AGA: EUFMD/RG/02

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

Çesme, Izmir, Turkey

17 – 20 September 2002

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS Rome, 2002

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TABLE OF CONTENTS

Page INTRODUCTION ……………………………………………………………… 1 Adoption of the Agenda ………………………………………………………….. 3 Item 1 Information on current FMD situation in the world and reports on outbreaks ………………………………………………………………… 4 Item 2a FMD control: epidemiology, surveillance, control measures: focus on endemic zones …………………………………………………………… 5 Item 2b FMD control: epidemiology, surveillance and control measures: focus on epidemic incursions ………………………………………………….. 7 Item 3 Pathogenicity and transmission …………………………………………. 10 Item 4 Virus characterisation …………………………………………………… 11 Item 5 Diagnostics – virus detection ……………………………………………. 13 Item 6 Diagnostics – antibody detection ………………………………………. 14 Item 7 FMD vaccines and vaccination ………………………………………… 17 Item 8 Closed Session …………………………………………………………. 19 1. Information on recent and future activities relating to the Caucasus, Turkey, Greece and Bulgaria 2. Matters arising from the 67th Executive Committee meeting, 25-26 April 2002 2.1 Capacity of FMD Reference Laboratories during crisis situations 2.2 Review of “The minimum requirements for importation into Europe of live animals, fresh meat and offal of the bovine species” 2.3 Development of Reference Sera 2.4 Objectives of Phase XVII 2.5 Guidance on the use of r values 2.6 Design of surveillance schemes, in particular through use of tests for antibodies to NSPs 2.7 Workshop on the internal quality control of ELISAs 2.8 Risk analysis tools 3. Matters raised by the Secretariat or Members 3.1 Future of closed or open meetings 3.2 Procedures for writing reviews for EUFMD 3.3 EUFMD website 3.4 Distribution of information 3.5 Standardisation of papers for EUFMD meetings 3.6 Involvement of epidemiological modellers 3.7 Audio and video-taping of meetings

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3.8 Support for attendance of FMD diagnostic laboratory personnel from countries not free of FMD in the region 4. Next Meeting Adoption of the report ………………………………………………………… 24 Closing remarks ………………………………………………………………. 24

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LIST OF APPENDICES

Page Appendix 1 ……………………………………………………………………………….. 25 David Paton Update on the global FMD situation Appendix 2 ……………………………………………………………………………….. 33 N.J. Knowles and P.R. Davies Molecular epidemiological studies on recently isolated foot-and-mouth disease viruses Appendix 3 ……………………………………………………………………………….. 46 Patrick J. O' Reilly, Michael O’Connor, Ann Harrington, Sally Gaynor and Dianne Clery Foot-and-Mouth Disease in Ireland; history, diagnosis, eradication and serosurveillance Appendix 4 ……………………………………………………………………………….. 58 Nigel P. Ferris, Geoff H. Hutchings, Scott M. Reid and Brenda Newman Laboratory virological investigations relating to the 2001 foot-and-mouth disease outbreak in the United Kingdom Appendix 5 ……………………………………………………………………………….. 59 Soren Alexandersen, R. Paul Kitching, Leonard M. Mansley, and Alex I. Donaldson Clinical and laboratory investigations of five outbreaks during the early stages of the 2001 foot-and-mouth disease epidemic in the United Kingdom Appendix 6 ………………………………………………………………………………... 67 Michael Thrusfield Initial analysis of the 2001 UK foot-and-mouth disease epidemic in Dumfries and Galloway, Scotland Appendix 7 …………………………………………………………………...................... 69 Emiliana Brocchi, S. Grazioli, F. Fallacara, M. Bugnetti, S. Bellini, and F. De Simone Update of Swine vesicular disease in Italy during 2002: epidemiology and diagnosis Appendix 8 ……………………………………………………………………………….. 74 Nilay Ünal The foot-and-mouth disease situation in Turkey Appendix 9 ………………………………………………………………………………. 76 A.N. Bulut, C. Çokçalýþkan, and B. Alpay A serosurvey following the autumn-2001 vaccination campaign in Thrace Region of Turkey Appendix 10 ……………………………………………………………………………… 87 A.N. Bulut, C. Çokçalýþkan, and B. Alpay A serosurvey to trace non-structural proteins to FMDV conducted with the sera from Thrace Region of Turkey

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Page

Appendix 11 ……………………………………………………………………………… 92 S. Aktaþ, U. Parlak and F. Özyörük Genetic characterisation of type O and A viruses isolated from outbreaks between 2000-2002 in Turkey Appendix 12 ……………………………………………………………………………… 98 Hagai Yadin FMD control in endemic areas Appendix 13 ……………………………………………………………………………… 103 François Moutou and Benoit Durand Modelling of foot-and-mouth disease: a comparison of models Appendix 14 ……………………………………………………………………………… 109 Simon J. Barteling Problems in stamping-out and rendering practice: suggestions for improvements and alternatives Appendix 15 ………………………………………………………………………………. 113 Simon J. Barteling and Paul Sutmoller Culling versus vaccination: challenging a dogma in veterinary (FMD) science Appendix 16 ………………………………………………………………………………. 122 Chris Griot and Lukas Perler Evaluation of acceptance of alternative FMD eradication strategies in Switzerland Appendix 17 ………………………………………………………………………………. 125 Soren Alexandersen, Melvyn Quan, Ciara Murphy, Jeannette Knight and Zhidong Zhang Studies of quantitative parameters of virus excretion and transmission in pigs and cattle experimentally infected with foot-and-mouth disease virus O UK 2001 Appendix 18 ……………………………………………………………………………… 141 Zhidong Zhang, Ciara Murphy, Melvyn Quan, Jeanette Knight and Soren Alexandersen Quantification and duration of foot-and-mouth disease virus RNA in bovine esopharyngeal fluid as determined by real-time RT-PCR Appendix 19 ……………………………………………………………………………… 150 Melvyn Quan, Louise Matthews, Ciara M. Murphy, Zhidong Zhang, Jeanette Knight, Mark E.J. Woolhouse, and Soren Alexandersen Modelling of viraemia in pigs infected with foot-and-mouth disease virus Appendix 20 ……………………………………………………………………………… 160 Ciara M. Murphy, Zhidong Zhang, Melvyn Quan, Jeanette Knight and Soren Alexandersen Expression of inflammatory and antiviral cytokines in pigs experimentally infected with foot-and-mouth disease

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Appendix 21 ……………………………………………………………………………… 174 Aldo Dekker, A. Bouma and M.C.M. de Jong Foot-and-mouth disease virus transmission between individually housed calves Appendix 22 ……………………………………………………………………………… 179 P.W. Mason, J.M. Pacheco, Q.-Z. Zhao and N.J. Knowles Comparisons of the complete genomes of Asian, African and European isolates of a recent foot-and-mouth disease virus type O pandemic strain (PanAsia) Appendix 23 ……………………………………………………………………………… 190 Neeraj Aggarwal, S. Cox, R.J. Statham, and P. V. Barnett Characterisation of monoclonal antibodies against foot-and-mouth disease vaccine strain C1 Oberbayern and their reactivity with field isolates Appendix 24 ……………………………………………………………………………… 194 S. Grazioli, F. Fallacara and E. Brocchi Monoclonal antibodies against FMDV type Asia 1: Preliminary characterization and potential use in diagnosis Appendix 25 ……………………………………………………………………………… 203 Scott M. Reid, N.P. Ferris, G.H. Hutchings and S. Alexandersen Diagnosis of foot-and-mouth disease virus by automated RT-PCR Appendix 26 ……………………………………………………………………………… 210 Scott M. Reid, N.P. Ferris and G.H. Hutchings Comparison 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 Appendix 27 ……………………………………………………………………………… 220 Nilgün Özdural, Aysel Candaþ, and Melahat Cengiz Diagnosis of A22 Mahmath and O Manisa FMD viruses from field samples: Development of a latex agglutination test kit Appendix 28 ……………………………………………………………………………… 226 David Paton, Robert M. Armstrong, L.S. Turner, P.A. Hamblin, M. Corteyn, D. Gibson and J. Anderson FAO Collaborative study Phase XVII: Standardisation of FMD antibody detection Appendix 29 ……………………………………………………………………………… 235 Esther Blanco Lavilla, Jordi X. Feliu, Francisco Sobrino and Antonio Villaverde Specific detection of antibodies against FMD by engineered ß-galactosidase enzymatic sensors

