Open session of the standing technical committee of the EUFMD- 2002

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

TABLE OF CONTENTS Page INTRODUCTION ………………………………………………………………

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Adoption of the Agenda …………………………………………………………..

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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 ……………………………………….

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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 i

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3.7 Audio and video-taping of meetings 3.8 Support for attendance of FMD diagnostic laboratory personnel from countries not free of FMD in the region Next Meeting

Adoption of the report …………………………………………………………

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Closing remarks ……………………………………………………………….

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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 iv

Page 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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Page 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 vi

Page 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

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areas and International Veterinary Health Certificates are issued only for such products. Recording and identification of large ruminants started in September 2001. 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 2

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 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: Item 2a: Item 2b: Item 3: Item 4: Item 5: Item 6: Item 7: Item 8:

Information on current FMD situation in the world and reports on outbreaks FMD control: epidemiology, surveillance, control measures: focus on endemic zones FMD control: epidemiology, surveillance and control measures: focus on epidemic incursions Pathogenicity and transmission Virus characterisation Diagnostics - virus detection Diagnostics - antibody detection FMD vaccines and vaccination Closed Session

The Agenda was adopted as proposed.

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Item 1: Information on current FMD situation in the world and reports on Outbreaks 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. 4

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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 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.

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Vaccine manufacturers and the WRL should collaborate closely to ensure that antigenic characterisations of field viruses includes comparisons with all available vaccine strains.

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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 5

independent vaccine control were started in the Bornova Veterinary Control and Research Institute in Izmir. 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.

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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.

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The test used for the detection of NSP antibodies revealed that the probability of active virus circulation in Thrace is very low.

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FMD viruses circulating in Turkey seem to be covered by current vaccine strains.

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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.

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Recommendations •

SVD control should be based on serological survey and on the detection of virus in faeces by immune-PCR.

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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.

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Surveys for the detection of antibodies against NSPs and confirmatory tests should be continued to detect possible incursions into the Thrace region.

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Turkey should continue to characterise FMD viruses from new outbreaks and also continue to send samples to WRL, Pirbright.

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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.

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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 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 8

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, 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. 9

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 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 (OPfluid) 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.

10

Conclusions •

The number of animals kept together in direct contact may influence the incubation period and the efficiency of spread.

•

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. 11

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 nonneutralising 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.

12

•

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 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. 13

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. 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 14

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 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 seroconversion 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 wellcharacterised 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 CHEKITFMD-3ABC ELISA in comparison with an in-house test of the WRL. It appears that this inhouse 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 15

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. 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.

16

•

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.

•

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 17

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 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. 18

•

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.

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 seromonitoring 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 19

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. 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 20

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 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.

21

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. 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 timeavailable 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

22

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 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.

23

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 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.

24

Appendix 1

Update on the Global FMD Situation

David Paton, OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease, Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK A series of maps show the known and presumed distribution of FMD globally. The known distribution is an under-representation of the true situation, being based on official reports to OIE (including updates from the monthly reports of the OIE SEAFMD Regional Coordination Unit), OIE records and on the basis of samples received at the FAO/OIE World Reference Laboratory for FMD (see Table 1 below). There are no recent reports of outbreaks due to FMD serotypes C or SAT 3.

Table 1. OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease Cumulative Report for January - August 2002 COUNTRY BHUTAN BOTSWANA HONG KONG (PRC) IRAN IRAQ PALESTINIAN AUTHORITY KUWAIT LAOS LEBANON MALAYSIA PAKISTAN SAUDI ARABIA SINGAPORE SOUTH KOREA THAILAND UNITED KINGDOM VIETNAM TOTAL

No. of samples 39 28 14

FMD virus serotypes O 20

A

C

SAT 1

9

2 9 2 3 17 37 9 2 10 228 13 527

2 7 1 2 4 2

1

SAT 3

5

6

14 98 2

SAT 2

1

11

9

12 69

23

NVD (b) 15 23 8 4 87 1 2 1

1 2

2 1

Asia 1 4

SVDV (a)

1

5

(a)

swine vesicular disease virus

(b)

no foot-and-mouth disease, swine vesicular disease or vesicular stomatitis virus detected

25

6

9 35 9 228 1 423

Countries in which FMD was reported, 2001

Countries in which FMD was reported

WRL

OIE/FAO World Reference Laboratory

DECEMBER 2001

Countries in which FMD was reported, 2002

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported SEPTEMBER 2002

Conjectured Status of FMD 2001

Endemic Intermediate, sporadic Free with vaccination Free. Virus present in Game Parks Free

WRL

SEPTEMBER 2001

OIE/FAO World Reference Laboratory

Conjectured Status of FMD 2002

Endemic Intermediate, sporadic Free with vaccination Free. Virus present in Game Parks Free

WRL

OIE/FAO World Reference Laboratory

JUNE 2002

Distribution of FMD type O 2001

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

DECEMBER 2001

Distribution of FMD type O 2002

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

SEPTEMBER 2002

Distribution of FMD type A 2001

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

DECEMBER 2001

Distribution of FMD type A 2002

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

SEPTEMBER 2002

Distribution of FMD type Asia 1 2001

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported DECEMBER 2001

Distribution of FMD type Asia 1 2002

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported SEPTEMBER 2002

Distribution of FMD type SAT 2 2001

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

DECEMBER 2001

Distribution of FMD type SAT 2 2002

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

SEPTEMBER 2002

Distribution of FMD type SAT 1 2001

WRL

OIE/FAO World Reference Laboratory

Countries in which FMD was reported

SEPTEMBER 2001

Appendix 2

Molecular Epidemiological Studies on Recently Isolated Foot-and-Mouth Disease Viruses N.J. Knowles and P.R. Davies OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease, Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom.

Abstract Phylogenetic analyses of VP1-coding sequences from viruses isolated from recent outbreaks of foot-and-mouth disease (FMD) are described. The PanAsia strain of FMD virus type O continues to be widespread throughout Asia, although other type O strains are also cocirculating in many countries. A new type O strain has been detected in India and the Middle East. Apparently new strains of types A and Asia 1 have been found in Iran and their possible origin is being investigated. Introduction FMD situation in 2002 Foot-and-mouth disease remains endemic in many parts of Asia, Africa and South America. However, the following specific reports of outbreaks were reported to the OIE: In February 2002, a single outbreak occurred in cattle at Wafra in Kuwait. Samples were received by the WRLFMD. Also in February 2002, a single outbreak of FMDV SAT 2 occurred cattle in the Matsiloje extension area, Francistown District, Botswana. Samples were received by the WRLFMD. In May and June 2002, 16 outbreaks of FMDV type O occurred in pigs in the Republic of Korea. Samples were received by the WRLFMD. In May 2002, a single outbreak of FMDV (type not known) occurred in cattle in Njelwa (Mbala district), Zambia. Samples were NOT received by the WRLFMD. In June 2002, three outbreaks of FMDV SAT 2 were detected in cattle in Midlands province. Subsequently, in August 2002, two outbreaks occurred in cattle in Manicaland and Masvingo provinces, adjacent to the Save Wildlife Conservancy; these also proved to be SAT 2. Samples were NOT received by the WRLFMD. In July 2002, 21 outbreaks of FMDV type O occurred in cattle, sheep and goats in the western part of Mongolia. Samples were NOT received by the WRLFMD. Also in July 2002, two outbreaks of FMDV O in sheep and goats were reported in the Palestinian Autonomous Territories. Samples were received by the WRLFMD. 33

Additionally, the World Reference Laboratory for FMD isolated viruses from samples collected in 2002 from the following countries: Bhutan (types O and Asia 1), Hong Kong (type O), Iraq (type A), Lebanon (type O), Malaysia(types O and A), Pakistan (types O, A and Asia 1), Saudi Arabia (type O), Thailand (type A) and Vietnam (type O). This paper presents phylogenetic analyses carried out on the VP1-coding sequences of viruses isolated from some of the above outbreaks. Materials and Methods Viruses. All the virus isolates were obtained from the WRLFMD strain collection either as 10% epithelial suspensions or as cell culture passaged material. RNA was extracted directly from these samples using RNeasy spin-columns (Qiagen) as per the manufacturer=s instructions. Oligonucleotide primers. Oligonucleotide primers with a Cy5 amidite fluorescent dye for use with the ALFexpressJ automated sequencer were purchased from a commercial source (Amersham Pharmacia Biotech, Sweden). Unlabelled primers used for PCR were purchased from Cruachem (UK). The sequences of the primers have been described previously (Knowles and Samuel, 1995). RT-PCR. Reverse transcription-polymerase chain reaction (RT-PCR) was performed essentially as described by Knowles and Samuel (1995) using the following primer sets (amplicon sizes are shown in parentheses): for type O - ARS4/NK61 (1301 bp); for type A – 1C562/NK61 (863-866 bp) or 1C612/NK61(813-816 bp); for type Asia 1 – 1C505/NK61 (908-914 bp) or 1C616/NK61 (797-803 bp). Cycle sequencing. fmolJ DNA sequencing kits (Promega, UK) which use the cycle sequencing method described by Murray (1989) were used according to the manufacturer=s protocol with the following amendments: approximately 80 fmoles of cDNA template was used in the reactions and 1.5 pmoles of Cy5 amidite-labelled primer. The reactions were heated to 95C for 2 minutes and subjected to 30 cycles of the following programme on a thermal heating block (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 (Amersham Pharmacia Biotech, Sweden) and cooled to 4C. The reactions were heated to 95C for 3 min prior to loading on an ALFexpressJ DNA Sequencer (Amersham Pharmacia Biotech, Sweden). The software, ALFwin Sequence Analyser v2.10.06 (Amersham Pharmacia Biotech, Sweden), was used to process the data which was then exported as an ASCII text file, aligned manually and analysed using the EpiSeq v2.0 suite of computer programs (N.J. Knowles, unpublished). Phylogenetic analyses. Nucleotide sequences were analysed 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 binary tree was constructed according to sequence relatedness across the chosen part of the VP1 gene (see figure legends) using the Neighbor-joining algorithm (Saitou and Nei, 1987) as implemented in the computer program NEIGHBOR (part of the PHYLIP 3.5c phylogeny package; Felsenstein, 1993). The subsequent unrooted tree was plotted using TreeView v1.6.6 (Page, 1996). 34

Results and Discussion FMDV type O The PanAsia strain (Knowles et al., 2000; Knowles et al., 2001b) continues to predominate having been isolated during 2001 and 2002 from many countries throughout Asia (Fig. 1; Hemadri et al., 2002; S. Aktas, personal communication, 2002). However, a new lineage, related the PanAsia strain, has evolved (probably in India) and begun to replace it (Hemadri et al., 2002). This strain has also been found in some of the Gulf States (Oman, United Arab Emirates, Bahrain and Saudi Arabia) in 2001 and Bhutan in 2002 (Fig. 1; Knowles et al., 2001a). Recently, Hemadri et al. (2002) reported a comparison of the complete VP1 sequences of 71 FMD type O viruses isolated in India between 1962 and 2001. They detected four distinct lineages, one of which was the PanAsia strain. This strain was widely distributed being found in 17 Indian states. New outbreaks in the Republic of Korea were also due to the PanAsia strain, but the viruses appear to be distinct from previous lineages present in that country in 2000 (Fig. 1). Analyses of two South Korean 2000 isolates suggests that there may have been two slightly different PanAsia lineages introduced in that year (Fig. 1), however, further sequencing of South Korean isolates from that epizootic would be required to determine if this was the case. All of the type O virus isolates that have examined from Iran and Iraq appear to belong to the PanAsia strain, although there is some sequence variation (Fig. 2). Further sequencing of viruses from Iran is in progress. In south-east Asia four distinct lineages appear to be co-circulating, i) the PanAsia strain (ME-SA topotype); ii) the Cam-94 strain (SEA topotype); iii) the Mya-98 strain (SEA topotype); and iv) the May-94 strain (SEA topotype) (Fig. 3). The PanAsia and Cam-94 strains occur throughout SE Asia, however, the other two appear to have more restricted distributions with the Mya-98 strain occurring in Myanmar, Thailand and Malaysia and the May-94 strain occurring in Malaysia and Thailand. Interestingly, the May-94 strain has not been detected since 1996. The Cathay topotype continues to be isolated from pigs in Hong Kong (Fig. 3), which has, so far, remained free of the PanAsia strain (although more isolates remain to be examined). FMDV type A The type A virus which caused extensive outbreaks in Argentina, Uruguay and southern Brazil during 2001 were closely related to each other and part of a larger group of viruses which have been isolated in Argentina and Paraguay since the late 1970’s (Fig. 4). The 2001 outbreak viruses were distinct from the type A virus which caused a number of cases in Argentina in 2000 (Fig. 4). Recent type A virus isolates from Iran fall into two distinct genetic lineages. Six isolates from 2000 fall within the Middle East-South Asia topotype (which also contains the classical A22 subtype viruses) (Fig. 5). Two isolates form a group which is not closely related to any other type A group so far examined (Fig. 5). Type A virus isolates recently received from Pakistan are currently being sequenced to ascertain their relationship with the Iranian viruses. Tosh et al. (2002) recently reported the complete VP1 sequences of 83 viruses isolated in India between 1977 and 2002. They were able to divide these viruses into four major 35

genotypes, but none were closely related to the new virus from Iran (data not shown). A single type A isolate received from Malaysia in 2002 is related to viruses which have occurred in both Malaysia and Thailand since the mid-1990’s (Fig. 6). FMDV type Asia 1 Two type Asia 1 virus isolates received from Iran during 2001 were examined. They were closely related to each other but were completely distinct from all other Asia 1 sequences residing in our database (Fig. 7). Sequencing of a third isolate from Iran and another from Pakistan is in progress. FMDV type SAT 2 An isolate from the SAT 2 outbreak in Botswana was found to be closely related to one of the Zimbabwe isolates from August 2001 (ZIM/1/2001) isolated from an outbreak near Bulawayo, but it was dissimilar to another virus (ZIM/13/2001) isolated in September 2001 from the Lupane Area, Jotholo North Diptank (near the Hwange National Park) (Fig. 8). Many other virus isolates remain to be sequenced and it is intended that future results will be made more accessible via the WRLFMD website: www.iah.bbsrc.ac.uk/virus/picornaviridae/aphthovirus/. Acknowledgements We would like to thank Dr. Sinan Aktas (Ankara, Turkey) for providing sequence data on recent type O and type A viruses from Turkey, Dr. Wilna Vosloo (Onderstepoort, South Africa) for the Zimbabwe 2001 sequences and Drs. Jorge Lopez (Panaftosa, Brazil) and Maria Elisa Piccone (INTA, Argentina) for South American sequence data. We would also like to thank Dr. David Paton for presenting this paper in our absence. References Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle. Hemadri, D., Tosh, C., Sanyal, A. and Venkataramanan, R. (2002). Emergence of a new strain of type O foot-and-mouth disease virus: its phylogenetic and evolutionary relationship with the PanAsia pandemic strain. Virus Genes 25: 23-34. 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. Rome: FAO. Appendix 8: 45-53. Knowles, N.J., Samuel, A.R., Davies, P.R., Kitching, R.P., Venkataramanan, R., Kanno, T., Scherbakov, A.V., Drygin, V.V., Zhao, Q.-Z. and Xie, Q.-G. (2000). Emergence of a pandemic strain of foot-and-mouth disease virus serotype O. Report of the Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-andMouth Disease, Borovets, Bulgaria, 5-8 September, 2000. Rome: FAO, Appendix 1: 20-31.

36

Knowles, N.J., Davies, P.R. and Samuel, A.R. (2001a). Molecular epidemiology of foot-and-mouth disease virus: the current situation in Europe and 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, Island of Moen, Denmark, 12-15 September 2001. Rome: FAO, Appendix 3: 38-47. Knowles, N.J., Samuel, A.R., Davies, P.R., Kitching, R.P. and Donaldson, A.I. (2001b). Outbreak of foot-and-mouth disease virus serotype O in the UK caused by a pandemic strain. Veterinary Record 148: 258-259. Murray, V. (1989). Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Research 17: 8889. Page, R.D.M. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358. Saitou, N. and Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425. Tosh, C., Sanyal, A., Hemadri, D., and Venkataramanan, R. (2002). Phylogenetic analysis of serotype A foot-and-mouth disease virus isolated in India between 1977 and 2000. Archives of Virology 147: 493-513.

37

Cathay

O/HKN/6/83

Euro-SA

O1/BFS 1860/67 O/IND/R2/75 O1/Manisa/TUR/69 O/IRN/52/2001

ME-SA

O/IRN/15/97 O/IRN/16/97 O/IRN/16/2001 O/IRN/41/2001 O/IRN/9/99 O/IRN/24/99 O/TAW/2/99 O/SAR/1/2000 O/UKG/12/2001 O/IRN/2/2000 1%

O/IRQ/26/2000 O/IRQ/27/2000

PanAsia strain

O/IRQ/30/2000 O/IRQ/29/2000 O/IRQ/31/2000 O/IRN/1/2000 O/IRN/16/2000 O/IRN/17/2000

Fig. 2. Genetic relationships between recent FMD type O viruses from Iran and Iraq and reference viruses. The tree was based on a comparison of complete or nearly complete VP1-coding sequences.

