992). Generation of a
subtype-specific neutralization epitope in foot-and-mouth disease virus of a different subtype. J
Gen. Virol. 73: 213-216.

Kitson, J.D.A., McCahon, D. and Belsham, G.J. (1990) Sequence analysis of monoclonal
antibody resistant mutants of type O foot-and-mouth disease virus: evidence for the involvement
of the three surface exposed capsid proteins in four antigenic sites. Virology, 179: 26-34.

Knowles, N.J. and Samuel, A.R. (1994). Polymerase chain reaction amplification and cycle
sequencing of the 1D (VP1) gene of foot-and-mouth disease viruses. Paper presented at the
Session of the Research Group of the Standing Technical Committee of the European
Commission for the Control of Foot-and-Mouth Disease, Vienna, Austria. 19-22 September
1994. Rome: FAO. Appendix 8. 45-53

Leippert, M., Beck, E., Weiland, F. and Pfaff, E. (1997). Point mutations within the ∃G-∃H loop
of foot-and-mouth disease virus O1K affect virus attachment to target cells. J. Virol. 71:
1046-1051.
 75

Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jackson, T., King, A., Lea, S., Lewis,
R., Newman, J., Parry, N., Rowlands, D., Stuart, D. and Fry, E. (1993). Structure of a major
immunogenic site on foot-and-mouth disease virus. Nature 362: 566-568.

Orlando, R.A. and Cheresh, D.A. (1991). Arginine-glycine-aspartic acid binding leading to
molecular stabilization between integrin αVβ3 and its ligand. J. Biol. Chem. 266: 19543-19550.

Parry, N., Fox, G., Rowlands, D., Brown, F., Fry, E., Acharya R., Logan, D. and Stuart, D.
(1990). Structural and serological evidence for a novel mechanism of antigenic variation in
foot-and-mouth disease virus. Nature 347: 569-572.

Pfaff, M., Tangemann, K., Máller, B., Gurrath, M., Máller, G., Kessler, H., Tip, R. and Engel, J.
(1994). Selective recognition of cyclic RGD peptides of NMR defined conformation by αIIbβ3,
αVβ3 and α5β1 integrins. J. Biol. Chem. 269: 20233-20238.

Rieder, E., Baxt, B. and Mason, P. (1994). Animal derived antigenic variants of foot-and-mouth
disease virus type A12 have low affinity for cells in culture. J. Virol. 68: 5296-5299.

Samuel, A.R. (1997). Genetic and antigenic studies on foot-and-mouth disease virus type O. PhD
Thesis. University of Hertfordshire, Hatfield, UK. 249 pp.

Samuel, A.R., Knowles, N.J., and Carpenter, W.C. (1988). Possible constraints in the use of
restricted monoclonal antibody panels for epidemiological studies of foot-and-mouth disease
virus. Report of a Session of the Research Group of the Standing Technical Committee of the
European Commission for the Control of Foot-and-Mouth Disease. Prague, Czechoslovakia
20-23 September 1988. Rome: FAO. Appendix 21: 131-135.
 76
Table 1 . Table of Cy5 amidite labelled primers used for ALF Express sequencing.
Primer Virus Sense Gene Primer Seq. (5'-3')
NK61 Universal Negative 2B GACATGTCCTCCTGCATCTG
MH2 Universal Positive 1A CACACAACCAACACCCAGAACAAT
L463R Universal Positive L ACCTCCRACGGGTGGTACGC
NK72 Universal Negative 2A GAAGGGCCCAGGGTTGGACTC
ARS3 Type O Negative 1C CATGTAACGCGCCTTCGCGTC
ARS4 Type O Positive 1C ACCAACCTCCTTGATGTGGCT
ARS10 Type O Negative 1C TGGTTGGAGGAACTACACCGA
ARS13 Type O Positive 1C GACGCGAAGGCGCGTTACATG
ROD2 Type O Negative 1D AACTGAGTAAGCACCCTGTCCC
ROD5 Type O Positive 1B TTTTGAGGATGTGAGCGGACC
1D-628F Type O Negative 1D GTTGGGTTGGTGGTGTTGT
1D-564R Type O Positive 1D CARATAACACACACGGGAAAGC
1D-386R Type O Negative 1D GTGTTTGACATGTGCTTTG
1C-329R Type O Negative 1C TTGAACACTTTCCCGTA
R= A or G

Table 2. Characteristics of the Mabs use to define the five neutralizing antigenic sites of
O1/Kaufbeuren/FRG/66 (Kitson et al., 1990; Crowther et al., 1993).
MAbs Antigenic Trypsin Denatured Substitution Critical Structural
Site Sensitive Virus Residues Feature

B2 Site 1 Yes Yes L→S VP1144 βG-βH loop VP1

D9 Site 1 Yes Yes L→S, L→R, VP1144, VP1148, βG-βH loop VP1
K→M VP1154
C6 Site 2 No No V→G, T→N, VP270,VP271, βB-βC loop VP2
D→H+ VP273
C9 Site 2 No No V→F, S→C, VP270, VP272, βB-βC loop VP2
F→L, S→H VP275, VP277
C8 Site 3 No No T→P, VP143, VP144 βB-βC loop VP1
P→T,L,Q
14EH9 Site 4 - No E→V,K VP358 βG1 VP3
OC3 Site 5 Yes No Q→H VP1149 βG-βH loop VP1
 77
Monoclonal Antibodies
Viruses D9 C6 C8 EH9 OC3 B2 C9
site 1 site 2 site 3 site 4 site 5 site 1 site 2
O1/Laus/66
O/CAR/3/89 ND
O/LAO/1/88
O/LAO/1/82
O/SAU/8/88
O/MAY/5/92 ND
O/UGA/3/72 ND
O/MYA/1/96
O/ERI/1/96
O/MYA/13/89
O/HKN/1/96
O/HKN/6/95
O/TAI/2/91
O/IRN/38/95 ND
O/JOR/2/95
O/UGA/5/96 ND
O/TAN/3/96 ND
O/AFG/6/96 ND

Figure 1. Results of antigenic profiling by ELISA with MAbs (Samuel et al., 1991). Black filled
squares are where the reactivity of the field isolate is >60% of the homologous viruses reactivity
with the MAb; grey filled squares are 25%-60% of the homologous reaction; white squares are
<20% of the homologous reactivity; ND is not tested.
 78 O1/Kauf/66
O/IRN/38/95
O/JOR/2/95
O/UGA/3/72
O/TAI/2/91
O/SAU/8/88
O/AFG/6/96
O/LAO/1/82
O/MYA/13/89
O/LAO/1/88
O/MAY/5/92
20.7% O/MYA/1/96
O/UGA/5/96
O/ERI/1/96
O/TAN/3/96
O/CAR/3/89
O/HKN/6/95
O/HKN/1/96

20 18 16 14 12 10 8 6 4 2 0
% Nucleotide difference (nt 1570-2205)
Figure 2. Dendrogram depicting the genetic relationships between FMDV
isolates of serotype O based on the VP1 nucleotide sequences.
 79
140 150 160
O1KAUF66 YNGECRYNRNAVPNLRGDLQVLAQKVARTLPTSFNYGAIKA
HKN95-06 ---SSK-GDTSTN-V----------AE-A------F-----
HKN96-01 ---SSK-GDTSTN-V----------AE-A------F-----
IRN95-38 ---N---GEGP-AAV----------A---------------
JOR95-02 ---N-K-GESP-T-V----------A---------------
SAU88-08 ---N-E-GESH-A-V-------S--AE-A------------
LAO82-01 ---N-K-AQSPPA-V----------A--P------------
LAO88-01 ---N-K-AEGSLT-V----------A--P------------
MAY92-05 ---N-K-AERSLT-V----------A--P------------
MYA89-13 ---N-K-AQGPLA-V----------A--P--N---------
MYA96-01 ---N-K-AEGSLA*V----------AT-P------------
TAI91-02 ---N-K-GDG--T-V------V---A--*------------
UGA96-05 ---N---GVTP-T-V------V---A---------------
ERI96-01 ---N---GETP-T-V--------------------------
AFG96-06 ---N-K-SDG-LT-V----------AT--------------
TAN96-03 ---N-K-VNTP-T-V-----A--R-AV--------------
CAR89-03 ---S-K-S-V----V---------RA-*-------F-----
UGA72-03 ---N-K-GTAP-T-V----------A---------------

Figure 3. Antigenic sites 1 and 5, GH loop VP1.

70 80 130 140
O1KAUF66 KTHLFDWVTSDSFGRCHLLELPT VAMVPELYSIQKRELYQLTLFPH
HKN95-06 ----------------------- -------C--S------------
HKN96-01 ----------------------- -P-----C*-S------------
IRN95-38 -**--------Q----------- -------C---------------
JOR95-02 --*--------P----------- -------C---------------
SAU88-08 -----------P----------- -------C-M------------*
LAO82-01 -----------P-----------
LAO88-01 -----------P----------- -----*-C--L------------
MAY92-05 --------AG-P----------- -------C---------------
MYA89-13 ----------------------- -------C-LKE-----------
MYA96-01 -----------P----------- -------C---------------
UGA96-05 -----------P----------- -------C--N------------
ERI96-01 -----------P----------- -------C---------------
AFG96-06 ---*-------P----------- -------CF--------------
TAN96-03 -----------P-------GC-- ---*---CF---G---------*
CAR89-03 -----------P----Y------ ---I---C---------------
UGA72-03 ----------------------- -------C---R-------*---

Figure 4. Antigenic site 2, BC loop VP2.

 80
30 40 50 60
O1KAUF66 TDVSFIMDRFVKVTPQNQINILDLMQIPSHT
HKN95-06 ---A--L------K-KG-V-V-------A--
HKN96-01 --IA--L------K-KE-V-V-------A--
IRN95-38 ------L--------KD---V-----T-A--
JOR95-02 ------L--------KD--TV-----T-A--
SAU88-08 ------I-------*KD---------T-A--
LAO82-01 ---A--L--------KD---V-----T-A--
LAO88-01 ------L--------KD---V-----T-A--
MAY92-05 ------L----Q---KD---V-----T-R--
MYA89-13 ---A--L--------KD---V-----T--Y-
MYA96-01 ------L--------KD---V-----T-A--
TAI91-02 ------L--------KD---V-----A-A--
UGA96-05 ------L--------LEGT-V-----T-A--
ERI96-01 ------L--------KD-T-V-----T-A-K
AFG96-06 -----*L-------HKT-F-V-A---T-A--
TAN96-03 K-A*-TL---------*-*-V-----*-AQ-
CAR89-03 K-R---L---------E---V-----T-A--
UGA72-03 ------L---------D---V-----T-ABB

Figure 5. Antigenic site 3, BC loop VP1.

50 60 70
O1KAUF66 ACPTFLRFEGGVPYVTTKTDS
HKN95-06 ----N-H-D-D----------
HKN96-01 ------H-D-D----------
IRN95-38 ----------A-----*-V--
JOR95-02 ------H---D-------A--
SAU88-08 ------H---D----------
LAO82-01 ------H-D-D----------
LAO88-01 ------H---D----------
MAY92-05 ------H---D----------
MYA89-13 ------H--------------
MYA96-01 ------H---D----------
TAI91-02 ------H---D----*---**
UGA96-05 ------H---D----------
ERI96-01 ------H---D-----*----
AFG96-06 ------H*--D----------
TAN96-03 ------H---D----------
CAR89-03 ------H---D----------
UGA72-03 ------H---D----------

Figure 6. Antigenic site 4.

 Appendix 8

Early pathogenesis of foot-and-mouth disease in pigs: a quantitative time-course study

using a newly developed TaqMan RT-PCR assay.

Soren Alexandersen, Martin B. Oleksiewicz, and Alex I. Donaldson

Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, GU24 ONF,
UK.

Summary
Countries normally free from foot-and-mouth disease (FMD) invariably apply the
“stamping out” policy when outbreaks occur. Increasingly, countries are including the option
of emergency vaccination in their national contingency plans as an adjunct to the “stamping
out”. However, emergency FMD vaccines are less effective for pigs than other species.
Rectifying that deficiency requires a more detailed knowledge of the pathogenesis of FMD in
pigs as well as the immune mechanisms involved in disease and protection. The objective of
the present study was to determine the early pathogenesis of FMD in pigs by a quantitative
time course study using a quantitative TaqMan RT-PCR method (Oleksiewicz, Donaldson,
and Alexandersen; submitted for publication) and by titration for FMD virus on primary
bovine thyroid cells. The data and their significance will be discussed.

Introduction
An increasing number of countries are considering the use of emergency FMD
vaccination as an adjunct to “stamping out” and many have included that option as an
additional strategy in their contingency plans. Emergency FMD vaccine is deployed with the
objectives of dampening down infection in the immediate area of the outbreak and containing
it within the zone of vaccination. The event most likely to compromise those objectives
would be the infection of pigs since that species can generate plumes of airborne virus which
could then spread disease beyond the vaccination zone.
The response of pigs to FMD vaccines is more variable and generally less efficient
than that of other livestock species 19. Thus, there is a need to improve the efficacy of FMD
vaccines for pigs based on a more detailed knowledge of the pathogenesis of infection as well
as the mechanisms involved in natural and artificial immunity in that species. Therefore, the
objective of the present study was to determine the early pathogenesis of FMD in pigs by a
time course study using our newly developed quantitative TaqMan assay and to correlate
these findings with results obtained using virus isolation in cell culture and histopatological
examination of selected tissues.

Materials and methods

Animals. Four donor, 8 recipient and 2 non-infected control Landrace crossbred Large White
pigs, weighing 20-30 kg, were used. Specimens collected during previous experiments were
also examined. Blood samples were taken each day from two recipient pigs (no. UD94 and
UD95) to monitor the progression of infection. Every 24 hours from day 1 to 4 post exposure
(days post exposure = dpe) two recipient pigs were randomly selected and killed. At 4 dpe
two freshly introduced, un-exposed pigs were included as non-infected controls. At necropsy,
one series of tissues was immersed immediately in RNAlater (Ambion, Austin, TX, USA) for
the RT-PCR analysis and another series was snap-frozen and stored at –70 0C for virus
titration. Sera were stored at –20 0C. Selected tissues were fixed in 10% neutral buffered
formalin for 24 hours, paraffin embedded, sectioned, stained with haematoxylin and eosin and
examined by light microscopy.

Virus. A stock preparation of FMDV strain O1 Lausanne Sw/65 was used. It had been
passaged once in cattle and then grown in porcine IB-RS-2 cells 6;7. For the experiments, a
pig-adapted inoculum was made by passaging the original stock virus sequentially through a
series of 3 pigs. The experimental inoculum was a 10% (w/v) suspension of epithelial tissue
from a single pig with multiple vesicular lesions. The inoculum had a titre of 108.7
TCID50/ml in primary bovine thyroid (BTY) cells 20 and 107.2 TCID50/ml in IB-RS-2 cells.

Infection of donor pigs and contact exposure of recipients. Each of four donor pigs
received approximately 0.5 ml of a 1:10 dilution of the suspension by the
intradermal/subdermal route in the heel bulbs 2 of the left fore foot. Two pigs were inoculated
on day 3 and two more on day 2 before being used as donors. The eight recipient pigs were
exposed to virus by moving them into the isolation room containing the four donor pigs for a
2 hour period, i.e. direct contact exposure. After exposure, the recipient pigs were returned to
their respective individual cubicles, two animals per room, to prevent direct contact between
room-mates.

Assay for virus and antibodies. Tissue suspensions and serum samples were assayed in
primary bovine thyroid cells and the specificity of cpe confirmed by ELISA 9;10;14;18. Serum
samples were assayed for antibodies to FMDV by liquid-phase-blocking-ELISA 15;16.

Quantitative RT-PCR. A quantitative reverse transcription polymerase chain reaction (RT-

PCR) method was used to determine the amount of FMDV RNA in extracts of RNA from
serum and tissue samples. Four 4-fold RNA dilutions and a minimal Ct method (scoring the
lowest Ct value, i.e. highest virus RNA content, obtained in the four dilutions) were used in
combination with specific primers and a TaqMan fluorogenic probe designed specifically for
the O1 Kaufbeuren/O1 Lausanne strains of FMDV as detailed elsewhere (Oleksiewicz,
Donaldson, and Alexandersen; submitted for publication).

Results

Quantitative RT-PCR results and calculation of equivalents to infectivity titres

(TCID50). We assayed the serum samples and all the tissue samples in this study by
quantitative RT-PCR and converted the Ct values to estimated TCID50. The results are
shown as the average of 2 pigs killed at each time point (Fig. 1 and 2 A, B, C & D). In
general, the results differed very little between individual pigs (usually less than 2 to 10-fold
variation). The data are shown on a log10 scale to be comparable with the way infectivity data
are usually depicted. The discriminating power of the assay is 5-10 fold and of the specific
instrument used is 5-fold. Therefore, a log10 representation of the data is considered
appropriate.
The kinetics of the viraemia results plotted as infectivity titres in BTY cell cultures and as
titres converted from TaqMan data are shown in Fig. 1. Viremia peaked at around 104
TCID50/ml at 3 dpe and decreased to around 103 TCID50s/ml at 4 dpe.
Figures 2A-D show that significant amounts of virus could be detected in several tissues as
early as 1 dpe. However, the concentrations usually were low on 1 and 2 dpe and then rose on
3 and 4 dpe. Some tissues, including liver, spleen, lung and bronchial lymph node, were very
low in virus concentration on 1 and 2 dpe, and then increased on 3 and 4 dpe. However, the
levels did not exceed those found in corresponding blood samples. Two sites in the lung were
examined; the frontal lobe and the accessory lobe. The viral content of each were found to be
very similar. Respiratory tract (nasal mucosa and trachea) samples were slightly higher in
virus content on 1 and 2 dpe and also increased at 3 and 4 dpe. Tissues of the pharynx
(ventral floor of pharynx, soft palate, and tonsil) were consistently intermediate in virus
concentration from day 1 and were probably the initial sites of viral deposition and
replication. The concentration of virus in tongue epithelium and skin samples from the
coronary band of the foot started at a low to medium level and then increased to very high
levels on 4 dpe. Sites with vesicular lesions had the highest virus concentration, but
macroscopically normal tongue and skin samples also had relatively high levels. Thus, these
epithelial tissues are thought to be major sites of virus replication.

Clinical signs, viraemia and seroconversion. No clinical signs were evident in recipient
pigs until 3 and 4 dpe. At 3 dpe they had painful feet and small vesicles on the coronary
bands of the feet and on the snout which at 4 dpe were more pronounced. At the time of
necropsy at 3 and 4 dpe of donor and recipient pigs vesicular lesions were evident on all four
feet, the gingival mucosa, the tongue and the snout. No gross pathological lesions were
observed in internal organs.
Histopathological examination of hematoxylin and eosin stained sections confirmed
the presence of vesicular lesions in the epithelia of the feet and tongue but not in normal skin
and tongue epithelia. Histopathological examination did not indicate any significant
cytopathogenic or inflammatory changes in any internal organs associated with the FMD virus
infection.
Testing for antibody on serum samples taken during the experiment revealed, that the
one day 4 pig (UD94) had an ELISA antibody titre of 1:362 on day 4 while the other day 4
pig (UD95) had a titre of 1:128. This last pig (UD95) had a borderline titre of 1:45 (1:45
being the cut-off point) on day 2. All other sera were negative for specific antibodies.

Discussion

The TaqMan RT-PCR assay had a quantitative range of more than 8 orders of
magnitude and the results correlated strongly with infectivity titres obtained from virus grown
in cell culture preparations. The results indicated that the method could be used satisfactorily
with clinical specimens also; the samples being serum or tissue samples. However, for
certain serum and tissue samples estimations by RT-PCR were approximately 2 log units
higher than those by virus titration, depending on the actual sample. This is most likely due to
the presence of low levels of FMDV neutralising antibodies in such samples. Nevertheless,
estimation of FMDV contents in tissue samples based on TaqMan RT-PCR and standard
curves gave consistent results, irrespective of antibodies being present .
The time course showed that the appearance of vesicular lesions was coincident with
peak viraemia and high concentrations of virus in sites where there were clinical lesions.
Histopathological abnormalities were found only in areas where there were macroscopic
lesions. However, samples of tissue from histopathologically normal areas in the tongue and
skin contained significant amounts of virus, albeit 1-2 log units less than similar sites with
macroscopic lesions. Relatively high titres of FMDV have been described previously in
macroscopically normal skin from cattle although microscopic lesions, i.e. microvesicles,
were observable in the sections 11-13.
 The dose which infected the recipient pigs exposed by contact was not determined but
since the donor pigs had severe generalised disease it can be presumed that the recipient pigs
would have inhaled virus associated with particles in a wide size range. Consequently, all
parts of their respiratory tract would most likely have been exposed to infectivity 17. While
the recipient pigs are most likely to have been infected by the aerogenic route the possibility
that additional routes were involved e.g. the alimentary route, cannot be excluded.
Nevertheless, the results indicate that the initial distribution of FMDV in pigs is determined in
part by the predilection of virus for certain sites in the pig as has been established for cattle 3.
Only tissues from the pharynx (ventral floor of pharynx, soft palate, and tonsil) had
intermediate concentrations of virus fluctuating from 104 to 106 TCID50s/g thoughout the
studied period (day 1 to 4), i.e. a level 100-fold or more higher than the other tissues at day 1
and 2. In contrast, the virus concentrations in other tissues were low initially and for certain
tissues increased sharply on 3 and 4 dpe. Thus, our data are consistent with tissues of the
pharynx being the most likely initial sites of viral replication or deposition. Whether the
FMDV replicates initially in the pharynx or reaches that site by aerogenous or haematogenous
routes is currently unknown. However, our hypothesis, which would explain the differences
observed between the kinetics of FMDV replication and accumulation in pigs infected by
natural compared to artificial routes of infection, is that natural FMD virus infection in pigs is
initiated by the deposition of inhaled virus in the pharynx, in particular the soft palate and the
tonsil. After passage through local lymph nodes, the virus enters the bloodstream at a low,
not yet measurable, level of infectivity. This initial viraemia results in the infection of a few
cells in the stratified squamous epithelia, perhaps including Langerhans cells 4 resulting in a
significant amplification of virus, then higher viraemia results in the infection of a greater
number of epithelial cells. In susceptible hosts this cycle continues until sufficient epithelial
cells are infected in predilection sites to cause the development of the vesicular lesions and
clinical signs typical of the disease or until controlled by the host response.
The lungs yielded only low amounts of virus indicating that they probably play a
minor role in FMD virus replication. This is in agreement with earlier studies on the source of
airborne FMDV excreted in the breath of infected pigs which showed that in the early stages
of infection most airborne virus originates from the upper part of the respiratory tract but as
infection proceeds the lower respiratory tract becomes more involved 8.
Although the data presented here suggest that there was little or no replication of
FMDV in the lungs during the 1 to 4 day period of investigation, the possibility that
replication occurs in lung epithelia or macrophages later on in infection cannot be excluded.
Replication in the lungs has been suggested by others based on in situ hybridisation and virus
isolation 1;21. However, the actual levels of replication and/or accumulation at those sites
were not determined. In our experiment, the virus concentrations were near maximal and
vesicular lesions were evident from 3 dpe, making that time the most likely point of peak
airborne virus excretion 5. This is consistent with our hypothesis that stratified squamous
epithelia cells, especially those in the skin, the oral mucosa, and the pharynx are those
primarily responsible for the amplification of virus. Thus, airborne virus excretion by the pigs
in our experiments most likely originated from stratified squamous epithelial cells and
subsequently release of particles into the lumen of the respiratory tract and the transport in
exhaled breath through the trachea, larynx, pharynx, nasal cavities and mouth. However, the
respiratory epithelia as well as lungs may play an indirect role in this excretion, because the
large surface area may generate aerosols of virus produced elsewhere and transported to these
sites by muco-ciliary movement or derived from the bloodstream.
Lymph nodes and the spleen appeared to accumulate virus at 3 and 4 dpe. This virus
was most likely produced elsewhere and filtered from the lymph and blood. The liver
contained virus at a level which could be explained by the concentration of virus in the
bloodstream.

Acknowledgments

We thank Martin Broomfield and Natasha Smith for their careful management of the
experimental animals. Geoffrey H. Hutchings, Linda Turner, Nigel P. Ferris, and Teli Rendle
are thanked for excellent technical assistance. Sue Hacker at IAH, Compton is thanked for
preparing and providing histopathological sections. R. Paul Kitching made helpful comments
on the manuscript. We gratefully acknowledge the financial support provided by the UK
Ministry of Agriculture, Fisheries and Food and by the Danish Ministry of Food, Agriculture
and Fisheries.

References

1. Brown, C. C., H. J. Olander, and R. F. Meyer. 1995. Pathogenesis of foot-and-

mouth disease in swine, studied by in-situ hybridization. J Comp Pathol 113:51-8.

2. Burrows, R. 1966. The infectivity assay of foot-and-mouth disease virus in pigs. J

Hyg (Lond) 64:419-29.

3. Burrows, R., J. A. Mann, A. J. Garland, A. Greig, and D. Goodridge. 1981. The

pathogenesis of natural and simulated natural foot-and-mouth disease infection in
cattle. J Comp Pathol 91:599-609.

4. David, D., Y. Stram, H. Yadin, Z. Trainin, and Y. Becker. 1995. Foot and mouth
disease virus replication in bovine skin Langerhans cells under in vitro conditions
detected by RT-PCR. Virus Genes 10:5-13.

5. Davidson, F. L. Alternative strategies for foot-and-mouth disease control in pigs: a

thesis submitted in partial fulfilment of the requirements of the University of
hertfordshire for the degree of Doctor of Philosophy. 1-192. 6-6-1997. Pirbright
Laboratory, Pirbright Laboratory. PhD thesis.

6. De Castro, M. P. 1964. Behaviour of the foot and mouth disease virus in cell cultures:
Susceptibility of the IB-RS-2 cell line. ARQS. INST. BIOL. S. PAULO 31:63-78.

7. De Castro, M. P. and R. C. B. Pisani. 1964. The chromosome complement of the IB-

RS-2 swine cell line susceptible to the foot and mouth disease virus. ARQS. INST.
BIOL. S. PAULO 31:155-166.

8. Donaldson, A. I. and N. P. Ferris. 1980. Sites of release of airborne foot-and-mouth

disease virus from infected pigs. Res. Vet. Sci. 29:315-319.

9. Ferris, N. P. and M. Dawson. 1988. Routine application of enzyme-linked

immunosorbent assay in comparison with complement fixation for the diagnosis of
foot-and-mouth and swine vesicular diseases. Vet. Microbiol. 16:201-209.
10. Ferris, N. P., H. Powell, and A. I. Donaldson. 1988. Use of pre-coated
immunoplates and freeze-dried reagents for the diagnosis of foot-and-mouth disease
and swine vesicular disease by enzyme-linked immunosorbent assay (ELISA). J.
Virol. Methods 19:197-206.

11. Gailiunas, P. 1968. Microscopic skin lesions in cattle with foot-and-mouth disease.
Arch. Gesamte Virusforsch. 25:188-200.

12. Gailiunas, P. and G. E. Cottral. 1966. Presence and persistence of foot-and-mouth

disease virus in bovine skin. J. Bacteriol. 91:2333-2338.

13. Gailiunas, P. and G. E. Cottral. 1967. Survival of foot-and-mouth disease virus in

bovine hides. Am. J. Vet. Res. 28:1047-1053.

14. Hamblin, C., R. M. Armstrong, and R. S. Hedger. 1984. A rapid enzyme-linked

immunosorbent assay for the detection of foot-and- mouth disease virus in epithelial
tissues. Vet. Microbiol. 9:435-443.

15. Hamblin, C., I. T. Barnett, and J. R. Crowther. 1986. A new enzyme-linked

immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth
disease virus. II. Application. J. Immunol. Methods 93:123-129.

16. Hamblin, C., I. T. Barnett, and R. S. Hedger. 1986. A new enzyme-linked

immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth
disease virus. I. Development and method of ELISA. J. Immunol. Methods 93:115-
121.

17. Hatch, T. F. and P. Gross. 1964. Pulmonary deposition and retention of inhaled
aerosols, p. 45-68. Academic Press, London.

18. Roeder, P. L. and P. M. Blanc Smith. 1987. Detection and typing of foot-and-mouth
disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable
technique for primary diagnosis. Res. Vet. Sci. 43:225-232.

19. Salt, J. S., P. V. Barnett, P. Dani, and L. Williams. 1998. Emergency vaccination of
pigs against foot-and-mouth disease: protection against disease and reduction in
contact transmission. Vaccine 16:746-54.

20. Snowdon, W. A. 1966. Growth of foot-and mouth disease virus in monolayer cultures
of calf thyroid cells. Nature 210:1079-1080.

21. Terpstra, C. 1972. Pathogenesis of foot-and-mouth disease in experimentally infected

pigs. Bull Off Int Epizoot 77:859-74.
 Viraemia determined by titration in BTY
and calculated from TaqMan data
Average of 2 pigs in BTY Average of 2 pigs, TaqMan
calculations

1e+10
1e+09
1e+08
1e+07
1000000
TCID50/ml

100000
10000
1000
100
10
1
0.1
0.01
0 1 2 3 4
Day

Tissues

VFPH Soft pal. Tonsil Nas. muc

1e+10
1e+09
1e+08
1e+07
1000000
TCID50/g

100000
10000
1000
100
10
1
0.1
0 1 2 3 4
Day

Tong. w/ Tong. w l Foot les. Skin w/o

1e+10
1e+09
1e+08
1e+07
1000000
TCID50/g

100000
10000
1000
100
10
1
0.1
0 1 2 3 4
Day
 FIGURE LEGENDS

FIG. 1. Viraemia in pigs after infection. Titres are given on a log10 scale. The average of 2
pigs at each time point (symbols) was determined by infectivity titrations in BTY cells and
calculated from TaqMan data. The lines connecting the symbols are drawn as a higher order
polynomial interpretation of the data.

FIG. 2. Viral content in tissues calculated from TaqMan data. Titres are given on a log10
scale. Actual plotted data from the experiment consist of the average calculated from 2 pigs
and are depicted as symbols. The lines connecting the symbols are drawn as a higher order
polynomial interpretation of the data.
A) Results for ventral floor of pharynx (VFPH), soft palate, tonsil and nasal mucosa.
B) Results for tongue without lesions, tongue with lesions, skin of foot with lesions and skin
of foot without lesions.
 88

Appendix 9

IgA response of cattle to FMDV infection in probang and saliva

samples

Amadori M.1 , Haas B.2, Moos A.2 and Zerbini I. 1

1)Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia,

Brescia, Italy, and
2) Bundesforschungsanstalt für Viruskrankheiten der Tiere, Tübingen,
Germany.

SUMMARY
Two groups of cattle, previously employed in potency tests of O1
Manisa vaccines, were kept in the isolation stables after intradermolingual
challenge infection; they included both vaccinated (protected) and
unvaccinated, control animals. Probang samples were collected from day
36 to day 234 p.i. in the 1st group and from day 22 to day 169 p.i. in the 2nd
group. Saliva samples were collected from day 113 to day 234 p.i. in the
first group and from day 71 to day 162 p.i. in the 2nd group. Samples were
frozen and later employed in a kinetic ELISA for IgA antibody to O1
Manisa FMDV. Virus isolation (plaque test) and PCR were carried out on
the same probang samples. Two 1/1 dose vaccinated steers + one control
were IgA-positive at day 22 p.i., which was probably due to transudation
of blood IgA into the probang sample . There was a striking correlation
between virus detection and IgA response in the case of 3 vaccinated
animals, as opposed to the unvaccinated control, which was virus-negative
and IgA-positive. This points at the IgA response being correlated with
silent virus re-circulation among convalescent ruminants. In practice,
re-circulating virus could be checked by the immune system, but the event
could anyhow trigger a transient IgA response. Such a tenet would be
confirmed by the repeated virus detection in the head close to the
IgA-positive, virus-negative one. There was a complete discrepancy in one
animal between the results of the virological (+) and Ab tests (-) in
probang samples; however, plenty of saliva samples were IgA-positive.
Interestingly, the mucosal IgA response to virus structural proteins seemed
to be much more resilient than the serum antibody response in
FMD-convalescent cattle. In this respect, a drop of the mucosal IgA
response to borderline values could indicate the absence of a proper
 89

antigenic stimulus, i.e. the absence of a carrier state and/or nearby

infected animals. Owing to the above, we conclude that tests for mucosal
antibody to FMDV can provide useful information about the virological
status of convalescent ruminants and, in particular, about silent virus
re-circulation in the aftermath of an outbreak. Such a strategy is definitely
more reliable and robust than virus isolation and PCR tests on probang
samples.

INTRODUCTION
In a scenario of a major outbreak of Foot-and-Mouth Disease in Europe,
emergency vaccination may be resorted to as an adjunct to stamping out to
control the spread of infection ( 9 ). Under such conditions, it is
necessary to detect those animals (ruminants) which get infected after
vaccination and become persistent virus carriers; the latter may in fact give
rise to new flare-ups of the disease in the aftermath of an epizootic. Owing
to the above, a great research effort has been devoted to the establishment
of tests for antibody to FMDV non-structural proteins (NSPs), which are
only induced in principle by virus replication in the host. In this respect,
the test for Ab to the 3ABC polyprotein ( 4 ) is the most reliable single
indicator of response to NSPs on a herd or group basis ( 9 ). Therefore,
such a test can be recommended to identify virus-positive farms, but not
single virus-positive animals inside the farms. In fact, there is usually
very limited replication of FMDV in animals given a potent vaccine; as a
result, the latter may be negative for Ab to NSPs; this is further
demonstrated by the established correlation between repeated virus
isolation and Ab response to NSPs in vaccinated and challenge infected
cattle ( 8 ). Such an outcome of poor response to NSPs is extremely
likely, since the emergency vaccines to be used in Europe are very potent
(≥ 6PD50). Owing to the above, absolute differentiation of infection from
vaccination is not possible by serological means alone. On the other hand,
virus isolation from probang samples suffers major constraints and PCR
protocols are not suited for large-scale screening campaigns; these
protocols are in fact laborious and prone to give false positive results. An
interesting alternative is the detection of convalescent ruminants by
assessment of the IgA mucosal antibody response to FMDV ( 2 ). This
may be in fact a more robust approach, since FMD-vaccinated cattle do
not mount an IgA response in saliva, as opposed to vaccinated and infected
cattle ( 2 ); furthermore, there is evidence that such a response is rather
 90

stable and not intermittent like virus excretion in probang samples ( 2 ).

On the basis of these assumptions, our study aimed at establishing the
correlation between the carrier state and the mucosal IgA response after
vaccination and subsequent infection.

MATERIALS AND METHODS

Animals. Two groups of cattle, employed in potency tests of O1 Manisa

vaccines according to the European Pharmacopoea, were kept after
intradermolingual challenge infection; they included both vaccinated
(protected) and unvaccinated, control animals:

1st Group of cattle:

Infected on 12.04.99.
Numbers 9156 and 9165: unvaccinated controls (diseased).
Numbers 6257 and 6845: vaccinated with 1/1 dose (protected).

2nd Group of cattle:

Infected on 17.05.99.
Number 81195: unvaccinated control (diseased)
Number 87919: vaccinated with ¼ dose (protected)
Numbers 1367 and 1368: vaccinated with 1/1 dose (protected)

Sampling. Oesophageal-pharyngeal fluid (probang samples) was

collected using a probang cup. Samples were poured into plastic tubes and
stored at -70°C as soon as possible. Probang samples examined by the
plaque test were treated with a mixture of 1,1,2-trichlor-trifluoroethane
(«Arcton») and chloroform (3:1) by shaking for 15 minutes at 4°C. After
centrifugation, the aqueous phase was either stored again at -70°C or
examined immediately. Probang samples examined by RT-nPCR were
usually not treated with Arcton, but mixed with extraction buffer
immediately after thawing.
In order to obtain saliva samples from cattle in a reproducibile and
convenient way, a sampling system originally developed for humans
(Salivetten, Sarstedt No.51.1534) was used. It consists of a cylindrical
cotton plug and two plastic tubes. The cotton plug was put into the buccal
cavity for some seconds, after which it was saturated with saliva. It was
held by two fingers in order to prevent cattle from swallowing it. The plug
 91

was then put back into the inner tube, contained in the centrifugation tube.
Saliva was centrifuged (4,000 rpm, 5 minutes) through a hole in the
bottom of the inner tube and then recovered from the centrifugation tube.
Saliva samples were stored at -20°C.
Probang samples were collected from day 36 to day 234 p.i. in
the 1 group and from day 22 to day 169 p.i. in the 2nd group.
st

Saliva samples were collected from day 113 to day 234 p.i. in the
first group and from day 71 to day 162 p.i. in the 2nd group.

Cell Suspension Plaque Test (PT). BHK21-CT cells, a special

clone of BHK21 cells selected for optimal sensitivity to FMDV, were used
for the cell suspension plaque test on probang samples as previously
described ( 1, 7 ).

RT-nPCR on probang samples. Total RNA was extracted as

described by Chomczynski and Sacchi ( 3 ), with minor
modifications. Primers were derived (5) from the genome of strain O1
Kaufbeuren, spanning from N-terminus of polymerase 3D to C-terminus of
3A:
-external primers:
3C1 antisense CGCTCTTCCACATCTCTGGT ;
3A1 sense CCACAAGCTGAAGGACCCT.
- internal primers:
3C2 antisense GAGTGAGTGCCGACGATGAA;
3B3 sense CGCTTTGAAAGTGAAAGCTA.
Reverse transcription was carried out at 42 °C for 60 min in a reaction
volume of 25 µl containing 50 mM Tris-HCl, pH 8.3, 50 mM Kcl, 10 mM
MgCl2, 10 mM DTT, 0.5 mM spermidine, 1mM dNTP (each), 20 U AMV
reverse transcriptase (Promega), 20 U Rnasin (Promega), 100 ng of primer
3C1 and 12 µl RNA. The cDNA was either stored at -20°C or amplified
immediately. PCR was performed in a reaction volume of 50 µl of 20 mM
Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 1 mM of each dNTP, 1
U Taq polymerase, 100 ng each of primers 3C1 and 3A1 and 5 µl of
cDNA. PCR was carried out on a Trio-Thermoblock TM (Biometra ® )
using the following programme: 1) 1 min at 94 °C, 2) 30 sec at 92°C, 3) 30
sec at 60°C, 4) 1.5 min at 75°C, 5) 5 min at 75°C; steps 2) to 4) for 30
cycles. The PCR gave rise to a product of approximately 900 bp. This (1
µl volume) was amplified by a nested PCR with the internal primers, using
 92

the same protocol. PCR Products were electrophoresed on 1.5 % agarose

gels at 95 V and stained with 5 µg/ml ethidium bromide. All samples
showing a DNA band of the expected size (600 bp) were considered
positive.

Mucosal antibody. The IgA and total Ig response to FMDV was

assessed by kinetic ELISA, as previously described ( 2 ). The
threshold values were unified for the two tests: 0.05 ∆ OD (Ag-coated –
blank wells) together with 2 mOD/sec x 10-3 ∆ OD slope (Ag-coated –
blank wells). Samples reaching either threshold value were scored dubious
(±); they represent true borderline samples. By a comparative examination
of the test results, it was shown that the highest sensitivity could be
achieved by calculating the time/OD slope after the 0.1 OD405 value;
therefore, the largest contribution of the spurious, non-specific side-effects
probably takes place at the beginning of the colour reaction and is later on
the wane.

RESULTS
Results are shown in tables 1-8. They report the results of the tests for
IgA antibody to O1 Manisa and indicate the samples scored positive in
the Plaque Test, in PCR and also in the test for total Ig.

A) PROBANG SAMPLES, 1st Group of cattle

Number 9165, always PCR and Plaque Test-negative, was always

IgA-negative.

Number 9156, repeatedly PCR and Plaque test-positive, was always

IgA-negative, albeit with higher, borderline values at days 50 and 134 p.i.

Number 6257, always PCR and Plaque Test-negative, was three times
IgA-positive, i.e. at days 120, 183 and 204 p.i. Notice however that
virological data of this latter sample are actually lacking.

Number 6845, PCR-positive at days 57 and 71 p.i., remained IgA-positive

with wide fluctuations of the response till day 234 p.i. The significant ∆
OD values at days 43, 106, 169, 225 and 234 p.i. were not confirmed by
 93

the corresponding ∆ OD slopes; this result is probably due to alternate

peaks of response, with a drop to borderline values in between.

B) PROBANG SAMPLES, 2nd Group of cattle

Number 1367. Plaque test-positive at 22 and 29 days p.i. and

PCR-positive at 106 and 120 days p.i. It remained IgA-positive with very
high values till day 169. There was only one negative sample at day 57 p.i.

Number 1368. Plaque test-positive at 57 and 113 days p.i. and

PCR-positive at 113 days p.i. It was intermittently IgA-positive over the
whole experiment; from day 113 p.i. onwards the Ab response remained
stable. The Ab kinetics is very interesting; this animal was IgA-positive
at day 22 p.i. (following intradermolingual infection), then again at day
57 p.i. (Plaque test +) and day 113 p.i. (PCR+, Plaque test+). There is
obviously a strict correlation in this case between virological/PCR data and
results of the IgA test.

Number 81195. Always Plaque test and PCR-negative. It was repeatedly

IgA-positive with peaks at day 57 p.i. and again at day 155 p.i. A
fluctuating time course of the Ab response is evident, with peaks followed
by drops; this could be correlated with virus excretion by the other
infected cattle of the group.

Number 87919. Plaque test-positive at 22 and 43 days p.i.; also

PCR-positive at day 43 p.i. It shows two peaks of IgA activity at days 64
and 78 p.i.; it remains then IgA-positive from day 106 p.i. on . Notice that
the time course of the IgA response is almost parallel to that of steer 1368.

C) SALIVA SAMPLES, 1st group of cattle.

Number 9165, always PCR and Plaque Test-negative, was IgA-positive at

days 113, 127, 190 and 234 p.i.; the response at days 127 and 234 was
close to the threshold value. Once again, these transient IgA responses
could be the result of the exposure to the nearby infected cattle.
 94

Number 9156, repeatedly PCR and Plaque test-positive, was

continuously IgA-positive from day 120 to day 148 p.i., with a clear
correlation with virus detection in this same phase. The response was
fluctuating later on.

Number 6257, always PCR and Plaque Test-negative, was continuosly

IgA-positive from day 113 to day 155, with the exception of day 120
sample; the probang sample of the same day was however IgA-positive.
From day 176 onwards the animal turned positive again, a final major peak
being detected at day 234 p.i.

Number 6845, PCR-positive at days 57 and 71 p.i., was continuosly

IgA-positive from day 120 to 225 p.i. with very high mean titres and a
substantial agreement with the data of probang samples. Interestingly, both
the 1st and the last saliva samples were negative and a parallel decrease of
the IgA response occurred in the last phase of the experiment in both
probang and saliva samples.

D) SALIVA SAMPLES, 2nd group of cattle.

Number 1367. Plaque test-positive at 22 and 29 days p.i. and

PCR-positive at 106 and 120 days p.i. Always IgA-positive with the
highest titres observed in this study, in good correlation with the data of
the probang samples.

Number 1368. Plaque test-positive at 57 and 113 days p.i. and

PCR-positive at 113 days p.i. Always IgA-positive, with fairly high mean
titres, in good correlation with the data of the probang samples.

Number 81195. Always Plaque test and PCR-negative. It showed two

waves of IgA response, i.e. from day 71 to day 92 and, again, from day
127 to day 155 p.i. This is strikingly in agreement with the two peaks of
IgA response in probang samples at days 57 and 155 p.i.

Number 87919. Plaque test-positive at 22 and 43 days p.i.; also

PCR-positive at day 43 p.i. Always IgA-positive, with fairly high mean
titres and minor decreases of the Ab activity at days 99 and 134 p.i. The
 95

rise of the IgA response after day 99 p.i. was coincident with the steady
IgA activity in probang samples.

DISCUSSION
On the basis of our data, testing saliva for FMDV-specific IgA
instead of probang samples for the presence of virus would have two main
advantages. First, virus excretion in probang samples was shown once
again to be intermittent: samples were negative over weeks and then
became positive again; on the contrary, the IgA response in saliva of
carrier animals was much more constant and usually remained above the
cut-off level during periods when no virus could be detected. Secondly, the
throughput of an ELISA is obviously much higher than that of any
virological test or PCR protocol.
With regard to the 2nd group of cattle, it is interesting to note that the
two 1/1 dose vaccinated steers + one control (81195) were IgA-positive at
day 22 p.i., which is probably due to transudation of blood IgA into the
probang sample . There was a striking correlation between virus
detection and IgA response in the case of the 3 vaccinated steers, as
opposed to the unvaccinated control, which was virus-negative and
IgA-positive. This points at the IgA response being correlated with silent
virus re-circulation among convalescent ruminants. In practice,
re-circulating virus could be checked by the immune system, but the event
could anyhow trigger a transient IgA response. Such a tenet would be
confirmed by the repeated virus detection in the head close to the
IgA-positive, virus-negative animal (81195). A persistent IgA response
would suggest instead the insurgence of a true carrier state.
In the 2nd group of cattle, the results obtained in the IgA test on
saliva samples were consistent with those of the probang samples. On the
contrary, the situation was ill-defined in the 1st group of cattle. So, for
instance, in the case of steer 9156 there was a complete discrepancy
between the results of the virological (+) and Ab tests (-) in probang
samples. However, plenty of saliva samples were IgA-positive; in this
respect, two aspects deserve due attention:
- probang samples are characterised by a low IgA/IgG1 ratio and are
prone to blood contamination;
- different immuno-secretory compartments could be related to probang
and saliva samples, respectively, which could be differently stimulated
during transient exposure to infectious virus and the carrier state.
 96

The virological status of the animals was not adequately represented

by the IgA response in probang samples in the case of animals 9156 and
87919 (1st and 2nd group, respectively); an adequate picture was provided
instead by saliva samples.
In a scenario of emergency vaccination against FMD, it has been
suggested to collect blood samples from vaccinated animals in order to
investigate the Ab response to non-structural proteins (9); samples should
be collected about one month after the completion of the emergency
vaccination (9). The tests for mucosal antibody could complement the
assays on sera. Regrettably, no saliva samples were collected in this study
in the period 30 to 60 days p.i., which should span the above post
vaccination phase; in fact, no reliable means of collection was available
at that time in our labs for saliva.
Interestingly, the mucosal IgA response to virus structural proteins
seems to be much more resilient than the corresponding serum antibody
response in FMD-convalescent cattle; the latter often show a long-lasting
plateau of the Ab titre, which is barely affected by new exposures to a
FMDV strain of the same serotype. In this respect, a drop of the mucosal
IgA response to borderline values could indicate the absence of a proper
antigenic stimulus, i.e. the absence of a carrier state and/or nearby
infected animals.
Owing to the above, we conclude that tests for mucosal antibody to
FMDV can provide useful information about the virological status of
convalescent ruminants and, in particular, about silent virus re-circulation
in the aftermath of an outbreak. Furthermore, such an ELISA can
conveniently match virological tests for FMDV, which can only be
performed in high security laboratories. However, at least during screening
campaigns after a FMD epidemic, it can be justified to perform ELISAs in
regional laboratories. Because not all of them can perform a kinetic ELISA
with the existing equipment, it will be investigated if a non-kinetic ELISA
can be used as a screening tool, so that only non-negative samples should
be re-tested by kinetic ELISA.Such a strategy is definitely more reliable
and robust than virus isolation and PCR tests on probang samples. This
way, the NSP-negative, virus-carrier animals (8) could possibly be
identified and adequate control actions be taken in the farm; this can be
also inferred by comparing the paper by Archetti et al (2) with that of
Mackay et al (6), both dealing with the same group of FMD vaccinated
and infected cattle. To achieve this validation, future studies should be
 97

focussed on saliva samples collected in the period 30 to 60 days after

contact infection of vaccinated and non-vaccinated animals.

ACKNOWLEDGEMENTS
The skilful technical assistance of A. Cristiano and S. Hengst is
gratefully acknowledged.
REFERENCES
1) Ahl R. (1974), Arch. ges. Virusforschung, 46, 302-314
2) Archetti I.L. et al.(1995), J. Clin. Microbiol. 33, 79-84.
3) Chomczynski P and Sacchi N (1987), Anal. Biochem., 126, 156-159.
4) De Diego M. et al (1997), Arch. Virol., 142, 2021-2033.
5) Krebs O. and Marquardt O. (1992), J. Gen.. Virol., 73, 613-619
6) Mackay D.K.J. et al (1999), Rpt. Sess. Res. Grp. Stan.Tech.Eur.Comm.
Cont. FMD, Maisons-Alfort, France, Appendix 12.
7) Moss A. and Haas B (1999), J. Virol. Meth. 80, 59-67.
8) Sørensen K.J. et al. (1998). Rpt. Sess. Res. Grp. Stan.Tech.Eur.Comm.
Cont. FMD, Aldershot, UK, 1998, Appendix 21.
9) "Strategy for emergency vaccination against FMD", Report of the
Scientific Committee on Animal Health and Animal Welfare, European
Commission, DGXXIV, Brussels, 10.03.1999
 98

CONCLUSIONS

- The mucosal IgA response can be activated by an ongoing infection

of either the animal under study, or nearby infected animals.

- In this respect, the IgA immuno-secretory system could sense the

silent virus re-circulation among convalescent ruminants.

- The course of the response is different in probang and saliva

samples because of the exposure to blood contamination, the IgA/IgG1
ratio and the connection with distinct immuno-secretory
compartments. Saliva samples would be more indicative of a genuine
secretory IgA response.

- The mucosal IgA response of convalescent cattle to FMDV

structural proteins is by far more resilient than the serum Ab
response.

- The IgA response tends to drop in the absence of a proper antigenic

stimulus; this may be either a carrier state or nearby infected
animals.

- The detection of infected cattle by assessment of the virus-specific

mucosal IgA response is definitely more reliable and robust than
virus isolation and/or PCR tests on probang samples.

- The IgA tests could complement the tests for serum Ab to NSPs and
possibly solve the problem of the virus-positive/NSP-negative cattle
described in some reports.

- The kinetic ELISA can be used on a large scale. This demands the
development of a simple software aimed at saving the readings of
many plates and pasting the OD results of fixed positions in the
plates into time/OD series; proper statistical calculations
(regression and correlation) would then ensue to determine the
proposed threshold values of the reaction.
 99

Table 1. Number 9165 (1st group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

36 (-) 0.029* 0.5

43 (-) 0.022* 0
50 (-) 0.038* 0.5
57 (-) o 0.028* 0
64 (-) 0.012* 0
71 (-) o 0.039* 1
78 (-) o 0 0
85 (-) 0.027* 0
92 (-) 0.003 0
99 (-) o 0.020 1
106 (-) o 0.034 1
113 (-) 0.024 1 113 (±) 0.073 1.5
120 (-) 0.017 0.5 120 (-) 0.025 1
127 (-) o 0.013 0 127 (±) 0.048 2
134 (-) o 0.023 0.5 134 (-) 0.012 -0.5
141 (-) o 0.012 0 141 (-) 0.010 0
148 (-) 0.008 0 148 (-) 0.030 1
155 (-) 0.005 0.5 155 (-) 0.019 1
162 (-) 0.009 0.5 162 (-) 0.034 1.5
169 (-) 0.019 1 169 (-) -0.008 0.5
176 (-) 0.007 0 176 (-) 0.006 0
183 (-) 0.027* 1 183 (-) 0.046 1
190 (-) 0.002 0 190 (+) 0.098 3
197 (-) 0.014 0 197 (-) 0.023 0
204 (-) 0.014 0 204 nd nd
211 (-) 0.009 0.5 211 (-) 0.012 0
218 (-) 0.011 0.5 218 (-) 0.018 1
225 (-) 0.013 0 225 (-) 0.044 1.5
234 (-) 0.016 1 234 (±) 0.048 3
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 100

Table 2. Number 9156 (1st group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

36 (-) -0.026 -1
43 (-) 0.011* 0
50 (-) 0.046 1.5
57 (-) o v 0.015* 0.5
64 (-) 0.001 0
71 (-) o 0.003 0.5
78 (-) v 0.028 0.5
85 (-) 0.020 0.5
92 (-) 0.018 0
99 (-) v 0.016 0
106 (-) v 0.005 0
113 (-) -0.006 0 113 (-) 0.017 1.5
120 (-) -0.019 -1 120 (+) 0.077 3.5
127 (-) v 0.033* 1 127 (+) 0.064 2
134 (-) v 0.044* 0 134 (+) 0.094 3
141 (±) v 0.054 1 141 (+) 0.084 3
148 (-) 0.003 0 148 (+) 0.057 3
155 (-) -0.004* -1 155 (-) 0.016 0.5
162 (-) 0.031 1 162 (+) 0.063 3
169 (-) -0.035 -0.5 169 (-) 0.007 -0.5
176 (-) -0.004 -0.5 176 (+) 0.065 2
183 (-) 0.012 0.5 183 (-) 0.049 1.5
190 (-) 0.038 1 190 (-) 0.021 0.5
197 (-) 0.012 1 197 (-) 0.031 1
204 (-) 0.015 0.5 204 nd nd
211 (-) 0.027 1 211 (+) 0.068 2.5
218 (-) 0.019 1 218 (-) 0.034 0
225 (-) 0.011 0 225 (+) 0.100 4
234 (-) 0.028 1 234 (-) 0.044 1
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 101

Table 3. Number 6257 (1st group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

36 (-) 0.021 0
43 (-) 0.004 0
50 (-) 0.016 0
57 (-) o 0.018 0
64 (±) 0.050 0
71 (-) o 0.028 0.5
78 (-) o 0.011 0
85 (-) 0.008 -0.5
92 (-) 0.015 0
99 (-) o 0.019 0
106 (-) o 0.020 0.5
113 (-) 0.014 0.5 113 (+) 0.188 5
120 (+) 0.054 2.5 120 (-) 0.039 0.5
127 (-) o 0.010* 0 127 (±) 0.097 1.5
134 (-) o 0.031 0.5 134 (±) 0.061 1
141 (-) o 0.018 1 141 (+) 0.177 5
148 (-) 0.018 0.5 148 (+) 0.079 2
155 (-) 0.022 0 155 (+) 0.152 3
162 (-) 0.006 0 162 (±) 0.039 2
169 (-) 0.005 0.5 169 (-) 0.008 0
176 (-) 0.011 0 176 (+) 0.092 2
183 (+) 0.109* 2 183 (+) 0.092 3
190 (-) 0.005 0 190 (±) 0.047 2
197 (-) 0.007 0.5 197 nd nd
204 (+) 0.091 2 204 nd nd
211 (-) 0.016 0 211 nd nd
218 (-) 0.001 0 218 (+) 0.080 2.5
225 (-) 0.002 0 225 (±) 0.044 2
234 (-) 0.009 0 234 (+) 0.241 6
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 102

Table 4. Number 6845 (1st group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

36 (-) 0.036* 1
43 (±) 0.054 1
50 (-) 0.039 1
57 (+) o v 0.064* 2
64 (-) 0.027* 0
71 (+) v 0.109* 3
78 (+) o 0.066* 2
85 (+) 0.123* 3.5
92 (-) 0.040* 1
99 (-) o 0.047* 0
106 (±) o 0.052 1
113 (-) 0.023* 0 113 (-) 0.044 1
120 (-) 0.083 2 120 (+) 0.132 4
127 o Nd nd 127 (+) 0.204 5.5
134 (-) o 0.018* 0 134 (+) 0.552 14.5
141 (+) o 0.080 2 141 (+) 0.590 15.5
148 (-) 0.042* 1 148 (+) 0.619 16
155 (+) 0.061* 2 155 (+) 0.252 7
162 (-) 0.029* 0.5 162 (+) 0.376 10
169 (±) 0.051 1 169 (+) 0.070 2
176 (-) 0.025* 0 176 (+) 0.406 10
183 (-) 0.020 0 183 (+) 0.508 13
190 (+) 0.065* 2 190 (+) 0.370 10
197 (+) 0.234* 6 197 (+) 0.289 8
204 (+) 0.218 6 204 nd nd
211 (+) 0.139* 3 211 (+) 0.227 6
218 (-) 0.041* 0.5 218 (+) 0.223 5.5
225 (±) 0.060 1 225 (±) 0.053 1.5
234 (±) 0.065* 1.5 234 (-) 0.024 1
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 103

Table 5. Number 1367 (2nd group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

22 (+) o v 0.196 6
29 (+) v 0.309 8
36 (+) 0.094 2.5
43 (+) o 0.437* 11
50 (+) 0.342* 9
57 (-) o 0.031 1
64 (+) 0.572* 13.5
71 (+) 0.298* 7 71 (+) 0.744* 17.5
78 (+) 0.714* 17 78 (+) 0.483 12
85 (+) 0.760* 18 85 nd nd
92 (+) 0.519* 12.5 92 (+) 0.539 14
99 (+) 0.607* 15 99 (+) 0.405 10
106 (+) v 0.822* 20.5 106 (+) 0.497* 12.5
113 (+) o 0.619* 15 113 (+) 1.030* 24.5
120 (+) v 0.168* 4 120 (+) 0.692* 17.5
127 (+) 0.556* 14 127 (+) 0.251 5.5
134 (+) 0.515* 13 134 (+) 0.526 12.5
141 (+) 0.945* 23 141 (+) 0.078* 3
148 (+) 0.997* 24.5 148 (+) 0.810* 20
155 (+) 1.018* 25 155 (+) 0.566 13.5
162 (+) 0.797* 20 162 nd nd
169 (+) 0.961 24
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 104

Table 6. Number 1368 (2nd group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

22 (+) o 0.059 2.5

29 (-) o 0.014 0.5
36 (-) 0.033 1
43 (-) o 0.022 1
50 (-) 0.024 1
57 (+) v 0.067 2
64 (+) 0.085* 2
71 (-) 0.028 1 71 (+) 0.228 5.5
78 (-) 0.036 1.5 78 (±) 0.049 2
85 (+) 0.130* 4 85 (+) 0.108 3
92 (-) 0.037 1 92 nd nd
99 (±) 0.048 2 99 (+) 0.296 7.5
106 (-) o 0.034 1 106 (±) 0.062 1
113 (+) v 0.069 2 113 (+) 0.143 4.5
120 (+) o 0.067 2 120 nd nd
127 (+) 0.086 2.5 127 (+) 0.145 4
134 (+) 0.077 2.5 134 (+) 0.069 2
141 (+) 0.048 2.5 141 (+) 0.202 5
148 (+) 0.060 2 148 (+) 0.211 5
155 (+) 0.065 2 155 (+) 0.404* 10
162 (+) 0.169 5 162 (+) 0.152* 4.5
169 (+) 0.111 3.5
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 105

Table 7. Number 81195 (2nd group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

22 (+) o 0.059* 2.5

29 (+) o 0.058 2
36 (-) 0.025 1
43 (+) o 0.111 3
50 (-) 0.019 1
57 (+) o 0.515* 14.5
64 (-) 0.019 0.5
71 (+) 0.112 3.5 71 (+) 0.133* 4
78 (-) 0.010 1 78 (+) 0.123 4
85 (+) 0.094 3 85 (+) 0.131* 4
92 (+) 0.057 2 92 (+) 0.084 3
99 (-) 0.011 0 99 (-) 0.003 0
106 (-) o 0.011 0 106 (-) 0.026 1
113 (-) o 0.034 1 113 (-) 0.018* 1
120 (-) o 0.019 0 120 (+) 0.067 2
127 (+) 0.089 3 127 (±) 0.045 2
134 (+) 0.059 2.5 134 (±) 0.042 2
141 (±) 0.047 2 141 (+) 0.179 5
148 (±) 0.044 2 148 (+) 0.159 4
155 (+) 0.930 3 155 (+) 0.082 3
162 (+) 0.139 4 162 (-) 0.036 1
169 (-) 0.018 0
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive.
Results ≥ threshold values are printed in bold
 106

Table 8. Number 87919 (2nd group). IgA response to FMDV O1 Manisa

Probang samples Saliva samples

Post-infection day ∆ OD 405nm ∆ OD slope Post-infection day ∆ OD 405nm ∆ OD slope

(score) (mOD/sec x (score) (mOD/sec x
10-3) 10-3)

22 (-) v 0.013* 0
29 (-) o 0.017* 1
36 (-) 0.009 0.5
43 (-) v 0.025* 1
50 (-) 0.029* 1
57 (-) o 0.019 0
64 (+) 0.066* 2
71 (+) 0.053* 2 71 (+) 0.205* 6
78 (+) 0.154 4.5 78 (+) 0.090 2
85 (-) 0.029 1 85 (+) 0.106 2
92 (-) 0.031* 0.5 92 (+) 0.139* 3.5
99 (-) 0.020 1 99 (+) 0.054 2
106 (±) o 0.053* 1.5 106 (+) 0.077* 2
113 (+) o 0.055* 2 113 (+) 0.092* 2
120 (-) o 0.029* 0.5 120 (+) 0.141 4
127 (+) 0.063 2 127 (+) 0.109* 3
134 (-) 0.048 1.5 134 (±) 0.052 1
141 (+) 0.055* 2 141 (+) 0.121* 3
148 (-) 0.012 0.5 148 (±) 0.043 2
155 (+) 0.096* 3 155 (+) 0.181* 5
162 (+) 0.074 2.5 162 (+) 0.135* 3.5
169 (+) 0.060* 2
Score: + IgA- positive; - IgA- negative; ± IgA-dubious
o: plaque assay/pcr positive assay in the same group; v: plaque assay/pcr positive assay in the same animal;
* total Ig positive;
Results ≥ threshold values are printed in bold
 107
Appendix 10

The role of sheep in the epidemiology of foot-and-mouth disease and proposals for
control and eradication in animal populations with a high density of sheep.

Alex I Donaldson

Institute for Animal Health, Pirbright, Woking, Surrey GU24 ONF, England

Summary

This paper highlights the role of sheep in the epidemiology of foot-and-mouth disease
(FMD), drawing on examples from disease episodes in which sheep or sheep products have
been implicated as the source of infection both in transboundary epidemics and spread within
countries.

Disease control strategies are proposed, based on the epidemiology of FMD in sheep, and
directed at achieving specific objectives under different situations where sheep are the
dominant species in the livestock population.

Clinical signs of FMD in sheep

The clinical signs of foot-and-mouth disease (FMD) in sheep under natural conditions have
been described by Zaikin (1959) and Littlejohn (1970) and are illustrated in the AVIS
multimedia suite (AVIS 1999). It is of considerable epidemiological importance that the
clinical signs of FMD in sheep are frequently mild or inapparent (Geering 1967; Donaldson
and Sellers 2000).