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Appendix 30 …………………………………………………………………………….... 242 L. Jacobs, G. Chénard, T. Jelsma, and K. de Clercq Modification and further validation by different laboratories of a FMDV type O Specific ELISA for detection of antibodies in all susceptible species Appendix 31 ………………………………………………………………………………. 248 Bernd Haas and Karl J. Sorensen Comparison of ELISAs for the differentiation of infection from vaccination by detection of antibodies to the non-structural protein 3ABC of foot-and-mouth disease virus Appendix 32 ……………………………………………………………………………… 257 R.M. Armstrong, Z. Zhang, N Aggarwal, P.A. Hamblin and S.J. Cox Detection of IgG against the foot-and-mouth disease Virus (FMDV) non-structural polyprotein 3ABC in cattle and sheep Appendix 33 ………………………………………………………………………………. 272 B.M. deC. Bronsvoort, J. Anderson, V.N. Tanya, R.P. Kitching, and K.L. Morgan Evaluation of the 3AB CHEKITTM for an African cattle population with endemic foot-and-mouth disease Appendix 34 ………………………………………………………………………………. 283 L. Schalch, D.E. Rebeski, H. Samaras, G. Lozano, B. Thuer, C. Schelp Recently generated data with the CHEKIT-FMD-3ABC ELISA kit and methods to monitor the operational performance of a 3ABC ELISA Appendix 35 ………………………………………………………………………………. 303 Scott Liu, Tseng Yuan Chang, Alan M. Walfield, Mei Lun Zhang, Kenneth K. Sokoll, Shih Ping Chen, Ming Chang Li, Yeou Liang Lin, Ming Hwa Jong, David Yang, Nancy Chyr, and Chang Yi Wang Synthetic Peptide-based serosurveillance and vaccine system for FMD Appendix 36 ………………………………………………………………………………. 321 Kris De Clercq and David K.J. Mackay Regulations of FMD vaccines within the European Union Appendix 37 ………………………………………………………………………………. 327 Lukas Bruckner and Christian Griot FMD vaccines: Potency testing in the target species Appendix 38 ………………………………………………………………………………. 328 Gülhan Aynagöz, A. Naci Bulut, and Aydin Coþkuner Potency control of FMD vaccine in the field during 2001-2002 Appendix 39 ………………………………………………………………………………. 331 Fuat Özyörük, Ünal Parlak, Gülhan Aynagöz, and Hidayet Bozoğlu Monoclonal antibody based detection of 3A containing non-structural proteins In A1(OH)3 adjuvanted foot-and-mouth disease vaccines

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Appendix 40 ……………………………………………………………………………… 336 Eliana Smitsaart, N. Mattion, G. Mazzuca, B. Robiolo, E. Maradei, J. Filippi, A. Sadir, A. Falczuk, J. La Torre, A. Pedemonte, R. D’Aloia, O. Periolo, G. Cadenazzi, E. Palma, and R. Bellinzoni Foot-and-mouth disease in Argentina: Development of vaccines for emergency, control and eradication of the disease Appendix 41 ………………………………………………………………………………. 349 S.I. Deliloglu Gürhan, M. Mustafaev Akdeste, Z. Mustafaeva, Akdeste, G. Aynagöz, G. Ünver, Nilay Ünal, and N. Çelik Preparation of Synthetic Peptide FMD vaccine with newly developed Antigen-Polymere conjugates to be used as imunogen and vaccine in veterinary medicine Appendix 42 ………………………………………………………………………………. 358 David Paton Proposals for completion of Phase XVII and for Phase XVIII Appendix 43 ………………………………………………………………………………. 360 List of participants

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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 in Çesme, Izmir, Turkey, from 17 to 20 September 2002. The meeting was chaired by Dr Kris De Clercq (Belgium). Members of the Group present were: Drs. Aldo Dekker (the Netherlands), Franco De Simone (Italy), Alex Donaldson (UK), Chris Griot (Switzerland), Bernd Haas (Germany), Per Have (Denmark), Vilmos Palfi (Hungary), Ms Nilay Unal (Turkey) and Hagai Yadin (Israel). Apologies were received from Dr Francois Moutou (France) and Dr J.M. Sanchez-Vizcaino (Spain) who were unable to attend. Dr Keith Sumption, Secretary of the EUFMD opened the meeting by welcoming all participants to the meeting. It was pleasing to see such an excellent turnout and thanked especially those who had travelled so far to attend the meeting. A welcome was also conveyed to the representatives of international organizations present, in particular, Dr Schudel from OIE and Dr Füssel from the EC. Special gratitude was conveyed to the Government of Turkey, in particular Dr Hüseyin Sungur the Director General of the General Directorate of Protection and Control of Turkey, and his team, who have worked so hard on the arrangements for hosting this Session of the Research Group and for arranging such an attractive venue. Dr Sungur was invited to take the floor. Dr Sungur welcomed all the participants to his country and was honoured to host this session in Turkey. He expressed the hope that this meeting will contribute to the control of FMD, and therefore the development of the livestock sector and food security in all countries. He indicated that individual efforts of countries in the control of contagious animal diseases are not always sufficient to protect their livestock from such diseases. He went on to say that in this respect, FMD is one of the best examples to use and highlighted the need for regional, or even global, cooperation for disease control. He stated that it is mainly for this reason that some countries and international organizations have developed several regional animal health projects. He used EUFMD as an example, which was established in 1954 with the help of FAO. Dr Sungur briefly informed the meeting of the measures taken by Turkey in its efforts to control FMD. The FMD Institute has been recently modernised and vaccine production conditions have improved sufficiently to obtain protection against FMD by the vaccination of at least 80% of the susceptible population. Vaccine demand has been met through local production and when required, through importation. Intensive preventive vaccination campaigns have been organised to take place twice a year. A Vaccine Control Laboratory has been established in Bornova near Izmir where modern diagnostic methods have been introduced. The illegal movement of animals has been minimised by the intensification of border control, and very strict measures such as quarantine, disinfection and preventive vaccination have been introduced to control FMD outbreaks. Meat, milk and poultry products for exportation are only obtained from FMD-free areas and International Veterinary Health Certificates are issued only for such products. Recording and identification of large ruminants started in September 2001.

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Dr Sungur took this opportunity to congratulate Dr Keith Sumption on his new position as Secretary of the EUFMD. He assured the meeting that Turkey will continue to contribute to the future studies in the field of FMD epidemiology, diagnosis and vaccine production to the best of its ability. On closing, Dr Sungur thanked all the participants for attending this meeting and wished the meeting success. He hoped that the participants would find some time during the very tight schedule of the meeting at least to benefit from Çesme, one of the most attractive tourist regions of Turkey. Dr Keith Sumption once again took the floor and on behalf of FAO conveyed the apologies of Mr Doumandji, the FAO Representative in Turkey, who at the last minute, was unable to attend. He recalled the long history and good relationships of the Government of Turkey with the EUFMD Commission, which is recognised by FAO. The work undertaken over many years has been very important in reducing the risk of FMD introduction into other member states of EUFMD. FAO recognises the efforts of the Turkish Authorities to promote animal health and disease control in the Eurasian region. The cooperation between Turkey and Iran through a FAO TCP project, may be followed by further cooperative transboundary disease actions. The involvement of a Turkish expert in an EC funded expert mission to Iran scheduled in October is a good sign of this. The expertise, experience and regional position of Turkey should be of great importance to the regional FMD control programme. FAO also recognises the efforts made by the Government of Turkey in the Tripartite action (EUFMD, EC and OIE) involving 3 countries in the southern Balkans (Turkey, Greece and Bulgaria). This work is ongoing and important for EUFMD members. The joint application for a TCP project involving these 3 countries has been submitted to FAO and it is hoped that approval will be given in order to begin activities in 2002. On closing, in his capacity as the new Secretary of the EUFMD he thanked everybody for their warm welcome into the Commission and is honoured to be able to work in support of the Research Group. He reminded the meeting of the importance of the Research Group to the Commission as this Group represents a very significant proportion of the research-active FMD scientists in the world. In addition, the Sessions of the Research Group are not only scientific conferences since the recommendations of the meetings feed back to the CVOs of the 33 Member States of the EUFMD for scientific guidance. The floor was given to Dr Kris De Clercq, Chairman of the Research Group, who on behalf of the Group welcomed all to the meeting. He thanked the Turkish Authorities for the excellent organization of the meeting. Most participants will know how much work goes into preparing such a meeting, especially with such a high number of participants and the hard work is much appreciated. He pointed out that at the RG level the collaboration with the SAP FMD Institute is good. The information received from the laboratory is of the utmost importance to the RG. In the Session of the Research Group held in Borovets in 2000, nobody had any idea of the events which took place in 2001 which were to change the world. Not only did 2001 bring back FMD but also the event which took place on 11 September created a possible tool for agro-terrorism. Many meetings, one of which was held in FAO HQs in February, were held