A/BRA/1/2001 (W) A/URU/1/2001 (W) A/Chapaleufu/LP/ARG/2001 (I) A/Rivadavia/BA/ARG/2001 (I) A/Union/COR/ARG/2001 (I) A/ARG/3/2001 (W) A27/Cundinamarca/COL/76 (U) A24/Cordoba/ARG/90 (I) A/Corrientes/ARG/92 (I) A/PAR/82 (I) A27/COL/67 (W)

A/BA/Arg/81 (I)

A81/URU/79 (I) A/Pehuajó/BA/ARG/92 (I) A/Ayacucho/BA/ARG/90 (I) A/San Ignacio/ARG/90 (I) A/25 de Mayo/BA/Arg/87 (I) A/Castellanos/SF/Arg/87 (I)

A/97245/COL/97 (P) A/96929/COL/97 (P)

A/Castellanos/SF/Arg/87 (A) A/ARG/1/2000 (W) A/ARG/2000 (I)

A/96928/COL/97 (P) A32/VEN/70 (P)

A/Utracan/LP/Arg/87 (I)

A24/Cruzeiro/BRA/55 (W) A/13753/RGdS/BRA/97 (P) A/6562/RGdS/BRA/97 (P)

A76/ARG/76 (W)

A/105325/BOL/98 (P) 1%

A/ARG/79 (U)

A/105330/BOL/98 (P) A/105331/BOL/98 (P)

Sequences determined at:

A/Venceslau/BRA/76 (U)

A/106455/PER/99 (P)

A/106453/PER/99 (P)

W = WRLFMD P = PANAFTOSA I = INTA U = PIADC A = University of Buenos Aires

Fig. 4. Genetic relationships between recent FMD type A viruses from South America and reference viruses. The tree was based on a comparison of complete or nearly complete VP1-coding sequences.

European/South American topotype

Indian/Middle Eastern (A22-like) topotype Iran-99 topotype

A10/HOL/42 A24/Cruzeiro/BRA/55 A5/Allier/FRA/60

A/IRN/32/2001 A/IRN/34/2001 A/IND/17/77 VS* A/IND/300/94* A/ALB/4/96 A/MCD/1/96 A22/IRQ/24/64 A22/Mahmatli/TUR/65 A/IRN/30/2000 A/IRN/7/2000 A/IRN/8/2000 A/IRN/12/2000 A/IRN/4/2000 A/IRN/10/2000 A/IRN/87 A/IRN/22/99 A/IRQ/24/2002 A/IRQ/33/2002

A/TUR/1/2000

Iran-96 topotype

1%

ALB, Albania BRA, Brazil FRA, France HOL, Holland IND, India IRN, Iran IRQ, Iraq MCD, Macedonia SAU, Saudi Arabia TUR, Turkey

A/TUR/11/2002* (Aktas)

A/IRN/28/99 A/IRQ/17/2000 A/TUR/4/2000 A/TUR/3/2000* (Aktas) A/TUR/1/2000* (Aktas) A/TUR/33/2001* (Aktas) A/TUR/7/2001* (Aktas) A/TUR/17/2001 A/TUR/9/2002* (Aktas) A/TUR/4/2001* (Aktas) A/TUR/5/2001* (Aktas) A/TUR/8/2001* (Aktas) A/TUR/10/2002* (Aktas) A/TUR/6/2001* (Aktas) A/TUR/2/2000* (Aktas) A/TUR/21/2001* (Aktas) A/IRN/4/98 A/IRN/17/96 A/IRN/1/97 * not WRLFMD reference numbers A/TUR/38/98 A/TUR/12/97 A/TUR/14/98 A/TUR/19/98

Fig. 5. Genetic relationships between recent FMD type A viruses from Iran, Iraq and Turkey and reference viruses. The tree was based on a comparison of partial VP1-coding sequences (nt 469-639).

A10/HOL/42 A24/Bilbao/SPA/73 A24/Cruzeiro/BRA/55 A5/Allier/FRA/60

Euro-SA topotype

ME-SA topotype

A/PHI/1/77 A22/IRQ/24/64

A/SKL/TAI/73

1%

A/PHI/2/79

A/SAU/16/95 A/ALB/1/96 A/MCD/1/96 A/GS/LX/CHA/62 A/NM/XZ/CHA/64 A/NM/EL/CHA/60

ALB = Albania BRA = Brazil CHA = P.R. China FRA = France HOL = Holland IRQ = Iraq MAY = Malaysia MCD = Macedonia PHI = Philippines SAU = Saudi Arabia SPA = Spain TAI = Thailand

A/PHI/10/75 A/PHI/1/76

A/XJ/KT/CHA/58

A15/Bangkok/TAI/60 A/TAI/183/86 A/NPT/TAI/86 A/TAI/179/86 A/TAI/186-2/86 A/TAI/118-1/87 A/TAI/25-1/88 A/MAY/48/95 A/MAY/28/96 A/MAY/69/95 A/MAY/71/95 A/MAY/14/97 A/MAY/10/97 A/MAY/16/97 A/MAY/9/97 A/MAY/12/97 A/MAY/13/97 A/MAY/15/97 A/TAI/1/97 A/TAI/2/97 A/TAI/6/99 A/TAI/7/99 A/MAY/2/2002 A/TAI/2/99 A/TAI/2/98 A/TAI/3/99 A/TAI/1/99

Fig. 6. Genetic relationships between recent FMD type A viruses from Malaysia and Thailand and reference viruses. The tree was based on a comparison of partial VP1-coding sequences (nt 469-639).

IND/63/72

0.1%

PAK/1/54 Bangkok/TAI/60 IRN/4/2001 IRN/11/2001 TUR/15/73

ISR/1/84 N1214/Armenia/USSR/84 N1181/Azerbaijan/USSR/83 BAR/1/85 LEB/1/84 ISR/3/89 GRE/1/84 LEB/3/83 PAK/3/98 TUR/10/99 TUR/8/99 IRN/58/99 GRE/2/2000 TUR/6/2000 IND/43/95 IND/51/95 IND/40/95 IND/233/95 IND/26/95 SAU/39/94 SAU/40/94 BAN/1/87 IND/9/89 (1988) NEP/8/87 NEP/36/87 NEP/58/88 NEP/28/90 (1989) BHU/1/86 NEP/23/85 WBN/IND/117/85 NEP/19/86 (1985) YEM/2/80 (1979) HKN/20/80 YEM/15/79 IND/10/82 IND/91/79 IND/60/79 SAU/32/92 IND/8/79

Fig. 7. Genetic relationships between recent FMD type Asia 1 viruses from Iran and reference viruses. The tree was based on a comparison of partial VP1-coding sequences (nt 469-633).

RWA/2/97

ZIM/13/2001 [Onderstepoort] ZIM/7/83

BOW/1/98 (buffalo 1) BOW/7/98 (buffalo 10) ZIW/4/97 (MUK-48) buffalo ZIM/1/97 ZIM/2/97 ZIM/4/97 ZIM/3/97

KEN/3/57 BOT/13/2002 ZIM/1/2001 [Onderstepoort]

RHO/1/48

1%

SAU/2/2000

BOT = Botswana BOW = Botswana wildlife KEN = Kenya RHO = Northern Rhodesia (Zambia) RWA = Rwanda SAU = Saudi Arabia ZIM = Zimbabwe ZIW = Zimbabwe wildlife

BOW/15/98 (buffalo 29) BOW/4/98 (buffalo 7)

Fig. 8. Genetic relationships between recent FMD type SAT 2 viruses from Botswana and Zimbabwe and reference viruses. The tree was based on a comparison of partial VP1-coding sequences .

Cathay Euro-SA WA EA SEA

ME-SA

1%

TOPOTYPES ME-SA, Middle East-South Asia SEA, South-east Asia EA, East Africa WA, West Africa Euro-SA, Europe-South America Cathay, China

O/HKN/4/2001 O/HKN/1/99 O/VIT/3/97 O/PHI/7/96 O/TAW/81/97 O1/Kaufbeuren/FRG/66 O/ARG/3/2000 O/ARG/38779/2000 [INTA-ARGENTINA] O/ARG/38867/2000 [INTA-ARGENTINA] O/URU/1/2000 O/MAU/19/2000 O/ALG/1/99 O/GHA/5/93 O/K83/79 (Kenya) O/UGA/5/96 O/TAI/2/2000 O/TAI/4/99 O/CAM/6/99 O/VIT/2/97 O1/Manisa/TUR/69 O/NEP/12/2000 O/OMN/7/2001 O/BAR/1/2001 O/BHU/7/2002 O/IND/83/2001 [IVRI-MUKTESWAR] O/IND/116/2001 [IVRI-MUKTESWAR] Ind2001 strain O/IND/96/2001 [IVRI-MUKTESWAR] O/UAE/6/2001 O/OMN/4/2001 O/SAU/3/2001 O/SAU/11/2001 O/KUW/3/97 O/BAR/2/97 O/UAE/7/97 O/SAU/2/97 O/TUR/2/2000 O/TUR/7/2000 O/KUW/1/98 O/BHU/2/2002 O/MAY/1/2001 O/BHU/1/98 O/BHU/2/98 O/SAU/13/2001 O/SAU/2/99 O/QTR/3/99 O/BAR/6/99 O/SAU/38/98 O/IRQ/30/2000 O/IRN/16/2000 O/TUR/5/2000 O/TUR/8/2000 O/TUR/2/2001 O/TUR/15/2001 O/MOG/2000 [ARRIAH-RUSSIA] O/SKR/1/2002 O/SKR/2/2002 O/SKR/2000 [PAIDC-USA] PanAsia strain O/Tibet/CHA/3/99 [LANZHOU-PRC] O/BAR/8/98 O/IRN/9/99 O/UAE/4/99 O/UAE/1/2000 O/UAE/2/2000 O/UAE/3/2000 O/Tibet/CHA/1/99 [LANZHOU-PRC] O/JPN/2000 [NIAH-JAPAN] O/SAR/1/2000 O/NET/1/2001 [LELYSTAD] O/IRL/134/2001 O/FRA/1/2001 O/UKG/12/2001 O/1734/Ussuriysk/RUS/2000 [ARRIAH-RUSSIA] O/SKR/1/2000 O/Tibet/CHA/2/99 [LANZHOU-PRC] O/TAW/2/99 O/Hainan/CHA/4/99 [LANZHOU-PRC] O/CAM/2/2000 O/LAO/2/2000 O/MAY/2/2000 [ ] sequence provided by laboratory indicated O/TAI/1/2000

Fig. 1. Genetic relationships between recent FMD type O viruses and reference strains. The tree was based on a comparison of complete VP1-coding sequences.

Euro-SA Cathay Hkn94

O1/Kaufbeuren/FRG/66 O/HKN/6/83 O/VIT/3/97 O/HKN/4/2001 O/HKN/9/99 O/HKN/4/99 O/HKN/7/98 O/HKN/10/99 O/HKN/1/99 O/HKN/22/99 O/HKN/7/96 O/HKN/20/96 O/HKN/5/97 O/HKN/6/97 O/HKN/3/98 O/HKN/10/98 O/VIT/2/99 O/VIT/3/99 O/VIT/6/99 O/HKN/16/96 O/PHI/5/95 O/TAW/111/97 O/TAW/13/98 O/TAW/36/97 O/TAW/83/97 O/TAW/81/97 O/TAW/4/99 O/PHI/13/95 O/PHI/7/96 O/PHI/8/98 O/PHI/19/98 O/PHI/25/98 O/PHI/1/99 O/PHI/5/99

O/MYA/13/89 O/BUR/2/89 O/BUR/6/89 O/CAM/11/94 O/CAM/12/94 O/CAM/3/98 O/CAM/6/99 O/CAM/1/98 O/CAM/2/98 O/VIT/1/97 O/VIT/2/97 Cam94 O/VIT/7/97 O/VIT/9/97 O/VIT/6/97 O/VIT/1/99 O/MAY/1/2002 O/TAI/8/99 O/LAO/1/98 O/LAO/4/98 O/TAI/189/87 O/MAY/5/2001 O/MAY/3/2001 O/MAY/2/2001 O/MAY/4/2001 Mya98 O/MYA/2/2000 1% O/MYA/4/98 O/MYA/1/98 O/MYA/7/98 O/MYA/5/99 O/TAI/4/99 O/TAI/5/99 O/MAY/6/99 O/MAY/4/99 O/MAY/8/99 O/MAY/17/96 O/TAI/2/2000 O/MAY/1/2001 O/MYA/7/99 O/SKR/2000 [PIADC] PanAsia TOPOTYPES O/Tibet/CHA/3/99 [LVRI] ME-SA, Middle East-South Asia O/SKR/2/2002 O/CAM/2/2000 SEA, South-east Asia O/CAM/1/2000 Euro-SA, Europe-South America O/CAM/4/2000 NVRQS = National Veterinary Research and Cathay, China O/LAO/1/2000 Quarantine Service, Rep. of Korea. O/LAO/2/2000 ARRIAH = All-Russian Research Institute for O/VIT/12/99 O/VIT/17/99 Animal Health, Russian Federation. O/SKR/1/2000 PIADC = Plum Island Animal Disease Center, O/SKR/2000 [NVRQS] USA. O/VIT/23/99 NIAH = National Institute for Animal Health, O/Tibet/CHA/2/99 [LVRI] O/TAW/2/99 Japan. O/Hainan/CHA/4/99 [LVRI] LVRI = Lanzhou Veterinary Research Institute, O/TAI/1/2000 P.R. China. O/MAY/1/2000 O/MAY/2/2000 O/MAY/3/2000 O/JPN/2000 [NIAH] O/TAI/9/99 O/Tibet/CHA/1/99 [LVRI]

SEA

ME-SA

Fig. 3. Genetic relationships between recent FMD type O viruses and reference strains. The tree was based on a comparison of partial VP1-coding sequences (nt 301-639).

Appendix 3

Foot and Mouth Disease in Ireland; History, Diagnosis, Eradication and Serosurveillance Patrick J O’Reilly1; Michael.O’Connor1; Anne Harrington1; Sally Gaynor2; Dianne Clery1 1

Central Veterinary Research Laboratory Abbotstown Castleknock Dublin 15 Ireland Department of Agriculture and Food Kildare Street Dublin 2 Ireland

2

Abstract Foot and Mouth disease first appeared in Ireland during the 19th Century and persisted for about half of that century. However during the 20th Century the disease occurred occasionally with total devastation during its visitations in a primarily agriculture based economy. The 21st century started with a recurrence of the disease. This means by which it came to the island of Ireland are described as are the measures for its diagnosis, control and eradication. It represents the first time in Ireland when modern diagnostic methods proved their value with this disease, which historically has been eradicated on clinical and epidemiological control principles. Introduction On the 22 March 2001 Foot and Mouth Disease (FMD) was confirmed in the Republic of Ireland (ROI) after a lapse of sixty years. This article summarizes the history of the disease in Ireland, the diagnosis of the disease on this occasion and its eradication. The rationale and methods of serosurveillance, both to facilitate eradication and to establish that the country had regained its status as a country free from FMD without-vaccination, are discussed. History of the Disease in Ireland FMD was first introduced into Ireland in 1839 probably from Great Britain (GB) where it had been introduced in the same year (Ferguson 1842; Anon 1869; Dun 1850). Contagious Bovine Pleuropneumonia also appeared in the country in the same year and may have been introduced with the same animals imported by the Cloncurry family into Cork from the Netherlands. The family however later denied that such an event had ever occurred (Anon 1873). FMD reappeared in 1869 being conveyed into Counties Antrim and Down via Southampton and the Channel Islands and spread to Co Cork and Waterford by November. Outbreaks began to wane until a further introduction in 1871, with calves imported from Bristol via Dublin to Castlepollard, Co. Westmeath. It continued to persist until 1877 (Anon 1877). The Select Committee on Contagious Diseases XI of 1873 concluded, that attempts to control the disease in Ireland and Great Britain had been unsuccessful. They recommended that the sale of diseased animals in public places or the carriage of infected animals by rail should be prohibited and that the Privy Council should cease issuing orders for the control of the disease. The disease reappeared in 1883 with 1397 outbreaks in Eastern Ireland and a small number in Ulster and Munster. This was eradicated in 1884 following movement controls and stamping out, which was used in Ireland for the first time. There were no further outbreaks until 1912 when the disease was diagnosed in animals in a slaughter plant in Liverpool. Infection, which was identified by backward tracing, was confirmed on a farm in Swords, Co. Dublin on 30 June. The disease infected 382 animals on 68 herds in seven counties and 46