The role of sheep in the transboundary spread of FMD

Sheep have often been implicated as disseminators of FMD virus, both between and within
countries. Examples of the involvement of sheep in the transboundary spread of FMD virus
include: the introduction of FMD into Canada by sheep imported from the UK in 1875
(Krystynak 1987); the 1978 and 1983 type A epidemics in Morocco (Donaldson 1999); and
the 1994 type O epidemic in Greece (Tsaglas 1995). Infected sheep imported from South
America were the most likely cause of the 1978 epidemic in Morocco (Dr H G Pereira,
personal communication; Bakkali 1982). In the 1983 Moroccan epidemic infected sheep
from Spain entered Morocco through the Spanish enclave of Ceuta and caused a chain of
outbreaks extending from the north of the country to Agadir in the southwest. Clinical
disease was identified only in cattle but a serological survey showed that many sheep and
goats had also been infected (Donaldson A I - unpublished results). The 1994 type O
epidemic in Greece was linked to the smuggling of sheep from Asiatic Turkey onto the Greek
island of Lesbos (Tsaglas 1995). FMD was not diagnosed initially on Lesbos, however,
when sheep were transferred to mainland Greece cattle became infected and the disease was
recognised. Yet another example of transboundary spread was the North African epidemic
of 1989-92. The epidemic started during the winter of 1989 in Tunisia and then swept
westwards into Algeria and Morocco. The majority of the spread was attributed to the
 108
uncontrolled movement of large numbers of sheep, especially around the time of religious
festivals when there was a surge in the demand for sheep meat (Samuel et al. 1999).

Excretion of FMD virus by sheep

Although FMD in sheep is often clinically silent, the amount of FMDV excreted by infected
sheep is significant, especially in the very early stages of infection during the viraemic phase.
Burrows (1968) found virus in oesophageal-pharyngeal samples, blood, milk, rectal swabs,
preputial swabs and vaginal swabs for up to 5 days before a clinical diagnosis of FMD could
be made. The usual mechanism by which infection is transmitted from sheep to other
species, most commonly cattle, is thought to be the aerogenic route i.e. infectious droplets and
aerosol particles excreted in the breath of infected sheep being inhaled by cattle. This is the
consequence of several determinants: firstly, sheep and cattle are often grazed together or are
brought into close proximity at markets; secondly, infected sheep excrete considerable
quantities of FMD virus in their exhaled breath from around 1 day before they show signs of
disease and for up to 4-5 days later (Sellers and Parker 1969; Donaldson et al. 1970); and
thirdly, cattle are highly susceptible to infection by airborne FMD virus (Donaldson et al.
1988).

A steep decline in virus excretion from sheep occurs around the fourth or fifth day of clinical
disease, the time when a circulating antibody response first becomes detected and presumably
results from immune clearance (Gibson et al. 1984; Pay 1988; Cox et al. 1999). Thereafter
the potential for sheep to transmit infection declines dramatically. Sheep, like other
ruminants, may become carriers. Around 50% of convalescent sheep may become
persistently infected for up to 9 weeks, and a small number of animals may carry virus for up
to 9 months (Burrows 1968; McVicar and Sutmoller 1969; Salt 1993). Carrier viruses
isolated from sheep have been found to have retained their pathogenicity for sheep, pigs and
cattle (Khukhorov et al. 1973; McVicar et al. 1968). Carrier sheep held under experimental
conditions have generally failed to transmit FMD but there are a couple of reports of
transmission of sub-clinical infection. In Germany carrier sheep did not transmit infection to
in-contact cattle but sub-clinical infection did result in one in-contact sheep (Geering 1967).
Similar experiments in India failed to transmit infection from carrier sheep to in-contact sheep
submitted to physical or chemical stress (Sharma 1978). There are apparently no
authenticated examples of carrier sheep being the origin of outbreaks although it has been
speculated that they may have been the source of the 1983 outbreak in Denmark (Dr E
Stougaard, personal communication).

Sheep products and the spread of FMD

Sheep products have been the origin of FMD outbreaks. For example, contaminated frozen
lamb (Aon-the-bone@) from Argentina was blamed as the source of the 1967-68 UK type O
epidemic, the largest ever recorded in the UK with a total of 2,364 outbreaks (Anon 1969).
This experience led the UK authorities to radically change the regulations for the importation
of meat from South America. These included a ban on all imports of unprocessed sheep and
 109
pig meat. Beef was accepted provided it was de-boned and the pH checked post-mortem to
ensure that it was sufficiently acid to inactivate FMD virus. Additional requirements were
imposed to ensure that cattle destined for slaughter were fully vaccinated. These procedures,
though viewed at the time by South American exporters as being very labourious and
expensive, allowed the trade in beef to continue without compromising the security of the
UK. The success of the policy over many years persuaded countries in other parts of the
world to follow suit and had a major impact in the global liberalisation of trade in beef.

Immunisation of sheep and evidence that FMD infection in sheep can be self-limiting

Sheep can be readily immunised against FMD with vaccines formulated either for routine
prophylactic or emergency use. Potent, oil-formulated, emergency vaccines can induce a
protective immunity in sheep against challenge by airborne homologous FMD virus within 4
days of vaccination (Barnett and Cox 1999; Cox et al. 1999). In circumstances where the
objective is to create an immune belt, for example when ring vaccination is implemented
around an outbreak or when a buffer zone is established to protect a disease free area, then
the vaccination of the sheep in the population is essential. However, experience from Kenya
and Uruguay suggests that the vaccination of sheep in control programmes in endemically
infected countries or zones is not justified and that a more effective strategy is to use available
vaccine in the cattle population to achieve the highest possible coverage in that species. In
Kenya, where the cattle population was routinely vaccinated but suffered occasional
outbreaks, Anderson et al. (1976) found in follow-up investigations that there was an almost
complete absence of virus carriers in both the sheep and goat populations, although there was
close contact with clinically infected cattle. It was, however, evident from the high
proportion which were seropositive that they had been exposed. Similarly, before the
eradication of FMD in Uruguay, where the sheep to cattle ratio is 2.6:1 and the two species
often graze together, the vaccination of sheep was abandoned as being both impractical and
uneconomical. Instead, available vaccine was used to increase the vaccination coverage of
the cattle population. This strategy was highly successful - outbreaks declined to zero in the
cattle population and there was no evidence of the continued circulation of virus in the sheep
population.

It can be concluded from these findings that in mixed cattle-sheep populations where the
cattle are immunised but suffer occasional outbreaks that infection in the sheep will be
self-limiting i.e. the Ro value will fall to less than 1.0 and so infection will not be sustained.
Ro, the basic reproduction rate, may be defined as the number of secondary cases arising
from a single primary case in the absence of any constraints on the spread of infection
(Macdonald 1952). Therefore, if Ro>1 each primary case will, on average, result in more
than one secondary case and infection will spread through the population. Conversely, if
Ro<1, each primary case will, on average, produce less than one secondary case and infection
will die out (see Haydon et al. 1997).

Even in mixed cattle-sheep populations where the cattle are fully or partly susceptible, there is
circumstantial evidence that infection in the sheep can be self-limiting. In the Greek 1994
epidemic a total of 95 outbreaks occurred over a five month period. The morbidity rate in
 110
sheep flocks declined during the course of the epidemic. At the start serological results
showed that around 65% of sheep in positive flocks had seroconverted but later in the
epidemic this dropped to around 5% (Mackay et al. 1995). Total stamping-out was employed
at the start of the epidemic but changed to partial stamping-out later (Tsaglas 1995).

Similarly, the 1989-92 North African epidemic began with a high attack rate, severe disease
among adult animals and a high mortality rate among lambs. However, as the epidemic
progressed the rate of mortality declined to a negligible level and serological investigations
detected only small clusters of seropositive animals in several flocks, suggesting that attack
rates within flocks had also fallen. In comparison with the epidemic in Greece these changes
occurred more slowly and were more closely associated with the implementation of control
measures such as vaccination (Samuel et al. 1999).

Laboratory investigation of the possibility that FMD infection in sheep can be

self-limiting

Experiments are currently in progress at IAH, Pirbright designed to test the hypothesis that
FMD outbreaks in sheep populations may be self-limiting. The progression of disease and
infection is being monitored as virus is transmitted from inoculated donor sheep through
groups of recipient sheep serially exposed by contact. The aims are to quantify the parameters
relating to virus transmission and virulence and to determine whether these determinants
change with serial passage. It has been found that the dose of virus administered to donor
sheep was critical and influenced their ability to transmit infection and the severity of disease
in both them and recipient in-contact animals. The results have demonstrated that the
relationship between the dose of virus, the quantity of virus replicated, the infectiousness of
the host and the rate of contact is complex (Gareth Hughes, unpublished results). The
influence of the strain of virus and host genetic factors are additional parameters which should
be examined to obtain a more complete picture.

Variation in virulence of sheep-adapted strains of FMD virus

 111
While host genetic factors are probably major determinants in the wide variability of the
severity of clinical signs manifested by sheep during FMD outbreaks it is likely that some of
the effect is due to inherent differences in the pathogenicity of the field strains involved.
There are numerous reports from the field of strains causing severe disease in sheep. For
example, the SAT 1 serotype during 1962-63 epidemic in the Middle East (Mackowiak 1970;
Nazlioglu 1972) and the A22 serotype in Iran (Hedjazi et al. 1972). In Botswana the SAT
1 virus strains were very virulent in sheep and goats, whereas the SAT 3 strains were not
(Falconer 1972). The strain which caused the 1989-92 epidemic in North Africa was very
virulent in sheep. The start of the epidemic in Tunisia coincided with the lambing season and
over 50,000 lambs succumbed. Cattle were also affected but the morbidity rates were low and
there was almost no mortality in that species. When the disease spread into Algeria and
Morocco sheep were predominantly affected. In Algeria the attack rates for the different
species were: sheep 95%; goats 3%; and cattle 3%. In Morocco the rates were: 92.7%; 7%
and 0.3%, respectively. The decline in the attack rate for cattle late in the epidemic was
attributed in part to the fact that in Morocco the cattle had been vaccinated previously, this was
not the explanation for Tunisia where the cattle had not been vaccinated before the start of the
epidemic (Samuel et al. 1999).

A feature common to the majority of these epidemics was that the strains of virus were exotic
to the country or region, the productivity losses among the sheep population were high, mostly
due to lamb mortality, and so a strong case could be made on economic grounds for
vaccinating the sheep population. By contrast, in situations where strains of low virulence are
circulating, for example when strains are endemically present, the lambs are likely to be
protected by maternal antibody, the economic losses will be lower and so the argument for
vaccinating the sheep will be weaker.

Proposed strategies for control and eradication

Based on the foregoing information a series of actions are proposed for the control and
eradication of FMD in animal populations with a high density of sheep (Table 1).

Acknowledgements

Paul Kitching and Soren Alexandersen are thanked for their helpful comments on the
manuscript.

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survey for antibody to FMD virus, Greece 1994. Report to FAO, 1995.

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des Epizooties 73, 703-714 (in French).

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responsible for epidemics of foot-and-mouth disease in North Africa. Epidemiology and
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of Hygiene 67, 671-7.
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European Commission for the Control of Foot-and-Mouth Disease, 5-7 April 1995, FAO,
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maintenance conditions. Veterinariya 36, 32-34.
Table 1: Proposed actions to control and eradicate FMD under different situations in animal populations with a high density of
sheep.

SITUATION OBJECTIVES ACTIONS

Buffer zone Create an immune belt to protect an Apply routine mass vaccination to all cloven-hoofed livestock* in the
FMD-free, non-vaccinated country/zone defined zone. Identify the vaccinated animals within the zone and restrict
from the risk of spread from an infected their movement by physical barriers and check-points. Prevent the
area. movement of animals and potentially infected animal products to the free
country/zone.
Surveillance zone Create an early warning zone between an Identify all cloven-hoofed livestock* in the zone and control their
FMD-free, non-vaccinated country/zone movement by physical barriers and check-points. Carry out regular
and a potentially infected area. clinical and serological surveys to determine the infectivity status of all
livestock* in the zone. Take measures to eradicate virus if infected
animals are identified.
Country or zone in Reduce the prevalence and economic Apply strategic routine mass vaccination to the large ruminant livestock
which FMD is losses due to FMD so that ultimately the species#, vaccinating the high producing animals as the first priority but
endemic strategy can be changed from disease extending the programme to other large ruminants according to available
control to virus eradication. resources. If virus continues to circulate in the food chain i.e. in pigs,
then include that species in the vaccination programme.

Control an outbreak caused by an endemic If the incidence of disease is high and there is no fund for paying
strain of virus. compensation, apply movement restrictions to the infected premises and
vaccinate all livestock* in the surrounding at risk area.

If the incidence of disease is low and a compensatory fund is available

apply stamping out and zoo-sanitary measures to the infected premises
and movement restrictions in the surrounding area. Verify that virus is
not circulating before removing the disease control restrictions.

Control an outbreak caused by an exotic Seek regional and/or international assistance and apply stamping out and
serotype or strain of virus. movement restrictions. Obtain an appropriate vaccine and vaccinate all
the livestock* in the surrounding at risk area.

115
Country or zone which Eradicate the virus following an outbreak. Apply total stamping out and zoo-sanitary measures to the infected
is free from FMD and premises. Establish protection and surveillance zones and restrict
does not vaccinate movement. Undertake a serological survey in the surveillance zone* to
determine whether virus is circulating. Maintain movement restrictions
until the absence of circulating virus has been verified.

Contain the area of infection and reduce Apply emergency vaccination to all livestock* in a zone beyond the at risk
the probability of spread beyond the at area. Identify and record all vaccinated animals. Maintain restrictions
risk zone. Appease public opinion against on the movement of all livestock* and animal products from the
the slaughter of animals. vaccinated zone until serological and clinical surveys have confirmed that
virus is not circulating.

* sheep included

# sheep not included

116
 117

Appendix 11

Possible Introduction of FMDV with sheep intestines from FMD countries

Bernd Haas*1 and Matthias Kramer*2

Federal Research Centre for Virus Diseases of Animals

*1
D-72076 Tübingen, Paul-Ehrlich-Str. 28
*2
D-16868 Wusterhausen, Seestr. 55

Abstract
In 1974 Boehm and Krebs showed that cleaned and salted intestines from FMD
infected sheep, used as casings for sausages, still contained virus. Lactic or
citric acid inactivated the virus within 5 minutes. However, usually no such
treatment is performed. The risk of introducing FMD into Europe by importing
sheep intestines is discussed.

Importation of intestines for casings

Germany imports about 70 000 tons of intestines for casings per year. Also Italy and Spain
import thousands of tons per year, and a significant part of this commodity comes from
non-EU-countries. According to an EUROSTAT statistic, in 1997 the EU imported 31959 tons
of intestines (commodity 050400, large and small intestines, including sheep and pig intestines,
bladders and stomach also included) from China, 18 536 tons from Brazil, 1256 tons from Iran,
550 tons from Pakistan and 378 tons from India. Even if some consignments from China may
have consisted of material originating in Europe, which had only been processed in China, there
are still thousands of tons of intestines per year entering Europe from FMD countries.

Neither the OIE International Animal Health Code nor Council Directive 92/118 EEC nor
Decision 94/187 (veterinary certificates for the importation of casings from third countries)
require any treatment that would reliably destroy FMDV. Neither washing nor salting or
drying, as required by Decision 94/187 inactivates the virus.

Treatment of intestines
The first operation in handling intestines (Grace et al.) is 'running', that is separating them from
the mesentery by means of a hand-operated knife. The next step is to run the intestine through a
'stripper' comprising large rollers (which resemble a laundry wringer), to squeeze out the
residues within the intestines. This step requires the use of a great quantity of water to wash the
casings and to keep the operation clean. The casing should then be soaked in water for
approximately 30 minutes at 38-42°C. In some areas of the world, casings then go through a
fermentation cycle. Intestines which have not been fermented are run through a crushing
machine and soaking tank. This breaks the intermucosal membrane and separates it from the
rest of the intestine. Next, the intestine goes through a mucosa stripper, which looks and acts
essentially like the 'manure' stripper above. Potable water is used to keep the operation sanitary.
Any remaining string-like material and mucosa are removed by rolling. After cleaning, the
casings are graded, salted and packed into barrels. Alternatively, they may be dried before
packing.

Temperature conditions and pH-values during production of sausages

 118

Sausages can be classed into three main groups according to production technology:
The first group, the cold smoked raw sausages, will not be heated. The pH will reach 4,5 to 6,0.
The second group, sausages destined for heating in simmering water, will be heated to about 80
°C for 45 – 60 minutes. It can´t be ruled out that some of these sausages reach the consumer as
half finished products before having been heated.
The third group are sausages prepared from precooked meat. Their pH will be about 6.2 to 6.6.
Filling the meat into the casings will usually be followed by a heating step. (Lücker, pers.
communication 2000; Gareis, pers. communication, 2000)

Experimental data on the survival of FMDV in casings

In 1975 Böhm and Krebs published an experiment, in which 5 sheep were infected intranasally
and perorally with O1Kaufbeuren virus. On days 2,3 and 4 p. inf. one animal was killed and the
organs were stored frozen for virological examination. Two animals were slaughtered on day 3
p. inf. following normal abattoir procedures. The small intestines of these two animals were
treated the way normally used to produce casings for sausages. The small intestine of the sheep
killed on days 2,3, and 4 contained up to 2.5 log 10 ID50 of FMDV per ml, irrespective of the
treatment (emptying, washing, removing the mucosa). Similar results were recorded for the two
animals slaughtered the normal way.
Salted as well as unsalted small intestines after 14 days still contained 2.5 log ID50 /ml of
FMDV. However, no virus could be found after treatment with either lactic acids or citric acids.
The mildest conditions used in the experiment was the application of a 0,5% solution of one of
these organic acids for 5 minutes, which inactivated the virus.

Discussion of risks
The probability of agent entry is determined by a country factor and a commodity factor
multiplied by the number of import units. Taking into account, that sheep infection may often
be overlooked and considering the facts mentioned above, is can be concluded that FMDV may
enter Europe in imported casings. However, the probability that FMDV present in imported
casings survives the production of sausages and is still present in finished products is small as
long as the usual procedures are properly followed. Thus, the risk that it reaches susceptible
animals via illegal feeding of sausages with swill is small, but not zero. However, the virus may
infect susceptible animals via offal from sausage production, trucks, fomites or staff.
Considering the amount of casing imported per year, it is recommended to study the feasibility
of measures to reduce the probability of agent entry, e.g. acid treatment of the casings or
restricting the countries from which casings can be imported.

Literature:

Gracey, Collins, Huey (eds.) Meat Hygiene, 10th ed., Saunders, London (page 133)

Böhm, H.O. und Krebs, H.; Nachweis von Maul- und Klauenseuche-Virus in Organen krank
geschlachteter Schafe (Detection of FMD-Virus in Organs of Slaughtered Infected Sheep) Berl.
Münch. Tierärztl. Wschr. 87, 410-412 (1974)
 119

Appendix 12

PECULIARITIES OF FUNCTIONING OF THE ANTI-FMD BUFFER ZONE FOR CIS

COUNTRIES.

V.M. Avilov, V.M. Zakharov, A.A. Gusev, S.A. Doudnikov, N.A. Yariomenko,
A.M. Rakhmanov, A.K. Karaulov.

I. Introduction
II. The epizootic situation in CIS countries and adjacent territories
A. The whole situation in former USSR
B. Russia
C. Transсaucasian Region
D. Central Asian Region
E. The extreme east region of Russia
III. The buffer zone in CIS countries – the history of the question
IV. The functioning of the pilot project for supporting the anti-FMD buffer zone in 1999 and
2000
V. The proposals and prospects
VI. Conclusions

I. FMD is the most important problem of livestock breeding because it severely

reduces the productivity of the meat and milk industries, losses in young stock, calves, lamb, and
piglets, and embargoes on the export of live animals and fresh meat to the FMD-free countries.
Even the sporadic FMD cases caused the economic loss due to additional restrictions and
quarantine measures, and the cost of additional tests and ring vaccination.
The FMD control program is basis on the complex of control measures, movement control
coupled with mass vaccination in countries with sporadic and/or endemic disease.
The blanket compulsory vaccination is necessary for FMD eradication in regions with vast
land borders adjacent to endemic regions and in mountain/steppe regions with transhumance. The
total FMD eradication and further prosperity are impossible without vaccination in such regions.
The global FMD eradication is not a realistic objective, that is why from 1996 FAO have
promoted control of FMD in regions supporting the international attempts and cooperation between
countries on a regional basis as the additional measures to national FMD control programs.

II. The FMD epizootology is almost a mirror image of the economic structure on the
post-Soviet space. There are three groups of countries/territories:
FMD-free group, FMD control regions, FMD endemic group.
A. There are two regions in former USSR (Baltic and Western) which are FMD-free since
1987.
B. Four FMD outbreaks were recorded in the territory of Russia in recent 10 years:
1990 - Tumen Region, type A, pigs, cattle;
1993 - Vladimir Region , type A, cattle;
1995 - Moscow Region, type 0, pigs;
2000 - Primorsky Territory, type 0, pigs.
 120

In all cases the disease was eradicated in primary foci of infection as a result of carrying
out stamping-out policy and ring vaccination of all susceptible animals at a radius up to
30 km.
It is worth describing in detail these last cases of the disease in Russia when FMD
vaccines including emulsion vaccines for pigs were used with a positive result both in
FMD outbreaks and in zones of risk.
During the FMD type outbreak in January 1990 on two small neighbouring farms (a
pig-farm and a cattle-farm) of the Tumen Region emergency vaccination of all FMD
susceptible animals including 50 000 pigs was carried out in the zone of risk. After the
extermination of all cattle (60 head) and pigs (46 head) involved in outbreaks, new cases
of FMD were not recorded in the Region. So, the immunization of animals including
pigs in the zone of risk provided protection against FMD.
Tumen Region was FMD-free and animals were not subjected to vaccination against
FMD for 20 years. As it turned out, shortly before the outbreak the timber enterprise
received hay from FMDV type A22 unfavourable area of Uzbekistan.
In the second case (Vladimir, 1993) the disease occurred due to the technogenic disaster
at the biofactory.
During the FMD type A22 outbreak in June 1993 in one of the settlements of the
Vladimir Region all FMD susceptible animals including 1500 pigs were vaccinated
directly on the spot and in the area under threat . After the extermination of an affected
animal and the remaining 16 contact animals (cattle, sheep, pigs) FMD was eradicated
in the primary focus of infection and did not spread. So, in that case the vaccination of
animals including pigs protected them against FMD.
The next FMD type O outbreak occurred on a pig-farm in the Moscow Region in June
1995. Five thousand and eight hundred pigs of different ages were housed in 15
buildings. The pig-farm was located very close to the meat-processing plant where meat
from China and South-Eastern Asia was processed. About 200 FMD affected sows and
more than 50 dead piglets were found in one of those buildings.
After FMD confirmation all sick animals were slaughtered and destroyed and the rest
were urgently immunized using the emulsion vaccine. Following the vaccine
application FMD affected pigs continued to be identified in 4 buildings for 6 days and
clinical signs were present in 290 pigs. In vaccinated pigs the course of the disease was
mild. In the same building 180 suckling piglets died. Owing to the vaccination and
induction of immunity in pigs other FMD cases were not recorded in 11 buildings of the
same pig-farm. Thus, the use of the emulsion vaccine protected the pig population
against FMD in 11 buildings located close to 4 affected ones. Three hundred and fifty
thousand of pigs of different ages were immunized with the emulsion vaccine in the
zone of risk and on other farms of the Moscow Region and no FMD cases were
registered.
The sequencing of the virus gene VP1 showed that the agent is closely related to those
of the pan-Asian group. The similar agent was isolated from meat processed in the plant.
All aforementioned information demonstrates close correlation between the outbreak
and introduction of the FMD agent with meat from the East-Asian Region.
Now about the last FMD type 0 outbreak. The FMD was suspected on the 15th of April
2000 in pigs on a pig-farm in Elitnoye village, Ussuriysk District, Primorsky Territory.
(The Territory was FMD free since 1964). The pig-farm is located close to the motor
road from China (the distance along the motor road from the pig-farm to the Chinese
border is about 70 km). The pathological material was collected from sick pigs and
delivered by air in the ARRIAH (Vladimir). On the basis of testings (serological tests
and PCR) and clinical signs the FMD type O1 was diagnosed. FMD notification was
sent to the OIE on the 17th of April 2000. In the bio-assay the agent caused in pigs and
 121

cattle FMD-specific clinical signs. By 17.04.2000 625 sows and piglets at the age of 1-3
months out of 965 pigs on the pig-farm were affected, 111 animals died. The supposed
date of infection is 10.04.2000. The pig-farm is located in the zone of regular
vaccination of cattle against FMD virus types O and A, that's why , because of the FMD
outbreak the revaccination of cattle and vaccination of pigs and small ruminants against
FMDV type O were urgently carried out in the Primorsky Territory (124,4 thousand of
cattle were revaccinated; 84,5 thousand of pigs and 26,1 thousand of small ruminants
were vaccinated). It should be noted that in the zone of risk around the affected pig-farm
the ring vaccination was carried out in pigs twice with an interval of 12-14 days and in
other zones of the Primorsky Territory the pigs were immunized once.
The quarantine measures were applied, movement control including control of animals
in preserves was introduced , quarantine and other preventive measures were introduced
along the border.
By 25.04.2000 299 pigs died and the remaining animals (736 pigs) were killed and
buried in the trench on the farm. New FMD outbreaks were not recorded. Since the
quarantine was lifted in 25.04.2000, the FMD situation in Russia is remaining
unfavourable; the information about this was sent to the OIE on 25.04.2000. The
eradication of this outbreak costed 8,7 min roubles (more than 300000 US dollars).
Thus, the FMD favourable situation in the Russian Federation was achieved owing to
the application of a complex of antiepizootic measures and, first of all, the use of FMD
vaccines, including emulsion ones, for vaccination of pigs.
The sequencing of the isolated agent showed that it belonged to the 0-pan-Asian
FMDV group circulating at present in the territory from Japan to Mongolia.
C. The Transсaucasian region is endemic for FMD. In Georgia there was the only one year
(1994) from the last ten years without FMD outbreaks, Armenia was FMD-free for only
three years from ten.
In 1996-97 all Trans Caucasian countries (Armenia, Azerbaijan, Georgia) had been
affected with FMD epizootic caused by FMDV type O.
From 1998 the FMD had been registered in Armenia and Georgia caused by FMDV
type A. The isolates from the both countries according to investigations conducted in
ARRIAH have been to be genetically different from other type A isolates and to be
antigenically distinct and outside the immune cover provided by existing vaccine
strains. Their relationship with Iran-96 isolate was revealed.
Today the territory of Georgia and Armenia are FMD unfavourable on FMDV type
Asia-1.
D. The FMD situation in Central Asian region remains aggravated and uncertain.
Nevertheless, the territory of Kazakhstan, Kyrgyzstan and Turkmenistan was
endemic for FMD.
An FMD outbreak in cattle and swine was detected in March, 1997 in two south
regions of Turkmenistan, boarding with Iran. The FMDV type O was recognized by
assay in ARRIAH (August,1999).
In 1997 another two FMD outbreaks were detected in Oshskiy region in Kyrgyzstan. In
October-November 1998 FMD outbreaks of type O in cattle were registered in three
settlements in Chuyskay region, and in July 1999 FMD was detected in 279 cattle and
298 sheep in Talasskay region.
Djambulskay and Alma-Atinskay regions (boarding with Kyrgyzstan and China) and
South-Kazakhstan and Kzyl-Ordinskay regions (boarding with Uzbekistan) were
unfavouable regions on FMDV type O in Kazakhstan during summer-autumn 1998.
The animals affected with FMDV type O were revealed again in 1999 in
Kzyl-Ordinskay region. Today FMDV type O is circulated among cattle and sheep
in five regions of Kazakhstan, including East-Kazakhstan and Karaganda regions,
 122

that is very dangerous for Russia, because animals in north regions of Kazakhstan and
boarding regions of Russia are not vaccinated against FMD and they have no strong
immunity to FMDV. The reasons of FMD unfavorable situation in this region were:
- shortcoming in access to an effective laboratory service capable of rapidly diagnosing
the disease
- shortage of vaccines and lack of a vaccination strategy
- a weak and ineffectual level of the FMD control measures
- inadequate control for movement of animals and products of animal origin
- lack of effective-acting veterinary legislation.
Transcaucasian and Central Asian countries have close relationships with countries of
Near East region, which are endemic for FMD types A, O and Asia-1. And such
unfavourable epizootic situation is constantly affected the CIS territory and even
European countries. FMDV type Asia-1 had moved to the west across Turkey territory
from December 1999, in summer 2000 it was near Bosporus, and now it is introduced in
Europe.
E. There is a persistent threat of FMDV introduction into the extreme east region of Russia
due to close economic cooperation with China, Vietnam, Malaysia and Thailand, which
are FMD endemic, and with South Korea, Japan and Taiwan, where the FMDV was
introduced in 1999-2000.
The preventive vaccination has been carried out for 20 years in Russian Federation
along boarders with China and Mongolia. So this region had an FMD favourable
situation.
The introducing of pig-adapted FMDV type O in the extreme east region of Russia is
very dangerous due to intensive trading links and vast land boarders in this region.
Particularly, the last FMD outbreak in Primorski Krai (April,2000) supports this point of
view, because the index case had appeared in non-vaccinated animals at the pig-farm.
But owing to control and quarantine measures and permanent cattle vaccination in this
region the disease was not allowed to spread and it was eradicated at the prime focus.

III. Accounting the epizootology of FMD at Near East (Turkey, Iran, Afghanistan)
during the USSR existence the anti-FMD buffer zone was created and supported on the territories of
all republics of Central Asia, Trans Caucasian and Northern Caucasus. Owing to that only the
sporadic cases of FMD had been registered in the Russia. The maximum number of FMD outbreaks
in the USSR during 1980-s had been 11 and they been registered mainly in Central Asian region.
After the break up of the USSR the general level of control measures and specific
prophylaxis had been decreased dramatically due to the economic situation. Certainly, the result
was the deterioration of FMD situation. The constant increasing of the number of FMD outbreaks is
seen in Transcaucasian (from 1 – 2 outbreaks during the 5 years period up to dozen outbreaks in a
year) and, similarly, in Central Asian region (from a single outbreak per year up to multiple
outbreaks recently).
Nevertheless the Russian Federation continues its national programme on maintainance of
FMD buffer zone in Transcaucasian region using about 12 million doses of bivalent type A and
O FMD vaccine annually. At present it allows to maintan favoulable situation on FMD in the
region. The last case of FMD was reported in the vicinity of Northern Caucasia region in 1983.
However because of the reduction of financial potentialities of Veterinary Service of the
Russian Federation the possibility of proper maintaining of this immunization zone is
problematical .
Destraction of existing buffer zone in Northern Caucasia region presents serious threat not
only to CIS-countries, but also to European countries. Additional factors, complicating the
situation in CIS-countries are:
 123

-intensive contacts of the countries of Transcaucasia and Central Asia with the FMD
enzootic territories of Turkey and Iran
-smuggling of animals
-tourism and pilgrimage
-mass ethnic migrations
-increased food dependence on foreign countries
-ethnic and military conflicts in the region.
In order to provide and maintain favourable situation on FMD in the territory of
CIS-countries and to prevent FMD introduction into Europe SVOs of CIS-countries (Azerbaijan,
Armenia, Georgia, Kazakhstan, Kirghizia, Russia and Uzbekistan) made a request in O.I.E and
FAO in October 1998 for financial support of project on FMD buffer zone creation.
Total number of livestock in the countries included in initially planned buffer zone,
number of animals subject to vaccination and required volume of vaccine were estimated.
As the cost of one dose of bivalent vaccine is US $ 0.35 total cost of FMD vaccine for
these purposes is US$ 15.4 million. Envisaged period of programme implementation is 3-5 years.
This idea was supported at XVIII Conference of O.I.E. Regional Commission for Europe
(Prague, Czechia, September 1998).
The problems of buffer zone organization and mechanizm of its financing were discussed
in detail at the Meeting of O.I.E. FAO and EC representatives with the participation of SVOs of
Azerbaijan, Armenia, Georgia, Kazakhstan, Russia, Turkmenistan, Uzbekistan and Ukraine
(Vladimir, Russia, November 1998) and also at the 62d Session of Executive Committee of FAO
European Commission of the Control of FMD (Oslo, Norway, 1998).
In the process of further consideration of the problem of financing FMD buffer zone in
CIS-countries (Brussels, Belgium, Fedruary, 1999; Moscow, Russia, March 1999; Rome , Italy,
April 1999) the inclusion of the countries of Transcaucasia and adjacent regions of the Russian
Federation only in buffer zone were envisaged at first stage due to large expenditures on the initial
variant of the project. Therewith Iran and Turkey were considered to be the most dangerous with
respect to virus introduction into Russia and then into European countries.

IV. The programme for FMD control in Transcaucasian region was worked out for the
realization of this project with the O.I.E./FAO/EC financial and scientific support. It has been
implemented since May,1999.
At first stage, May 1999-May 2000, the following measures were taken:
- ARRIAH delivered 1065 thousands of doses of bivalent (A-O) FMD vaccine to
Azerbaijan, Armenia and Georgia.
- -The volume of delivered vaccine was: Azerbaijan –350 thousands of doses (8 regions),
Armenia – 250 thousands of doses (5 regions), Georgia –300 thousands of doses (20
regions) and Russia –150 thousands of doses (1 Autonomous Republic).
- 80% of vaccine volume were used for immunization of cattle and small ruminants in the
regions adjacent to the south borders of the above- mentioned countries.
- Blood sampling was carried out for serologic testing in 2 regions of Armenia, 6 regions
of Azerbaijan and 8 regions of Georgia before the immunization.
- Repeated blood sampling was performed 4 months after the immunization
(March,2000).
- ARRIAH delivered supplies for blood sampling and for collecting and transportation
of pathologic material for virus typing.
- Republic Laboratories were provided with the kits for FMDV typing. The training
courses on collecting /transportation of pathologic material and on FMDV typing, using
complement fixation reaction were held.
 124

Antibodies to FMDV nonstructural proteins were detected in serum samples from great
number of animals tested before immunization. This fact causes great concern and suspicions on
FMDV circulation in the territory of Transcaucasia.
In autumn 1999 there was an outbreak of the disease in Adzharia (Georgia) caused by
FMDV type A, which involved 6 villages of 2 regions.
At the second stage, since May 2000 up to date:
- ARRIAH delivered 1000 thousands of doses of bivalent type A and O FMD vaccine to
Azerbaijan, Armenia and Georgia.
- The vaccine has been already used in Azerbaijan (300 thousands of doses, 8 regions). In
Armenia and Georgia it will be used for autumn vaccination campaign.
At the same time our concern about complicated epizootic situation in Transcaucasian
region has found confirmation during the project implementation:
- Two types of FMDV, type O and type Asia-1 circulate in the territory of Georgia
simultaneously.
- FMDV type Asia-1 circulates in the territory of Armenia.
- The FMD control measures are obviously inadequate. We have every reason to believe
that this will lead to the further FMD spreading over the territory of the region and
outside it.
According to the results of field visit in Transcaucasian region in July 2000 the main
disadvantages of pilot programme, implemented in Transcaucasia are:
- inadequate provision countries of the region with vaccine. Volume of vaccine provided
under the programme conditions constitutes 7% of national requirements in Azerbaijan,
19% in Armenia, about 30% in Georgia and less than 1.5% in Russia.
- inappropriate diagnosis
- ineffectual level of control and quarantine measures
- absence of the legislation on FMD prophylaxis and control including legal rights and
duties of both veterinarians and animal owners
- insufficient attention of veterinary services and livestock owner to the problem of
FMD control measures.

The great concern is caused by emergence of FMDV type Asia-1 in Georgia and Armenia
as vaccination against this type of FMDV has not been planned and carried out.

V. Spreading of FMDV type Asia-1 to the territory of Balkan Peninsula and Transcaucasia
necessitates solution of the problem of prompt additional delivery of vaccine against FMDV type
Asia-1 for ring vaccination and elimination of emerging foci of FMDV type Asia-1.
Since FMDV of exotic type, SAT-2, spreads to the north ( from Africa to Near East)
reserve of vaccine for ring vaccination in the zone of potential FMDV SAT-2 introduction,
elimination of focus /foci and maintenance of favourable situation on FMDV of exotic type is
required. This problem is subject to consideration.
We should consider the question of inclusion of Central Asian countries especially
Kazakhstan in FMD buffer zone due to the spreading of FMDV type O over the territory of
Kazakhstan to the north and FMD enzootic situation in Central Asian region.
The national programme supporting the anti-FMD buffer zone in Northern Caucasia region
and in extreme east region of Russia has been implemented for several years by the Russian
Federation. But due to economic reasons we are afraid that in 2001 Russia will fail to support this
national programme and that can aggravate the epizootic situation. So the Veterinary Department
of Russia applies for finding the possibility to strengthen the buffer zone in the countries of
former USSR.
According to the results of serological investigations and epizootic surveillance in
Transcaucasion region it will be necessary to change the seromonitoring strategy. In the aim of
 125

active control of epizootic situation it is appropriate to change this strategy and to collect blood
samples not only in the region, where the vaccination is performed according to the programme, but
to carry out the seromonitoring at all territories of the countries (Azerbaijan, Armenia, Georgia) on
the basis of stratification/randominisation.

VI. The FMD-situation along the south Russian border became worse last time , that
together with economic problems in these countries leads to the decrease of effectiveness of
veterinary measures aimed to the maintenance of FMD –favourable situation. In such situation the
probable FMD prognosis for the next year is rather anxious. We believe that it is appropriate to
enhance the responsibility of the regional veterinary officers together with the attention and
control from side of international organizations FAO/O.I.E./EC for measures to be taken.
 Appendix 13

Results of Seromonitoring in FMD Buffer Zone in the CIS Countries

T.A.Fomina, V.M.Zakharov, A.A.Gusev, N.E.Kamalova, S.R.Kremenchugskaya, O.A.Schekotova
All-Russian Research Institute for Animal Health, Vladimir, Russia

Summary
In total 2654 samples of cattle blood sera and 617 samples of sheep blood were studied for the presence of
FMD types A and O virus antibodies and for the presence of nonstructural polypeptides 3ABC during
spring-autumn 1999-2000 in Georgia, Armenia, Azerbaijan and in Volga region of Russian Federation.
In these Republics the FMD epidemiological situation is controlled by emergency vaccination of contact
animals using bivalent A-O vaccine supplied by ARRIAH.
About 48% of the examined animals had FMD type A antibodies and 41% - FMD type O antibodies.
During the expedition in 1999 89 animals had positive reaction with 3ABC antigene in three
Transcaucasian Republics (60 samples in sheep) and 67 animals had positive reaction (55 – in cattle and 12 – in
sheep) in 2000.
During the examination of 283 blood sera samples taken in three regions of Kalmykia and Krasnodar
Territory (202 – from cattle and 81 – from sheep) virus-specific antibodies to FMD types A and O virus were also
found. Positive reacting animals with nonstructural polypeptides 3ABC were not found in the above mentioned
regions of Russian Federation.

Introduction
Identification of infected animals in the herds and their differentiation from vaccinated livestock is one of
the tasks of effective control of FMD. This identification is also important in examination of small cattle sera
because the animals may have subclinical FMD and may infect the susceptible animals even if the agent present in
their organisms in very small amount (1, 2, 5, 9).
To solve this problem one can use the method of identification of nonstructural proteins antibodies (NP),
which are not formed in the blood after vaccination. Not less than 6 NP which may cause the formation of
antibodies found in reconvalescent sera (3A, 2C, 3AB, 3AC, 3D, 3ABC) are known (2, 4, 5, 7, 8, 9).
One of them, 3D or VIA antigene, connected with RNA-polymerase activity of FMD virus, independent
of agent’s type, was used at the end of 80-ies in different laboratories of the world to differentiate convalescent
animals in agar gel immunodiffusion test (5, 9). However, this method had very low sensitivity could give false
positive results, especially in the cases of revaccination or in the cases when concentrated vaccines were used. That
is why many FMD laboratories of the world use ELISA with recombinant 2C or 3ABC antigene, controlling the
antibodies formed against these antigenes in blood sera of different animals. This makes the control of
epidemiological situation more adequate.
The aim of this work was to carry out FMD seromonitoring in order to study the epidemiological situation
in Transcaucasian Republics and in the regions of Russian Federation that have the border with FMD unfavourable
regions and with great economic significance.
To reach this aim we used ELISA, virusneutralization test (VNT) and 3ABC trapping-ELISA.

Materials and Methods

Principles of blood sera sampling
During our business trip in 1999-2000 in order to take the blood samples we based on the following
issues:
- information about FMD outbreak in this region;
- regions that were included in the National Programme on FMD vaccination;
- regions having borders with FMD endemic zones where emergency vaccination is carried out;
- geographical position of the region, i.e. situation to the borders with Iran and Turkey;
- regions of Russian Federation were included into the study because they have borders with
Transcaucasian Republics (Krasnodar Territory and Abkhazia) and because they are situated in the
zone of military conflicts (Kalmykia, Dagestan, Chechnya). Besides, great amount of cattle is has
been sold from the regions of Kalmykia to the neigbouring Astrakhan, Volgograd and Rostov regions
of Russian Federation.
The blood was taken at random (10-15% from the tested animals) before immunization in accordance
with the plan of buffer zone functioning measures and also in 6-10 months after vaccination.
 Sera testing
All the sera were scrinning for the presence of FMD types A and O virus in liquid-phase blocking ELISA.
Positive in ELISA samples were studied in VNT for the above mentioned strains. The sera were considered
positive if the titre of antibodies was equal or above 1 : 16 in ELISA and in VNT. 746 samples (30%) were
examined for the presence of antibodies against nonstructural 3ABC polypeptides of FMD virus using modified
variant of ELISA developed in Istituto Zooprofilattico Sperimentale Della Lombardia e dell’Emilia-Romagna,
Brescia, Italy, and the kit of components kindly provided by E. Brocchi and F. De Simone for differentiation of
postvaccinal and postinfectional antibodies. Index of optical density more than 0,2 was considered positive for this
method.

Results
Antibodies against structural proteins
In total 990 blood samples from cattle and 236 samples from sheep were tested in Georgia, Armenia and
Azerbaijan in 1999. Among them 249 (25,15%) FMDV type A samples were found in cattle and 11 (4,6%) – in
sheep; FMDV type O – 257 (25,9%) and 54 (22,8%), respectively. Moreover, higher percent of positive samples
(41,4 and 40,5) was found in animals in Armenia. In Georgia these figures were 11,7 and 16,3% for FMDV types
A and O, in Azerbaijan – 8,5 and 21,6%, respectively, in animals of both species.
These data are shown in Table 1.
During the expedition in 2000 1566 blood samples of cattle and sheep in Georgia, Armenia, Azerbaijan
and also in Kalmykia and Krasnodar Territory of Russian Federation were taken after usage of bivalent FMD A-O
vaccine produced in ARRIAH. FMDV type A antibodies in Georgia were found in 56,6% of animals and 34,8% -
FMDV type O antibodies. In Armenia these figures were 72,5 and 70,5%, respectively, in Azerbaijan – 78,4 and
55,2%. Results of these studies are shown in Table 2.
Results of the study of FMD immune status of animals in the regions of Russian Federation using ELISA
showed that 80 and 66,6% of animals with positive reaction for FMD types A and O in Krasnodar Territory caused
by prophylactic vaccination of animals with vaccine produced by ARRIAH. In VNT these figures were 90,0 and
83,3%, respectively.
In Kalmykia these figures in cattle were 60,8% and 51,9% for FMDV types A and O, respectively.
Results of this study are shown in Table 3.

Antibodies to nonstructural polypeptides of FMD virus

In 1999 because of reduced number of components for 3ABC-ELISA 228 samples from cattle and sheep
were tested. Among them 88 positively reacting samples were found, moreover, 32,0% formed sheep.
Postinfectional FMDV antibodies were found in 4 localities (2 regions) of Georgia and in 3 localities
(Nakhichevan Autonomous Republic) of Azerbaijan.
In 2000 518 samples were studied for the presence of antibodies against NP of FMD virus in three
Transcaucasian Republics and in some regions of Russian Federation. None of positive samples were found in
Krasnodar Territory and in three regions of Kalmykia.
Among cattle and sheep 39 positive samples were found in Georgia, 16 - in Armenia and 11 - in
Azerbaijan. Half (21) of positive samples in Georgia are from Adigensk Region where in December 1999 an FMD
outbreak caused by type A virus (it was typed in ARRIAH) was registered. OIE Regional Reference Laboratory
for FMD in Vladimir didn’t receive samples from Armenia and Azerbaijan for typing.

Discussions
Transcaucasian countries are very often the zone of FMD introduction into southern part of Russian
Federation and then into Europe. Historical analysis showed that during USSR times a very effective method of
control for FMD using FMD vaccines, veterinary specialists’ training and so on, existed. When USSR collapsed
these methods became weaker, specially, very small percent of livestock was vaccinated. Taking into account all
mentioned above it is obvious that buffer zone organization in this region under the control of ARRIAH is an
extremely important moment.
Nonstructural proteins presence during FMD virus reproduction and their absence after vaccination may
become a reliable method for identification of animals with antibodies formed as a result of convalescence or
carriage of viruses from postvaccinal antibodies (1, 2, 3, 4, 5, 6, 7, 8, 9). In order to eradicate the infection one of
the most important conditions to solve the question about necessity of vaccination of animals against FMD in the
herds where live agent is circulating, available sensitive method is essential. Such method should give an
opportunity to identify the infected animals in vaccinated population. Modified variant of ELISA is such a method.
With the help of this method one can identify the antibodies against nonstructural proteins of FMD virus 3AB,
3ABC, 3D and others.
Distribution of sera positive for FMD virus in different regions of Transcaucasus and Russian Federation
was different and depended on the fact whether the animals were vaccinated or infected with the virus.
From the given results one can see that in 1999 not very high percent of immune animals in all three
Transcaucasian Republics was found among cattle and sheep, i.e. 25 and 11% for A type and 25 and 24% for O
 type, respectively. The highest percent of positive samples was identified in Ararat Region of Armenia (70,2 –
74,7%). This fact can be explained by the following: annually Armenia bought in ARRIAH 800 thousand doses of
FMD vaccine.
In 2000 after realization of buffer zone programme and usage of bivalent A-O vaccine this index
significantly increased in all Republics up to 56,6 – 78,4% for type A and 34,8 – 70,5% for type O, respectively.
As for the data on nonstructural proteins, identified with the help of 3ABC-ELISA, 88 positively reacting
animals were found. Among them 4 samples were found in Georgia, 41 – in Armenia and 43 – in Azerbaijan. That
can be explained by the fact that in August 1998 FMD outbreak caused by A type virus which in antigenic relation
is quite different from productional A22 strain (r1=0,22) was registered.
In 2000 the situation in Armenia and Azerbaijan became better, from 426 examined animals 16 and 11
positively reacting animals were found. In Georgia the number of positively reacting animals increased up to 39.
Perhaps, that can be explained by the FMD outbreak in Adigensk and Goriysk Regions caused by O type.
So, as a result of fulfilled work one can make a conclusion that seromonitoring carried out with the help of
ELISA and VNT gives an objective analysis of epidemiological situation in the region under control. In this
monitoring identification of antibodies against FMD virus nonstructural polypeptides, carried out with the help of
3ABC-ELISA which gives an opportunity to identify reconvalescent animals and carriages of virus in vaccinated
population, plays very important role.

Table 1: Results of FMD seromonitoring in Transcaucasian Countries, July 1999

Region Species of Total Positive A% Positive O% 3ABC

animals number of positive/total
samples number
Azerbaijan
Astarinsk region cattle 115 0 0 0/5
small cattle 25 0 0 0/0
Dzhulphinsky region cattle 45 24,4 13,3 4/6
small cattle 15 0 0 0/0
Shakhbuzsky region cattle 68 35,2 44,1 17/26
small cattle 20 10,0 0 1/1
Babeksky region cattle 50 0 14,0 5/14
small cattle 71 0 45,0 17/23
Sharursk region cattle 21 0 9,5 0/2
small cattle 13 0 7,7 0/1
Ordubadsky region small cattle 16 12,5 6,3 0/1
Georgia
Adzharia cattle 73 9,6 16,4 0/19
Borzhom region* cattle 54 31,4 22,3 1/7
Ahalkalak region cattle 13 7,7 0/0 -
small cattle 8 0 -
Gardabansk region cattle 44 3 8 0/8
Mtskhetsky region cattle 45 0 4,4 0/7
small cattle 13 0 7,6 0/0
Akhaltsikhsky region* cattle 45 22,2 42,2 3/15
Ozurgetsky region cattle 30 0 0 3/15
Armenia
Artashatsky region* cattle 90 18,8 28,8 2/10
small cattle 156 33,9 23,7 0
Ararat region* cattle 178 74,7 70,2 28/66
small cattle 61 14,8 32,7 11/17
Note: * - regions where animals with positive reaction in 3ABC-ELISA were found

Table 2: Results of Seromonitoring in Transcaucasian countries, April 2000

 Region Species of Number of A, % O, % Number of Positive
animals samples 3ABC reacting
3ABC
Georgia
Goriysk region Cattle 46 82,6 17,4 32 11
Adzharia Cattle 107 64,5 35,5 34 5
Gardabansk region Cattle 56 51,8 28,6 15 0
Small cattle 60 6,7 3,3 13 2
Adigensk region Cattle 56 78,6 87,5 41 21
Armenia
Aparansk region Cattle 80 63,8 71,3 30 5
Small cattle 20 30,0 100,0 20 6
Ararat region Cattle 93 74,2 67,7 10 0
Small cattle 7 42,9 85,7 0 0
Echmiadzinsk region Cattle 79 69,6 39,2 19 0
Small cattle 20 85 45 20 0
Abovyansk region Cattle 69 82,6 95,6 6 0
Small cattle 10 60 80 10 0
Shiraksk region Cattle 80 85 78,8 13 5
Azerbaijan
Shakhbuzsk region Cattle 64 84,3 50 25 0
Sharursk region Cattle 56 75 55,4 32 0
Small cattle 15 80 33,3 0 0
Babeksk region Cattle 88 63,6 40,9 27 2
Ordubadsk region Cattle 57 56,1 63,2 14 5
Small cattle 13 76,9 92,3 13 4
Dzhulphinsk region Cattle 45 75,6 44,4 15 0
Astarinsk region Cattle 162 84,6 54,9 37 0

Table 3: Animal blood sera study for the presence of FMD antibodies in Krasnodar Territory of Russian
Federation and in Kalmykia using ELISA

Region Species of Number of A22 O1

animals samples
Number of % Number of %
positives positives
Kalmykia
Gorodovikovsk region Cattle 45 38 84,4 25 65,7
Chernozemelsk region Cattle 97 32 32,9 31 31,9
Small cattle 60 3 5 0 0
Priyutnensk region Cattle 30 26 86,6 24 80,0
Small cattle 21 5 23,8 1 4,7
Krasnodarsk Cattle 30 27 90 25 83,3
Territory

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 131
Appendix 14

Sensitivity of primary cells immortalised by oncogene transfection for the isolation of

foot-and-mouth disease virus

N.P. Ferrisa, G.H. Hutchingsa, H.J. Moulsdaleb and J.B. Clarkec

a
Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF,
UK
b
European Collection for Cell Cultures, Centre for Applied Microbiology & Research,
Porton Down, Salisbury, Wiltshire SP4 0JG, UK
c
Q-One Biotech Ltd., Todd Campus, West of Scotland Science Park, Glasgow G20 0XA, UK

Summary

Primary cells of calf thyroid (CTY), calf kidney (CK) and piglet kidney (PK) have been
immortalised by oncogene transfection and their susceptibility to infection by foot-and-mouth
disease (FMD) virus (and swine vesicular disease [SVD] virus where appropriate) has been
compared with cultures of primary CTY cells and those of a permanent cell line of pig kidney,
IB-RS-2, which are routinely employed for vesicular virus diagnosis. To date 65 immortalised
cell lines (27 CTY, 20 CK and 18 PK) have proved stable upon repeated cell culture passage and
many support the growth of FMD (and SVD, in the case of PK cell lines) virus but none exhibit
either the degree of sensitivity or the specificity for all FMD virus serotypes exhibited by primary
CTY and IB-RS-2 cell cultures.

Introduction

The OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD) uses a
combination of virus isolation in cell culture and ELISA (Ferris and Dawson, 1988)
supplemented by RT-PCR (Reid et al., 1999) for the diagnosis of FMD. Primary calf thyroid
(CTY; Snowdon, 1966) cells and those of a permanent line of pig kidney (IB-RS-2; De Castro,
1964) are routinely employed for this purpose. Cell culture is more sensitive than ELISA for
antigen detection while primary CTY cells are generally the most sensitive system for the
detection of small quantities of FMD virus, and which are commonly encountered in submitted
clinical samples of poor quality. The use of IB-RS-2 cells help in the differentiation of SVD
from FMD and are often essential for the isolation of porcinophilic strains of FMD virus.
However, the population of FMD virus insensitive fibroblasts in the CTY cell monolayer rapidly
outnumber the sensitive epithelial cells upon cell culture passage necessitating the regular supply
of fresh calf thyroid glands from which to prepare primary cell cultures for diagnostic use. This is
expensive, labour intensive and requires skill and experience to produce suitable CTY cell
monolayers for use. The quality of IB-RS-2 cell batches has often been poor which may be
associated with cells being permanently infected with hog cholera virus. To try to circumvent
these problems, attempts have been made to produce permanent cell lines of CTY and other
primary cells by transfection using plasmids containing the SV40 large-T antigen oncogene or by
a Tetracyclin-inducible gene expression system encoding the oncogene human cmyc and to
evaluate the sensitivity of resulting cultures for FMD (and SVD, where appropriate) virus
propagation. It is required that such cell lines should retain their differentiated characteristics,
most importantly to include high susceptibility to FMD virus replication and to maintain stability
upon prolonged cell culture passage.
 132

Materials and methods

Cell immortalisation

Cells were disaggregated from calf thyroid glands, calf or pigley kidneys by dispase and attempts
were made to transfect the cells with the plasmids pUK42, pUKEδt and pUKEori (Kreuzberg-
Duffy and MacDonald, 1994) containing the SV40 large-T antigen oncogene by standard calcium
phosphate precipitation technique. Cell colonies resistant to G418 were isolated using ring
cloning techniques and sub-cultured into cell lines. An alternative Tetracyclin-inducible gene
expression system encoding the oncogene human cmyc
using a strontium phosphate transfection technique was also employed with CTY cells.

Growth of FMD and SVD virus in immortalised and other cells

Ten percent suspensions of vesicular epithelium representing each of the seven serotypes of FMD
virus were prepared as previously described (Ferris and Dawson, 1988). Other FMD virus
suspensions resulted from passage in BHK-21 cell cultures. Cell culture grown antigens and
epithelial suspensions were mixed with an equal volume of glycerol and resulting stocks were
subsequently stored at -20oC. SVD virus (resulting from IB-RS-2 cell culture passage) was
additionally used for comparing immortalised PK cell lines with IB-RS-2 cell lines. Ten-fold
dilution series of FMD (or SVD) virus were made in 0.04 M phosphate buffer and inoculated
onto cell monolayers (washed with phosphate buffered saline and overlaid with 2 ml of serum-
free Eagle=s maintenance medium) grown in plastic cell culture tubes (0.2 ml per tube, 3-5 tubes
per serial ten-fold dilution) and subsequently rolled continuously at 37oC. The cell cultures were
examined microscopically for evidence of cytopathic effect (CPE) daily for up to five days post
inoculation and 50% tissue culture infective doses (TCID50/ml) calculated. The specificity of
resulting random cell culture supernatant antigens was confirmed by ELISA (Ferris and Dawson,
1988)..

Results

Cell immortalisation

The only plasmid containing the SV40 large-T antigen which successfully transfected cells was
the plasmid pUK42 and 23 CTY, 20 CK and 18 PK cell lines have been established. A further 4
CTY cell lines have resulted from the Tetracyclin-inducible gene expression system encoding the
oncogene human cmyc. These cell lines have proved to be stable after subculture, many up to 30
passages.

Growth of FMD and SVD virus in immortalised and other cells

The sensitivity to virus infection of the immortalised cell lines in comparison with primary CTY
cells and those of the permanent cell line of IB-RS-2, as indicated by titres of virus stocks of each
of the seven serotypes of FMD virus (and SVD virus, where appropriate), are listed in Tables 1 to
5.

It can be seen from Table 1 that all the immortalised CTY cell lines were susceptible to FMD
virus serotypes O, A and C although the titres of the virus stocks achieved were lower in
 133
comparison with primary CTY and IB-RS-2 cells. However, there was marked variation between
cell lines in their susceptibility to virus of other types and to strains within a serotype, as
illustrated by the titrations of two type Asia 1 virus strains (and also demonstrated by the
susceptibility of immortalised PK cell lines to propagate two strains of SVD virus; Table 3)

There were instances of comparable sensitivity for detection of certain FMD viruses between the
immortalised cell lines of PK (SAT3 virus; Table 3), those of CTY immortalised by the
tetracycline-inducible gene expression system (SAT1 and SAT2 viruses; Tables 4 and 5) and the
control cultures of primary CTY and IB-RS-2 cells. However, the general pattern of lower virus
sensitivity and susceptibility to breadth of virus types of immortalised cell lines resulted.

Discussion

The investigation has shown that permanent cell lines of bovine and porcine cells can be
established by oncogene transfection which can retain the ability to propagate FMD virus.
However, none of the created immortalised cell lines have either the degree of sensitivity or the
breadth of susceptibility to different FMD virus serotypes and strains (or SVD virus) exhibited by
the current cells (primary CTY and IB-RS-2 cells) routinely employed for vesicular virus
diagnosis.

The publication of Clarke and Spier (1980) demonstrated that whilst some FMD virus strains
may grow strongly in a range of cell lines, other strains may be more fastidious, growing better in
some but less so or not at all in the same cell lines. Conversely, some cell lines can support
multiplication of a range of FMD virus strains whilst others can only support a few of these. The
immortalised cell lines currently described appear to fall into this latter category.

This present study and other work carried out at the European Collection for Cell Cultures,
Centre for Applied Microbiology & Research, Porton Down and elsewhere (Dr David Lewis,
personal communication) suggest that the techniques of oncogene transfection are largely
incompatible with maintenance of cell differentiation to include virus susceptibility.

Acknowledgements

The authors are grateful to Mrs Christine Reynolds and Mr Geoff Pero for maintenance of the
cell cultures. The work was supported financially by the UK Ministry of Agriculture, Fisheries
and Food (Project number SE 1113).
 134
References

Clarke, J.B. and Spier, R.E. (1980). Variation in the susceptibility of BHK populations and
cloned cell lines to three strains of foot-and-mouth disease. Archives of Virology 63, 1-9.

De Castro, M.P. (1964). Comportamento do virus aftoso em cultura de células : susceptibilidade

da linhagem de células suinas IB-RS-2. Archivos do Institúto Biológico, Sâo Paulo 31, 63-78.

Ferris, N.P. and Dawson, M. (1988). Routine application of enzyme-linked immunosorbent assay
in comparison with complement fixation for the diagnosis of foot-and-mouth and swine
vesicular diseases. Veterinary Microbiology 16, 201-209.

Kreuzberg-Duffy, U. and MacDonald, C. (1994). Analysis of SV40 early region expression in

immortalised mouse macrophage cell lines. In: Animal cell technology : Products for today,
prospects for tomorrow. Editors: Spier, R.E., Griffiths, J.B. and Berthold, W. Butterworth-
Heinemann Ltd., Oxford, pp. 80-82.

Reid, S.M., Hutchings, G.H., Ferris, N.P. and De Clercq, K. (1999). Diagnosis of foot-and-mouth
disease by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in
clinical samples. Journal of Virological Methods 83, 113-123.

Snowdon, W.A. (1966). Growth of foot-and-mouth disease virus in monolayer cultures of calf
thyroid cells. Nature 210, 1079-1080.
 135
Table 1
Titrations of cell culture grown FMD virus in routine cells of primary calf thyroid and IB-RS-
2 cells and cell lines of calf thyroid cells immortalised by plasmid pUK42 containing the
SV40 large-T antigen oncogene

Cell FMDV serotype

system
01 A24 C3 SAT1 SAT2 SAT3 Asia 1 Asia 1
Manisa Cruzeiro Indaial BOT 4/80 ZIM 5/81 ZIM 4/81 PAK 1/54 CAM 9/80

CTa/2 5.53b 5.2 6.53 - - 6.2 - 6.53

CT/3 5.53 5.2 6.87 - - - - 6.87

CT/4 5.53 5.53 6.87 6.2 - 5.53 - 6.87

CT/5 5.2 6.53 6.87 - - - - 6.53

CT/6 6.2 5.53 6.53 - - 6.87 4.2 6.87

CT/10 -? 5.2 6.2 3.2 - 5.2 -? 6.53

CT/11 5.53 5.87 6.2 4.53 - 5.87 - 6.53

CT/12 6.53 5.2 6.53 5.53 - 5.87 - 6.87

CT/13 5.2 4.87 5.87 2.53 - 4.2 - 4.2

CT/14 5.2 5.87 6.53 - - - - 6.87

CT/21 5.2 5.87 5.53 4.87 6.53 6.2 3.53 6.53

CT/22 5.87 5.2 6.53 5.2 5.87 6.2 3.2 6.2

CT/23 5.53 5.2 6.53 6.2 4.53 6.2 3.87 6.53

CT/24 5.2 4.53 4.53 3.87 2.87 - - 3.53

CT/25 5.2 5.2 3.87 3.2 - 4.2 - -

CT/26 6.2 5.87 6.53 5.2 5.53 6.53 3.2 6.2

CT/27 5.53 6.53 6.2 5.53 6.2 6.53 4.53 -

CT/28 5.87 5.53 6.87 5.2 4.87 6.2 ? -

CT/29 5.2 5.2 5.53 - - - - 2.53

CT/30 5.2 4.87 5.53 - - - - -

CT/31 5.2 5.87 6.2 - - - - -

CT/32 3.53 5.87 4.87 - - - - -

CT/33 5.2 5.53 6.2 - - 3.53 - 5.2

CTYc 7.21 7.07 8.65 8.0 8.07 8.21 8.53 8.4

IB-RS-2d 6.96 6.87 8.2 7.99 7.57 8.07 8.29 8.06

a
CT, calf thyroid cell line immortalised by plasmid pUK42 containing the SV40 large-T antigen oncogene
b
Virus titre, log10 TCID50/ml
c
CTY, primary calf thyroid cells
d
IB-RS-2, permanent cell line of pig kidney cells
 136
Table 2
Titrations of cell culture grown FMD virus in routine cells of primary calf thyroid and IB-RS-
2 cells and cell lines of calf kidney cells immortalised by plasmid pUK42 containing the
SV40 large-T antigen oncogene

Cell FMDV serotype

system
01 A24 C3 SAT1 SAT2 SAT3 Asia 1 Asia 1
Manisa Cruzeiro Indaial BOT 4/80 ZIM 5/81 ZIM 4/81 PAK 1/54 CAM 9/80

CKa/1 5.53b 6.53 6.2 6.87 6.53 7.2 4.53 6.53

CK/2 5.53 5.53 6.53 6.2 4.2 6.53 4.53 6.87

CK/3 3.53 5.53 6.2 6.87 6.2 6.87 2.53 6.53

CK/4 3.87 5.87 6.87 5.2 5.2 6.2 2.53 5.87

CK/5 5.2 4.2 6.53 7.2 6.87 7.2 5.87 6.87

CK/6 5.2 6.2 6.2 6.87 6.87 7.2 6.2 6.53

CK/7 5.2 5.2 6.53 6.53 6.53 7.2 5.2 6.87

CK/8 4.53 5.2 6.53 6.87 6.87 6.87 4.87 6.87

CK/9 - 4.2 6.2 - - - - -

CK/10 2.87 5.53 6.87 - - - - -

CK/11 5.2 - 5.2 - - - - -

CK/12 5.2 4.87 5.2 6.87 5.87 6.53 2.87 6.53

CK/13 3.53 4.2 5.2 - - 3.87 - 2.87

CK/14 - - 3.2 - - - - -

CK/15 4.87 - 4.53 - - 6.2 3.53 -

CK/16 3.2? 3.2? 5.53 - 2.0 2.53 - -

CK/21 3.53 - 5.2 - 5.53 - - -

CK/22 4.2? - 6.2 3.53 - 4.53 - 2.53

CK/23 - 4.2? 5.2 - - - - -

CK/24 - - 5.2 - - - - -

CTYc 6.47 6.98 8.14 7.54 7.69 8.1 7.07 8.6

IB-RS-2 d
6.17 7.01 7.95 7.59 7.36 8.15 6.64 8.14

a
CK, calf kidney cell line immortalised by plasmid pUK42 containing the SV40 large-T antigen oncogene
b
Virus titre, log10 TCID50/ml
c
CTY, primary calf thyroid cells
d
IB-RS-2, permanent cell line of pig kidney cells
 137
Table 3
Titrations of cell culture grown FMD virus and SVD virus in routine cells of primary calf
thyroid and IB-RS-2 cells and cell lines of pig kidney cells immortalised by plasmid pUK42
containing the SV40 large-T antigen oncogene

Cell FMDV serotype SVDV

system
O1 A24 C3 Indaial SAT1 SAT2 SAT3 Asia 1 SVDV SVDV
Manisa Cruzeiro BOT 4/80 ZIM 5/81 ZIM 4/81 PAK 1/54 UKG 27/72 ITL 1/94

PK/8a 4.2b 5.2 6.2 -? 6.53 -

PK/11 - - - - - - - - -

PK/12 3.87 4.2 6.2 4.2 3.53 6.2 4.53 -? -?