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on this in 2001 and 2002. As an outcome of these many meetings, our diagnostics were debated, especially the NSP test and more generally the validation of tests. We will consequently have to focus on the urgent development of reference sera which cannot be done by one laboratory alone. Therefore, a closer network of laboratories will have to be established in the near future. EUFMD will surely play a major role in this. Other important items were slaughter policy and vaccines which will also be presented at this meeting. On the subject of vaccines, Dr De Clercq will inform the meeting of progress made in 2001/02 concerning the Eur.Pharm. and other documents established by EMEA and OIE. The year 2002 brought other changes to the Commission. Dr Yves Leforban left his position as Secretary to return to France. He acted as Secretary for a period of 8 years during which he did an outstanding job. The position was taken up by Dr Keith Sumption as of 1 September 2002. Dr De Clercq extended a warm welcome to the new Secretary. Dr De Clercq presented individually each member of the Research Group present to the meeting and presented apologies from Drs Moutou and Sanchez-Vizcaino who were unable to attend. Dr De Clercq then gave the floor to Prof. Reinhard Ahl, Chairman of the Sub-Committee of Animal Health of the Scientific Committee for Animal Health and Welfare of the EU Commission in Brussels. Professor Ahl conveyed the regards of the Committee to the participants and organisers of this meeting. He pointed out that a number of questions had arisen from the recent epidemics of FMD in different parts of the world which in part, are difficult to solve by experimental studies alone, since the introduction of FMD cannot be predicted with complete certainty. New experience may lead to resolving some of the problems, but he indicated that we should be aware of the limit to our science. Adoption of the Agenda The Chairman proposed that the following Agenda should be adopted. Item 1: Information on current FMD situation in the world and reports on outbreaks Item 2a: FMD control: epidemiology, surveillance, control measures: focus on endemic zones Item 2b: FMD control: epidemiology, surveillance and control measures: focus on

epidemic incursions Item 3: Pathogenicity and transmission Item 4: Virus characterisation Item 5: Diagnostics - virus detection Item 6: Diagnostics - antibody detection Item 7: FMD vaccines and vaccination Item 8: Closed Session The Agenda was adopted as proposed. Item 1: Information on current FMD situation in the world and reports on Outbreaks

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Dr David Paton, representing the World Reference Laboratory (WRL), gave a brief overview of the global FMD situation (Appendix 1). Foot-and-mouth disease remains endemic in many parts of Asia, Africa and South America. Since there is limited reporting, our picture of the disease situation worldwide is incomplete. So far in 2002, 103 viruses from 17 countries were submitted to the WRL for analysis. Type O was received from 13 countries, type A from 4 countries, type Asia 1 from 3 countries, whereas SAT 2 was received from 1 country. These figures compare with 179 viruses received from 29 countries in 2001. He presented molecular epidemiological studies (Appendix 2) showing that in the last few years, a succession of different type A viruses have been recorded in Iran and Iraq. A group of Iranian viruses from 2001 fall in a unique phylogenetic cluster with a position intermediate to the European/South American, and Indian/Middle Eastern topotypes. Two Iraqi isolates from 2002 form a new lineage within the Iran96 topotype. For these groups, as well as A22-like Iranian viruses from 2000, there appear to be few suitable vaccine strains. Dr Patrick O’Reilly described the way FMD was introduced into Ireland (Appendix 3). He summarised aspects of FMD diagnosis, control, eradication and serosurveillance in Ireland during 2001. Mr Nigel Ferris described the enormous number of samples that had to be processed in the Pirbright laboratory (UK) which severely taxed the laboratory resources, and highlights the need for contingency planning (Appendix 4). Laboratory-based methods to confirm clinical diagnoses for secondary cases were too slow. This caused problems, particularly for diagnosing the disease in sheep, which may not become obviously ill. Clinical and laboratory investigations by staff from the Institute for Animal Health (IAH), Pirbright, of five foot-and-mouth disease (FMD) outbreaks during the early stages of the UK 2001 epidemic were described by Dr Soren Alexandersen (Appendix 5). He discussed the epidemiology of these cases with emphasis on the appearance of clinical signs, the incubation periods, the role of airborne transmission, and their temporal and spatial links. A description and some provisional analyses of the epidemic in South West Scotland, were given by Dr Michael Thrusfield (Appendix 6). The traditional control techniques (slaughter of infected premises and dangerous contacts, and patrolling) were implemented rapidly throughout the epidemics course, although the novel extent of the pre-emptive cull competed detrimentally for scarce resources. The estimated dissemination rate dropped below the value of one by the third week of March (before pre-emptive culling began). Three smaller peaks in the epidemic curve followed this date, but were caused by “sparks” some distance from the initial focus. This highlights the dangers of interpreting simple summary parameters, such as the estimated dissemination rate, outside their geographical context. Conclusions • The detection, in the Middle East, of new type A strains that are poorly covered by

existing vaccine strains gives reason for concern. Any military conflict could exacerbate this risk.

• No laboratory in the World has the resources immediately available to deal with a major

epidemic. Contingency plans should be developed so that testing capacity can be scaled

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up as quickly as possible. • It remains controversial whether the peak of the UK epidemic had already been passed

before novel control measures were introduced. A fuller analysis of the data is awaited. Recommendations • Further steps should be taken to encourage the submission of samples to WRL so as to

give a fuller picture of the world situation. • Vaccine manufacturers and the WRL should collaborate closely to ensure that antigenic

characterisations of field viruses includes comparisons with all available vaccine strains.

• High throughput laboratory vırus detection tests and field tests should be developed and validated with high priority.

Item 2a: FMD control: epidemiology, surveillance, control measures: focus on endemic

zones Under this item six papers were presented, the first dealt with SVD in Italy while 5 of the papers reviewed the FMD situation and control measures in the Middle East, Turkey and Israel. Dr Franco De Simone dealt with the SVD epidemiology and diagnosis in Italy where the disease is endemic in southern regions (Appendix 7). In the last 6 months a zoo-epidemic situation occurred. A total of 166 outbreaks were reported lasting from February to July. These recent outbreaks were characterised by lack of clinical signs as observed also during the past years. This forms a serious obstacle for field diagnosis and makes clinical examination useless. He showed that the most reliable virological test is the Immune-PCR performed on faeces suspension. The antigenic profile of virus isolates from this epidemic performed by monoclonal antibodies was comparable. This profile was comparable to the fourth chronological antigenic type that appeared in 1992 and remained unchanged during the last decade. The first paper regarding FMD in Turkey, presented by Dr Nilay Ünal reviewed the outbreaks during 2002 (Appendix 8). Three serotypes O1 Manisa, A Aydin98 and Asia 1 were circulating in 2002. No outbreaks due to Asia1 have been notified since April 2002. In general, the number of outbreaks (29) decreased in comparison to previous years and no outbreaks have been notified in the Thrace region since June 2001. Trivalent vaccine produced by the Sap Institute was used for the routine bi-annual vaccination. In addition, in the Thrace region, trivalent vaccine provided by EUFMD was used and a serological survey was carried out. Vaccination rate in the Thrace region gained 97% for cattle and 73% for small ruminants. Regarding vaccine production at the SAP Institute, production systems and technology were improved and 22 million monovalent vaccine doses were produced in 2002. Studies for independent vaccine control were started in the Bornova Veterinary Control and Research Institute in Izmir.

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Dr. Naci Bulut summarised the serosurvey performed after FMD vaccination in Thrace during autumn 2001 (Appendix 9). In total 4061 sera taken on day 0, 28, 60 and 120 pv, were examined by LPB ELISA. The protection level indicated by the test was acceptable after 28 and 60 days but insufficient after 120 days. He then reported on a sero-survey for NSP antibodies by 3ABC ELISA conducted on the same sera (Appendix 10). In total, 1310 sera were tested of which 16 (1.22%) were found positive all of them from different herds. This gave no significant indication of circulating virus in the region. Dr. Sinan Aktas presented a paper on the genetic characterisation of FMD type O and A viruses in Turkey (Appendix 11). The results showed that type A viruses isolated from Turkey between 2000-2002 were closely related to A Iran 96. Regarding the O type although slightly different type O viruses were isolated in Turkey, these were antigenically found to be related to the vaccine strain O Manisa. The last paper presented by Dr. Hagai Yadin reviewed the policy of FMD control for prevention and emergency situations in an FMD endemic region (Appendix 12). This policy consists mainly of annual vaccination of all livestock including sheep, goats and pigs. The imported vaccine is checked upon arrival for serological response. An annual serological surveillance is performed to check the annual vaccination campaign. In case of an FMD outbreak, animals are vaccinated and risk animals are placed under quarantine. Conclusions • Due to the lack of clinical signs, the laboratory diagnosis of SVD is based on examining

faeces samples instead of epithelial tissues. The VI test is affected by the possible loss of virus infectivity and the presence of entero-viruses other than SVDV that may grow more quickly than SVDV. The Immune PCR assay developed at the Brescia Reference Centre circumvents these difficulties.