persisted until 17 November. A Royal Commission of inquiry investigated the handling of the disease (Anon 1913). One of the side issues was the diagnosis of Armagh disease, which causes peeling of the superficial epithelium of the tongues of cattle and is caused by an undetermined infectious agent. Experiments demonstrated that it could be transmitted to susceptible animals when challenged with homogenized epithelium. No signs occurred when the challenge dose was first passed through a Berkfeld filter. This disease has no clinical significance except that it can be confused with FMD. FMD was again confirmed on 30 January1914 with outbreaks occurring in Kildare, Dublin, Cork and Tipperary (Anon 1915). There were in all 76 outbreaks with 957 animals being affected and 4180 slaughtered. Trade restrictions were removed on 9 September. The origin of the disease was not determined. On 17 January 1921 FMD was confirmed on a premises in Co. Wicklow, which together with a neighboring premises were slaughtered out. No further outbreaks were recorded until 16 May when six farms were detected in southwestern Wexford and a total of 169 animals slaughtered. The origin of these infections was not determined and no connection between the two outbreaks could be established (Anon 1922). FMD was confirmed on a farm in Wexford town on 17 February 1928 and on two adjoining farms on 22 March. One hundred and seventy nine animals were destroyed. It was suggested at the time that meat imported from South America might be the source of the infection. Trade restrictions were removed on 3 August. . On the 19 June 1931, FMD was confirmed in Downpatrick, Co. Down, NI on the farm of a Mr Hutton from whence it spread to Westmoreland, Lancaster and Yorkshire North Riding. The origin of the infection was not determined, but the introduction of animal feed from the continent was believed to have been the source (Anon 1931b). Trade restrictions were removed on 23 August (Anon 1931; Anon 1931a; Anon 1932). On the16 January1941 FMD was diagnosed at Merklands Wharf Glasgow in cattle exported from Co. Derry NI. On the same date it was confirmed in Eglinton, NI and within a few days in Claudy, Campsie, Thermoyle, Every and Derry City and on 20 January in Bridestown, Kilmanagh Irish Free State (now the ROI). Subsequently the disease affected 556 farms in the Republic of Ireland, of which 5,912 cattle, 143 sheep and 198 pigs were diagnosed as infected and a total of 42,047 animals were destroyed including 27,942 cattle, 10,187 sheep, 3,310 pigs and 608 goats (Anon 1942; O’ Brien 1941). 2001 Outbreak The most recent outbreak was diagnosed in a single holding in Co. Louth in the ROI (Outbreak number FMD 2001/1) on 22 March 2001 in a sheep flock where the disease was first suspected on clinical grounds on 20 March. FMD came to the Island of Ireland (IOI) with a truckload of about 349 sheep imported into NI on 19 February. These had been assembled as two lots consisting of Lot A and Lot B purchased in Longtown Market, Carlisle on 15 February. Lot A went to a lairage at Penrith and Lot B went to Lockerbie in Scotland from 15 to 18 February. Lot A subsequently went to Broadway Market Carlisle for health certification and thence to Lockerbie to collect the other sheep and to unload some. The consignment crossed to NI on 19 February. Initial information indicated that some sheep were dropped off at Killeen and the rest went to Meigh, Co. Armagh NI where 21 sheep were dropped off with the remainder going for slaughter in Athleague, Co Roscommon in the ROI. FMD was confirmed 47

in Meigh, NI (a distance of approximately 2km from the border with Ireland) on 1 March. FMD was also confirmed at the lairage at Lockerbie. The disease in Britain was first confirmed in a meat plant in Essex on 20 February. The index case was believed to be a swill feeding pig herd at Tyne on Wear, Heddon on the Wall, Northumberland where FMD was confirmed on 23 February (Case No 4). The date of infection here was estimated as being 2 February. Outbreak number 6 probably became infected by aerial spread from index case no 4. Sixteen sheep from this flock were sent to Hexham Market on 13 February. Ten of these were brought to Longtown Market Carlisle on 15 February (Gibbens et al 2001). Thus these sheep movements and probably transport of one of them to NI constituted the source of the Meigh outbreak. Following the confirmation of the outbreak in Northern Ireland on 1 March further epidemiological tracings from the infected holding at Meigh were carried out by staff of DAFRD, including, the Special Investigation Unit (SIU) and the National Bureau of Criminal Investigation (NBCI) of the Irish Police force (The Gardai). Seven premises, including two abattoirs and five farms, had received animals from the single consignment of sheep imported into Northern Ireland on 19 February. No evidence of FMD was found in these five farms, despite extensive clinical and serological examination of all susceptible species present on the holdings, and clinical examination of animals on contiguous holdings. Materials and Methods Diagnosis of the disease was based on reported clinical suspicions of the disease followed by inspections and laboratory testing. Virus isolation and identification and virus neutralization tests were performed at the IAH Pirbright Laboratory but extensive serological screening was carried out at the Central Veterinary Research Laboratory, Abbotstown using reagents supplied by Pirbright. The techniques used were the Antigen Capture ELISA (ACE) (Ferris and Dawson.1988) (Roeder, LeBlanc Smith, 1987), the Liquid phase Blocking ELISA (LPBE), (Hamblin, Barnett & Hedger (1986); Hamblin Kitching Donaldson Crowther and Barnett (1987) and virus isolation. Tests were performed in compliance with Office International Epizooties (OIE) Manual of Diagnostic Tests and Vaccines (Anon 2000). Samples were transported to the IAH, Pirbright as described by Kitching and Donaldson (1987) and in compliance with International Airline Transport Association (IATA) rules (Anon 2002). Suspect Investigations State Veterinary Officers from local Veterinary Offices investigated suspect cases reported by veterinary practitioners or members of the public. Where samples were to be taken or where a second opinion was sought, officers from one of the six Regional Veterinary Laboratories (RVLs) or from the Central Veterinary Laboratory were deployed. One hundred and sixty four suspects cases were investigated from 25 of the 26 counties between 21 February and 20 July. Animals in fifty two of these cases were sampled and the samples sent to IAH, Pirbright for virus isolation and/ or serology. Thus 31.2 per cent of clinical cases required laboratory testing to rule out FMD as determined by clinical opinion. Table 1 illustrates the number of suspected cases by county, the species involved and the proportion of cases where sampling occurred. Differential Clinical Diagnoses Recorded Seventy-six suspect cases involved cattle, 80 sheep, 3 pigs and five goats. In 4 cases the species were not recorded and in three cases sheep and cattle presented and in one sheep and goats presented. In excess of 50,000 animals were present on these holdings at the time of suspicion.

48

Specific clinical diagnoses were offered in 66 cases. These are shown in Table 2. However the making of a specific diagnosis to explain the presenting signs does not always rule out the concurrent presence of FMD. Thus even where a specific diagnosis was made, samples to rule out FMD were sometimes taken. The diagnoses which predominated were Orf,: Foot Rot; Malignant Catarrhal Fever; Mucosal Disease and Papular Stomatitis. Often where diagnoses were not made the lesions and their locations were described. A summary of these is shown on Table 3. These convey the clinical signs presented to clinicians. Mouth lesions predominated; these lesions were described in 105 cases and limb lesions in 60 cases no lesions were recorded in 29 cases, although a clinical diagnosis was made in some. Lesions in both the foot and the mouth were described in 70 cases. The nature of the lesions were blisters, erosions, ulcers abrasions, trauma and abscesses. Diagnosis Mc Manus et al (2001) and Murphy (2001) described the clinical signs of the one FMD case recorded (Outbreak number FMD 2001/1). The lesions presented as erosions on the dental pad and adjacent gums, which were demarcated and were between 5 to 25 mm in diameter, extending onto the gums and medial aspects of the lips. The feet of the affected animals were hot and painful. Collapsed vesicles and wetness was seen on the coronary band; inter-digital erosions were also seen. Three lambs aged one to two days old died in the 20 hours prior to examination and 12 more died during examination of the group of 97 ewes. The oldest lesions were estimated to be five days old. FMDV serotype type “O” was confirmed on 22 March by the ACE on two of the five epithelial samples. Virus was isolated from all five samples on cell culture and from one of five heparin samples taken from an animal, which was seronegative by LPBE. The remaining four samples had LPBE titres ranging from 724 to > 2048 and VN titres of 178. Seventeen out of 28 contemporaneously taken samples were antibody positive by LPBE with titres ranging from 90 to >2048. Fifteen of 16 samples taken from sheep on an adjacent land parcel (Proleek) in the same ownership were positive by LPBE with titres ranging from 362 to > 2048 and VN titers 32 to > 90. No evidence of viraemia was substantiated from any of the blood samples. This was considered to be the index case and clinical lesions compatible with FMD infection were seen at the time of slaughter in some animals. It was considered that two cycles of disease might have occurred on this land parcel. Sheep were moved from Proleek to Broughattin about two weeks prior to disease confirmation there. Using median periods for incubation period and infectious periods it was estimated that infection of this site might have been between 25 February and 1 March. The virus strain was confirmed by nucleotide sequencing of part of the genomic region coding for virus protein 1 (VP1) (693 bp) as Pan Asian type “O” indistinguishable from strains isolated in GB and NI (Knowles, Davies, Samuel, 2001 Personal communication; Knowles, Samuel, Davies, & Kitching, 2001). Prevention Details of preventative and control measures applied are described elsewhere (Costelloe, Gaynor, Gaynor, McAteer, & O'Reilly, 2002). Immediate restrictions were placed on the importation of live animals from FMD infected EU member states. Additionally, nation wide movement controls were instituted on 28 February. All animals imported from GB since 1 February, from France since 16 February and from the Netherlands since 20 February (i.e. the probable dates that infection occurred in these countries) were traced. The records of slaughter of 78 consignments of slaughter pigs from NI and two consignments of sheep from GB were verified. Cinical examinations were carried out on the holdings of destination of two consignments of cattle from both France and The Netherlands. Clinical and serological examinations were performed prior to slaughter on two consignments of sheep from 49

GB. All examinations revealed negative results. Increased intensity of measures operated at the 141 checkpoints on the 400 km border with NI in addition to surveillance and publicity at 12 major ports and 8 major airports with international connections. Control Measures and Eradication Following the confirmation of the outbreak in Meigh control measures were introduced in accordance with Directive 85/511/EEC in that portion of the protection and surveillance zones in the Republic of Ireland. These included a census, clinical examinations, surveillance, testing and culling of sheep and goats within the zones. The disease, suspected on the infected holding on 20 March, was confirmed on 22 March. The holding was divided into 7 land parcels with total stock numbers of 113 cattle and 447 sheep and was located 8 km south of the Meigh premises and within the extended protection zone of that outbreak. The area was on the Cooley Peninsula bounded to the south and east by sea and bordering NI to the North. Half of the restricted area consisted of mountain, which provided common grazing for sheep and a number of family groups of wild goats and deer. The holding had been subject to the nation wide ban on movement of susceptible species on 28 February and movement control census and surveillance measures introduced according under the EEC Directive following the outbreak in NI. The affected animals on the land parcel at Broughattin were slaughtered on 20 March. As a precaution the animals on the remaining land fragments were slaughtered within 24 hours. A cull of all susceptible species within one kilometer of the infected premises was carried out within 36 hours. This cull was later extended from 26 March to include all wild goats and deer on the Cooley Peninsula and extended to include all sheep in the same area from 4 April. A total of 53,000 animals was clinically examined and slaughtered and 4,400 sampled during the precautionary cull in Co Louth. Most of this was performed in a disused meat plant in the area. Carcasses were transported in sealed containers for rendering. The product of this was later incinerated. Clinical examination and serology of the remaining sheep goat and cattle holdings, within the surveillance zone, was in accordance with Council Decision 2001/295/EC. Safeguard measures were introduced to control the movement of livestock and animal products in accordance with decision 2001/234/EC as amended by Commission Decision 2001/267/EC. A precautionary cull of all susceptible species on the holdings traced from the Meigh outbreak was also carried out following clinical and serological examination. Control measures introduced to protect against the spread of virus by milk initially included on-farm acidification of milk in the infected area. Subsequently designated milk tankers with filters fitted to the exhaust were used to transport milk for processing into milk powder. Total prohibition was applied to the holding of markets, to the assembly of susceptible species, to inseminator operated artificial insemination, to sheep shearing etc. A total ban on swill feeding was implemented (from 28 March) and is still in force. Origin of the Outbreak The immediate source of the infection and the connection with Britain has been described above. What has not yet been determined is how the animals on the holding in Broughattin or Proleek became infected. Investigations in this respect are continuing.

50

Serological Surveillance Targeted Surveillance At the first announcement of FMD in Britain reagents were sourced from the Institute of IAH, Pirbright to permit the screening of samples for antibody using the LPBE. Fortunately, a member of staff had attended a training course on FMD diagnosis in Pirbright in the year prior to this outbreak. The samples taken for laboratory testing for diagnosis and surveillance are recorded in Table 4. In the tracing and eradication phase of the outbreak targeted surveillance testing was performed, which included the screening of 23,902 samples from 1,012 flock and herd units in 18 counties. Most of these arose in the surveillance testing in the initial protection and surveillance zones around Meigh and subsequently in the zones surrounding Proleek /Broughattin. The remaining samples arose from the tracings related to imported and exported stock and tracings associated with illegal or suspected illegal movements. Over 4,400 of these samples were taken during the precautionary cull immediately prior to slaughter. Screening of these samples resulted in 463 samples being sent to Pirbright for confirmation (using the LPBE and VNT) of which 39 proved to be positive and four suspicious. Most of the positive samples arose from the two land fragments already referred to. Four other samples gave rise to positive results on animals sampled in contiguous flocks during the ensuing cull following disease confirmation. One of ten samples taken from a third parcel of land in the same ownership gave an LPBE titre of 1448 but was negative to VNT. One sample of 61 from a neighboring flock to the first confirmed fragment at Broughattin gave an LPBE titre of 362 but was also negative on VNT. Three other samples with LPBE and VNT titers respectively of 2048 and 64: 181 and 22: 256 and 32 relate to a single animal of twelve sampled on a contiguous farm and two wild goats on a contiguous area of commonage to the infected farm. These samples were all taken at the time of the cull. The animals, which gave suspect reactions, were all negative when these animals were retested. Random Survey It was apparent that there were difficulties in establishing the presence of infection in sheep with the “O” pan-Asian topotype. This was compounded by the extensive systems of management in use in this species, the possibility of illegal trading and the difficulties in identifying individual animals. It became imperative therefore to perform an extensive random survey to show, that the remaining 38,325 flocks in the country, containing 7 million sheep, were free of the disease. The survey design was based on, and exceeded, the recommendations for serological surveillance in the ‘Guide to the establishment and maintenance of FMD-free zone or country’, which was circulated at the OIE/Food and Agriculture Organization (FAO) International Scientific Conference on FMD on 17/18 April 2001. A computer-generated, random survey was conducted in two phases. Phase 1 concentrated on flocks in 18 counties, which were at greatest risk of having sheep introduced from Northern Ireland or Great Britain. A total of 3,639 of 18,388 flocks was sampled. Proportional weighting was given such that flocks with more than 100 sheep were five times more likely to be selected than those with 20 sheep. Each epidemiological unit within each selected flock was sampled using a formal randomized technique that would give 95 per cent confidence of detecting at least one seropositive animal if a prevalence of 5 per cent or greater existed.

51

Phase 2 concentrated on the lower risk flocks in the remaining counties. A total of 308 of 19,937 flocks was sampled, giving 95 per cent confidence of detecting seropositive flocks if they were present at a prevalence of 1 per cent or greater. The within-flock sampling was as described above. All sampled sheep were individually identified. The survey period was between 8 May and 25 July using the LPBE at the Central Veterinary Research Laboratory, Dublin. A total of 159,868 samples from 3,957 flocks from all 26 counties was tested. Of these, 1,232 samples were sent to IAH, Pirbright for confirmatory testing (Table 6). This yielded 11 singleton inconclusive samples. All samples from these animals were found to be negative on resampling, with the exception of one, which was again inconclusive. This animal was slaughtered. A total, including clinical suspect investigations, of 184,324 samples was tested. Of these 2,249 samples were sent to Pirbright in addition to 174 tissue samples and 380 blood samples from primary suspect investigations and 1,695 samples arising from the screening program for confirmatory testing The screening of samples at the CVRL made the transportation of samples to IAH Pirbright practicable. The prospect of Pirbright meeting all of the ROI testing requirements, in addition to their own requirements and the logistics of sample transportation, would have been impracticable. ROI was again fortunate, considering the problems in GB and NI, that sufficient reagents to meet the additional requirements of both Ireland and France could be accommodated by the Pirbright Laboratory (Kitching 2001). Resolution The checkpoints around Co. Louth were removed on 19 April (the date on which the EU safeguard measures lapsed),leaving the surveillance and protection zones intact. Controls were removed from the latter zones in turn, on 30 April following completion of the clinical and serological surveillance required by Commission Decision 2001/295/EC. The OIE conditions for restoration of the status of Ireland as a FMD free country without vaccination were met on 22 June and this status was confirmed by OIE on 19 September 2001. Acknowledgements The authors would like to acknowledge their colleagues in the State Veterinary services both on the laboratory and the field side, who contributed so much to the desired outcome. In particular the tremendous spirit and contribution by the laboratory staff is acknowledged. They would also like to acknowledge the assistance of the staff of the IAH Pirbright in particular Dr Alex Donaldson and Dr Paul Kitching for the supply of reagents to perform the screening tests, report and interpretations of samples submitted to Pirbright and for their advice at crucial times. References

Anon (1869) Eczema Epizootica- Foot and Mouth Disease. The Veterinarian XL11, 852. Anon (1877) Parliamentary Papers LXVIII 345 (Signed by Hugh Ferguson) Anon (1913) Report on foot-and-mouth Disease in Ireland in the Year 1912 by the Department of Agriculture and Technical Instruction for Ireland. pp.1-68. Printed under the authority of His Majesty's Stationery Office by Browne and Nolan, Ltd., Dublin. Anon (1931) Foot and mouth disease National Control imposed. Veterinary Record XI No’s 26 & 31 695 and 843.