PK/36 - - - - - - - - -

PK/38 - - - - - - - - -

PK/39 - - - - - - - - -

PK/41 4.2 5.2 6.53 ? 3.2 6.53 4.2 ? ?

PK/42 3.87 4.2 7.2 6.2 5.53 8.2 4.53 - -

PK/43 5.53 4.53 6.53 4.53 4.2 7.87 7.2 6.87 -

PK/44 3.2 - - - - - 4.2 7.2 -

PK/45 6.2 3.2 6.53 4.2 4.2 7.53 5.53 6.87 3.53

PK/46 6.87 4.87 7.87 - - - 6.53 7.2 -

PK/47 4.87 ? ? ? ? 7.53 5.2 3.2 -

PK/48 5.2 - 6.2 ? ? 7.2 3.2 - -

PK/49 - 4.87 7.2 4.2 4.87 7.53 - - -

PK/50 6.2 3.87 6.87 4.2 3.87 8.2 5.2 6.53 3.87

PK/51 5.53 3.87 6.87 4.2 3.53 7.53 - 5.2 -

PK/60 3.53 3.87 7.53 4.2 3.2 7.53 - 3.87 -?

CTYc 6.87 7.01 8.28 8.2 8.2 8.2 8.45

IB-RS-2d 7.07 7.01 7.78 8.2 7.87 7.53 8.07 7.91 3.98

a
PK, pig kidney cell line immortalised by plasmid pUK42 containing the SV40 large-T antigen oncogene
b
Virus titre, log10 TCID50/ml
c
CTY, primary calf thyroid cells
d
IB-RS-2, permanent cell line of pig kidney cells
 138
Table 4
Titres of cell culture grown FMD virus in routine cells of primary calf thyroid and IB-RS-2
cells and cell lines of calf thyroid cells immortalised by a Tetracyclin-inducible gene
expression system encoding the oncogene human cmyc

Cell FMDV serotype

system
O1 A22 C3 SAT1 SAT2 SAT3 Asia 1
Manisa IRQ 24/64 Indaial BOT 1/68 ZIM 5/81 ZIM 4/81 PAK 1/54

CTYa 1 5.87b 4.53 5.7 7.95 6.53 5.53 4.53

CTY 2 4.2 3.2 5.45 7.45 7.2 4.53 4.53

CTY 4 5.53 4.2 5.7 7.87 6.53 5.2 4.53

CTY 5 5.53 3.87 5.53 8.53 7.2 4.87 4.87

CTYc 6.4 7.65 8.4 7.73 8.2 7.87 8.4

IB-RS-2d 6.53 6.53 7.4 8.31 7.53 7.2 7.73

a
CTY, cell line of calf thyroid cells immortalised by a Tetracyclin-inducible gene expression system encoding the oncogene human cmyc
b
Virus titre, log10 TCID50/ml
c
CTY, primary calf thyroid cells
d
IB-RS-2, permanent cell line of pig kidney cells

Table 5
Titres of epithelial suspensions of FMD virus in routine cells of primary calf thyroid and IB-
RS-2 cells and cell lines of calf thyroid cells immortalised by a Tetracyclin-inducible gene
expression system encoding the oncogene human cmyc

Cell FMDV serotype

system
O A C3 SAT1 SAT2 SAT3 Asia 1
BRA 1/92 IRN 7/97 Resende TAN 44/99 ERI 9/98 BEC 1/65 MAY 14/92

CTYa 1 3.87b 4.53 4.2 5.2 5.53 3.53 4.53

CTY 2 3.53 5.2 4.53 4.53 6.2 3.87 5.2

CTY 4 4.87 4.2 4.2 5.53 5.87 3.87 4.87

CTY 5 3.87 4.53 5.2 4.53 6.2 2.53 4.53

CTYc 6.73 7.73 6.53 7.59 7.2 6.87 6.53

IB-RS-2d 5.98 6.73 4.87 6.98 5.98 5.87 6.4

a
CTY, cell line of calf thyroid cells immortalised by a Tetracyclin-inducible gene expression system encoding the oncogene human cmyc
b
Virus titre, log10 TCID50/ml
c
CTY, primary calf thyroid cells
d
IB-RS-2, permanent cell line of pig kidney cells
 139

Appendix 15

Interferon and Foot-and-Mouth Disease Virus

Reinhard Ahl, formerly of the

Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany

Introduction:

Interferons belong to cytokines. They are glycoproteins with multifaceted signal effects on
cellular functions among which the antiviral effects belong to the early and non-specific
defence mechanisms of organisms against infections. Type I interferons are divided in
leukocyte interferon (interferon alpha) which is based on a family of at least 12 genes, and
interferon beta. Whereas man and mouse possess only one gene for interferon beta, ungulates
have up to 5 different genes for this type of interferon which can be produced by almost all
somatic cells in vivo and in vitro (Vilcek and Sen, 1996).

Research in interferons has proceeded now for more than 40 years, and this system has been
used successfully to elucidate the mechanism of action of cytokines. Three cascades of events
are necessary to establish an interferon induced antiviral state within cells:

- the cascade for transcriptional induction of the interferon genes preceding interferon
synthesis after exposure to virus or another inducer like poly I:C,
- the signal transduction pathway taking place when interferon gets in contact with the
specific cell receptor leading to activation of other inducible genes, and finally
- the chain of events when in interferon exposed cells the antiviral pathways are activated
after infection.

These pathways have largely been elucidated. In order to achieve a strictly regulated transient
response only proteins are involved in activation of interferon synthesis as well as subsequent
gene induction by interferon, but not small signal transduction molecules like cAMP. Progress
in knowledge has enabled researchers to design spectacular experiments to demonstrate the
importance of the interferon system as a defence mechanism against viral infections. Some
recent research highlights have illustrated the importance of the interferon system:

Whereas monocytes were considered for a long time to be significant producers of interferon
alpha in blood, it has recently been found that the main interferon alpha producing cells are the
precursors of type 2 dendritic cells able to produce up to 1000 times more interferon than other
blood cells after virus challenge in vitro (Siegal et al., 1999). After maturation, type 2 dendritic
cells stimulate T-helper cells to induce antibody production.

A Swiss group (Müller et al., 1994) generated genetically modified mice lacking the receptor
for interferon alpha and beta on the cell surface. Those animals were unable to cope with virus
infections. The same was shown with cell cultures derived from interferon type I receptor-free
mice. This experimental system was also used by M.Grubman and co-workers (Chinsangaram
et al., 1999) to demonstrate poor interferon production in bovine embryonic kidney cells after
infection with virulent A12 FMDV, as compared to a leader protease free variant, A12-LLV2.
 140

The specificity of the antiviral response was shown by the preparation of a mutant of the
interferon dependent 2’-5’ oligoadenylate dependent RNase (RNase L) which retained 2-5A
binding activity while lacking RNase activity. When expressed in murine cells it suppressed the
function of the normal 2-5A dependent RNase L, and the cells were unresponsive to the
antiviral activity of interferon for EMC virus (Hassel et al., 1993). The 2-5A synthetase –
RNase L pathway was shown elsewhere to be the most important one in the interferon mediated
inhibition of picornaviruses.

We were stimulated to work on interferon when in view of the variety of FMDV strains we
thought that interferon might be one factor responsible for the observed differences in virulence
and pathogenicity, as also indicated by R.F. Sellers (1964). Our approach was to use different
strains and mutants of FMDV for investigating the induction of and sensitivity to interferon in
secondary bovine kidney (BK) cells. Recent reports led me to present our rather old results
which were published in part and may be compared with those published recently by M.
Grubman and co-workers (see Chinsangaram et al, 1999).

Results and Discussion:

Bovine interferon beta inhibits the replication of FMDV in a strictly dose dependent manner,
and FMDV has a relatively high interferon sensitivity (Ahl and Rump, 1976). It was of interest
to see if interferon was able to influence the effect of substances and factors which by
themselves affect the growth of FMDV. It was found that in single cycle experiments interferon
beta increased the effect of non-optimal incubation temperature on virus growth, but the results
varied depending on the virus strain. In cell cultures pre-treated with a low dose of interferon
the number of infectious centres, i.e. cells producing virus, was reduced. Virus growth was
delayed but the rate of virus production and the amount of virus produced per cell was
unaltered. The inhibitory effect of guanidine, a well known inhibitor of picornavirus
replication, was increased by interferon at optimal growth temperature, but reduced at
sub-optimal temperature (Ahl, 1974). Likewise, intercalating drugs which are able to bind to
RNA like ethidium bromide, enhance the antiviral effect of interferon to FMDV.

Our work was encouraged by the detection and isolation of a mutant of strain O1 Lombardy
(O1L) of FMDV, O1Lif, which induced only a slight and sometimes transient cytopathic effect
and produced minute plaques in monolayer cultures of BK cells, presumably due to a strong
interferon induction or a higher sensitivity to interferon. It was not before FMDV-sensitive cell
lines like BHK-21 and IB-RS-2 which are unable to produce interferon, became available that
we could work satisfactorily with this mutant. We undertook an analysis of the biological
properties of the FMDV mutant O1Lif. The results were summarised in a doctorate thesis
(Rump, 1975). It was shown that the mutant differs in several markers from wild type O1L. It
proved to be more sensitive to uv- light irradiation, the growth cycle was slowed by 30 minutes,
and it was more sensitive to interferon at sub-optimal temperature. We could demonstrate that
the mutant replicated in BK monolayer cultures without inhibiting cellular protein synthesis,
but the reason for this was unknown. In the light of recent research it is reasonable to assume
that this could be due to a defect in the leader protease gene (Strebel and Beck, 1986; Piccone et
al., 1995).

A small series of experiments in animals showed that the mutant O1Lif was avirulent for pigs
infected intranasally by spray with a dose of 109 PFU of the virus. The pigs developed high
antibody titres and were immune to a challenge with wild-type O1L applied four weeks later.
Cattle infected intranasally with the mutant virus showed symptoms of a transient rhinitis and
 141

sporadic small vesicles at the entrance of the nostril. Samples of nasal mucus contained high
concentrations of interferon. The animals developed antibody titres of 1:512 against FMDV
strain O1L.

Using this virus and other interferon inducers like NDV, BTV or Poly I:C, we looked for
substances which were able either to enhance or to inhibit the production of interferon or its
action in our system. We thought that this could be useful for diagnostic work with FMDV in
cell cultures and for the assessment of the interferon system in the pathogenesis of FMD.

Guanidine hydrochloride, at 0.1 – 0.4 mg/ml (1.05 – 4.2 mM) in medium was found to enhance
the synthesis of interferon in FMDV infected BK cells, even when added after completion of
the viral growth cycle. This effect was therefore independent of the inhibition of FMDV
replication by the drug. The enhancement was specific in that it was not shown with
guanidine-resistant mutants and also not with other viruses like NDV or BTV. This finding
suggests that viral protein 2C which is the target for guanidine (Saunders and King, 1982) is
somehow involved in the regulation of interferon synthesis in FMDV infected BK cells, but the
mechanism underlying the effect of guanidine on interferon synthesis remains to be solved.
Different functions have been assigned to protein 2C in picornavirus infected cells (Pfister and
Wimmer, 1999), however, the mechanism of action of guanidine at the cellular level is still
unknown (Cho and Ehrenfeld, 1991). Unfortunately, the mutant O1Lif has not been analysed at
the genome level by cDNA sequencing which could give more information with regard to its
mutation(s).

We then looked for inhibitors of interferon production. In virus infected cells (FMDV, NDV)
interferon is predominantly produced “late” after infection, i.e. starting in BK cells at about 5
hours after infection and decreasing after 12 hours, provided the cells are not destroyed by the
virus. However, cells induced to produce interferon after treatment with a low dose of
interferon (primed cells) or cells induced with poly I:C switch to an “early” interferon
production cycle starting immediately after induction and slowly decreasing after 3 hours.

We found that inhibitors of S-adenosylmethionine-mediated methylation like cycloleucine

(l-aminocyclopentanecarboxylic acid) at a concentration of 0.5 - 2 mg/ml (3.87 – 15.48 mM)
blocked the synthesis of virus induced “late” interferon. The same result was obtained with
another methylation inhibitor, a combination of adenosine, L-homocysteine thiolactone and
erythro-9[3-(2-hydroxynonyl)]-adenine (Ahl, 1981).

Contrary to this, we found that the synthesis of “early” interferon was inhibited by retinoic acid
and retinal, the acid and aldehyde, respectively, of vitamin A. These drugs are known to exert
pleiotropic effects on cells and to promote the differentiation of embryonic cells as well as to
inhibit the cellular protein kinase C. In order to equilibrate the BK cells with the drug
concentration in medium cultures were pretreated with retinal at 20 µg/ml (0.065 mM) for
about two hours (Ahl, 1982).

A combination of cycloleucine and retinal almost abolished the production of interferon beta in
BK cells and enhanced the cytopathic effect induced by FMDV.

When BK cells were induced to produce interferon after induction by Poly I:C or by the mutant
O1Lif and shortly after infected with wild-type O1L, a decreased amount of interferon was
produced. This showed that wild-type FMDV could specifically inhibit the production of
interferon. Chinsangaram et al. (1999) arrived at the same conclusion working with virulent and
modified A12 virus.
 142

With the tools in hand we were able to produce high titre interferon preparations (up to 100,000
units per ml) using FMDV O1Lif plus guanidine, NDV strain CG, or BTV as inducers. From
this we started a programme to purify bovine interferon beta by a two step chromatography on
Blue Sepharose and Phenyl Sepharose. Peak fractions contained up to 6 million units of
interferon per ml with a 400-fold purification. This purified material was then used to inoculate
mice, rabbits and a goat in order to induce antibodies against bovine interferon beta (Ahl and
Gottschalk, 1986). Animals reacted after several injections of interferon with the formation of
high antibody titres (up to 1:50,000). This anti-interferon serum had an enhancing effect on the
replication of FMDV in BK cells. Growth and plaque formation of the mutant O1Lif as well as
wild-type O1L was considerably improved.

In summary, methods and tools have been made available to effectively reduce in BK cells the
effect of interferon which may interfere with virus isolation. This could be of importance for a
diagnostic laboratory when a homologous cell system is used for isolation of field virus
showing a moderate or low pathogenicity. It cannot be excluded that recently occurring FMDV
strains which appeared in far east Asia belong to this type of virus.

References:

Ahl, R. (1974) Arch. Gesamte Virusforsch. 46, 302-314

Ahl, R. (1981) J. Interferon Res. 1, 203-218
Ahl, R. (1982) Develop. Biol. Standard. 50, 159-166
Ahl, R. and Gottschalk, M. (1986) Methods in Enzymology 119, 211-220
Ahl, R. and Rump, A. (1976) Infect. Immun. 14, 603-606
Chinsangaram, J., Piccone, M.E., and Grubman, M.J. (1999) J. Virol. 73, 9891-9898
Cho, M.W., and Ehrenfeld, E. (1991) Virology 180, 770-780
Hassel, B.A., Zhou, A., Sotomayor, C., Maran, A., and Silverman, R.H. (1993) The EMBO
Journal 12, 3297-3304
Müller, U., Steinhoff, U., Reis, L.F.L., Hemmi, S., Pavlovic, J., Zinkernagel, R.M., and
Aguet, M. (1994) Science 264, 1918-1921
Pfister, T., and Wimmer, E. (1999) J. Biol. Chem. 274, 6992-7001
Piccone, M.E., Rieder, E., Mason, P.W., and Grubman, M.J. (1995) J. Virol. 69, 5376-5382
Rump, A. (1975) Thesis, Veterinary School, Hannover
Saunders, K., and King, A.M.Q. (1982) J. Virol. 42, 389-394
Sellers, R.F. (1964) J. Immunol. 93, 6-12
Siegal, F.P., Kadowaki, N., Shodell, M., Fitzgerald-Bocarsly, P.A., Shah, K., Ho, S.,
Antonenko, S., and Liu, Y.-J. (1999) Science 284, 1835-1837
Strebel, K., and Beck, E. (1986) J. Virol. 58, 893-899
Vilcek, J., and Sen, G.C. (1996) Interferons and other cytokines, , in Fields Virology
(B.N. Fields, D.M. Knipe, and P.M. Howley, eds.) Lippincott-Raven Publishers,
Philadelphia, PA., p. 375-399
148

Appendix 16

Type-independent detection of foot-and-mouth disease virus by ELISA using monoclonal

antibodies that bind to the aminoterminus of capsid protein VP2.

Brigitte Freiberg, Bernd Haas, Barbara Höhlich, Armin Saalmüller, Eberhard Pfaff and
Otfried Marquardt

Bundesforschungsanstalt für Viruskrankheiten der Tiere

Paul-Ehrlich-Straße 28, D-72076 Tübingen, Germany

Foot-and-mouth disease (FMD) is no longer endemic in Europe, but is occasionally being

introduced, for instance in July 2000 to Greece. Primary diagnosis consists in the notice of
symptoms. The suspect has to be confirmed by laboratory diagnosis which consists in virus
propagation, detection of FMD antigen by ELISA and of viral genomes by RT-PCR. In case that
seroconversion has already happened, the detection of FMD virus-specific antibodies adds to the
diagnostic tools.

While type-independent detection of viral genomes by RT-PCR is easily feasible, the detection of
FMD antigen in a sample by conventional ELISA is laborious, because FMD virus occurs as
seven distinct serotypes which require the use of specific reference sera. The diagnosis of FMD
antigen has substantially been improved as will be described below.

Panels of monoclonal antibodies (mAbs) were generated using the FMD isolates A22 Iraq/1964
(Freiberg et al., 1999), Asia1 Shamir-Israel/1989 (Marquardt et al., 2000) and SAT1
Zimbabwe/1989 (B. F., unpublished). Virus used for immunization was propagated in BHK cells,
concentrated by cesium chloride gradient centrifugation and inactivated with binary ethylene
imine as it is done to produce vaccine.

The mAbs were analyzed with regard to neutralizing activity and sensitivity of their epitopes to
treatment with trypsin. Thereby, non-neutralizing mAbs were identified in each panel that bind to
trypsin-sensitive epitopes (Tab. 1).

Table 1
149

Reaction of monoclonal antibodies with trypsin-treated FMD virus

mAbs neutralizing mg trypsin/4 mg virus (15 min/37oC)
activitya 0 20 200 400__
Asia1 panel
15F7 - 2.11b 1.01 0.72 0.47
16D6 - 2.33 0.40 0.21 0.21
16H12 + 2.57 2.57 2.28 2.19
SAT1 panel
13A6 - 1.53 0.19 0.17 0.19
20F8 - 1.21 0.99 0.84 0.55
10H2 + 1.92 1.89 1.60 1.08
A22 panel
9B4 - 1.26 0.16 0.15 0.13
18G11 - 2.01 2.14 1.36 1.22
2B1 + 2.26 1.85 0.12 0.11

a: -, no; +, yes in a plaque reduction assay; b: antigen dilution that accounts an OD value of 0.5 in
ELISA; bold, mAbs that bind to intertypically conserved epitopes (Tab 2).

The data summarized in Table 1 show that mAbs of each panel bind to trypsin-resistant epitopes.
This implies that preparation of the antigen used for immunization and in the ELISA resulted in
intact virus particles, because decayed FMD virus does not exhibit trypsin-resistant epitopes.
Furthermore non-neutralizing mAbs were contained in each panel.

All mAbs were investigated by ELISA for reactivity with all available FMD virus isolates.
Thereby, one non-neutralizing mAb was identified in each panel that reacted with all FMD virus
samples (Tab. 2). The samples are representative for six virus types and 25 separate lineages of
evolution as shown by nucleotide sequencing. Briefly, the ELISA was performed as follows.
Microtiter plates were coated with a mixture of sera from guineapigs immunized with the FMD
virus types A, C, O, Asia1, SAT1 and 2. Then, inactivated FMD virus was added in duplicate in
serial dilution, beginning with log10 = 1 in row A and ending with log10 = 3.1 in row H.
Thereafter, each of the pretitrated mAbs was added. Binding of the mAbs was detected by labeled
anti-mouse antibodies. Compared were the reactivities of those antigen dilutions which resulted
in OD values of 0.5, because this compensates differences in antigen concentration.

Table 2
150

Identification of mAbs that bind to all kinds of FMD virus

mabs from the Asia1 panel SAT1 panel
A22 Iraq panel___
FMDV isolates 15F7 16D6 16H12 13A6 20F8 10H2
9B4 18G11 2B1
A5 Bernbeuren/84 2.53 2.25 0 2.50 0 0 2.53 2.33 0
A10 Holland/42 1.88 0 0 1.88 0 0 1.70 1.91 0
A22 Pirbright 2.42 0 0 2.59 1.69 0 2.47 2.82 2.99
A24 Cruzeiro 1.44 1.96 0 1.45 0 0 1.20 0.89 1.78
A Castellanos/87 2.50 0 0 2.45 0 0 2.33 0 0
A Saudi Arabia/92 2.45 0 0 2.62 0 0 2.50 2.64 0
A Turkey 10/92 2.17 0 0 2.22 0 0 2.09 2.54 0
A Turkey 16/96 2.12 2.49 0 2.43 0 0 2.22 2.81 0
A Albania/96 2.20 0 0 2.16 0 0 2.12 2.62 0
A Malaysia/97 1.89 2.16 0 1.88 0 0 1.63 2.07 0
A Iran 2/97 2.12 0 0 2.13 0 0 2.12 2.34 0

Asia1 Shamir/89 2.48 2.88 2.96 2.47 0 0 2.34 0 0

Asia1 Bangladesh/87 2.74 2.94 0 2.79 0 0 2.65 0 0
Asia1 Bangladesh/96 2.54 2.06 2.06 2.55 0 0 2.54 0 0
Asia1 Thailand 5/96 2.68 3.10 0 2.59 n.d. 0 2.46 0 0

C1 Oberbayern 2.56 0 0 2.52 0 0 2.33 0 0

O1 Kaufbeuren/66 2.55 0 0 2.55 0 0 2.49 0 0

O1 Manisa TR/69 2.61 0 0 2.62 0 0 2.50 0 0
O Bangladesh 21/87 2.42 0 0 2.44 0 0 2.25 0 0
O Greece/96 2.86 0 0 2.77 0 0 2.74 0 0
O Bangladesh 3/96 3.13 0 0 3.06 0.92 0 3.09 0 0
O Iran 16/97 2.82 0 0 2.83 0 0 2.78 0 0
O Algeria 1/99 2.17 n.d. 0 2.49 n.d. 0 2.34 0 0

SAT1 Zimbabwe/89 1.76 0 0 1.87 1.60 2.21 1.25 0 0

SAT2 Zimbabwe/86 1.51 0 0 1.54 0 0 1.30 0 0

The non-neutralizing mAbs 15F7, 13A6 and 9B4 reacted with heterologous virus, other
non-neutralizing mAbs reacted to different extent with heterologous virus, but neutralizing mAbs
(Tab. 1) reacted rarely with heterologous virus. This is consistent with the mechanism of escape
from neutralization by codon-changing mutation fixation. No neutralization epitope can be
expected to be of intertypically conserved sequence.
The conclusion that the epitopes for the mAbs 15F7, 13A6 and 9B4 are intertypically conserved
was supported by the identification of their residence at the N-terminus of capsid protein VP2.
This was revealed by scanning peptides by ELISA (Tab. 3). As far as analyzed is this protein part
151

of conserved sequence. The lysines at positions two and three and the arginine at position 13 may
be substrates for trypsin.
Table 3
Identification of the intertypically conserved epitopes on FMD virus
Peptide sequence Designation/location 15F7 13A6
9B4

DKKTEETTlLEDRIl VP2 N-terminus 2.48 2.52

2.52
ETTlLEDRIlTTRng VP2 second 0 1.39
0

Capital letters in the peptide sequences indicate intertypically conserved residues, lowercase
letters variable positions. Putative trypsin cleavage sites are indicated in bold italics. Numbers are
explained in Tables 1 and 2.

It is evident that the epitope of mAb 13A6 is different from those of the other mAbs, because it is
represented by both peptides. Also the epitopes for the mAbs 9B4 and 15F7 are not identical
although being present on the same peptide, because their variable heavy chains differ in
sequence (Fig. 1).

Figure 1
Alignment of the heavy chain sequences of the mAbs 15F7, 13A6 and 9B4
. . . . . . .
LFLLTGTTGVHSEIQLQQSGPKLVKPGASVKVSCKASGYSFPDYNIYWVKQSHGNSLEWIGFIDPNNDEI 15F7
A...........H....D............T.A...SLTTTY.....R..Q......W...Y.G.T 13A6
...MAVVI.IN..V......AE..RS.....L..T...FNIK..YMH....RPEQG.....N...E.GDT 9B4
leader FW1 CDR1
FW2 CD

HYSQKFKGKATLTADKSSSTAFMHLNSLTSEDSAVYYCTRGLATYWGQGTLVTVSAARTT 15F7
I.N..........V.......Y..........TG....I.RMG 13A6
E.APR.Q....M...T..K..HLQ.S......T.....NA.Y-Y......TL...S.K..PPPVYPLAPG 9B4
R2 FW3 CDR3
FW4 constant

References:
Brigitte Freiberg, Mostafizur M. Rahman & Otfried Marquardt (1999). Genetical and
immunolo-gical analysis of recent Asian type A and O foot-and-mouth disease virus isolates.
Virus Genes 19, 167-182.
152

Otfried Marquardt, Mostafizur M. Rahman & Brigitte Freiberg (2000). Genetic and antigenic
variance of foot-and-mouth disease virus type Asia1. Arch. Virol 145, 149-157.
 152

Appendix 17

SEROTYPING OF FMDV WITH COAGLUTINATION TEST AS AN

ALTERNATIVE TO CFT AND ELISA

Gülnur ÜNVER1 , Feray ALKAN2

1
FMD Institute, PO Box 714, 06044 Ankara, TURKEY (correspondent address).
2
A.U., Faculty of Veterinary Medicine, 06110 Ankara, TURKEY.

ABSTRACT
In this study, the coagglutination test (COAT) was compared with ELISA and CFT, which are applied in Ankara
FMD Institute (Şap Enstitüsü) for diagnosis of FMD. The main point of the work was the application of the
COAT, using coaaglutination conjugate prepared by coupling the guinea pig anti- A22 Mahmatlı 65 and
O1Manisa 69 FMD viruses hyperimmune serum IgGs and protein A fragment of the inactivated Staph. aureus
Cowan I strain. Totally 120 cattle vesicular tongue epithelium and 270 sheep oesophageal-pharyngeal fluid (O/P)
samples were tested with COAT. The results of the CFT, ELISA and COAT applied to vesicular tongue
epithelium samples for validation of COAT indicated that the sensitivity of the test was high, whereas, the
specificity was low. On the other hand, COAT was also applied to O/P samples directly and to the culture fluids
of CPE produced samples following three blind cell culture passages. Consequently, the sensitivity and
specificity of COAT were determined rather high for both the O/P samples. It was concluded that COAT could
be used as a rapid, simple and economical technique in the diagnosis of the FMD and determination of the
carrier animals especially in the field surveys.

INTRODUCTION

One of the constrictive factors in the control of foot and mouth disease (FMD) in developing
countries is the long lasting diagnosis period. “Diagnosis period” means the time curse
between the first notification by the farmer and informing the field veterinary service about
the typing by the laboratory.

Due to rapid spreading nature, any suspicious case in the field should be considered as FMD
until the confirmation with a laboratory test. Although several reliable diagnostic tests such as
complement fixation test (CFT), enzymelinked immunosorbent assay (ELISA) and
polymerase chain reaction (PCR) already exist; none of them are applicable in the field
conditions.

FMD virus detection by coagglutination test (COAT) was first described in 1994 (9). Protein
A of Staph.aureus cellular membrane is used as solid phase immunosorbent linked to Fc
portion of most of the mammalian IgG and agglutinates. By this way it is used for the
detection of bacterial and viral antigens. Because of this peculiarity, it is used for the detection
of several bacterial and viral antigens (5,7,8,12.).

The present report describes the applicability of COAT in the field for rapid diagnosis of
FMDV and detection of carrier animals in comparison with CFT and ELISA.

MATERIAL & METHODS

Cell cultures: BHK21 cell-line (BHK21 An30 from Animal Cell Culture Collection of FMD
Institute, Ankara) was used for the cultivation of A and O type vaccine virus strains. Primary
 153

feotal lamb kidney (FLK) cells were used for virus isolation from oesophageal-pharyngeal
(O/P) fluid of the sheep.
Viruses: (a) Vaccine virus strains: A22 Mahmatlı 65 and O1Manisa 69 strains grown in
BHK21 cell-line were used for guinea pig hyperimmune serum production and as positive
control in the tests. (b) Field viruses: 120 vesicular cattle tongue epithelium samples collected
from the FMD suspicious animals. They were pounded in sterile mortar with sterile sand,
diluted in 9ml PBS with 1% gentamycin and centrifuged at 40 C 3500rpm for 30 min. The
supernatant was kept at -700 C in 1ml portions. (c) O/P fluid: Probang samples collected 2–3
months after the last vaccination from 270 sheep which were routinely vaccinated with O+A
bivalent FMD vaccine twice a year. These viruses either used directly or produced in FLK
cells with 2-3 blind passage (6).

Preparation of Staph.aureus Suspension: Staph.aureus Cowan I strain (A.U.Fac.Vet.Med.

Microbiology Dep.,Ankara) grown on blood agar was activated in brain-heart infusion broth
medium and inoculated to the tryptic soy broth (1%). Following 24 hours incubation in 370 C
the culture was centrifuged 30 minutes at 3500rpm.The sediment was washed 2 times by
centrifugation in PBS (0.03M phosphate, 0.12M NaCl, pH7.3) 15 minutes at 3500rpm.
Isolated bacteria were inactivated with 0.5% formaline sol. in PBS for 3h. in room
temperature and washed 3 times with PBS by centrifugation. Pure Staph.aureus was diluted
10% in 0.02% sodium azide containing PBS and kept at 40 C. The protein content of the
suspension was determined at 525nm wave length spectrophotometrically (1,9,11).

Conjugation of the Antibodies: Guinea pig hyperimmune sera against A22Mahmatlı and
O1Manisa strains of FMDV in three different quantities (125μl, 250μl and 500μl) was added
into 2ml of Staph.aureus suspensions (1/10, 1/100 and 1/200 dilutions respectively) and kept
in 370 C for 1.5h then centrifuged for 15min. at 3500rpm. Pellet was resuspended with 0.02%
sodium azide containing PBS, washed twice by centrifugation. As the last step, the conjugate
was resuspended in 2ml PBS with 0.02% sodium azide and 0.25% Tween20 then kept at 40 C.
Control conjugates were also prepared by the same procedure with FMDV antibody-free
guinea pig sera.

Specificity and Sensitivity Determination of Coagglutination Conjugates: The previously

described techniques were used for that purpose (9).

Coagglutination Test (COAT): Vesicular tongue epithelium samples and O/P fluid samples
were typed by using conjugates prepared with either A22Mahmatlı or O1Manisa hyperimmune
sera or normal guinea pig sera. Test was performed as follows: 25μl conjugate and 25μl test
sample mixed with a stick on a glass slide, kept at room temperature for 5–10 min. The
agglutination reaction was macroscopically inspected under indirect light on a dark
background (9).

In the case of non-specific reaction the samples were kept overnight at 40 C following mixing
with Stph.aureus. Next day they were centrifuged 30min.at 40 C, 3500rpm. The supernatants
were used as antigen in COAT. (11).

Complement Fixation Test (CFT): This test was applied for typing of the cattle samples
according to the conventional technique of Kolmer as described elsewhere.
 154

Indirect Sandwich Enzymelinked Immunosorbent Assay (ELISA): This test was used for
typing all of the samples as well as the cell culture isolated virus in accordance with the
previously published technique (10).

Sensitivity and Specificity of the Tests: The sensitivity and specificity of the tests, which
used for virus detection was calculated with the following formula (2):
Chi square (x2)
Relative sensitivity of the test = A/A+B
Relative specificity of the test = D/C+D

A = number of positive samples in both test,

B = number of samples negative in the first test but positive in the second,
C = number of samples positive in the first test but negative in the second,
D = number of negative samples in both test.

RESULTS

Preparation of the Conjugate: The titre of the guinea pig hyperimmune sera was 1/480 for
both serotypes. The adsorbans of Staph.aureus Cowan I strain in 1/10, 1/100 and! /200 were
3.625, 1.210 and 0.696 respectively. There was non-specific reaction with 1/10 dilution and
1/200 dilution didn’t react with the antigen. Whereas, the conjugates containing 250μl
hyperimmune serum and 1/100 Staph.aureus suspension were sensitive and specific for both
A and O types. In the case of conjugate preparation with 250μl hyperimmune serum the
results were not accomplished, also 500μl of serum performed false positive reactions with
negative control antigens (Table 1)

Table 1. Specificity of the anti- A22 Mahmatli and anti- O1 Manisa conjugates.
Viruses grown in BHK21 cell culture Control Antigens
O1 Manisa A22 Mahmatli PBS BHK21
*Conjugates
G S G S G S G S
Anti-O1 3+ 0.30 - - - - - -
Anti-A22 - - 3+ 0.22 - - - -
Control - - - - - - - -
.G : The strength of the coagglutination (3+ indicates the higher positive reaction).
S : The period passed until the initial coagglutination reaction (minutes, seconds).
* : Conjugates produced with 250μl guinea pig sera.

Complement Fixation Test (CFT): 113 out of 120 (94.16%) cattle vesicular-tongue
epithelium samples were typed with CFT. 25 (20.83%) of them were A and 84 (70%) were O
type. 4(3.33%) samples were positive for both A and O (Table: 2).

Enzymelinked Immunosorbent Assay (ELISA): 106 out of 120 (83.33%) cattle tongue
epithelial samples were typed. 26 (4.81%) of them were A and 66 (55%) were O type.14
(11.66%) samples were positive for both A and O types (Table: 2). 13 out of 270 (4.81%) O/P
fluid samples were typed with ELISA. 1 (0.37%) of them was A and 8 (2.96%) of them were
O type and 4 (1.48%) were A+O (Table: 3).

Coagglutination Test (COAT): 115 out of 120 (95.83%) cattle tongue epithelium samples
were typed. 28 (23.33%) of them were A, 73(60.83%) of them were O and 14 (11.66%) of
them were A+O (Table: 2). 16 out of 270 (5.92%) of the O/P fluid samples were typed with
 155

COAT. 1 (0.37%) of them was A, 12 (4.44%) were O and 3 (1.1%) were A+O. Non-specific
reaction was detected in 9 O/P fluid samples with COAT. They were tested following
overnight Staph.aureus treatment. After that 1 sample was O type positive, 7 were negative
and 1 sample was still non-specifically reacting. Tissue culture passages of the O/P fluids
were also tested with COAT. 18 out of 270 (6.66%) samples were typed. 1 (0.37%) was A, 13
(4.81%) were O and 4 (1.48%) were A+O (Table: 3).
Table 2. Typing results of the 120 vesicular cattle tongue epithelial samples with CFT, ELISA and COAT.
CFT ELISA COAT
Serotypes A O A+O A O A+O A O A+O
Positive results 25 84 4 26 66 14 28 73 14
(20.83%) (70%) (3.33%) (4.81%) (55%) (11.66%) (23.33%) (60.83%) (11.66%)
TOTAL 113 106 115
(94.16%) (83.33%) (95.83%)

Table 3.Comparison of the typing results of CFT and ELISA with COAT applied to vesicular tongue epithelial
samples.
Number of Samples CFT ELISA COAT
104 + + +
6 + - +
3 - - +
3 + - -
2 - + +
2 - - -
TOTAL 120 113 106 115

Table 4.Typing results of 270 O/P fluid samples with ELISA and COAT.
ELISA COAT *COAT
Serotypes A O A+O A O A+O A O A+O
Positive results 1 8 4 1 12 3 1 13 4
(0.37%) (2.96%) (1.48%) (0.37) (4.44%) (1.1%) (0.37%) (4.41%) (1.48%)
TOTAL 13 16 18
(4.81%) (5.92%) (6.66%)
ELISA: ELISA applied to the samples following 2-3 blind passages in FLK cell culture.
COAT: Direct application of COAT to O/P fluid samples.
*COAT: COAT applied to the samples following 2-3 blind passages in FLK cell culture.

Sensitivity and Specificity of the Tests: In comparison of COAT, CFT and ELISA with
cattle tongue epithelial samples, COAT was found as higher sensitivity but lower specificity.
However for O/P fluid samples COAT was more sensitive and specific than CFT and ELISA
(Table: 5, 6, 7, 8).
Table 5.Comparison of the typing results of CFT and ELISA with COAT applied to vesicular tongue epithelial
samples.
Number of Samples CFT ELISA COAT
104 + + +
6 + - +
3 - - +
3 + - -
2 - + +
2 - - -
TOTAL 120 113 106 115
 156

Table 6.Comparison of the typing results of ELISA with COAT applied to O/P fluid samples
Number of Samples ELISA COAT *COAT
12 + + +
1 + - -
4 - + +
2 - - +
251 - - -
TOTAL 270 13 16 18
ELISA: ELISA applied to the samples following 2-3 blind passages in FLK cell culture.
COAT: Direct application of COAT to O/P fluid samples.
*COAT: COAT applied to the samples following 2-3 blind passages in FLK cell culture.

Table 7 Sensitivity and specificity of CFT and ELISA in comparison with COAT for typing of vesicular tongue
epithelial samples.
CFT ELISA
+ - S% S’ % + - S% S’ %
COAT (+) 103 6 28.57 97.34 103 9 45.45 98.11
COAT (-) 10 1 3 5
Total 113 7 28.57 97.34 106 14 45.45 98.11
S % : Relative specificity.
S’ %: Relative sensitivity

Table 8. Sensitivity and specificity of ELISA in comparison with COAT for typing of O/P fluid samples
ELISA
+ - S% S’ %
COAT (+) 12 253 98.44 92.30
COAT (-) 1 4
*COAT (+) 12 252 100 88.88
*COAT (-) 1 5
Total 13 257
ELISA: ELISA applied to the samples following 2-3 blind passages in FLK cell culture.
COAT: Direct application of COAT to O/P fluid samples.
*COAT: COAT applied to the samples following 2-3 blind passages in FLK cell culture.

DISCUSSION

Rapid diagnosis of FMD is very important factor in control of the disease. The covansional
techniques such as ELISA, CFT and PCR can only be applied in a laboratory designed for this
purpose. Sampling, transferring the specimens to the laboratory, testing and information needs
at least 2 days. If the matter is detection of the carrier animals this period prolong up to 15
days because of the tissue culture passages.

The potential application of COAT for the direct diagnosis of FMD from infected cells and
tissues were described previously (9). In this study COAT was applied to the vesicular
epithelium samples from the animals with FMD symptoms and O/P fluid of the sheep. It was
aimed to standardise the criteria of the reagent preparation for COAT and estimate the
sensitivity and specificity of the test.
 157

The second target of this study was to apply the COAT directly to the samples taken from the
susceptible animals for rapid diagnosis in the field.

Preparation and standardisation of the coagglutination conjugate against FMDV has an

important role in COAT’s sensitivity. In the production of hyperimmune serum the animal
species is important. Binding capacity of the total IgG fraction to Protein A is variable
depending on the animal species. Guinea pig IgG1 and IgG2 both strongly bind to protein A
whereas the other immunoglobulins do not bind (3). In this study by using guinea pig
hyperimmune sera, IgG purification step could be neglected.

It seems there is no problem in the susceptibility of CFT or ELISA when applied to the
freshly isolated vesicular epithelial samples. Cellular breakdown material conversely effects
ELISA. The elapsed lesions, samples transported or stored in poor conditions may cause false
reactions in ELISA. Although, cross receptions are common for CFT, it can rarely happen
with ELISA. The samples, which contain high amount of 12S subunit and virus infection
associated (VIA) antigen, can be diagnosed as positive with CFT but negative with ELISA.
Just contrary some samples which contains high amount of 146S antigenic particles may
positively react with ELISA but in CFT they may not show any reaction. The reason is the
ELISA can detect even very small amount of 146S particles in the sample. So, it is
recommended to apply both CFT and ELISA in the diagnosis laboratories for FMD (4).

In this study 10 cattle tongue vesicular epithelial samples were typed as O and 3 were A
serotype of FMDV with COAT but they were not detected with ELISA. Similar results were
obtained with O/P fluid samples. This can be attributed to the higher sensitivity of COAT to
12S subunits than ELISA (9).

The non-specific reactions of the O/P fluid samples could be removed with 1 exception by
Staph.aureus treatment, which is in agreement with the findings of the other authors (11).

In the present study COAT was found highly sensitive and specific for O/P fluid samples and
sensitive but weakly specific for vesicular epithelial samples.

CONCLUSIONS

1- Preparation of conjugate for COAT is an easy procedure,

2- The practitioners can apply COAT even in the field conditions. It doses not need a
sophisticated laboratory.

3- FMDV detection and typing can be done in a few minutes with COAT.

4- COAT can be used in the primary diagnosis of FMDV for both infected animals with
vesicular lesions and especially with O/P fluids of the carrier animals. But it should be
confirmed with CFT and/or ELISA in the well-equipped diagnostic laboratories.
 158

REFERENCES

1- Akay, Ö., Izgür, M., Esendal, Ö., Çetin, C. (1990): Serogroupping of the streptococcus
strains isolated from cow milk, Turkish Scientific&Research Council, Veterinary
Medicine&Animal Husbandry Research Group, Project No. VHAG-670.
2- Bailey, NT. (1981): Statistical methods in biology.2nd ed. Northumberland Press Ltd.
London.
3- Goding, WC. (1978): Use of Staphylococcal protein A as an immunological reagent. J.
Immunol. Methds, 20 241-253.
4- Hamblin, C., Armstrong, RM., Hedger, RS (1984): A rapid enzymelinked immunosorbent
assay for the detection of foot and mouth disease virus in epithelial tissues. Vet.
Microbiology, 9,435-443.
5- Kessler, SW. (1975): Rapid isolation of antigens from cells with a staphylococcal protein
A antibody adsorbent parameters of the interaction of antibody-antigen complex with
protein. The Journal of Immunology, 6, 1617-1624.
6- Kitching, RP and Donaldson. AI. (1987): Collection and transportation of specimens for
vesicular investigation. Rev. sci. tech. Off. Int. Epiz, 6. 263-272.
7- Kronvall, G. (1973): A rapid single agglutination method for typing pneumococci by
means of specific antibody adsorbed to protein A containing staphylococci. J.Med.
Microbiol.6, 187-190.
8- Mishell, BB., Shigi, S. (1980): Selected methods in cellular immunology, W.H.Freeman
and Company New York, ISBN 0-7167-1106-0.
9- Montassier, H.J., Araujo, J.P., Pinto, A.A. (1994): Rapid coagglutination test for the
detection and typing of foot and mouth disease virus. Journal of Virological Methods.50,
29-42.
10- Roeder, PL, Smith, LB (1987): Detection and typing of FMDV by ELISA a sensitive,
rapid and reliable technique for primary diagnosis. Reseach in Veterinary Science, 43,
225-232.
11- Skaug, K. Figenskhau, J., Orstavik, I. (1983): A rotavirus staphylococcal co-agglutination
test. Achta Path. Microbiol. Iummunol. Scand., 175-178.
12- Suksanong, M., Dajania, S. (1977): Detection of Hemophylus influenza type B antigens in body fluids using
specific antibody coated staphylococci. Journal of Clinical Microbiology. 81-85.
 159

Appendix 18

Evaluation of a chromatographic strip test for foot-and-mouth disease virus

antigen detection

Scott M. Reida, Anke Brüningb, Nigel P. Ferrisa, Geoffrey H. Hutchingsa and Lennart Åkerblomc

a
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey,
GU24 ONF, UK
b
Present address: University of Leeds, School of Biology, Miall Building, Leeds, Yorkshire,
LS2 9JT, UK
c
SVANOVA Biotech, Uppsala Science Park, Glunten, S-751 83 Uppsala, Sweden

Summary

Routine diagnosis of foot-and-mouth (FMD) virus is carried out at the WRL by the combined use
of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase
chain reaction methods. These techniques require dedicated and expensive laboratory facilities
and skilled personnel. A rapid test for the detection of FMD virus antigen using ClearviewTM
chromatographc strip test technology was developed and evaluated at the OIE/FAO World
Reference laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright. The strip test
devices rapidly (after 10 minutes test incubation) and specifically detected FMD viral antigen in
nasal swabs and probang samples from naturally or experimentally infected animals, in epithelial
suspensions of clinical material submitted to the WRL for diagnosis and in their cell culture
passaged supernatant fluids. The devices were more sensitive then the ELISA for the diagnosis of
all seven serotypes of FMD virus in the suspensions of epithelium, nasal swabs and probang
samples and matched the sensitivity of the ELISA for detection of the virus in cell culture. These
results indicate that such strip test devices could potentially achieve a more immediate diagnosis
at the site of a suspected FMD outbreak to allow control procedures to be effected more rapidly.
Such pen-side diagnosis would be particularly relevant to FMD control programmes operating in
endemic regions such as South East Asia and would increase disease awareness in other regions
where disease control may be limited.

Keywords: Rapid diagnosis; Foot-and-mouth disease; Chromatographic strip test

 160

Introduction
Routine laboratory diagnosis of the vesicular viruses of foot-and-mouth disease (FMD), swine
vesicular disease, vesicular stomatitis and vesivirus (which includes vesicular exanthema of
swine virus and San Miguel sea lion virus) largely depends upon the shipment of the epithelium
(the preferred diagnostic specimen) or vesicular fluid from the scene of the suspected outbreak to
the diagnostic laboratory. Transport costs and other delays in transmission of the samples to the
laboratory and often compounded by poor communication and harsh geographical terrain,
inappropriate pH of transportation medium and adverse environmental temperatures during
shipment.

In the WRL for FMD, 70-80% of the positive specimens are diagnosed by ELISA on suspensions
of clinical material; the remaining 20-30% of field samples being diagnosed positive after virus
amplification in cell culture. The latter procedure may require up to four days for completion
(two cell passages each of two day durations) and thereby delay the implementation of correct
control procedures. Virus isolation also requires laboratory cell culture facilities which can be
difficult and expensive to maintain. RT-PCR procedures, while extremely useful as a diagnostic
tool in conjunction with ELISA and virus isolation (Reid et al., 1998; Reid et al., 1999; Reid et
al., 2000, in press), require skilled personnel as well as PCR-dedicated, contamination-free
laboratory space and specialised equipment such as thermocycler machines.

Field diagnosis would avoid problems associated with transportation of samples to the laboratory
and would be especially useful in endemic situations for a faster diagnosis of vesicular disease
and to increase disease awareness. ClearviewTM technology (Patent No. 01291 194 B1, 1988)
was introduced by Unipath Ltd. (Bedford, UK) for the determination of pregnancy in women and
similar technology was used later for the detection of Chlamydia antigen in endocervical swabs.
These novel solid-phase chromatographic strip tests were reliable, sensitive and easy to use at
home or in the clinic. Moreover, results could be achieved from small sample volumes within 15
min. The Chlamydia test was later developed for the diagnosis of Chlamydia antigen in sheep
swabs (Wilsmore and Davidson, 1991). A similar, rapid diagnostic test for the detection of
rinderpest virus (RPV) in lachrymal fluids of cattle using ClearviewTM technology was described
by Brüning et al.(1999) allowing rapid pen-side diagnosis of rinderpest. The technology is
applicable for the diagnosis of diseases of other animal pathogens. This paper describes the
development of a prototype chromatographic strip test device based on a monoclonal antibody
having broad but specific reaction to FMD viruses and its evaluation for the detection of FMD
virus antigen by the laboratory based test of ELISA.

Materials and methods

Selection of monoclonal antibody (Mab) and determination of the optimum antibody
concentration on microparticles and nitrocellulose

A panel of monoclonal antibodies (Mabs) against FMD virus subtype A24 was screened by
 162

ELISA (Ferris and Dawson, 1988) for their reactivity against homologous and heterologous
serotypes of FMD virus and against strains of swine vesicular disease (SVD) virus. One Mab
(designated C1a ) had a high reactivity against four of the FMD virus antigen serotypes (O, A, C
and Asia 1) but no cross- reactivity with SVD virus (data not shown) and was chosen for
incorporation into the chromatographic strips, as the prototype pen-side devices were intended
for the universal rather than serotype-specific detection of FMD virus. The C1a Mab was purified
from ascitic fluid on a protein A (Amersham-Pharmacia, Sweden) column before use. To find the
optimal concentration of Mab on microparticles and on the nitrocellulose membrane, different
concentrations of the Mab in the range 0.2-1.5 mg/ml were tested.

Conjugation of Mab C1a to latex microparticles, adsorption of Mab to nitrocellulose and

adsorption of latex/Mab conjugate to glass-fibre filters

The procedures for the conjugation of the Mab C1a to latex microparticles (Seradyn, IN, USA),
adsorption of the Mab to the nitrocellulose membrane (Hi-flow membrane, Millipore, USA) and
the adsorption of the latex/Mab conjugate to glass-fibre filters (Whatman, UK) were similar to
the corresponding procedures as described for the development of the devices for the detection of
RPV (Brüning et al., 1999) .

Virus strains and sample preparation

Epithelial suspensions (ES) were prepared from current and historical field samples which had
been submitted to the WRL for FMD virus diagnosis and were representative of each of the
seven FMD virus serotypes (Ferris and Dawson, 1988). Oesophageal-phayngeal samples
(Aprobangs@) from African buffalo containing FMD viral antigen of serotypes SAT1, SAT2 or
SAT3 were tested alongwith supernatant fluids of strains of each of the seven FMD virus
serotypes after their propagation in either primary calf thyroid or a permanent cell line of porcine
kidney cells (IB-RS-2).

Epithelial suspensions were also prepared from pigs two days after intra-dermal inoculation of
the heel pad with FMD virus O1 Lausanne and from sheep 8 to12 days after either direct
inoculation or contact-infection with FMD type O GRE 23/94. Nasal swabs were collected from
the first group of pigs and from another set of pigs seven days after aerosol exposure to a low
dose of the O1 Lausanne strain and suspensions prepared in a similar manner to the epithelia.

To test the virus specificity of the devices an epithelial suspension of SVD virus and cell culture
grown isolates (SVD virus, vesicular stomatitis [VS] virus and vesivirus) of the other vesicular
viruses were used as was a cell culture isolate of a porcine enterovirus (PEV). Epithelial
suspensions of field samples in which no FMD virus had been detected by the routine diagnostic
methods of ELISA and virus isolation in cell culture were also tested with the pen-side devices.
These suspensions were denoted as >no virus detected= (NVD) samples. Supernatant fluids from
 163

non-infected calf thyroid cell cultures and suspensions prepared from normal bovine epithelium
were used as negative controls.

Test principle and operation

The operation and test principle of the prototype devices used in the evaluation is essentially the
same as that employed for the ClearviewTM RPV prototype devices as described by Brüning et al.
(1999) with 150 Φl sample being added to the sample pad of the device containing the air-dried
antibody-labelled microparticles. As previously described, the test and control lines are scored on
the progressive scale of negative (no line visible), +/- (possible line - repeat test)), + (just visible
line), ++ (clearly visible line), +++ (strongly visible line) 10 minutes after addition of the sample
to the pad.

ELISA
The epithelial suspensions, probangs and cell culture isolates of the FMD virus samples were
tested by the indirect sandwich ELISA as described by Ferris and Dawson (1988) and the nasal
swabs collected from the pigs seven days after exposure to the strain O1 Lausanne were tested for
antibody to FMD by the liquid phase blocking ELISA of Hamblin et al. (1986).

Results
Optimum antibody concentrations
To optimise the test system, a set of latex conjugates containing different amounts of Mab were
analyzed using nitrocellulose membranes to which different concentrations of Mab had been
applied. The strongest reaction without any false positive result was seen with the Mab at a
concentration of 1.0 mg/ml on the latex and 1.5 mg/ml on the nitrocellulose membrane.

Strip test device and ELISA results on epithelial suspensions, probangs and cell culture isolates
of field samples

The results obtained by the strip test devices and ELISA on the ES and cell culture supernatant
fluids of virus from all seven serotypes of FMD, from probang samples, other vesicular viruses
of SVD, VS and vesivirus and from the PEV sample are summarised in Table 1. The strip
devices detected FMD viral antigen in more samples than the ELISA and all seven FMD
serotypes were detected by the devices. No result (>void=) was obtained from six of the
epithelial suspensions as the glycerol preservative in these samples prevented the devices from
working. A similar situation would not occur in the field as the addition of glycerol would only
take place once the samples were in the laboratory and ready for long term storage. All FMD cell
culture virus isolates were detected 100% efficiently by both techniques. The devices did not
cross-react with antigen from swine vesicular disease, vesicular stomatitis, vesivirus and PEV.
 164

No positive results were achieved from the NVD samples on the devices or from the negative
control samples.

Strip test device and ELISA results on epithelial suspensions from animals experimentally
infected either with FMD virus serotype O1 Lausanne or GRE 23/94

The pigs infected with O1 Lausanne had some clinical signs of FMD at the time ES were
collected (two days post-infection; S. Alexandersen, personal communication) and the prepared
ES gave strong positive results in both the strip test device and ELISA procedures. This
contrasted with the results of ES collected from clinically infected sheep 8-12 days after infection
with FMD type O GRE 23/94 which gave strong positive results by the strip test devices but not
by ELISA (data not shown).

Strip test device and liquid phase blocking ELISA results of nasal swabs from experimentally
infected pigs

Strong positive results with the strip test devices were achieved from three of the donor pigs but
not from the fourth which correlated with the FMD disease status of these animals (S.
Alexandersen, personal communication). The strip test devices scored positive on the nasal
swabs from the second group of pigs which indicated that these tests were detecting the virus in
the nasal passages seven days post infection with a large aerosol dose of the O1 Lausanne FMD
virus. No antibody was detected from this second group (data not shown).

Conclusion
The pen-side test described by Brüning et al. (1999) for the detection of RPV antigen in the field
is rapid and specific and in addition to speed, accuracy and sensitivity, is robust and inexpensive
and easy to carry out by unskilled personnel. A similar test was developed and evaluated in the
laboratory for the universal diagnosis of FMD virus antigen. Such a test for FMD would be
especially useful in areas endemic for FMD virus such as India where the cost of diagnostic
equipment or facilities prevent widespread application.The prototype pen-side test with the C1a
Mab was evaluated at the WRL on ES and cell culture isolates of clinical samples and on ES and
nasal swabs from experimentally infected animals as a prelude to more extensive field trialing.
The nasal swabs, from pigs, were typical of the type of clinical sample obtainable from an animal
at the scene of a suspected outbreak but original material like gum scrapings would also be
suitable.

This evaluation showed these pen-side devices to have better sensitivity than ELISA on ES and
 165

comparable sensitivity to ELISA for detection of cell culture isolates of clinical samples (Table
1). All seven serotypes of FMD virus were detected without cross-reactivity against the other
vesicular viruses of SVD, VS and vesivirus and also against PEV. The devices performed well
on the suspensions prepared from the epithelium of the experimentally infected pigs and sheep
where the results correlated exactly with the clinical signs shown by the animals. This was also
apparent with the nasal swabs taken two days post infection from the pigs.

The pen-side devices performed well in the laboratory and compared well with the routine
diagnostic methods of ELISA and virus isolation. The tests are rapid and simple to perform with
high reproducibility and could undergo trials in the field but these tests are subjective and should
be performed in an environment with adequate lighting. It would be interesting to look at the
performance of the devices on pre-clinical animals to determine whether a specific diagnosis of
FMD could be achieved in advance of the clinical signs of the disease. Mabs can be produced for
serotype-specific pen-side diagnosis of FMD virus antigen as well as the diagnosis of SVD virus
antigen. Ultimately, prototype devices could be developed for antibody detection of FMD in the
field. These devices, if sensitive and specific enough, could then be used in advance of the
current laboratory-based serological tests.

Acknowledgements
The authors would like to thank Dr Alex Donaldson, Professor Soren Alexandersen and Mr
Gareth Hughes from the Institute of Animal Health, Pirbright for supplying the epithelium
samples and nasal swabs from the experimentally infected sheep and pigs, Mrs Linda Turner
(Institute for Animal Health, Pirbright) for carrying out the liquid phase blocking ELISA on the
nasal swabs and the Institute for Animal Health, Compton for supplying the bovine epithelium
tissue for the preparation of the negative control. This work was supported financially by the
Ministry of Agriculture, Fisheries and Food, UK.

References
Brüning, A., Bellamy, K., Talbot, D. and Anderson, J. (1999) A rapid chromatographic strip test
for the pen-side diagnosis of rinderpest virus. J. Virol. Methods 81, 143-154.

Ferris, N. P. and Dawson, M. (1988) Routine application of enzyme-linked immunosorbent assay

in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular
diseases. Vet. Microbiol. 16, 201-209.
 165

Hamblin, C., Barnett, I. T. R. and Hedger, R. S. (1986) A new enzyme-linked immunosorbent

assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I.
Development and method of ELISA. J. Immunol. Methods, 93, 115-121.

Reid, S. M., Forsyth, M. A., Hutchings, G. H. and Ferris, N. P. (1998) Comparison of reverse
transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation
for the routine diagnosis of foot-and-mouth disease. J. Virol. Methods 70, 213-217.

Reid, S. M., Hutchings, G. H., Ferris, N. P. and De Clercq, K. (1999) Diagnosis of

foot-and-mouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral
RNA in clinical samples. J. Virol. Methods 83, 113-123.

Reid, S. M., Ferris, N. P., Hutchings, G. H., Samuel, A. R. and Knowles, N. J. (2000) Primary
diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol.
Methods, in press.

Wilsmore, A. J. and Davidson, I. (1991) Clearview= rapid test compared with other methods to
diagnose chlamydial infection. Vet. Rec. 128, 503-504.
 7

Table 1
Summary of the pen-side test and ELISA results on epithelial suspensions and cell culture supernatant
fluids of FMD field samples and other vesicular viruses, a porcine enterovirus and on clinical samples
where no virus was detected by the routine methods

Ratio of number of samples positive to number tested per FMD virus and source of antigen

ESa Cell culture

Serotypeb ELISA Pen-side diagnosisc ELISAd Pen-side diagnosis

O 40/54 42 (+4)/54 9/9 9/9

A 5/15 11(+1)/15 2/2 2/2

C 5/10 6/10 4/4 4/4

SAT1 4/11 7 (+1) /11 6/6 6/6

SAT2 8/16 8 (+2) /16 3/3 3/3

SAT3 1/7 2/7 3/3 2 (+1)/3

Asia 1 12/15 13/15 2/2 2/2

SVDVe 0/1 0/1

VSVf 0/2

Vesivirus 0/1

PEVg 0/1

NVDh 0/6 0/6

a
ES, epithelial suspension
b
Serotype of FMD virus unless otherwise stated.
c
Test lines scored as negative (no line visible), +/- (unclear so repeat test)), + (just visible line), ++ (clearly visible
line) or +++ (strongly visible line) ten minutes after addition of the sample to the pad. Figure in bracket is a +/-
result. The pen-side results recorded as >void= (Table 1) were classified in this table as negative.
d
All cell culture isolates were typed by ELISA.
e
SVDV, swine vesicular disease virus.
f
VSV, vesicular stomatitis virus.
g
PEV, porcine enterovirus.
h
NVD, no virus detected by ELISA or virus isolation in cell culture.
 167
Appendix 19

The development of an antigen capture reverse transcription polymerase chain reaction

method for foot-and-mouth disease virus antigen detection

Scott M. Reid, Geoffrey H. Hutchings and Nigel P. Ferris

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey,
GU24 ONF, UK

Summary

Several protocols were developed and evaluated for the capture of foot-and-mouth disease
(FMD) virus antigen on a microtitre plate to enable RNA extraction and reverse transcription
to be carried out in the plate wells prior to PCR amplification of the cDNA product. One
protocol enabled the FMD viral antigen from suspensions of clinical samples to be captured
in the well by coating with a cocktail of type-specific guinea pig anti-FMD virus immune
sera. The viral RNA was extracted by heating the plate for five minutes at 900C and was
subsequently detected by PCR amplification with universal primer sets. In an alternative
protocol, specific primer sets were able to serotype FMD viruses of types O, A, C and Asia 1
after individual wells were coated with homologous type-specific guinea pig anti-FMD virus
immune serum. Both protocols were specific for FMD virus and their sensitivity was the
same as that of the RT-PCR currently used to supplement the routine methods of ELISA and
virus isolation in cell culture for diagnosis at the OIE/FAO World Reference Laboratory for
FMD, Pirbright. This antigen capture (immunocapture) RT-PCR methodology is seen as a
prototype to the development of antigen capture RT-PCR or PCR-ELISA methods involving
combinations of labelled reagents such as biotin with streptavidin or digoxigenin with
anti-digoxigenin to enable the entire protocol to be carried out in a microtitre plate well.
These systems could increase the sensitivity of the RT-PCR for FMD diagnosis and would
have the additional advantage of being objectively analysed by use of an ELISA plate reader
linked to a computer.