• Serosurveillance in Thrace indicated that the duration of protection following FMD

vaccination was shorter then 120 days which indicates that booster vaccination is required at least 3 times the first year and twice a year thereafter.

• The test used for the detection of NSP antibodies revealed that the probability of active

virus circulation in Thrace is very low. • FMD viruses circulating in Turkey seem to be covered by current vaccine strains. • The policy of FMD control in an endemic area should consist of strict surveillance and

vaccination, including vaccine control and sero-surveillance. In case of an outbreak, quarantine and emergency vaccination is carried out.

Recommendations • SVD control should be based on serological survey and on the detection of virus in

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faeces by immune-PCR. • For the future a more potent FMD vaccine should be applied in the Thrace region

with a longer protection period. A training programme for the organisation and execution of a serosurvey should be organised to improve future serosurveys.

• Surveys for the detection of antibodies against NSPs and confirmatory tests should

be continued to detect possible incursions into the Thrace region. • Turkey should continue to characterise FMD viruses from new outbreaks and also

continue to send samples to WRL, Pirbright. • The policy of FMD control as adapted in Israel can form a model for endemic

areas.

Item 2b: FMD control: epidemiology, surveillance and control measures: focus on epidemic incursions

The first paper under this heading was presented by Dr Michelle Remond on behalf of Dr Francois Moutou who was unable to attend. The paper describes the different models which have been used to help decision-making during FMD epidemics (Appendix 13). Prior to the UK 2001 epidemic a few models had been used for operational purposes during epidemics, however, the majority had been used as training tools. The UK 2001 epidemic stimulated the development of new models, including deterministic differential equation models, detailed microsimulation models and stochastic/deterministic models. In the paper these were compared using information from publications and their strengths and weaknesses as decision support tools were reviewed. Models had a major influence on the introduction of novel disease control strategies in the UK during the early stages of the 2001 epidemic even though the parameters on which they were based were collected during the 1967-68 UK epidemic when farming conditions were very different. During the course of the 2001 UK epidemic when contemporary data became available the models were refined and improved. Following this presentation, Dr Michael Thrusfield reviewed the history of the development of models in the field of biology. He informed the audience that the polarisation of views between the quantifiers and non-quantifiers about the appropriateness of models seen during the UK 2001 epidemic was not new as a similar debate occurred when models were first introduced several centuries ago. He described the different types of models and the approaches, deductive or inductive, used for their development. Models can be used for retrospective or present-time analysis or for future prediction. Clearly, the value of models is dependent on the accuracy of the input data. Satisfying that requirement is most challenging for predictive models. In the context of the application of models to FMD, Dr Thrusfield underlined the difficulty of modelling the evolution of the epidemic due to the complexity of farm management systems and differences between livestock species in respect to their susceptibility and amplification of virus. Dr Simon Barteling presented 2 papers on the procedures for managing outbreaks, including biosecurity and vaccination. The first paper (Appendix 14) dealt with the problems of containment of disease on suspicious and outbreak farms. Principles and technologies developed for high-containment laboratories were discussed and, also whether they could be

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implemented on suspicious and outbreak farms in addition to the current bio-containment practices. For transport and rendering of infected carcasses a closed system was proposed with the rendering process starting already during transport. The practicality of reducing the concentration of airborne virus in infected farms by spraying with acid solution or by the installation of high efficiency air filtration systems was discussed. It was pointed out that spraying water equivalent to a heavy rainfall shower was required to produce a significant reduction in airborne virus concentration. Using acid sprays in the same way might not be practical. However, the effects of an evapourable acid such as acetic acid is not known and might well be evaluated as well as the effect of citric acid spray on the FMDV presence on surfaces. His proposal that the vaccination of animals on large suspicious or infected premises could assist disease control during an epidemic was also challenged since premises containing infected animals would be difficult to properly clean and disinfect and therefore efficient decontamination might be impossible. However, the present possibilities to use protective vaccination or suppressive vaccination must be kept open. Dr Barteling’s second paper considered contiguous culling and, as an alternative, emergency vaccination (Appendix 15). Although the simplicity of the contiguous-cull has the advantage of rapid decision-making it has as a main disadvantage of the associated risk of further spread of the disease. The other drawback is the associated large number of animals that have to be culled and the environmental problems as well as the human dramas. Emergency vaccination has the disadvantage that it takes time (organisation plus the 4-5 days before protection starts). The spread of disease during this time should be anticipated for estimating the size of the vaccination zone. When carried out by well-trained vaccinators, the risk of associated spread of disease can be minimised. Dr Barteling stated that the use of vaccination is very much hampered by concerns about the risk of (vaccinated) carriers and by the belief that “vaccination suppresses the symptoms but does not eradicate the disease”. Dr Barteling claimed that during the last 15 years of vaccination – when carried out with a qualified vaccine – campaigns were always successful and there were no indications of ongoing or re-occurring disease, he recommended that this belief does not hold true. Therefore, he concluded that the associated “punishment” on export trade should be discontinued. During the discussion which followed Dr Barteling’s argument in favour of vaccination was disputed. Several participants pointed out that in the event of outbreaks vaccination alone will not control the disease - additional measures, including movement stand-still and zoo-sanitary procedures are essential. In regard to the consequences for trade of vaccination not everybody agreed with Dr Barteling’s opinion that surveillance can be employed as effectively in a vaccınatıng country as ın an FMD-free non-vaccinating country even when supported by testing for antibodies against non structural proteins (NSP). However, when vaccination is carried out on a limited scale – just to control an outbreak – both methods of eradication must be considered. Dr Chris Griot presented a paper on the evaluation of eradication strategies including the acceptance of any future vaccination of livestock in case of an FMD outbreak in Switzerland (Appendix 16). The evaluation involved a roundtable discussion with the livestock producers,

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state veterinary services, consumer organisations, animal rights activists and food retailers. The aim was to inform all participants about the basic aspects of FMD, current eradication strategies and the alternative possibilities. The main conclusions drawn from these discussions were a) stamping out of infected herds is accepted to be the basic tool for eradication; b) mass prophylactic vaccination is not conceived to be an option for the protection of animals; c) suppressive vaccination is rejected; and, d) protective vaccination is accepted by all stakeholders. The consumer organisations as well as the food retailers seemed to be willing to accept the marketing of products from vaccinated animals although there was some hesitation expressed by food retailers to sell products from FMDV vaccinated animals. Dr Alex Donaldson informed the meeting that the “minimum conditions for the importation into Europe of live animals fresh meat and offal of the bovine species” are under revision. Conclusions

• Models can be a valuable tool to aid the decision-making processes but in the absence of

strong data they should not be used to drive control policy. • Policy discussions on disease eradication strategies with stakeholders and consumer

organisations should be initiated before any FMD “crisis” arises. • The use of highly sensitive fully validated NSP tests would allow veterinary services to

establish whether virus was still circulating following a vaccination campaign and compensate for the reduced effıciency of clinical surveillance resulting from vaccination.

• The devastating consequences of the UK 2001 epidemic have stimulated consideratıons of

disease control strategies less reliant on extensive cullıng. Recommendations • The possibility of applying increased biosecurity to farms, transport of carcasses and

rendering plants during FMD emergencies using containment principles from biosecure laboratories should be investigated.

• Refine existing models and apply them as a decision support process after peer review. • It is recommended to investigate whether principles applied in the high-containment

laboratory can – in addition to existing procedures – also be applied on suspicious and outbreak farms. The development of a completely closed system for transporting carcasses which, on arrival at rendering plants enter air locks, should be investigated.