52

Anon (1915) Report of Proceedings under the Diseases of Animals Act for the Year 1914. Printed under the Authority of His Majesty's Stationery Office by Alexander Thom & Co LTD, Middle Abbey Street, Dublin. Anon (1922) Proceedings under the Diseases of Animals Acts. Report by the Department of Agriculture and Technical Instruction for Ireland. London: His Majesty's Stationery Office. Anon (1931a) Department of Agriculture Report of Proceedings under the Diseases of Animals Acts for the Year 1928. pp.1-4. Dublin: The Stationery Office. Anon (1931b) Foot-and-mouth disease. National control imposed. Veterinary Record 11, 695 Anon (1932). First Annual Report of the Minister for Agriculture, 1931-1932. pp.112-115. Dublin: Stationery Office. Anon (1942) Eleventh Annual Report of the Minister for Agriculture, 1941-1942. pp.121-130. Stationery Office.

Dublin:

Anon. (2002) Infectious substances shipping guidelines, 3rd Ed. International Air Transport Association, Montreal and Geneva Anon (2000) Foot and Mouth Disease. In OIE Manual of Standards and Diagnostic Tests and Vaccines 2000 4th Edition. Publ. Office International des Epizooties December 2000 Part 2 Section 2.1 Chapter 2.1.1. Commission of the European Communities (2001) Commission Decision 2001/234/EC of 22 March 2001 concerning certain protective measures with regard to foot-and-mouth disease in Ireland. Off. J. Eur. Communities, L 84, 62-68 Commission of the European Communities (2001) Commission Decision 2001/267/EC of 3 April 2001 amending Decision 2001/234/EC concerning certain protective measures with regard to foot-and-mouth disease in Ireland. Off. J. Eur. Communities, L 94, 26 Commission of the European Communities (2001) Commission Decision 2001/295/EC of 10 April 2001 laying down the measures to be carried out before releasing the restrictions applied in accordance with Article 9 of Council Directive 85/511/EEC. Off. J. Eur. Communities, L 100, 35-37 Council of the European Communities (1985) Council Directive 85/511/EEC of 18 November 1985 introducing Community measures for the control of foot-and-mouth disease. Off. J. Eur. Communities, L 315, 11-18 Costelloe, J. A., Gaynor, M. C., Gaynor, S., McAteer, W. J., & O’Reilly, P. J., (2002). Control of Foot and Mouth Disease: Lessons from the experience in Ireland. Revue Scientifique et Technique- Office International des Epizooties in press. Dun, Finlay. (1850) On Murrain, or the Vesicular Epizootic. The Veterinarian XXIII, 485-495. Ferguson, H. (1842) A popular Lecture on the Prevailing Epizootic. The Veterinarian xv 575-592. Ferguson, N.M., Donnelly, C.A. & Anderson, R.M. (2001) The foot-and-mouth Epidemic in Great Britain: Pattern of spread and impact of interventions. Science 292, 1155-1160

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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 Gibbens, J. C., Sharpe, C. E., Willesmith, J. W., Mansley, L. M., Michalopoulou, E., Ryan. J. B.M., Hudson, M (2001) Descriptive Epidemiology of the 2001 foot-and-mouth disease epidemic in Great Britain: the first five months. Veterinary Record 149 729-743. Hamblin, C., Barnett, I.T.R. & Hedger, R. S. (1986) A new enzyme linked immunosorbent assay (ELISA) for the detection of antibodies against foot and mouth disease virus 1. Development and method of ELISA. Journal of Immunological Methods 93, 115-121 Hamblin, C., Kitching, R. P., Donaldson, A. I., Crowther, J. R., & Barnett, I. T. R., (1987) Enzyme –linked immunosorbent assay (ELISA) for the detection of antibodies against foot and mouth disease virus.3. Evaluation of antibodies after infection and vaccination Epidemiology. Infection 99 733 744.1 Kitching, P. (2001) FMD- Lessons for the Laboratory. Cattle Practice BCVA 9 no 4 231-233 Kitching,. R. P. and Donaldson, A. I. (1987) Collection and transportation of specimens for vesicular virus investigation Rev. sci. tech. Off. Int. Epiz 6 (1) 263-272. Knowles, N. J., Samuel, A. R., Davies, P. R. & Kitching, R. P. (2001) Outbreak of foot and mouth disease virus serotype “O” in the UK caused by a pandemic strain. Veterinary Record 148, 258-259 Knowles N.J., Davies,P.R., Samuel,A.R., (2001) Personal Communication McManus, T., McConville, J., Collery, P. & Murphy, F. (2001) Diagnosis of the first two outbreaks of FMD in Ireland. Veterinary Record 148, 486-487 Murphy, F. (2001) Observations on FMD-infected sheep. Veterinary Record 148, 791 O’Brien, T.F. (1941) Notes on the behavior of the Foot and Mouth Virus during the 1941 epizootic in Eire. Veterinary Record 55, 341-343 O’Reilly, P. J. and Kitching, P. (1992) Foot and Mouth Disease: A Review and implications post 1992. Irish Veterinary News 14 No 7 pp 7-12 Roeder, P. L., LeBlanc –Smith, P. M. (1987) Detection and Typing of Foot and Mouth Disease Virus by Enzyme Linked Immunosorbent Assay; a sensitive, rapid and reliable technique for primary diagnosis Research in Veterinary Science 43 225-232

54

TABLE 1

Suspect Cases Reported and Sampled up to 20 July County Carlow Cavan Clare Cork Donegal Dublin Galway Kerry Kildare Kilkennyα Laois Leitrim Limerickα Longford Louthα Mayo Meath β Monaghan Offaly Roscommon Sligo Tipperary Waterford Westmeath Wexford Wicklow Total

Bovine Cases

Porcine Cases 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1

Caprine Cases 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 2 0 1 0 0 0 0 0 0 0

Unknown Cases 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 1 0 0 1 1

Total Cases 7 3 7 13 6 7 3 1 1 6 3 1 3 0 23 8 12 7 5 9 3 11 3 6 11 5

Sampled

4 2 4 10 4 3 1 1 0 3 2 0 3 0 7 1 3 5 1 4 1 9 1 3 4 0

Ovine Cases 3 1 2 2 2 3 2 0 1 4 1 1 1 0 16 7 8 1 2 4 2 2 2 3 7 3

2 0 0 4 2 0 1 0 0 6 3 1 0 0 13 1 4 1 1 0 0 5 2 1 3 2

28.6 0 0 30.8 33.3 0 33.3 0 0 100 100 100 0 0 52.5 12.5 33.3 14.3 20 0 0 45.5 66.7 16.7 27.3 40

76

80

3

5

4

164

52

31.7

α Both sheep and cattle presented in one case β Both sheep and goats presented in one case

55

%

Table 2

Specific Diagnoses Recorded

Specific Diagnoses Recorded Abortion Barley Poisoning Calf Diphtheria Foot rot Foot and Mouth Infectious Bovine Rhinotracheitis Joint Ill Listeriosis Malignant Catarrhal Fever Mastitis Misc Foot Conditions Mouth Damage Mucosal Disease Orf Papular Stomatitis

No Occurrences

Diagnoses

No Occurrences

1 1 4 7 1 1

Pasteurellosis Perinatal death Photosensitization Pneumonia Sheep Hepatitis Sheep Scab

1 1 1 4 1 1

2 1 3 1 1 1 4 12 4

Tetany Trauma Ulcer on Abomasum Uraemia Wear Respiratory disease Mastitis Diarrhoea None

1 5 1 1 1 1 2 1 98

Total Diagnoses

66

Total Cases

164

56

Table 3

Anatomical Location of Recorded Lesions/ Clinical Signs Mouth Associated Lesions

No

Limb and other Associated Lesions

No.

Stomatitis Tongue Lips Gingiva Dentition Dental Pad Hard Palate Retropharyngeal Muzzle Rhinitis Salivation Vesicles/Blisters

2 33 11 14 1 8 8 1 5 4 18 7

Lame Foot Interdigital Coronary Band Joint

42 38 7 7 4

No With Limb Lesions

60

Fever

32

No. with no Mouth Lesions No. with Mouth Lesion

50 105

Abscesses Scald Limb Vesicle/ Blisters No with no Limb Lesions No. with Both Mouth & Limb Lesions

5 3 4 96 70

Table 4

Summary of Samples Collected for Laboratory Analysis Samples

Number

Pirbright

Positive

Suspect

Targeted Survey (Sera) Random Survey (Sera) Suspects (Sera) Suspects (Tissues) Total

23,902

463

20 γ

4

No. Counties of origin 18

159,868

1232

0

11

26

261(234)α 158 184,189

261 (52) β 158 (52) β 2114

21 γ 6γ 47

0 0 15

17 17

α Number of Animals Samples β Number of Cases Sampled γ all positive samples relate to infected premises and contiguous farms as described in text

57

Appendix 4

Laboratory virological investigations relating to the 2001 Foot-and-Mouth disease outbreak in the United Kingdom Nigel P Ferris, Geoff H Hutchings, Scott Reid and Brenda Newman Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK ABSTRACT An unprecedented number of diagnostic samples were submitted to IAH, Pirbright Laboratory for the investigation of FMD virus subsequent to the first submissions which were received from an abattoir near Brentwood, Essex on 19 February 2001. A total of 15,574 samples were to be received from around the UK as a consequence of the UK 2001 outbreak and a further 297 from Ireland. This contrasts with the normal complement of 500-800 samples which are annually received from overseas under the activities of the World Reference Laboratory for FMD (fortunately, only 322 in 2001). Major problems had to be addressed as to how these sample could be processed. Volunteer help was enlisted from other groups and departments within Pirbright, while others arrived from our sister IAH Laboratory at Compton besides other organisations elsewhere. Over 50 people were ultimately to be involved in sample processing often for differing lengths of time. A small number assisted the three core staff members for sustained periods - 1-2 months or for regular, intermittent 1-2 week spells. However, more commonly volunteers came for sole 1-2 week periods or helped when they could. An overlapping shift system was organised to allow continual 24 hour cover, 7 days a week from the end of February until early June when work at night discontinued. Laboratory diagnosis for the bulk of the outbreak was essentially reliant upon the use of the antigen detection ELISA in combination with virus isolation in cell culture. Samples originating from all species type were inoculated onto primary calf thyroid (CTY) cells, while porcine samples were additionally inoculated onto IB-RS-2 cells. The capacity to generate the large numbers of CTY cell cultures required for diagnosis (often 3-4,000 tubes per week) was accomplished through the tissue culture department at IAH, Compton assuming responsibility for this activity from May until the end of the year. Primary lamb kidney cells were made available as a reserve. Approximately 90% of positive epithelial suspensions were typed by ELISA, allowing results to be reported within hours of sample receipt. However, the remaining positive samples (epithelial but also blood, probang, milk samples etc) were only recognised following virus amplification in cell culture. Additionally, negative samples could only be defined after several days culture in cells (some 4-5 days for epithelium and 3 for blood). The consequence of the UK Governments adoption of the 24/48 hour culling policy for animals on infected/contiguous premises towards the end of March meant that the majority of subsequent outbreaks were confirmed by clinical diagnosis alone. If the period between sample collection and laboratory receipt (normally 0.5 to 1 day) is also considered then the limitations of the described diagnostic procedures having a critical role in FMD control decisions are apparent. Rapid, on farm diagnosis will be required in the future to quickly investigate the clinical suspicion of disease to ensure that the presence of virus is quickly identified and speedily eliminated.

58

Appendix 5

Clinical and laboratory investigations of five outbreaks during the early stages of the 2001 Foot-and-Mouth disease epidemic in the United Kingdom Soren Alexandersen1*, R Paul Kitching1, Leonard M Mansley2 and Alex I Donaldson1 1 Institute for Animal Health, Pirbright, Woking, Surrey, GU24 0NF, UK 2 Department for Environment, Food and Rural Affairs (DEFRA), Animal Health Divisional Office, Carlisle, UK

SUMMARY 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 are described. The first outbreak, confirmed on 20 February 2001, was at an abattoir in Essex which specialised in the processing of culled sows and boars. On 23 February the disease was confirmed at a pig farm in Northumberland. This holding contained waste-food-fed cull sows and boars and was probably the origin of the epidemic. Other premises investigated were a waste-food-fed cull sow/boar pig unit in Essex, probably infected by contact with either the Essex abattoir or the Northumberland pig farm, two sheep and cattle farms, one of which was located close to the Northumberland pig unit, and probably infected by airborne virus plumes from it, and another in Devon which was probably infected by sheep originating from Northumberland. In addition, analyses of the risk of airborne spread from the premises to other holdings in the area and to the nearby continent were performed in collaboration with meteorologists in the UK and Denmark. These results will be reported in detail elsewhere. INTRODUCTION This paper describes the results of clinical and laboratory investigations conducted by staff from the Institute for Animal Health (IAH), Pirbright at the start of the UK 2001 epidemic and gives an initial assessment of potential airborne spread. The main objective was to estimate the time when infection was probably introduced onto the farms. This was done by determining the age of the oldest lesions in those animals left to examine, calculating the date when clinical disease commenced on the farm and then subtracting 4-14 days from this date, the assumed incubation period for airborne farm-to-farm spread (Sellers and Forman 1973). A 2-14 day within-farm incubation period was used for all other predictions for example, if transmission resulted from direct contact with an infected animal. These estimations were compared with an analysis of laboratory results of samples collected from the holdings. The procedures for the ageing of lesions and the collection of samples were the same throughout the study and were done according to criteria established at IAH, Pirbright. First outbreak visited FMD 1, near Brentwood, Essex The first suspicion of FMD was at an abattoir near Brentwood, Essex that specialised in the processing of culled sows and boars. On Monday 19 February 2001 the official veterinary surgeon (OVS) observed 27 sows to be lame. The sows’ feet were found to have vesicular lesions in the inter-digital clefts and around the coronary bands. No mouth or snout vesicles were detected. The affected animals had arrived on Friday 16 February from two sources, one group 59

was from a farm in Buckinghamshire and a second group was from a farm on the Isle of Wight. Temperatures were recorded from 15 animals from the Buckinghamshire farm and they ranged between 38 and 400 C. Clinical examination of animals from both farms revealed ruptured vesicles on the coronary band and the inter-digital area. The animals from the Buckinghamshire farm were in the more serious condition and were reluctant to stand up. A group of boars that arrived at the abattoir on Sunday 18 February from Yorkshire contained an animal that was acutely lame and had a ruptured vesicle at the interdigital space of one foot. Samples were collected and sent to IAH, Pirbright for investigation. Pigs from FMD 4, Northumberland, (see later), delivered to the abattoir around midnight on 15/16 February and slaughtered on 16 February had not been recorded by the OVS to have any signs of FMD. A specialist from IAH, Pirbright visited the abattoir during the afternoon/evening of Tuesday 20 February. A series of trotters, snouts and tongues, collected post mortem from 5 sows from the farm in Buckinghamshire and with the most advanced lesions were presented for examination. All the trotters had lesions, typical of a vesicular disease, around the coronary bands, extending to the heel bulbs on eight of 20 trotters. The majority of the lesions were deep erosions with loose epithelium at the edges. Three snouts and two tongues had vesicular lesions at different stages ranging from unruptured vesicles to shallow erosions. The ages of the lesions were first estimated to be mainly 3-4 days of age with a few of 4-5 days. In the lairage many pigs were seen to be lame and recumbent. Vesicular lesions were apparent on the feet, snouts and mammary glands. All of the 22 sows that had arrived on Friday 16 February from the Isle of Wight had vesicular lesions, aged 2 to 3 days. Two of eight pigs examined in this group had temperatures above 400C. Lesions, aged 2-day and 1-day old respectively, were also seen in pigs delivered on 18 and 19 February. Three boars delivered on Sunday 18 February had vesicular lesions on all four feet and were recumbent. A group of 101 fattener pigs, delivered on 19 February, and being slaughtered during the visit, was inspected ante mortem. Two animals were found to have suspected very early vesicular lesions on the coronary bands of a single foot. The time from delivery to the abattoir until these lesions were found was estimated to be around 33 hours. The total number of pigs with lesions at the time of the visit was 47. Two steers, present in the lairage since Thursday 15 February, were inspected. Neither was febrile but one had a suspected early vesicular lesion (<1 day) at the bulb of a heel. The clinical signs in the sows and boars were typical of early acute generalised FMD. The virus isolate was evidently highly contagious for pigs since two animals admitted to the lairage on 19 February had early vesicular lesions when examined 33 hours later. The presence of lesions estimated to be 4-5 days in pigs delivered on 16 February suggested the possibility of an even shorter incubation period and/or an over-estimation of the age of the lesions. Second outbreak visited FMD 4, Heddon-On-The-Wall, Northumberland Suspected cases of FMD were seen on this farm, a licensed waste food-feeder, on 22 February when the farm was visited by an official veterinarian due to the tracing activities in relation to pigs delivered to the Essex abattoir at Brentwood. Twenty out of twenty blood samples from adult pigs taken on 22 February and submitted to IAH, Pirbright, were found strongly positive for antibodies (titre above 1:2048 in LPBE) to FMD virus (results available 23 February). The farm was visited on 24 February and around 500 of the total 527 pigs examined. The farm consisted of housed cull sows and boars, a few family groups as well as a number of growers and bacon pigs. A collection of 248 blood samples was taken at this visit, mainly from the adult sows and boars as 60