Keywords: Antigen capture, Immunocapture, RT-PCR, FMDV serotyping, Diagnosis

Introduction

Routine diagnosis of FMD viral antigen is carried out at the OIE/FAO World Reference
laboratory for FMD (WRL), Pirbright using a combination of ELISA and virus isolation in
cell culture (Ferris and Dawson, 1988). These methods have recently been backed-up, but not
replaced by RT-PCR procedures involving universal primers for the detection of all seven
serotypes (Reid et al., 1998; Reid et al., 2000, in press) and serotype-specific primers (Reid et
al., 1999). The RT-PCR method with the universal O/A/C/Asia 1 primer set (1F/1R) was as
sensitive as virus isolation and more sensitive than ELISA for primary diagnosis of the
serotypes O, A, C and Asia1 but was less sensitive than virus isolation and ELISA for the
detection of the SAT serotypes in clinical samples (Reid et al., 2000, in press).

Increased sensitivity in RT-PCR procedures for serotypic detection of FMD viral RNA in
clinical samples could be achieved by using alternative strategies or formats. Nested PCR
 168
formats have been developed for the serotyping of FMD virus (Moss and Haas, 1999; S. M.
Reid, unpublished results) and for the detection of swine vesicular disease ([SVD], Lin et al.,
1997). Callens and De Clercq (1999) used a PCR-ELISA kit (Boehringer Mannheim) for the
sensitive detection of PCR products of SVD virus. Despite their high sensitivity, the nested
procedures have a high risk of contamination (Lin et al., 1999) which could prohibit the use
of this format for routine diagnosis while the expense of PCR-ELISA kits would also be
restrictive. Antigen capture or immunocapture-PCR systems have been reported for the
detection of plant viruses and subviral pathogens (Nolasco et al.,1993) and plum pox
potyvirus (Olmos et al.,1997). Suryanarayana et al. (1999) described an antigen capture
RT-PCR system for the serotyping of FMD virus which was similar to the method described
by Jensen et al. (1990) for epidemiological studies on human hepatitis A virus.

The aim of this study was to develop improved antigen capture RT-PCR formats for both
detection and serotyping of FMD viral RNA in clinical samples. These formats avoid the
handling of the RNA extracted from the samples and allow several samples to be tested
simultaneously and economically without contamination. As their initial development
involved the universal and serotype-specific primers currently used in RT-PCR diagnosis of
field samples at the WRL for FMD (Reid et al.,1999; Reid et al., 2000, in press), the
sensitivity of the antigen capture RT-PCR methodology could readily be compared to that of
the current RT-PCR procedure for detection or serotyping of clinical samples.

Materials and methods

Virus samples

Cell culture supernatants were prepared after propagation of a selection of epithelial

suspensions of clinical samples (Ferris and Dawson, 1988) in calf thyroid and/or IB-RS-2
(permanent line of porcine kidney cells). Epithelial suspensions and cell culture supernatants
of the other vesicular disease viruses of SVD, vesicular stomatitis (VS) and vesivirus were
also tested to check the specificity of the antigen capture RT-PCR formats for FMD virus.
Non-infected cell culture supernatant was used as the negative control.

Coating of microtitre plates with anti-FMD virus guinea pig immune sera or hyperimmune
rabbit sera

Fifty Φl of anti-FMD virus type-specific guinea pig immune sera against each of the
serotypes O, A, C and Asia 1 was coated on to separate wells of a flat-bottomed microtitre
plate (Maxisorp, NUNC, UK) overnight at 40C. Coating of each well with a cocktail of
guinea pig immune sera against the serotypes O, A, C and Asia 1 as a group or against all
seven serotypes as a group was also tried. Anti-FMD virus hyperimmune rabbit serum against
each of the O, A, C and Asia 1 serotypes was similarly coated on to separate ELISA plate
wells either individually or as a cocktail of the serum.

Antigen capture RT-PCR

 169
After coating, the microtitre plates were machine washed three times (>Wellwash= 5 or 5000
models, Denley, UK) with phosphate buffered saline (PBS) buffer. Blocking with 1% (w/v)
ovalbumin (Sigma, UK; Anon, 1996) was adopted. Dried skimmed milk (>marvel=, 1% w/v)
was used as a blocking agent on one occasion. Blocking was initially performed by
completely filling the well and incubating overnight at 40C but blocking for 2 hr 370C was
found to be suitable as was using a blocking volume of 70 Φl per well.

If a cocktail of rabbit or guinea pig immune sera was used for coating, 50 Φl of sample was
added to the well and either a universal primer or a cocktail of specific primers added to the
PCR amplification reaction mix for the sample but when the specific primers were used
individually, separate PCR amplification mixes had to be prepared for each sample depending
on the number of primer sets employed. In plates coated with separate type-specific rabbit or
guinea pig anti-FMD virus serum, 50 Φl of sample was added to the well coated with the
immune serum against each serotype and if the specific primers were used individually in
PCR amplifications, these were added to the PCR reaction mixes to match with the serotype
specificity of the coating antibody. With this latter approach, each sample could be tested in
four PCR serotype-specific reactions corresponding to four pairings of serotype-specific
coating antibody with specific primer set for serotypes O, A, C and Asia 1.

After addition of samples, the plates were shaken for 1 hour at 370C then washed three times
in PBS as before. RNA extraction was then carried out in the microtitre plate well by a
similar method to that described by Suryanarayana et al. (1999). Twenty Φl of reverse
transcription reaction mix as used previously (Reid et al. 1999) was added to each well and
the plate incubated for 5 min at 900C on a water bath. After cooling to room temperature for
20 min, five Φl of a mix containing one Φl Moloney-murine leukemia virus reverse
transcriptase (Gibco Life Technologies) and 10 pmol of a random hexanucleotide mix
(Promega) was added to each well and the contents subjected to reverse transcription for 45
min at 370C (Reid et al., 1998; 1999) on a water bath.

The 1F/1R and P1/P2 primer sets were designed for the detection of all seven serotypes of
FMD virus as described previously (Amaral-Doel et al., 1993; Reid et al., 2000, in press). The
specific primers sets P33/P38, P33/P87-P92, P33/P40 and P33/P74-P77 for the detection of
the serotypes O, A, C and Asia 1 respectively, were as listed previously (Vangrysperre and De
Clercq, 1996; Callens and De Clercq, 1997). All primers were synthesized by Cruachem Ltd.,
UK.

Thirty Φl of a PCR reaction mix as used previously (Reid et al., 1999) and 10 pmol each of
the respective primers was added to a labelled tube and 20 Φl of cDNA transferred from the
microtitre plate into the corresponding tube. The contents were mixed by a short spin and
PCR amplification carried out on a PTC-100TM thermocycler machine (MJ Research, USA)
using the programme previously used with the universal primer sets (Reid et al., 2000, in
press) and the programme: 940C for 5 min, 1 cycle; 940C for 1 min, 580C for 1 min, 720C
for 2 min, 20 cycles; 720C for 7 min, 1 cycle with the specific primers (either individually or
as a cocktail) that had been employed previously (Reid et al., 1999). RT-PCR products were
detected by electrophoresis on a 1.5 % agarose gel.
 170
All of the FMD virus samples tested in the antigen capture RT-PCR formats were also tested
in diagnostic RT-PCR protocols with the specific primer sets P33/P38, P33/P87-P92,
P33/P40, P33/P74-P77 (Reid et al., 1999; Reid et al., 2000, in press) and with the primer sets
1F/1R and P1/P2 (Reid et al., 2000, in press).

Results

Coating of separate wells with guinea pig immune serum to the FMD serotypes O, A, C and
Asia 1

With the cocktail of specific primers, serotype Asia 1 cell culture supernatants produced false
positive bands after incubation in wells coated with the heterotypic guinea pig immune sera.
This was seen to a lesser extent with some of the type O samples but other type O and Asia 1
samples were specifically detected with this format. Blocking reduced the number and/or
strength of false positive bands (data not shown).

When the specific primer sets were used separately and were added to the PCR amplification
mixes to correspond to the serotype-specificity of the guinea pig immune serum for well
coating, perfect results were achieved when the blocking step was used. Serotype-specific
diagnosis was achieved on cell culture supernatant fluids of serotypes O, A and Asia 1
following incubation in wells coated with the guinea pig immune serum of the same serotype
specificity. No cross-reactivity was apparent when the same samples were tested with the
heterotypic coat antibody/primer combinations. A small degree of cross-reactivity was seen
between serotypes O and Asia 1 when blocking was not employed. When coated on to the
separate wells against each serotype, the rabbit immune serum produced false positive bands
from type O and Asia 1 cell culture supernatant fluids and weaker bands from the true
positive samples (data not shown).

Coating of wells with a cocktail of guinea pig immune sera to the FMD serotypes O, A, C and
Asia 1 as a group or to all seven FMD serotypes as a group

The 1F/1R and P1/P2 primer sets in the antigen capture RT-PCR matched the sensitivity
achieved by the diagnostic RT-PCR with these primers (Reid et al., 2000, in press) for
detection (but not serotyping of) FMD virus cell culture supernatant fluids of all serotypes
and no cross-reactivity was seen with viruses of the other vesicular diseases of SVD, VS and
vesivirus. Promising results were also achieved in the antigen capture RT-PCR with the
primer set P33/P38 which again matched the sensitivity and specificity of the diagnostic
RT-PCR with the same primer set for detection of type O cell culture supernatants. False
positive results were produced in wells coated with the cocktail of rabbit immune sera against
the serotypes O, A, C and Asia 1 as a group (data not shown).

Conclusion
The antigen capture RT-PCR formats presented here were predominantly performed in
ELISA plates unlike the method described by Suryanarayana et al. (1999) but the procedure
for RNA extraction, namely: heating of the sample for five minutes at 900C was repeated as it
was efficient, fast and suitable for samples in ELISA plates.

An antigen capture RT-PCR format for universal FMD virus detection had the same
sensitivity as the diagnostic RT-PCR protocol involving the same primer sets (Reid et al.,
2000, in press) on cell culture supernatant fluids and did not cross-react with the other
 171
vesicular viruses of SVD, VS or vesiviruses. This was found with another antigen capture
RT-PCR format for serotype-specific diagnosis of samples where the wells were coated with
individual type-specific guinea pig immune sera instead of a cocktail and the specific primers
were added to the PCR amplification mix to correspond to the serotype specificity of the
immune serum used to trap the viral antigen in the well. The guinea pig immune sera were
more sensitive and specific than the rabbit hyprerimmune sera for capturing FMD virus on to
the solid phase. Type specific rabbit antisera had been used to coat the solid phase
(microcentrifuge tubes) in the antigen capture RT-PCR of Suryanarayana et al. (1999) so that
non-specific capture of FMD virus may have taken place in that system. This would tend to
cast some doubt as to the true specificity of their procedure for the serotyping of FMD virus.

The antigen capture RT-PCR formats presented here with the guinea pig immune sera can be
evaluated more extensively with ES of clinical material which would further test the
suitability of the methods for primary diagnosis of FMD virus. The format for
serotype-specific diagnosis of samples can be developed for the detection of the SAT
serotypes in clinical material including oesophageal-pharyngeal scrapings (probangs).
However, the sensitivities of these formats for diagnosis or serotyping of FMD virus or for
differential vesicular virus diagnosis could be increased greatly if the entire protocol was
performed in an ELISA plate and involved the interaction of labelled reagents such as biotin
with streptavidin, digoxigenin with an anti-digoxigenin conjugate or if fluorescence detection
methods were used (Nolasco et al., 1993). As long as the specificity was satisfactory, the
antigen capture RT-PCR results could then be objectively analysed by use of an ELISA plate
reader linked to a computer rather than the more subjective analysis of RT-PCR products on
agarose gels.

Acknowledgements

This work was supported financially by the Ministry of Agriculture, Fisheries and Food, UK.

References
Amaral-Doel, C. M. F., Owen, N. E., Ferris, N. P., Kitching, R. P. and Doel, T. R. (1993)
Detection of foot-and-mouth disease viral sequences in clinical specimens and
ethyleneimine-inactivated preparations by the polymerase chain reaction. Vaccine 11,
415-421.

Anon. (1996) FMD ELISA kit. Liquid Phase Blocking Enzyme Immunoassay for detection of
antibodies to foot-and-mouth disease virus. Bench Protocol, February 1996. Joint FAO/IAEA
Programme. Animal Production and Health, Animal Production Unit, Agriculture
Laboratory, Agency=s Laboratories Division, Seibersdorf, Austria.
Callens, M. and De Clercq, K. (1997) Differentiation of the seven serotypes of
foot-and-mouth disease virus by reverse transcriptase polymerase chain reaction. J. Virol.
Methods 67, 35-44.

Callens, M. and De Clercq, K. (1999) Highly sensitive detection of SVDV based on a single
tube RT-PCR system and DIG-ELISA detection. J. Virol. Methods 77, 87-99.

Ferris, N. P. and Dawson, M. (1988) Routine application of enzyme-linked immunosorbent

assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine
vesicular diseases. Vet. Microbiol. 16, 201-209.
 172

Jensen, R. W., Siegl, G. and Lemon, L. S. (1990) Molecular epidemiology of human hepatitis
A virus defined by an antigen-capture polymerase chain reaction method. Proc. Natl. Acad.
Sci. USA 87, 2867-2871.

Lin, F., Mackay, D. K. J. and Knowles, N. J. (1997) Detection of swine vesicular disease
virus RNA by reverse transcription-polymerase chain reaction. J. Virol. Methods 65, 111-121.

Moss, A. and Haas, B. (1999) Comparison of the plaque test and reverse transcription nested
PCR for the detection of FMDV in nasal swabs and probang samples. J. Virol. Methods 80,
59-67.

Nolasco, G., de Blas, C., Torres, V. and Ponz, F. (1993) A method combining
immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses
and subviral pathogens. J. Virol. Methods 45, 201-218.

Olmos, A., Cambra, M., Dasai, M. A., Candresse, T., Esteban, O., Gorris, M T. and Asensio,
M. (1997) Simultaneous detection and typing of plum pox potyvirus (PPV) isolates by
Heminested-PCR and PCR-ELISA. J. Virol. Methods 68, 127-137.

Reid, S. M., Forsyth, M. A., Hutchings, G. H. and Ferris, N. P. (1998) Comparison of reverse
transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus
isolation for the routine diagnosis of foot-and-mouth disease. J. Virol. Methods 70, 213-217.

Reid, S. M., Hutchings, G. H., Ferris, N. P. and De Clercq, K. (1999) Diagnosis of

foot-and-mouth disease by RT-PCR: evaluation of primers for the serotypic characterisation
of viral RNA in clinical samples. J. Virol. Methods 83, 113-123.

Reid, S. M., Ferris, N. P., Hutchings, G. H., Samuel, A. R. and Knowles, N. J. (2000) Primary
diagnosis foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol.
Methods, in press.
Suryanarayana, V., Madanamohan, B., Bist, P., Natarajan, C. and Tratschin, J. D. (1999)
Serotyping of foot-and-mouth disease virus by antigen capture reverse
trtanscriptase/polymerase chain reaction. J. Virol. Methods 80, 45-52.

Vangrysperre, W. and De Clercq, K. (1996) Rapid and sensitive polymerase chain reaction
based detection and typing of foot-and-mouth disease virus in clinical samples and cell
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symptomatically related viruses. Arch. Virol. 141, 331-344.
173

Appendix 20

RT-PCR probing-ELISAs for the diagnosis and typing of foot-and-mouth disease using
our newly developed SNAP (Simple and Aqueous Phase) hybridization

Soren Alexandersen, Morag A. Forsyth*, Scott M. Reid, Graham J. Belsham

Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, GU24 ONF, U.K.
* Presenting Author

SUMMARY
A RT-PCR / ELISA detection system has been developed that targets a relatively
conserved region within the RNA genome of all seven serotypes of foot-and-mouth disease
(FMD) virus. Using a rapid hybridisation step with labelled oligonucleotides, the assay is
highly sensitive, specific and easy to perform. Rapid preliminary genotyping of FMDV was
made possible using a modification of the assay, based on multiple hybridisation
oligonucleotides and targeting a highly variable region of the FMDV genome. The method
could be modified for diagnosis and strain differentiation in any system amenable to PCR
amplification.

INTRODUCTION
Because clinical diagnosis of foot-and-mouth disease (FMD) can be difficult,
particularly in sheep and goats where clinical signs are often mild, definitive diagnosis must
be carried out in specialized laboratories. Rapid, sensitive and specific techniques such as
reverse transcription polymerase chain reaction (RT-PCR) assays have been developed for
the diagnosis of FMDV infection. Assays for the “serotyping” of FMDV by PCR have been
developed but they are very labour intensive[7;12].
The aim of the current study was to develop an assay suitable for routine FMDV diagnosis
which was highly sensitive and specific. This was achieved by including a novel Simple aNd
Aqueous Phase (SNAP) hybridization step giving optimal specificity within an easy and fast
assay. This RT-PCR ELISA assay was adapted to target a more variable region of the virus
and by using a panel of oligonucleotide probes, it was possible to do preliminary genotyping
of FMDV isolates.

MATERIALS AND METHODS

Virus. Samples were original suspensions prepared from epithelial lesions and cell culture
grown virus stored at the OIE/FAO World Reference Laboratory for FMD (WRL) at
Pirbright. All virus samples had previously been serotyped by antigen-detection ELISA [9].
Total RNA was extracted by the TRIzol method (BRL, Gibco Life Technologies).
PCR amplification. RT-PCR was carried out using different combinations of primers based
on sequences within two regions, the Internal Ribosome Entry Site (IRES) [4], primers IRES
1 – 4, and the VP1 (1D) region, primers P1 and P2 [1] (Table 1). Upstream primers were
labelled with Dig or FITC and downstream primers were unlabelled or labelled with biotin.
The numbering system is based on an alignment of the following sequences from GenBank
(accession Numbers X00871, AJ007347, AJ007572, X74812, AF154271 and M10975).
SNAP capture probes. The oligonucleotides used as SNAP capture probes for the IRES
region are shown in Table 1. For the VP1 region, a number of oligonucleotides were designed
based on available data in public databases (GenBank), produced as the complementary
strand and purchased already labelled with biotin.
174

SNAP hybridization and ELISA procedure. Biotinylated SNAP capture probes (10-20
pmol) were aliquoted into the wells of a PCR microtitre plate. The PCR products (2-10 µl)
were added, the plates covered, heated at 95°C and cooled to the optimised annealing
temperature. As the multiple capture probes had variable melting temperatures, the optimal
annealing temperature varied from 40oC to 65oC. After annealing, the ELISA blocking
buffer (PBS / 0.05% Tween 20 / 0.1% bovine serum albumen) was immediately added to the
wells, mixed and the material transferred to a streptavidin-coated ELISA plate. After
incubation at 37°C, the plate was washed and the captured product detected by anti-Dig-POD
or anti-FITC-POD conjugate (Boehringer Mannheim) on a spectrophotometer at wavelength
405nm.

Designation sequence (5’ to 3’) Location

P1 CCTACCTCCTTCAACTACGG Corresponding to FMDV nt 3737-3756

P2 GAAGGGCCCAGGGTTGGACTC Complementary to FMDV nt 3955-3935

IRES1 CCTGGTCTTTCCAGGTCTAGA Corresponding to FMDV nt 667-687

IRES2 CCTCCTTGGTAACAAGGACCC Corresponding to FMDV nt 790-810

IRES3 CCTTCTCAGATCCCGAGTGT Complement of FMDV nt 987-968

IRES4 CCTATTCAGGCGTAGAAGCTT Complement of FMDV nt 1042-1022

TABLE 1. Primers used for the RT-PCR. Certain primers were also used as SNAP
capture probes, see text.

RESULTS
Direct RT-PCR ELISA for FMDV and development of SNAP hybridization. Originally,
an RT-PCR / ELISA system had been developed for the detection of RNA from all seven
serotypes of FMD virus using primers from conserved regions of the IRES region (within the
5’-untranslated region [UTR]). Although sensitive, the system was not sufficiently specific
for routine application [10]. To improve specificity, a hybridization step (SNAP) using an
internal oligonucleotide was introduced (Figure 2).
Optimization of the IRES RT-PCR for use with SNAP hybridization. The method was
optimized using primers IRES2 and IRES4 together with a high temperature “hot start” PCR
(Amplitaq GOLD, PE Applied Biosystems). The sensitivity of the assay was improved
100-fold compared to the original protocol [10] giving a final sensitivity of 0.2 TCID50 for
the combined RT-PCR oligo-ELISA.
The RT-PCR SNAP ELISA results with all seven serotypes of FMDV are shown in Figure 3.
The IRES primers are specific for sequences that are highly conserved between different
FMDV isolates. They have previously been shown to work well with samples from
experimentally infected animals [10]. Data obtained using other IRES primers indicate that
this part of the genome is very suitable for the RT-PCR diagnosis of FMDV from clinical
samples submitted to the WRL (Reid et al, in press, 2000).
Development of an assay to distinguish different FMDV strains. For FMDV, the VP1
(1D) region is highly variable and is thought to play a major role in defining serogroup
specificity. The region has been extensively sequenced and isolates classified based on their
VP1 sequence [3;11]. Primers designed for this region, such as P1 and P2 [1], have had
varied success in RT-PCR assays for serotyping FMDV isolates [5;13]. The conserved
primers, P1 and P2, amplify a 216 bp fragment of VP1 and detect most type O, A and Asia 1
175

isolates of FMDV as well as some C, SAT 1, SAT 2, and SAT 3 types (Reid et al., in press,
2000). An assay was developed to distinguish between virus strains using a combination of
these primers and internal hybridization probes, The SAT serotypes were excluded from the
primer design due to lack of sufficient sequence information. Alignment of all sequences
indicated that within the 216 nucleotide sequence amplified by the P1/P2 primers, there were
several regions potentially useful for the mapping assay. One region was most conserved
while another region was more variable making it more specific for particular isolates. As
examples we designed capture probes capable of distinguishing the 1997 Taiwan-like type O
isolates [2] from Kaufbeuren-like type O isolates, as well as isolates resembling Asia 1, and
strains of A22 or C3.
Mapping of the VP1 region using a multiple array SNAP-hybridization-ELISA.
Initially studies using the dig-labelled P1 and unlabelled P2 primers proved unsuccessful.
Although the PCR products gave bands of the expected size (216bp), the SNAP capture
ELISA with each of the 8 oligonucleotides was negative, giving only background signal in all
wells. The concentration of the unlabelled PCR primer was reduced giving an asymetric PCR
product as it was assumed that the internal oligonucleotides could not hybridize to the
conventional double-stranded product during the SNAP procedure. When the SNAP ELISA
was repeated the internal oligonucleotides now hybridized to the asymetric PCR products.
Using the multiple probe ELISA, 59 samples including controls were mapped. 27 serotype O
isolates were analyzed and all were classified as serotype O in the SNAP assay. Of the ten
type A isolates analyzed, six of these could be classified as A type (positive with the A22
probe) while four were classed as type A or C. Nine type C samples were analyzed and all
except one were classified as type A or C. Type C isolates that did not produce visible bands
in gel electrophoresis could be detected by this method. One isolate also had a weak type O
reaction that could easily be removed by the inclusion of competitor oligonucleotides where
the specific probe is mixed with a similar concentration of the other unlabelled probes. As
these probes compete for binding to the target, only the probe with the highest sequence
similarity binds efficiently producing a serotype-specific signal. Eight Asia 1 type samples
were analyzed; seven were characterized as Asia 1 type (one had borderline reaction) and one
sample was negative. The Asia 1 isolates reacted weakly with most of the SNAP capture
probes independent of type design. However, when competitor probes were included, the
reactions with the Asia SNAP probes were predominant. The isolates could therefore be
typed as serotype Asia 1. The weak reaction with the Asia probes may indicate that the
isolates tested are rather distant from the Asia 1-like sequences used for the probe design.
Seven SAT samples were also analyzed although no specific primers were designed for this
purpose. Five samples were negative in the SNAP assay (consistent with no SAT specific
SNAP probes), but two isolates (SAT1 NIG 20/75 and SAT2 RHO 10/80) showed a modest
type A or C reaction. Interestingly, this reaction could not be inhibited by the addition of
competitor probes, which indicated that these two isolates are highly related to A and C
isolates in this particular region of the genome (data not shown). All negative control
preparations were negative in the assay.

DISCUSSION
We have developed RT-PCR assays which, when combined with SNAP probing with
internal oligonucleotides in an ELISA format, can be used in the diagnosis of FMD and
preliminary genotyping of the virus. The assays are highly sensitive, specific, fast, and easy
to perform. The assay developed for detecting conserved sequences in the IRES region of the
FMDV genome worked with all seven serotypes of FMDV. It was highly sensitive and
specific, detecting as little as 0.2 TCID50s or around 20 molecules of FMDV RNA. This
assay which requires only standard RT-PCR and ELISA equipment has great potential as a
robust diagnostic method for the detection of all FMDV serotypes.
176

A modified assay was developed targeting a highly variable region of the FMDV genome, the
VP1 (1D) region, by using previously described primers for RT-PCR in conjunction with a
selection of internal SNAP probes. Sequences of 25 FMDV isolates spanning this region
and accessible in the public databases (GenBank) were grouped by serotype and aligned. The
alignments of all sequences indicated that the P1/P2 primer region contained areas potentially
useful for the mapping assay and this was confirmed by the data obtained. Although the assay
could be made even more specific for the selected serotype by including unlabelled
competitor probes, mapping could consistently be achieved without these inhibitors. In
conclusion, the multiple SNAP probing can be used to give preliminary typing characteristics
of the isolate that could be important if short term control measures were required. However,
more SNAP probes should be designed and tested.
Other RT-PCR ELISA assays are available, both for general use (PCR Applications
Manual, Boehringer Mannheim, Mannheim, Germany, 1995) and for detection of FMDV and
swine vesicular disease (SVD) virus [6;8]. The advantage of our SNAP assay is the
significant reduction in time taken for the hybridization step, reducing it from three hours to
five minutes. The reason for the high speed and efficiency of the SNAP hybridization step is
the use of solution hybridization at a relatively high temperature and the very high
concentrations of both product and capture probe. This combination gives very fast
hybridization, while subsequent steps inhibited possible low temperature, non-specific probe
binding by dilution of the reaction mix.
The SNAP assay for FMDV can be developed as a general diagnostic assay and has
been recently adapted for detection of two morbilliviruses, rinderpest virus and peste des
petits ruminants virus. Therefore application of this method could contribute to fast, easy and
specific diagnoses as well as easy, preliminary typing of the pathogens received in the
clinical microbiological laboratory.

ACKNOWLEDGMENTS
We thank Geoff H. Hutchings and Nigel P. Ferris for assistance. Alex I. Donaldson and Paul R. Kitching made
helpful comments to the work. The research was supported in part by the UK Ministry of Agriculture,
Fisheries and Food (Project No. SE1113) and the Danish Ministry of Food, Agriculture, and Fisheries.

REFERENCES
1. Amaral-Doel, C. M., N. E. Owen, N. P. Ferris, R. P. Kitching, and T. R. Doel. 1993. Detection of
foot-and-mouth disease viral sequences in clinical specimens and ethyleneimine-inactivated preparations by
the polymerase chain reaction. Vaccine 11:415-21.

2. Beard, C. W. and P. W. Mason. 2000. Genetic determinants of altered virulence of Taiwanese

foot-and-mouth disease virus. J. Virol. 74:987-991.

3. Beck, E. and K. Strohmaier. 1987. Subtyping of European foot-and-mouth disease virus strains by
nucleotide sequence determination. J Virol 61:1621-9.

4. Belsham, G. J. 1993. Distinctive features of foot-and-mouth disease virus, a member of the picornavirus
family; aspects of virus protein synthesis, protein processing and structure. Prog Biophys Mol Biol
60:241-60.

5. Callens, M. and K. De Clercq. 1997. Differentiation of the seven serotypes of foot-and-mouth disease
virus by reverse transcriptase polymerase chain reaction. J Virol Methods 67:35-44.

6. Callens, M. and K. De Clercq. 1999. Highly sensitive detection of swine vesicular disease virus based on
a single tube RT-PCR system and DIG-ELISA detection. J. Virol. Methods 77:87-99.
177

7. Callens, M., K. De Clercq, and M. Danes. 1998. Transmission of foot-and-mouth disease virus between
contact sheep and contact pigs: detection of infected animals. Session of the Research Group of the
Standing Technical Comittee, European Commission for the Control of Foot-and-Mouth Disease
1998:129-138.

8. Callens, M., K. De Clercq, M. Gruia, and M. Danes. 1998. Detection of foot-and-mouth disease by
reverse transcription polymerase chain reaction and virus isolation in contact sheep without clinical signs of
foot-and-mouth disease. Vet Q 20 Suppl 2:37-40.

9. Ferris, N. P. 1987. Development and use of Elisa in the control of foot-and-mouth disease.
IAEA-Proceedings 348:65-77.

10. Forsyth, M. A., G. J. Belsham, E. Felipe, and D. K. Mackay. 1998. Detection of FMDV in nasal swabs
and oesophago-pharyngeal fluids using a reverse-transcription polymerase chain reaction reaction. Session
of the Research Group of the Standing Technical Comittee, European Commission for the Control of
Foot-and-Mouth Disease 1998:88-92.

11. Marquardt, O. and B. Haas. 1998. VP1-coding sequences of recent isolates of foot-and-mouth disease
virus types A, O and Asia1. Virus Genes 16:185-93.

12. Reid, S. M., M. A. Forsyth, G. H. Hutchings, and N. P. Ferris. 1998. Comparison of reverse
transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the
routine diagnosis of foot-and-mouth disease. J Virol Methods 70:213-7.

13. Reid, S. M., G. H. Hutchings, N. P. Ferris, and K. De Clercq. 1999. Diagnosis of foot-and-mouth disease
by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples. J. Virol.
Methods 83:113-123.
 P1-2A P2 P3
5' UTR Lab 2A 3' UTR
Lb 1A 1B 1C 1D 2B 2C 3A 3B 3C 3D
CCn
AAA
IRE S

1D
2A 2B

IRES1 IRES2 P1
P2
IRE S3 IRES4

FIGURE 1. Schematic representation of the FMDV genome outlining the principal

features. The positions of the IRES region and the P1/P2 primer region are shown as
well as the areas used for SNAP probes (shading).

cDNA and primers

PCR

PCR product

SNAP
+
SNAP hybrid

ELISA

POD
Detection

FIGURE 2. Schematic representation of the SNAP procedure. The primers used for the
PCR step are indicated ( Dig or FITC-label), as is the SNAP oligonucleotide ( biotin-
labelled) used for capture of the specific product onto the streptavidin (S) coated ELISA
plate. Final detection is in a standard ELISA format using peroxidase-labelled (POD)
antibody.
 5 ul RNA 1 ul RNA Control
probe

3.00

2.40

1.80
OD

1.20

0.60

0.00
O1 BFS A22 C1 SAT 1 SAT 2 SAT 3 Asia 1 neg SVDV ddH2O

FIGURE 3. SNAP probing ELISA. RT-PCR amplification was performed using the
optimized protocol on the IRES region with FITC-labelled IRES2 primer and
unlabelled IRES4 primer. The SNAP capture probe was IRES3-biotin. As a control
capture probe P2-biotin was used. RNA samples were derived from cells infected
with all seven FMDV serotypes (O, A, C, Asia 1, and SAT 1, 2, 3) and various
negative controls (uninfected cells, swine vesicular disease (SVD) virus infected cells
and distilled water) were also included. Two samples from each isolate were analyzed
using 1 or 5 l of total RNA isolated from cell cultures.

2
 181
Appendix 21

Evaluation of RT-PCR procedures for diagnosis of clinical samples of foot-and-mouth

disease virus (serotypes O, A, C and Asia 1) under the European Union Concerted Action
Group Project Pl 98-4032

Scott M. Reid, Geoffrey H. Hutchings and Nigel P. Ferris

Institute for Animal Health, Pirbright laboratory, Ash Road, Pirbright, Woking, Surrey, GU24
ONF, UK

Summary

A collaborative study initiated from the first annual European Union (EU) Concerted Action
Group meeting (project PL 98-4032, 5-7 May 1999, Federal Research Centre for Virus Diseases
of Animals, Tübingen, Germany) was organised between the Veterinary and Agrochemical
Research Centre in Brussels, the Federal Research Centre in Tübingen and the OIE/FAO World
Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright. Each laboratory
was supplied with 40 epithelium samples of FMD virus positive clinical material (ten each of the
serotypes O, A, C and Asia 1) and the supernatant fluids resulting from their passage in cell
culture to evaluate the sensitivity and specificity of locally employed RT-PCR procedures. The
40 samples were supplied unlabelled with respect to FMD virus serotype. In the WRL, FMD
virus was detected (but not serotyped) in 36 of the 40 epithelial suspensions and in 39 of the 40
cell culture supernatant fluids by a diagnostic RT-PCR protocol using a universal O/A/C/Asia 1
primer set (1F/1R). These primers also detected 36 of the 40 epithelial suspensions in a prototype
antigen capture (immunocapture) RT-PCR but variable results were achieved in a diagnostic
RT-PCR using serotype-specific primers. The latter primers were sensitive on the FMD virus
type Asia 1 samples but less sensitive on virus samples of the other serotypes. The sensitivities of
the universal O/A/C/Asia 1 primer set and the serotype-specific primers in the diagnostic
RT-PCR mirrored results previously achieved on other virus samples while the antigen capture
format was suitable for diagnosis of FMD virus and could be further developed for
serotype-specific detection. FMD virus in all of the cell culture supernatants was detected by a
universal primer/probe set for FMD using a quantitative PCR machine. The results will be
compared with those from the other laboratories to evaluate and improve existing PCR protocols
designed for FMD virus diagnosis.

Keywords: RT-PCR, FMDV serotyping, Diagnosis, Antigen capture, Immunocapture

Introduction

Diagnosis of foot-and-mouth disease (FMD) virus by reverse transcription polymerase chain

reaction (RT-PCR) procedures have been widely documented and have involved universal
primers for the detection of all seven serotypes of the virus (Amaral-Doel et al., 1993) and
specific primers for the detection of each serotype (Vangrysperre and De Clercq, 1996: Callens
 182
and De Clercq, 1997). Although these published RT-PCR methods, were successful in the
detection or serotype-specific diagnosis of FMD virus, none have been extensively evaluated on
enough clinical samples to cover the sequence variation within each serotype and their suitability
for primary diagnosis of FMD virus using epithelial suspensions (ES) or original vesicular fluids
of clinical samples have not been fully determined. This was addressed in three studies by Reid
et al. (1998; 1999; 2000, in press) at the OIE/FAO World Reference Laboratory for
Foot-and-Mouth disease (WRL for FMD), Pirbright where universal or serotype-specific primer
sets were used in RT-PCR procedures on ES and cell culture supernatant fluids prepared from
large panels of positive FMD virus specimens of each serotype.

The study carried out by Reid et al. (1999) had used the same specific primer sets as had been
used in the earlier work of Vangysperre and De Clercq (1996) and Callens and De Clercq (1997).
However, the RT-PCR protocols used at the WRL differed from those in the earlier work in that
significantly lower concentrations of primer were used which could have accounted for
differences in the performance of the RT-PCR procedures between the laboratories. The higher
concentrations of primer used in the earlier studies would not have been practical at the WRL
given the number of RT-PCR=s routinely performed there. In order to achieve a higher
sensitivity for diagnosis of FMD virus by RT-PCR, alternative formats have been developed such
as the nested PCR format of Moss and Haas (1999) and PCR-ELISA (Callens and De Clercq,
1999). These formats have a high sensitivity for the diagnosis of field samples but also have a
high risk of contamination and are expensive to carry out.

Many laboratories use RT-PCR procedures for the diagnosis or serotyping of FMD virus but the
protocols are not standardised and the inter-laboratory variation has not been investigated. A
collaborative study initiated from the first annual European Union (EU) Concerted Action Group
meeting (project PL 98-4032, 5-7 May 1999, Federal Research Centre for Virus Diseases of
Animals [BFAV], Tübingen, Germany) was organised between the Veterinary and Agrochemical
Research Centre in Brussels, BFAV Tübingen and the OIE/FAO World Reference Laboratory for
Foot-and-Mouth Disease (WRL for FMD), Pirbright. This blind trial would provide an
assessment of the sensitivity and specificity of the RT-PCR procedures employed by the
laboratories within the EU and should lead to improvements in the diagnosis of FMD virus by
RT-PCR. The results from the RT-PCR procedures on the clinical samples obtained at the WRL
for FMD are presented and discussed here.

Materials and methods

Preparation of the unlabelled samples for the blind trial in the collaborating laboratories

Ten epithelium samples each of the FMD virus serotypes O, A, C and Asia 1 were chosen from
the WRL reference bank of positive submitted specimens along with the supernatant fluids
prepared from the propagation of epithelial suspensions (ES) of these samples in cell culture at
the time of receipt (and stored in PBS/glycerol at -200C since then). Fresh cell culture supernatant
fluids were prepared in calf thyroid and/or IB-RS-2 cell culture for a minority of the 40 samples
 183
where the original cell culture supernatants were not available. These were aliquotted for
distribution to the collaborating laboratories (including the WRL for FMD) but were not labelled
in terms of the strain or serotype of the sample. At the WRL for FMD, suspensions were
prepared from the epithelium tissues in phosphate buffer (Ferris and Dawson, 1988) and stored at
-200C until use. An ES was similarly prepared from bovine epithelial tissue from a non-infected
animal to act as a negative control. The identity of the samples was not revealed until all the
testing had been carried out so that a truly blind trial was performed.

RNA extraction and reverse transcription

Total RNA was extracted from the samples, negative control ES and non-infected cell culture
with TRIzol reagent7 (Gibco Life Technologies; Simms et al., 1993) according to the
manufacturer=s protocol. Five Φl of RNA was subjected to reverse transcription by following the
longer of the two protocols (final reaction volume of 20 Φl) as previously described (Reid et al.,
1999).

Synthetic oligonucleotide primers and RT-PCR

The universal O/A/C/Asia 1 primer set (1F/1R) was designed for the intended diagnosis of all
seven FMD virus serotypes. In a previous evaluation this set had been found to be sensitive in
RT-PCR for the primary diagnosis of the serotypes O, A, C and Asia 1 but could also detect the
SAT serotypes (Reid et al., 2000, in press). The specific primers P33/P38/P87-P92/P40/P74-P77
were designed for the diagnosis of the serotypes O, A, C and Asia 1 (Vangrysperre and De
Clercq, 1996; Callens and De Clercq, 1997) and were evaluated in RT-PCR procedures (Reid et
al., 1999; Reid et al., 2000, in press). A forward primer, reverse primer and probe were also
designed from conserved sequences of the FMD viral genome for use in a quantitative PCR
machine.

The RT product from each sample was tested with the 1F/1R primer pair as before in a diagnostic
RT-PCR procedure (Reid et al., 2000, in press). Similarly, the RT product from each sample was
tested with a cocktail of the specific primers P33/P38/P87-P92/P40/P74-P77 in RT-PCR
procedures as before (Reid et al., 1999) using both thermocycler amplification programmes,
namely : (1) 940C for 5 min, 1 cycle; 940C for 1min, 580C for 1 min, 720C for 2 min, 20 cycles;
720C for 7 min, 1 cycle and (2) 940C for 5 min, 1 cycle; 940C for 1min, 590C for 1 min, 720C for
2 min, 20 cycles; 720C for 7 min, 1 cycle.

Antigen capture RT-PCR (Immunocapture RT-PCR)

Each sample was tested in a prototype antigen capture RT-PCR method for FMD virus diagnosis
involving PCR amplification with the 1F/1R primer set after the samples had been captured in a
microtitre plate well coated with a cocktail of guinea pig anti-FMD immune sera against all
seven serotypes. A prototype antigen capture RT-PCR for serotype-specific diagnosis was also
used to test ten of the epithelial suspensions after the samples were added to wells coated
 184
individually with serotype-specific guinea pig anti-FMD immune serum and the specific primer
sets P33/P38, P33/P87-P92, P33/P40 and P33/P74-P77 (for the diagnosis of serotypes O, A, C
and Asia 1 respectively) were used in separate PCR amplifications so that the serotype specificity
of the primer matched that of the coating antibody. The procedures for the antigen capture
RT-PCR are given elsewhere in these proceedings.

TaqMan7 RT-PCR

All of the cell culture supernatant fluids were tested in a prototype TaqMan7 RT-PCR in a
GeneAmp75700 thermocycler machine (PE Biosystems, UK) which can be used for quantitative
studies but was used in this trial to give a positive or negative result for FMD viral RNA in each
sample. Briefly, one Φl of RT product was added to 24 Φl of a PCR mix containing forward
primer, reverse primer and probe in a 96-well optical reaction plate (PE Biosystems, UK) and
PCR amplification for 50 cycles was carried out in the thermocycler machine.

Results

RT-PCR
The results from the RT-PCR on the 40 epithelial suspensions and cell culture supernatant fluids
with the1F/1R primer set and the cocktail of specific primers P33/P38/P87-P92//P40/P74-P77
are summarised for each serotype in Table 1. Only one sample was not detected either as an ES
or cell culture supernatant with the 1F/1R primer set which detected every other cell culture
supernatant and 36 of the 40 ES samples including all of the serotype O and Asia 1 samples. This
mirrored the results from a previous evaluation with this primer set (Reid et al., 2000; in press)
on other FMD virus samples where better results were also achieved on type O, A and Asia 1
samples than on type C samples. The specific primers successfully detected both ES and cell
culture supernatant fluids of type Asia 1 samples using both thermocycler programmes but were
less successful for detection of the other serotypes which again mirrored previous results with
these primers (Reid et al., 1999). The annealing temperature used in the thermocycler programme
was particularly significant in the performance of the specific primers for detection of the
serotype A strains as the sensitivity of the primers dropped as the annealing temperature was
raised from 580C to 590C (Table 1).

Antigen capture RT-PCR

The performance of the 1F/1R primer set on the 40 ES samples in the antigen capture RT-PCR
were identical to those achieved by this primer set in the (diagnostic) RT-PCR (Table 1). These
methods were therefore of equal sensitivity for primary diagnosis of the O, A, C and Asia 1
serotypes with this primer set. Table 1 also shows the results of the antigen capture RT-PCR with
the specific primers on ten of the ES samples. These results were again similar to those achieved
with the same primers in the (diagnostic) RT-PCR but the serotype A strains were not detected in
the antigen capture format.

TaqMan7 RT-PCR
 185

All of the cell culture supernatant fluids were detected (but not serotyped) by the TaqMan7
RT-PCR (Table 1). This methodology is at an early stage of development and is currently subject
to patent considerations.

Conclusion
When the 1F/1R primers were used in the prototype antigen capture RT-PCR format for
FMD virus detection on the 40 ES samples, the results were identical to those from the
diagnostic RT-PCR with the same primer set. FMD virus was detected in all but one of the 40
samples so that disease would have been reported had the samples been submitted for diagnosis
from a suspected outbreak. Attempts to specifically detect the strains of the serotypes O, A and C
were less successful which suggested a lack of sensitivity of the primers for the detection of the
homologous serotype. The prototype antigen capture format for serotype-specific diagnosis was
unable to detect the type A strains but otherwise had similar sensitivity to the diagnostic RT-PCR
with the specific primers. An evaluation of these specific primers by Reid et al. (1999) concluded
that additional or alternative primers were required to improve the detection of the serotypes O
and C. However, the sensitivity of the RT-PCR for the detection or serotyping of FMD virus in
field samples may be improved to greater extent by development of alternative formats such as
the antigen capture RT-PCR and TaqMan7 RT-PCR.

Highly promising results were achieved with the TaqMan7 RT-PCR which detected all 40 cell
culture supernatant fluids with a universal primer and probe set designed from conserved
sequences in the FMD virus genome. The sensitivity of this system was 100% on the cell culture
supernatant fluids of the EU samples but the ES of the samples will have to be tested to provide a
more thorough evaluation of this method for FMD virus diagnosis. However, this method is no
more time-consuming than the diagnostic or antigen capture RT-PCR methods and only one
microlitre of RT product is required for the testing of each sample (cf. five micolitres with the
diagnostic RT-PCR) so that the tests can be performed in duplicate or triplicate.

This study was a true blind trial but it was known beforehand that all 40 samples were positive
for FMD virus which therefore imparted a bias to the way in which the testing was performed.
With this prior knowledge, more tests can be performed on the samples in order to >achieve= the
positive result. Such a luxury is not normally available.

AcknowledgementsThe authors would like to thank Professor Soren Alexandersen (Institute for
Animal Health, Pirbright) for the design of the primers and probe and for technical assistance
with the TaqMan7 RT-PCR. This work was supported financially by the Ministry of Agriculture,
Fisheries and Food, UK.

References
Amaral-Doel, C. M. F., Owen, N. E., Ferris, N. P., Kitching, R. P. and Doel, T. R. (1993)
Detection of foot-and-mouth disease viral sequences in clinical specimens and
ethyleneimine-inactivated preparations by the polymerase chain reaction. Vaccine 11, 415-421.
 186

Callens, M. and De Clercq, K. (1997) Differentiation of the seven serotypes of foot-and-mouth

disease virus by reverse transcriptase polymerase chain reaction. J. Virol. Methods 67, 35-44.

Callens, M. and De Clercq, K. (1999) Highly sensitive detection of swine vesicular disease based
on a single tube RT-PCR system and DIG-ELISA detection. J. Virol. Methods 77, 87-99.

Ferris, N. P. and Dawson, M. (1988) Routine application of enzyme-linked immunosorbent assay

in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular
diseases. Vet. Microbiol. 16, 201-209.

Moss, A. and Haas, B.(1999) Comparison of the plaque test and reverse transcription nested PCR
for the detection of FMDV in nasal swabs and probang samples. J. Virol. Methods 80, 59-67.

Reid, S. M., Forsyth, M. A., Hutchings, G. H. and Ferris, N. P. (1998) Comparison of reverse
transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation
for the routine diagnosis of foot-and-mouth disaease. J. Virol. Methods 70, 213-217.

Reid, S. M., Hutchings, G. H., Ferris, N. P. and De Clercq, K. (1999) Diagnosis of

foot-and-mouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral
RNA in clinical samples. J. Virol. Methods 83, 113-123.

Reid, S. M., Ferris, N. P., Hutchings, G. H., Samuel, A. R. and Knowles, N. J. (2000) Primary
diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol.
Methods, in press.

Simms, D., Cizdziel, P. E.and Chomczynski, P. (1993) TRIzolTM : a new reagent for optimal
single-step isolation of RNA. Focus 15, 99-102.

Vangrysperre, W. and De Clercq, K. (1996) Rapid and sensitive polymerase chain reaction based
detection and typing of foot-and-mouth disease virus in clinical samples and cell culture isolates,
combined with a simultaneous differentiation with other and/or symptomatically related viruses.
Arch. Virol. 141, 331-344.

Table 1
Summary of the results from the blind trial using the diagnostic RT-PCR, antigen capture
RT-PCR and TaqMan7 RT-PCR methods
Ratio of number of samples tested positive to number of samples tested
for each FMD virus serotype
 187
Primer set/ O A C Asia 1
RT-PCR format
ESa ES ES ES
ccb cc cc cc
Diagnostic RT-PCR

1F/1R 10/10 10/10 9/10 7/10 10/10 10/10

10/10 9/10

Cocktail of specific 5/10c 4/10c 3/10c 2/10c 4/10c 9/10c 8/10c

primers 2/10c
P33/P38/P87-P92/P40/
P74-P77

Cocktail of specific 5/10d 1/10d 1/10d 1/10d 3/10d 8/10d 8/10d

primers 4/10d
P33/P38/P87-P92/P40/
P74-P77

Antigen capture
RT-PCR

1F/1R 10/10 NTe 9/10 7/10 10/10 NT

NT NT

Cocktail of specific 1/1c 0/2c 0/1c 2/2c

primers NTc NTc NTc NTc
P33/P38/P87-P92/P40/
P74-P77

Cocktail of specific 0/1d 0/1d NTd 2/2d

primers NTd NTd NTd NTd
P33/P38/P87-P92/P40/
P74-P77

TaqMan7 RT-PCR NT NT NT NT
10/10 10/10 10/10 10/10

a
ES, epithelial suspension.
b
cc, cell culture supernatant fluid.
c
PCR amplification with themocycler programme : 940C for 5 min, 1 cycle; 940C for 1min, 580C for 1 min, 720C for
2 min, 20 cycles; 720C for 7 min, 1 cycle.
d
PCR amplification with themocycler programme : 940C for 5 min, 1 cycle; 940C for 1min, 590C for 1 min, 720C for
2 min, 20 cycles; 720C for 7 min, 1 cycle.
e
NT, not tested.
DIAGNOSIS OF PERSISTING INFECTIONS OF FOOT-AND-MOUTH DISEASE VIRUS IN CATTLE:
IGA ELISA, VIRUS ISOLATION AND RT-PCR.
P. Moonen, L. Jacobs, H. Costa, A. Crienen, and R. S. Schrijver
Institute for Animal Science and Health, Department of mammalian virology, Lelystad, The Netherlands

Key words: foot-and-mouth disease, persisting infection, diagnosis, RT-PCR, IgA ELISA

Introduction Results
Foot-and-mouth disease (FMD) is one of the most The results of the VI are shown in figure 1. The
important diseases from both a veterinary as well as animals vaccinated with the highest dose (nrs 1315-
an economic point of view. Outbreaks with devastating 1319) shed virus for a longer time period than animals
economic consequences still occur and remain a vaccinated with lower doses. Using RT-PCR more
terrible threat to countries that have eradicated the samples were found positive than using VI.
disease and to those that never had the disease. The
main concern nowadays is how to prevent introduction Figure 1: Results of the virus isolation and PCR in
and, in case of an outbreak how to prevent spread of probang samples. Positive isolations and PCRs are
the virus. In this respect a critical issue is the depicted +, periods of intermittent positive findings are
occurrence of carrier animals and their risk in
transmitting the virus (3, 6). Carrier animals are carriers -->

10-mei
17-mei
25-mei

16-aug
23-aug
30-aug

13-sep
20-sep
27-sep

16-nov
23-nov
30-nov
currently defined as animals from which FMDV can be

15-mrt
22-mrt
29-mrt

12-apr
19-apr
26-apr
23-feb
24-feb

14-jun
21-jun
28-jun

11-okt
18-okt
25-okt
3-mei

2-aug
9-aug

6-sep

1-nov
9-nov
12-jul
19-jul
26-jul
1-mrt
8-mrt

5-apr

1-jun
7-jun

4-okt
5-jul
1315 + + + + + + + +

isolated from oropharyngeal fluid (OPF) more than 28

VI

P CR + + + + +
1316 VI + + + + + + + + + + + +

days after infection. However, virus isolation (VI) from

P CR + + + + + + + +
1317 VI + + + + + + + +
P CR + + + + + + + + + + + + +

carriers is intermittent. To improve detection of carrier 1318

1319
VI

P CR
+

+
+
+
+ + + +
+ + + +
+
+
+ +

+
+

+
+
+

+ +
+ +
+
+ +
+

animals, virus isolation, polymerase chain reaction

VI

P CR + + + + + + + + + + + + +
1320 VI + + + + + + + + + + +

(PCR), IgA ELISA and virus neutralisation test (VNT)

P CR + + + +
1321 VI + +
+

were compared. For that purpose animals were

P CR

1322 VI + + + + + + + + + + + + + + + +
P CR + + + + + + + + + + + + + + + +

infected with FMDV Atur 14/98 and samples were

1323 VI + + + + +
P CR + + + + + + + + + + + +
1324 VI + + + + + + + + +

taken at regular intervals during an eight months 1325

P CR

VI
+
+
+
+ + + + + + +
+ +
+ + + + + + +
+

+
+ +
+ +
+ +
+ + + + +

period after infection.

P CR

1326 VI + + + +
P CR + + + + + + + + + + + +
1327 VI + + + + +
P CR + + + + + + + + + + +
1328 + + +

Materials and methods

VI

P CR + + + + + + + + + + + +
1329 VI + + +

Fifteen cattle were vaccinated with Atur 14/98: five

P CR + + + + + + + + + + +
1330 VI +
P CR + +

animals with a full dose (nrs 1315-1319), five animals 1331 VI

P CR
+
+
+
+ + + + + + + + + +
+
+ +

with an 1/4 dose (nrs 1320-1324) and five animals with

1979 VI

P CR

1/16 dose (nrs 1325-1329), two animals were not shaded light grey for VI and dark grey for PCR.
vaccinated. After three weeks all animals were
infected with Atur 14/98. Two months after infection a Of the 465 samples tested in VI and PCR, 35 were
sentinel animal was placed between the infected positive in both assays, 127 were positive in PCR but
cattle. The animals were sampled weekly; serum negative in VI and 43 were positive in VI and negative
samples were tested in an IgA ELISA and VNT; in PCR, 260 samples were negative in both assays
sputum samples, collected using a probang sampler (Fig. 2).
and diluted 1:1 in EMEM +2% FBS +2% antibiotics, Within 10 days after infection neutralisation titres in all
were tested in a PCR and after treatment with 1,1,2- animals raised from 1 10log after vaccination, to
trichlorotrifluoroethane (5) in a virus isolation and IgA comparable levels of ca. 3.5 10log. Representative
ELISA. profiles of neutralisation titres and IgA responses
VI was performed in eightfold in microtiter plates by detected in serum and probang samples are shown in
adding 50 µl sputum to 150 µl porcine kidney cells. figure 3.
After three days incubation the plates were read
microscopically and, after staining with amidoblack, Figure 2: Comparison of reactions of probang samples
macroscopically. Supernatants were additionally in virus isolation and PCR.
tested in an IDAS ELISA for antigen content. RT-PCR
as performed using primers and hybridisation probes VI + VI - total
selected on the 3D nucleotide sequence of ATur 14/98
and were analysed using Lightcycler® technology PCR + 35 127 162
(Roche). The isotype specific IgA ELISA was done
essentially as described by van Zaane and IJzerman PCR - 43 260
(6). The VNT was done as described by van Maanen Total 78
et al. (2).
 Figure 3: Results of PCR, virus isolation, virus neutralisation test, IgA ELISA on serum and probang samples.
ELISA data are indicated on the right Y-axis, the other data on the left Y-axis. Animals are representative for the
whole experiment

1315 1320

4 1 4 1
P CR P CR
3.5 3.5
0.8 0.8
3 VI VI
3
2.5 0.6 2.5 0.6
VNT titres

VNT titres
VNT VNT

OD450

OD450
2 2
1.5 0.4 0.4
IgA in 1.5 IgA in
s e rum s e rum
1 1
0.2 0.2 IgA in
IgA in
0.5 s putu 0.5 s putu
m m
0 0 0 0
0
6
13
20
34
54
75
96
116
137
158
179
200
217
238
260

0
6
13
20
34
54
75
96
116
137
158
179
200
217
238
260
days post infection days post infection

1325 1330

4 1 P CR
4 1 P CR
3.5
3.5
0.8 VI
0.8 VI 3
3
2.5 0.6

VNT titres
2.5 0.6 VNT
VNT titres

OD450
VNT
OD450

2
2
1.5 0.4 IgA in
1.5 0.4 IgA in
s e rum
s e rum 1
1 0.2
0.2 IgA in
IgA in 0.5 s putu
0.5 s putu m
m 0 0
0 0

17

34

61

89

116

144

172

200

224

253
0

17

34

61

89

116

144

172

200

224

253

days post infection

days post infection

In serum, shortly after infection, IgA levels raised, and

decreased within 10 to 20 weeks to baseline levels. In
sputum IgA levels were hardly detected. Only in a few References
cases OD450 values higher than 1 were reached 1. Archetti IL, Amadori M, Donn A, Salt JS, and Lodetti E.
The sentinel animal was negative for VI, PCR, VNT Detection of foot-and-mouth disease virus-infected cattle
and IgA in probang during the entire period of by assessment of antibody response in oropharyngeal
sampling. fluids. J. Clin. Microbiol. 1995; 33: 79-84.
2. Maanen, C v, Terpstra C. T. Comparison of a liquid
phase blocking sandwich ELISA and a serum
Discussion neutralisation test to evaluate immunity in potency tests
of foot-and-mouth disease. 1989; 129: 111-119
VI is the standard method to detect carriers of FMDV, 3. Mezencio JMS, Babcock G, Kramer E, and Brown F.
however, other techniques may be more suited, such Evidence for the persistence of foot-and-mouth disease
as assessment of specific antibody responses as virus in pigs. Vet. J. 1999; 157: 213-217
suggested by Archetti et al (1) or detection of viral 4. Reid SM, Forsyth MA, Hutchings GH, and Ferris NP.
RNA by RT-PCR (4). We found that detection of IgA Comparison of reverse transcription polymerase chain
responses in serum were not suitable to detect reaction enzyme linked immunosorbent assay and virus
carriers. Although IgA antibodies were detected up to isolation for the routine diagnosis of foot-and-mouth
150 days post infection, correlation between the rate disease. J. Virol. Meth. 1998; 70: 213-7.
5. Sutmoller P, and Cottral GE. Improved techniques for
of decline of the antibodies titres and the presence of the detection of foot-and-mouth disease virus in carrier
virus in probang samples was not clear. IgA detection cattle. Arch. Ges. Virusforsch. 1967; 21: 170-7.
in probang samples was, in our hands, difficult. No, or 6. Van Bekkum JG, Frenkel HS, Frederiks H, and Frenkel
only low levels were detected, and no correlation was S. Observations on the carrier state of cattle exposed to
found between virus detection in VI or RT-PCR and foot-and-mouth disease virus. Tijdschr. Diergeneesk.
reaction in the IgA ELISA. 1959; 20; 1159-64.
RT-PCR seemed to be a more suitable technique to 7. Van Zaane, D., and IJzerman, J.J. 1984. Monoclonal
detect carriers of FMDV. The number of positive antibodies against bovine immunoglobulins and their use
in isotype-specific ELISAs for rotafirus antibody. J. Imm.
samples found in RT-PCR was approximately 17% Meth., 72; 427-441.
higher than the number of samples found in VI. Only 8
% of the samples was positive in both, VI and the RT-
PCR. Approximately 9% of the samples was positive
in VI and negative in RT-PCR. This can be due to the
low virus content of the sample and the smaller
volume of the sample (10x) tested in the RT-PCR
compared to VI. Approximately 27% of the samples
was positive in the RT-PCR and negative in the VI. If
virus is neutralised, it will not be detected in the VI but
will be detected in the RT-PCR. In conclusion: RT-
PCR is more sensitive than VI for detection of FMDV
in probang samples and moreover more easy to
perform and less time consuming
 190

Appendix 23

Spray-drying of inactivated FMDV antigens for diagnostic use

Amadori M.

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia,

Brescia, Italy,

SUMMARY
The production of inactivated, spray-dried FMD vaccines has been
described by Russian research workers for a long time . We were therefore
stimulated to exploit such a technology to obtain preparations of
inactivated FMDV antigens for diagnostic use. The latter usually consist
of a large proportion of degraded 12S virus particles; yet, they are
perfectly suited to work in the established serological tests for Ab to
FMDV . Our study was carried out using a laboratory scale spray-drier
which includes a main cabinet (control panel, inlet air filter, blower, top
chamber, peristaltic pump) and a spray assembly (main chamber, cyclone,
collection bottle). The apparatus enables the fine setting of the main
working parameters: drying temperature, air flow, compressor air pressure,
peristaltic pump speed. The preliminary tests were carried out on an
inactivated Cardiovirus preparation as a test model; by using a preserving
solution of skim milk + NaCl at different concentrations we could
demonstrate almost complete recovery of both EMCV particles, i.e.
complete virions and empty capsids. Later on we took to horse serum
instead of skim milk, because of the more favourable behaviour of the
sugar in the presence of high exhaust air temperatures. As for FMDV, the
tests were carried out on BEI-inactivated O1 Lausanne and A5 Parma
FMDV. In the presence of adequate additions of horse serum and saline,
an inactivated virus suspension with high antigen content could keep its
usual properties in the serological tests after the spray-drying process,
despite an increased concentration of 12S particles. In particular, the
dose/response curve of the Ag in ELISA tests for 12S and 146S/12S
particles was not significantly different from that of the usual frozen Ag
preparations. Furthermore, Ab-negative and Ab-positive bovine sera gave
the same results with both conventional and spray-dried antigens. The
possible major advantages of this procedure in terms of simplicity and ease
 191

of use, convenience for the diagnostic laboratory and suitability for a

process of stepwise harmonisation of the procedures among the National
FMD Laboratories are put forward and illustrated.

INTRODUCTION
The process of stepwise harmonisation of the procedures among the
national FMD laboratories demands the provision of suitable reagents for
large-scale ring tests, aimed at evaluating the reliability and the
reproducibility of the investigation procedures for both virus and antibody
detection. With regard to the latter, the availability of large homogeneous
batches of FMDV inactivated antigens is of utmost importance; on the one
hand, it is arguable that such large batches should be made available to the
regional laboratories in a scenario of extensive serological surveillance in
the aftermath of a FMD outbreak; on the other hand, they would certainly
conducive to the ongoing accreditation process within a quality assurance
scheme ( 8 ). One practical solution is represented by the freeze-drying of
FMDV antigens. Infectious FMD viruses, inactivated antigens and even
vaccines (6, 10) can be freeze-dried without substantial detrimental effects
on their biological properties. Furthermore, shipment of freeze-dried virus
antigens is possible without the need for refrigeration, and their reactivity
with bovine convalescent antisera in the liquid phase blocking sandwich
ELISA is not unduly affected ( 11). Diagnostic kits may also conveniently
include reference freeze-dried antisera ( 9 ). However, freeze-drying is a
laborious procedure, since it may extend over 2-3 days and demands the
filling and plugging of a large number of ampoules. In this respect, spray-
drying may represent a convenient alternative. It must be stressed that the
production of inactivated, spray-dried FMD vaccines has been described
by Russian research workers for a long time; mono- or polyvalent FMD
vaccines were obtained, which showed high potency and a shelf life not
shorter than 5 years at 4°C; the stability during spray-drying was different
between A22 and O1 FMD viruses; both could be also formulated into
potent emulsified vaccines for pigs (13,14,15). On the basis of these
favourable results, we were therefore stimulated to investigate such a
technology to obtain preparations of inactivated FMDV antigens for
diagnostic use.
 192

MATERIALS AND METHODS

Apparatus. Our study was carried out using a laboratory scale spray-
drier (LAB-PLANT model SD/05), which includes a main cabinet (control
panel, inlet air filter, blower, top chamber, peristaltic pump) and a spray
assembly (main chamber, cyclone, collection bottle). The apparatus
enables the fine setting of the main working parameters: drying
temperature, air flow, compressor air pressure, peristaltic pump speed.