• It is recommended that the development and full validation of improved tests for

serological surveillance, in particular the NSP tests, is supported. Item 3: Pathogenicity and transmission This item started with a presentation by Dr Soren Alexandersen about quantitative parameters of virus excretion and transmission (Appendix 17). The purpose of this study was to provide quantitative disease parameters that could improve models predicting the spread of FMD

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virus. Transmission of FMD virus within a group of cattle resulted in more severe lesions in the contact infected animals than in the inoculated animals. Airborne virus excretion starts with the occurrence of clinical signs and fall within the viraemic period. Clinical disease appears approximately 3-4 days later in the contact cattle under the conditions used. In pigs, the time of development of clinical disease in contact pigs seems to be related to the number of pigs kept together and that virus transmission is more efficient when more animals are kept together. Dr Alexandersen concluded that real time RT-PCR on nasal swabs is a promising tool for the detection of FMD. Based on the estimated half-life of virus RNA excreted it may take up to 30 days before the concentration falls below 1 RNA copy per ml of sample. Dr Zhidong Zhang studied the temporal patterns of viral load in esopharyngeal fluid (OP-fluid) in FMDV infected cattle (Appendix 18). The experiments showed no correlation between virus growth rate, doubling time or virus peak levels and whether cattle became carriers or non-carriers. In contrast, the viral load clearance rate (half-life) early after peak levels was significantly slower in carriers than in non-carriers indicating that an early event determines whether or not a carrier state is established. Albeit that the clearance in OP-fluid was faster in non-carriers than in carriers, the clearance was slower than observed in the blood or in nasal or mouth swabs. Dr Melvyn Quan described efforts to develop a mathematical model of FMD virus infection in pigs (Appendix 19). In the two infection experiments performed, including groups given a high, medium and low dose of virus, he found statistical significant differences between the first time active vireamia was detected in the groups. He was able to fit a model partly explaining the results, but more research has to be performed on the validity of the model. Dr Ciara Murphy described studies of inflammatory and antiviral cytokines in pigs experimentally infected with FMD virus (Appendix 20). After a short introduction on the role of the different cytokines she showed results of two infection experiments and both experiments showed a complicated pattern of activation and degradation of mRNAs isolated from PBMCs. More research is needed to clarify the results and to define the role of the different cytokines in the innate immune response to FMD virus. Dr Aldo Dekker presented a paper evaluating transmission of FMDV between individually housed calves (Appendix 21). The study used FMDV type O from the 2001 outbreaks in the Netherlands. In experiments 1-4 a single calf were inoculated intranasally and were housed at a 1 metre distance from a naïve calf and in experiments 5-6 a single intranasally inoculated calf housed between two naïve calves. Although the calves were housed side by side in experiment 5-6, the calves could only with difficulty get in direct contact. All inoculated calves became infected but none of the other calves showed any signs of infection, i.e. no rise in temperature, no vesicular lesions and serologically negative. Conclusions • The number of animals kept together in direct contact may influence the incubation period

and the efficiency of spread.

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• Real time RT-PCR on nasal swabs is a promising tool for the detection of FMD. • A difference in the viral load in OP-fluid samples of carrier and non-carrier cattle was

observed already early in infection, i.e. shortly after peak levels. • It was possible to fit a preliminary mathematical model to experimental data of viraemia

levels in pigs but more work is needed to optimise the model and to test several interesting hypotheses.

• Preliminary work of cytokine mRNA levels and activation of innate immunity in FMDV

infected pigs is interesting but future work is needed to clarify and extend the findings. • Transmission of FMDV between calves may be limited when separated physically. Recommendations • Efficiency and speed of transmission of FMDV is variable and highly dependant on direct

or indirect contact intensity and on housing conditions. More studies under varying conditions and using several different strains of virus will provide a better understanding of the epidemiology of FMD and such data are urgently required for development of realistic simulation models of disease spread. Also modelling of virus infection in the animals and studies of pathogenesis can help in refining epidemiological models.

• More research aimed at understanding the mechanisms of the carrier state should be

encouraged. Item 4: Virus characterisation Dr. Soren Alexandersen presented the paper by Mason et al (Appendix 22) on behalf of Nick Knowles on the sequence analysis of 5FMDV type O PanAsia strains from separate geographical regions based on the comparisons of complete genomes. These analyses revealed a remarkable conservation of all portions of the genomes among the PanAsia virus isolates and provided confirmation of the close relationship between the viruses responsible for South African and UK outbreaks. The complete genome analysis indicated that only minor changes in the genome of FMDV can dramatically alter virulence in animals. Dr. Neeraj Aggarwal presented a paper on the characterisation of five monoclonal antibodies against FMDV vaccine strain C1 Oberbayern (Appendix 23). Based on their reactivity with five field isolates of type C viruses it was concluded that the MAbs represented 4 different epitopes 2 of which were neutralising. Due to the limited number of MAbs and unknown correlation between antigenic sites and in vivo protection, it was not possible to assess the suitability of the vaccine strain against the field isolates. It was concluded that a much larger panel of well characterised MAbs are needed to eventually replace the current characterisation based on establishment of r values using polyclonal antisera. Dr. Emiliana Brocchi reported on the characterisation of 24 Mabs raised against FMDV type Asia 1 (Appendix 24). Ten type specific MAbs identified at least 3 different sites involved in neutralisation, while 14 non-neutralising MAbs showed different features of reactivity with also heterologous FMDV types. MAbs against 2 of the 3 neutralising sites, as well as all non-

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neutralising MAbs, broadly reacted with 11 field isolates tested, suggesting the target sites are well conserved and stable, whilst one neutralising site seemed specific for the homologous strain. One isolate (Cambodia 3/93) appeared considerably changed being not recognised by any of the neutralising MAbs. Carefully selected MAbs proved to have high potential as diagnostic reagents. Sandwich ELISAs for antigen detection have been developed, specific for Asia 1 type or universal for all types tested (Asia, O, A, C) depending on the combination of Mabs selected as catching and conjugated antibody. A SPCE using a neutralising MAb as competitor proved to work in principle. It detected the candidate reference sera (Phase XVII) correctly, although improvements of sensitivity and specificity may be achieved through further studies. Following the presentation of papers a panel discussion was held for Items 3 and 4. Dr. Soren Alexandersen raised the following points: the need for more full length sequence data in addition to the partial sequence data of VP1; the need for more work on excretion, transmission and virulence studies for emerging FMD strains, so that it would be possible to predict the outcome of an infection if the disease entered the country. Dr. Aldo Dekker mentioned that the selection of vaccine strains should be done by determination of r values and epidemiological studies by sequencing. He stressed that more work is necessary on r values and the selection of reference sera. Dr. Emiliana Brocchi stated that sequencing is important mainly for epidemiological studies. Antigenic characterisation can be better achieved using a well-characterised panel of MAbs directed against known sites. MAbs profiling can also be a valuable tool, helping in the appropriate selection of vaccine strains. To this purpose MAbs to neutralising sites are appropriate considering that virus neutralisation is a potent, well known mechanism of protection in FMD. Dr. Sinan Aktas stated that the r values are important but the actual titres of antibodies induced by vaccine strains against field viruses is also very important for decision making. He also supported Dr. Brocchi for the usefulness of monoclonal antibody profiling. Dr. David Paton stressed the importance of standardised determination of r-values and pointed out the limited supply of post-vaccine sera. In this respect close cooperation between WRL and vaccine manufacturers is important. Conclusions • Sequencing studies conducted on FMD PanAsia virus isolates from distant geographical

regions confirmed the suitability of this method for analysing relationships between isolates and for establishing links to previous outbreaks.

• Partial sequence analysis provides useful information for most epidemiological studies.

The comparison of full genome sequences of isolates may provide additional information to be used for virus characterisation.

• Although sequence data are useful for establishing genetic relationships, they do not

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provide sufficient information on antigenicity. • Antigenic characterisation of viruses using polyclonal sera (r values) and monoclonal

antibody profiling provides essential data which complements genetic analysis. Recommendations • New field isolates should continuously be characterised antigenically (ELISA and VNT)

for the determination of r values against existing vaccine strains. • A panel of reference sera for antigenic characterisation should be prepared, updated as

necessary and be made available. • Given the large number of MAbs produced against FMDV in various laboratories efforts

should be directed for establishing common panels of MAbs that can be used for the antigenic characterisation of field isolates in addition to the determination of r values. One or more Institutes should be designated to coordinate information on MAbs produced in various laboratories and to produce MAbs against other strains in order to complete the panels, when necessary.

• More complete genome sequences should be obtained for better characterisation of FMD

viruses. Item 5: Diagnostics - virus detection Mr Scott Reid presented a paper on the diagnosis of FMD by real-time, automated RT-PCR (Appendix 25). The main objective of his work was to accelerate laboratory diagnosis in order to avoid situations where herds have to be stamped out on the basis of clinical suspicion alone. Improvements were made on the nucleic acid extraction from test samples in order to increase the speed, flexibility and capacity and of RT and PCR procedures without harming the assay sensitivity. PCR results can now be achieved on a 96-well plate by 2 people within a day. Definitive diagnostic results can be achieved on first passage cell culture supernatant fluids. Mr Scott Reid presented another paper (Appendix 26) on the comparison of RT-PCR procedures for diagnosis of clinical samples of FMD virus (serotypes O, A, C and Asia 1) under the European Union Concerted Action Group Project PL 98-4032. RT-PCR results from the 7 participating laboratories using locally-employed procedures were presented. Each laboratory had been supplied with 40 unlabelled epithelial suspensions (10 each of the serotypes O, A,C and Asia1) and five laboratories had also received cell culture grown virus preparations. A variety of procedures were used including conventional RT-PCRs with universal and serotype-specific primer sets, nested PCR, a novel PCR with restriction enzyme digestion and quantitative methods (not automated). Some primer sets were used in more than one laboratory. Dr Nilgün Özdural presented a paper (Appendix 27) on the development of a latex agglutination test kit for the detection of A22 Mahmatli and O1 Manisa FMD antigens which is intended for use in the field. The test is highly sensitive and therefore special attention should be given to the time of observation of agglutination.