well as representative pigs from each pen with piglets, growing pigs and bacon pigs. Vesicular lesions of varying age were found on the feet and snouts. Tongue lesions were not a major feature. Approximately 450 out of the 500 pigs examined i.e. 90% had signs of FMD. The estimated age of the lesions ranged from 1 to 2 days in a few pigs up to 12 days (4% of affected pigs). The age distribution of the lesions indicated that most were 9 to 10 days old (45% of affected pigs) with a smaller group aged at 2 days (12%). These findings correlated well with the results of laboratory investigations that showed that 8.5% of the samples were virus positive and 88% antibody positive. The finding of lesions 12 days of age indicated that disease (lesions) had been present at least since 12 February 2001. Subtracting from that a likely maximum incubation period of 14 days, the infection could have been introduced onto the farm on 29 January or perhaps a few days earlier given the potential variability of the estimate of age of lesions. Third outbreak visited FMD 6, Ponteland, Northumberland On 25 February experts from IAH-Pirbright visited a farm at Ponteland (Prestwick), Northumberland, located 5 km north-north-east of premises FMD 4. This was a mixed farm consisting of around 100 housed pedigree beef cattle, 320 grazing sheep and 30 housed hogs. On 21 February a veterinarian was called to the farm and diagnosed acidosis among a group of 18 month old Limousin heifers. On the 22 February 25 animals were ill, sore mouths and tongue lesions were noted and suspected FMD was reported. At the visit on 25 February all of the cattle, consisting of 38 heifers (18-28 months) and 37 bull-beef (10-16 months) housed in four pens in one cattle shed and 22 young stock (6-12 months) housed separately were examined immediately after the animals had been slaughtered. The group of 38 medium sized heifers was located in the south-western part of the shed and was the group in which clinical signs had first been noticed; the 37 bull-beef animals were penned separately on the opposite side of this shed and some were beginning to show early clinical signs of disease. The group of 22 younger stock were housed separately and showed no clinical or post-mortem signs of FMD. No clinical signs had been reported in the sheep grazing away from the farm buildings but one small group of 11 Blue Face Leicesters, grazed and housed at nights close to the south western corner of the cattle shed, had been treated for “foot-scald" by foot bathing on 10 and 20 February according to information obtained at the visit. The majority of the sheep (237 animals) were bled in connection with the visit. The laboratory results (available on 26 February) showed, that the remaining 11 Blue Face Leicester sheep, located just outside the cattle shed, were all positive for antibodies while of the other 226 sheep (mainly cross-bred ewes located separately) only 4 sheep (2%) were positive for antibodies and another 8 sheep (3%) were virus positive. Some of these Blue Face Leicester sheep were kept together at night with a group of Texel sheep in the same pen (close to the other Leicesters and close to the cattle), before 16 of these sheep went to Hexham Market on 13 February. Lesions found in the cattle consisted of vesicles and deep erosions on the tongue, the dental pad, gingiva and lips. The lesions were estimated to be from 1 to 9 days old, i.e. twelve bulls and a single heifer had lesions of 1-3 days whilst one bull and the rest of the heifers had lesions of 3 days and up to 9 days in a single heifer. Foot lesions were not a major finding although some heifers had obvious lesions (estimated age of 3-4 days). This reflected the general lack of lameness observed, possibly associated with the soft bedding on which they were kept. All of the heifers examined in the south-western part of the shed had clinical signs of FMD, while of the 61

bulls at the other side of the shed only 13 of the 37 bulls (35%) examined showed lesions. Assuming that the heifer with the 9-day old lesion was the index case, the most likely period for the first clinical signs of FMD would have been around 16 February, and the introduction of the infection may have been as early as 2 February. Alternatively, if the group of Blue Face Leicester sheep were infected before the cattle and had 2 day old lesions when treated for "foot-scald" on 10 February then the introduction of the infection could have been between 25 January and 4 February. However, if the underlying condition on 10 February was not FMD but was foot-rot, whereas on 20 February it was FMD, then the period during which FMD virus was introduced would have been between 4 and 14 February. Fourth outbreak visited FMD 7, Highhampton, Devon Movement of sheep from the Ponteland farm mentioned above occurred on 13 February through Hexham market in Northumberland, before stopping off at Longtown market near Carlisle on 15 February en route to Devon (Vet Record 3 March 2001). Therefore, FMD 7, Highampton, Devon, which had received sheep from Ponteland was investigated on 26 February by experts from IAH-Pirbright. The farm had a total of 730 sheep and 200 cattle. Of approximately 500 sheep examined, 23 (5%) had clinical signs of FMD. Lesions were between 1 and 6 days old. Ninety-eight of approximately 100 cattle had clinical signs and the lesions were between 1 and 6 days of age. A total of 148 sheep sera were collected, representing all groups. Of these, 12 (8%) were antibody positive (by virus neutralisation) and 36 (25%) were virus positive. Out of 22 rams tested from the pen where the sheep bought in from Northumberland had been placed, 10 were viraemic (45%), however of the total of 60 rams examined in this pen, only 7 (12%) had clinical signs. The cattle on this premises had clinical disease from at least 20 February 2001 which, for a 4 to 14 between-farm incubation period would indicate that virus was introduced between 6 and 16 February. The results indicate that (1) 88% of the virus positive rams were not showing clinical signs, and (2) transmission of FMD between the sheep had been relatively slow, compared to the cattle. Fifth outbreak visited FMD 5, Canewdon, Essex This holding, a licensed swill feeding premises near Canewdon, Essex situated approximately 30 km from the FMD 1 in Brentwood, was visited on Monday 26 February. The holding contained 522 pigs, mainly cull sows and boars. Around 50 sows were examined out of the total 522 pigs (approximately 200 sows and 322 slaughter pigs). The rest of the pigs had already been moved to the incineration site. Around 160 sows had been in groups in twelve pens within one shed. Pigs in two of these pens had old lesions and in one pen early lesions. Some pens were unaffected. Few signs were observed in the slaughter pigs located separately. The pigs were killed on 23 February and left in their pen until on 26 February. They consisted of: 1) a pen with 17 sows where lesions were first observed, 2) another pen (19 sows) where the pigs had shown fewer clinical signs and finally 3) a pen (14 pigs) containing “fat sows”. Lesions at various stages of development were found on the feet and snout. The majority of pigs in the first pen (1) had FMD lesions with an estimated age of 7-8 days. In pen (2) only a very few animals had lesions (of less than 7-8 days) while the “fat sows” in pen (3) had more acute lesions with an estimated age of 4 days. 62

This farm had animals with FMD for 7-8 days at the time of slaughter on 23 February, which indicates that there were animals with clinical disease on this farm from 15 or 16 February. ADDITIONAL LABORATORY RESULTS FMD 1, the abattoir near Brentwood, Essex was diagnosed by tests on pig epithelial tissue samples as positive for FMD type O by initial and repeat ELISA tests on 20 February 2001. Subsequent isolates of FMD virus serotype O were obtained from each of the five premises. Serum samples were tested for antibodies against FMD virus in the liquid-phase-blocking-ELISA. Sera giving suspected positive or inconclusive results were tested by virus neutralisation. All the diagnostic techniques were carried out in accordance with the methods described in the Manual of Standards for Diagnostic Techniques and Vaccines, l’Office International des Epizooties (OIE; 2000). Nucleotide sequencing of the VP1 coding region of an isolate from the Essex abattoir (FMD1) showed that the virus belonged to the type O PanAsian strain (Knowles et al. 2001). ANALYSIS AND PREDICTION OF AIRBORNE SPREAD Three of the five premises investigated (FMD 1, 4, 5) contained pigs and that raised concerns about the risk of airborne spread both in the local area and more distantly, including the nearby continent. The risk of airborne disease spread from these three premises was analysed, both in real time based on a “worst case scenario” (Donaldson et al. 1882a and 1982b) and later based on more detailed virus emission data of the UK 2001 virus (Alexandersen and Donaldson 2002, Alexandersen et al. 2002, Donaldson and Alexandersen 2001, Donaldson et al 2001), using atmospheric dispersion models - the Gaussian and NAME-models (Gloster et al. 1981, Gloster et al. 1982, Ryall and Maryon 1998) operated by the Met Office, UK, and the RIMPUFF/LINCOM (Mikkelsen et al. 1984, Mikkelsen et al. 1997)) and DERMA models (Sorensen 1998) developed and operated by the Riso National Laboratory, Roskilde, and the Danish Meteorological Institute, Copenhagen, respectively. Both the local-scale RIMPUFF and the long range DERMA model have previously been used for the retrospective estimation of the dispersion of FMD virus (Sorensen et al. 2000, Sorensen et al. 2001), and the calculations performed corresponding to the current epidemic show that DERMA gave similar results to the long range NAME model. The methods used and the results obtained are being submitted for publication elsewhere (Gloster et al. 2002, “The 2001 epidemic of foot-and-mouth disease in the United Kingdom – airborne transmission of virus from Burnside Farm, Heddon-on-the-Wall, Northumberland”). DISCUSSION Both laboratory and field observations showed that the UK virus was highly virulent for pigs. Infected pigs developed severe clinical signs and in some pigs the incubation period was as short as 33 hours. Lesions seen in the mouth of cattle at FMD 6, Ponteland and FMD 7, Highampton Devon were severe and extensive. Clinical disease was seen in only a few sheep examined by us in the field, however, after careful examination of a group of animals it was possible to find sufficient vesicular epithelial tissue for laboratory diagnosis. In experiments performed later in which sheep were infected with the UK virus by inoculation or by contact all the animals developed foot lesions, however, no mouth lesions were observed. This points to the importance of making a complete examination of sheep, including the feet. The data presented suggest that the first outbreak of FMD in the UK 2001 epidemic occurred at premises FMD 4, a pig farm near Heddon, Northumberland. The farm was a waste food-feeding 63

premises and it can be speculated that if the virus was in the waste food then that could have been the mechanism by which the pigs became infected. Investigations at FMD 4, Heddon, indicate that FMD lesions had been present at least since 12 February and that virus could have been introduced as early as late January or the beginning of February 2001. Based on the history that the farm had sent cull pigs to the abattoir near Brentwood, Essex on 8 and 15 February there is a very high probability that the pigs which were delivered on 15 February (arrived around midnight 15/16 February) were the source of the outbreak there. Pigs with FMD are potent emitters of airborne virus (Sellers and Parker 1969, Donaldson et al. 1970) and considering the number of pigs affected on the pig farm at Heddon, the quantity of airborne virus released would have been high. The number of pigs found to be affected at the Canewdon pig farm was 40 out of the 50 animals examined, however, as more pigs on that farm were at risk it is possible that others were also infected and so the amount of airborne virus excreted may have been high. Since fewer animals were affected at the abattoir in Essex, the amount of virus excretion would have been lower. Maximum excretion from the Heddon pig unit would likely have occurred at an estimated peak around 15 to 19 February with a continuing significant release throughout the period from 12 February or earlier until 24 February when the pigs were killed. Predictions were made based on estimations that a small number of pigs were already excreting virus from around 3 February. Simulation modelling of airborne spread from the Heddon farm using excretion values for the UK 2001 showed that the risk of long distance airborne spread was limited. However, short range models suggested a considerable risk of airborne transmission to cattle and perhaps sheep located within 5-10 km of the Heddon pig unit (to be described in Gloster et al. 2002, “The 2001 epidemic of foot-and-mouth disease in the United Kingdom – airborne transmission of virus from Burnside Farm, Heddon-on-the-Wall, Northumberland”). Thus cattle and/or sheep on the farm in Ponteland were most probably infected by windborne virus from the Heddon farm as the Ponteland farm fell under the plume of virus and despite exhaustive enquiries by MAFF/DEFRA no other links could be established (J. M. Scudamore, personal communication). The movement of sheep from Ponteland on 13 February through Hexham market in Northumberland, before stopping off at Longtown market near Carlisle on 15 February en route to Devon (FMD 7, Highampton, Devon), was the probable mechanism by which the virus was spread to Devon (Vet Record 3 March 2001). It has been proposed that before the first case was confirmed at the abattoir in Essex on 20 February 2001, that infection had already spread further and was present on farms in Essex, Northumberland, Cumbria, Devon, Dumfries & Galloway, and Ireland. At least 57 premises may have been infected at that time (J. M. Scudamore, personal communication). The spread of infection by sheep moved off the Ponteland farm on 13 February, means that virus gained entry before 13 February 01. Simulated airborne excretion from the Heddon pig unit in the period from 3 to 12 February indicated that the meteorological conditions were favourable for local spread on several days although the predicted levels of airborne virus emission were low (to be described in Gloster et al. 2002, “The 2001 epidemic of foot-and-mouth disease in the United Kingdom – airborne transmission of virus from Burnside Farm, Heddon-onthe-Wall, Northumberland”). If virus was transmitted to the Ponteland farm by the wind then either cattle or sheep could have been infected. Since clinical disease was more advanced in the medium-sized heifers then it is probable that either this group of animals or the sheep located outside were infected first. It may be relevant that a group of 17 Blue Face Leicester sheep were treated on 10 February for “foot64

scald". Some of the 11 sheep remaining in this group after the departure of 6 cohorts on 13 February became acutely lame on 20 February and were foot-bathed. At the time of slaughter they were all FMD antibody positive. If the underlying condition was not FMD on 10 February but was on 20 February then the period during which virus was introduced would likely have been between 6 and 16 February. If the origin of infection on the Ponteland farm was airborne virus it is more likely that the cattle were infected first since they are more susceptible to infection by airborne virus and disease was widespread in cattle at the time of the visit. Nevertheless, it seems most likely, independently of whether the infection was initially introduced to cattle or sheep, that the Blue Face Leicester sheep mixed with the Texel sheep and sold from the Ponteland farm on 13 February distributed the infection to many parts of the UK and subsequently abroad. In conclusion, the primary outbreak, most likely the Heddon pig farm, probably resulted from the feeding to pigs of waste food containing or contaminated with FMD virus. Pigs taken from this premises to the abattoir in Essex on Thursday 15 February (arriving around midnight) spread infection to other pigs held in the lairage during the week-end. The presence of disease was seen on Monday 19 February when the pigs were being slaughtered. Airborne transmission is considered to be the most likely mechanism for spread from the Heddon pig farm to the mixed (sheep and cattle) farm at Ponteland. Sheep from this farm moving through a local market first and then through other markets probably spread the virus to a large sheep and cattle herd at Highampton in Devon. The mechanism of introduction of virus to the pig unit in Canewdon, Essex, located about 30 km away from the Brentwood abattoir is still under investigation by DEFRA. Our analysis is based on data collected at the time of the visits early in the epidemic. It is likely that a more detailed picture of the sequence of events and the mechanisms involved will be constructed as more data and additional information become available. ACKNOWLEDGEMENTS We thank Eleni Michalopoulou, Craig Kirby and David Harris from the DEFRA Regional Office, Essex for their help on infected premises during the field visits. The work was supported by the UK Department for the Environment, Food and Rural Affairs (DEFRA), Meteorological Office, UK and the Danish Ministry of Transport. REFERENCES

Alexandersen, S., Brotherhood, I., & Donaldson, A.I. (2002), Natural aerosol transmission of footand-mouth disease virus to pigs: minimal infectious dose for strain O1 Lausanne. Epidemiology & Infection, vol 128:301-312, 2002. Alexandersen, S., & Donaldson, A.I. (2002), Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs. Epidemiology & Infection, vol 128:313-323, 2002. Donaldson, A. I. & Alexandersen, S. (2001), "The relative resistance of pigs to infection by natural aerosols of foot-and-mouth disease virus", Vet.Rec., vol. 148, 600-602. Donaldson, A. I., Alexandersen, S., Sorensen, J. H., & Mikkelsen, T. (2001), "The relative risks of the uncontrollable (airborne) spread of foot-and-mouth disease by different species", Vet.Rec., vol. 148, 602-604. Donaldson, A. I., Ferris, N. P., & Gloster, J. (1982b), "Air sampling of pigs infected with foot-andmouth disease virus: comparison of Litton and cyclone samplers", Res Vet Sci, vol. 33, 384-5. Donaldson, A. I., Gloster, J., Harvey, L. D., & Deans, D. H. (1982a), "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., vol. 110, 53-57.