Antigens. Preliminary experiments were carried out on a BEI-inactivated

Cardiovirus preparation. Subsequent experiments were performed on BEI-
inactivated O1 Lausanne and A5 Parma FMD viruses. These were
supplemented with either skim milk or horse serum in the presence of
variable final concentrations of NaCl. The pH of these preparation varied
between 7.4 and 7.8 .Small aliquots were dried under vacuum for a rough
estimate of the total dry content; this enabled us to calculate an expected
recovery for each sample. All antigens had been supplemented with 0.5%
sodium thiosulfate (final) after BEI treatment.

Spray-drying. 200 to 500 ml of inactivated antigen were used at a time.

After repeated preliminary testing, the following range of conditions was
adopted:
 Sample temperature: 4 to 20 °C (excursion during the process).
 Process temperature: 130 or 150 °C
 Exhaust air temperature: 80 to 90 °C
 Pump speed: 500 to 700 ml/hour
 Compressor pressure: 1.8 to 2.0 bar
 Air-flow: 60 to 65 m3/hour

Tests on dried antigens. At the end of each experiment, the powder was
weighed and the obtained recovery was compared with the expected one.
Aliquots of O1 Lausanne FMDV were reconstituted with distilled water at
a precise weight to volume ratio and tested by sandwich ELISA with
couples of monoclonal antibodies reacting with 146S and/or 12S particles
( 1 ). After reconstitution with water, both O1 Lausanne and A5 Parma
FMDV spray-dried antigens were employed for antibody detection by a
monoclonal antibody-based competition ELISA, as previously described (
 193

5 ); tests were carried out on reference post-vaccination and post infection

bovine sera in parallel with the usual antigens, stored in small aliquots at -
70°C .

RESULTS
Cardiovirus. This was chosen as a test model in the preliminary phase of
this study. The inactivated antigen was obtained from a high-titred virus
preparation (≥ 109 TCID50/ml). It was supplemented in the 1st experiment
with 10% skim milk and 3% NaCl and dried at 130°C. Due to the low
recovery in the collection bottle, the two following experiments were
carried out at 150 °C in the presence of 10% skim milk + 0.5% NaCl (2nd
test) and 8% skim milk (3rd test). The three dried Ag preparations were
reconstituted with distilled water to the initial volume and tested by
sandwich ELISA with couples of monoclonal antibodies recognising both
complete and empty capsids; the two antigenic forms were well
preservedin the 3 tests without an appreciable loss of reactivity, the OD/Ag
dilution curves being very similar to that of the control untreated antigen.
A slightly better recovery of complete virions was observed in the 1st test.

FMDV. On the basis of these favourable results, we decided to undertake

the experiments on FMDV. All of these were carried out at 150°C with the
addition of horse serum instead of skim milk because of problems linked
to early clogging of the nozzle and coagulation of lactose. We reasoned
that optimal recovery of 146S particles was not a priority, since the usual
ELISAs for detection of Ab to FMDV can work in the presence of
antigens characterised by a large degradation of 146S particles into 12S
particles.
The 1st set of experiments were carried out on a batch of O1 Lausanne
FMDV antigen, showing a high initial infectious titre (8.4 TCID50/ml,
log10). The best results in terms of both dry content and Ag recovery were
obtained in the presence of 10% (final) horse serum and a final Ag
dilution 1:2 in saline (0.42% NaCl, final). It must be stressed that the
OD/Ag dilution curves in the ELISAs for total Ag (figure 1a) and for 12S
Ag (figure 1b) were very similar to those of the reference standard antigen
kept at -70°C . Furthermore, the results of the Ab tests on reference bovine
 194

sera were almost identical by using these same two antigens (see figures
2a and 2b).
The 2nd set of experiments was carried out on a batch of A5 Parma FMDV,
showing a much lower initial infectious titre (7.5 TCID50/ml, log10). In
this case, the antigen preparation before spray-drying had a dramatically
reduced reactivity in the test system and gave rise to Ab titres of some
post infection bovine sera, which were lower than those obtained with the
reference antigen preparation; an equal or even lower reactivity was shown
by the spray-dried preparations; the best results were obtained in the
presence of 12.5% horse serum and 0.42 to 0.85% (final) NaCl, in close
agreement with the above results on O type FMDV.

DISCUSSION
Spray-drying is largely used in the pharmaceutical industry; the
applications in the field of microorganisms are instead scanty but anyway
significant; spray-drying has been described for microorganisms as diverse
as Streptococcus bacteriophages (7) and lactic acid bacteria ( 17 ).
Interesting experiences have been also described in the field of
vaccinology; in particular, spray-drying can be successfully used to
prepare microparticles containing entrapped protein antigens ( 2,3 ); such
microparticles were very efficient e.g. for immunoglobulin delivery to the
respiratory tract (4) and for inducing a strong immune response in guinea-
pigs to diphteria toxoid ( 12 ). There is in practice a large body of evidence
that proteins can retain stability and antigenic properties during spray-
drying, thus giving rise to a product with an extended shelf life. In our
laboratory, a notable concentration of 146S particles was shown e.g. in a
dried A22 FMD vaccine prepared 11 years before at ARRIAH, Vladimir
(Amadori M., unpublished results). In this respect, the experience of the
Russian research workers in the FMD field suggests that temperature and
residual humidity play a major role: in the presence of a residual humidity
≤3% the shelf life is not less than 5 years at 2-8 °C and the product can
work better than the same one kept at room temperature for one year only
(16). A further reduction of residual humidity may be obtained by vacuum
dehydration of concentrated powders: in this case the moisture content can
 195

be reduced to less than 1%, while the structure and properties of FMD
virus are retained ( 18 ).
The substantial effectiveness of spray-drying for obtaining suitable
preparations of FMDV antigens for diagnostic use has been confirmed in
our study, although further data are needed before reaching final
conclusions. It must be stressed that the laboratory spray-drier used in this
study does not allow for a complete recovery of the powders; hence
qualitative rather quantitative data should be taken into account. In our
experience, the real bottleneck of the procedure was represented by the
process temperature: at 150 °C the flow rate should not exceed 500-600
ml/hour, provided that the compressor pressure and the air flow are within
the aforementioned limits; if not, condensed moisture accumulates in the
main chamber, which is an adverse prognostic factor for the recovery and
the final quality of the product. Another crucial point is the NaCl
concentration; on the one hand, a certain addition of salt is badly needed
to increase the recovery of powder in the cyclone; on the other hand,
there is evidence that high NaCl concentrations are detrimental to the
stability of inactivated 146S particles and, to a lesser extent, of 12S
particles as well. This may be due to the inverse relationship between ionic
strength and isoelectric point of FMD virus particles ( 19 ); notice that
ionic strength was also increased in our experiments by the addition of
sodium thiosulfate after the BEI treatment. Finally, the initial Ag
concentration could possibly play a major role: good results were obtained
on high-titred virus suspensions only, and it is arguable that a preliminary
5 to 10-fold concentration before drying could be actually of use; anyhow,
a preliminary ELISA on the inactivated virus suspension could be
predictive of the final results of the spray-drying procedure.
An adequately standardised procedure could be very convenient: 10
litres/day of inactivated FMDV antigen could be easily processed by one
technician using a combined ultrafiltration and spray-drying scheme with
a simple laboratory apparatus; then, the powder should be stored at 2-8°C
protected from humidity; at the beginning of each week, a certain amount
of powder could be reconstituted with cold distilled water at a defined
weight to volume ratio and the antigen used in the next 4 – 5 days. Such a
working scheme could be very convenient and conducive to the
reproducibility of the Ab tests; therefore, it would be instrumental to the
process of accreditation and quality assurance ongoing in many national
FMD laboratories ( 8 ).
 196

ACKNOWLEDGEMENTS
The skilful technical assistance of G. Passador, I. Reda and M.
Scaramuzza, as well as the precious co-operation of the serology
laboratory of the Italian National Reference Centre for Vesicular Diseases
are gratefully acknowledged.

REFERENCES

1. Amadori M. et al. (1994), Vaccine, 12, 159-166.

2. Baras B. et al. (2000), J. Microencapsul., 17 (4), 485-498.
3. Baras B. et al. (2000), Vaccine, 18 (15), 1495-1505.
4. Bot A.I. (2000), Pharm. Res., 17 (3), 275-283.
5. Brocchi E. et al. (1990), Rpt. Sess. Res. Grp. Stan.Tech.Eur.Comm.
Cont. FMD, Lindholm, Denmark, 25-29 June, Appendix 14.
6. Butchaiah G. and Rao B.U. (1988), Rev. Sci. Tech. Off. Int. Epiz., 7
(2), 347-356
7. Chopin M.C. (1980), J. Dairy Res., 47 (1), 131-139.
8. De Clercq K. And Donaldson A.I. (1997), Rpt. Sess. Res. Grp.
Stan.Tech.Eur.Comm. Cont. FMD, Poiana-Brasov, Romania,
Appendix 19.
9. Ferris N.P. et al. (1988), J. Virol. Methods, 19 (3-4), 197-206.
10. Ferris N. P. et al. (1990), J. Virol. Methods, 19 (1), 43-52.
11. Ferris N.P. et al.(1990), J. Virol. Methods, 30 (2), 183-195.
12. Johansen P. et al. (1999), Vaccine, 18 (3-4), 209-215.
13. Kravchenko V.M. et al. (1991), Proc. Int. Conf. "Towards a new
strategy to combat FMD", Vladimir, Russia, pp 39.
14. Kravchenko V.M. et al. (1991), Proc. Int. Conf. "Towards a new
strategy to combat FMD", Vladimir, Russia, pp 40-41.
15. Kravchenko V.M. et al. (1991), Proc. Int. Conf. "Towards a new
strategy to combat FMD", Vladimir, Russia, pp 45.
16. Kurlova N. P. et al. (1991), Proc. Int. Conf. "Towards a new strategy
to combat FMD", Vladimir, Russia, pp 46-47.
17. Mauriello G. et al. (1999), J. Food Prot., 62 (7), 773-777.
18. Ponomarev A.P. et al. (1996), Vopr. Virusol., 41 (5), 218-221.
19. Vande Woude G.F. (1967), Virology, 31 (3), 436-441.
 197
Appendix 24

A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease

virus

N.P. Ferrisa, A.N. Bulutb, T. Rendlea, F. Davidsona and D.K.J. Mackayc

a
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey,
GU24 0NF, UK
b
FMD Institute, PK 714, 06 044 Ulus, Ankara, Turkey
c
Veterinary Medicines Directorate, Woodham Lane, New Haw, Addlestone, Surrey
KT15 3NB, UK

Summary

A solid-phase competition ELISA (SPCE) has been devised for the measurement of
antibodies to foot-and-mouth disease (FMD) virus. The assay uses polyclonal antisera and
inactivated purified 146S antigens of FMD virus and was compared with the liquid-phase
blocking ELISA (LPBE) and virus neutralisation test (VNT) on a range of serum sets. The
SPCE exhibited equal test sensitivity as the LPBE and VNT and a better specificity of
reaction than the LPBE when using test sera at a 1:5 dilution and a cut-off point of 30
percentage inhibition of antigen. The assay thus retains the sensitivity and ease of use of the
LPBE whilst being more robust and specific and offers an improvement for FMD virus
antibody detection.

Introduction

The OIE prescribed tests for foot-and-mouth disease (FMD) serology are the virus
neutralisation test (VNT; Golding et al., 1976) and the liquid-phase blocking ELISA
(LPBE; Hamblin et al., 1986). The LPBE has since been adopted by a large number of
laboratories worldwide (Mackay et al., 1994, 1998) to replace the VNT for routine
screening because it is quicker, more reproducible, correlates well with the VNT and does
not suffer from the biological variability that is inherent in the VNT. However, the test is
not without its problems. The percentage of animals giving false-positive results varies
according to the animal population particularly in animals which are stressed and can be as
high as 4% (Haas, 1994). For this reason, it is usually recommended that low-positive
LPBE results are confirmed by VNT, particularly for the purposes of international trade
(Anon, 1996). The assay requires training and experience to yield reproducible results and
is not particularly 'robust'. A major factor for the lack of robustness of the LPBE has been
in the variable stability of inactivated antigens employed in the procedure.

We decided that it would be worthwhile to examine a solid-phase competition ELISA

 198
(SPCE) for FMD virus serology using polyclonal antiserum reagents (as used by the LPBE)
and inactivated, purified 146S antigens of FMD virus which have been observed to be
remain stable after long term (years) storage at 4oC (N.P. Ferris, unpublished results)

Materials and methodsMaterials and methodsMaterials and methodsMaterials and

methods

Viruses

FMD virus types O1 BFS 1860, A22 IRQ 24/64 and C1 Noville were grown on monolayers
of BHK-21 cells, inactivated with 0.001 M binary ethyleneimine (BEI; Bahnemann, 1990),
146S antigens purified according to the method of Ferris et al., 1984 and stored at 4oC for
subsequent use in the SPCE.

Test Serum Samples

Four sets of positive sera were examined : i) reference sera obtained from cattle between 21
and 28 days after either infection or vaccination with single, defined strains of FMD virus
of known origin; ii) sera collected sequentially following either infection (type O1 BFS) or
vaccination (types A22 and C1) with FMD virus; iii) sera generated for the FAO
Collaborative Studies Phases XIII (Mackay et al., 1994) and XV (Mackay et al., 1998)
and iv) 'problem' sera : sera from cattle or water buffaloes which had been found to be low
positive by LPBE and/or VNT on serological testing of animals prior to international trade
but in which there was no recorded history of previous vaccination or infection.
Additionally, negative sera from the UK, Canada or elsewhere which originated from
animals with no history of exposure to FMD virus and which had been tested by LPBE
and/or VNT with negative results.

Virus neutralisation test and Liquid-phase blocking ELISA (LPBE)

Virus neutralisation tests (VNT) were performed in tissue culture grade microtitre plates
using the method described by Golding et al. (1976). The LPBE was carried out as
described by Hamblin et al. (1986).

Solid-phase competition ELISA (SPCE)

The final procedure adopted for the solid-phase competition ELISA (SPCE) was as follows
(50 μl reagent volumes were used throughout, ELISA plates [Nunc Maxisorp
immunoplates] incubated for 1 hour in 37oC on a rotary shaker, unless otherwise stated,
and plates washed three times with phosphate buffered saline [PBS, pH 7.4] containing
0.05% Tween 20 after each incubation step). Plates were coated with an optimal dilution of
rabbit antiserum to FMD virus in carbonate/bicarbonate buffer, pH 9.6 and incubated
 199
o
overnight at +4 C. Next an optimal dilution of 146S antigen of FMD virus homologous to
the rabbit antiserum was diluted in PBS containing 0.05% Tween 20 and phenol red
indicator (PBST) and added to each well. Duplicate, two-fold dilutions of each test serum
(from an initial dilution of 1:2.5) in PBST containing 10% normal serum of the species
under test and 5% normal rabbit serum (blocking buffer 3) were performed in plates.
Immediately homologous guinea pig antiserum, diluted to the optimal concentration in
blocking buffer, was added to each well. After plate incubation, an optimal dilution of
rabbit anti-guinea pig immunoglobulins conjugated to horse radish peroxidase in blocking
buffer was added to each well. Substrate (0.05% H2O2)/chromogen (orthophenylene
diamine) in citrate/phosphate buffer, pH 5.0 was next added. After 15 min incubation the
reaction was stopped by adding 1.25 M sulphuric acid. The optical density (OD) of each
well was read by using a spectrophotometer (Dynatech) with a 492 nm filter. Antibody
titres were expressed as the last dilution of serum showing 30% inhibition of OD compared
to the mean OD of the reaction control wells where serum was absent.

Blocking Buffers

Three different blocking buffers were evaluated using defined positive and negative sera: i)
blocking buffer 1 - PBS with 0.05% Tween 20 containing 5% skimmed milk powder
("Marvel"); ii) blocking buffer 2 - PBS (pH 7.4) with 0.1% Tween 20 and containing 0.3%
normal serum of the species under test and iii) blocking buffer 3 PBS (pH 7.4) with
0.05% Tween 20 and containing 10% normal serum of the species under test and 5%
normal rabbit serum.

Results

Differentiation of positive from negative sera

To evaluate the performance of the test, 57 positive reference sera for type O and 120
negative sera were titrated in the SPCE for antibody to FMD virus type O1 BFS 1860 in a
dilution series from 1:2.5 to 1:80. The results were analysed in terms of percentage
inhibition of antigen at each serum dilution (Fig. 1). The dilution at which there was
optimal differentiation of positive from negative sera was 1:5. All positive sera had more
than 30% inhibition and all negative sera less than this value. At this dilution there was no
overlap in the two populations using blocking buffer 3, in contrast to considerable overlap
of the positive and negative populations using blocking buffers 2 and 3 (results not shown).
Blocking buffer 3 was therefore used for all subsequent work.

Having established a provisional cut-off for the test, large numbers of negative sera were
then examined at a single dilution of 1:5 (753 sera against type O1 and 216 against types
A22 and C, of which 113 sera were examined against all 3 serotypes). Frequency
distributions showed the percentage inhibitions of the negative sera to be normally
 200
distributed (Fig. 2). For a normal population, establishing a cut-off at the mean + 3
standard deviations (SD's) should mean that less than 1% of the values exceed the cut-off.
For the negative populations studied here, the mean % inhibitions for types O, A and C
were 9, 10 and 10% respectively with standard deviations of 6% in each case. This gave
cut-off values of 27, 28 and 28% for types O, A and C respectively. To simplify
interpretation and to ensure a high level of specificity, the cut-off was therefore set at 30%
for all serotypes. Using this cut-off, the specificity of the assays for types A and C was
100% (zero positive out of 216). For type O, the specificity was 99.3% as 5 out of 753 sera
scored positive at the single dilution of 1:5. However, all 5 sera were negative in a titration
ELISA (i.e. titre <1:5) raising the specificity of the overall testing procedure to 100% for
this population.

Correlation with LPBE

End point titres were determined in the SPCE and LPBE for positive reference sera for
each of the serotypes O, A and C (Fig. 3). Statistically significant correlations were
measured for each serotype and all sera positive by LPBE were also positive by SPCE.
This result also shows that the SPCE detects antibody to a wide variety of strains within
each serotype. For each serotype a single strain of FMD virus was used as the antigen in
ELISA, whereas a wide variety of strains from within each serotype was used for infection
or vaccination of cattle to produce the reference sera.

The dynamics of the antibody responses to infection (type O) and vaccination (types A and
C) showed that antibody titres were similar irrespective of measurement by either the
LPBE or SPCE (results not shown). Titres in the low positive/doubtful range were
measured at 7 days post vaccination (dpv) and all animals were strongly seropositive by 14
dpv.

The sensitivity and reproducibility of the SPCE were compared directly with the LPBE and
the VNT. Five two-fold dilutions of a type O reference serum were prepared in normal
negative bovine serum such that the estimated end point titre in the SPCE was "bracketed"
in either direction. These sera were then randomly labelled from A to E and repeatedly
examined blind by SPCE, LPBE and by VNT, together with the original positive reference
serum (N; Table 1). The SPCE and the LPBE had the same sensitivity, both scoring the
1:160 dilution (B) positive and the 1:320 dilution (D) negative. The VNT was slightly less
sensitive than either ELISA scoring the 1:160 dilution (B) doubtful (i.e. titre between 1:16
and 1:32) but the 1:80 dilution (E) positive (i.e. titre >1:45). In terms of reproducibility,
both ELISA's generally had lower coefficients of variation (CV) than the VNT. The CV's
of the sera in the SPCE were similar to but slightly higher than those in the LPBE.

Sera from two FAO Standardisation Exercises were examined by SPCE, LPBE and the
VNT. In the Phase XIII Exercise (Table 2), panels of four reference sera for each of the
 201
strains O1 BFS 1860, A22 IRQ 24/64 and C1 Noville were created which varied in strength
from weak positive through to strong positive. Although the absolute values varied
between assays, there was good correlation between the three assays. All the reference sera
correctly scored positive in the SPCE with the exception of RS-4 for type O1 BFS 1860
which was negative by half a dilution step (titre 1:3.5; cut-off 1:5). However, this serum
was also doubtful by VNT with a titre of 1:32 (cut-off in VNT 1:45).There was also good
correlation between the results of all three tests in examination of the Phase XV
provisional reference sera (results not shown).

Using as antigen either the homologous strain of FMD virus with which the animal was
infected or a heterologous strain of the same or a different serotype, there was a high
degree of cross-reactivity between the two strains of type O in each of the 3 assays but the
extent of cross-reactivity was greatest in the SPCE (results not shown). Some degree of
cross-reactivity between serotypes was observed in all three assays for all three serotypes.
In all cases, there was less cross-reactivity between serotypes in the SPCE than in the
LPBE or VNT, suggesting that the SPCE is more serotype specific than the other two tests.

Of the 73 of 85 >doubtful sera= which were positive in the LPBE for antibody to type O,
only 3 were positive in the SPCE, one of which was also positive by VNT (Table 3).
Likewise for antibody to type A, only 4 out of 48 sera which were LPBE positive were also
positive by SPCE and two of these were also VNT positive. Assuming that these sera were
actually from FMD naive animals, which could not be proven beyond doubt, then the
SPCE was considerably more specific than the LPBE for these 'doubtful' sera and was only
slightly less specific than the definitive VNT.

Discussion

Analysing the results overall, higher concentrations of positive sera were required to
achieve a given percentage inhibition in the SPCE than in the LPBE. This meant that the
cut-off in the SPCE was at a lower dilution (1:5) than in the LPBE (1:40; Hamblin et al.,
1986) and at a lower percentage inhibition (30% as opposed to 50%). Nevertheless, using
this cut-off value, the sensitivities of the two ELISA's were almost identical (Table 1).
Only some sera with the lowest positive titre by LPBE (1:45) just scored negative (1:3.5) in
the SPCE. The SPCE was at least as sensitive (Table 1) and certainly more specific (Table
3) than the 'gold standard' VNT.

Any new test for antibody to FMDV must score correctly reference sera which are
recognised as international standards. The SPCE scored the provisional FAO reference sera
for FMD virus types O1 Manisa, A22 IRQ 24/64 and C1 Noville correctly (Table 2) and
showed good correlation with both the LPBE and the VNT for panels of positive reference
sera to FMD virus type O1 BFS 1860, A22 IRQ 24/64 and C1 Noville. When using serology
for diagnosis of FMD it is useful to have a test which is as broadly cross-reactive between
 202
strains of a given serotype, but as serotype-specific as possible. Examining selected
reference sera by SPCE, LPBE and VNT showed that the SPCE most closely matched
these criteria. Although positive titres were recorded against serotypes other than the one
used to infect the animal, their heterologous titres were universally low, and lower in
comparison, than those recorded in the LPBE and the VNT. The ability of the SPCE to
detect antibody generated by exposure to a wide variety of strains within a serotype is best
shown in Fig 3. For all 3 serotypes, all reference sera collected between 21 and 28 days
after either infection or vaccination with any strain within a serotype gave a positive
reaction in the SPCE using as antigen purified FMD virus of the homologous serotype.
This figure also shows a strong correlation between titres in the SPCE and in the LPBE
(and the r values would have been higher if end point titres had been determined for all sera
having an SPCE titre of greater than 1:160). The fact that some sera had a high titre by
LPBE, but a low titre by SPCE and vice versa suggests that the population of antibodies
measured in the two tests were not identical. This is to be expected as the LPBE measures
both antibody which prevents binding of the antigen in the liquid phase to the immobilised
rabbit antiserum and antibody which blocks the detecting guinea pig antiserum. In contrast,
the SPCE relies only on competition between the test serum and the detecting guinea pig
antiserum.

The objective of the work reported here was to develop an ELISA for antibody to FMD
virus which retained the sensitivity and ease of use of the LPBE whilst being more robust
and, hopefully, more specific. A sensitive, specific and reliable SPCE has been developed
to measure antibody to FMD virus to fulfill the objective. Work is now in hand to adopt the
assay for use with the other FMD virus serotypes and to evaluate the test in day-to-day use
in the WRL for FMD.

A full presentation of this work is in preparation for submission to the Journal of

Virological Methods.

Acknowledgements

This work was supported financially by the UK Ministry of Agriculture, Fisheries and
Food (project number SE 1113). The authors wish to thank members of the International
Vaccine Bank, Pirbright Laboratory and Dr J. Salt for provision of test sera.

ReferencesReferencesReferencesReferences

Anon. (1996). OIE Manual of Standards for Diagnostic Tests and Vaccines. Lists A and B
diseases of mammals, birds and bees. Chapter 2.1.1. Foot and Mouth Disease, pp. 47-56.
Bahnemann, H.G. (1990). Vaccine 8, 299-303.
Ferris, N.P., Donaldson, A.I., Barnett, I.T.R. and Osborne, R.W. (1984). Revue Scientific et
Technique de l'Office International des Epizooties 3, 339-350.
 203
Golding, S.M., Hedger, R.S. and Talbot, P. (1976). Research in Veterinary Science 20,
142-147.
Haas, B. (1994). Report of the Session of the Research Group of the Standing Technical
Committee of the European Commission for the Control of Foot-and-Mouth Disease,
Vienna, 19-22 September, 1994, 124-127.
Hamblin, C., Barnett, I.T.R. and Hedger, R.S. (1986). Journal of Immunological Methods 93,
115-121.
Mackay, D.K.J., Rendle, T. and Armstrong, R.M. (1994). Report of the Session of the
Research Group of the Standing Technical Committee of the European Commission for
the Control of Foot-and-Mouth Disease, Vienna, 19-22 September, 1994, 128-145.
Mackay, D.K.J., Rendle, T., and Kitching, R.P. (1998). Report of the Session of the Research
Group of the Standing Technical Committee of the European Commission for the
Control of Foot-and-Mouth Disease, Aldershot, UK, 14-18 September, 1998, 148-157.
Mackay, D.K.J., Bulut, A.N., Rendle, T., Davidson, F. and Ferris, N.P. In preparation for
submission to the Journal of Virological Methods.

Table 1
Two-fold dilutions (shown in brackets) of a positive serum (N) diluted in normal bovine
serum and repeatedly tested by SPCE, LPBE and VNT.

Test Sera
C (1280) A (640) D (320) B (160) E (80) N (neat,
undiluted
serum)
SPCE 0.00a 0.00 0.47 0.80 1.07 2.50
0.00b 0.00 0.11 0.08 0.08 0.15
0%c 0% 23% 10% 7% 6%
LPBE 1.34 1.34 1.59 1.80 2.17 3.29
0.00 0.00 0.09 0.00 0.09 0.17
0% 0% 6% 0% 4% 5%
VNT 0.83 0.92 1.18 1.39 1.72 3.13
0.13 0.21 0.09 0.32 0.24 0.09
15% 23% 7.6% 23% 14% 3%
a
Mean titre (n=5 for SPCE and LPBE; n=3 for VNT) of each serum as logarithmic value
b
Standard deviation (SD)
c
Co-efficient of variation
Number in parenthesis shows the serum dilution
 204
Table 2
Examination of the provisional reference sera from the FAO Phase XIII Standardisation
Exercise by SPCE, LPBE and VNT

Phase XIII RS-1 RS-2 RS-3 RS-4

O1 BFS 1860
SPCE 452a 2.66b 80 1.90 14 1.15 3.5 0.54
LPBE 2048 3.31 1024 3.01 90 1.95 45 1.65
VNT 912 2.96 468 2.67 51 1.71 32 1.50

Phase XIII RS-1 RS-2 RS-3 RS-4

A22 IRQ 24/64
SPCE 113 2.05 80 1.9 57 1.76 20 1.30
LPBE 1448 3.16 724 2.86 362 2.56 64 2.26
VNT 513 2.71 123 2.09 42 1.63

Phase XIII RS-1 RS-2 RS-3 RS-4

C1 Noville
SPCE 905 2.96 160 2.20 57 1.76 7 0.85
LPBE 2048 3.31 1024 3.01 362 2.56 45 1.65
VNT 1230 3.09 224 2.35 78 1.89 32 1.51
a
Left hand columns : arithmetic titre
b
Right hand columns : logarithmic titre

Table 3
A panel of 85 >problem= sera were selected for examination by SPCE, LPBE and VNT on
the basis of previous reactivity in the LPBE for import/export testing and assumed to
originate from FMD virus naive cattle

FMD virus Test result Test

SPCE LPBE VNT
O1 BFS 1860 Number positive 3 73 1
Number negative 82 12 84
Specificity (%) 96.4 14.1 98.8
A22 IRQ 24/64 Number positive 4 48 2
Number negative 81 37 83
Specificity (%) 95.2 43.5 97.6
205
 1:2.5 dilution 1:20 dilution
50 45
40
40 35
30

Frequency
Frequency

30 25
neg neg
20 pos
20 pos
15
10
10
5
0 0
-25 -10 5 20 35 50 65 80 95 -25 -10 5 20 35 50 65 80 95

% Inhibition % Inhibition

1:5 dilution 1:40 dilution

40 50
35
30 40
Frequency

25
20 neg Frequency 30
neg
15 pos
20 pos
10
5
10
0
-25 -10 5 20 35 50 65 80 95
0
% Inhibition -25 -10 5 20 35 50 65 80 95

% Inhibition

1:10 dilution 1:80 dilution

50 50

40 40
Frequency

30 30
Frequency

neg
neg
20 pos
20 pos

10
10
0
-25 -10 5 20 35 50 65 80 95 0
-25 -10 5 20 35 50 65 80 95
% Inhibition
% Inhibition

Fig. 1. Frequency distribution of positive and negative sera in relation to test serum dilution and percentage
inhibition of FMD virus type O1 BFS 1860 in the SPCE using blocking buffer 3
 Frequency distribution in SPCE Buffer 3 Frequen
1:2.5 dilution
50 45
40
40 35
30

Frequency
Frequency

30
neg 25
20
20 pos
15
10
10
5
0 0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95 -25 -15 -5

% Inhibition

Frequency distribution in SPCE Buffer 3 Frequenc

1:5 dilution
40 50
35
30 40
Frequency

25

Frequency
20 neg 30

15 pos
20
10
5
10
0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95
0
% Inhibition -25 -15 -5

Frequency distribution in SPCE Buffer 3 Freque

1:10 dilution
50 50

40 40
Frequency

30 30
Frequency

neg

20 pos
20

10
10
0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95 0
-25 -15 -5
% Inhibition
ncy distribution in SPCE Buffer 3
1:20 dilution

neg
pos

5 5 15 25 35 45 55 65 75 85 95

% Inhibition

cy distribution in SPCE Buffer 3

1:40 dilution

neg
pos

5 15 25 35 45 55 65 75 85 95

% Inhibition

ency distribution in SPCE Buffer 3

1:80 dilution

neg
pos

5 15 25 35 45 55 65 75 85 95

% Inhibition
 a)

350

300

Frequency 250

200

150

100

50

0
-25

-10

20

35

50

65

80

95
Percentage inhibition

b)

80

70

60

50
Frequency

40

30

20

10

0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95

Percentage inhibiton

c)

80
70
60
50
Frequency

40
30
20
10
0
-25

-15

-5

15

25

35

45

55

65

75

85

95

Percentage inhibition

Fig. 2: Frequency distribution of negative sera at a 1:5 dilution to percentage

inhibition of FMD virus type a) O1 BFS 1860, b) A22 IRQ 24/64 and
c) C1 Noville
 Frequency distribution in SPCE Buffer 3 Frequen
1:2.5 dilution
50 45
40
40 35
30

Frequency
Frequency

30
neg 25
20
20 pos
15
10
10
5
0 0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95 -25 -15 -5

% Inhibition

Frequency distribution in SPCE Buffer 3 Frequenc

1:5 dilution
40 50
35
30 40
Frequency

25

Frequency
20 neg 30

15 pos
20
10
5
10
0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95
0
% Inhibition -25 -15 -5

Frequency distribution in SPCE Buffer 3 Freque

1:10 dilution
50 50

40 40
Frequency

30 30
Frequency

neg

20 pos
20

10
10
0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95 0
-25 -15 -5
% Inhibition
ncy distribution in SPCE Buffer 3
1:20 dilution

neg
pos

5 5 15 25 35 45 55 65 75 85 95

% Inhibition

cy distribution in SPCE Buffer 3

1:40 dilution

neg
pos

5 15 25 35 45 55 65 75 85 95

% Inhibition

ency distribution in SPCE Buffer 3

1:80 dilution

neg
pos

5 15 25 35 45 55 65 75 85 95

% Inhibition
 a)

4.5

3.5
LPBE log titre

2.5

2
y = 1.1021x + 1.2577
1.5 R² = 0.6373

1
0.5 1 1.5 2 2.5
SPCE log titre n= 57

b)

4.5

3.5
LPBE log titre

2.5

2
y = 1.0123x + 1.2882
R² = 0.6534
1.5

1
0.5 1 1.5 2 2.5

SPCE log titre

n=78
c)

4.5

3.5
LPBE log titre

2.5

2 y = 0.9492x + 1.0121
R² = 0.6114
1.5

1
0.5 1 1.5 2 2.5

SPCE log titre n=61

Fig. 3: Correlation between the SPCE and LPBE of mean antibody titres using FMD virus
type a) O1 BFS 1860, b) A22 IRQ 24/64 and c) C1 Noville
 Frequency distribution in SPCE Buffer 3 Frequen
1:2.5 dilution
50 45
40
40 35
30

Frequency
Frequency

30
neg 25
20
20 pos
15
10
10
5
0 0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95 -25 -15 -5

% Inhibition

Frequency distribution in SPCE Buffer 3 Frequenc

1:5 dilution
40 50
35
30 40
Frequency

25

Frequency
20 neg 30

15 pos
20
10
5
10
0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95
0
% Inhibition -25 -15 -5

Frequency distribution in SPCE Buffer 3 Freque

1:10 dilution
50 50

40 40
Frequency

30 30
Frequency

neg

20 pos
20

10
10
0
-25 -15 -5 5 15 25 35 45 55 65 75 85 95 0
-25 -15 -5
% Inhibition
ncy distribution in SPCE Buffer 3
1:20 dilution

neg
pos

5 5 15 25 35 45 55 65 75 85 95

% Inhibition

cy distribution in SPCE Buffer 3

1:40 dilution

neg
pos

5 15 25 35 45 55 65 75 85 95

% Inhibition

ency distribution in SPCE Buffer 3

1:80 dilution

neg
pos

5 15 25 35 45 55 65 75 85 95

% Inhibition
 208

Appendix 25

Animal Production and Health Section of the Joint FAO/IAEA Programme of Nuclear
Techniques in Agriculture (Vienna) Coordinated Research Project on: “The use of
non-structural proteins of foot-and-mouth disease virus (FMDV) to differentiate
between vaccinated and infected animals.

J. R. Crowther

Summary

The countries in Latin America have committed themselves to have Foot-and-Mouth

virus eradicated from the continent by the year 2009. Based on the experience of FMD
eradication in Uruguay, OIE recently accepted a new category “FMD free with vaccination”.
The need of a test that accurately separates vaccinated from FMD infected animals became
critical. Laboratories in S. E Asia have also acquired better expertise in technologies facilitating
the improved diagnosis of FMD and measurement of antibodies, e.g., through a five year
FAO/IAEA CRP “Use of immunoassay technologies for the diagnosis and control of
foot-and-mouth disease in Southeast Asia” completed in 2000), the results of which have been
published in a TECDOC. (IAEA-TECDOC-1150). The immune status of animals is often
unclear in this region and must be elaborated to allow a better knowledge of the factors involved
when considering vaccination and movement control. programmes. The rapid comparative
examination, development and standardisation of an accepted, world-wide test, for
differentiating vaccinated and infected animals would be greatly beneficial to all authorities
involved in FMD control.
The overall objective of this CRP is to improve the effectiveness of national and
international FMD control and eradication campaigns through the application of an assay that
is able to distinguish antibodies due to vaccination from infection. More specific research
objectives include: the validation of an ELISA based technology for the detection of
antibodies against non-structural proteins of FMD virus under different epidemiological
conditions: the selection of the most appropriate antigen (3ABC, 2A), antigen expression
system (baculovirus, E. coli) and ELISA system (competitive, indirect); the comparison of
different diagnostic methods (ELISA, EITB, PCR) suitable for screening and confirmation
and to determine diagnostic criteria (cut-off values); the collection and definition of sera and
analyse results from different, critical epidemiological regions e.g. - FMD free areas without
vaccination, FMD free areas with vaccination, FMD infected areas with vaccination
The expected research outputs include provision of comparative data examining
several candidate tests already available, validated assay(s) with full protocols, which can
distinguish between FMD vaccinated and FMD infected livestock including cattle, sheep
goats and pigs, an improved understanding of epidemiological situation in defined areas
within South America and S.E. Asia and an Internationally sanctioned sustainable assay(s)
developed with External Quality Assurance (EQA) and Internal Quality Assurance(IQC ).
The CRP has been in existence for over a year and a status report will be given.
 209

Appendix 26

DETECTION OF ANTIBODIES AGAINST NON-STRUCTURAL PROTEINS OF FMD

VIRUS IN BULGARIA

GEORGI GEORGIEV, EMILIA VELEVA, ALBENA DIMITROVA, YANKO IVANOV

National FMD and exotic viral infections laboratory – Sofia , Bulgaria

Foot-and-mouth disease (FMD) is an economically devastating disease of cloven-hoofed animals

and rarely existing on the territory of a Balkan Peninsula countries, The causative agent is aphthovirus
belonging to the Picornaviridae family for which seven serotypes have been described. Current
serological tests used in laboratory practice detect only anibodies directed to the structural FMD virus
(FMDV) proteins. Antibodies directed to the capside proteins of FMDV are induced by both –
inactivated (from vaccines) and live viruses (infection , carrier animals) therefore it is not possible to
differentiate the origin of the antibodies using the conventional current Liquid Phase Blocking (LPB)
ELISA.
Several laboratories recently developed a 3 ABC ELISA tests for detection of antibodies directed
to the non-structural FMD virus proteins (NSP) and this is the great advantage for differentiation
vaccinated from infected animals (1,2,3,).
It is of great importance for the countries using vaccination programs in their strategy for control
FMD is to recognize the infected or carrier from vaccinated animals. For Bulgaria that has more than
300 km. border with Turkey, which use vaccination policy against FMD infection it is important to
have a high sensitive and exact techniques for early detection the FMD infection. In Turkey there are a
new circulating strains of FMD viruses, including not only European O,A,C types, but also exotic
strains such Asia1 and A- Iran- 86 . Using conventional LPB ELISA one sample should be tested to
each current FMD- virus serotype. A new common NSP ELISA test is a great advantage for a
serosurveillance and gives the possibility to investigate a massive population of susceptible animals
using one test.

The aim of this article is to report our results on 29 sera from the 1993 outbreak and 61 sera
from our serosurveillance, using the 3ABC ELISA developed in Brescia (1).

MATERIALS AND METHODS

ELISA: The LPB ELISA was used as described by Hamblin et al. (4). De Diego et al. (1)
described the protocol used for the NSP ELISA.
SERUM SAMPLES: 29 archive serum samples from bovine origin, collected during the
Simeonovgrad outbreak in 1993 and preliminary estimated as FMD O-1 type positive were investigated
together with another 41 bovine, 17 ovine and 3 caprine serum samples from the field, accepted in the
laboratory during July-August 2000 period.

RESULTS

The Brescia 3ABC ELISA test was used in our routine laboratory practice. Serum samples
from a archive collection of the Bulgarian FMD laboratory and sera from animals located nearby the
border zone of Greece and Turkey were investigated. The sera collected from infected animals during
the FMD outbreak in Simeonovgrad in 1993 year were positive by both - conventional O1 Manisa LPB
ELISA and 3 ABC ELISA tests. The current investigated sera from animals located near the border
zone were negative by using Brescia 3 ABC NSP ELISA (Table 6).

CONCLUSIONS:

Early detection of antibodies against 3 ABC non structural FMD proteins is of a vital
importance for Bulgaria which is FMD free country, not practicing vaccination since 1991 year and
bordering with Turkey which is endemic for the disease. This could be achieved by introduction of the
new 3 ABC ELISA to the non-structural proteins of FMD.
This is a useful tool for the control of FMD and could replace the conventional LPB
ELISA in the serosurveillance at high risk zones where virus migration is expected.
 210

LITERATURE:

1. De Diego M., E. Brocchi, D.Mackay, F. De Simone ( 1997) The non-structural polyprotein 3

ABC of FMDV as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle.
Arch. Virol. 142, 2021-2033.
2. Mackay D., M.A. Forstyth, P.R.Davies, A.Berlinzani, G.J.Beilsham, M.Flint, M.D.Ryan (1998 )
Differentiating infection from vaccination in FMD using a panel of recombinant non-structural
proteins in ELISA. Vaccine(16) 5 ,446-459.
3. Rodriguez A., J.Dopazo, J.C.Saiz, F.Sorbino (1994) Immunogenicity of non-structural proteins of
FMDV differences between infected and vaccinated swine. Arch. Virol. 136, 123-131.
4. Hamblin, C., Barnett, I.T.R. and Hedger, R.S. (1986a) A new enzyme-linked immunosorbent assay
(ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and
method of ELISA. J. Immunol. Methods 93, 115-121.

Table No 1. Results from investigated archive serums and serum samples from the
field using Brescia 3 ABC NSP ELISA reagents

Final result +/
OD NET of
Serum samples T/P category
investigated Result
origin RATIO
serums
29 archive bovine
serums from
Simeonovgrad > 0.2 + > 0.15 29 +/ strong positive
outbreak 1993
protocol 25.07.2000
Bovine 2, Ovine 3
and Caprine 2 sera
from Borislavtzi
village, Haskovo < 0.2 - <0.1 -/ negative
region
Protocol
06.08.2000
Bovine 10,Ovine 14
and caprine 1 serum
samples from
< 9.2 - <0.1 -/ negative
Kardjali region
Protocol
24.08.2000
 211

Appendix 27

IMPROVED INDIRECT ELISA BASED ON THE 3ABC

POLYPROTEIN FOR DIFFERENTIATINF INFECTION
FROM VACCINATION IN FOOT-AND-MOUTH DISEASE.

Esther Blanco, Marisa Arias and Jose Manuel Sanchez-Vizcaíno

CISA-INIA, Valdeolmos, Madrid 28130. Spain
Tel: +34 91 620 23 00
Fax:+34 91 620 22 47
e-mail: vizcaino@inia.es

SUMMARY
In an attempt to circumvent the specificity problems founded until now in the
current diagnosis test available to differentiate infected from vaccinated animals , and
develop a convenient, fast and simple test, we have explored the suitability of an
indirect ELISA based on a recombinant 3ABC protein excised from a preparative SDS-
polyacrylamide gel. In this communication we report promising preliminar results
obtained in a study that includes experimental and field sera from non-infected,
vaccinated and infected animals of different species (cattle, pigs and sheep). As a part of
this study we analysed field sera from sheep collected in Morocco during the 1999
FMD outbreak, pig sera from China and Philippines and cattle sera from Argentina.

Modifing protocol of purified 3ABC protein allowed to increase the specificity

and sensibility of the ELISA to about 98%. So, with this improved test the
differentiation between infected and vaccinated animales is more clear, and the
confirmation of the results by Immunoblotting test is avoided. Furthermore, the ELISA
developed is more simply than the other described before, is less time consuming and it
isn’t neccesary to use monoclonal antibodies. Therefore this ELISA is a suitable test to
large scale samples and could be very advisable to serological surveys.
Our results testing sheep sera collected in Morocco during the outbreaks
declared in 1999, indicate the capacity of the ELISA describe here to detect
subclinically infection in small ruminants. Therefore, this test could be very advisable
too, during serological surveys when any outbreak has been declared.
 212

INTRODUCTION
Identifying animals that have been infected with foot-and-mouth disease virus
(FMDV) is of considerable importance because it is well established that infected cattle
and sheep frequently become carriers of the virus and consequently may become the
source of new outbreaks of the disease (1,2,5). Furthermore in small ruminants, sheep
and goat, the course of the disease is characterised by very mild or absent clinical signs,
so FMD can spread unnoticed (9). This situation is compromised by the difficulty in
distinguishing infected animals from those that have been vaccinated against the disease
since both groups contain neutralizing antibodies in their sera. Moreover, asymptomatic
carrier animals can be found in vaccinated herds.

Consequently, efforts have been directed to the development of diagnostic tests

that can distinguish infected animals from those that have been vaccinated. The
approach has been to identify antibodies against virus-specific proteins that are present
only in infected animals. As the vaccines use currently consist in viral particles
inactivated where the non-structural proteins are not present or in very few
concentration, the FMDV non-structural proteins meets this criterion .Their potential
use to differentiate infection from vaccination was first report by
radioimmunoprecipitation (RIP). As this technique is not well suited to screening large
numbers of samples, different strategies were investigated, as Immunoblotting test (IB)
and mainly ELISA (4,6,8).

Among the FMDV non-structural proteins, different studys suggested that the
polyprotein 3ABC was apparently the most immunogenic in pigs (8) and cattle (6). This
virus-specific protein has been expressed in several heterologous systems (i.e. E. coli
and insect cells) and used for the development of ELISAs to discriminate the sera from
vaccinated and infected animals. These tests present specificity problems that make
interpretation of results difficult, thus limiting their efficacy. It has been shown that in
many cases the specificity problems are associated with the presence in the sera samples
of antibodies against expression vector antigens (proteins from E. coli or insect cells)
that co purify with recombinant products.

Therefore our approach has been to improve the purification system of

recombinant proteins in an attempt to increase the sensitivity and specificity of the
diagnosis test based on the use of this proteins. The results exposed in this
communication indicate that the ELISA develop using a purified 3ABC protein allows
not only to distinguish clearly between vaccinated and infected animals, also is very
useful alternative method to detect subclinically infection, mainly in small ruminants.
 213

MATERIAL AND METHODS

• Sera
In order to evaluate the capacity of the new ELISA developed to differentiate
between infected and vaccinated animals to FMDV, sera from the three main hostess of
the virus, cattle, pig and sheep, were analyzed.
Sheep sera were collected in Morocco, from infected regions (Oujda, Khouribga
and Beni-Mellal) during the outbreaks declared in 1999 and from the provinces in the
north (Tanger) after to start the vaccination campaignt. Negative sheep sera to FMDV
were collected from different region in Spain, a FMD free country.
Cattle sera from experimental infected animals with the three main serotypes A,
O and C, were obtained in experiments carry on in Argentina. Sera from vaccinated
cattle with a trivalen vaccine were collected also in Argentina, as well as the negatives
controls from naive cattle.
The validation of the ELISA test in pig was performed testing pig sera collected
in Philippines and China from farms genetically controled and vaccinated with a
comercial vaccine to FMDV type O. Experimental infection of pigs were performed in
our lab, in order to collect sera from infected animals to different days post-infection.
These sera allowed to perform a cinetic of antibodies response to 3ABC protein, in
those pigs.
• Antigens
Inactivated FMDV, isolate O1-Manisa, supplied by WRCL in Pirbright (7) was
used to perform LPBE for detection of antibodies against FMDV structural proteins.
Recombinant 3ABC polypeptide from FMDV, isolate O1-Kaufbeuren expressed
in E. coli 537 (10) was used to perform ELISAs for detection of antibodies against
FMDV non structural proteins. In this system, the 3ABC polypeptide is expressed as a
fusion protein with the N-terminal part of MS2 polymerase, under the control of the
inducible lambda PL promoter. The recombinant 3ABC expressing E. coli was kindly
provided by F. Sobrino. The protein was obtained from heat induced bacterial cultures
and semipurified by an adaptation of the method described by Strebel et al., 1987 (10).
The specificity of the semipurified material was assayed by immunoblotting using 2C2
monoclonal antibody kindly provided by E. Brocci.
 214

A new batch of protein was further purified excising the protein from a
poliacrilamida gel of 12,5% and eluting it in a CO3NH4 buffer.
• ELISA
The LPBE was performed as described in the protocol supplied by the World
Reference Central Laboratory (WRCL) in Pirbright, UK (7). Samples were tested in
two-fold serial dilutions spanning from 1/25 to 1/3200. All the sera were tested by
duplicates and the standard deviation were always lower than 5%. The cut off was
established following standard procedures (7).The negative controls gave always an
OD620 above 0.9. The end point dilution titres were expressed as the inverse of the log
of the serum dilution that gave the same OD620 as the negative control at a 1/25 dilution.
Sera samples were tested to 3ABC recombinant protein in two-fold serial
dilutions spanning from 1/25 to 1/3200. All the sera were tested by duplicates and the
standard deviation were always lower than 5%. Detection of antibodies bound to 3ABC
was performed by addition of anti-sheep, anti-pig IgG or protein-A (for cattle samples)
conjugated to horseradish peroxidase (Sigma) . 200 µl of 80,6 mM 3-
dimethylaminobenzoic acid (Sigma D-1643): 1,56 mM methyl-2-benzothiazolinone
hydrazine hydrochloride monohydrate (Sigma M-8006) (1:1) and 0,0075% H2O2 were
used as substrate. Reactions were stopped by adding 50 µl of 3N H2SO4 and
absorbances were measured at 620 nm (A620). The negative controls gave always an
OD620 below 0.3.

RESULTS

Sheep Samples.
The presence of antibodies to FMDV structural proteins was analysed in 345
sera collected from areas where FMD outbreaks were declared during the 1999
epizootic in Morocco. 23 out of 299 sera tested were identified as seropositive to
FMDV. None of the seropositive sheep had shown clinical signs consistent with FMD,
and therefore had not been diagnosticated as FMDV-infected.
Figure 1A shows the antibody titres to FMDV structural proteins detected in the
29 sheep sera collected from the FMDV-infected flock in Oujda. The 23 seropositive
sera displayed a wide range of antibody titres to FMDV type O structural proteins. The
graphic 1B shows the titer of antibodies to 3ABC non-structural protein detected in the
 215

same flock. The differences in the sensibility of the ELISA performed with the
semipurified (graphic up) or purfied protein (graphic below) are clear. When the
purified protein was used all the seronegatives detected to FMDV structural proteins
were also negatives to 3ABC protein. The cut-off can be establish clearly in a OD 620 of
0.4.

3 LPBE (structural proteins)

FMDV type O titre

2,5

2
(log10)

1,5

0,5

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 25 26 27 28 29 30
Sheep
3ABC (old purification
1,8

1,4

1,0

0,6
OD620

0,2

1,8 3ABC (new purification

1,4

0,6

0,2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 25 26 27 28 29 30

Sheep

Figure 1.- Comparison of the to FMDV and to 3ABC in a flock exposed to

FMD infection during the 1999 outbreaks declared in Morocco.
The first graphic shows the antibody titres to FMDV structural proteins. The
graphics below compare the OD620 measured in the 3ABC recombinant ELISA using
the old purified protein (semi-purified) or the new purified protein (excised from gel).
 216

The clare bars correspond to seronegatives sera to FMDV structural proteins. The line
indicate the cut-off of the assay.
In order to validate the utility of the ELISA described to differentiate vaccinated
and infected sheep, 170 sera from sheep vaccinated in the North of Morocco during the
vaccination campaing starting last year were analyzed by the 3ABC-ELISA. The Figure
2 show the distribution of OD620 values obtained testing 50 infected , 170 vaccinated
and 50 negatives sheep sera.

40
35 Infected
30
25
20
15
10
5

7
0,2 0,2- 0,4- 0,6- 1,0 1,0- 1,2- 1,4- 1,6-
6 Vaccinated
5
Frecuency (%)

4
3
2
1

60

50
Negatives
40

30

20

10

0
0-0,2 0,2-0,4 0,4-0,6 0,6-0,8 0,8-1,0 1,0-1,2 1,2-1,4 1,4-1,6 1,6-1,8

OD intervals
Figure 2.- Frecuency distribution of OD620 values obtained testing infected,
vaccinated and naive sheep sera.
The sera were analyzed by duplicates in a dilution of 1:25 and using as antigen the
3ABC protein excised from gel.
 217

Pig Samples.

Two pigs Landrance White Large were inoculated with 105 pfu of FMDV type
C-S8 developing characteristic clinical signs of the disease. Serum samples were
collected from time 0 (before inoculation) untill 96 days post infection, bleeding the
animal every day in the first week and once a weel in th others. The antibodies to 3ABC
are detected as soon as 4 days post infection and the maximum titer are reached between
9 and 10 days post-infection (Figure 3). 63 days post-infection the pigs were reinfected
with the heterologous FMDV type O-BFS and the animals developed again clinical
signs.
After the reinfection the Humoral immune response to 3ABC protein increase,
being the maximum titer of antibodies 69 days post-infection , higher that those
obtained in the maximum peak after infection.
pig 1
pig 2
25

15
OD620

0,5
Reinfection
with O-BFS
0
0 3 9 15 21 27 33 39 45 51 57 63 69 75 81 87 93
Days post-infection
Infection
with C-S8

Figure 3.- Cinetics and duration of the antibody response to 3ABC in pigs
experimentally infected and reinfected with FMDV. The arrows indicate the time of
infection and reinfection with the viruses.
 218

165 vaccinated pig sera collected in Philippines and China were analyzed to
3ABC (Figure 4). The OD620 values obtained in these sera were very similar to those
obtained testing 50 sera from naive pigs located in Spain.

60

50 Vaccinated
40

30

20

10

80
70 Negatives
60
50
40
30
20
10
0
0-0,2 0,2-0,4 0,4-0,6 0,6-0,8 0,8-1,0 1,0-1,2 1,2-1,4 1,4-1,6 1,6-1,8

Figure 4.- Frecuency distribution of OD values obtained analyzing pig sera from
vaccinated and naive animal.

Cattle Samples.
Cattle sera from animals inoculated experimentally in Argentina with the FMDV
types 01-Campos, A24-cruzeiro and C3-Argentina 85, were used as positives controls in
the 3ABC-ELISA. These sera were collected at 7 days post infection or 1 year post-
infection (Figure 5). All of them were positives to 3ABC showing a wide range of titers.
Sera from cattle vaccinated with a trivalent vaccine, prepared with the three
serotypes related before were also tested by the 3ABC-ELISA. The vaccinated sera
analyzed were classified in two groups: one with animal vaccinated with one dose and a
boost, and another one multivaccinated more than twice. All these sera from vaccinated
cattle displayed a OD620 values to 3ABC less than 0,4 (Figure 5).
 219

DISCUSSION

The results exposed in this communication shows that the use of a suitable
purified 3ABC-protein allow to develop a symple diagnosis test as ELISA capable to
discriminated between infected and vaccinated animal. The test described here
improved the diagnosis methods described until now increasing the sensibility and
specificity. Furthermore, the interpretation of the results with this ELISA is easier, and
suggest that it application to routine diagnosis could be facilitate.
The study performed studiying sera from infected areas in Morocco has allowed
to validate this ELISA to applicate it in field conditions. Since the sheep sera samples
collected in this infected areas were collected before to start the vaccination campaing,
and that they present antibodies to FMDV structural proteins without be present clinical
signs of the disease, the results to 3ABC consitute a confirmation of the existance of
subclinically infections in the small ruminants population. Thus, these results indicate
 220

the high potential of the 3ABC-ELISA to be used even more in absence of clinical
symptomatology.
The cinetics of antibody response to 3ABC in pigs infected with FMD seems to
be shorter than those found in other hostess (as cattle). Further work is in progress to
confirm this pattern of reactivity to 3ABC protein in sera from infected pigs, in order to
check how long is possible to detect antibodies to this protein. The detection of
antibodies to 3ABC even more after a heterologous infection confirm the capacity of
this protein highly conserved among the 7 serotypes of FMD to be recognaized by
animal infected with different serotypes.
In spite of the number of cattle sera analyzed is little, these preliminary results
obtained testing sera from multivaccinated animal indicate that the improving of the
protein used as antigen allow to improve also the sensibility of the ELISA to distinguish
between infected and multivaccinated cattle. This fact can be very advisory to adapted
the ELISA to control and serosurveillance of the countries where an outbreak has been
declared and have to star a vaccination programm.
Further work would have to be perform in order to determine the possible
application of the test also to detect the carrier state in the susceptible animal to FMD.