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Conclusions • Once fully validated, real-time, automated RT-PCR could support the ELISA tests for the

detection of FMDV in epithelial suspensions and largely remove the necessity for virus isolation in cell culture for the confirmation of secondary cases.

• The comparison of the RT-PCR procedures confirmed the high sensitivity of RT-PCR

methods. Nested and novel PCR procedures had a higher sensitivity than conventional RT-PCRs with universal primers, but only quantitative methods scored all samples positive on epithelial suspensions.

• The simplicity and sensitivity of the latex agglutination test (LAT) makes it a good

candidate for a pen-side test in endemically infected areas. Recommendations • More validation work should be carried out on the automated RT-PCR and the system

should be optimised for the testing of probangs and milk. • A more extensive comparison of RT-PCR procedures should be performed. Thus, more

experimental details such as the position of the assay cut-off, the use of positive and negative controls and details of assay validation need to be made available. Also the specificity of the RT-PCR procedures should be further investigated.

• The specificity of the LAT kit should be more thoroughly evaluated. Item 6: Diagnostics - antibody detection Dr Kris De Clercq gave an overview of plans for an EU project for the production of FMD reference sera. The recent outbreaks in Europe and the need to develop and validate new assay systems, including those for the detection of NSP antibodies, have highlighted the importance of developing a panel of such reagents as quickly as possible. The project should involve a consortium of laboratories and an agency specialising in the storage, aliquoting and distribution of such materials. The EU Commission will have to tender the work for competitive bids. Drs David Paton and Bob Armstrong presented results from the FAO Phase XVII serology standardisation exercise (Appendix 28). The reference sera prepared in Phase XV against O Manisa, A22 and C Noville have been adopted by OIE. A new set of reference sera have been prepared against O SKR, A Iran 96 and Asia 1 Shamir. For each strain, a candidate strong, weak and cut-off serum was prepared and distributed, along with a negative serum, to 9 other laboratories. Reagents and a protocol were also supplied for solid phase competition ELISA (SPCE) for sera type O. Results from all the laboratories were presented, including analyses by virus neutralization test (VNT), liquid phase blocking ELISA (LPBE), SPCE, NSP tests and other in-house assays. The view was expressed that the weak and cut-off sera need to be slightly strengthened. Dr Esther Blanco presented a paper (Appendix 29) on the engineering of ß-galactosidase

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enzymatic sensors for the species-independent detection of strain specific antibodies against VP1 of FMDV by way of antibody-concentration dependent modulation of the activity of the enzymes expressing 12 copies of the viral peptide. Research is planned for the engineering of sensors for the detection of antibodies against NSP. During the 2001 crisis it became evident that quick and high throughput diagnosis of sero-conversion was necessary and therefore a quick and simple MAb based ELISA (Ceditest FMDV type O, Cedi-Diagnostics) was developed for the detection of antibodies against FMDV O in all species. The test was evaluated in a number of European FMD laboratories. From the data reported by Dr Liesbeth Jacobs (Appendix 30) it appears that results are highly comparable to SPCE conducted at VAR and IAH-P but further improvement is needed with regard to pig samples. Steps are being taken to develop equivalent tests for other species and other FMD virus strains. Two papers were presented on studies comparing in-house NSP-tests with a commercially available NSP-test (CHEKIT-FMD-3ABC ELISA, Bommeli). In the first case Dr Bernd Haas reported on the comparison made on the base of well-characterised samples from animals vaccinated and later on infected during an outbreak and of a panel of samples from potency trials (Appendix 31). As a result of the studies, the in-house competitive ELISA of the Danish laboratory appeared to be superior in sensitivity to the CHEKIT-FMD-3ABC ELISA. However, the sensitivity of the latter could be improved by reducing the cut-off value without significantly reducing its specificity. A second comparative study was carried out by Mr Robert Armstrong (Appendix 32) on samples including those of vaccinated and challenged cattle and sheep using the CHEKIT-FMD-3ABC ELISA in comparison with an in-house test of the WRL. It appears that this in-house test is again superior to the CHEKIT-FMD-3ABC ELISA in sensitivity. However, the data should be interpreted with care and take into account the sampling intervals and the different approaches to deciding on the cut-offs. The in-house test comprises 5 steps and is therefore laborious. In a field study carried out by Dr Mark Bronsvoort in Cameroon (Appendix 33) endemically infected with multiple serotypes of FMD, the results obtained by testing a statistical number of cattle blood samples with the CHEKIT-FMD-3ABC ELISA and the Danish C ELISA were compared with the VNT. The comparative sensitivity (with VNT) of both NSP tests was low in the studied bovine population where FMD is endemic. Suggestions were made to make a sensitivity comparison between both NSP tests before making final conclusions. Dr Lucas Schalch presented additional data (Appendix 34) to further validate the test at the established cut-off value, and to provide a method for monitoring the operational performance of the test. The additional data presented concerned in particular sera from experimentally and challenged cattle and sheep, from single and multiple vaccinated cattle and pigs and various samples of antibody negative animals. Validation results on specificity, sensitivity, repeatability and reproducibility were presented. The kinetic of antibodies against NSP was followed and certain infected animals were scored negative by 133 dpi. Dr Lucas Schalch also explained the charting method as an essential tool for day to day performance validation of laboratory tests.

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Following on from earlier developments of peptide based ELISAs for the detection of 3B NSP, Dr Scott Liu, presented a confirmatory system including 3-ELISA using peptides from NSP 3A and 3B (Appendix 35). There is a confirmatory 3B blocking ELISA and a confirmatory 3A ELISA which were established in order to increase sensitivity of the 3B NSP-ELISA and simultaneously to reduce the number of false positive results. However, true sensitivity of the test needs to be established especially using sera from vaccinated and challenged animals of various species and of known status. Conclusions • Plans will be finalised for an EU project for the production of FMD reference sera. • New candidate reference sera have been assessed under phase XVII but some

strengthening of weak positive and cut-off sera are required. • The development of enzymatic sensors for FMD diagnosis is at an early state of

development but deserves further investigation. • The commercially available test kit “Ceditest®FMD” for the detection of antibodies

against O1 FMDV is promising. • The CHEKIT-FMD-3ABC ELISA may be used with increased sensitivity in ruminants

when read with modified cut-off. Further work is necessary for the validation. • Animals vaccinated with a highly potent vaccine and challenged at the peak of immunity

and which become infected do not all necessarily develop antibodies to NSP. These findings confirm that NSP testing can only be carried out for surveillance purposes on a herd level.

• Based on European data, the competitive NSP-ELISA developed in Denmark has a very

high sensitivity. The Pirbright in-house NSP test performs well but is laborious. • The peptide-based ELISA (UBI) has sufficient specificity but has incompletely

characterised sensitivity. Recommendations • Laboratories are encouraged to develop a consortium to undertake the EU FMD reference

serum project. • The constitution of the new reference sera tested under phase XVII should be finalised. • Future FAO serology standardisation should make use of reagents developed from the

above-mentioned EU project. They should also look closely at the standardisation and internal quality control practiced within participating laboratories.

• Development of enzymatic sensor should be pursued in particular with regard to NSP.