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Donaldson, A. I., Herniman, K. A., Parker, J., & Sellers, R. F. (1970), "Further investigations on the airborne excretion of foot-and-mouth disease virus", J Hyg (Lond), vol. 68, 557-64. Gloster, J., Blackall, J., Sellers, R. F., & Donaldson, A. I. (1981), "Forecasting the spread of footand-mouth disease", Vet Rec, vol. 108, 370-374. Gloster, J., Sellers, R. F., & Donaldson, A. I. (1982), "Long distance transport of foot-and-mouth disease virus over the sea", Vet Rec, vol. 110, 47-52. Knowles, N. J., Samuel, A. R., Davies, P. R., Kitching, R. P., & Donaldson, A. I. (2001), "Outbreak of foot-and-mouth disease virus serotype O in the UK caused by a pandemic strain", Vet Rec., vol. 148, 258-259. Mikkelsen, T., Larsen, S. E., & Thykier-Nielsen, S. (1984), "Description of the Riso puff diffusion model", Nuclear Technology, vol. 67, 56-65. Mikkelsen, T., Thykier-Nielsen, S., Astrup, P., Santabarbara, J. M., Sorensen, J. H., Rasmussen, A., Robertson, L., Ullerstig, A., Deme, S., Martens, R., Bartzis, J. G., & Pasler-Sauer, J. (1997), "MET-RODOS: A comprehensive atmospheric dispersion module", Radiat.Prot.Dosim., vol. 73, 4556. Ryall, D. B. & Maryon, R. H. (1998), "Validation of the UK Met Office's NAME model against the ETEX dataset.", Atmospheric Environment, vol. 32, 4265-4276. Sellers, R. F. & Forman A. J. (1973). The Hampshire epidemic of foot-and-mouth disease, 1967. J.Hyg., vol. 71, 15-34. Sellers, R. F. & Parker, J. (1969), "Airborne excretion of foot-and-mouth disease virus", J Hyg (Lond), vol. 67, 671-7. Sorensen, J. H. (1998), "Sensitivity of the DERMA long-range dispersion model to meteorological input and diffusion parameters", Atmos.Environ., vol. 32, 4195-4206. Sorensen, J. H., Jensen, C. O., Mikkelsen, T., Mackay, D. K., & Donaldson, A. I. (2001), "Modelling the atmospheric dispersion of foot-and-mouth disease virus for emergency preparedness", Phys.Chem.Earth, vol. 26, 93-97. Sorensen, J. H., Mackay, D. K., Jensen, C. O., & Donaldson, A. I. (2000), "An integrated model to predict the atmospheric spread of foot-and-mouth disease virus", Epidemiol.Infect., vol. 124, 577-590.

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Appendix 6

The Foot-and-Mouth disease epidemic in Dumfries and Galloway, Scotland, in 2001 Michael Thrusfield †

The 2001 epidemic of foot-and-mouth disease (FMD), in the UK, caused by the PanAsia strain, a genetic ‘sublineage’ of the ME-SA topotype (Knowles and others 2001), and comprising 2030 confirmed outbreaks (Infected Premises: IPs), marked the end of the country’s longest inter-epidemic period in recent history. Several regions in Great Britain were affected, but the most severely affected were Cumbria (with 893 IPs), and the adjacent Dumfries and Galloway (D&G) (with 177 IPs). The control of FMD in D&G initially followed the well-tried methods for ‘stamping out’ the disease, namely, slaughter and disposal of all susceptible animals on IPs and on premises considered to be at risk of being exposed to infection (so-called ‘Dangerous Contacts’), movement restrictions, veterinary inspections of livestock on premises adjacent to IPs, and veterinary epidemiological investigations to identify potential sources and spread of infection. These methods were supplemented, later, with a pre-emptive cull of all susceptible animals on premises contiguous to IPs, and culling of all small ruminants (essentially, sheep) and pigs on premises within a 3km radius of IPs, the rationale being that a pre-emptive approach would remove animals incubating disease, and also, possibly, remove sub-clinically affected animals (notably sheep, in which clinical signs can be mild) that might act as a cryptic reservoir of infection, as well as denying the virus new hosts. This policy resulted in over 18 000 cattle and 47 000 sheep being killed on the contiguous cull, and over 350 000 sheep on the 3km cull - in addition to some 61 000 cattle and 151 000 sheep slaughtered on IPs. (Pigs did not figure noticeably in the epidemic.) The first confirmed IP in D&G was recorded on 1st March 2001 (over a week after the first confirmed case in the UK: Gibbins and others, 2001), with initial ‘seeding’ from Longtown Market, Cumbria, just over the English/Scottish Border. The epidemic peaked on 21st March, a day before the 3km, and two days before the contiguous, culls began. Preliminary studies of the Estimated Dissemination Rate (Miller, 1976) indicated that this first dropped below the value 1 around 21st March. The response of veterinary field personnel was quick from the start, with report-ofsuspect-IPs to end-of-slaughter times of less than a day being achieved within the first week of the epidemic. Responses to reported suspect cases and the necessary tracing of potential sources/spread were similarly rapid. The last case was documented on 25th May 2001 (the last in the UK on 30th September). Post-epidemic serological surveillance with 10km zones around IPs, and Department of Veterinary Clinical Studies, University of Edinburgh, Royal (Dick) School of Veterinary Studies, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG UK.

†

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more extensive regional surveillance in Southern Scotland, found no evidence of continued presence of FMD virus.

References Gibbens, J.C., Sharpe, C.E., Wilesmith, J.W., Mansley, L.M., Michalopoulou, E., Ryan, J.B.M. & Hudson, M. (2001) Descriptive epidemiology of the 2001 FMD epidemic in Great Britain: the first five months. Veterinary Record 149, 729-743 Knowles, N.J. Samuel, A.R., Davies. P.., Kitching, R.P. & Donaldson, .J. (2001) Outbreak of foot-and-mouth disease virus serotype O in the UK caused by a pandemic strain. Veterinary Record, 148, 258-259 Miller, W. (1976) A state-transition model of epidemic foot and mouth disease. In: New Techniques in Veterinary Epidemiology and Economics. Proceedings of an International Symposium, Reading, UK, July 12th – 15th 1976. Pp. 56-72

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Appendix 7

Swine Vesicular Disease in Italy during 2002: epidemiological and diagnostic aspects Brocchi E., Grazioli S., Fallacara F., Bugnetti M., Bellini S., and De Simone F.

Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna, via A. Bianchi 7, 25124, Brescia Italy

Epidemiology Circulation of SVD virus, in spite of the eradication programme implemented since 1995, is still observed in Italy. The 2002 SVD epidemic originated in southern regions and progressively affected also central and northern ones. Epidemiological data and remarks on relevant diagnostic tests are reported below. Figure 1 shows the epidemic curve of SVD: the maximum wave of outbreaks was registered during March and April, then the number of outbreaks slowly declined during following months and steeply dropped at the beginning of summer; the last outbreak of this epidemic was notified on 20th July. Figure 1: Epidemic curve of SVD outbreaks in Italy during 2002

Table 1 shows the regions where the outbreaks occurred, the farm typology, the number of outbreaks per region and the number of animals involved in the outbreaks. A final 166 outbreaks have been notified during the period January-August 2002, involving 10312 pigs. All animals present in outbreaks have been stamped out. SVD has been notified in many Italian regions although with different degrees of diffusion. One hundred fifty out of 166 outbreaks (90%), occurred in southern (Campania, Basilicata, Calabria) and central (Abruzzo, Molise, Lazio and Toscana) regions; the most affected region was Calabria, with a final 102 outbreaks. In these regions 1812 pigs were involved, representing 17% of all animals, with a mean of 12 animals/outbreak. Sixteen outbreaks (10%) have been recorded in northern regions (Lombardia, Emilia-Romagna, Veneto and Piemonte), where animals involved were 8146 (83%), with a mean of 509 animals/outbreak. The highest number of pigs involved in northern Italy reflects the different typology and size of herds with respect to southern Italy. 69

Concerning farms category, 77% (129) of outbreaks were detected in fattening herds which had had an epidemiological connection with an infected dealer’s premise following purchase of pigs: the majority were very small fattening herds, consisting of few heads fattened for family consumption. Only 7% (11) of outbreaks were detected in breeding farms, while 16% (26) were identified in dealers’ premises during the routine surveillance activities, consisting of laboratory examination of environmental faeces samples. Out of the 26 dealers’ premises found infected, 24 were distributed in southern and central regions. As observed in recent years, dealers’ premises played an important role for SVD spreading. All outbreaks occurred subclinically and virus was isolated or detected in faeces samples. Only on one occasion, SVD virus has been isolated from vesicles observed in one pig. Beside eradication and surveillance activities recommended by EU legislation, further policy actions have been enforced in southern regions in order to control the infection, decrease the possible danger originated by the commercial activity of dealers’ premises and also to discourage the illegal movement of pigs. Table 1 Region Campania Calabria Basilicata Molise Abruzzo Lazio Toscana Veneto Lombardia E. Romagna Piemonte TOTAL

Fattening Farms 7 94 3 2 14 1 6 1 1 129

Breeding Farms 2 1 1 1 1 4 1 11

Dealers’ Premises 8 7 3 1 3 1 1 1 1 26

N. Outbreaks 17 102 7 3 18 2 1 1 11 1 3 166

N. Animals 314 1132 34 70 159 32 25 747 4973 1083 1643 10 312

Diagnostic aspects Due to the lack of clinical symptoms that makes the clinical examination useless, demonstration of SVDV circulation and consequently the confirmation of the disease can only be achieved indirectly, by the detection of specific antibodies in serum samples, or directly by virus identification in faeces, where SVDV can be generally found up to 20-30 days post infection. Virus Isolation (VI) test is the recommended virological assay for confirmation of the disease; according EU guidelines (2000/428/EC) ELISA and PCR for SVDV antigen and genome detection respectively have the same diagnostic value as VI, provided that also presence of seropositive animals or clinical signs or epidemiological connection with a confirmed outbreak is associated. Besides the lack of clinical symptoms, additional difficulties have been found for confirmation of the disease due to the inadequate performance of the reference virological assay. VI test is performed on IBRS-2 or equivalent cell line that is susceptible to SVD enterovirus, as well as other enteroviruses currently present in pig faeces (Fallacara et al., 2000). The isolation of SVD virus from faeces often requires 2 to 4 passages and may be adversely affected by other interfering enteroviruses. Growth of the latter may exceed the growth of SVD virus and samples containing 70

both viruses may result negative for SVD virus. Moreover faeces, especially when collected in the environment, may undergo adverse conditions for viral viability. As alternative approach to VI assay, an immune-PCR was previously developed, based on a preliminary immune-capture of SVDV from faeces suspensions by a specific monoclonal antibody coated to ELISA plates: this strategy enabled to prevent false-negative reactions due to inhibitors of Taq polymerase in faeces. The immune-PCR was extensively validated on a large number of field samples, showing advantages in terms of sensitivity, rapidity and simplicity with respect to VI test (Fallacara et al. 2000). On occasion of the last epidemic, efforts have been made in order to ascertain the impact of adverse conditions in results of VI test. For this purpose both VI test and I-PCR have been used to detect SVD virus in faeces samples submitted to virological examination; the two assays have been applied simultaneously on several samples, as shown in figure 2 Figure 2: Examination of faeces samples by VI test and by I-PCR simultaneously or separately during first half of 2002.

VI test was the most used assay during the first semester, VI and I-PCR were used simultaneously during the second trimester, while I-PCR was the test of choice from July. The exact number of tests performed with the I-PCR during the first half of the year is shown in table 2: 1628 out of 2638 (62%) samples submitted to diagnosis during the considered period have been examined by I-PCR. Table 2: Number of samples examined by I-PCR during the 2002 epidemic of SVD Month January February March April May June July August Total

N° of faeces samples submitted to diagnosis 177 311 459 447 279 538 253 174 2638 71

N° of samples examined by I-PCR 1 83 92 219 268 538 253 174 1628

Table 3 shows comparative results obtained with VI and I-PCR assays. Table 3

Month January February March April May June July August Total

Samples examined in I-PCR

Samples positive in I-PCR (homogenate)

1 83 92 219 268 538 253 174 1628

1 28 8 2 13 10 1 63

CPE observed in VI (1 to 3 passages) Presence ELISA pos. 1 8 1 2 4 3 19

ELISA neg. 19 1 1 21

Absence 1 6 9 6 1 23

Sixty-three out of 1628 faeces samples (homogenates) resulted positive to I-PCR; using VI test only 19 showing cytopatic effect (CPE) in infected cell cultures were successively identified as SVDV positive using an ELISA assay, whilst 44 could not be confirmed by VI. Twenty-three out of 44 homogenates did not show CPE after three culture passages, possibly due to presence of SVD notinfectious viral particles. Other 21 samples did show CPE but the infected supernatants were not recognised as SVDV in the ELISA. These 21 false-negative VI results could have been the consequence of co-occurring enteroviruses, which adversely affected SVDV isolation. On the other hand, the incidence of enteroviruses other than SVDV in swine faeces submitted to VI test during 2002 was approximately 25%, that confirmed figures previously observed (Fallacara et al., 2000). In conclusion 70% (44 out of 63) of samples positive by I-PCR scored negative when examined by the reference VI assay. The specificity and higher sensitivity of the I-PCR are supported by the fact that all suspensions positive to this assay originated from animals belonging to seropositive herds or epidemiologically connected with previously confirmed SVD outbreaks. On the contrary none samples positive in VI but negative to I-PCR was observed. In conclusion, the I-PCR proved to be the most reliable virological test, when performed on faeces homogenates. Finally, the tissue culture fluids from 24 representative SVD viral isolates collected during the 2002 epidemic were examined by means of a well characterized panel of monoclonal antibodies produced at the Italian Reference Centre (Brocchi et al. 1997, Borrego et al. 2002). These isolates showed antigenic profiles identical or very close to those of the viruses collected since 1992, proving that they belong to the fourth chronological group of SVD virus that has been circulating unchanged during the last decade (1992-2002) (Borrego et al. 2000). . References

Borrego B, Carra E, Garcia-Ranea JA and Brocchi E, (2002) “Characterisation of neutralization sites on the circulating variant of Swine Vesicular Disease virus: a new site is shared by swine vesicular disease and the related Coxsackie B5 virus”. J Gen Virology, 83: 35-44. Brocchi E, Zhang G, Knowles Nj, Wilsden G, Mccauley Jw, Marquardt O, Ohlinger Vf, De Simone F, (1997) “Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-94”., Epidemiol Infect. - Vol. 118. - p 51-61.

72

Borrego B, Bugnetti M, Berlinzani A, De Simone F, Brocchi E, (2000) “Stability of the antigenic profile of Swine Vesicular Disease virus isolates collected during 8 years (1992-1999).” Proceedings of the 5th International Congress of the European Society for Veterinary Virology, Brescia 27-30 August 2000, 169-170. Fallacara F, Pacciarini M, Bugnetti M, Berlinzani A, Brocchi E, (2000) “Detection of Swine Vesicular Disease virus in faeces samples by immune-PCR assay”. Proceedings of the 5th International Congress of the European Society for Veterinary Virology, Brescia 27-30 August 2000) 173-174.

73

Appendix 8

The Foot-and-Mouth disease situation in Turkey Nilay Ünal FMD Institute (SAP Institute), Ankara, Turkey

In 2002, up to September, a total of 29 outbreaks have been reported, 16 due to type O; 11 due to type A and 2 due to type Asia-1 (Table 1). Although these three types (O,A and Asia1) are circulating in Turkey, it has not seen any outbreak due to type Asia 1 since April 2002. No FMD outbreak has been reported in Thrace Region since June 2001. These types were identified at Şap Institute. Samples from fields were tested by CFT, antigen ELISA and virus isolation in tissue culture. The PCR method was also applied to selected samples. Table 1. OUTBREAKS MONTH Type O A Asia1 January 1 February 1 March 2 1 April 2 1 May 3 1 June 4 2 July 4 4 August 1 2 0 TOTAL 16 11 2

Total 1 1 3 3 4 6 8 3 29

SUSCEPTIBLE CASES DEATH Bovine Ovine Bovine Ovine Bovine Ovine 16 52 519 215 1045 4187 2104 2519 10657

1100 1013 2113

16 3 7 30 51 141 279 36 563

180 3 183

3 5 36 2 46

0

The vaccination programme in Turkey was as follows: -

Turkey aims to vaccinate 80 % of bovine population in the country. All over the country bi-annual vaccination (spring and autumn campaign) of large ruminants with a trivalent vaccine is carried out. In Thrace and Marmara Sea Region single vaccination of small ruminants with trivalent vaccine in a year is carried out. For vaccination the FMD trivalent vaccine containing serotypes O1 Manisa, A Aydın 98 (A Iran96) and Asia1 produced by Şap Institute is used. In 2001 autumn and 2002 spring campaign, the vaccines donated by EUFMD/EC were used in Thrace. Vaccination rate in the 2002 spring campaign was about 97 % in large ruminant, 73% in small ruminant in Thrace and 70% in Anatolia.

In 2001 autumn campaign in Thrace, 1.1 million doses of trivalent vaccine provided by EUFMD/EC was used. Following the vaccination, a serological survey was carried out in four

74

different groups. Sera were tested by LPB-ELISA and also CHEKIT 3ABC ELISA (Bommeli/Intervet) for detecting NSP antibodies.

Vaccine Production The situation for FMD vaccine production in Şap Institute is favourable and the quantity of vaccine is sufficient to cover the needs for spring and autumn campaign. A total of 21,950,000 cattle doses of monovalent FMD vaccine have been produced in 2002. In the last two years, in Şap Institute, for improvement of vaccine quality,many studies were carried out. Some of them as follows; -

For antigen concentration, crossflow concentration system was tested and virus concentrated in 100 times, then stored in – 70°C. The studies to decrease of FMD vaccine dose from 5 ml to 3 ml for large ruminants and from 2 ml to 1 ml for small ruminants were completed and vaccine protocol was updated. In addition to the safety and potency test in laboratory, every vaccine batch has been regularly tested in the field for herd immunity levels.

Vaccine Control In 2002, the initial studies for independent vaccine control was started in Bornova Veterinary Control and Research Institute. The cell culture and inactive antigen required for testing were provided from Şap Institute and stored after proliferation. At the same time mutual tests started for standardization of test between two institutions. Animal Identification For animal identification, implementation of identification of all bovine animals in Turkey was started in September 2001. Within this framework, a computerised database system was established at Genaral Directorate of Protection and Control. All 81 provincial Directorates and most District Directorates have internet connections and e-mail addresses at present. Within the existing system, about 6 million cattle out of over 10 million bovine animals have been tagged and registered. About 4 million cattle and approximately 750,000 bovine animal holdings have been recorded into the computerized database.