REFERENCES
1. Barnett,P.V. and Cox,S.J., “The role of small ruminants in the epidemiology
and transmission of foot-and-mouth disease”. Vet.J., 1999; 158, 6-13
2. Callis,J.J. “Evaluation of the presence and risk of foot-and-mouth disease
virus by commodity in international trade”. Rev.sci.tech.Off.int.Epiz, 1996,
15 (3), 1075-1085.
3. Cox,S.J., Barnett,P.V., Dani,P., and Salt,J.S. “Emergency vaccination of
sheep against foot-and mouth disease: protection against disease and
reduction in contact transmission”.Vaccine, 1999; 17: 1858-1868.
4. De Diego,M., Brocci,E., Mackay.D, and De Simone.F. “The non-structural
polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in
ELISA to differentiate infected from vaccinated cattle”. Arch Virol, 1997;
142: 2021-2033.
5. Leforban,Y. “Prevention measures against foot-and-mouth disease in Europe
in recent years”. Vaccine, 1999; 17: 1755-1759.
 221

6. Mackay,D.K.J., Forsyth,M.A., Davies,P.R., Berlinzani,A., Belsham,G.J.,

Flint,M and Ryan,M.D. “Differentiating infection from vaccination in foot-
and-mouth disease using a panel of recombinant, non-structural proteins in
ELISA”. Vaccine, 1998; 16: Nº5, 446-459.
7. O.I.E/FAO/WHO. “ Manual of Standards for diagnostic tests and vaccines”.
1996.
8. Rodriguez.A., Dopazo.L., Saiz.J.C. and Sobrino.F. “Immunogenicity of non-
structural proteins of foot-and-mouth disease virus: differences between
infected and vaccinated swine”. Arch.Virol, 1994; 136: 123-131.
9. Sharma, SK. “Foot and Mouth disease in sheep and goats”. Vet.Res.J. 1981;
4 (1):1-21.
10. Strebel.K., Beck.E., Strohmaier.K. and Schaller.E. “Characterisation of foot-
and-mouth disease virus vene products with antisera against bacterially
synthesized fusion proteins”. J.Virol. 1986, 57, 983-991.
 222
Appendix 28

INDIRECT ELISA USING RECOMBINANT VP1 CAPSID PROTEIN FOR THE SERODIFFERENTIATION OF
FOOT-AND-MOUTH DISEASE VIRUS INFECTED ANIMALS
M. Wenger,, M. A. Hofmann, C. Moser, J. -D. Tratschin, M. A. Hofmann Formattato
Institute of Virology and Immunoprophylaxis Mittelhäusern, Switzerland

Key words: foot-and-mouth disease virusFMDV, serodifferentiation, recombinant VP1, ELISA

Introduction The new PCR products corresponding to the exact VP1 were
Foot-and-Mouth mouth Disease disease (FMD) is a highly then cloned into the bacterial pBAD/Thio-TOPO expression
contagious viral disease resulting in high tremendous vector plasmid (Invitrogen) and sequenced.. The different f
economical losses. In order to minimize the risks of an NH2 - thioredoxin - VP1 - V5 epitope - 6x His tag - COOH Formattato
eventual FMD outbreak in disease-free countries, due to fusion proteins as well as NH2 - thioredoxin - V5 epitope - 6x
animal imports, rapid and reliable screening of sera from His tag - COOH control proteins corresponding to the three
susceptible animals for the presence of FMD virus (FMDV) serotypes were then expressed in E. Colicoli and analysed by
antibodies is necessary. With the increasing number of resulting in a fusion protein containing (N'- to C'-end):
importations for example of „exotic“ ruminants like lamas and thioredoxin- VP1- V5 epitope- 6x His-tag. Besides this, we
alpagas and with the fact that some species show very expressed the control protein: thioredoxin- V5 epitope- 6x His-
discrete or no clinical signs, the need for rapid and reliable tag under the same conditions. SDS PAGE.-Pages were
screening tests becomes more and more urgent. performed with the different proteins to check if the estimated
The reasons reasons for the development of this a novel molecular weights corresponded.
FMDV FMDV antibody ELISA are multiplemanifold: (i)to have After having lysed the E.Coli, a first purification with the
a test with increasing theed sensitivity compared to the Rrecombinant proteins for wereO1Manisa purified by affinity
currently used liquid phase blocking ELISA (12), (ii), to have chromatography and with the control protein was then
availability of a simple, inexpensive test for rapid screening performed under denaturing conditions using a Ni-chelate
which allowsing diagnosis within one day, (iii), to reliable columnresin. and The purified fusion and control proteins
differentiation of e serotype- specific antibodies, and (vi) and were coated on ELISA plates. Indirect ELISA was performed
to have a safe test which can be used outside of a using bovine an ELISA was tried by coating in parallel
containment laboratory. columns: mock (=coating buffer alone), thioredoxin- V5
The VP1capsid protein gene is a highly variable region of the epitope- 6xHis-tag and thioredoxin- VP1(O1Manisa)- V5
FMDV genome and is known to contain the two major epitope- 6xHis-tag (the two latter having been purificated
antigenic sites of the virus (23). By expressing in parallel VP1 under the same denaturing conditions). A positive and a
from of various FMDV serotypes in bacterias and insect cells negative FMDV reference control sera and (diagnostics
and then performing an ELISA using these respective reference sera) were then added after having washed the
recombinant proteins as antigens, we expect to be able to plate and HRPO-conjugated monoclonal anti -bovine IgG
differentiate antibodies against the different FMDV serotypes. antibodies were added as detecting antibodies. ABTS with
In analogy withBased on our experiences with recombinant H2O2 was then added to induce the reaction. The results were Formattato
the ELISA antigens used in the ELISA for the detection of read at 405 nm. .
antibodies against classical swine fever virus (4), and PRRS In addition to parallel to these constructsexpression in E. coli, Formattato
virus (31), we expressed the VP1 capsid protein as a fusion two more constructs for each serotypes were made (one with
protein in E. coli as well as , but in addition, we tried and one without Kozak sequence). Each of these constructs
constructs with and without Kozak in the a baculovirus containing thioredoxin- VP1- V5 epitope- 6xHis-tag. The, the
expression system. Bac-to-Bac system (Gibco BRL) was then used to obtain
generate a recombinant baculoviruses containing expressing
the respective serotype-specific thioredoxin- VP1- V5 epitope-
Material and methods 6xHis-tag.thioredoxin-VP1 fusion proteins.
The viralFMDV strains A22(Iraq), O1(Manisa) and C1(Noville)
were obtained from the FMD World Reference Laboratory, in Results
Pirbright, UK. BHK-21 cells were used for virus propagation. The exact VP1 sequences encoding VP1 of each the FMDV
BHK cells were infected and virus isolated from cell culture serotypes A22, O1, and C1 was defined by comparing with
supernatant. other VP1 sequences found in thewere obtained from viral
RNA was TRIZOL® (Gibco BRL) extracted with TRIZOL®, and RNA by RT/PCR and subsequent cloning into a plasmid Formattato
RT-PCR was performed with two universal VP1-external vector. The VP1 sequences were determined and compared
flanking primers (sense primer in the VP3 gene and reverse to the respective sequences found in GenBank Genebank or
primer in the 2A2B gene). T, the PCR products was were then inin the sequence databasebank from of the FMD World
cloned into the pCR-XL-TOPO vector (Invitrogen). in order to Reference Laboratory, in Pirbright, UK in order to confirm the
obtain clear signals in the sequencing gels. serotype.
After having determined theThe exact complete VP1 The VP1 coding sequences were then subcloned into a
sequence of each of the three serotypes was determined and prokaryotic expression vector to obtain a fusion protein
specific thanks to sequencing primers localized in the VP3- including bacterial thioredoxin, viral V5 epitope, and a polyHis
(forward primer) and the 2A2B- (reverse primer) gene, PCR tag. Analysis by All the definitive constructs were controlled by
primers corresponding to the exact 5‘- and 3‘- ends of VP1 PCR, restriction analyzes and sequencing.
(specific primers forof each serotype) were designed and After having performed SDS PAGE -Page, we could
PCR performed. The respective PCR products seerevealed that all the fusion proteins from the differentof all
 223
three serotypes migrated athad the expected molecular our novel ELISA.
weight of approx. 40 kDa. The fusion proteins expressed from We have not yet had time to produce proteins in the
E. coli proved to be soluble and could be purified by nickel baculovirus sytem, but with our constructs in different sytems,
chelate affinity chromatography as shown by SDS PAGE. and we could show the differences of solubilty and expression
the control thioredoxin at approx. 16 kDa. level of the same fusion protein expressed in procariotic and
So far, the indirect ELISA was performed with the fusion eucariotic systems.
protein of serotype O1. Analysis of bovine sera derived from Considering the baculovirus system alone, the importance of
experimental infections with FMDV showed that this fusion the Kozak sequence for the protein expression level could be
protein reacts specifically with sera from animals infected with demonstrated by comparing to recombinant baculoviruses
serotype O1. No reaction with We could show the solubility of diverging only in the fact that one has the Kozak sequence
our fusion proteins and the effectiveness of the purification in and the other not.
other SDS-Pages.
The ELISA was performed once and with only one serotype,
but the OD between mock and control thioredoxin were
similar, so that we would not be obligedthe control protein
expressed from the vector alone (thioredoxin - V5 epitope - 6x
His tag) was observed indicating that the respective fusion
proteins can be used as an ELISA antigen. References
to remove the thioredoxin after purification. On the other 1. Denac H., Moser C., Tratschin J.-D., Hofmann M.A. 1997. An
indirect ELISA for the detection of antibodies against porcine
hand, there was a clear difference of OD between the positive
reproductive and respiratory syndrome virus using recombinant
and the negative bovine sera. nucleocapsid protein as antigen. J. Virol. Methods 65: 169-1813.
Recombinant baculoviruses for VP1 expression in the Using 1. 2. Hamblin, C., Barnett, I.T.R., Hedger, R.S. 1986. A new enzyme-
the Bac-to-Bac system have been constructed and linked immunosorbent assay (ELISA) for the detection of antibodies
characterized. The properties as well as the yield of the against foot-and-mouth disease virus. J. Immunol. Meth. 93, 115-
expressed proteins are compared to the respective proteins 121.OIE-Manual...
obtained from the bacterial expression system., the definitive 23. McCullough, K. C., Crowther, J. R., Carpenter, W. C., Brocchi, E.,
plasmid constructs were again controlled by PCR, restriction Capucci, L., De Simone, F., Xie, Q., McCahon, D. 1987. Epitopes on
Foot-and Mouth Disease Virus Particles. I. Topology. Virology 157,
analyzes and sequencing and the cosmid was checked by
516-525.Review paper...
performing PCRs with different primers combinations. 3. Denac H., Moser C., Tratschin J.-D., Hofmann M.A. 1997. An
indirect ELISA for the detection of antibodies against porcine
reproductive and respiratory syndrome virus using recombinant
Discussion nucleocapsid protein as antigen. J. Virol. Methods 65: 169-1813.
In response to the unsatisfactory performance of the liquid 4. Moser, C., Ruggli, N., Tratschin, J.D., Hofmann, M.A. 1996.
phase blocking ELISA (2) it is the aim of the present study to Detection of antibodies against classical swine fever virus in swine
develop a new FMDV antibody ELISA. It is based on the use sera by indirect ELISA using recombinant envelope glycoprotein E2.
Vet. Microbiol. 51, 41-53.
of the viral structural protein VP1 as a recombinant protein
expressed either in its authentic form or as a fusion protein.
Expression of VP1 as a fusion protein should allow its
expression in a soluble form, as well as efficient affinity
purification. Furthermore, it might also facilitate the exposure
of the conformational epitopes of VP1 when it is coated as a
fusion protein on microtiter plates.
For this purpose, VP1 was expressed as a thioredoxin-
histidine tag fusion protein in different expression systems.
Although expression in E. coli resulted in efficient recovery of
purified VP1, the baculovirus system is considered as an
alternative. Thus, the two expression systems will be
compared in terms of quantity as well as quality of the
recombinant protein in the ELISA.
In order to obtain a soluble protein, to ensure good purification
possibilities and eventually to have a better antigen
presentation on the ELISA plates, we decided to express the
VP1 as fusion protein.
Until now, we only performed once the ELISA has been
carried out and with VP1 from FMDV serotype O1 only, one
serotype, but we consider the obtained results as are
encouraging. and until July, besides western blots to show
that the proteins migrating at 40 kDa are effectively our
interest fusion proteins, The final goal is towe will have done
new runs ofset up a combined ELISA with using the
respective fusion proteins from all three serotypes coated on
different columns of the same microtiter plate. So we shall
have more datas to see if it is possible. This would then
represent a convenient and rapid test for the to
serodifferentiation ofe antibodies against FMDV serotypes O,
A, and C.then go into the optimization and validation steps of
 224
Appendix 29

FAO Collaborative Study Phase XVI :

Establishing Reference Standards for FMD Serology

P.Kitching, T.Rendle, B.Newman,

Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF

Introduction

The FAO Collaborative Study Phase XV established reference sera prepared against the foot-and-
mouth disease virus serotypes A, O and C. These were :

Negative sera: negative by virus neutralization test (VNT) and ELISA to all FMDV serotypes.

Cut-off sera: serum against each of the three serotypes which was positive approximately 50% of
the times it was tested by VNT (and negative the remainder), and which gave similar results by
ELISA.

Low Positive sera: serum against each of the three serotypes which was consistently positive by
VNT and ELISA.

Strong Positive sera: serum against each of the three serotypes which was strongly positive by
VNT and ELISA , although still within the normal dilution series used in these tests, so that a
titre could always be derived.

These sera were supplied to 30 participating laboratories (Table 1a) together with a panel of 12
Atest@ sera, the objective being to calibrate the test in routine use within the laboratory using the
reference sera, and then to derive a result for the Atest@ sera. Laboratories were requested to
establish their own secondary standard sera against the primary standards supplied in order to
minimise their direct use. It is intended to have the primary standard sera available from the
World Reference Laboratory for as long as possible in order to supply additional FMD
laboratories not involved in this collaboration.

The participating laboratories were asked to carry out an initial screening test (spot test) on all 12
sera, and then titrate each by ELISA and/or VNT. Each serum could then be classified as
negative, cut-off (CO), low positive (LP) or high positive (HP).

Materials and Methods

Each laboratory was sent:

- 6 x 0.5ml vials of each of three reference sera for each of the three strains of
FMD virus O1 Manisa, A22 Iraq and C1 (54 vials in total).
- 10 x 0.5 ml negative reference serum.
- 3 x 0.1ml positive original (PO) serum for each of the three strains of FMD virus.
- 1 x 10ml negative serum
- A panel of 12 Atest sera@
 225

Laboratories were provided with a protocol to prepare secondary standards and calibrate their
routine diagnostic tests. With these calibrated tests the 12 Atest sera@ were examined, titrated
and classified.

Results

Twenty-three laboratories responded by supplying results (Table 1b).

The classification of the 12 Atest sera@ is given in Table 2, two were type O sera, three were type
A, five were type C and one was negative. Some of these sera gave hetrologous titres, lower than
the homologous titres, although in some instances the reverse was found, such as with serum 8, a
low positive type C, was classified as a low positive type A and cut-off type C by one laboratory.
Serum 11, a low positive type A, was classified by one laboratory as high positive for type A and
type O and low positive type C. Approximately half of the laboratories reporting results had
facilities for carrying out the VNT.

Spot Test

The results of the spot test are shown in Table 3, and graphically on Tables 4,5 and 13 There was
a high test sensitivity for identifying positive sera, except for the type C sera 7,8 and 9, but there
was considerable variation of test specificity between laboratories. As this is a screening test, a
number of false positive results can be tolerated, as the intention would be to titrate positive sera.
However, positive sera missed in the spot test would no longer be considered, leading to
potentially dangerous situations in which animals with high antibody titres could be moved
across international borders.

In this study, laboratories were requested to titrate spot test positive sera in ELISA and VNT.
Those laboratories which missed positive sera on the spot test therefore also failed to register
ELISA and/or VNT titres.

ELISA and VNT

The results are given in Tables 6-11 and summarized in Table 12. When available, titres have
also been given, but not converted to Units of Antibody (Uab) as used in Phase XV; here they are
included to show the variations in absolute titres between laboratories. The summary (Table 12)
does not show the level of heterologous reaction reported by some laboratories, although this can
be seen in Tables 5-10.

Serum Classification

Table 11 also summarizes the classification of the sera according to High Positive (HP), Low
Positive (LP), Cut-Off (CO) and Negative (N), for ELISA and VNT. There is good correlation
between the ELISA and VNT results, with most of the inconsistencies being with the type C sera,
7, 8 and 9.

Discussion
 226
The reference sera identified in Phase XV were successfully distributed to 30 participating
laboratories, together with the 12 Atest sera"; 23 laboratories submitted results (Table 16). The
reference sera were used to calibrate the ELISA and VNT=s in routine use and to prepare
secondary standards. The results of the Atest sera@ (Table 12) indicate a high level of
consistency between the laboratories, although there are a few notable exceptions.

As expected, it was not the high positive or negative sera which caused the most difficulty, but
the low positive and cut-off sera. These sera are always likely to provide the most problems to
diagnostic laboratories involved in certifying animals for international trade. There was good
correlation between the ELISA and VNT results for the Atest sera@, although these did not fully
represent those sera giving false positive results by ELISA frequently encountered in
import/export testing. For these the availability of the VNT is a considerable advantage in order
to help in their identification. A problem previously addressed in these collaborative studies has
been the use by different national laboratories of antigens derived from different strains of
FMDV, and in this study the use of different antigens could account for some of the variation.
However, some of the variability may also be due to fundamental properties of the liquid phase
blocking ELISA. This test has been the OIE recommended test for the last 10 years, with little
concession to improvement in technology. It may be coincidence that the only laboratory to score
100% on the spot screening test was using a competition ELISA, but it could also indicate that is
time to consider an alternative to the LPBE.

Laboratories were also requested to summarise their experience in making reliable and consistant
secondary reference sera. However, few reported on this.

Finally, it is necessary to decide on the objective of the next Phase, Phase XVII. Phases XV and
XVI have shown that standard sera can be produced which successfully provide consistency
between laboratories. Phase XVII could address the quality of the secondary standards produced
by the participating laboratories, again by using a set of "test sera". Alternatively, replacements
for the LPBE could be assessed.

Acknowledgements

This collaboration was funded by the EUFMD. We are grateful to all laboratories for their
participation, and those staff of the IAH involved in the preparation and distribution of the
reagents.
Table 1a: List of participants in FAO Collaborative Study Phase XVI

Albania Institute of Veterinary Research, Tirana 10, Albania

Austria Bundesanstalt fur Virusseuchen- bekämpfung bei Haustieren, Emil Behring-Weg 3,A-1233 Wien
Belgium Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels UKKEL
Bulgaria National Veterinary Services, Ministry of Agriculture & Food Industry, 15P Slavejkov Blv, 1606
Sofia
Croatia Croatian Veterinary Institute, 10000 Zagreb, Savska cesta 143, P.P. 883
Czech Republic State Veterinary Institute, Sidlistni 136/24, 165 03 Praha 6, Lysolaje
Denmark State Veterinary Institute for Virus Research, Lindholm DK 4771, Kalvehave
FR Yugoslavia Faculty of Veterinary Medicine. Dept for Infectious Diseases of Animals & Bees, Bul. JNA 18,
11000 Belgrade, FR of Yugoslavia
France CNEVA Alfort, Laboratoire Central de Recherche Vétérinaire, 22 rue Pierre Curie, BP 67, 94703
Maisons-Alfort
France CNEVA, 31 Avenue Tony Garnier BP 7033, 69342 Lyon Cedex 07, France
Germany Federal Research Centre for Virus Diseases of Animals, Paul Ehrlich Strasse 28, D-72076 Tüebingen
Greece Institue for FMD, 25 Neapoleos Str, 153 10 Ag Paraskevi, Athens
Hungary Central Veterinary Institute, Tabornok u2, 1149 Budapest
Iran G.D. for Diag. & Cntrl. of Drug and Biological Products, C.V.L. Vali-Asr Ave., S. J. Asad Abidi
St. P.O. Box 14155-6349, Tehran,
Israel Kimron Veterinary Institute, c/o Ministry of Agriculture, PO Box 12, Beit-Dagan, 50200
Italy Istituto Zooprofilattico Sperimentale della Lombardia e dell'Amilia, Via A Bianchi 7, 25125- Brescia
Latvia National Veterinary Laboratory, Lejupes Str. 3, RIGA LV 1067, Latvia
Lithuania National Veterinary Laboratory, Lithuanian Veterinary State Services, Moletu pl 86, 2021, Vilnius
Netherlands Institute for Animal Science and Health, ID-DLO Mammalian Virology, Houtribweg 39, P.O. Box
65, NL-8200 AB Lelystad, Netherlands
Poland National Veterinary Research Institute, Foot-and-Mouth Disease Department, Wodna Str. 7, 98-220
Zdunska Wola
Romania I.N.M.V. Pasteur, Sos. Giulesti nr. 333, R-7000 Bucharest
Russia All-Russian Foot-and-Mouth Disease Research Institute, 600900 Jur'Evets, Vladimir, Fed. Rep. of
Russia
Slovak Republic State Veterinary Institute, PO Box 14/D, 949 01 Nitra
Slovenia University for Microbiology and Parasitology, University of Ljubljana, Veterinary Faculty,
Gerbiceva 60, 61000 Ljubljana
Spain Centra de Investigacion en Sanidad Animal, CISA-INIA, 28130 Valdeolmos, Madrid
Sweden National Veterinary Institute, Box 7073, S 75007 Uppsala
Switzerland Institut für Viruskrankeheiten und Immunoprophylaxe, Sensemattstrasse 293, CH-3147
Mittelhäusern
The FYRO Macedonia Veterinary Institute Skopje, Veterinary pop Trajkov 5-7, 91000 Skopje
Turkey FMD Institute (SAP Enstitüsü), PO Box 714, 06044 Ulus, Ankara
United Kingdom Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Nr Woking, Surrey GU24
ONF
 Table 1b: List of responding laboratories in FAO Collaborative Study Phase XVI

Austria Bundesanstalt fur Virusseuchen- bekämpfung bei Haustieren, Emil Behring-Weg 3,A-1233 Wien
Belgium Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels UKKEL
Bulgaria National Veterinary Services, Ministry of Agriculture & Food Industry, 15P Slavejkov Blv, 1606 Sofia
Czech Republic State Veterinary Institute, Sidlistni 136/24, 165 03 Praha 6, Lysolaje
Denmark State Veterinary Institute for Virus Research, Lindholm DK 4771, Kalvehave
France CNEVA Alfort, Laboratoire Central de Recherche Vétérinaire, 22 rue Pierre Curie, BP 67, 94703
Maisons-Alfort
Germany Federal Research Centre for Virus Diseases of Animals, Paul Ehrlich Strasse 28, D-72076 Tüebingen
Hungary Central Veterinary Institute, Tabornok u2, 1149 Budapest
Israel Kimron Veterinary Institute, c/o Ministry of Agriculture, PO Box 12, Beit-Dagan, 50200
Italy Istituto Zooprofilattico Sperimentale della Lombardia e dell'Amilia, Via A Bianchi 7, 25125- Brescia
Latvia National Veterinary Laboratory, Lejupes Str. 3, RIGA LV 1067, Latvia
Lithuania National Veterinary Laboratory, Lithuanian Veterinary State Services, Moletu pl 86, 2021, Vilnius
Poland National Veterinary Research Institute, Foot-and-Mouth Disease Department, Wodna Str. 7, 98-220
Zdunska Wola
Romania I.N.M.V. Pasteur, Sos. Giulesti nr. 333, R-7000 Bucharest
Russia All-Russian Foot-and-Mouth Disease Research Institute, 600900 Jur'Evets, Vladimir, Fed. Rep. of
Russia
Slovak Republic State Veterinary Institute, PO Box 14/D, 949 01 Nitra
Slovenia University for Microbiology and Parasitology, University of Ljubljana, Veterinary Faculty, Gerbiceva
60, 61000 Ljubljana
Spain Centra de Investigacion en Sanidad Animal, CISA-INIA, 28130 Valdeolmos, Madrid
Sweden National Veterinary Institute, Box 7073, S 75007 Uppsala
Switzerland Institut für Viruskrankeheiten und Immunoprophylaxe, Sensemattstrasse 293, CH-3147 Mittelhäusern
The FYRO Macedonia Veterinary Institute Skopje, Veterinary pop Trajkov 5-7, 91000 Skopje
Turkey FMD Institute (SAP Enstitüsü), PO Box 714, 06044 Ulus, Ankara
United Kingdom Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Nr Woking, Surrey GU24
ONF
Total 23
Table 2: Phase XVI test sera definition

02/08/00
Final number Serotype Purpose Origin

1 O High positive O Manisa, 14-day post vaccinated bovine serum

2 C 1:15 C dilution series C Oberbayern, 21-day post infected bovine serum
3 - Negative Normal Adult Bovine Serum
4 C 1:30 dilution series C Oberbayern, 21-day post infected bovine serum
5 A High positive A22 Iraq, 10-day post challenge bovine serum: Diluted 1:12
6 A Low positive A22 Iraq, 10-day post challenge bovine serum: Diluted 1:45
7 C 1:240 C dilution series C Oberbayern, 21-day post infected bovine serum
8 C Neat low positive C Oberbayern, 6-day post infected bovine serum
9 C 1:120 C dilution series C Oberbayern, 21-day post infected bovine serum
10 C 1:60 dilution series C Oberbayern, 21-day post infected bovine serum
11 A Neat positive A Tur 2/98, 21-day post infected bovine serum
12 O Low positive O Manisa, 7-day post vaccinated bovine serum
Table 3: FMD test sera results by SPOT TEST

Samples tested for FMD type O Samples tested for FMD type A Samples tested for FMD type C
Laborator 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
1
2 + - - - - - - + - - + + - - - - + + - - - - + - - + - + - - - - - + - -
3 + - - - - - - - - - - + - - - - + + - - - - + - - + + + - - + - + + - -
4 + - - - - - - - - - - - - - - - + + - - - - - - - + - + - - - - +/- +/- - -
5
6 + + - - - - - - - - + + - - - - + + - - - - + - - + - + - - - - - + + -
7 + - - - - - - - - - - + - - - - + + - - - - + - - + - + - - + + + + - -
8 + - - - - - - - - - - + - - - - + +/- - + - - + - - + - - - - + +/- +/- + - -
9
10 + + - + - - - - - - + + - - - - + + - - - - + - - + - + - - - - + + + -
11
12
13 + + - + - - + + + + + + + + - + + + - + - - + + ND ND ND ND ND ND ND ND ND ND ND ND
14
15 + + - + - - - + + + + + + + - - + + - - - - + - + + - + + - + + + + + +
16 + + - + + + + + - + + + - + - - + + - - - - + - + + - + - - - + + + - -
17
19 + + - - - - - - - - + + - - - - + + - - - - + - - + - + - - - - - + + -
21 + - - - - - - - - - - - - - - - + + - - - - + - - + - + - - - - - - - -
22 + + - - - - - - - - + + - - - - + - - - - - + - - + - + - - - - - - - -
23 + - - - - - - - - - - + - - - - + - - + - - + - - + - + - - - - + + - -
24 + +/- - - - - - +/- - - + + +/- + - + + + +/- + - - + + - + - + - - - - - + - -
25
26 + - - - - - - - - - + + + - - - + + - - - - + - ND ND ND ND ND ND ND ND ND ND ND ND
27 + + - +/- - - - + - - + + - - - - + + - +/- - - + - - + - + - - - +/- +/- + + -
28
29 + - - - - - - - - - - + - - - - + + - - - - + - - + - + - - - - - + - -
30
31 + + - - - - - - - - + + - - - - - - - - - - - - + - + - - - - - - + + -
+ Result 19 9 0 4 1 1 2 5 2 3 12 17 3 4 0 2 18 15 0 4 0 0 17 2 3 16 2 15 1 0 4 3 6 14 6 1
+/- Result 0 1 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 2 3 1 0 0
- Result 0 9 19 14 18 18 17 13 17 16 7 2 15 15 19 17 1 3 18 14 19 19 2 17 14 1 15 2 16 17 13 12 8 2 11 16
Labs test 19 19 17
Actual O C NEG C A A C C C C A O O C NEG C A A C C C C A O O C NEG C A A C C C C A O
Table 4:

F M D T e s t
N u m b e r o f L a b s s c
1 9
1 7
1 5
1 3 (19 labs tested)

O
1 1 (19 labs tested)

A
9 (17 labs tested)

C
7
5
3
1
1 . O H3 .P N E 5 G. A 7H . P C 19 :. 2 C 4 0 1 1 : 1 1 . 2 A0 L P
2 . C 14 :. 1 C 5 1 6 : . 3 A0 L8 P
. C 1L 0P . C 1 1 2 : . 6 O0 L P

T e s t S e r u m
Table 5:

F M D T e s t
N u m b e r o f L a b s s c
1 9
1 7
1 5
1 3 (19 labs tested)

O
1 1 (19 labs tested)

A
9 (17 labs tested)

C
7
5
3
1
1 . O H3 .P N E 5 G. A 7H . P C 19 :. 2 C 4 0 1 1 : 1 1 . 2 A0 L P
2 . C 14 :. 1 C 5 1 6 : . 3 A0 L8 P
. C 1L 0P . C 1 1 2 : . 6 O0 L P

T e s t S e r u m
Table 12: Clasification of test sera with homologous serotype
SERUM 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
C O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP C 1:120 C 1:60 A LP O LP
L
A
S Result
N HP HP N HP HP/LP LP CO/N CO/N CO LP/CO HP LP

E HP 2,3,4,6,7 2,3,4,6, except 2,3,6,7, 2,4,6, 10. 15. 2,3,6,7, 10,23.

,8,10,13, 7,8,10, 31 (C) 8,10,15, 10,13, 8,10,13,
14,15,19, 14,15, 24,27, 21,24, 14,15,
L 22,23,24,
26,27,29,
19,23,
24,25,
30. 27,30. 19,21,
23,24,
30,31. 27,29, 26,27,
I 30. 29,30.
LP 21. 21,22. 4,14,19, 3,7,8, 2,4,6,7, 7,8. 2,3,6,7, 4,22, 2,3,4,6,
S 23,25,
29
14,15,
19,23
13,14,
15,21,
8,10,23,
24,27.
8,15,19,
22,26,
25,26, 25,26, 27,29,
A 29 27,30. 30.
CO 21,22. 22 8,19,23, 7,8,15, 7,8,10, 3,6,10, 4,14,19, 7,13,14,
24,29. 14,15, 14,15, 22,25, 24,25.
27. 22,23, 29,30,
27,30. 31.
N All labs/ 3,22. 3,6,10, 3,6,19, 19,21. 21. 21,31.
All 14,19, 21,22,
types 21,22, 30.
SERUM 1. 2. 3. 4. 5. 6. 7.27, 30. 8. 9. 10. 1. 12.
C O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP C 1:120 C 1:60 A LP1 O LP
L
A
S Result
N HP HP N HP HP/LP LP CO CO LP HP HP LP
V HP 2,3,6,7,
8,12,13,
2,3,7,8,
12,14,
3,7,8,
12,14,
2,14,23,
25,27.
13. 3,7,8,
12,14,
7,12,13,
14,23,
8,13.

14,23, 23,27, 27. 23. 27,29.

N 25,27,
29.
29.

T LP 2,23,29. 7,8,12,
13,29.
2,7,12,
14,25,
8,12. 7,14. 3,7,8,
12,14,
27. 8. 2,3,6,12
,23,29.
27. 27.

CO except 8,23,29. 3,14,27. 8,12,27. 23. 2,29. 2. 7,14,25,

3 (C) 27.

N All labs/ 7. 3.
All
types
Table 13: Clasification of test sera with homologous serotype
SERUM

S C
L 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
A O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP C 1:120 C 1:60 A LP O LP
P S
N
2,3,4,6,7 2,3,4,6, except 2,3,4,6, 2,3,4,6, 2,3,4,6, 3,7,8, 7,15,16. 3,7,10, 2,3,6,7, 2,3,6,7, 2,3,6,7,
O ,8,10,13,
15,16,19,
7,8,10,
15,16,
3,31 (C) 7,10,
15, 16,
7,8,10,
13, 15,
7,10,13,
15,16,1
15. 15,16,
23,
8,10,15,
16,19,
8,10,13,
15, 16,
8,10,13,
15,16,1

T
21,22,23, 19,21, 19, 16,19, 9,21,24, 23,24, 19,21, 9,22,23,
+ 24,26,27, 22,23, 21,22, 21,22, 26,27,2 27,29, 22,23, 24,26,2
29,31. 24,27, 23,24, 23,24, 9. 31. 24,26, 7,29,31.
29. 27, 29. 26,27, 27, 29.
29.

T 8. 8,27. 4,8,27. 4.

+/-
E
31. All labs/ 8,31. 31. 22,23, 2,4,6, 2,3,4,6, 2,6,19, 21,22. 4,31. 4,21.
S All
types
31. 10,16,
19,21,
10,19,
21,22,
21,22,
24,29,
-
T 22,23,
24,27,
23,24,
29,31.
31.

29,31.
Table 6 : FMD test sera results by ELISA type O

1 2 3 4 5 6 7 8 9 10 11 12
Laboratory Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class
1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2 3.17 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N 0.00 0.00 1.79 CO 2.09 LP
3 3.19 HP 1.50 N 1.20 N 1.20 N 1.20 N 1.68 N 1.68 N 1.68 N 1.50 N 1.20 N 2.58 LP 2.58 LP
4 2.80 HP 0.00 1.64 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.69 CO 1.95 LP
5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
6 3.10 HP 1.00 LP 0.70 N 1.40 CO 0.20 N 0.20 N 0.20 N 1.30 CO 0.90 N 0.90 N 1.60 LP 1.80 LP
7 1.93 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.95 CO
8 2.70 HP 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.68 LP
9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10 3.36 HP 1.95 LP 1.59 N 1.66 N 1.59 N 1.59 N 1.59 N 1.59 N 1.59 N 1.59 N 2.11 HP 2.20 HP
11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
13 3.00 HP 2.10 HP 0.09 N 1.50 LP 0.90 N 0.75 N 0.90 N 1.05 CO 0.90 N 1.20 CO 2.10 HP 1.95 CO
14 2.43 HP 1.26 CO 0.95 N 0.95 N 0.95 N 0.95 N 0.95 N 0.95 N 0.95 N 0.95 N 1.36 CO 1.28 CO
15 1.18 HP 1.20 LP 0.00 N 0.60 CO 0.00 0.00 0.00 0.60 CO 0.60 CO 0.60 CO 1.50 LP 1.20 LP
16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
19 2.90 HP 1.60 CO 0.89 N 1.50 N 0.89 N 0.89 N 0.89 N 1.50 N 0.90 N 1.00 N 1.80 CO 2.00 LP
21 1.63 LP 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N
22 2.40 HP 1.68 LP 1.19 N 1.50 CO 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.81 LP 1.68 LP
23 2.58 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.28 HP
24 2.70 HP 1.20 N 0.00 0.00 0.00 0.00 0.00 1.20 N 0.00 0.00 1.81 CO 1.81 CO
25 0.00 HP 0.00 CO 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 CO
26 2.94 HP 1.19 N 1.28 N 1.24 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.55 CO 1.82 LP
27 3.33 HP 1.94 LP 0.00 1.57 CO 0.00 0.00 0.00 1.59 CO 0.00 0.00 1.91 LP 2.12 LP
28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
29 2.25 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.37 LP
30 3.16 HP 1.95 LP 1.34 N 1.65 CO 1.34 N 1.34 N 1.34 N 1.35 N 1.34 N 1.35 N 1.95 LP 1.95 LP
31 3.31 HP 1.81 LP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.81 LP 1.50 N
32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
HP 20 1 0 0 0 0 0 0 0 0 2 2
LP 1 7 0 1 0 0 0 0 0 0 7 12
CO 0 3 0 5 0 0 0 4 1 2 6 5
N 0 5 12 7 11 11 11 11 11 10 2 2
Labs tested 21 16 12 13 11 11 11 15 12 12 17 21
Actual O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP nt C 1:120 C 1:60 A LP True O LP
Table 7: FMD test sera results by ELISA type A

1 2 3 4 5 6 7 8 9 10 11 12
Laboratory Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class
1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2 0.00 0.00 0.00 0.00 2.01 HP 1.64 LP 0.00 0.00 0.00 0.00 2.60 HP 0.00
3 1.20 N 1.20 N 1.19 N 1.20 N 1.98 LP 1.20 N 1.19 N 1.19 N 1.19 N 1.19 N 2.11 HP 1.20 N
4 0.00 0.00 1.64 0.00 2.10 HP 1.18 LP 0.00 1.69 CO 0.00 0.00 1.95 LP 0.00
5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
6 0.80 N 1.30 CO 0.80 N 1.10 N 1.90 HP 1.60 LP 0.80 N 1.10 N 0.80 N 0.90 N 2.30 HP 1.00 N
7 0.00 0.00 0.00 0.00 1.70 LP 1.30 LP 0.00 0.00 0.00 0.00 1.91 HP 0.00
8 1.19 N 1.19 N 1.19 N 1.19 N 1.50 LP 1.38 CO 1.20 N 1.50 LP 1.20 N 1.20 N 2.20 HP 1.20 N
9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10 1.59 N 1.59 N 1.59 N 1.59 N 2.40 HP 2.10 HP 1.59 N 1.59 N 1.59 N 1.59 N 2.90 HP 1.59 N
11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
13 1.50 LP 1.50 LP 0.09 N 0.90 CO 1.80 HP 1.50 LP 0.09 N 0.90 CO 0.45 N 0.75 CO 2.10 HP 0.90 CO
14 0.95 N 0.95 N 0.95 N 0.95 N 1.87 LP 1.43 LP 0.95 N 0.95 N 0.95 N 0.95 N 2.28 HP 0.95 N
15 0.90 LP 0.90 LP 0.00 0.00 1.20 LP 0.90 LP 0.00 0.00 0.00 0.00 1.81 HP 0.00
16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
19 1.10 N 1.40 N 0.89 N 1.40 N 2.20 LP 1.90 CO 0.90 N 1.40 N 1.00 N 1.20 N 2.80 HP 1.00 N
21 0.59 N 0.59 N 0.59 N 0.59 N 2.50 HP 2.00 LP 0.59 N 0.59 N 0.59 N 0.59 N 2.60 HP 0.59 N
22 1.19 N 1.19 N 1.19 N 1.19 N 1.68 CO 1.20 N 1.19 N 1.50 CO 1.19 N 1.19 N 2.10 LP 1.19 N
23 0.00 0.00 0.00 0.00 1.68 LP 1.08 CO 0.00 1.98 HP 0.00 0.00 1.98 HP 0.00
24 1.30 CO 1.50 LP 0.00 1.50 LP 1.95 HP 1.20 CO 1.20 CO 1.95 HP 0.00 0.00 3.31 HP 1.81 LP
25 0.00 0.00 0.00 0.00 0.00 LP 0.00 LP 0.00 0.00 0.00 0.00 0.00 0.00
26 1.65 CO 1.19 N 1.19 N 1.19 N 2.25 LP 1.85 LP 1.34 N 1.33 N 1.19 N 1.19 N 3.01 HP 1.28 N
27 0.00 0.00 0.00 0.00 2.26 HP 1.88 LP 0.00 1.83 CO 0.00 0.00 2.73 HP 0.00
28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
29 0.00 0.00 0.00 0.00 1.65 LP 1.20 CO 0.00 0.00 0.00 0.00 1.86 HP 0.00
30 1.34 N 1.34 N 1.34 N 1.65 CO 2.26 HP 1.95 LP 1.34 N 1.34 N 1.35 N 1.65 CO 2.86 HP 1.65 CO
31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
HP 0 0 0 0 9 1 0 2 0 0 17 0
LP 2 3 0 1 10 12 0 1 0 0 2 1
CO 2 1 0 2 1 5 1 4 0 2 0 2
N 9 9 11 9 0 2 11 8 11 9 0 9
Labs tested 13 13 11 12 20 20 12 15 11 11 19 12
Actual O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP nt C 1:120 C 1:60 A LP True O LP
Table 8: FMD test sera results by ELISA type C

1 2 3 4 5 6 7 8 9 10 11 12
Laboratory Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class
1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2 0.00 2.48 HP 0.00 1.99 HP 0.00 0.00 0.00 0.00 0.00 1.70 LP 0.00 0.00
3 1.20 N 2.53 HP 1.19 N 2.05 HP 1.19 N 1.19 N 1.19 N 1.19 N 1.45 CO 1.75 LP 1.20 N 1.19 N
4 0.00 2.40 HP 1.64 N 1.18 LP 0.00 0.00 0.00 0.00 0.00 1.69 CO 0.00 0.00
5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
6 1.00 N 2.50 HP 0.20 N 2.10 HP 0.20 N 0.20 N 1.00 N 1.10 N 1.30 CO 1.60 LP 1.60 LP 1.00 N
7 0.00 2.20 HP 0.00 1.87 HP 0.00 0.00 1.18 CO 1.26 CO 1.52 LP 1.68 LP 0.85 N 0.00
8 1.20 N 2.28 HP 1.19 N 1.98 HP 1.19 N 1.19 N 1.38 CO 1.57 CO 1.70 LP 1.68 LP 1.20 N 1.20 N
9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10 1.59 N 2.15 HP 1.59 N 2.26 HP 1.59 N 1.59 N 1.59 N 1.62 CO 1.68 CO 1.92 LP 1.66 CO 1.59 N
11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
14 0.95 N 2.24 HP 0.95 N 1.86 LP 0.95 N 0.95 N 1.00 N 1.25 CO 1.23 CO 1.48 CO 1.23 CO 0.95 N
15 0.90 CO 2.40 HP 0.00 1.81 HP 0.60 CO 0.00 0.90 CO 1.20 CO 1.20 CO 1.81 HP 1.20 CO 0.90 CO
16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
19 1.50 N 2.50 HP 0.89 N 2.00 LP 1.10 N 0.89 N 1.10 N 1.40 N 1.40 N 1.60 CO 1.90 CO 1.30 N
21 0.59 N 1.34 LP 0.59 N 0.84 CO 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N 0.59 N
22 1.20 N 2.10 LP 1.19 N 1.50 CO 1.19 N 1.19 N 1.19 N 1.19 N 1.50 CO 1.50 CO 1.50 CO 1.50 CO
23 0.00 2.58 HP 0.00 2.28 LP 0.00 0.00 0.00 0.00 1.68 CO 2.28 LP 0.00 0.00
24 0.00 2.40 HP 0.00 1.95 HP 0.00 0.00 0.00 0.00 0.00 1.50 LP 0.00 0.00
25 0.00 0.00 HP 0.00 0.00 LP 0.00 0.00 0.00 0.00 0.00 0.00 CO 0.00 0.00
26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
27 0.00 2.43 HP 0.00 2.24 HP 0.00 0.00 1.17 N 1.40 CO 1.46 CO 1.78 LP 1.88 LP 0.00
28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
29 0.00 1.95 HP 0.00 N 1.65 LP 0.00 0.00 0.00 0.00 0.00 1.20 CO 0.00 0.00
30 1.34 N 2.86 HP 1.34 N 2.11 HP 1.34 N 1.34 N 1.34 N 1.35 N 1.64 CO 1.65 CO 1.65 CO 1.35 N
31 2.70 HP 0.00 2.10 HP 0.00 0.00 0.00 0.00 0.00 0.00 1.81 CO 1.50 CO 0.00
32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
HP 1 16 1 9 0 0 0 0 0 1 0 0
LP 0 2 0 6 0 0 0 0 2 9 2 0
CO 1 0 0 2 1 0 3 6 9 8 7 2
N 9 0 11 0 9 9 9 6 2 1 4 8
Labs tested 11 18 12 18 10 9 12 12 13 19 13 10
Actual O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP nt C 1:120 C 1:60 A LP True O LP
Table 9: FMD test sera results by VNT type O

1 2 3 4 5 6 7 8 9 10 11 12
Laboratory Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class
1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2 3.01 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.90 N 0.00 0.00 1.20 CO 1.81 LP
3 3.31 HP 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 1.19 N 2.11 LP
4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
6 2.80 HP 1.20 CO 0.60 N 0.90 N 0.20 N 0.20 N 0.20 N 1.00 N 0.60 N 0.90 N 1.20 CO 1.70 LP
7 2.56 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.51 CO
8 2.71 HP 1.00 N 0.60 N 0.60 N 0.60 N 1.04 N 0.78 N 0.00 0.60 N 0.60 N 0.60 N 1.90 HP
9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12 0.00 HP 0.00 N 0.00 N 0.00 N 0.00 N 0.00 N 0.00 N 0.00 N 0.00 N 0.00 N 0.00 LP 0.00 LP
13 3.00 HP 1.20 CO 0.09 N 0.45 N 0.45 N 0.45 N 0.09 N 1.20 CO 0.60 N 0.75 N 1.80 HP 1.80 HP
14 3.22 HP 1.18 N 1.18 N 1.18 N 1.18 N 1.18 N 1.18 N 1.18 N 1.18 N 1.18 N 1.18 N 1.95 CO
15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
23 2.41 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.81 LP
24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
25 2.40 HP 0.70 N 0.60 N 0.60 N 0.60 N 0.60 N 0.60 N 0.60 N 0.60 N 0.60 N 0.00 1.00 CO
26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
27 2.40 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.20 CO
28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
29 2.55 HP 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.38 LP
30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
HP 12 0 0 0 0 0 0 0 0 0 1 2
LP 0 0 0 0 0 0 0 0 0 0 1 6
CO 0 2 0 0 0 0 0 1 0 0 2 4
N 0 5 7 7 7 7 7 6 7 7 3 0
Labs tested 12 7 7 7 7 7 7 7 7 7 7 12
Actual O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP nt C 1:120 C 1:60 A LP True O LP
Table 10: FMD test sera results by VNT type A

1 2 3 4 5 6 7 8 9 10 11 12
Laboratory Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class
1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2 0.00 0.00 0.00 0.00 2.11 HP 1.51 LP 0.00 0.00 0.00 0.00 1.05 CO 0.00
3 1.19 N 1.19 N 1.19 N 1.19 N 0.00 0.00 1.19 N 1.19 N 1.19 N 1.19 N 0.00 1.19 N
4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
7 0.00 0.00 0.00 0.00 1.20 LP 0.90 LP 0.00 0.00 0.00 0.00 1.65 HP 0.00
8 0.00 0.00 0.00 0.00 1.50 LP 1.30 CO 0.00 0.00 0.00 0.00 1.50 LP 0.00
9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12 0.00 N 0.00 N 0.00 N 0.00 N 0.00 LP 0.00 LP 0.00 N 0.00 N 0.00 N 0.00 N 0.00 HP 0.00 N
13 0.09 N 0.45 N 0.09 N 0.09 N 1.65 LP 1.80 HP 0.09 N 0.45 N 0.09 N 0.09 N 1.95 HP 0.09 N
14 1.18 N 1.18 N 1.18 N 1.18 N 2.29 HP 1.98 LP 1.18 N 1.18 N 1.18 N 1.18 N 2.49 HP 1.18 N1
15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
23 0.00 0.00 0.00 0.00 1.81 HP 1.20 CO 0.00 0.00 0.00 0.00 2.11 HP 0.00
24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
25 0.60 N 0.60 N 0.60 N 0.60 N 1.88 HP 1.60 LP 0.60 N 0.60 N 0.60 N 0.60 N 0.00 0.60 N
26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
27 0.00 0.00 0.00 0.00 1.93 HP 1.69 LP 0.00 0.00 0.00 0.00 2.12 HP 0.00
28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
29 0.00 0.00 0.00 0.00 1.84 LP 1.51 CO 0.00 0.00 0.00 0.00 1.87 HP 0.00
30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
HP 0 0 0 0 5 1 0 0 0 0 7 0
LP 0 0 0 0 5 6 0 0 0 0 1 0
CO 0 0 0 0 0 3 0 0 0 0 1 0
N 5 5 5 5 0 0 5 5 5 5 0 5
Labs tested 5 5 5 5 10 10 5 5 5 5 9 5
Actual O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP nt C 1:120 C 1:60 A LP True O LP
Table 11: FMD test sera results by VNT type C

1 2 3 4 5 6 7 8 9 10 11 12
Laboratory Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class Titre class
1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2 0.00 2.41 HP 0.00 1.81 LP 0.00 0.00 0.00 0.00 0.00 1.20 CO 0.00 0.00 0.00
3 1.19 N 2.28 HP 1.20 CO 2.11 HP 1.19 N 1.19 N 1.20 CO 1.19 N 1.50 LP 1.98 HP 1.19N 1.19 N
4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.90N
7 0.00 1.95 HP 0.00 1.65 HP 0.00 0.00 0.78 N 1.04 LP 1.04 LP 1.51 HP 0.00 0.00 0.00
8 0.00 2.71 HP 0.00 2.28 HP 0.00 0.00 1.50 LP 1.05 CO 1.62 LP 2.10 HP 0.60N 0.00
9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.59N
11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
12 0.00 N 0.00 HP 0.00 N 0.00 HP 0.00 N 0.00 N 0.00 LP 0.00 CO 0.00 LP 0.00 HP 0.00N 0.00 N
13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.20CO
14 1.18 N 2.85 HP 1.18 N 2.51 HP 1.18 N 1.18 N 1.38 CO 1.55 LP 2.37 LP 2.37 HP 1.18N 1.18 N
15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.60CO
16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
17 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
19 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00N
21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.59N
22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.19N
23 0.00 2.71 HP 0.00 1.26 LP 0.00 0.00 0.00 0.00 1.51 CO 2.41 HP 0.00 0.00 0.00
24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
25 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.19N
27 0.00 2.18 HP 0.00 1.98 HP 0.00 0.00 1.34 CO 1.34 CO 1.80 LP 1.65 LP 0.00 0.00 0.00
28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
29 0.00 2.20 HP 0.00 1.98 LP 0.00 0.00 0.00 0.00 0.00 1.35 CO 0.00 0.00 0.00
30 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.35N
31 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
HP 0 9 0 6 0 0 0 0 0 6 0 0
LP 0 0 0 3 0 0 2 2 6 1 0 0
CO 0 0 1 0 0 0 3 3 1 2 0 0
N 3 0 2 0 3 3 1 1 0 0 4 3
Labs tested 3 9 3 9 3 3 6 6 7 9 4 3
Actual O HP C 1:15 NEG C 1:30 A HP A LP C 1:240 C LP nt C 1:120 C 1:60 A LP True O LP
 241

Appendix 30

Comparison of FMDV neutralisation tests using three different cell lines:

validation of the new FAO reference sera

P. Moonen, G.K.W. Miedema, F. van Hemert-Kluitenberg, G. Chenard, A. Dekker

Abstract:
To minimise the variation between laboratories doing FMDV serology it was decided
to select a set of reference sera that can be used to define the cut-off in each laboratory. At
the time of selection already serious doubts were expressed about the level of the cut-off
serum, it was felt that the specificity of the test would be too low. For this reason we validated
the specificity of the FMDV virus neutralisation test (VNT) against type A22Iraq, O1Manisa
and C1Noville, using three different cell lines and compared the results with those obtained
using the liquid phase blocking ELISA (LPBE). The results show that with the currently
selected cut-off sera over 2.4% false positive results are found (≥ 11 out of 464). In case of
an outbreak, but also in import- and export screening, such a high number of false positive
reactions is not acceptable. The titres of the cut-off sera are therefore too low. Therefore,
new cut-off sera have to be produced that have a higher titre. For type A the titre should be
0.60 higher, for type O 0.45 and for type C 0.15 (10log). This adjustment reduces the number
of false positive reactions found in this study to zero.

Introduction:
The neutralisation test for foot-and-mouth disease (FMD) serology is one of the
prescribed tests for international trade of the Office International des Epizooties (OIE). For
this test BHK (Macpherson and Stoker, 1962), IBRS-2 (de Castro, 1964), lamb- or pig-kidney
cells can be used (Donaldson et al., 2000). If the same serum is tested in tests using
different cells the resulting neutralisation titre is different. To minimise the variation between
laboratories it was decided to select a set of reference sera which can be used to define the
cut-off in each laboratory (FAO, 1998). At the time of selection of the new reference sera the
choice of cut-off serum was criticised, the titre of the cut-off serum seemed to low, which
would result in an unacceptable number of false positive reactions. It was agreed that the
different labs should use the selected reference sera in their standard test and report the
results on the next meeting. Because of the differences in antibody titre found between
different cell lines used we validated the specificity of the FMDV virus neutralisation test
(VNT) against type A22Iraq, O1Manisa and C1Noville, using three different cell lines and
compared the results with those obtained using the liquid phase blocking ELISA (LPBE).

Materials and methods:

Sera
Reference sera were obtained from the World Reference Laboratory in Pirbright. The
cut-off, weak positive and strong positive sera were dilutions from the same positive serum
diluted in negative serum. For type A22Iraq, the dilution factor of the cut-off, weak positive and
strong positive was respectively, 1:100, 1:50 and 1:10. For type O1Manisa, the dilution
factors were respectively, 1:48, 1:12 and 1:4, and for type C1Noville: 1:50, 1:25 and 1:12. A
 242

total of 464 sera of Dutch and German cattle were collected at the slaughterhouse in
Leeuwarden. 3.75 % of the Dutch cattle slaughtered on that day were born before 1991, the
year the last FMDV vaccination was carried out. Sera from the control cattle of 14
vaccination challenge experiments collected 7 - 8 days after infection.

Cells
Primary porcine kidney cells were produced by chopping the kidney of a 6 - 10 week
old specific pathogen free piglet into 2 to 3 mm3 pieces and trypsinising these pieces at 37° C
with 0.06% trypsin diluted in phosphate buffered saline. After 0.5 to 1 hour trypsin treatment,
the cells were centrifuged at approximately 250 g, put into suspension using buffer
containing Hank's salts, supplemented with 0.035% NaHCO3, 0.5% lactate albumin
hydrolysate, 5% foetal bovine serum and antibiotics, and seeded in glass flasks. The primary
porcine kidney cells were used in the VNT 2 to 21 days after primary culture and were
designated secondary porcine kidney (PK-2) cells. BHK and IBRS-2 cells were grown in
Eagles MEM supplemented with protein hydrolysate, 5% foetal bovine serum and antibiotics.

Virus neutralisation test

The virus neutralisation test was performed as described in the OIE "Manual of
standards for diagnostic tests & vaccines" (Donaldson et al. 2000). Before testing the
complement in the serum was inactivated by heat treatment at 56°C for 30 minutes. In a
microtitre plate two-fold serial dilutions of the serum, starting with undiluted serum, were
mixed with 100 TCID50 of FMD virus. After 1 hour incubation at 37 °C, 5% CO2 in a
humidified atmosphere, the cells were added. After 3 days the plates were dipped in citric
acid and the monolayers stained with an amido-black staining solution. The plates were read
macroscopically. The end-point titre of each serum was expressed as the logarithm of the
reciprocal dilution that prevented virus growth for more than 50% in 50% of the wells. In each
test the virus concentration was determined and a test was considered correct if the amount
of virus used was between 30 and 300 TCID50 per well. For each cell line a different batch of
virus was used, which had been passaged on that cell line at least two times. For calculation
a 10log titre of <0.3 was changed into 0.

Liquid phase blocking ELISA

The LPBE was performed as described in the OIE "Manual of standards for
diagnostic tests & vaccines" (Donaldson et al., 2000). Briefly, in a microtitre plate 50 µl of
two-fold serial dilutions of the serum were mixed with 50 µl of a predetermined dilution of
inactivated FMD viral antigen. After an overnight incubation at 4 °C, 50 µl of the mixture was
transferred to an ELISA plate coated with rabbit immunoglobulins directed to the same
serotype of FMD. The ELISA plate was incubated for 1 h at 37 °C on a rotary shaker. After
washing with tap water containing 0.05% Tween 80, 50 µl of a predetermined dilution of
guinea-pig immunoglobulins was added. After 1 h incubation and washing, the plate was
incubated with a predetermined dilution of rabbit anti-guinea-pig conjugated antibodies
(DAKO®). After this last hourly incubation the plate was washed and incubated for 15 minutes
with 100 µl of a chromogen/substrate solution (0.4 mg/ml OPD in buffer pH 5, containing
0.05% H2O2 (30%). After 15 minutes the colour reaction was stopped by addition of 100 µl of
a 0.5 M H2SO4 dilution. Sera, 64 for type A, 45 for type O and 169 for type C that were
negative in the initial dilution tested were omitted from the calculation of the mean titre. For
type C strain C1Detmold was used in the LPBE as antigen.

Results
Table 1 shows the mean titres and standard deviation of the different sera in all
tests. Based on the results of the various reference sera and their dilution we calculated the
average titre of the cut-off serum (Table 1). The mean titre of the calculated cut-off and the
mean titre of the cut-off as determined in each test differed between 0.007 and 0.22 (10log).
 243

Because the calculated cut-off was determined on more observations we used this one to
determine which sera were positive or negative. First we calculated the number of false
positive sera if the 464 sera were tested against three types of FMD virus (FMDV) (Table 2).

Table 1: Mean titres of all sera: (X̄ = mean, σ = standard deviation)

VNT on secondary Porcine kidney cells
Serum A22Iraq O1Manisa C1Noville
X̄ σ X̄ σ X̄ σ
Cut-off1 0.28 0.21 0.43 0.26 0.66 0.11
Cut-off 0.30 0.30 0.55 0.23 0.70 0.09
Weak positive 0.65 0.09 1.10 0.23 0.90 0.00
Strong positive 1.20 0.26 1.35 0.30 1.30 0.17
Negative sera 0.01 0.07 0.02 0.13 0.02 0.15
Negative sera, max. titre 1.05 N.A. 1.35 N.A. 1.35 N.A.
VNT on BHK cells
Serum A22Iraq O1Manisa C1Noville
X̄ σ X̄ σ X̄ σ
Cut-off 1
0.71 0.26 0.99 0.18 1.27 0.43
Cut-off 0.75 0.15 0.98 0.11 1.28 0.74
Weak positive 1.28 0.31 1.58 0.32 1.57 0.53
Strong positive 1.50 0.15 2.10 0.21 1.88 0.32
Negative sera 0.02 0.11 0.10 0.22 0.24 0.35
Negative sera, max. titre 1.20 N.A. 1.35 N.A. 1.35 N.A.
VNT on IBRS-2 cells
Serum A22Iraq O1Manisa C1Noville
X̄ σ X̄ σ X̄ σ
Cut-off 1
0.90 0.35 0.92 0.48 1.26 0.17
Cut-off 1.00 0.23 1.13 0.11 1.25 0.17
Weak positive 1.35 0.15 0.90 N.A. 1.70 0.09
Strong positive 1.65 0.52 2.10 0.64 1.75 0.17
Negative sera 0.24 0.25 0.10 0.23 0.07 0.23
Negative sera, max. titre 1.35 N.A. 1.35 N.A. 1.35 N.A.
Liquid phase blocking ELISA
Serum A22Iraq O1Manisa C1Noville/Detmold
X̄ σ X̄ σ X̄ σ
Cut-off1 1.39 0.30 1.91 0.23 1.77 0.13
Cut-off 1.58 0.32 2.13 0.25 1.88 0.11
Weak positive 1.80 0.21 2.48 0.15 2.03 0.11
Strong positive 2.10 0.21 2.83 0.06 2.25 N.A.
Negative sera 1.19 0.24 1.57 0.33 0.95 0.28
Negative sera, max. titre 1.35 N.A. 1.95 N.A. 2.25 N.A.
1
Titre calculated on the basis of the results of all reference sera and their dilution factor.

In total only 22 sera were found positive in one of the neutralisation tests. Of these
22 sera 13 were positive for one serotype on only one cell line, 3 were positive for only one
serotype but on two or more cell lines. The remaining 6 sera were positive for two or more
serotypes on two or more cell lines. Of the 22 VNT positive sera 15 were also positive for
one or more types in the LPBE. These 22 sera were tested in the neutralisation test for
antibodies against A10Holland, O1BFS and C1Detmold; the strains used in the vaccination
 244

campaigns before 1991. A total of 12 sera had a neutralisation titre against the vaccine
strains that was higher than the titre against the heterologous strain.

Table 2: Total number of sera with a titre greater than or equal to one of the cut-off1 sera
when tested for antibodies against type A, O and C.
VNT on secondary porcine kidney cells 11
VNT on BHK cells 13
VNT on IBRS-2 cells 13
Liquid phase blocking ELISA 117
1
Titre calculated on the basis of the results of all reference sera and their dilution factor.

After adjusting the cut-off in our test with 0.60 (10log) for type A, 0.45 (10log) for type
O and 0.15 (10log) for type C, no false positive reactions were found in the VNT on BHK and
IBRS-2 cells (Table 3). After adjustment of the cut-off also no false positive reactions were
found in the LPBE (Table 3).

Table 3: Number of false positive sera after adjusting the cut-off level in the test.
A22Iraq O1Manisa C1Noville
Adjustment (10log) + 0.60 + 0.45 + 0.15
VNT on PK-2 cells 1 4 7
VNT on BHK cells 0 0 0
VNT IBRS -2 cells 0 0 0
LPB ELISA 0 0 0

In 14 vaccination challenge experiments performed over the last 10 years, an

average homologous neutralisation titre determined on PK-2 cells of 2.41 (σ = 0.39) was
found in the control cattle 7 to 8 days after challenge. The maximum neutralisation titre
was 3.00 and the minimum 1.50.

Discussion
Foot-and-mouth disease is a very important disease of livestock. For this reason
almost every country checks animals when imported to their country. Based on the results of
serological tests in different laboratories it was clear that problems could arise if a standard
dilution in the test is used as cut-off. For this reason it was decided that a panel of
international reference sera had to be produced to standardise the results obtained in
different laboratories (FAO, 1998). Observations that the cut-off sera consequently scored
negative in our test indicated that the specificity of the neutralisation test would be too low
using the new cut-off sera. Different laboratories use different cell-lines for the neutralisation
test, previous standardisation exercises showed that the results differed between
laboratories, which was probably caused by the difference in cells used. For this reason we
set up three different neutralisation tests, using three different cell lines, to determine the titre
of the cut-off sera, and the specificity of the tests using a set of 464 field sera from
non-infected cattle.
The VNT titres found using BHK or IBRS-2 cells were comparable (Table 1), but
significantly higher than when PK-2 cells were used in the VNT. This result confirms the
results of the previous study where different laboratories using different tests tested the same
sera (Mackay et al., 1998). Not only the titres of the reference sera are higher, but also the
titres of the negative field sera, which resulted in almost the same number of false positive
reactions (Table 2). This shows that the system of using reference sera as cut-off is valuable.
The fact that 15 of the 22 VNT positive sera were also positive for one or more types in the
 245

LPBE indicates that interferon or other cytokines, which can prevent virus growth in cells, are
not responsible for the false positive reaction. Studies on FMDV false positive sera in a newly
developed monoclonal antibody based ELISA showed that the reaction co-precipitated with
the proteins precipitated with saturated ammonium sulphate (Chenard, personal
communication). This indicates that antibodies cause these false positive reactions, some of
the cattle have been vaccinated and might still have some low levels of FMDV specific
antibodies (3.75 % of all Dutch cattle slaughtered were born before 1991, the last year FMDV
vaccination was carried out in the Netherlands and Germany). In a study in Saudi Arabia the
antibody titres after vaccination had a half-live of approximately 41 days. Titres induced by
vaccination ten years ago should have vanished (Woolhouse et al., 1996). Terpstra et al.
(1990), however, showed that antibody titres stayed almost the same from 1 year to 3 years
after three consecutive vaccinations. For this reason we tested the 22 sera that had a
neutralising antibody titre against one of the three serotypes in for neutralising antibodies
against A10Holland, O1BFS and C1Detmold, the three strains that were included in the
vaccine used in the Netherlands. Twelve of the 22 false positive reactions were possibly
caused by previous vaccination. But 3, 2 and 4 of the false positive reactions on respectively
PK-2, BHK and IBRS-2 could not be attributed to vaccination. The purpose of the cut-off sera
is not to be able to detect cattle that have been vaccinated 10 years ago, but to be able to
detect a more recent infection.
Validation of the neutralisation test for Swine Vesicular Disease (SVD) virus on 80
negative sera in our laboratory resulted in a mean titre of 1.14 with a standard deviation of
0.31 (10log). The European reference serum for SVD serology (cut-off serum ERS 01.04.93)
has a mean titre of 2.19 (Dekker, 2000) which is higher than the average titre of the negative
sera plus 3 times the standard deviation (1.14 + 3 x 0.31 = 2.07). This indicates that less
than 1% of the negative serum has a neutralisation titre above the titre of the cut-off serum,
in fact only 5 out of 10.000 sera were found false positive the last two years when testing
over 1.2 million sera (Dekker, 2000). In the case of the FMDV neutralisation tests, using the
newly defined cut-off sera, we find over 2.4% false positive results (≥ 11 out of 464). In case
of an outbreak, but also in import- and export screening, such a high number of false positive
reactions is not acceptable. The titres of the cut-off sera are therefore too low. During the last
session of the research group of the European commission for the control of foot-and-mouth
disease (14 - 18 September 1998, Aldershot, United Kingdom) it was agreed that the
neutralisation titre of the cut-off should be 1.35 (10log), between OIE criterion of being
negative (≤ 1.05) and positive (≥1.65). Considering that most other laboratories use BHK or
IBRS-2 cells, the titre of the cut-off serum should be approximately 1.35 on these cell lines.
But in all serotypes the titre of the cut-off serum is lower. Therefore, new cut-off sera have to
be produced that have a higher titre, and should be validated using a large panel of negative
field sera. For type A the titre should be 0.60 higher, for type O 0.45 and for type C 0.15
(10log). This adjustment reduced the number of false positive reactions to zero (Table 3).
The mean antibody titres found in cattle 8 days after infection were 2.41, which is
much higher than the titres found in the cut-off sera. If the cut-off sera would be adjusted as
proposed above, the titre on PK-2 cells would be 0.88, 0.90 and 0.81 for type A22, O1 and C1
respectively, which is still much lower than the lowest titre found in unvaccinated cattle after
infection (1.50). The adjustment will therefore not influence the sensitivity of the test, but will
be a huge step forward with regard to the number of false positive reactions. After selecting
new cut-off sera, the neutralisation tests have to be validated more extensively with regard to
the sensitivity of the test also. For the validation of the sensitivity a large set of sera of FMDV
infected cattle, sheep and pigs are needed, preferably collected during an outbreak.

Acknowledgements
We like to thank the staff of the slaughterhouse Brada in Leeuwarden for the co-operation in
collecting bloodsamples.
 246

References:
De Castro, M.P. 1964. Behaviour of the foot-and-mouth disease virus in cell cultures:
susceptibility of IB-RS-2 line. Archivos do Instituto Biologico São Paulo, 31; 63-78
Dekker, A. 2000. Pathogenesis, diagnosis and epizootiology of swine vesicular disease.
Thesis, University Utrecht.
Donaldson, A.I., Kitching, R.P. and Barnett, P.V.. 2000. Foot and mouth disease. In Manual
of standards for diagnostic tests & vaccines, OIE. http://www.oie.int/
FAO. 1998. Report of the session of the research group of the European commission for the
control of foot-and-mouth disease. 14 - 18 September 1998, Aldershot, United
Kingdom. FAO, Rome, 1998; 1-17.
Mackay, D.K.J., Rendle, T. and Kitching, R.P. 1998. FAO collaborate study on FMD antibody
detection. European commission for the control of foot-and-mouth disease, session of
the research group of the standing technical committee. 14 - 18 September 1998,
Aldershot, United Kingdom, 148-157.
Macpherson, I., Stoker, M. 1962. Polyoma transformation of hamster cell clones, an
investigation of genetic factors affecting cell competence. Virology, 16; 147-151.
Terpstra, C., van Maanen, C., van Bekkum, J.G. 1990. Endurance of immunity against
foot-and-mouth disease in cattle after three consecutive annual vaccinations.
Research in Veterinary Science, 49; 236 - 242.
Woolhouse, M.E.J., Haydon, D.T., Pearson, A. 1996. Failure of vaccination to prevent
outbreaks of foot-and-mouth disease. Epidemiology and Infection, 116; 363 - 371.
 247

Appendix 31

Longevity of the antibody response in pigs and sheep following a single

administration of high potency emergency FMD vaccines

S. J Cox and P. V. Barnett

Institute for Animal Health, Pirbright Laboratory,

Ash Road, Pirbright, Surrey GU24 0NF, U.K.