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• The ELISA test kit “Ceditest” should also be developed for other serotypes and strains. • In relation to NSP tests more data on sensitivity in all target species, including sheep and

pigs, are needed from animals vaccinated and subsequently challenged. • Consideration should be given to increase sensitivity in the 3ABC-Chekit test, possibly by

modifying inter alia the cut-off value. • The transfer into commercial production of the Danish in-house SND test is strongly

encouraged. • Laboratories are encouraged to implement the charting methods for day by day

performance check. Item 7: FMD vaccines and vaccination Dr Kris De Clercq presented a report (Appendix 36) of the progress made by the Committee for Veterinary Medicinal Products (CVMP) ad hoc group, a working group comprised of members of the Immunological Working Party of the CVMP, of the Research Group of the EUFMD, OIE, Pharm.Eur., EU and at a later stage the FMD vaccine manufacturers tasked with preparing guidelines on the requirements for FMD vaccines. Recent recommendations for FMD control strategy make vaccination more likely and recent experience suggests that the general public and the farming industry would expect any vaccine that is used to be fully authorised. The guidelines currently being prepared propose possible solutions to many of the technical challenges presented by FMD vaccines and it is to be hoped that after the consultation period they will provide valuable advice to manufacturers. Dr Chris Griot reported that in Switzerland (Appendix 37) a batch of A Iran 96 antigen stored in the vaccine bank had been formulated into a vaccine lot and tested for potency in cattle with strain A Iran 99 as challenge. The experiment showed that the vaccine was able to protect against A Iran 99 which differs antigenically from A Iran 96. Dr Gülhan Aynagöz showed how the efficacy of FMD vaccine batches was assessed by testing serum samples taken from cattle in the field (Appendix 38). Blood sampling was done before and 21 or 28 days after vaccination. Based on LPB ELISA results it was estimated that more than 80% of vaccinated animals were protected against FMD homologous strains. The study is ongoing. Along with the application of assays able to detect antibodies against non-structural proteins (NSP) of FMDV it is important that antigens prepared for vaccines are free from contaminating NSP. Remarks on this matter were presented by Dr Fuat Özyörük (Appendix 39) who used an amplified Western Blot method to detect NSP 3A, 3AB and 3ABC in fluids eluted from Al (OH)3 adjuvanted vaccines. Anti 3A monoclonal antibody (2C2) produced in IZSLER, Brescia was used to demonstrate the presence of 3A, 3AB and 3ABC NSP at a detection limit of 11.5 ng/lane. Contaminating NSP 3 AB was sedimented together with cellular debris and could not be detected in vaccines tested. The paper presented by Dr Eliana Smitsaart (Appendix 40) described the development and performance of oil adjuvanted FMD vaccine used in recent emergency and mass vaccination

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campaigns in Argentina. Newly emerged virus strains of type A were characterised by in-vitro neutralisation assays and in-vivo protection studies, in addition to 2 dimentionally CFT, partial sequencing and MAb profiling. These studies lead to the incorporation of two new field strains of type A in the O1 Campos – A24 Cruzeiro vaccine. Dose-response studies including challenge in cattle were performed in order to determine the required amount of antigen of the new field strains in the vaccine. After two rounds of vaccination of the whole herd with the finally composed vaccine, immunity levels above 80% protection were achieved. The potency was reflected by the dramatic decrease in the number of FMD outbreaks reported. The contribution of the national vaccine bank was recognised. Dr Scott Liu described progress towards the development of a peptide vaccine based on the VP1 G-H loop and incorporating a T helper epitope. The peptide has now been evaluated in pigs and, to a lesser extent, in cattle. Dr Ismet Gürhan presented data on the production of a synthetic peptide vaccine combined with synthetic polymeric adjuvants (Appendix 41). Two amino acid sequences of VP1 protein of strain A Aydin 98 were conjugated to four locally synthesised polymeric complexes to prepare vaccines. Following preliminary studies such as in vitro cytotoxicity, in vivo toxicity in mice and guinea pigs, peptide specific antibody response in mice, and guinea pig potency tests, a so-called VAC2 was selected as a candidate vaccine for further studies. The results indicated that VAC2 is able to protect guinea pigs against type A FMDV challenge, but it did not induce sufficient specific antibody against either the synthetic peptides or intact virus in cattle. The antibody level estimated with both LPB ELISA and NT was not at an acceptable level for protection experiments. Quantitative as well as qualitative modifications such as addition of T-cell epitope sequences or increasing the quantity of the peptide in the vaccine may be useful to induce a stronger immune response in cattle. Conclusions • The Research Group fully supports the important initiative of the CVMP ad hoc group on

the regulations of FMD vaccines which, when it achieves its objectives, will be a major step forward in supporting FMD disease control not only in the EU but world-wide.

• Modern vaccine batches with a strong potency are able to cover only partially related field

strains which have recently emerged. • Vaccines applied in the field which contain antigens of recent field strains have a higher

potential to be effective than heterologous antigens of type A after single vaccination. • Non-structural proteins can be sufficiently removed during processing from antigenic

preparations so that there is only a minimal risk that NSP have an antigenic potential in vaccinated animals.

• A synthetic peptide vaccine developed in Turkey, adjuvanted with synthetic polymer is

capable of inducing an immune response in laboratory animals and to protect against homologous challenge. However, so far, it has not induced sufficient specific antibodies in cattle. The result of the UBI synthetic peptide vaccine looks promising.

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Recommendations • The RG supports the effort to harmonise the activities of the CVMP and other

organisations and to supplement the monograph of the European Pharm. With guidelines in order to cover the specific requirements for FMD vaccines.

• In view of the limited capacities in the FMD institutes or laboratories it is recommended

that the results of vaccine potency tests which include heterologous challenge be reported, where possible, to EUFMD and further be distributed to other FMD vaccine laboratories.

• Studies with synthetic peptide vaccines should be continued if it can be expected that a

specific immune response in ruminants and/or pigs is achieved. • A simple procedure such as should be developed to detect low levels of residual NSP in

antigen preparations. Item 8: Closed Session The members of the Research Group, Prof. Dr Reinhard Ahl, the Representative of the EC Scientific Committee, Dr Alf Füssel, the Representative of the European Commission and Dr David Paton, the Head of the WRL Pirbright met in a closed meeting on 19 September. Dr Kris De Clercq, Belgium, chaired the meeting. The EUFMD Secretariat was represented by Keith Sumption. The agenda circulated at the start of the meeting was approved. It was agreed that the issue of procedures to reduce risk from sausage casings should be re-considered at a later stage. The following items were examined at the meeting: 1. Information on recent and future activities relating to the Caucasus, Turkey, Greece

and Bulgaria The Secretary reported that the joint proposal to FAO from Turkey, Greece and Bulgaria for a TCP for strengthening surveillance and control of FMD and other exotic diseases had been received by FAO and that a follow-up meeting is planned for 25 October 2002, in Athens. The timing is also opportune for a meeting of the Tripartite (EUFMD/OIE/EC) to discuss the situation in the region and particularly the plans for sero-monitoring in Turkish Thrace. The current Session of the Research Group had provided a good opportunity to discuss sero-monitoring post-vaccination in Turkish Thrace in 2003 and guidelines for this would be prepared by the Secretariat before the Tripartite meeting. The Secretary reported that it is anticipated that OIE would organise a follow-up meeting of the Tripartite (EUFMD/OIE/EC) Group in Paris on 4 November 2002 to discuss the plans for the vaccination campaigns for Spring 2003, and associated sero-surveillance activities. A longer term vision and strategy for the control of FMD in the region was required and this would be the main subject of the meeting. The meeting would be timely in relation to the development of a global framework for the control of trans-boundary animal diseases under development by FAO. Dr Füssel reported that the EC would support a further 200,000 doses above that previously agreed, bringing the intended supply to the Caucasus region of 1,200,000 doses of oil-adjuvanted vaccine, of which 200,000 would be reserved for emergency use.

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The Secretary also informed the Group that he would represent EUFMD in a Mission to Iran, on 5-15 October 2002. There would be a strong association in the mission of those currently or previously involved in the EUFMD Commission by the participation of Dr Per Have (representing the EU), and Yves Leforban, representing France, and Dr Tony Garland representing the WRL. It was agreed that strengthening surveillance in the region was of great importance since much of the concern for EUFMD, and work for EUFMD Research Group members concerns the need for identification of suitable vaccines and diagnostic reagents (reference sera) required in response to the threat of emergent virus strains from this region. 2. Matters arising from the 67th Executive Committee meeting, 25-26 April 2002 2.1 Capacity of FMD Reference laboratories during crisis situations Dr Donaldson reported that reviews were ongoing of requirements for diagnostic capacity during FMD crises. It was suggested that Dr Garland would be invited to the Executive Committee meeting to report on this matter. Dr Have mentioned the contingency development plans including capacity figures. It was agreed that collation of information on the diagnostic capacity of the FMD laboratories in the EUFMD member countries, and the level of activity predicted under crisis situations would be very important to the identification of potential problems and solutions. It was noted that in 2001 dramatic increases in submissions had occurred to laboratories across Europe and that there may be limited “spare” capacity in the region to enable countries to undertake additional tests in support of others in a crisis. It was proposed that Dr Garland be invited to the Group to report on this matter. Dr Have mentioned that contingency plans were under development in Denmark which will include figures on capacity for FMDV diagnosis. The Secretariat will check OIE reports for similar information. It was recommended that the Secretariat should establish a system for recording and reporting the level of submissions to the FMD laboratories on a yearly basis. It was also agreed that information should be collated on the number of submissions received during March and April 2001 as an indicator of the possible workload of laboratories during a crisis. 2.2 Review of “The minimum requirements for importation into Europe of live animals, fresh meat and offal of the bovine species” Dr Donaldson reviewed the background to this issue, and the comments received from members of the Research Group concerning the document, prepared by the EU in 1993. He noted that in 2001 there was disparity between the export restrictions faced by the FMD affected countries in the EU compared to those in South America. The differences between recommended standards in the OIE Animal Health Code and the standards required in the EU were discussed. The group recognised that increased stringency of measures intended to reduce risk could have serious consequences for trade for EUFMD members in the event of an outbreak and therefore increase the potential agricultural impact. The group also recognised that the original minimum requirements had been highly effective but that the epidemiological situation was now different, particularly in situations where vaccination had been suspended and later resumed and the immunity level of animals at slaughter would therefore be less certain. Dr Füssel highlighted that the latter situation is reflected in recently modified EU import conditions for fresh meat. The basis for the time-temperature requirements for heat treatment of meat, and milk