75

Appendix 9

A Serosurvey Following the Autumn-2001 FMD Vaccination Campaign in Thrace Region of Turkey Bulut, A. N., Çokçalışkan, C. and Alpay, B. FMD Institute (Sap Enstitutusu), PO Box: 714, 06044 Ankara, Turkey

Abstract A serosurvey was carried out after the autumn vaccination campaign in Thrace region of Turkey in 2001. A total of 4061 sera were collected at days 0, 28, 60 and 120 from cattle and small ruminants from three different groups of which each were consisted of 35 epidemiological units. These sera were examined by Liquid-Phase Blocking ELISA at a single dilution of 1:100.The dilution, which was accepted as threshold level for protection. Although the protection level was in acceptable levels for all three types (type O, A and Asia1) at days 28 and 60, it was declined for all types particularly type O at day 120. To assess the potency of the vaccines used this vaccination campaign, 30 large and 30 small seronegative ruminants were vaccinated in the field experimentally and they were bled sequentially at days 0, 14, 28,42,56 and 120. Sera collected from those animals were tested by LPB ELISA in two fold dilution series to determine endpoint titres. It was found that the percentage of protection levels was over %95 for all types. Although the ratio of positivity was variable unit-to-unit and this variability affected real expected protective levels, this vaccination campaign can be accepted as successful. Keywords: Vaccination; Foot and mouth disease; Protection; Serology 1. Introduction Foot-and-mouth disease (FMD) is economically the most important disease of domestic livestock. Countries which are recognised as being free from FMD gain enormous economic advantage from their ability to trade freely in livestock and their products. In the other hand, countries, which are epidemic or endemic with FMD, combat with the disease to take under the control. This disease threatens the livestock remains endemic in Turkey. For that reason, Turkey which was undertook vaccination campaign and surveillance in order to manage achievement for as being free from FMD. Therefore the trivalent FMD vaccine (O1 Manisa, A96 Iran and Asia1) donated by the EU was used in the Turkish Thrace including the Anatolian part of Istanbul and Canakkale provinces for the autumn 2001 campaign. A serosurvey was carried out after this vaccination to assess this vaccination policy and monitor situation of general antibody surveillance in Thrace. This work is indicated this surveillance results. 2. Materials And Methods 2.1. Test Sera Four different groups of samples were used as test sera: 1st group- a total of 35 villages and 30 large and small ruminants from each village were selected and sera were collected at 0and 28 days post vaccination (dpv). 76

2nd group- the same amount of animals but from different 35 villages were selected and the blood sera were collected at 60 dpv. 3rd group- the same amount of animals but from different 35 villages were selected and the blood sera were collected at120 dpv. 4th group- was selected to measure the protective level of the vaccine in the field experimentally. For this purpose 30 seronegative cattle and 30 seronegative sheep were vaccinated and were bled sequentially at days 14, 28,42,56,70 and 120 and were tested by LPB-ELISA. Sera, which were collected at day 0 of the first group, were tested at a single dilution of 1:32. At day 28 of the first, second and third groups sera were examined at a single dilution of 1:100, which was accepted as protective level by LPB-ELISA. In the fourth group sera were tested by LPB-ELISA, which was carried out with two fold dilutions to determine endpoint titres. 2. 2. Virus strains FMD virus types O1 Manisa, A96 Iran and Asia-1 were grown on monolayers of BHK-21 cells in 175 cm2 tissue culture flasks. When 100% CPE was observed, tissue culture supernatants were harvested, clarified and inactivated with 0.001 M binary ethyleneimine (BEI) at 37 °C for 24 h (Bahnemann, 1990). 2. 3. Liquid-phase blocking ELISA The liquid-phase blocking ELISA was carried out as described by Hamblin et al. (1986a). Titres were expressed as the last dilution of serum at which 50% of wells showed a 50% level of inhibition compared with the virus control. ELISA titres of 1:45 or more were considered positive. 2. 4. Virus neutralisation test Virus neutralisation tests were carried out in tissue culture grade microtitre plates using the method described by Golding et al. (1976). Antibody titres were expressed as the reciprocal of the final dilution of serum in the virus-serum mixture, which was capable of neutralising an estimated 100 TCID50 of virus at the 50% end-point titre estimated according to the method of Ka¨ rber (1931). VNT titres of greater than or equal to 1:45 were considered positive. 3. Results 3.1. The first group sera To evaluate the prevaccination antibody levels, a total of 1080 sera from 540 cattle and 540 small ruminants in the first group were tested by the LPB ELISA at a single dilution of 1:32. The ratio of positive sera was 13% for types O, 23% for type A and 12.5% for type Asia1. The results were shown in Tables 1a, 1b, 1c and 1d. In the same group, at day 28, a total of 1045 sera from 520 cattle and 526 small ruminants were examined by the LPB ELISA at a single dilution of 1:100, which was accepted as the protective level. The positive sera levels in total for O1, A, and Asia-1 were 75%, 67%, and 63% respectively. Detailed data were shown as total in Table 2a, by animal species in Table 2b, by ages in table 2c, by provinces in table 2d. 3.2. The second group sera Sera from the second group were tested to detect the protective level 2 months after vaccination. A total of 945 sera were examined at a single dilution of 1:100 by LPB ELISA. 77

The rate of the protection to O, A, and Asia-1 were 72%, 78%, and 63% respectively (Table 3a, 3b, 3c and 3d). 3.3. The third group sera In total 990 sera from the third group were tested by LPB ELISA in single dilution of 1:100 to measure the protection level 120 days post vaccination. The percentage of the protection against O, A, and Asia-1 were 28%, 51%, and 54% respectively (Table 4a, 4b, 4c and 4d). 3.4. The fourth group sera/Evaluation of the vaccines applied in the campaign In the fourth group, to evaluate the vaccines used in this vaccination campaign, 60 cattle and sheep were vaccinated and sera were bled sequentially at days 14, 28, 42, 56, 70 and 120 were tested by LPB-ELISA in two fold dilution series to estimate the endpoint titres for all three serotypes. Although titres of days 14, 28, 42, 56 and 70 were within satisfied limits, protection rates for types O, A and Asia-1 which were significantly dropped at day 120 for cattle 40%, 60% and 63% and for sheep 37%, 63% and 67% respectively. These data from representative sera is shown in fig. 1a,b and Table.5. 3.5. Results of randomly selected some sera examined by virus neutralisation test (VNT) To compare the results of ELISA with VNT, 160 sera, which were initially tested, by ELISA were selected from day 120 and examined by VNT in single dilution. The results of the two tests were in good correlation (Table.6.). 3.7. Analysing data from epidemiological units individually The results of individual epidemiological units for cattle and sheep were analysed to determine the variation between these units. Standard deviation and coefficient of variation are found very high percentage than expected (Table.7). Therefore, this variation affected negatively protection level in each group, in other word differences of ratio of positivity unit to unit decline post vaccination level of immunity. 4. Discussion The objective of the work reported above was to determine the antibody levels after the autumn 2001 vaccination campaign and to evaluate the vaccination policy in Turkish Thrace Region. This serosurvey described here fulfilled these mentioned objectives. Protective antibody levels of sera collected at day 0 against types O, A and Asia-1 were 13%, 23% and 12.5% respectively. Since the animals in Thrace have been vaccinated once every six months, these results should have been much higher than determined. Same animals were bled again at day 28 of vaccination and 75%, 67% and 63% protection rate were determined for types O, A and Asia-1 respectively. The results of large ruminants were slightly higher compared to the results of small ruminants. These results should have been higher, because in experimental group (group 4) the results were better. These lower results can be explained by vaccination and sampling errors. When the individual units were examined, this can be seen. While over 12 sera were positive in significant number of units, in some other units less than 2 positive animals determined. This result can only be explained by vaccination failure or sampling mistake. Although the units were different at days 28 and 60, the results were almost same in these two groups. These results showed that the protection rate was quite stable during the first two months of the vaccination. Again the results of large ruminants were slightly higher compared to the results of small ruminants. When individual units were examined, great variation was observed. 78

On the other hand significant decrease were observed against all three serotypes at day 120, particularly for type O (28% type O, 51 % type A and 54% type Asia-1). Although variation between units was observed at day 120, only this variation was not enough to explain this decrease in protection levels. These decrease were also determined in experimental group (group 4). Although the protection rates until day 70 were very good, significant decrease were observed at day 120 of experimental group sera, particularly for type O this decrease was very dramatic (40% from 84%). The results of the group 4 were given in table 5. These results were higher compared to the results obtained in the field. But when the effects of individual unit variation were removed, the results were much better comparable. On the other hand, the decrease seen at day 120 was quite similar to that obtained in the field study. To confirm the results of LPB ELISA, 160 sera which were tested by LPB ELISA were also tested by VNT and the results were found quite similar. Conclusions The results of days 0 and 120 showed that the vaccines used in vaccination campaign did not give enough protection for 6 months. But with regular vaccination ( 3 times for the first year and twice annually after the age of one) better protection level can be obtained. The protection rates of the three serotypes were not same, type O being the least protective one and much potent vaccine should be used for type O. A training programme for the organisation and execution of the serosurvey is essential to obtain better results and to decrease variation between epidemiological units.Vaccination teams should be trained to minimise vaccination failures. Acknowledgements Vaccine, which was used in this vaccination campaign, was donated by EU. The authors wish to thank members of Sap Institute particularly Dr. Sinan Aktas and Lab. Technicians, who are Oktay Tezal and Yusuf Demir. References

Bahnemann, H,G., 1990. Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine. Vaccine 8, 229-303. Bulut, A. N., Parlak, Ü. And Ünal N. 2001. Report of the Session of the Research Group of the Atanding Technical Committee of the European Commission for the control of Foot-andMouth Disease, 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 Hamlin, C.,Barnett, I.T.R., 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. Hamlin, C.,Barnett, I.T.R., Hedger, R.S., 1986b. 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. Karber, G., 1931. Beitrag zur kollektivan Behandlung pharmakologischer Reihenversuche. Arch. Exp. Pathol.. Pharmak. 162, 480-483.

79

FMD Serotypes

Positive

%

Negative

%

O

137

13

943

87

A

252

23

828

77

ASIA-1

135

12.5

945

87.5

Table 1a: Cumulative results of the sera collected from the animals in group1 before vaccination

Large Ruminants Positive FMD Serotypes 95 O

(540)

Small Ruminants (540)

%

Negative

%

Positive

%

Negative

%

18

445

82

42

7

498

93

A

144

27

396

73

108

20

432

80

ASIA-1

92

17

448

83

43

7

497

93

Table 1b: LPB-ELISA results of the sera collected from the animals in group 1 before vaccination: Distribution of results by animal species.

Large Ruminants

(540)

Small Ruminants (540)

0-1 (168)

1-2 (185)

%

+

%

+

%

+

%

+

%

11

+ 19

%

O

+ 19

10

57

30

16

9

9

4

17

10

A

26

15

30

16

88

47

19

11

31

15

58

34

ASIA-1

22

13

24

13

46

24

8

4

17

8

18

10

FMD

>2 (187)

0-1 (172)

1-2 (200)

>2 (168)

Serotypes

Table 1c: LPB-ELISA results of the sera collected from the animals in group 1 before vaccination: Distribution by ages (+)=positive, %= percentage of positivity

Large Ruminants O

FMD Types

A

(540)

Small Ruminants (540)

Asia-1

O

A

Asia-1

Provinces⇓

+

%

+

%

+

%

+

%

+

%

+

%

Çanakkale(45)

13

29

16

35

6

13

5

11

14

31

5

11

Edirne (165)

16

10

40

24

25

15

9

5

27

16

13

8

İstanbul (75)

11

15

17

22

9

12

8

11

18

24

3

4

K.eli (135)

15

11

31

23

12

9

12

9

41

30

14

10

Tekirdağ(120)

40

33

40

33

40

33

8

7

8

7

8

7

Table 1d: LPB-ELISA results of the sera collected from the animals in group 1 before vaccination: Distribution of results by provinces (+)=positive, %= percentage of positivity

FMD Serotypes

Positive

% 80

Negative

%

O

781

75

265

25

A

704

67

342

33

ASIA-1

660

63

388

37

Table 2a: Cumulative results of the sera collected from the animals in group1 28 days postvaccination

Large Ruminants Positive FMD Serotypes 391 O

(520)

Small Ruminants (526)

%

Negative

%

Positive

%

Negative

%

75

128

25

389

74

137

26

A

356

69

164

31

348

66

178

34

ASIA-1

345

66

175

34

313

60

213

40

Table 2b: LPB-ELISA results of the sera collected from the animals in group 1, 28 days post vaccination: Distribution of results by animal species

Large Ruminants

(520)

Small Ruminants (526)

0-1 (161) +

%

1-2 (177) +

%

> 2 (182) +

%

O

87

54

140

79

165

A

76

47

126

71

Asia-1

65

40

131

74

FMD Types

0-1(168) +

%

90

106

154

85

149

82

1-2(198) +

%

63

161

87

52

75

45

> 2(158) +

%

81

122

77

132

67

129

82

134

68

104

66

Table 2c: LPB-ELISA results of the sera collected from the animals in group1, 28 days post vaccination: Distribution by ages (+)=positive, %= percentage of positivity

Large Ruminants O

FMD Types

A

(520)

Small Ruminants (526)

Asia-1

O

A

Asia-1

Provinces⇓

+

%

+

%

+

%

+

%

+

%

+

%

Çanakkale

26

58

22

49

21

48

30

67

29

65

33

73

Edirne

133

81

122

74

116

71

132

85

127

82

108

69

Istanbul

63

84

61

81

54

72

48

64

46

61

29

39

Kırklareli

100

76

91

70

95

73

98

75

94

72

85

65

Tekirdağ

70

67

60

57

59

56

81

68

52

43

58

48

Table 2d: LPB-ELISA results of the sera collected from the animals in group 1 28 days post vaccination: Distribution by provinces (+)=positive, %= percentage of positivity

Positive

%

81

Negative

%

O

680

72

265

28

A

733

78

212

22

ASIA-1

591

63

354

37

Table 3a: Cumulative results of the sera collected from the animals in group2, 60 days post vaccination

Large Ruminants Positive FMD Serotypes 380 O

(495)

Small Ruminants (450)

%

Negative

%

Positive

%

Negative

%

77

115

23

300

67

150

33

A

399

81

96

19

334

74

116

26

ASIA-1

311

63

184

37

280

62

170

38

Table 3b: LPB-ELISA results of the sera collected from the animals in group 2, 60 days post vaccination: Distribution of results by animal species

Large Ruminants 0-1 (171) +

%

1-2 (158) +

%

O

127

74

118

A

131

77

Asia-1

88

52

FMD Types

(495)

Small Ruminants (450)

> 2(166) +

%

75

135

127

80

96

61

0-1(138) +

%

81

82

141

85

127

77

1-2(163) +

%

60

116

87

63

76

55

> 2(149) +

%

71

102

69

126

75

121

81

103

63

101

68

Table 3c: LPB-ELISA results of the sera collected from the animals in group2, 60 days post vaccination: Distribution by ages (+)=positive, %= percentage of positivity

Large Ruminants O

FMD Types

A

(495)

Small Ruminants (450)

Asia-1

O

A

Asia-1

Provinces⇓

+

%

+

%

+

%

+

%

+

%

+

%

Çanakkale

26

87

28

94

17

57

17

57

29

97

14

47

Edirne

81

77

81

77

81

77

74

71

79

75

78

74

Istanbul

54

60

60

67

66

73

58

65

65

72

75

83

Kırklareli

98

82

95

79

65

54

87

73

88

74

64

53

Tekirdağ

121

81

135

84

82

51

64

61

73

70

49

47

Table 3d: LPB-ELISA results of the sera collected from the animals in group 2, 60 days post vaccination: Distribution by provinces (+)=positive, %= percentage of positivity

Positive

%

82

Negative

%

O

277

28

713

72

A

504

51

486

49

ASIA-1

538

54

452

46

Table 4a: Cumulative results of the sera collected from the animals in group3, 120 days post vaccination

Large Ruminants Positive FMD Serotypes 127 O

(495)

Small Ruminants (495)

%

Negative

%

Positive

%

Negative

%

26

368

74

150

30

345

70

A

261

53

234

47

243

49

252

51

ASIA-1

265

54

230

46

273

55

222

45

Table 4b: LPB-ELISA results of the sera collected from the animals in group 3, 120 days post vaccination: Distribution of results by animal species

Large Ruminants

(495)

Small Ruminants (495)

0-1 (161) +

%

1-2 (140) +

%

> 2 (164) +

%

0-1(149) +

%

1-2(153) +

%

> 2(156) +

%

O

32

20

38

27

37

23

37

25

45

30

53

34

A

66

41

76

54

95

58

59

40

70

46

95

61

Asia-1

72

45

73

52

98

60

74

50

80

52

104

67

FMD Types

Table 4c: LPB-ELISA results of the sera collected from the animals in group3, 120 days post vaccination: Distribution by ages (+)=positive, %= percentage of positivity

Large Ruminants O

FMD Types

A

(495)

Small Ruminants (495)

Asia-1

O

A

Asia-1

Provinces⇓

+

%

+

%

+

%

+

%

+

%

+

%

Çanakkale

0

0

9

60

3

20

3

20

10

67

9

60

Edirne

18

14

51

43

52

43

27

23

58

48

56

46

Istanbul

44

37

87

73

94

78

22

18

39

33

78

65

Kırklareli

4

9

12

27

20

45

15

33

18

40

23

51

Tekirdağ

61

31

102

52

96

49

83

43

118

61

107

55

Table 4d: LPB-ELISA results of the sera collected from the animals in group 3, 120 days post vaccination: Distribution by provinces (+)=positive, %= percentage of positivity

83

LOG. TITRES

CATTLE 2,9 2,7 2,5 2,3 2,1 1,9 1,7 1,5 1,3 1,1

Type O Type A Typa Asia-1

0

14

28

42

56

70

120

DAY

Fig. 1.a. Results of the sequentially bleeding sera collected from cattle in group 4; 0,14,28,42,56,70 and 120 days. Log titres mean average of 30 sera endpoint titres as log.