Summary

Ideal vaccines should be capable of stimulating a potent and long-lasting immunity, after a
single vaccination. Conventional FMD vaccines, however, require frequent re-vaccinations to
maintain antibody at protective levels. Consequently the search for more effective FMD
vaccines continues to occupy research teams world-wide. Studies using high potency
emergency FMD vaccines, particularly those incorporating the ‘ready-to-formulate’ oil
adjuvant Montanide ISA 206, have shown not only rapid sero-conversion but maintenance of
a protective antibody response for at least 6 months in sheep and 4.7 months in pigs, after a
single vaccination. Such long-lasting immunity, which is likely to overcome the need to
revaccinate, would be invauable in the control of an FMD outbreak. These high potency
vaccines are worthy of further investigation, to monitor exactly how long protective
immunity can be maintained following a single application, as this may provide not only
benefits for the control procedures used in FMD-free countries but also a more efficient and
cost effective means of maintaining herd immunity in endemic areas.

Introduction

Much research and effort has been directed towards the improvement of vaccines to FMD
and to the understanding of host immune responses to natural FMD infection in order to
develop vaccines which promote potent long lasting immunity ( as reviewed by Barteling
and Vreeswijk, 1991, Doel, 1991 , 1996). However, depending on type and quality, the
widely used conventional, low potency FMD vaccine formulations are often unable to
provide an immunity that lasts longer than 6 months. Consequently livestock need to be
re-vaccinated, depending upon the epidemiological situation, between one and three times a
year for protection to be maintained. Ruminants require a primary vaccination followed by a
booster vaccination at 3-4 weeks. Pigs generally only require a single dose of oil adjuvanted
vaccine, which can result in a long duration of immunity (Anderson et al., 1971), but more
often they require boosting at approximately 6 monthly intervals.

The main requirements of emergency vaccines are to rapidly induce protective immunity and
reduce local virus replication thus limiting virus spread and transmission during an outbreak
of FMD. In recent years, studies at the International Vaccine Bank (IVB), located at IAH
Pirbright laboratory, using cattle (Doel et al., 1994, Salt et al., 1995), pigs (Salt et al., 1997)
and sheep (Cox et al., 1999), have provided much data confirming the ability of such
vaccines to prevent disease and transmission of FMDV. These studies, however, were
interested in early immune events and were frequently terminated after 28 days so that the
duration of the response remained unknown. As the need for a vaccine for both ruminants
and pigs that confers long lasting immunity, ideally after a single vaccination remains,
several recent additional studies have been performed using the same emergency vaccine
formulations to investigate the longevity of the antibody response in sheep and pigs. In
 248

sheep, both oil and aqueous vaccine formulations were investigated where as the pig study
evaluated the total neutralising antibody and some of the different classes of antibody
present following vaccination with oil adjuvanted vaccine.

Materials and methods

Vaccine formulations incorporating FMDV O1 Lausanne or C1 Oberbayern inactivated

antigen as a water-in-oil-in-water (W/O/W) emulsion with Montanide ISA 206 (Seppic,
Paris) were used in pigs. Studies in sheep used formulations incorporating FMDV O1
Manisa and A22 Iraq inactivated antigen as W/O/W or oil-in-water (O/W) emulsions with
either Montanide ISA 206 or ISA 25 (Seppic, Paris) respectively or an aqueous
(Al/OH3)/saponin formulation. All the vaccines were prepared from antigen concentrates
held currently by the IVB over liquid nitrogen and are highly potent with PD50 values of 41,
≥112, ≥112 and 75 for O1 Lausanne, C1 Oberbayern, O1 Manisa and A22 Iraq respectively
when tested as an Al(OH)3/saponin formulation. All vaccines used in sheep were
administered as a 1.0ml volume (equivalent to half of a bovine dose), by the intramuscular
route (W/O/W and O/W vaccines) into the right quadriceps mass or subcutaneously (aqueous
vaccine) over the left shoulder. Pigs received a 2.0ml volume (equivalent to 1 bovine dose)
by the intramuscular route immediately caudal to the ear.

Groups of three Large White crossbred Landrace pigs weighing 20-30 kg or three polled
Dorset Horn sheep aged between 6 and 12 months were immunised with vaccines as detailed
above. All animals were monitored daily for temperature and well-being for 10 days
following vaccination and bled regularly for serology up to 141 days (pigs) and 168 days
(sheep) post vaccination. Neutralising antibody titres against FMDV in serum were measured
by a microneutralisation assay, essentially as detailed by Golding et al. (1976). End-point
titres were calculated as the reciprocal of the last serum dilution to neutralise 100 TCID50 of
homologous FMDV in 50% of the wells. An antibody isotype analysis of the pig sera was
performed using an ELISA (IDAS) as described by Salt et al. (1996). Anti porcine isotype
reagents (anti IgM, IgG1, IgG2 and IgA) were supplied by Serotec, UK.

Results

Temperatures remained normal and no side effects were recorded following vaccination
with either the aqueous or oil formulations in the relevant target species. Neutralising
antibody responses for sheep vaccinated with A22 Iraq and O1 Manisa over 168 days are
shown in figure 1. All animals had seroconverted by seven days post vaccination, regardless
of adjuvant, and in most animals titres had peaked within 28 days. The sheep vaccinated
with Montanide ISA 206 oil formulation (fig 1, c and f ) maintained high titres of antibody
for the duration of the trial, particularly for the A22 Iraq vaccine. However, responses for the
vaccines formulated with either Al(OH)3/saponin (fig 1, a and d) or Montanide ISA 25 (fig 1,
b and e) demonstrated a gradual decline from peak antibody titres which leveled off after
66 days for most individuals.

Neutralising antibody titres for pigs vaccinated with either O1 Lausanne or C1 Oberbayern
are shown in figure 2. All pigs had seroconverted by seven days post vaccination with peak
titres generally being evident between 21 and 28 days post vaccination. Antibody isotype
analyses of sera from pigs vaccinated with either of the two serotypes demonstrated typical
profiles and are shown in figure 3. All animals responded to vaccination with a rapid IgM
response which was prolonged in animals vaccinated with O1 Lausanne. IgG1 responses
 249

were generally earlier and reached higher titres than IgG2. One animal (TX 89) appeared
unable to mount an IgG2 response and no IgA was detected from any animal at any point
after vaccination.

Conclusions

Conventional aqueous vaccines in ruminants generally promote protective immunity within

8-10 days following primary vaccination and a secondary injection is required to maintain
immunity at protective levels for about 6 months. Thereafter further re vaccinations at
regular intervals, depending upon epidemiological situation, are required to maintain
protective immunity. All the high potency vaccine preparations used in the sheep study
generated a rapid antibody response (confirming previous observations, Cox et al., 1999) and
in most sheep the antibody titres were maintained, at levels considered protective, for the
duration of the study. The sheep vaccinated with A22 Iraq serotype responded particularly
well following vaccination with all three formulations although sheep vaccinated with the
Montanide ISA 206 oil formulation were the only group to maintain peak titres for the
duration of the trial. Peak antibody titres in the sheep vaccinated with O1 Manisa vaccine
formulations were less dramatic and more variable but again the Montanide ISA 206 oil
formulated vaccine gave the best results. However, since the A22 Iraq vaccine formulations
gave a better response than the O1 Manisa formulations but had originally shown lower
potency as an aqueous formulation ( 75 and >112 respectively in cattle), maintenance of the
response cannot relate entirely to this or indeed the payload of antigen per dose (1.246 and
2.394 for A22 Iraq and O1 Manisa respectively). Variation between serotypes which display
differing levels of immunogenicity will inevitably contribute to the magnitude of the response
(Doel.,1996). Since the sheep only received a single injection of vaccine these results suggest
that higher potency vaccines may offer advantages over conventional aqueous vaccines
particularly the oil adjuvanted formulations. Oil adjuvanted vaccines of various types have
previously been shown to promote good protection in ruminants and pigs with immunity
lasting for more than six months (reviewed Barteling & Vreeswijk, 1991).

Depending on the formulation used, conventional oil adjuvanted vaccines that are used in
pigs normally only require a single injection to promote a protective immunity which
develops within approximately 8 days and lasts for about 6 months. Our study with C1
Oberbayern and O1 Lausanne Montanide ISA 206 oil formulated vaccines show that high
potency vaccines also promote neutralising antibody which is maintained at levels considered
protective for at least 4.7 months and compare well with the level of protective immunity
maintained in convalescent pigs following FMD infection (Doel., 1999). Highest titres were
evident between 21 and 28 days which was later than had previously been observed (Barnett
et al., 1996). Once peak titres had been achieved they were generally maintained although
some minimal decline was evident in the antibody levels of those pigs vaccinated with the O1
Lausanne formulation for the remainder of the trial period. Interestingly, Anderson et al
(1971) observed similar antibody decline in pigs after vaccination with a single oil emulsion
containing O1 Swiss 1/66 but not with similar formulations containing an A or C serotype.

Individual isotype-specific antibody responses were measured in the pig sera. As previously
shown by Ouldridge et al. (1982), and typical of primary responses, a rapid IgM response was
prominent 3-5 days following vaccination which peaked within 7 – 14 days post
immunisation. IgG responses were only generally detected 9 or more days post-vaccination.
This may have implications with regard to the role of the antibody response and the early
protective immunity achieved within 4 days of vaccination (Salt et al., 1997). Although
innate parameters are felt likely to be involved in such rapid protection (Cox et al., 1999), the
role of the antibody response cannot be dismissed. However, if antibody does have a function
 250

at the early stages following vaccination, then the isotype profiles seen in this study suggest
that IgM and not IgG plays an important role in early protection.

IgG1 appears to be the first of the IgG isotypes detected which in most cases peaked
around 28 days and was followed later by IgG2 ( apart from pig TX 90 which appeared
unable to mount an IgG2 response). Levels of IgG1 tended to be higher than IgG2 which is
consistent with previous reports for cattle ( Mulcahy et al., 1990) although the antibody
isotypes of different species and there functions are not fully understood and therefore do
not necessarily correlate with each other. Further studies are on going to monitor this primary
response for 6-9 months following which the qualitative aspects of the response in terms of
protection will be investigated.

In conclusion, these preliminary investigations in sheep and pigs have suggested that oil
adjuvanted high potency ‘emergency’ vaccines provide not only a rapid immune response but
also a long lasting immunity following a single application. Such attributes make them ideal
for use in FMD-free countries where emergency ring vaccination may be necessary, as
they are likely to overcome the need to re-vaccinate during an outbreak. Similar formulations
would also be more efficient, and possibly more cost effective, for maintaining herd
immunity in areas endemic for FMD by increasing the interval between re vaccinations.

Acknowledgements

This work was supported financially by MAFF, UK and EU (FAIR-PL97-3665).

References

Anderson, E.C, Masters, R.C and Mowat, G.N. (1971) Immune response of pigs to
inactivated foot-and-mouth disease vaccines. Res. Vet. Sci. 12, 342-350.

Barteling, S.J and Vreeswijk, J. (1991) Developments in foot-and-mouth vaccines. Vaccine 9,

75-88.

Barnett, P.V, Pullen, L., Williams, L. and Doel, T.R. (1996). International bank for
foot-and-mouth disease vaccine: assessment of Montanide ISA 25 and ISA 206, two
commercially available oil adjuvants. Vaccine 14(13), 1187-1198.

Cox, S.J., Barnett, P.V., Dani, P. and Salt, J.S. (1999) Emergency vaccination of sheep
against foot-and-mouth disease: protection against disease and reduction in contact
transmission. Vaccine 17, 1858-68.

Doel, T.R., Williams, L and Barnett, P.V. (1994) Emergency vaccination against
foot-and-mouth disease: rate of development of immunity and its implications for the carrier
state. Vaccine 12, 592-600.

Doel, T.R. (1996) Natural and vaccine-induced immunity to foot and mouth disease: the
prospects for improved vaccines. Rev. sci. tech. Off. Int. Epiz. 15(3), 883-911.

Doel, T.R (1999) Optimisation of the immune response to foot-and-mouth disease vaccines.
Vaccine 17, 1767-1771.
 251

Golding, S.M., Hedger, R.S. & Talbot, P. (1976). Radial immuno-diffusion and serum
neutralisation techniques for the assay of antibodies to swine vesicular disease. Res. Vet. Sci.
20, 142-147.

Mulcahy, G., Gale, C., Robertson, P., Iyisan, S., DiMarchi, R.D. and Doel, T.R. (1990).
Isotype responses of infected virus-vaccinated and peptide vaccinated cattle to
foot-and-mouth disease virus. Vaccine 8, 249-256.

Ouldridge, E.J., Francis, M.J. and Black, L. (1982). Antibody response of pigs to
foot-and-mouth disease oil emulsion vaccine: the antibody classes involved. Res. Vet Sci. 32,
327-331.

Salt, J.S, Williams, L., Statham, R and Barnett, P.V. (1995). Further studies on the rate of
development of protection in cattle given emergency vaccination against FMD. Report of
the session of the Standing Technical Committee of the European Commission for the
Control of oot-and-Mouth Disease, Moedling, Vienna, Austria, Appendix 17, 90-97.

Salt, J.S., Mulcahy, G. & Kitching, R.P. (1996). Isotype-specific antibody responses to
foot-and-mouth disease virus in sera and secretions of carrier and non-carrier cattle.
Epidemiol. Infect. 117, 349-360.

Salt, J.S., Barnett, P.V., Dani, P. and Williams, L. (1997) Emergency vaccination of pigs
against foot-and-mouth disease: protection against disease and reduction in contact
transmission. Vaccine 16(7), 746-754.
 SV71 SV80
3 3
SV72 SV81

2.5 SV73 2.5 SV82

a) 2
d) 2

1.5 1.5

1 1

0.5 0.5

0 0
0 7 14 21 28 66 70 112 168 0 7 14 21 28 66 70 112 168

SV77 SV86
3 3
SV78 SV87
SERUM ANTIBODY (LOG 10)

2.5 SV79 2.5 SV88

b) 2 e) 2

1.5 1.5

1 1

0.5 0.5

0
0 7 14 21 28 66 70 112 168 0
0 7 14 21 28 66 70 112 168

SV74
SV83
3
3
SV75 SV84

2.5 SV76 SV85

2.5

c) 2
f) 2

1.5 1.5

1 1

0.5
0.5

0
0 0 7 14 21 28 66 70 112 168
0 7 14 21 28 66 70 112 168

DAYS POST VACCINATION DAYS POST VACCINATION

Figure 1: Serum antibody rsponses in sheep following vaccination with FMDV type A22 Iraq
(a,b,c) and O1 Manisa (d,e,f) as measured by microneutralisation assay.
Vaccines adjuvanted with: Al(OH)3/saponin (a and d), Montanide ISA 25 (b and e) and
Montanide ISA 206 (c and f).
 a) 3
TX89

TX90

2.5 TX91
SERUM ANTIBODY (LOG 10)

1.5

0.5

0
0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 134 141
DAYS POST VACCINATION

b) 3
TX92

TX93

2.5 TX94
SERUM ANTIBODY (LOG 10)

1.5

0.5

0
0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 134 141
DAYS POST VACCINATION

Figure2: Serum antibody responses in pigs following vaccination with FMDV type C1 Oberbayern (a)
and O1 Lausanne (b) as measured by microneutralosation assay.
Vaccines adjuvanted with Montanide ISA 206.
 TX89
4
IgM
IgG1
3

TITRE (LOG 10)

IgG2

0
0 2 5 7 9 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 135 141
TX90
3.5
IgM
3
IgG1
2.5
TITRE (LOG 10)

IgG2
2

1.5

0.5

0
0 2 5 7 9 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 135 141
TX91
4
IgM
IgG1
3
TITRE (LOG 10)

IgG2

0
0 2 5 7 9 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 135 141
TX92
3
IgM
2.5 IgG1
TITRE (LOG 10)

2 IgG2

1.5

0.5

0
0 2 5 7 9 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 135 141
TX93
4
IgM
IgG1
3
TITRE (LOG 10)

IgG2

0
0 2 5 7 9 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 135 141
TX94
3.5
IgM
3
IgG1
2.5
TITRE (LOG 10)

IgG2
2

1.5

0.5

0
0 2 5 7 9 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 120 127 135 141
DAYS POST VACCINATION

Figure 3: Serum antibody isotype analyses as measured by IDAS ELISA. TX89 - TX91
vaccinated with C1 Oberbayern Montanide ISA 206 formulated vaccine and TX92 - TX94
with O1 Lausanne Montanide ISA 206 formulated vaccine.
 255

Appendix 32

Enhancement of the immune response induced by the inclusion of saponin in oil adjuvanted
vaccines against foot-and-mouth disease

E. Smitsaart(1), N. Mattion(1); J.L. Filippi(1); B. Robiolo(2) ; O. Periolo (2);

J. La Torre(2) and R.C. Bellinzoni(1)

(1)
Biogenesis S.A. Ruta Panamericana km 38,2. (B1619IEA) Garín. (Buenos Aires).
E-mail: esmitsaart@biogenesis.com.ar. (2)CEVAN- CONICET. Serrano 669, (1414 ) Buenos Aires. (Argentina)

ABSTRACT

The immunogenicity of oil formulations that include saponin as an immunomodulator in cattle,

swine and goats was evaluated. The specific immune response was examined by the detection of
total antibodies (Ab) against each one of the viral strains in the vaccine by Liquid-Phase ELISA.
The titres of Ab were related to the Expected Protection Percentage (EPP) and in each experimental
group the proportion of individuals with Ab levels compatible with protection (EPP>81%) was
determined. The specific titres of IgG1 and IgG2 isotypes in vaccinated cattle were measured by
ELISA. The induction of Ab anti-VIAA and against the polymerase 3D was evaluated by agar gel
immunodiffusion and ELISA, respectively. The results indicate that oil vaccines with saponin
induced 10 times higher total specific Ab levels, for each one of the viral strains at 30 days post-
vaccination (DPV) than formulations lacking saponin. In addition, the inclusion of this
immunomodulator in formulations with reduced antigen payload, elicited at 30 DPV, significantly
higher total antibody protection levels (P<0.005) than those obtained with formulations with high
140S antigen payload, and absence of the immunomodulator. The IgG isotype profiles induced in
cattle by these vaccines indicated that both IgG1 and IgG2 were higher in formulations with
saponin. In addition, these formulations did not elicited antibodies to non structural proteins in
vaccinated animals at 30 DPV. High antibody levels were also induced in pigs and goats after
vaccination with vaccines containing saponin. Furthermore, high and persistent immune response
induced by this formulation in calves with maternal antibodies was also detected. No adverse
effects were shown in the vaccinated animals of any species. The adjuvant effect of the
formulations containing saponin with no adverse reactions suggests its use as alternative for
emergency vaccination and for prophylactic campaigns.

Key words: foot-and-mouth disease; single emulsion vaccine, saponin, immune response,
emergency vaccines.
 256

INTRODUCTION

Either for prophylactic campaigns or for emergency vaccination, immunogens of rapid, persistent
and of high potency capable to be applied in all susceptible species are required.
Saponin has been extensively used incorporated into the aluminum hydroxide adjuvant for
immunization of susceptible species (1). However, it is well known that these vaccines are
ineffective in inducing adequate immunity in pigs (2).
Oil based vaccines have proved to give a longer duration of immunity, negligible interference with
maternal antibodies and ability to induce early protection both in cattle and pigs (3,4,5).
The aim of this study was to evaluate the efficacy of the saponin incorporated into the aqueous
phase of water-in-oil single emulsion oil adjuvanted vaccines as possible candidates for emergency
or prophylactic vaccines. Both tolerance and immune response following vaccination of cattle, pigs
and goats were examined. The immune response was evaluated by assessing both total and subclass
IgG specific antibodies to FMDV.

MATERIALS AND METHODS

Animals: One hundred and nine (109) Hereford breed cattle aged 18-24 months free of FMD
antibodies were used. They belonged to Patagonia region (south paralell 42°) of Argentina.
Twenty-two Duroc Jersey 60-day-old pigs and thirty (30) 30-day-old goats free of Ab of FMDV
were also used.
Thirty-five (35) calves born from vaccinated dams aged 30 to 90 days were also used.
Five non-vaccinated animals served as controls on each experiment.
Animals were kept during the whole experiment under grazing conditions on controlled pens.

Vaccines and vaccination: FMDV antigen of each of the four FMDV vaccine strains (O1 Caseros,
A Argentina A79, A Argentina 87 and C3 Argentina 85) were propagated in BHK21 cell suspension
cultures and inactivated with binary etilenimine. Antigen were concentrated and partially purified
with 7% (w/v) polyethylene glycol 6000. Vaccines were prepared as water-in-oil single emulsion.
Vaccines differed in their antigen and saponin content, both components included in the aqueous
phase of the oil vaccines (Table 1). Vaccines were prepared individually for each trial. All animals
were immunized by the intramuscular route with 2.0 ml dose, except goats vaccinated with vaccine
“N” that received 1.0 ml dose.

Detection of total antibodies (Ab) against FMDV: The Ab titres against each one of the viral
strains in the vaccine was examined by Liquid-Phase ELISA (6). The titres of Ab were related to
the Expected Protection Percentage (EPP) according to the Logist Transformation (7). In addition,
the proportion of individuals with Ab levels compatible with protection (EPP>81%) was
determined in each experimental group.

Detection of IgG1 e IgG2 isotypes against FMDV in vaccinated cattle: IgG1 and IgG2 Ab titres
against FMDV O strain were examined on serum samples from cattle of Trial 2 according to the
method previously described (8).

Detection of antibodies against VIAA and 3D protein: VIAA Ab and anti-3D Ab were examined
on serum samples collected at 35 DPV from cattle of Trial 2 and from pigs at 30 DPV. Both assays
were performed at Virology Institute (CICVyA-INTA) (9,10).
 257

RESULTS
Immune response in vaccinated animals:
Trial 1: Mean group Ab titres against each of the four FMDV strains induced by vaccine containing
saponin (vaccines “B”, “C”, “E”, “F”, “G”) were significantly higher (t student P<0.005) than those
induced by vaccines lacking immunomodulator (vaccines “A” and “D”). Vaccines with saponin
induced an early immune response at 30 DPV with Ab levels equivalent to that obtained at 60 DPV
with formulations lacking saponin. Furthermore, the inclusion of this immunomodulator in
formulations with antigen payload of 9.7 µg/dose, induced Ab levels higher than those obtained
with vaccines including 4 times higher 140S in absence of saponin (data not shown). The
percentage of cattle with protected Ab is shown in Fig 1. Vaccines containing saponin conferred Ab
titres compatible with protection at 30 DPV in the 80-100% of cattle, whereas those lacking saponin
induced protected Ab levels in the 45-55% of the vaccinated cattle.
Trial 2: the percentage of cattle with protective Ab titres is shown in Fig 2. At 35 DPV 100% of the
cattle vaccinated with vaccines including saponin (“H” and “I”) achieved protective Ab, whereas
25-75% of the cattle vaccinated with vaccine lacking saponin (“J”) achieved protective Ab titres
even in the presence of higher antigen mass content.
Trial 3-4: High immune response was also induced by these formulations in pigs and goats (Table
2).
Trial 5:Vaccines containing saponin induced positive immune response in calves in the presence of
high levels of maternal antibodies (Fig 3). Calves vaccinated at 30 days of age showed at 150 DPV
higher Ab titres than non-vaccinated controls. Percentage of calves with protective titres was higher
than 80% at 150 DPV, regardless the age at which vaccination was applied (data not shown).

Detection of IgG1 e IgG2 isotypes against FMDV in vaccinated cattle: IgG1 and IgG2 Ab titres
against FMDV O type are shown in Table 3. High levels of IgG1 and IgG2 were induced by
vaccines containing saponin, whereas low levels of both isotypes were elicited by vaccine lacking
saponin in agreement with the titres of total Ab detected. Vaccine containing saponin and higher
antigen payload (vaccine “H”) induced higher IgG2 than that with lower 140S content (vaccine
“I”).

Detection of antibodies against VIAA and 3D protein: Non detectable Ab against VIAA and 3D
protein were detected in the serum samples assayed.

DISCUSSION
The immunomodulator effect of saponin included in the water phase of water-in-oil vaccines was
evaluated in cattle, pigs and goats.
Oil vaccines with saponin induced 10 times higher total specific Ab levels, for each one of the viral
strains at 30 DPV than formulations lacking saponin. In addition, the inclusion of this
immunomodulator in formulations with reduced antigen payload, elicited at 30 DPV, significantly
higher total antibody protection levels (P<0.005) than those obtained with formulations with high
140S antigen payload, and absence of the immunomodulator. Moreover, the immune response
obtained at 30 DPV with vaccines containing saponin was as high as that achieved at 60 DPV when
vaccines lacking saponin were applied.
No effect on the Ab response was detected with increasing amount of saponin per dose (2, 3 or 6
mg/dose) (Fig 1). It remains to be investigated the immunomodulator effect by using lower amount
of saponin per dose.
The IgG isotype profiles induced in cattle by these vaccines indicated that both IgG1 and IgG2 Ab
titres were higher in formulations with saponin. Both isotypes were also induced in high levels by
oil vaccines including other immunomodulators (Mycobacterium sp and Avridine ) (12).
 258

Correlation between high levels of IgG1 and protection against challenge has been previously
demonstrated in cattle (8).
The superiority of oil vaccines in conferring neutralizing Ab and protection against challenge in
pigs has been well documented (2) Several authors have demonstrated significant correlation
between Ab detected at 30 DPV and protection against challenge in pigs (11, 13, 14). In Trial 3, at
30 DPV high Ab levels were detected. However, no differences were observed between vaccines
lacking and not the immunomodulator, probably due to the high antigen content into both
experimental vaccines.
The immune response in goats with these formulations was evident without causing untoward side
effects. Furthermore, the Ab response was persistent, at 6 months post-vaccination Ab titres against
each of the four vaccine strains were related to EPP above 93% (data not shown).
The results obtaining in calves (Trial 5) added more evidence to the advantages of oil vaccines in
conferring persisted and high immunity in the presence of maternal antibodies.
None of the vaccines used caused neither general nor local untoward side effects.
It is also desirable that immunogens to be used in prophylactic or emergency vaccination lack of
antigenic components that could induce Ab against non-structural proteins that serve as infection
indicators (10,15,16). The non-detectable Ab to 3D and VIAA suggests that even with vaccines
containing high antigen payload, the concentration antigen process did not lead to non-structural
protein concentration.
The adjuvant effect of the formulations containing saponin without causing adverse reactions
recommends its use as an alternative for emergency vaccination and for prophylactic campaigns as
universal vaccine.

Acknowledgements: The authors are grateful to Dr. D.Abaracón for his valuable suggestions and
support and to M. Iglesias, C. Devicenzo and M. Rodríguez (CEVAN) for their technical assistance.

REFERENCES
1-Barei, S et al. (1979). ZblVetB, 26:454-460
2-Cunliffe & Graves (1963) Can J Comp Med, 27: 193-197
3-Doel, T R Rev Sci Tech Off Int Epiz. 1996; 15 (3): 883-911
4-Salt, J; Barnett,P; Dani,P et al. (1998) Vaccine 7: 746-754
5- Späth, E J A., Smitsaart, E N , Casaro, A P E et al. (1995) Vaccine 13, 909-914
6-Robiolo, B, Grigera, P, Periolo, O, et al. (1995) Vaccine14:1346-1352
7- SENASA. Resolución 219/95, Boletín Oficial N° 28125, Disposición Oficial N° 03/96.
8-Capozzo , A V, Periolo, O, Robiolo, B et al. (1997) Vaccine 15: 624-630
9-Alonso Fernández, A; Sondhal, et al. (1984) Serie de Manuales Técnicos N°6 CPFA, Río de
Janeiro, OPS, OMS, 1-3
10-O’Donnell, V K; Boyle, D; Sproat, K et al. (1996) J Vet Diagn Invest 8:143-150
11-Augé de Mello, P; Gomes, I; AFernandez, A et al. (1978) Bltn Centro Panamericano Fiebre
Aftosa 31-3213-19
12-Pérez Filgueira, D M, Wigdorovitz, A, Zamorano, P I et al. (1999) Vaccine 17: 345-352
13-Anderson, E C; Masters, R C y Mowat, G N (1971) Res Vet Sci, 12: 342-350
14-Mc Kercher, P y Bacharach, H L (1976) Can J Comp Med 40:67-74
15-Garland, AM In: Report of the 32nd Sess of the European Commission for the Control of FMD
April 1997
16-Mackay, D, Forsyth, M et al. (1998) Vaccine 5:446-459
 259

Tables and Figures

Table 1a: Animals free of FMDV antibodies. Experimental design and vaccines used.
Trial Species Group N1 Vaccine 140S2 Saponine3 Bleedings (DPV)4
1 Cattle I 9 A 38.8 no 0, 30, 60
II 15 B 38.8 2
III 9 C 38.8 3
IV 9 D 9.7 no
V 15 E 9.7 2
VI 14 F 9.7 3
VII 9 G 9.7 6
2 Cattle I 10 H 30 3 0, 35
II 10 I 15 3
III 8 J 40 no
3 Pigs I 10 K 38 no 0, 30, 60
II 12 L 36.4 3
4 Goats I 10 J 40 no 0, 30
II 10 M 27 3 0, 30, 60
III 10 N 13.5 1.5 0, 30, 60

Table 1b: Calves born from vaccinated dams. Experimental design and vaccine used.

Trial Species Age group* N1 Vaccine 140S2 Saponine3 Bleedings (DPV)4

5 Calves 30 days 10 O 40 3 0, 30, 60, 90, 120, 150
60 days 10 O 40 3
90 days 10 O 40 3

1-Number of animals in each group. 2- Antigen payload (µg of 140S particles/dose of the mixture of the 4 FMDV
strains (O1 Caseros, A79, A87 y C85). 3-Saponin content (mg/dose). 4- Days post-vaccination.
* Five 19-110 day-old calves remained non-vaccinated as control of decay of maternal antibodies.

Table 2: Immune response in pigs and goats following vaccination with polyvalent oil vaccines
containing saponin

Trial Vaccine 140S1 Sapo- ELISA titres (log10) %n 3 EPP4

(species) (ug/ds) nin2 305 60 30 60 30 60
3 K 38 -- 2.32 3.21 100 100 97 97
(pigs) L 36.4 3 2.5 2.71 100 100 97 97
4 J 40 -- 2.14 ND 100 ND 96 ND
(goats) M 27 3 2.15 2.12 100 100 96 95
N 13.5 1.5 2.32 2,45 100 100 97 97

1-Antigen payload (µg of 140S particles/dose of the mixture of the 4 FMDV strains (O1 Caseros, A79, A87 and
C85). 2-Saponin content (mg/dose). 3- Percentage of animals with protective titres (for A79 strain log10> 1,74) 4-
Expected Protection Percentage (EPP) 5- Days post-vaccination. ND: not done
 260

Table 3: IgG1 and IgG2 isotype Ab titres against O type following vaccination with polyvalent
oil vaccines containing saponin (Trial 2)

Vac- 140S1 Sapo ELISA titres (log10)3 IgG1 (log10)4 IgG2 (log10)5
cine (ug/ds) nin2 06 35 0 35 0 35
H 30 3 mg <0.90 2.75 1.12 2.81 0.93 2.81
I 15 3 mg <0.90 2.89 1.07 2.82 0.78 2.1
J 40 -- <0.90 1.92 1.06 1.64 0.8 1.60

1- Antigen payload (µg of 140S particles/dose of the mixture of the 4 FMDV strains (O1 Caseros, A79, A87 and
C85). 2-Saponin content (mg/dose) 3-Total Ab titres against O type. 4-IgG1 Ab titres against O type 5- IgG2 Ab
titres against O type. 6-Days post-vaccination.

Figure 1: Percentage of cattle with protective Ab titres following vaccination with oil vaccines.
“A” y “D”: vaccines lacking saponin. “B”, “C”, “E”, “F” and “G”: vaccines including saponin.
“A”, “B”, and “C”: vaccines containing 38,8 µg 140S /dose.
“D”, “E”, “F” and “G”: vaccines containing 9.7 µg 140S/ dose.
 261

Figure 2: Percentage of cattle with Ab titres compatible with protection following vaccination (35
DPV) with oil vaccines containing and lacking saponin (Trial 2).
 262

Figure 3: Immune response of calves with maternal antibodies after vaccination. Polyvalent oil
vaccine with saponin. Line indicates ELISA antibody titre related with 81% of protection after
challenge (log10 1.84)
 263

Appendix 33
Experimental vaccination
Against FMDV with Immunocomplexes

H.Yadin 1, Dalia Chai 1, A. Bril 1, B. Gelman 1 and M. Amadori 2.

1.Kimron Veterinary Institute, Beit Dagan50250, Israel.

2. Instituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia, Brescia,
Italy.

Introduction

Vaccination against Foot and Mouth Disease is compulsory in Israel for all domestic
ruminants on annual, biannual or triannual basis. The aim of these vaccinations is to
enhance immunity against FMD for preventing huge virus amplification from primary
outbreaks and reduction the extent of secondary outbreaks. The most susceptible
animals on affected premises are the young calves and lambs, which possess maternal
antibodies and are reacting slowly or not at all to vaccination. In addition, the
duration of immunity is often limited in primo-vaccinated animals, which actually
further accrues to the effectiveness of the vaccination campaigns.
This work was based on the hypothesis that administration of antigen in complex
with specific antibody alters the process of B cell memory formation in germinal
centers, thus enhancing the immune response. Preliminary experiments in guinea-pigs
(Amadori et al, 1998) would support such a tenet. Practical challenging this
hypothesis in view of a future improvement of the immune response of young animals
was the aim of this work.

Materials and Methods

Animals - On a commercial fattening farm a group of 16 calves in the age of 3-4

months was divided into 4 subgroups. Each subgroup was vaccinated s.c. With 2 ml
of the corresponding vaccine of the subgroup.
Blood samples for Ab testing were collected just before vaccination day 0 (D 0), at
day 21 (D 21) and day 45(D 45) post vaccination.

Serum - A post vaccination serum was collected from a cow one year after the 4th
shot of a trivalenrt vaccine containing O1 Geshur Isr. 2/85, O1 Manisa, A22 Iran 87
and Asia 1 Shamir. The serum was tested for SN Ab as described in the OIE manual
and stored at –20°C in small aliquots; its SN titer was equal to 1:256.

Vaccines - A commercial vaccine, containing the antigens O1 Geshur

Isr. 2/85 and O1 Manisa in Al (OH) 3 adjuvant, was used. For each subgroup, vaccine
was formulated as follows:
• Group 1 : 9.8 ml aqueous Al(OH)3 vaccine + 200 µl PBS (control).
• Group 2 : 9.8 ml aqueous Al(OH)3 vaccine + 200 µl positive serum (1:50 ratio).
 264

• Group 3 : 9.8 ml aqueous Al(OH)3 vaccine + 100 µl positive serum +100 µl

PBS (1:100 ratio).
• Group 4 : 9.8 ml aqueous Al(OH)3 vaccine + 50 µl Positive serum +150 µl
PBS (1:200 ratio).

Serology - The antibody titers were evaluated by SN test using microplates with
monolayers of secondary pig kidney cells. Each serum sample was titrated in
duplicate against 100 TCID50/ well of O1 Geshur. Results were checked after 48 hours
according to OIE manual.

Results

The results of the SN tests are shown in table 1 and figure 1. There was evidence of a
bell-shaped dose/response effect of the immunocomplexes: a slight reduction of the
Ab response was observed at the 1: 50 ratio; a stronger inhibition was evidenced at the
1:200 ratio with a remarkable heterogenicity: two calves (5522 and 9336) did not
respond to the vaccine, one had a high and one a usual response, with a clear tendency
to a decrease on day 45 post vaccination; a very effective immunization was obtained
instead at the 1:100 ratio with a clear tendency to a further increase of the Ab titers at
day 45 post vaccination. The mean Ab titer of group 3 at day 45-post vaccination was
significantly different from those of the other groups (P<0.05 in variance analysis).
 265

TABLE 1

Ab response (SN titers*) of calves to different FMD vaccine formulations

Group treatment Calf Nr. D 0 vaccination D 21 P. vacc. D 45 P. vacc.

Gr. 1 9342 2.0 5.0 4.0
Conventional 7269 2.5 3.0 2.5
Vaccine 9340 2.0 6.5 5.5
9341 1.5 >7.0 4.0
Mean ± Sd 2.0 ± 0.41 5.4 ± 1.8 4.0 ± 1.2
Gr. 2 4180 2.0 3.5 3.5
Vaccine + 8105 2.5 - -
1/50 positive serum 5503 2.5 4.5 3.0
9326 <1 4.5 3.5
Mean ± Sd 1.75 ± 1.2 4.2 ± 0.58 3.33 ± 0.29
Gr. 3 2873 5.0 >7.0 >9.0
Vaccine +1/100 8106 2.0 3.5 4.0
positive serum 9335 2.0 6.0 5.5
5505 2.0 6.5 >7.0
Mean ± Sd 2.75 ± 1.5 5.75 ± 1.55 6.38 ± 2.14
Gr. 4 9327 2.0 4.5 3.0
Vaccine +1/200 9328 <1 5.5 4.0
positive serum 5522 5.0 3.0 4.0
9336 4.0 2.5 2.0
Mean ± Sd 2.75 ± 2.2 3.88 ± 1.38 3.23 ± 0.96

* SN titres were expressed as Log2 .

 266

FIGURE 1

Kinetics of Ab response in FMD-vaccinated calves

5
Gr. 1
4
Gr. 2
Gr. 3
3
Gr. 4
2
Lg 2

0
D 0 D. 21 PV D. 45 PV

Discussion

The immune response to a vaccine injected parenterally usually starts with the step
wise capture of antigen by dendritic cells, which transport it to the local draining
lymph node. This process goes along with a gradual change of the main features of
dendritic cells under the influence of Ag capture and of the local inflammatory
environment: they stop the intense pinocytotic activity and are now able to achieve a
high and stable expression of Ag/MHC II and co-stimulatory molecules on the cell
surface. Under such conditions they can now adequately attract T cells by chemokines
once arrived in the lymph node paracortex and stimulate them. Instead, free or
released Ag is usually deposited on specialised Follicular Dendritic Cells (FDCs) in
the form of Ag/Ab/C'3 complexes. In this case, the "natural" antibodies probably play
a role in naive animals, as opposed to specific Ig in primed animals. B cells recognize
 267

their target on such FdCs and become activated. Thus, they can secrete chemokines,
which attract the nearby activated T cells from the paracortex and give rise to a
cognate mutual recognition between B and T cells and to a Th function leading to the
maturation and differentiation of B cells; a part of these cells become Ab-secreting
with/without change of the expressed Ab isotype and mostly settle in the bone
marrow; another part is submitted to the germinal centre reaction, leading to isotype
switch and somatic hypermutation; this leads in turn to the appearance of memory B
cells with increased Ab affinity. The administration of Ag in the form of Ag/Ab
immunocomplexes is likely to interfere with the speficic phase of Ag recognition by B
cells; the size, type and stability of the immunocomplexes deposited on FDCs would
be probably different; as a consequence, there could be a longer persistence of the
germinal centre reaction; this could lead to the selection of high-affinity B cell clones
able to persist under condition of low Ag concentration, or to perform a long-term Ab
secretion in the bone marrow, as previously reported in other experimental models. A
direct role of immunocomplexes in the regulation of the GC reaction has been
demonstrated in a murine model (Nie X. et al. 1997): as compared to the control Ag,
there was a GC reaction characterised by a wider array of Ig populations and a
higher frequency of point mutations/Vh gene.
Notice however that the interactions of Ag and Ab may lead to quite different results
in the immunization; on the one hand, it is widely known that pre-existing maternal
antibody are quite inhibitory; the same holds true of Ab injected before the
administration of a rabies vaccine in mice (Schumacher C.L. et al, Vaccine), whereby
the magnitude and the duration of such suppression are related to concentration and
half-life of Ig, respectively. The inhibiting role of pre-formed antibodies has been also
indicated in models of mucosal immunization against Rotavirus in calves (Van Zaane
D., 1986); on the contrary an immunocomplex Rotavirus vaccine administered
parenterally (Hess R.G. et al., 1982) and an immunocomplex Hepatitis B vaccine
(McCluskie MJ et al, 1998) administered intranasally can be quite effective. It seems
therefore that the presence of large amounts of pre-formed antibodies are often
inhibiting, whereas a small amount of antibody complexed with antigen is generally
instrumental in generating a vigorous Ab response. A preferential interaction with the
low affinity Fc gammaRII (CD32) in the case of a large excess of pre-formed
antibodies could partly explain this apparent paradox, since Fc gammaRII-knock-out
mice mount a much more vigorous response to Ag/Ab complexes (Wernersson,
1999); notice however that F(ab’)2 fragments of antibodies can also block the Ab
response to sheep erythrocytes (Karlsson MC, 1999). The Ag/Ab ratios in the vaccine
are another crucial factor for the final outcome of the immunising procedure; thus, for
instance, a slight Ab excess was reported in the Rotavirus model (Hess et al.). Such a
tenet would be confirmed by our study, in which different ratios were tested at the
same time. The 1:100 ratio, which gave excellent results, might correspond the slight
Ab excess described by Hess et al. (1982) in the Rotavirus model.
Please notice that the antiserum was added to antigens adsorbed onto aluminium
hydroxide; this could affect the type, size and form of the resulting
immunocomplexes. The duration of the Ab response in the 1:100 group is unusual for
aqueous vaccines in primo-vaccinated animals: this is reminiscent of the Ab kinetic
after injection of W/O vaccines and points to a fundamental adjuvanticity of the
Ag/Ab complexes: this has been confirmed in a murine model whereby toxins fused
to S. aureus protein A and linked to antibodies to surface components of APC can
induce an Ab response without the need for adjuvants (Leonetti M, 1998).
 268

In the field of FMD, the technology of immunocomplexes could be possibly

exploited to achieve three goals:
1. A longer duration of the immunity conferred by aqueous vaccines.
2. A wider protection against heterologous strains, as previously suggested (Amadori
M, FAO, 1998).
3. An effective vaccination of young animals vìs-a-vìs maternal antibodies; this
mighy be the case of calf 2873 (see table 1).

This latter possibility is of paramount importance in the areas of prophylactic

vaccination characterised by high infectious pressure, where the highest vaccination
coverage is badly needed and repeated vaccinations of livestock are impractical; in
this respect, the use of immunocomplexes could complement that of oil vaccines,
which are less affected by maternally-derived antibody ( Spath et al, 1995 ).

Further investigations are needed to understand the correlation between type of

immunocomplexes and stimulatory vs inhibiting effect on the immune response in the
FMD field; in this respect, the guinea-pig model (Amadori M. 1998) could actually
provide a certain degree of prediction.

Acknowledgements
The skilful technical assistance of Mrs. Ida Reda is gratefully acknowledged.

References
Amadori, M. 1998: Influence of FMD vaccine potency and formulation on the
immune response to heterologous FMDV strains. European Commission for the
Control of Foot and mouth Disease, session of the Research Group of the standing
Technical Committee, Alderhot, UK . 14-18 September 1998, 261-265.

Hess, R.G., Bachmann,P.A., Eichhorn, W., Frahm, K., and Plank, P., 1982:
Stimulierung der laktogenen Immunitat des Rindes gegenuber Rotavirusinfekionen.
Fortsch. Vet. Med., 35,103-108.

Karlsson, M.C., Werersson, S., Diaz de Stahl, T., Gustavsson, S., Heyman, B., 1999:
Efficient IgG-mediated suppression of primary antibody responses in Fcgamma
receptor-deficient mice. Proc. Natl. Acad. Sci. USA. 2; 96(5); 2224-2249.

Leonetti, M., Thai, R., Cotton, J., Leroy, S., drevet, P., Ducancel, F., Boulain, J.C.,
Menez, A., 1998: Increasing immunogenicity of antigens fused to Ig-binding proteins
by cell surface targeting. J. Immunol. 160(8);3820-3827.

McCluskie, M.J., Wen Ym, Di Q, Davis HL., 1998: Immunization against hepatitis B
virus by mucosal administration of antigen-antibody complexes. Viral. Immunol.
11(4);245-252.

Nie, X., Basu, S., and Cerny, J., 1997: Immunization with immune complex alters the
reperoire of antigen-reactive <bcells in the germinal centers. Eur. J. Immunol.
27;3517-3525.
 269

Schumacher, C.L. Ertl, HC., Koprowski, H., Dietzschold, B., 1992: Inhibition of
immune responses against rabies virus by monoclonal antibodies directed against
rabies virus antigens. Vaccine;10(11);754-760.

Spath, E.,J., Smitsaart, E., Casaro, AP., Fodevila, N., Fernandez, F., Leunda, Mr.,
Compaired, D., Buffarini, M., Pessi, H., 1995: Immune response of calves to Foot-
and-Mouth disease virus vaccine emulsified with oil adjuvant. Strategies of
vaccination. Vaccine, 13(10); 909-914.

Van Zaaane, D., Ijzerman, J., De Leeuw, P.W., 1986: Intestinal antibody response
after vaccination and infection with rotavirus of calves fed colostrum with or without
rota virus antibodies. Vet. Immunol. Immunopathol. 11(1);45-63.

Wernersson, S., Karlsson, M.C., Dahlstrom, J., Mattsson, r., Verbeek, J.S.,
Heyman,B., 1999: Igg-mediated enhacement of antibody resposes is low in Fc
receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.
J. Immunol. 15;163(2);618-622.
 270

Appendix 34

Formaldehyde enhances BEI-inactivation rates of Foot-and-

Mouth Disease (FMD) virus by at least a ten-fold
Simon J. Barteling and Nazeem I. Cassim, Onderstepoort Veterinary Institute, Private
Bag X6, Onderstepoort 0110, Republic of South Africa

Summary

For FMD vaccine production, in general, inactivation of the FMD virus is carried out with
binary ethylenimine. Under optimal conditions, inactivation rates are in the range of 0.5 – 1.0
log10 per hour. In general, 48 hours of inactivation is required.
Formaldehyde, the ‘classical’ inactivating agent, inactivates at a rate of 0.3 logs per hour only.
Therefore, a significant contribution of formaldehyde to the inactivation of BEI can hardly be
expected. However, if formaldehyde is added at the start of the BEI-inactivation process,
inactivation rates are augmented to 2 – 3.5 logs per hour. This enables overnight inactivation
with even higher safety levels of the vaccines.
Also, it is known that formaldehyde cross-links viral proteins which stabilizes the antigen. In
general, some 146 S antigen is lost during inactivation with BEI. However, for all 5 SAT
vaccine strains no antigen losses were observed after the inactivation with the BEI-FA
combination. Even in case of the labile SAT2 Zim 7/83 strain, that deteriorates to a great
extent if inactivation is carried out with BEI only, no reduction in 146 S yields was observed
with the combination of inactivating agents.
The consequences for FMD vaccine production (antigen yields), the stability of the antigens
(and of vaccines), and the endurance of the immune response will be discussed.

Introduction

Virus inactivation and safety tests are the most critical steps in the production of inactivated
vaccines. In the case of foot-and-mouth disease (FMD) vaccines in particular, guaranteed
safety is essential because any occurrence of the disease will have great economic
consequences e.g. by a blockade of all export trade of animals and animal products.
Classical FMD vaccines as developed by Vallee, Schmidt, and Waldmann (Waldmann et
al.,1937) were inactivated by formaldehyde (FA). The “nature” virus, obtained from diseased
cattle was first adsorbed to Al(OH)3 gel and then inactivated with 0.02 % FA. Safety of
vaccines could at first only be tested on cattle tongue and later, in addition, in suckling mice.
When titration in tissue culture became possible, inactivation plots could not be made because
of the presence of the (toxic) Al(OH)3. Several studies (with non-adsorbed virus) showed that
at the FA concentration prescribed by Waldmann, inactivation plots were not linear and often
showed “tailing off”, which may cause incomplete inactivation (Wesslen and Dinter, 1957;
Graves, 1963; Barteling et al.,1983; Barteling and Woortmeyer, 1984). Consequently, large
vaccine batches may contain some surviving virus. However, in the field the vaccines
performed quite well, without causing outbreaks that clearly could be associated with
vaccination campaigns. However, in 1981 it was shown by King et al. that an outbreak strain
isolated in the UK, originating from Brittany (France), could well be vaccine related. By
comparing nucleotide sequences of outbreak strains and vaccine strains it was also shown by
 271

Beck and Strohmaier (1987) that the sequences of European outbreak strains were very
similar to those of vaccine strains suggesting that most of the outbreaks in Europe very likely
were caused by improperly inactivated vaccines or by viruses that had been escaped from
vaccine production laboratories.
Linear inactivation plots were obtained with acetyl ethylenimine (AEI) and other aziridins
(Brown et al.,1963; Bahnemann 1975) . Although it was shown that, under proper conditions,
FA-inactivation plots - of Al(OH)3-adsorbed virus - were linear as well (Barteling and
Woortmeyer, 1984), inactivation with aziridins became the method of choice. Binary
ethylenimine (BEI) is used in particular, because this method – developed by Bahnemann
(1975, 1990) – circumvents the direct handling of the very toxic other aziridins.
However, Aziridins lack the cross-linking and fixation activity of formaldehyde that made the
old-type Frenkel vaccines stable for 5 years and more (non-published observation).
Inactivation with BEI often results in a loss of some 10 – 30 % of the 146 S antigen
(observation at OVI). Therefore, for labile vaccine strains (like SAT2 Zim 7/83), the antigen
is first (partly) inactivated with BEI, fixed with formaldehyde, and then inactivation is
completed by the addition of another portion of BEI (Rowlands et al. 1972, Mowat 1973,
Rweyemamu 1989).
When studying the effect of the simultaneous addition of the two inactivants, we detected a
synergistic effect of FA to the inactivation by BEI. For all 5 (SAT) vaccine strains produced
at Onderstepoort, inactivation rates were enhanced by more than a 10-fold. Inactivation
reached a safe level within 8 hours and no losses in 146 S were observed. A polyvalent
vaccine prepared from the 5 BEI-FA-inactivated antigens induced (in cattle) satisfying levels
of neutralizing antibodies and of protection against one of the strains.

Materials and methods

- Virus antigen production and virus titration

Because it was difficult to mimic all aspects of large scale production on the small scale, the
new inactivation method was tested for all five vaccine strains on the production scale. This
was carried out during working weeks of 4 days when regular production – using only BEI –
was not possible.
The strains routinely produced at Onderstepoort are: SAT1-SAR9/81; SAT1-KNP196/91-1;
SAT2-KNP19/89-2; SAT2-ZIM7/83; and SAT3-KNP10/90-3. Virus was produced on roller
bottles as described by Panina et al. (1976) according to a strict weekly schedule.

- Inactivation, concentration and storage of the antigen

Clarified chloroform-treated virus culture was brought to a temperature of 30ºC and then
inactivated with 1 mM of BEI (Bahnemann 1990) or with a combination of 1mM BEI and
0.04% FA (BEI-FA).
With BEI only, samples were taken every hour during the first 6 hours and after 24 and
48hours. After 24 hours of inactivation the same quantity of BEI was added according to the
same procedure as before. Then, the inactivation mixture was transferred into a second
inactivation tank where inactivation was continued. After 48 hours inactivation was stopped
by the addition 1/10 vol. of a 20 % Na-thiosulfate solution.
Samples were collected in 1/10 volume Na-thiosulfate (to stop the inactivation by BEI) and
1/10 vol. of calf serum, chilled on ice, diluted for titration and/or stored away at – 70ºC for
back-up (no differences were observed between immediate titration and titration of the frozen
samples). Because the BEI-FA combination inactivates FMD very rapidly (> 2 log10 ID50 per
hour), samples were taken every 20 minutes during the first 3 hours. Sampling was also
 272

carried out at 6 hours, when the inactivating mixture was transferred into a second inactivation
tank, and at 24 hours, when the inactivation was stopped by the addition of Na-thiosulphate.
At the end of the inactivation (and for the FA-BEI also at 6 hours) a large sample of 200 ml
was taken for an additional safety control on BHK- and IBRS-roller bottles with 2 subsequent
blind passages each after 48 hours (modified after Anderson et al. 1970). The inactivated
antigen was chilled to 8ºC and then transferred to a cold room where it was concentrated by
ultra-filtration. The concentrate was stored away above liquid nitrogen.

- Preparation and testing of (sample) vaccines

An experimental vaccine was prepared from a mixture of all five FA-BEI-inactivated vaccine
strains. At the time of formulation, the (computerized) 146 S analysing system valued antigen
concentrations approximately 4 times too high. Unfortunately, this was detected only when
the vaccine was already injected in cattle. Therefore, the vaccine contained per (3 ml) dose 0.4
µg (approx.) of each of the vaccine strains with the exception of SAT3 KNP90/3 of which 0.8
µg was incorporated.
A quarter of a vaccine dose (0.75 ml) was injected intramuscularly in 5 cattle. Two cattle were
left as controls. Serum samples were taken weekly. At 2 weeks p.v. the serum samples were
immediately tested in a micro-neutralization test. At 3 weeks p.v. the cattle were challenged
with the strain showing the lowest antibody levels at 2 weeks p.v.(SAT 1 KNP 196/91-1).
Challenge was carried out by injecting 10.000 ID50 intra-dermolingually.
Cattle were observed for clinical disease for the following 8 days when they were slaughtered
and post mortem inspection was carried out for signs of clinical disease.

Results and discussion

A representative inactivation plot obtained with BEI is given in fig 1a.

In our experience, with BEI alone, inactivation rates vary between 0.4 and 1.0 log per hour
(see also Bahnemann, 1990).
At OVI inactivation is carried out for 48 hours. At 24 hours a second portion of BEI is added.
Thus, even at the lowest inactivation rates, sufficient safety can be guaranteed. The BEI-FA
plots obtained with the 5 vaccine strains that are routinely produced at OVI are given in
fig1(b-f). The inactivation rates were varying from 2.0 (SAT1-SAR9/81, fig 1b) to more than
3 logs per hour (SAT2-ZIM 7/83 and SAT2 KNP-19/89/2, fig 1d and 1e resp., table 1) Thus,
sometimes inactivation is over a 100-fold faster if FA is added.
It is difficult to say what causes this synergistic effect. Under optimal conditions, FA alone
inactivates at a rate of approx. 0.3 log/hr only (Barteling, 1984). Thus hardly any addition is to
be expected to the much faster inactivation of BEI. Aziridins attacks the nucleic acid. It seems
that the cross-linking action of FA makes the virus particle more accessible for the BEI
enabling a faster attack of the nucleic acid.
From these graphs it is clear that by the FA-BEI combination linear plots were obtained and
virus titers were found to be reduced at least more than 2 logs per hour. Thus already within 8
hours sufficient inactivation was reached for acceptable safety. In accordance, no surviving
virus could be detected in a large (200 ml) sample taken after 6 hours, as was the case for the
24 hr samples. It should be noted that in the micro-titer system undiluted and 10-fold diluted
samples were causing a cpe-like effect but this was due to the toxicity of the formaldehyde
and no virus could be propagated from these cups.
Thus inactivation can be carried out within the time span of a working day or just overnight in
stead of the 48 hours required for the inactivation with BEI alone. This gives greater
flexibility in weekly production schedules. E.g. in Onderstepoort a production batch could just
 273

be completed within 5 working days. Now, the rapid inactivation by FA-BEI allows
production within 4 days and production is no longer hampered by e.g. national holidays.
With BEI alone, antigen yields (of SAT-strains) were often reduced from 10 to 30 percent
during the 48 hours of inactivation. No reduction in 146 S antigen concentration was observed
after the 24 hr of inactivation with FA-BEI. Where conditions were otherwise identical, a
reduction with approximately half that observed with BEI could be expected. The optimal
yields are probably due to fixation of the antigen by the cross-linking action of FA (Barteling,
1984).
Considering the low quantities of antigen that were incorporated in the vaccine, the vaccine
performed quite well. At 2 weeks p.v., the lowest neutralising antibody response was against
SAT 1 KNP 91/1(with a mean titre of 1.8 log) and, therefore, this strain was selected as
challenge strain (table 2). Two animals were protected and one was partly protected,
indicating a protection level of approximately 50 % (1 PD 50). Because the injected vaccine
dose contained approximately 0.2 µg per FMD virus type, these results indicate that (for the
weakest antigen) per µg a protection level of approximately 5 PD50 can be expected which –
in our experience – is quite good.
Stability of the 146 S antigen is an important parameter in the selection of new vaccine strain.
By the cross-linking action of FA, which stabilizes the 146 S antigen, this parameter becomes
less critical, and, therefore the BEI-FA inactivation method will make the rapid introduction
of new vaccine strains more easy.
Because of the fixation of the antigen by the cross-linking action of FA, we expect the
vaccines to be of superior stability. This is particularly important for developing countries
(e.g. in Africa), where maintenance of cold chain conditions is not always possible. Whether
these vaccines induce a relatively long lasting immune response (as for the Frenkel vaccines)
remains to be confirmed.

Acknowledgement

We thank Mrs Erika Kirkbride, Mrs Brenda Botha, Mr. Anton Barnard, Mr. Philemon Dolo,
Mr. Lourens VanStaaden, and Dr. Comfort Piri greatly for their technical assistance and for
performing the animal experiments.

References

1.Waldmann O, Pyl G, Hobohm KO, Mohlmann H: Die Entwicklung des Riemser Adsorbatimpfstoffes gegen
Maul- und Klauenseuche und seine Herstellung. Zbl Bakt I Orig 148: 1, 1941
2. Graves JH: Formaldehyde inactivation of foot-and-mouth disease virus as applied to vaccine preparation.
Am J Vet Res 24: 1131, 1963
3. Weslen, T. and Dinter, Z. Then inactivation of foot-and-mouth disease virus by formali. Arch. Ges.
Virusforsch. 1957, 7, 394-4014.
4. Barteling SJ, Woortmeijer R, Visser N: Innocuity testing of foot-and-mouth disease vaccines. I.
Formaldehyde-inactivated alhydrogel vaccines. J Biol. Stand. 1983 11: 297,
5. Barteling SJ, Woortmeijer R: Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the
preparation of safe vaccine. Arch Virol 80: 103,
6. King AMQ, Underwood BO, McCahon D, Newman JWI, Brown F: Biochemical identification of viruses
causing the 1981 outbreaks of foot-and-mouth disease in the U.K. Nature 293: 479, 1981
7. Beck E, Strohmaier K: Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence
determination. J Virol 61: 1621,1987
8. Brown F, Hyslop NSG, Crick J, Morrow AW: The use of acetyl-ethyleneimine in the production of
inactivated foot-and-mouth disease vaccines. J Hyg (Camb) 1963b, 61: 337
9. Bahnemann HG: Binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application
for vaccine production. Arch Virol., 1975, 47: 47-56
 274

10. Bahnemann, H.G.: “Inactivation of viral antigens for vaccine preparation with particular reference to the
application of binary ethylenimine. 1999, Vaccine 8, 299—303
11. Rowlands et al. () – Stabilizing the immunizing antigen of foot-and –mouth disease virus by fixation with
formaldehyde. Arch. Ges. Virusforsch. 1972, 39, 274-283;
12. Mowat, G.N. Enhancement of immunizing potency of foot-and-mouth disease vaccine for cattle by
treatment of the antigen with formaldehyde. Arch.ges.Virusforsch. 1973, 41, 365-370
13. M M Rweyemamu et al. Effect of formaldehyde (FA) and binary ethyleneimine (BEI) on the integrity of
foot-and-mouth disease virus capsid. Rev. sci. tech. Off. Int. Epiz. 1989 8, 747-767
14. Panina, G.F. Animal cell culture and FMD virus production in a large scale insuatrial roller bottle plant.
Proc. First Gen. Meet. Eur. Soc. Anim. Cell Techn. (eds. Spier, R. and Van Wezel A.L.) RIVM, Bilthoven,
The Netherlands, 1976, 4

Table 1: Inactivation rates (with BEI-FA) of five SAT vaccine strains.

Strain Inactivation rate

SAT 1-SAR 9/81 2.0 log/hr

SAT 1- KNP 91/1 2.5 log/hr

SAT 2 – ZIM 7/83 3.1 log/hr

SAT 2 – KNP 89/2 3.3 log/hr

SAT 3 – KNP 10/90-3 2.2 log/hr

Table 2: Mean virus neutralisation titres at 2 weeks p.v and challenge results. The animals
were vaccinated with ¼ of a dose containing 0.4µg of the SAT 1 and SAT 2 strains and 0.8µg
of SAT 3.

Strain Mean titre

SAT 1 KNP 196/91/1 1.8

SAT 1 SAR 9/81/1 2.4

SAT 2 KNP 19/89/2 2.0

Sat 2 Zim 7/83/2 2.3

SAT 3 KNP 10/90/3 2.3

Challenge (at 3 w. p.v.) was carried out with SAT 1 KNP 196/91-1. Out of 5 animals 2 were protected and 1 was
partly protected (1 lesion).
 275

Text fig 1

Inactivation plots obtained with BEI alone (a) for one of the SAT vaccine strains and with
BEI-FA (b – f) for all 5 vaccine strains produced at OVI.

a) SAT1-SAR 9/81 - BEI b) SAT1-SAR 9/81 -BEI-FA

10 10
5 5
Titre log 10

Titre log
0 0

10
-5 -5
-10 -10
-15 -15
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)

c) SAT 1 - KNP 196/91-1 BEI-FA d) SAT 2 - ZIM 7/83 BEI-FA

10 10
5 5
Titre log 10
Titre log 10

0 0
-5 -5
-10 -10
-15 -15
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)

e) SAT 2 - KNP 19/89-2 BEI-FA f) SAT 3 - KNP 10/90-3 BEI-FA

10 10
5 5
Titre log 10
Titre log 10

0 0
-5 -5
-10 -10
-15 -15
0 5 10 15 20 25 0 5 10 15 20 25
Time (h) Time (h)
 276
Appendix 35

Validating the efficacy of emergency foot-and-mouth disease vaccines in sheep following

virus challenge using the non-structural polyprotein 3ABC MAT-ELISA

Paul Barnett, Morag Forsyth and Sarah Cox

Institute for Animal Health, Pirbright Laboratory,

Ash Road, Pirbright, Surrey GU24 0NF, U.K.

Summary

Recent studies, using sheep, have confirmed the rapidity of protective immunity following
vaccination with high potency emergency vaccines. However, because of the often inapparent
nature of the disease in this species much reliance was placed on the isolation of virus from blood
or oesophageal-pharyngeal (O-P) fluid samples as a measure of the vaccines protective ability,
rather than on absence of clinical signs. As an extension to these experiments we have used the
3ABC MAT-ELISA to detect non-structural protein antibody responses and an isotype specific
ELISA to detect local FMD specific IgA as tools for confirming viral replication following
challenge. Results indicate that besides discriminating infected from uninfected sheep, the 3ABC
MAT-ELISA may also be a useful alternative to the extensive sampling regime used to determine
the protection conferred by emergency FMD vaccines under challenge conditions. Detection of
local FMD specific IgA, however, was unreliable in identifying infected from vaccinated
animals.