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products, was discussed and it was agreed that current recommendations should be critically reviewed, since the validity of some of the published findings was questioned. The RG therefore proposes to review the risk associated with current heat inactivation methods for meat and milk. Dr Dekker was prepared to undertake this task. Other members should send all relevant data to him. 2.3 Development of Reference Sera The availability of reference sera was a concern to members and the proposal of the Chairman that he and Dr Paton prepare a proposal on production of additional reference sera was strongly supported. This proposal would be available in time for the Executive Committee meeting in November. 2.4 Objectives of Phase XVII The group recognised the high value of finishing the work of preparing the 3 new reference sera in this Phase. It was also recognised that the impact of the 2001 epidemic had affected progress under the Phase. Dr Paton also reported that the current financial support to WRL for Phase XVII does not cover full economic costs as there is insufficient to cover personnel costs. The group recognised that since the start of the Phase XVII, new tests had received recognition by the OIE, and that greatly increased additional support is required. The importance of selection of suitable sera was re-iterated and the further standardisation and proficiency studies (including specificity checks on negative serum panels) should be conducted using reference sera to A22, O Manisa and Asia1 (Shamir) as part of Phase XVIII. A proposal (Appendix 42) that a plan for Phase XVIII would be developed by Dr Paton and Dr De Clercq was supported. The meeting also recognised that the issue of virus detection tests is very important and currently not addressed in a co-ordinated manner by EUFMD. The group supported the proposal that ways and means to support an extension into this area be explored. 2.5 Guidance on the use of r values The group recognised that to provide more precise estimates of cross-protection between vaccine strains and epidemic viruses would require a series of large controlled heterologous challenge studies. The value of the current system is evident from the relatively few examples of lack of protection in the field when a high r value has been obtained. A review of the precision of the methods, and predictive value for protection would be helpful, to update the earlier review of Dr Paul Kitching. The problem of obtaining sufficient supply of suitable antiserum to seed vaccine virus strains was re-iterated; this is a limiting factor and affects methodology and range of vaccine strains for which r values can be determined. The group recommends to the Executive Committee that any tender for vaccines should include the supply of standard reagents to enable accurate prediction of the suitability for the vaccine.

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2.6 Design of surveillance schemes, in particular through use of tests for antibodies to NSPs The Group considered that it is extremely important that the EUFMD Research Group should have the opportunity to review relevant documents before international standards for surveillance, following stamping-out or vaccination, are agreed by the OIE or other bodies. The Research Group was concerned that they be involved in the process of review since the implications for EUFMD members are of great importance to the design of control programmes, and because improvement of performance of diagnostic tests should be made in the context of their potential use. The use of sampling schemes to detect a 1% prevalence rate might be extremely difficult to undertake and this would influence decisions on the use of emergency vaccination. The scientific basis for time-bound periods to regain freedom from infection was also recognised by the group as very questionable in light of development in surveillance methods. The Group recommended that freedom from infection be recognised after evaluation of the surveillance data generated and not be time-bound. For small outbreaks where vaccination was limited this would accelerate the return to pre-outbreak disease status. The proposed OIE standard for surveillance requirements in the establishment or regaining of recognition of FMD free zone or status was circulated for comments. However, the time-available to review the document was short and it was considered that the justification for the sampling schemes post-vaccination was not evident. The risk of circulating virus following epidemics is related to the risk of failure to detect infected or previously –exposed holdings or animals in the post-outbreak surveillance and that more attention be given to quantifying this risk. 2.7 Workshop on the internal quality control of ELISAs The Chairman introduced the item and the Group supported the proposal that he will explore further the use of workshops for those EUFMD members who might benefit. 2.8 Risk analysis tools The Secretary indicated that he would be prepared to help develop risk analysis tools to assist EUFMD members, through the development of an expert system for analysis or epidemiological studies on FMD that makes use of recent developments in FAO of databases on predicted livestock distribution across the globe, on trade patterns and livestock price data, and which could be integrated to enable better appreciation or estimation of risk at any given time. The first step would be to arrange a meeting of those who could contribute to such an expert system. The Group considered that such developments should be supported by EUFMD. 3. Matters raised by the Secretariat or members 3.1 Future of closed or open meetings

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The item was discussed and it was agreed that the next meeting should be closed, i.e. for Research Group members and invited representatives of international organisations, and invited speakers. The Group considered that the closed meeting would allow time to intensively review and discuss papers prepared by Research Group members. The representative of the EC re-iterated how important he considered the work of the Research Group and particularly that of the closed meeting. 3.2 Procedures for writing reviews for EUFMD The meeting approved the proposal of the Secretary that the Research Group considers the issue of best practice in commissioning and writing reviews, through the guidance of those with expertise in systematic medical reviews. 3.3 EUFMD website Dr Griot recommended that the EUFMD website be updated more frequently and with more information and up to date maps. The Secretary replied that he intended to obtain the monthly disease information reports from OIE and therefore to update the information monthly in future. The meeting agreed that official information only should be published on the web and that careful consideration be given to inclusion of strain typing information. 3.4 Distribution of information Dr Griot introduced this item and after discussion it was agreed that the Secretariat at present does not have the capacity to provide a reviewed or moderated FMD information system for members but that this area must be considered in future, since informal data sources are also important. 3.5 Standardisation of papers for EUFMD meetings Dr Griot suggested that instructions to authors should be used in future, and made available well in advance of open meetings but that there should remain flexibility to ensure that as much data is given, and therefore available to a wide readership, as possible. The proposal was supported. 3.6 Involvement of epidemiological modellers The Group supported the Chairman and the Secretariat in their efforts to ensure that epidemiological expertise including biomathematical modellers are represented and active in the Research Group meetings. 3.7 Audio and Video-taping of meetings The Group agreed that in the interests of openness and debate, audio and video-taping of meetings by attendees should not normally be permitted. The situation might arise in future, and has been an issue at other recent FMD meetings. 3.8 Support for attendance of FMD diagnostic laboratory personnel from countries not free of FMD in the region

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The representative of the EC agreed that support to ensure the participation of persons who could benefit from the meetings and who could assist in increasing understanding of FMDV situation in their region would be useful and the Group supported the proposal for the Secretariat to invite appropriate persons. 4. Next meeting Dr Griot gave details of the venue for the next meeting to be held from16 to19 September 2003, at Gerzensee, Switzerland. The Chairman thanked him on behalf of the Group for the effort to ensure that accommodation and venue would be available. The location for the 2004 meeting was undecided. The Group were interested to hear of the informal offer to hold the event in Canada, where the meeting could involve more scientists from the new world. However, reservations were expressed that we do not create difficulties for FMD researchers from the European region to attend. Adoption of the Report The draft report of the meeting was discussed by the Session and accepted with some amendments. Closing Remarks Dr Kris De Clercq, on behalf of the EUFMD and FAO conveyed the gratitude of all of the participants and Research Group members to the Government of Turkey for hosting the meeting. In particular, he requested the representatives of Turkey to express our thanks to Dr Sungur and his team who arranged the meeting in an extremely efficient manner. Special thanks were conveyed to Dr Zafer Zög, Liaison Officer in Turkey who dealt with all the practical arrangements of the meeting. Dr De Clercq also took the opportunity to thank the EUFMD Secretariat, in particular Dr Keith Sumption who started on 1 September and was immediately faced with the very difficult task of organising the meeting. Dr De Clercq also thanked all of the participants for their valuable contributions to the meeting. On closing the meeting he wished everybody a safe journey home and will look forward to seeing all again in 2004, perhaps in Canada. Before adjourning Dr Alex Donaldson proposed a vote of thanks to Kris De Clercq in all his activities in organising the present and past meetings.