Introduction

In recent years the response of animals to non-structural proteins (NSP) has been shown to be a
reliable indicator of FMD infection in host species and a means of differentiating between
vaccinated and infected livestock (Berger et al., 1990; Bergmann et al., 1993; De Diego et al.,
1997; Lubroth and Brown. 1995; Meyer et al., 1997; Neitzert et al., 1991; Rodriguez et al.,
1994;Silberstein et al., 1997) regardless of serotype. This has particular relevance to disease
control policy whereby sub-clinically infected vaccinated animals can be identified in order to
eliminate the potential spread of disease from so-called ‘carrier’ animals. Of the individual non-
structural proteins there is now general agreement that the detection of antibody to the
polyprotein 3ABC is the most specific, sensitive and reliable single indicator of previous
infection with FMD virus and discriminates regardless of source of vaccine, antigen
concentration, adjuvant or the number of applied immunisations (Mackay, 1998a). Although a
variety of assay methods using this non-structural protein have been explored, the 3ABC
monoclonal antibody trapping–ELISA (MAT-ELISA) has found particular favour, being a safe
and reproducible test that is ideally suited for large scale serological surveys (Diego et al., 1997;
Brocchi et al., 1998).

The accurate diagnosis of infection with FMDV is of great importance for both control and
eradication campaigns in FMD endemic areas and as a supportive measure to the stamping out
policy in FMD-free areas. Additionally, the ability to reliably detect infection in the face of
vaccination, particularly in species such as sheep in which the disease is often mild or vague
(Barnett and Cox, 1999), offers a valuable tool for examining the efficacy of emergency FMD
vaccines and their ability to confer sterile immunity.
 277
Oil and aqueous emergency vaccine formulations, produced at the International Vaccine Bank
(IVB) at Pirbright, have been shown to protect sheep against challenge with homologous FMDV
as early as 3 days post vaccination (Cox et al., 1999). For some virus strains, much reliance was
placed on the isolation or non isolation of virus from heparinised blood and oesophageal-
pharyngeal (OP) fluid samples as a measure of the vaccines efficacy. The importance of the
interval between vaccination and challenge and the level of viral replication in the oropharynx
was later substantiated along with the equal efficiency of the two adjuvant formulations used
(Cox et al., 1998).

As an extension to these studies we have examined responses against the non-structural protein
3ABC of FMD using the 3ABC MAT- ELISA with sheep sera samples from these early
protection trials, in which protection had previously been assessed by virus isolation in the
absence of clinical disease. Additionally, we also examined the levels of IgA in O-P fluid
samples as studies in cattle (Salt et al.,1996) suggested that the local production of FMD specific
IgA may be a useful differentiator of vaccinated and/or recovered convalescent cattle and
persistently infected carriers.

Materials and methods

3ABC MAT- ELISA

Sera (0 days post vaccination, day of challenge and 28 days post challenge) from sheep
vaccinated at various days pre-challenge with either FMDV antigen Asia1 India, formulated as a
water-in-oil-in-water (W\O\W) emulsion with Montanide ISA 206 (Seppic, Paris) or C1
Oberbayern, formulated as either W\O\W emulsion in Montanide ISA 206 or Al(OH)3/saponin
vaccine were assayed by ELISA for antibodies to the non-structural protein 3ABC. The assay
used to detect antibody to the non-structural proteins (NSPs) of foot-and-mouth disease (FMD)
virus was a monoclonal antibody trapping-ELISA (MAT-ELISA). The antigen was the poly
protein 3ABC, which had been expressed as a fusion protein attached to the carrier protein
glutathione S-transferase (GST) in E. coli. The GST-3ABC polyprotein was trapped by a
monoclonal antibody (Mab) to NS protein 3A which had been coated onto ELISA plates
overnight. Dilutions of the test sera were allowed to react, and antibody to GST-3ABC detected
using a horseradish peroxidase-conjugated anti-species antiserum raised in rabbits (DAKO).
Three selected control sera were included in the test, a strong positive (P1), a borderline positive
(P2) and a negative (N1) and results were expressed as optical densities (OD) or as a test to
positive ratio (T/P) based on the T/P of P1 being 1.00 (this was found to minimise plate to plate
and day to day variation). Samples were considered positive if OD’s and T/P’s were greater than
0.2.

Vaccination and challenge

The experimental protocol used for the vaccination of sheep and their subsequent challenge has
been previously reported (Cox et al., 1999). Briefly, all vaccines were administered as a 1.0 ml
volume (equivalent to half of a bovine dose) by the intramuscular (W\O\W vaccine) or
subcutaneously (Al(OH)3/saponin vaccine) route. Vaccinations were staggered to allow
simultaneous airborne challenge from infected donor pigs. Groups of non-vaccinated but
challenged control sheep were housed in separate boxes. Two susceptible in-contact sheep were
also housed with each group of challenged sheep. Sheep were examined daily for clinical signs of
FMD and pyrexia. Heparinised blood and O-P fluid samples were collected regularly and used
for the isolation of virus by the inoculation of primary calf thyroid (BTy) cells (Ferris and
Dawson, 1988).
 278

Isotype specific IgA antibody ELISA

Isotype specific IgA antibody responses in O-P fluid samples were measured by ELISA (IDAS)
as described by Salt et al. (1996) except that the test was performed as a spot test and the
sensitivity of the test improved by performing repetitive sample incubations at 37oC for 1 hour on
four consecutive occasions. Samples were considered positive or negative using a 0.4 OD cut-off
which had been previously determined using O-P fluid samples from known negative sheep.

Results

Tables 1-3 summarise the clinical data, O-P fluid virus status, 3 ABC MAT-ELISA and IgA
results for each animal. All animals exhibiting clinical signs of FMD and or viraemia were
detected positive by the 3ABC MAT-ELISA at 28 days post challenge. Samples from animals in
which no clinical signs were evident but where virus was detected from O-P fluid samples on two
or more occasions from 4 days post challenge also gave a positive result in the 3ABC MAT-
ELISA except for sheep TO77. One non vaccinated sheep (TG32) which showed no clinical
signs, viraemia or virus from O-P fluid samples but was shown to have FMDV neutralising
antibody by microneutralisation assay (data not shown) also gave a positive result in the 3ABC
MAT-ELISA.

The detection of IgA was more variable and frequently no local IgA was identified in animals
which had been shown to be virus positive for FMDV.

TABLE 1: OUTCOME OF CHALLENGE OF SHEEP VACCINATED WITH MONTANIDE ISA 206 W/O/W FORMULATED FMD
TYPE ASIA 1 INDIA VACCINE PRIOR TO CHALLENGE WITH HOMOLOGOUS FMDV.
ANIMAL DAYS VACC. CLINICAL SIGNS/ OP VIRUS 3ABC MAT ELISA IgA IDAS ELISA
PRIOR TO VIRAEMIA
CHALL. 0DPV DC 28DPC 4DPC 24/28PC
SV 17 10 +a 0.07b -0.02 0.76c -d +e
SV 18 -f NSg 0.04 0.01 NS -
SV 19 + 0.21 0.17 0.29 - +
SV 20* h - NS 0,03 0.06 - -
SV 41* L,P,V i + NS 0.03 1.74 - +
SV 42 6 + 0.01 0.00 0.66 - +
SV 43 - NS 0.01 0.01 - -
SV 44 - 0.00 0.01 -0.01 - -
SV 45* V + NS -0.01 0.95 - -
SV 46* L,P,V + NS -0.01 0.80 - +
SV 47 4 P - 0.00 -0.02 0.04 - -
SV 48 P - 0.02 0.01 0.02 - -
SV 49 P - 0.01 0.01 0.02 - -
SV 50* - NS 0.02 0.01 +/-j -
SV 51* p - NS 0.00 0.00 - -
SV 52 3 - 0.02 0.12 0.00 - -
SV 53 - 0.00 0.01 0.08 - -
SV 54 P - 0.01 0.01 0.17 - -
SV 55* P - NS 0.01 0.01 - -
SV 56* P,D,I k + NS 0.01 1.31 - -
SV 57 0 P,V + NS 0.01 0.69 - +
SV 58 V + NS 0.00 1.02 - -
SV 59 P,V + NS 0.01 0.68 - +
SV 60* - NS 0.01 0.02 - -
SV 61* - NS 0.00 0.00 - -
a
Virus detected in oesophageal-pharyngeal sample on two or more occasions from 4 days post challenge. b OD. c OD > 0.2 – samples positive
d
No IgA detected. e IgA detected. f No virus detected. g No sample. h In-contact. i L-lame, P-pyrexia, V-viraemia. j borderline positive. k D-diarrhoea, I-
inappetance

TABLE 2: OUTCOME OF CHALLENGE OF SHEEP VACCINATED WITH AQUEOUS FMDV TYPE C1 OBERBAYERN VACCINE
 279
PRIOR TO CHALLENGE WITH HOMOLOGOUS FMDV.

ANIMAL DAYS VACC. CLINICAL SIGNS/ OP VIRUS 3ABC MAT ELISA IgA IDAS ELISA
PRIOR TO VIRAEMIA
CHALL. 0DPV DC 28DPC 2DPC 27DPC
TG 18 11 -a 0.00b 0.00 -0.01 -c -
TG 19 - 0.01 0.00 0.01 - +d
TG 20 - 0.00 0.00 0.00 - -
TG 40*e Pf - NSg 0.00 0.00 - -
TG 41* P - NS -0.01 -0.1 - -
TG 21 7 p - 0.00 0.03 0.01 - +
TG 22 - 0.03 0.03 0.03 - -
TG 23 - 0.01 0.03 0.03 - -
TG 34* - NS 0.01 0.01 - -
TG 39* P - NS 0.00 0.00 - -
TG 24 5 p - 0.00 0.00 0.00 - -
TG 25 p +h -0.01 -0.01 0.36i - -
TG 26 p - 0.00 -0.01 -0.02 - -
TG 37* - NS -0.03 -0.01 - -
TG 42* P - NS -0.04 0.01 - -
TG 27 4 - 0.00 0.00 0.03 +/-j +/-
TG 28 - 0.01 0.01 0.12 - -
TG 29 - 0.05 0.00 0.13 - -
TG 36* - NS 0.01 0.00 - -
TG 38* - NS 0.00 -0.01 - -
TG 30 0 FL,P,Vk + NS 0.00 1.87 - -
TG 31 FL,P,V + NS 0.06 1.87 - +
TG 32 - NS 0.07 0.39 - -
TG 33* FL,P,V - NS 0.06 1.63 - +
TG 35* FL,P,V - NS 0.07 1.69 - -
a
No virus detected. b OD. c No IgA detected. d IgA detected. e In-contact. f Pyrexia. g No sample. h Virus detected in oesophageal-pharyngeal
sample on two or more occasions from 4 days post challenge . i OD >0.2 – sample positive j Borderline positive. k FL-foot lesions, P-pyrexia,
V-viraemia

TABLE 3: OUTCOME OF CHALLENGE OF SHEEP VACCINATED WITH MONTANIDE ISA 206 W/O/W FORMULATED FMDV
TYPE C1 OBERBAYERN VACCINE PRIOR TO CHALLENGE WITH HOMOLOGOUS FMDV.

ANIMAL DAYS VACC. CLINICAL SIGNS/ OP VIRUS 3 ABC MAT ELISA IgA IDAS ELISA
PRIOR TO VIRAEMIA
CHALL. 0DPV DC 28DPC 2DPC 27DPC
TO 66 11 -a 0.02b 0.02 0.02 -c -
TO 67 - 0.02 0.01 0.01 - -
TO 68 - 0.04 0.04 0.03 - -
TO 69* d - NSe 0.00 0.00 - -
TO 70* - NS 0.01 0.00 - -
TO 71 7 - 0.00 0.00 0.00 - -
TO 72 - -0.00 0.00 0.00 - -
TO 73 - 0.00 -0.01 0.00 - -
TO 74* - NS -0.02 -0.02 - -
TO 75* - NS -0.01 -0.01 - -
TO 76 5 Pf - -0.01 0.01 0.02 - -
TO 77 + 0.02 0.02 0.01 - -
TO 78 P - 0.01 0.01 0.01 - -
TO 79* FL,P,I,V g + h
NS 0.02 0.85 i - -
TO 80* FL,P,V + NS 0.01 0.67 - -
TO 81 4 - 0.01 0.00 0.00 - -
TO 82 - 0.00 0.00 0.00 - -
TO 83 - 0.01 0.00 0.00 - -
TO 84* - NS -0.00 -0.01 - +j
TO 85* - NS -0.00 -0.01 - -
TO 86 0 FL,P,V + NS -0.01 0.93 - -
TO 87 FL,P,V + NS -0.01 0.91 - -
TO 88 P,V + NS 0.03 0.71 - -
TO 89* FL,V + NS 0.03 0.94 +/-k -
TO 90* FL,V + NS 0.03 1.18 - -

a
No virus detected. b T/P ratio. c No IgA detected. d In-contact. e No sample. f Pyrexia. g FL-foot lesion(s), P-pyrexia, I-inappetance, V-
viraemia h Virus detected in oesophageal-pharyngeal sample on two or more occasions from 4 days post challenge . i T/P ratio >0.2 - sample
positive j IgA detected k borderline positive
 280

Conclusions

Previous reports have promoted the 3ABC MAT-ELISA as a reliable diagnostic assay for
distinguishing animals that have encountered live virus, whether they be vaccinated or not even
under circumstances where the animal had been repeatedly vaccinated (Mackay et al.1998,
Mackay, 1998b). There have also been reports that the test failed to detect 3ABC antibodies in
experimental subclinically infected cattle (Mackay et al., 1998) and sheep suspected of being
exposed to natural infection ( Brocchi et al., 1998). The assay is therefore considered only
suitable for use on a group or herd basis to detect FMDV infection. Sheep in which systemic
virus replication took place in this study were detected positive by the 3ABC MAT-ELISA at 28
days post challenge regardless of whether they were clinically or subclinically infected. Allowing
for the possibility of environmental contamination in the O-P samples taken shortly after
challenge, evidence for local virus replication was assessed on the basis of virus detection on two
or more occasions from 4 days post challenge. Apart from the C1 Oberbayern vaccinated sheep
TO77, under this criteria all sheep which exhibited local virus replication also gave a positive
result in the 3ABC MAT-ELISA. Since TO77 was housed with a group of sheep exhibiting
clinical signs the virus detected from O-P fluid samples in this animal may have been from the
environment and not true viral replication which would explain the absence of 3ABC antibodies.
One non vaccinated animal (TG32) which had shown no evidence of infection from virus
isolation also gave a positive result in the 3ABC MAT-ELISA. This animal, however, had
previously been shown to have FMDV specific neutralising antibody 6 or more days post-
challenge (Cox et al., 1999) confirming that it had been infected. All sheep that had been
vaccinated and protected from infection as demonstrated by lack of clinical signs and virus
isolation were negative for 3ABC antibodies. Our results from experimentally challenged
vaccinated, non vaccinated and in-contact sheep thus support the ability of the 3ABC-MAT
ELISA to accurately identify FMDV infection in sheep.

Additionally, the results show that the use of high potency ‘emergency’ vaccine does not impair
the ability of the test to identify where virus replication has taken place. In validating the efficacy
of such emergency foot-and-mouth disease vaccines in sheep great reliance has been placed on
virus isolation but owing to the difficulty encountered in isolating virus particularly from O-P
fluid samples it is often necessary to take a large number and range of samples to be sure of the
animals true virus status. These results, however suggest that the 3ABC-MAT ELISA may be a
useful alternative to the extensive sampling regime and virus isolation techniques presently used
for confirming viral replication in sheep following vaccination.

Salt et al. (1996) suggested that the appearance of FMDV specific IgA in mucosal secretions
could provide the basis of a test to differentiate vaccinated and/or recovered convalescent cattle
from FMDV carrier cattle. In this sheep study the IgA IDAS ELISA used as a spot test was
investigated as a means of identifying sheep that had encountered virus. Only 39% sheep shown
to have been infected by clinical signs and/or virus isolation were positive for local IgA at 28dpc.
Additionally, some uninfected but vaccinated sheep were local IgA positive. We conclude that
the IgA IDAS ELISA would therefore be unsuitable for identification of infected and recovered
convalescent sheep.
 281

Acknowledgements

This work was supported financially by MAFF UK (OC 9416 & SE 2807)

References

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(1998) Differentiating infection from vaccination in foot-and-mouth disease using a panel of
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 283

Appendix 36

Experimental FMD infections in pigs: a possible design for a PD50 experiment

Hanny Swam and Aldo Dekker
Institute for Animal Science and Health; ID-Lelystad
P.O. Box 65, 8200 AB Lelystad, The Netherlands

Introduction
During the last open session of the European Commissee for control of Foot and Mouth disease
(FMD) in 1998 in Aldershot there was a lot of discussion about the FMD monograph of the European
Pharmacopoeia in general and potency testing in particular. One of the items discussed was whether
a pig challenge test should be included in the European Pharmacopoeia or not.
In the meantime at ID-Lelystad we tried to develop a possible design for such a test in pigs.
The European Pharmacopoeia only describes a cattle PD50 : it says that at least 3 groups of at least 5
animals per group should be used. The cattle should be at least 6 months of age and free of
antibodies against FMD. The three different groups are vaccinated with different dose of vaccine,
preferably as said in the European Pharmacopoeia , by diminishing the volume of vaccine. The
experiment should always include 2 control animals which are not vaccinated. Three weeks after the
vaccination the animals are challenged with 10.000 ID50 cattle adapted challengevirus. Then the
animals are observed for 8 days. After these 8 days the animals are slaughtered : unprotected animals
show lesions at other sites than the tongue.
For pigs no PD50 test is described in the European Pharmacopoeia. When developing a design for
such a test the so called secondary contact infection should be taken into account : pigs that are
protected against the initial challenge virus may develop lesions due to this secondary contact
infection. Based on this individually housing would be preferred but is almost impossible under normal
experimental conditions.

Materials and Method

17 Pigs of at least 3 months of age and free of antibodies against FMD were used in each PD50
experiment. These animals were divided in 5 groups of each 3 animals and 2 control animals.The
different groups were housed seperately. The sequence of caretaking was from a full dose to half a
dose to ¼ dose etc. The control animals were the last group taken care of.
The different groups received different doses of vaccine by diminishing the volume of the vaccine: full
dose (2 ml); half dose (1 ml); ¼ dose: (0,5 ml) 1/8 dose (0,25 ml) and 1/16 dose (0,125 ml). The
control animals were not vaccinated.
Four weeks after the vaccination the pigs were challenged with 10.000 pfu (titrated on secundary pig
kidney cells) pig adapted challenge virus. The method used was the needle challenge method: the
challengevirus was injected in the left hindclaw of the pigs.
As soon as any clinical sign of FMD was visible, on other locations than that left hind claw, the animals
were removed from the group and slaughtered. 8 Days after the challenge the remaining animals were
moved to the slaughter place and judged for any sign of FMD.
All sera collected during the experiment (the pigs were bled on day 0,7,14, 21, 28 and 36) were tested
in the virusneutralisationtest.

Results
Using this design of 5 groups of each 3 animals which are housed seperately, two experiments were
performed.
The challenge results and the virusneutralisationtitres of the two experiments are shown in table 1 and
2.
In both experiments all animals vaccinated with a full, ½, ¼ and 1/8 dose were protected. However the
animals vaccinated with 1/16 dose all went down in both experiments. Also the control animals
showed clear signs of generalized FMD.
This resulted in both experiments in a PD50 of 0.18 ml (according to Reed and Muench).
 284

Table 1: serological and challenge results from experiment 1

challenge
Dose Animalnr 0 wkpv 1 wkpv 2 wkpv 3 wkpv 4 wkpv pm Protected?
2 ml 1245 <0.30 1.20 1.05 2.25 2.40 >2.40 yes
1410 <0.30 0.75 0.75 1.35 1.50 1.20 yes
1473 <0.30 <0.30 1.05 1.50 1.65 1.50 yes
Mean <0.30 0.65 0.95 1.70 1.85 1.80
1 ml 1456 <0.30 1.20 1.20 1.65 1.80 >2.40 yes
1457 <0.30 1.80 1.20 1.50 1.65 1.05 yes
1479 <0.30 0.60 0.30 1.35 1.50 1.65 yes
Mean <0.30 1.20 0.90 1.50 1.65 1.80
0.5 ml 1241 <0.30 0.90 1.05 1.65 1.65 2.10 yes
1256 <0.30 0.30 0.90 1.20 1.20 1.65 yes
1489 <0.30 0.75 0.75 1.50 1.65 >2.40 yes
Mean <0.30 0.65 0.90 1.45 1.50 2.15
0.25 1277 <0.30 <0.30 <0.30 <0.30 0.30 0.30 yes
ml 1282 <0.30 0.60 0.75 1.65 1.50 1.20 yes
1447 <0.30 0.60 0.45 1.35 1.35 1.50 yes
Mean <0.30 0.40 0.40 1.00 1.05 1.00
0.125 1272 <0.30 <0.30 0.45 0.75 1.35 >2.40 No; sec contact infection?
ml 1334 <0.30 <0.30 0.60 0.45 0.75 1.05 No; First animal with
clinical signs
1466 <0.30 1.05 0.60 1.35 1.50 >2.40 No; sec contact infection?
Mean <0.30 0.35 0.55 0.85 1.20 2.15

Table 2: serological and challenge results from experiment 2

challenge
Dose Animalnr 0 wkpv 1 wkpv 2 wkpv 3 wkpv 4 wkpv pm Protected?
2 ml 4971 <0.30 0.45 1.05 1.05 1.50 1.95 yes
4972 <0.30 0.75 0.90 1.05 1.05 1.50 yes
4973 <0.30 0.75 1.20 1.65 1.95 1.95 yes
Mean <0.30 0.65 1.05 1.25 1.50 1.80
1 ml 4974 <0.30 0.45 1.05 1.20 1.35 1.20 yes
4975 <0.30 0.30 0.45 0.60 0.75 0.75 yes
4976 <0.30 0.45 1.20 1.80 2.10 2.25 yes
Mean <0.30 0.40 0.90 1.20 1.40 1.40
0.5 ml 4977 <0.30 0.45 0.45 1.05 1.05 0.75 yes
4978 <0.30 <0.30 0.75 1.65 1.65 1.80 yes
4979 <0.30 <0.30 0.90 1.50 1.50 2.40 yes
Mean <0.30 <0.30 0.70 1.40 1.40 1.65
0.25 4981 <0.30 0.30 0.60 1.05 1.65 1.80 yes
ml 4982 <0.30 <0.30 0.60 1.65 1.80 1.80 yes
4983 <0.30 <0.30 0.75 1.05 1.05 1.65 yes
Mean <0.30 <0.30 0.65 1.25 1.50 1.75
0.125 4984 <0.30 <0.30 0.75 1.20 1.20 1.05 No; sec contact infection?
ml 4985 <0.30 <0.30 <0.30 <0.30 <0.30 <0.30 No; First animal with
clinical signs
4986 <0.30 <0.30 0.60 1.65 1.95 >2.40 No; sec contact infection?
Mean <0.30 <0.30 0.45 0.95 1.05 1.25

Discussion
Also in this experiment there was evidence for secondary contact infection; In experiment 1 animal
(number 1334 ; table 1) was the first animal to show clinical signs of FMD. These clinical signs were
seen 2 days after the needlechallenge took place.This was not amazing since this animal had at the
moment of challenge (4 weeks postvaccination) not a protective virusneutralisationtitre. However the 2
other animals from this group also developed clinical signs of FMD, about 2 days after animal 1334.
 285

These two animals (nr 1272 en 1466; table 1) had at the moment of challenge a protective titre. So
these animals were protected against the initial challenge but went down due to the secondary
contactchallenge. This is supported by the fact that the animals vaccinated with 1/8 dose had the
same virusneutralisationtitre at the moment of challenge and those animals were protected.
The same is seen in the second experiment: animal 4985 was the first to show clinical signs. This
animal didn’t develop at virusneutralisationtitre at all after the vaccination so again it was not amazing
that this animal went down. However, especially animalnumber 4986 (table 2) showed a protective
titre at the moment of challenge but also this animal went down a few days after 4985 was removed.
Again this is probably due to the secondary contact infection.
The PD50 result can be seriously influenced due to this secondary contactchallenge: if only 1 animal in
the 1/16 dose group shows clinical signs this would have resulted in a PD50 of 0.11 ml. If e.g. 5
animals per group were used, as in the cattle PD50, the result can vary from 0.07 ml (1 out of 5 animals
went down) to 0.25 ml (5 out of 5 animals went down). So the secondary contactchallenge may
influence the PD50 result seriously. By using more smaller groups instead of a few large groups this
influence is reduced.

Conclusion
It is possible to perform a PD50 test in pigs. However the influence of the secondary contactchallenge
should be taken into account. This can be done by making groups of a few animals instead of a few
groups with a lot of animals. The different groups should be housed separately. As soon as clinical
signs of FMD are visible the animals should be removed from the group.
 286

Appendix 37

The second report of the European Pharmacopoeia Working Group. Present and
future contacts with the European Pharmacopoeia and with EMEA.
Kris De Clercq,
on behalf of the European Pharmacopoeia Working Group.

The monograph on FMD vaccines of the European Pharmacopoeia, prescribes principles of preparation and
quality criteria for FMD vaccines. The issue of a substantial revision of the FMD vaccine Monograph was
first raised at the 1997 Session of the Research Group of the EUFMD in Poiana-Brasov, Romania.
Therefore an ad hoc Working Group was appointed to elaborate proposals for amendments. At the meetings
representatives from FMD Institutes, Scientific Committees, FAO and Industry were brought together.

Members of the Working Group

K. De Clercq
M. Amadori (Chairman of the meetings)
S. Barteling
B. Haas
Experts
R. Ahl (Representative of the EC Scientific Veterinary Committee)
G. F. Panina (Representative of the EC Scientific Veterinary Committee)
P. Barnett (IAH, Pirbright, representing the International Vaccine Bank)
Representatives of Private companies
T. Doel (Mérial)
H. Roedder (Bayer)
A. J. Breeuwsma (Intervet)
Secretariat EUFMD
Y. Leforban
J. Ryan (Rapporteur)

The Working Group made a document that indicates the limitations of the current monograph. In addition,
the document outlines the changes proposed and gives a Revised Monograph. Finally, this document
presents a balanced view of the issues on which there was not total agreement.

Critical changes: following critical changes should be part of any revision of the EP FMD vaccine
monograph:

A strong reduction of the in vivo tests (Animal welfare).

Safety test: detection of residual infectivity.
Replacement: by a sensitive cell system with an internal validation.

Potency test: the challenge of vaccinated animals.

Replacement: by tests based on serology if a correlation is proofed.
Reduction: in a dynamic link with a Quality Assurance System (GMP).
Refinement: a challenge test in pigs.

An update of production procedures with a specific reference to virus inactivation by a two-tank system
as described under GMP principles by means of 1st order inactivants.

The distinction between tests needed for licensing and those needed for batch release.

Replacing the Structure of the Monograph by a more systematic layout, in accordance with a good
manufacturing practice (GMP) approach:
 287

Structure current Monograph Proposed structure:

DEFINITION DEFINITION
PRODUCTION PRODUCTION
Validation of the inactivation procedure. Good Manufacturing Practice (GMP)
BULK INACTIVATED ANTIGEN High-containment production facilities
Inactivation Seed virus
Antigenicity New Strains
Safety Production of the virus
Sterility Inactivation of the virus.
Potency Concentration and purification of the antigen
IDENTIFICATION Storage of the antigen
TESTS Formulation of the vaccine
Safety IN PROCESS TESTS
Sterility Kinetics of inactivation
POTENCY Test for absence of residual virus
STORAGE Antigen content
LABELLING Identity
Immunogenicity of Antigen
TESTS OF FINAL PRODUCT
Sterility
Safety
Potency
DURATION OF IMMUNITY
EMERGENCY VACCINES
STORAGE
LABELLING

The Potency Controversy

It is arguable that the present system of injection of variable vaccine volumes instead of vaccine
dilutions in neutral buffer can give rise to artificially higher PD50 values. In practice, 3 PD50 under the
vaccine dilution system would correspond to 4.5 PD50 under the variable vaccine volumes.
Too strong an emphasis on high potency (6 instead of 3) may induce overconfidence in the vaccine to
the detriment of other aspects of paramount importance for the success of vaccination campaigns.
It may lead to higher vaccine prices and may prompt reduced usage of FMD vaccines in poorer
countries.

Present and future contacts

The European Pharmacopoeia secretariat and Expert Group

Although the procedure followed is not the normal one, the report is considered as a good basis to start
a discussion in the Expert Group. The document is distributed by the Secretariat to the members of the
Expert Group. The chairman of the EUFMD Research Group is invited on October 3 to present the
document. Proposed changes are welcome as long as they have a solid scientific basis and are well
documented. The proposed change of the Monograph structure will be difficult to accept as it has
consequences on other Monographs. Normally the items are presented in a general way without going
into so many details.

The EMEA

The legal basis for the European Centralised procedure for registration will be reviewed. There is a
proposal to make it applicable to all vaccines used for ‘wide prophylaxis’ on a European basis (rabies,
FMD, CSF). The Immunological Group of the CVMP in collaboration with DG SANCO and DG
Enterprises will propose guidelines. The chairman of the EUFMD Research Group is invited on
December 4 to present our document. In this way EUFMD could be involved in making the guidelines.
 288
Appendix 38

Preliminary Results of the Expert Elicitation Workshop on the Risk of

Introduction of FMD to Europe

Held in Borovets, Bulgaria

04-05 Sept. 2000

John Ryan & Lisa Gallagher

Introduction
During late 1999 and during the year 2000, the FMD situation world-wide deteriorated
significantly. The Executive Committee of EUFMD became increasingly concerned about the
increasing risks to Europe of FMD introduction and requested the Research Group of EUFMD to
analyse the risks facing Europe.
EUFMD had already begun building competencies in the relatively new and fashionable discipline
of Risk Analysis, and Dr. Susan Horst - a specialist in Risk Analysis from Wageningen University,
The Netherlands - had already presented her work to the Research Group. Therefore, based on this
specific request from the Executive Committee and trying to build upon the work of Dr. Horst, it
was decided to conduct an expert elicitation workshop immediately before the Research Group
Meeting in Borovets, Bulgaria on the 4-5 Sept. 2000.

Objectives
Although the workshop was planned to build upon work presented by Dr. H.S Horst at the Research
Group meeting in Maisons-Alfort 1999, it was decided that to scale up her entire model (i.e. dealing
with disease introduction, disease spread and economic consequences) from one country level to the
entire European level would be too optimistic given the time constraints. Therefore, the objective of
this workshop was to examine only the risk of Primary Introductions of FMD to Europe and it did
not examine spread within Europe or the economic consequences of FMD outbreaks.
In examining the Risk of Introduction of FMD to Europe, 3 key questions were framed:
Where in Europe is most likely to be affected?
From where outside Europe is the disease most likely to come from?
How or by what route is the virus most likely to be transmitted?
Finally we decided that it would be useful to generate a forecast of the number of primary outbreaks
of FMD that the experts believed would take place over the next 5 years.

Method
Due to time and resource constraints, it was decided to reduce the complexity as much as possible
and to use the elicitation of expert opinion to generate the answers to our key questions.

Expert Elicitation
The use of Expert Opinion in Risk Analysis is indicated when any of the following conditions are
true:
- "Data has never been collected in the past
- Data is too expensive to obtain
- Past data is no longer relevant
- Data is sparse
 289
- The area being modeled is new" 1
In the context of the questions we were trying to answer, all of the above indications for the use of
Expert Opinion were true.

Reduction of Complexity
In trying to reduce the complexity of the questions we would have to ask the experts, we decided to
aggregate countries into groups both inside and outside Europe and to limit the list of possible
routes of transmission. This was accomplished by allowing the experts themselves to decide on the
groups and list of routes to be considered in a pilot questionnaire that was circulated 6 weeks before
the workshop.
In the pilot questionnaire countries were grouped into 5 European Groups with countries that
represented possible sources of FMD were grouped into 8 source or external groups. The experts
were asked to agree or disagree with these groupings and suggest possible changes. Then for each
European group - source group pair, the experts were asked to rate the likelihood (from 0="not
possible" to 5="highly likely" to be a source of introduction) that a limited list of 14 routes would
be the route of introduction between that pair and to suggest alternative routes than the routes
proposed in the questionnaire. This was an effort to reduce the number of factors to be considered
for each European group - source group pairing, as we eliminated any routes that did not receive an
average of 1="highly unlikely" from the experts.
The results of the Pilot Questionnaire were as follows:
• the response rate was 50%
• 67% agreed & 17% disagreed with the proposed European Grouping much of the disagreements
centred on individual countries and as a result 4 countries were reclassified in the workshop.
• 83% agreed & 8% disagreed with the Source Groupings and 2 countries were reclassified as a
result
• 18% of routes were removed as a result of eliminating any routes did not score an average of
1="highly unlikely"
• In addition for some European group - source group pairings, new routes were suggested.
The Pilot questionnaire thus helped us frame our final questionnaires for the workshop itself. In
addition, a limited trial run took place in FAO HQ and the important feedback from that exercise
was also incorporated into the design of the workshop questionnaires.

Workshop
The workshop method itself used a modified Delphi Technique: where two iterations of the same
questionnaire took place with lengthy discussions on the results of questionnaire one taking place
before questionnaire two was completed. In the first iteration of the questionnaire, each expert
answered the questionnaire individually without the bias that could be introduced in a group
situation. Then lively group discussions took place as the results of the first questionnaire were
critically examined by the experts. This discussion forum raised many issues and was a means
whereby experts with unique knowledge of the risks could share them with the entire group before
all the experts completed the second questionnaire. This allowed individuals to re-evaluate their
responses after the group discussion and it would be expected that there would be a movement of
opinion towards consensus in the second questionnaire.

Questioning Method
The questioning method used was a direct elicitation of conditional probabilities e.g. Given that a
primary outbreak of FMD has occurred in the Balkan Group, what is the probability that the source

1
D Vose, 2000 “Risk Analysis a Quantitative Guide”
 290
of introduction was from the following groups:....list of Source Groups. Visual aids such as pie-charts
and bar charts were provided to help the experts elicit the probabilities. Conjoint Analysis (indirect
elicitation) was not used because many experts found the conjoint analysis questions too
conceptually difficult and it would have been impossible to organise given the time constraints.

Questions Asked
There were 4 levels of questioning:
1. For each of the European Groups - what is the probability of any primary introduction of FMD in
the next 5 years from any source and from any route.
2. For each of the European Groups (assuming an introduction has taken place) - what is the
probability that each source group was the source.
3. For each European group - source group pairing (assuming an introduction between the pair) -
what is the probability that each route was the route of introduction.
4. Finally, each expert was asked to predict the minimum, most likely and maximum no. of primary
outbreaks in each European group over the next 5 years.
The following is the list of countries in each of the 8 Source Groups:
Turkey 2 Caucasian and Asia Rest of Africa
Central Asian Afghanistan Angola Mozambique
Middle East Republics Bangladesh Benin Namibia
Bahrain Armenia Bhutan Botswana Niger
Egypt Azerbaijan Brunei Burkina Faso Nigeria
Iran Georgia Cambodia Burundi Rwanda
Iraq Kazakhstan China Cameroon Senegal
Israel2 Kyrgyzstan Hong Kong Central African Republic Sierra Leone
Jordan Tajikistan India Chad Somalia
Kuwait Turkmenistan Korea Dem.People's Rep Congo South Africa
Lebanon Uzbekistan Korea, Republic of Djibouti Sudan
Oman Laos Equatorial Guinea Swaziland
Palestinian NA Russia/Eastern Europe Malaysia Eritrea Tanzania
Qatar Belarus Mauritius Ethiopia Togo
Saudi Arabia Moldova Mongolia Gabon Uganda
Syria Russia Myanmar Gambia Zaire
UAE Ukraine Nepal Ghana Zambia
Yemen Pakistan Guinea
South America Philippines Guinea Bissau
North Africa Bolivia Sri Lanka Ivory Coast
Algeria Brazil Taiwan Kenya
Libya Colombia Thailand Lesotho
Morocco Ecuador Vietnam Liberia
Tunisia Peru Malawi
Venezuela Mali
The following is the list countries in each of the 5 European Groups:
“Islands” Balkans Eastern Europe Western Europe Southern Europe
Finland Albania Czech Republic Belgium Italy
Ireland Bulgaria Hungary Denmark Malta
Iceland Croatia Lithuania France Portugal
Norway Cyprus Poland Germany Spain
Sweden FR Yugoslavia Romania Luxembourg
United Greece Slovak Republic Netherlands
Kingdom Slovenia Latvia Switzerland
FYRO Macedonia Estonia Austria
Bosnia

2
Turkey and Israel are members of EUFMD but due to recent outbreaks of FMD and the fact that they are surrounded
by FMD endemic countries it was decided to include them as potential sources.
 291
The following is the list of routes used (not all routes were considered in each pairing based on the
results of the pilot questionnaire) - a definitions sheet accompanied the list of routes to clarify what
introductions were included under each heading:
Legal Import of Livestock Illegal Import of Livestock Legal Import of Meat
Legal Import of Other Animal Products Illegal Import of Animal Products Returning Livestock Trucks
Other Returning Trucks Swill from Boats Swill from Aircraft
Airborne Spread Wildlife (import/transboundary movement) Tourist/Immigrant Vehicles
Tourist/Immigrant Foodstuff Imported Bedding/Packing Material “Natural” Spread

Analysis of questionnaires
All completed
questionnaires were
entered in spreadsheets
and @Risk. From there
the different conditional
probabilities
(representing question
levels 1-3 above - see
figure 1 below) were
combined to assign a
probability to each route
in each European group
- source group pair for
each expert. The results
of the different experts
for each of these
probabilities were
treated as discrete
distributions and where
time permitted, Monte
Carlo simulations were Figure 1 Generation of Probabilities
run for each of these
probabilities and the results presented were the means and the 5th & the 95th percentiles.
The generation of such a large number (460) of discrete probabilities allows us to aggregate these
probabilities back up to answer any specific question we want to ask. In particular we can answer
the key questions (where will be affected? where will it come from? how will it be transmitted?) for
Europe as a whole...or for each group...even for each source group.
Finally, the prediction of the number of outbreaks over the next five years was generated by
combining the experts beta pert distributions i.e. their min, most likely & max.

Results
Where will be affected?
Group 5th %ile Mean 95th %ile
(%) (%) (%)
Balkans 40 59 90
Eastern Europe 5 23 50
Southern Europe 0 11 23
Western Europe 0 5 10
“Islands” 0 2 5
 292
Where will it come from?
External Group Mean
1 Turkey 41
2 Russia/Eastern Europe 13
3 Middle East 13
4 Caucasia and Central Europe 11
5 North of Africa 9
6 Asia 6
7 Rest of Africa 4
8 South America 3
How will it be transmitted?

Route Mean %
1 Illegal import of livestock 21
2 Illegal import of other animal products 15
3 Tourist/immigrant foodstuffs 11
4 Legal import of other animal products 6
5 Returning Livestock trucks 6
6 Legal import of meat 5
7 Swill from aircraft 5
8 Swill from boats 5
9 “Natural” spread 5
10 Tourist/immigrant vehicles 4
11 Other returning trucks 4
12 Legal import of livestock 4
13 Imported bedding/packing materials 3
14 Wildlife 3
15 Airborne spread 3

The top 10 possible introductions

European Source Route 5th %ile Mean 95th %ile

Group Group (%) (%) (%)
1 Balkans Turkey Illegal import of livestock 2 9 20
2 Balkans Turkey Natural Spread 0 5 20
3 Balkans Turkey Illegal import of other animal products 0 3 6
4 Balkans Turkey Wildlife 0 2 4
5 Balkans Middle East Illegal import of livestock 0 2 7
6 Balkans Turkey Tourist/immigrant foodstuffs 0 2 7
7 Balkans Turkey Airborne 0 2 4
8 Balkans Turkey Returning livestock trucks 0 2 3
9 Balkans Caucasia/Cen Asia Illegal import of livestock 0 1 8
10 Balkans Russia/East Eur Illegal import of livestock 0 1 3
 293
Top 5 introductions for the Balkans

Source Route Mean %

1 Turkey Illegal import of livestock 9.43

2 Turkey Natural spread 4.60
3 Turkey Illegal import of other animal products 2.90
4 Turkey Wildlife 2.34
5 Middle East Illegal import of livestock 2.02

Top 5 introductions for Eastern Europe

Source Route Mean %

1 Turkey Illegal import of other animal products 1.10

2 Turkey Illegal import of livestock 1.06
3 Turkey Tourist/immigrant foodstuffs 0.86
4 Russia & East Eur Illegal import of livestock 0.74
5 Caucasia & Cent Asia Illegal import of other animal products 0.69

Top 5 introductions for Southern Europe

Source Route Mean %

1 North Africa Tourist/immigrant foodstuffs 0.44

2 Middle East Tourist/immigrant foodstuffs 0.42
3 Turkey Illegal import of other animal products 0.40
4 Turkey Tourist/immigrant foodstuffs 0.39
5 North Africa Illegal import of livestock 0.39

Top 5 introductions for Western Europe

Source Route Mean %

1 Turkey Tourist/immigrant foodstuffs 0.28

2 Turkey Illegal import of other animal products 0.19
3 Middle East Tourist/immigrant foodstuffs 0.15
4 Turkey Illegal import of livestock 0.13
5 Turkey Swill from aircraft 0.12
 294
Top 5 introductions for the “Islands”

Source Route Mean %

1 Turkey Tourist/immigrant foodstuffs 0.11

2 Middle East Tourist/immigrant foodstuffs 0.10
3 Turkey Illegal import of other animal products 0.07
4 Russia/East Eur Illegal import of other animal products 0.06
5 Asia Tourist/immigrant foodstuffs 0.06

Prediction of No. of Outbreaks in 5yrs

Group 5th %ile Mean 95th %ile

Balkans 3 7 14
Eastern Europe 1 4 10
Southern Europe 1 3 7
Western Europe 0 1 3
“Islands” 0 1 2

In the first iteration of the questionnaire, there was huge variation in the answers between the
experts. This was to be expected as many of the participants were unfamiliar with this type of
workshop and questioning. There were also confusions as to what types of introductions were
included under each route of introductions.
In the second iteration of the questionnaire, there was good convergence in the results of the
different experts. This was not only due to becoming more familiar with the exercise and clearing
up some misunderstandings, but it was also the result of sharing of knowledge between the experts,
particularly when they were not familiar with the epidemiological profile of certain regions.
Although there were 20 experts participating from many countries across Europe, there was no
“weighing” of the experts opinions.

Discussion
The results clearly demonstrated the experts opinion of the greatest risks of FMD introduction
facing Europe. There was good convergence in opinion in the second iteration of the questionnaire
and on presentation of the results to the experts, there was no disagreement with any of the results
presented. Although the accuracy of the actual probabilities may be disputed, the ranking of the
risks are very robust, particularly because they represent the opinion of not just one expert but many
experts.
Such results will be very useful in directing the attention of decision makers and risk managers to
the main risks of introduction of FMD. The results of a workshop like this are certainly an
improvement on other methods such as informal analysis of risks by an individual or by a small
group from only one country or no analysis of the risks at all.
 295
Problems
A major limitation from the methodological point of view was that there was no formal data
presentation step to assist the experts in their deliberations. This was somewhat compensated by the
geographical spread of the experts and the details that surfaced during the discussions. The main
reason that no data was presented to the experts was because the overwhelming volume of data that
is required could not have been collected in the limited time available. To gather this data for future
exercises will be costly in time and resources and one has to question whether the extra accuracy
that more data would bring to the results justifies this extra investment.
Another problem was that the workshop was perhaps too ambitious, it was very long and very
difficult for the experts, there were difficulties with timekeeping and not enough time was allocated
to the discussions. This aspect could be improved if the workshop had been computerised, but this
would also require more time and resources.
The profile of the participating experts may have been too narrow, as it consisted mostly of
virologists. In future the profile of the experts could be widened to include more epidemiologists,
Veterinary Service Staff involved in the control of the livestock and meat industries, traders from
the meat and livestock industries and policy makers.
Some routes of introduction were not considered especially laboratory escapes. The point was made
that for some regions, this might pose the greatest risk. It was also argued that a laboratory escape is
not technically an introduction.

Conclusions
For everyone involved it was an extremely interesting and useful exercise and this was reflected by
the results of the feedback questionnaire that were very supportive and constructive.
There was a lot of data that required processing: 500 numbers x 20 participants x two questionnaires
= 20,000 data entries (10,000 in one day with results that night!) and this placed a lot of stress on
the timetable and the organisers - this could easily be solved by computerising the process for future
workshops.
A lot of methodological lessons were learnt during the workshop but this is unsurprising as no-one
has attempted a workshop like this before.
The results were surprisingly convergent, because in general expert elicitations are not a precise
science and by their very nature of collecting the opinions of diverse experts it is expected that there
would be divergence in opinion.
The results above are a written expression of the beliefs of the many experts. This document can
now be consulted and used as opposed to this knowledge remaining buried in the minds of the
experts. It is also an improvement on individual opinions alone. It is a useful tool for risk managers
who need to formulate overall disease prevention strategy without being able to conduct thousands
of import risk analyses and consult privately with many experts.
Although this workshop had some limitations as discussed in the "Problems" section, it is fair to say
that the method works, and would work much better when the lessons learned in this workshop are
implemented in future workshops.
It was an extremely cost effective workshop for EUFMD as it took advantage of the fact that the
experts had gathered in Borovets for the Research Group Meeting later that week. There is also no
guarantee that a more expensive workshop (i.e. more experts, computerisation and an expensive
data gathering process) would give results that lead to significantly better decision making.
Therefore there are cost-benefit considerations to be taken into account before trying to improve the
accuracy of the results.
 296
A full report on all the results will be prepared and presented to the Executive Committee of
EUFMD in Nov. 2000, and will be published on the EUFMD web-site in due course.

The Future?
Possibilities for future work in this area:
to repeat this exercise at the Research Group(RG) Meeting next year or in two years time?
to de-couple the workshop from the RG meeting and to include epidemiologists, decision makers
and vets and traders from the meat and livestock industries?
to try a validation exercise next year by focussing on a specific European group and comparing the
more detailed results with the results of this workshop?
to take a look at inter-European spread?

Acknowledgements
A huge THANK YOU to all the participants for their patience, diligence and hard work.
To the secretary of EUFMD, Dr. Leforban and the Chairman of the Research Group, Dr. De Clercq,
for their support and advice.
To Lisa for all her hard work and ideas.
 Appendix 39

List of Participants

Members of the Research Group

Dr. Kris De Clercq Dr. C. Griot

Chairman Director
CODA-CERVA-VAR Institute of Virology and
Groeselenberg 99 Immunoprophylaxis
B-1180 Ukkel CH3147 Mittelhäusern, Switzerland
Belgium Tel/fax: 41 031 848 9211/848 9222
Tel/fax: 32 2 3754455/2 3750979 e-mail: Christian.Griot@ivi.admin.ch
e-mail: kris.de.clercq@var.fgov.be
Dr. I. Gürhan
Dr. M. Amadori Head of the Laboratory/Cell Culture
Istituto Zooprofilattico Sperimentale Laboratory Manager
della Lombardia e dell'Emilia FMD Institute/ SAP Enst.
Laboratorio Ricerca Sviluppo PK 714, 06044, Ankara, Turkey
Prodotti Immunizzanti Tel/fax: 90 312 2873600/2873606
Via A. Bianchi 7/9, 25124-Brescia e-mail: ismetgurhan@yahoo.com
Italy
Tel/fax: 030-2290277/2425251 Dr. B. Haas
e-mail: Mamadori@bs.izs.it Head of FMD Diagnostic Laboratory
Federal Research Centre for Virus
Dr. S.J. Barteling Diseases of Animals, Paul Ehrlich
c/o Institute for Animal Science & Strasse 28,D-72076 Tübingen
Health Germany
(ID-DLO), P.O. Box 65, NL-8200 AB Tel/fax 49 7071 9670/967305/905
Lelystad, The Netherlands e-mail: Bernd.Haas@Tue.BFAV.DE
Tel/fax: 31 320 238238/228668
e-mail: simbar@scarlet.nl Dr. Y. Ivanov
Deputy Director General
Dr. A.I. Donaldson (Ex Officio) NVS, Head of FMD Laboratory
Head of Laboratory, AFRC/IAH 15 P. Slaveikov Blvd., Sofia, Bulgaria
Pirbright Laboratory, Ash Road, Tel/fax: 359 2 525256/9549593
Pirbright, Woking, GU24 ONF, U.K. e-mail: boikovet@mobikom.com
Tel/fax: 44 1483 232441/232448
e-mail: alex.donaldson.@bbsrc.ac.uk.
Dr. J.M. Sanchez-Vizcaino
Dr. M. Danes Director
I.N.M.V. Pasteur Center of INIA (CISA-INIA)
Sos. Giulesti nr. 333 28130 Valdeolmos (Madrid)
R-7000 Bucharest, Romania Spain
Tel/fax: 40 1 2206920/2206915 Tel/fax: 0034 91 6202216/6202247
e-mail: pasteur@fx.ro e-mail: vizcaino@inia.es
Dr. H. Yadin Dr. Ilian Boykovski
Kimron Veterinary Institute Animal Health Dept.
Head of FMD Laboratory National Veterinary Services
c/o Ministry of Agriculture 15A P. Slaveikov Blvd., Sofia 1606
P.O. Box 12, Beit-Dagan, Israel Bulgaria
Tel/fax: 972 3 9681619/9681788 Tel/fax: 359 2 9521345/9549593
e-mail: hagaiy@moag.gov.il e-mail: boikovet@mobikom.com
Julia.vet@mobikom.com

Observers from member countries Dr. Ilian Gechev

Director of Institute for Control on
Dr. Roland Silber Bioproducts
Head of FMD Department 1, Adam Mitskiewicz Blvd.
B.A.f. Virusseuchenbek. b.H Sofia, Bulgaria
MKS Abteilung/Emil Behringweg 3 Tel/fax: 359 2 263345/260045
A-1233-Vienna, Austria e-mail: ICVP@mobikom.com
Tel/fax: 0043 1 8021212/802121211
e-mail:silber@mks.batsb.at
Prof. Dr. Pavel Tekerlekov
Institute for Control on Bioproducts
Dr. Ilian Bachvarov 1, Adam Mitskiewicz Blvd.
Director General Sofia, Bulgaria
National Veterinary Services Tel/fax: 359 2 263345/260045
15A P. Slaveikov Blvd., Sofia 1606 e-mail: ICVP@mobikom.com
Bulgaria
Tel/fax: 359 2 9521345/9549593
e-mail: boikovet@mobikom.com Dr. Peter Antonov
Institute for Control on Bioproducts
1, Adam Mitskiewicz Blvd.
Dr. Boiko Likov Sofia, Bulgaria
Head of Dept. International relations, Tel/fax: 359 2 263345/260045
eurointegration and veterinary e-mail: ICVP@mobikom.com
legislation
National Veterinary Services Dr.Georgi Georgiev
15A P. Slaveikov Blvd., Sofia 1606 Head of National Laboratory for FMD
Bulgaria and other exotic diseases
Tel/fax: 359 2 9521442/9549593 Central Research Veterinary Institute
e-mail: boikovet@mobikom.com 15 P.Slaveikov Blvd., Sofia 1606
Bulgaria
Tel/fax: 359 2 383011/52 53 06
Dr. Pencho Kamenov
Animal Health Dept. Mrs. Emilia Veleva
National Veterinary Service National Laboratory for FMD and
15A P. Slaveikov Blvd., Sofia 1606 other exotic diseases
Bulgaria Central Research Veterinary Institute
Tel/fax: 359 2 9521345/9549593 15 P.Slaveikov Blvd., Sofia 1606
e-mail: boikovet@mobikom.com Bulgaria
Julia.vet@mobikom.com
Tel/fax: 359 2 383011/52 53 06
Dr. Milena Hesounova Dr. Vilmos Pàlfi
National Reference Laboratory for Central Veterinary Institute
FMD and Ves. Diseases 1149 Budapest, Tábornok Utca 2
State Veterinary Institute, Sidlistni 24 Hungary
165 03 Prague 6 Lysolaje Tel/fax 36 1 2527533/36 1 222 6069
Czech Republic e-mail: palfi@oai.hu
Tel/fax: 420 2 51031111/ 20920655
e-mail: SVUPRAHA@MS.ANET.CZ
Dr. Dianne Clery
Research Officer
Dr. K.J. Sorensen Virology, Department of Agriculture
Danish Veterinary Institute for Food and Rural Development, CVRL
Virus Research, Lindholm DK 4771 Abbotstown, Dublin 15, Ireland
Kalvehave, Denmark Tel/Fax: 353-1-6072778
Tel/fax: 45 55860231/55860300 e-mail: dianneclery@hotmail.com
e-mail kjs@vetvirus.dk

Dr. Jovan Bosnakovski

Dr. Michele Remond Veterinary Institute “Skopje”
AFSSA str. Lazar Pop Trajkov 5-7
22 rue Pierre Curie 91000 Skopje, FYR Macedonia
94703 Maisons Alfort, France Tel/fax: 389/91/115-125/114619
Tel/fax: 33 01 49771300/43 68 97 62 e-mail: vetinrum@lotus.mpt.com.mk
e-mail: m.remond@alfort.afssa.fr
Dr. Ivanco Naletoski
Veterinary Institute “Skopje”
Dr. Otfried Marquardt str. Lazar Pop Trajkov 5-7
Federal Research Centre for Virus 91000 Skopje FYR Macedonia
Diseases of Animals Tel/fax: 389/91/115-125/114619
Paul Ehrlich Strasse 28 e-mail: vetinrum@lotus.mpt.com.mk
D-72076 Tübingen, Germany
Tel/fax 49 7071 9670/967305/905
e-mail: marquardt@tue.bfav.de Dr. F.H. Reek
Head of FMD Vaccine Production and
Development
Dr. H. Roedder Institute for Animal Science and
Bayer AG Health (ID-Lelystad)
GB-TG M BIO Postbus 65,8200 AB Lelystad
51 368 Leverkusen, Germany The Netherlands
e-mail:helmut.roedder.hr@bayer-ag.de e-mail: f.reek@id.wag.ur.nl

Dr. Helen Reboutzskou Dr. Eblé Phaedra

FMD and Exotic Diseases Institute ID-Lelystad
Aghia Paraskevi Postbus 65
Athens, Greece 8200 AB Lelystad
Tel/fax: 0030 1 6010925/6082085 The Netherlands
e-mail: vasilikirousi@hotmail.com Tel/fax: 31 320 238238/238662
e-mail:p.l.eble@id-wag.ur.nl
Dr. H. Swam Dr. Flaviu Fileru
ID-Lelystad Assistant Researcher, Molecular
Institute for Animal Science & Health Biology
Postbus 65 I.N.M.V. Pasteur, Sos. Giulesti nr. 333
NL-8200 AB Lelystad R-7000 Bucharest, Romania
The Netherlands Tel/fax: 40 1 2206920/2206915
Tel/fax: 31 320 238673/238663 e-mail: pasteur@fx.ro
e-mail:h.swam@id.wag-ur.nl

Dr. Virgilia Popa

Dr. P.L.J.M. Moonen Senior Researcher, Molecular Biology
ID Lelystad I.N.M.V. Pasteur, Sos. Giulesti nr. 333
Dept of Mammalian Virology R-7000 Bucharest, Romania
Postbus 65, 8200 AB Lelystad Tel/fax: 40 1 2206920/2206915
The Netherlands e-mail: pasteur@fx.ro
Tel: 31 320-238680 Lab 31 320
238858
e-mail: P.L.J.M.Moonen@id.wag-ur.nl Dr. Monica Vanghele
Assistant Researcher, Molecular
Biology
Dr. O. Breeuwsma I.N.M.V. Pasteur, Sos. Giulesti nr. 333
Intervet Int.B.V. R-7000 Bucharest, Romania
P.O. Box 31,5830 AA Boxmeer Tel/fax: 40 1 2206920/2206915
The Netherlands e-mail: pasteur@fx.ro
Tel/fax: 31 485 587418/587491
E mail:
Marguerite.Hoogendijk@Intervet.akzonobel.nl Dr. Monique Wenger
Institute of Virology and
Immunoprophylaxis
Dr. Andrzej Kesy CH3147 Mittelhäusern, Switzerland
Department of Foot-and-Mouth Tel/fax: 41 031 848 9211/848 9222
Disease, National Vet Res Institute e-mail: monique.wenger@ivi.admin.ch
98-220 Zdunska Wola, Poland
e-mail: andrzejke@piwzp.invar.net.pl
Dr. Elisabeth Gallagher
Dr. Wieslaw Niedbalski Department of Risk Research
Department of Foot-and-Mouth Veterinary Laboratories Agency –
Disease, National Vet Res Institute Weybridge
98-220 Zdunska Wola, Poland New Haw, Addlestone, Surrey KT15
e-mail: wieslaw@piwzp.invar.net.pl 3NB, UK
Tel/fax: 44 1 932 357774 / 357445
e-mail: l.gallagher@vla.mafff.gov.uk
Dr. Daniela Botus
Junior Researcher Dr. Tim Doel
Molecular Biology Unit Site Manager, MERIAL
I.N.M.V. Pasteur, Sos. Giulesti nr. 333 Animal Health Ltd
R-7000 Bucharest, Romania Ash Road, Pirbright, Woking
Tel/fax: 40 1 2206920/2206915 GU24ONQ, Surrey, UK
e-mail: pasteur@fx.ro Tel/fax: 44 1483 238111/238102
e-mail: tim.doel@merial.com
Dr. Djordje Dobric Mr. Nick Knowles
Faculty of Veterinary Medicine IAH, Pirbright Laboratory
Bul Jna 18, 11080 Belgrade, Ash Road, Pirbright, Woking
FR Yugoslavia Surrey GU24 ONF, UK
Tel: 381 11 685080 Tel/fax: 44 1483 232441/232448
e-mail: nick.knowles@bbsrc.ac.uk

WRL
EU/EC
Prof. Soren Alexandersen (visiting
scientist at Pirbright Laboratory) Professor R. Ahl
Ash Road, Pirbright Federal Research Centre for Virus
Woking GU24 ONF, Surrey, UK Diseases of Animals
Tel/fax: 44 01483-231025/232448 Paul Ehrlich Strasse 28
e-mail: soren.alexandersen@bbsrc.ac.uk D-72076 Tüebingen, Germany
E mail: RFH.Ahl@t-online.de

Dr. Paul Kitching

IAH, Pirbright Laboratory FAO
Ash Road, Pirbright, Woking
Surrey GU24 ONF, UK Dr. M. Rweyemamu
Tel/fax: 44 1483 232441/232448 Senior Officer (Infectious
e-mail: paul.kitching@bbsrc.ac.uk Diseases/EMPRES)
Animal Production and Health
Division, FAO, Rome, Italy
Dr. Alan R. Samuel Tel/fax: 0039 06 57056772/57055749
FMD International Vaccine Bank e-mail: Mark.Rweyemamu@FAO.Org
Research & Development Section
IAH, Pirbright Laboratory
Ash Road, Pirbright, Woking IAEA
Surrey GU24 ONF, UK
Tel/fax: 44 1483 232441/232448 Dr. J. Crowther
e-mail: alan.samuel@bbsrc.ac.uk Wagramerstrasse 5
A-1400 Vienna, Austria
Tel/fax: 00431 2060/20607
Ms. Morag Forsyth e-mail: j.crowther@iaea.org
IAH, Pirbright Laboratory
Ash Road, Pirbright, Woking
Surrey GU24 ONF, UK
Tel/fax: 44 1483 232441/232448 Other Observers
e-mail: morag.forsyth@bbsrc.ac.uk
Dr. Dana Horska
National Reference Lab for FMD
Mr. Scott Reid State Veterinary Institute
IAH, Pirbright Laboratory Akademicka 3, 949 01 Nitra, Slovakia
Ash Road, Pirbright, Woking Tel/fax: 00421 87 6536520-3
Surrey GU24 ONF, UK /7336210
Tel/fax: 44 1483 232441/232448 e-mail: svunitra@svunitra.sk
e-mail: scott.reid@bbsrc.ac.uk
Dr. Eliana Smitsaart Dr. Sergei A. Doudnikov
Res & Dev Department ARRIAH, (All-Russian Research
Biogenesis Institute for Animal Health)
Ruta Panamericana km 38.2 600900 Jur’evets, Vladimir
B1619IEA- Garin (Buenos Aires), Federation of Russia
Argentina Tel/fax: 7-0922-260753/243675
Tel/fax: 54 3327 44 8300/ 44 8324 e-mail: sinko@arriah.elcom.ru
e-mail: esmitsaart@biogenesis.com.ar

Dr. Salah Hammami

Dr. C. Groocock Institut de recherche Vétérinaire de
APHIS-VS-NVSL, Veterinary Attaché Tunisie (IRVT)
US Embassy, Boltzmanngasse 16 Rue Djebel Lakhdar 1006
A-1091 Vienna, Austria La Rabta, Tunis,Tunisia
Tel/fax: 00 43 1 313 392951/392913 Tel/fax: 216 1 264321/569692
e-mail: cgroocock@csi.com e-mail: hammami.salah@iresa.agrinet.tn

Dr. Kenichi Sakamoto Secretariat

Department of Exotic Diseases, NIAH
6-20-1, Josui-Honcho, Kodaira-shi Animal Production and Health
187-0022 Tokyo, Japan Division, FAO, Rome, Italy
Tel/fax: 81 413 21 1441/423 25 5122 Fax no: 0039 06 570 55749
e-mail: skenichi@ed.affrc.go.jp
Dr. Yves Leforban,
Secretary, EUFMD
Dr. Toru Kano Tel: 0039 06 57055528
Department of Exotic Diseases, NIAH e-mail: yves.leforban@FAO.org
6-20-1, Josui-Honcho, Kodaira-shi
187-0022 Tokyo, Japan
Tel/fax: 81 423 21 1441/25 5122 Dr. John Ryan,
Associate Professional Officer,
EUFMD
Dr. Viatcheslav M. Avilov Tel: 0039 06 57053326
ARRIAH, (All-Russian Research e-mail: john.ryan@FAO.org
Institute for Animal Health)
600900 Jur’evets, Vladimir
Federation of Russia Ms Egiziana Fragiotta,
Tel/fax: 7-0922-260614/243675 Clerk Stenographer, IDG, AGAH,
e-mail: sinko@arriah.elcom.ru Tel: 0039 06 57052637
e-mail: egiziana.fragiotta@FAO.org

Dr. Tamara A. Fomina

ARRIAH, (All-Russian Research
Institute for Animal Health)
600900 Jur’evets, Vladimir
Federation of Russia
Tel/fax: 7-0922-260753/243675
e-mail: sinko@arriah.elcom.ru