LOG. TITRES

SHEEP 2,9 2,7 2,5 2,3 2,1 1,9 1,7 1,5 1,3 1,1

Type O Type A Typa Asia-1

0

14

28

42

56

70

120

DAY

Fig. 1.b. Results of the sequentially bleeding sera collected from sheep in group 4; 0,14,28,42,56,70 and 120 days. Log titres mean average of 30 sera endpoint titres as log

CATTLE (30) 84

DAY 14

DAY 28

DAY 42

DAY 56

DAY 70

DAY 120

TYPES

NPA

%

NPA

%

NPA

%

NPA

%

NPA

%

NPA

%

O

26

87

29

97

28

94

27

90

25

84

12

40

A

27

90

30

100

29

97

28

94

25

84

18

60

ASIA-1

27

90

29

97

27

90

25

84

23

76

19

63

SHEEP (30) O

26

87

28

94

26

87

25

84

23

77

11

37

A

27

94

28

94

28

94

26

87

24

81

19

63

ASIA-1

25

84

28

94

27

90

27

90

25

84

20

67

Table. 5. Results of the sequentially bleeding sera collected from sheep in group 4; 0,14,28,42,56,70

and 120 days. (NPA= Number of protected animal (total number of bleeding animals are 30 in each species)

ELISA(+)

%

VNT(+)

%

O

64

40

66

41

A

89

55

95

59

ASIA-1

99

62

106

66

Table.6. Comparison of results of some sera tested by ELISA and VNT

85

FMD TYPES AVERAGE MAX MIN STD DEVIATION COEFFIENT OF VARIANCE

FMD TYPES AVERAGE MAX MIN STD DEVIATION COEFFIENT OF VARIANCE

FMD TYPES AVERAGE MAX MIN STD DEVIATION COEFFIENT OF VARIANCE

Analysis of group 1, day 28 results O A Positive Negative Positive Negative 11 4 10 5 15 14 15 14 1 0 1 0 3 3 3 3 29 85 30 66 Analysis of group 2, day 60 results O A Positive Negative Positive Negative 11 4 12 3 15 12 15 9 3 0 6 0 3 3 2 2 24 62 21 73 Analysis of group 3, day 120 results O A Positive Negative Positive Negative 4 11 7 8 15 15 15 15 0 0 0 0 4 4 4 4 99 38 57 56

ASIA-1 Positive Negative 9 5 15 15 0 0 4 4 41 69

Positive 10 15 0 4 39

ASIA-1 Negative 5 15 0 4 68

ASIA-1 Positive Negative 8 7 15 15 0 0 4 4 53 64

Table.7. Data generated from analysis of each sera group in terms of number of positivity and negativity in epidemiological units. Positive and negative are indicated number of positive or negative sera results.

86

Appendix 10

A serosurvey to trace non-structural proteins to FMDV conducted with the sera from Thrace Region of Turkey Bulut, A.N., Çokçalışkan, C., Alpay, B. FMD Institute (Sap Enstitutusu), PO Box: 714, 06044 Ankara, Turkey

Abstract This paper summarises the results of 3ABC ELISA measuring antibodies to non-structural proteins (NSP’s) of foot-and-mouth disease (FMD) to differentiate infection from vaccination. For this purpose, in total 1310 sera, which were collected from cattle and small ruminants at day 28 and 60 post vaccination were tested by FMD-3ABC ready to use kit, which has been developed by Bommeli Diagnostics. 16 sera were detected as positive in total of those sera, 11 of which are from cattle and 5 from small ruminants. The ratio of positive samples in total was 1.22% (1.6% bovine and 0,7% ovine sera). After this work, a field active surveillance was conducted in epidemiological units where had been collected those positive sera, it was not defined any clinical infection. It was concluded that those animals detected positive relating to NSP antibody might be infected or sub-clinically infected in previous years and that antibody has been still remained as positive. Keywords: 3ABC ELISA, non-structural proteins, FMD 1. Introduction Foot-and-mouth disease (FMD) is highly contagious viral disease, which affects all clovenhoofed animals. It has an economically devastating impact affected countries, since trade barriers, which are imposed where the disease occurs. The detection of antibody to the polyprotein 3ABC of FMDV is the single most reliable indicator of infection (Diago, M de. et. al. 1997) all naive animals seroconvert to this protein following infection. It is known that vaccinated animals, which are exposed to infection, can become persistently infected with FMD without ever showing clinical signs (Mackay, D.K.J., et. al., 1998). Countries, where use vaccination to control FMD outbreaks, it is important to differentiate to antibody against structural protein from non-structural proteins (NSP’s) in order to discriminate virus which is circulated in field either clinical infection or persistence. In the study presented here, sera, which were collected from cattle and small ruminants in Thrace Region of Turkey, were examined by 3 ABC ELISA. 2. Material and Methods 2.1. Test sera Two different group samples were used as test sera: 1st group: in total 1046 sera in 35 villages and 30 large and small ruminants from each village were selected and sera were collected at day 28 post vaccination. 2nd group: 260 sera in 9 villages but different from group first were selected and the blood sera collected at day 60 post vaccination.

87

2.2. 3ABC ELISA Sera were tested by commercial ready to use ELISA kit (Bommeli Diagnostic/Intervet), which was donated EU for this serosurveillance. Test is direct ELISA and very simple to perform. The assays were made as described in the kits instructions. 3. Results A total of 1310 sera collected from 651 large ruminants and 659 small ruminants (of 1046 sera from day 28. and 264 sera from day 60) were tested by 3ABC-ELISA against 3ABC proteins in Thrace Region including Edirne, Tekirdag, Kirklareli, Istanbul and Canakkale Provinces. 16 sera were detected as positive in total of 1310 sera, which were collected from day 28. (14/1046) and day 60 (2/ 264). The ratio of positive samples in total was 1.22 % (1.6% bovine and 0,7% ovine sera) (Tables 1 and 2 and Fig.1). Sera, which were detected positive, collected from animals that their ages were more than 2 except two cattle one of them from Istanbul and the other from Tekirdağ province, which were between 1 and 2 old. Taking account each OD value of test serum, positive and negative control, it was estimated mean, maximum and minimum OD, standard deviation and coefficient of variation. Mean OD of positive control sera was 1.107. Detailed data is shown in table 3. 4. Discussion In this study reported here, it was reflected a statistical percentage in terms of NSP antibody in selected animals from whole animal population of Thrace Region. It was detected very low percentage which was1.22% in total 1310 animals as positive for antibody to FMD NSP. This percentage is almost correlated with previous NSP surveillance. Sera, which were determined as positive, were collected from animals in different provinces and we don’t have any detail about number of applied vaccine. But we have certainly known that those animals have not had any FMD infection during the last one year. Lubroth et. al. (1996) reported that when sera from animals repeatedly vaccinated in the field were examined, occasional positive or doubtful antibody levels to NSP were detected. Likewise Bergman et. al. (1993) found that older, repeatedly vaccinated animals gave occasional positive reaction to one or more bands in the EITB. These and other studies (Mackay, D., et. al.) indicate that the detection of antibodies to FMD NSP is not clearly an indication of previous infection. Therefore age and previous history of seropositive animals must always be taken into account when designing a planning of srosurveillance to trace viral activity. In the light of all these findings, it is thought that those animals detected as positive for antibody to FMD NSP might be: • The rate of positive animals was too low to indicate active virus circulation in the region. • Animals previously infected with FMDV in Anatolian and introduced to Thrace region. • Animals previously infected with FMDV in Thrace region and antibody remained until test time. • False positive. In this issue, since 14 animals of in total 16 detected as positive were more than two years old, it was supported this possibilities. Furthermore this possibility was proven by a field active surveillance, which was conducted in epidemiological units where had been collected those positive sera, in conclusion it was not identified any clinical infection.

88

Perhaps since vaccinated animals have been under the risk to become carriers, this is another complication for interpretation in our results. In any case, the last FMD outbreak was reported in Malkara district, Tekirdag Province in the Thrace Region on 29 June 2001, it has not been luckily occurred any outbreak since this time. 3 ABC ELISA used in this study is very easy to perform, reproducible and specific. Although test instruction was recommended that OD value of positive control should be less than 0.900, we obtained 1.107 as a mean OD value, which was slightly higher absorbance than recommendation. Also not only positive control, at the same time we generally had got high absorbance in whole plate wells. However, we have had an opportunity retest those positive sera and some negative sera, which were tested as negative our study, during the FAO workshop in Sofia, Bulgaria in March 2002, then those 16 sera were scored as positive and rest of 16 sera as negative with low absorbance like recommendation. So we have been sure that those sera have been proven as positive with repeated test. It was concluded that test conditions such as high room temperature and incubator condition might be caused such high absorbance. Data generated from analysis of results are indicated that in generally reproducibility of test seems very plentiful because of the obtained low standard deviation and coefficient of variation which they are assigned less pipetting error and test stability. In conclusion, this study was important to reveal the rate of animals previously contacted with FMDV in Thrace Region. But a further study is needed to clarify the real and implicit situation of the virus circulation in the region such as probang sampling and virus isolation. Acknowledgements ELISA kits, which were used in this study, were donated by the EU. The authors wish to thank members of the Sap Institute particularly Dr. Sinan Aktas and Lab. Technicians, who are Oktay Tezal and Yusuf Demir. References Bergman, I.E., et.al. (1993). Diagnosis of persistent aphtovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens. Am. J. Vet.Res. 1993; 54(6): 825-31 Diago M. De., et.al. (1997).The non-structural polyprotein 3ABC of FMDV as a diagnostic antigen in ELISA to differentiated infected from vaccinated cattle. Arch. Virol.; 142:2021-33 Lubroth, J., et.al. (1996). Cattle response to FMDV nonstructural proteins as antigens within vaccines produced using different concentrations.Res. Vet.Sci. 59:70-8 Mackay, D.J.K., et. al. (1998). Antibody to the nonstructural proteins of FMDV in vaccinated animals exposed to infection. Vaccine16(5) 446-59

89

Number of sera 1046 264 1310

Positive 14 2 16

SERA FROM DAY 28. SERA FROM DAY 60. TOTAL Table.1. The results of the 3ABC-ELISA

Provinces

Species

% %1.3 %0. 7 %1.22

Positive

Large Rum.

Small Rum.

Large Rum.

No.

No.

No.

%

No.

%

Edirne

161

169

0

0

1

1.29

Tekirdag

155

155

2

1.29

2

1.29

Kirklareli

165

165

2

1.22

1

0.60

Istanbul

125

125

5

4.0

0

0

Canakkale

45

45

2

4.44

1

2.22

TOTAL

651

659

11

1.67

5

0.76

1310

Small Rum.

16 (1.22%)

Table 2: The results of the 3ABC-ELISA: Distribution of results by provinces and animal species

mean

Standard deviation

Coefficient of variation (%) <%2 %2-5 %5-10 >%10 OD of negative control 0,189 1.2 80 15 3 2 OD of positive control 1,107 1.6 76 20 3 1 OD of test sera 51 29 15 5 Table.3: Data generated from analysis of each test plate wells OD value. Standard deviation value was given as mean of cumulative. Coefficients of variation values were shown in different category of as percentage.

90

690 640 590 540 490 440 390 340 290 240 190 140 90 40 -10

species large ruminants species small ruminants positive L. ruminants positive small ruminants

91

TOTAL(1310)

Canakkale(90)

Istanbul(250)

Kirklareli(330)

Tekirdag (310)

Edirne(320

Figure 1: The results of the 3ABC-ELISA

Appendix 11

Genetic characterisation of type O and A viruses isolated from Turkey between 2000-2002 Parlak Ü, Aktaş, S., Özyörük, F. FMD Institute, P.K. 714, Ulus, Ankara, Turkey

Introduction Foot and mouth disease is one of the most important diseases of livestock due to direct and indirect losses. FMD has been endemic in Turkey causing several outbreaks every year. The disease has been controlled by mass vaccination of susceptible animals, restriction of animal and animal product movements, quarantine and other zoosanitary measures. Introductions of new virus strains from neighbouring countries have caused several epidemics in the past. Therefore it is very important to characterise virus strains and to determine the relationship between field viruses and vaccine strains. The objective of this study was to characterise FMD type O and A viruses isolated from Turkey between 2000 and 2002. For this purpose, sequences of approximately 170 nucleotides at the 3’ end of the gene coding for capsid protein 1D were determined. Material and Methods Viruses 9 FMD type A virus isolates and 4 type O virus isolates from Turkey between 2000 and 2002 were used in this study (Tables 1 and 2). All viruses were adapted to BHK-21 cells and the clarified infected cell culture supernatants were used for extraction of RNA. Additional sequences used in the study obtained from Genbank. RNA Extraction, RT-PCR Total RNA was extracted using RNeasy kit (Qiagen) according to the manufacturer’s instructions. RT-PCR reaction was performed as described by Knowles and Samuel (1998). The primers used for RT-PCR were NK61, ARS4 and 1C562 (Table 3). The PCR products were purified using Promega Wizard Preps Kit. Cycle Sequencing The fmolTM DNA sequencing kit (Promega, U.K.) which utilises the method described by Murray (1989) was used and the manufacturer’s sequencing protocol using an end-labelled primer (NK72) was followed. Sequence analysis Sequence of 165 nucleotides at the 3’ end of the 1D gene of all samples subjected to phylogenetics analysis. For comparison type A and type O sequences from different regions reported earlier were also included in the study. Nucleotide sequences were aligned with CLUSTALX (Thompson et al., 1997), Phylogenetic analysis was carried out with the SEQBOOT, DNADIST and FITCH algoritms in the PHYLIP ver 3.5c package. The phylogenetic tree was constructed as described by Felsenstein (1993).

92

Table 3. List of primers and sequences used in this study. Primer

Virus

Sense

Gene

Primer Seq. (5'-3')

pNK61

Universal

Negative

P2B

GACATGTCCTCCTGCATCTG

pNK72

Universal

Negative

P2A

GAAGGGCCCAGGGTTGGACTC

pARS4

Type O

Positive

P1C

ACCAACCTCCTTGATGTGGCT

Type A

Positive

P1C

TGGTTGGAGGAACTACACCGA

1C562

Results and Discussion The nucleotide sequences of type O and A viruses used in this study and previously sequenced viruses were plotted as dendograms and shown in Figures 1 and 2. Viruses that have a difference of about 5% or less in nucleotide sequence were considered as closely related ( Samuel et al., 1988). All type A viruses sequenced in this study were closely related to each other and A/Iran/96 virus group. A/Iran/96 was first detected in Turkey at the end of 1997 (A/TUR/12/97). The nucleotide difference between these viruses and other type A viruses isolated previously from Turkey was over 18% (Aktas, 1998). The results of this study showed that type A viruses isolated from Turkey between 2000 and 2002 were belonging to A/Iran/96 group. The close genetic relationship between field isolates and the Turkish vaccine strain (same as A/TUR/40/98) and r values detected by antigenic characterisation ELISA, shown in brackets in Figure 1 and Table 3 ), confirmed that the current type A vaccine strain was suitable for Turkey. The sequencing results of type O viruses isolated from Turkey between 2000 and 2002 showed that there were three different genetic sublineages circulating in Turkey during this period. Similar result was found in a previous study (Aktas, 1998). Although these viruses were genetically different from each other, all of these type O FMD viruses could be considered as belonging to a single genotype since nucleotide differences between these viruses were less than 15% (Samuel et al., 1997). On the other hand antigenic study showed that these viruses were closely related to the vaccine strain, O Manisa 1969 (Table 3).

93

Table 1. List of type A viruses used in this study. Viruses A24//BRA/55 A10/HOL/42 A/IRQ/24/64 A/IND/17/77 A/IRN/87 A/IRN/17/96 A/MCD/3/96 A/SAU/23/99 A/TUR/12/73 A/TUR/2/80 A/TUR/7/85 A/TUR/3/89 A/TUR/9/91 A/TUR/3/92 A/TUR/3/95 A/TUR/8/96 A/TUR/11/97 A/TUR/12/97 A/TUR/25/98 A/TUR/40/98 A/TUR/1/00 A/TUR/2/00 A/TUR/3/01 A/TUR/4/01 A/TUR/5/01 A/TUR/6/01 A/TUR/7/01 A/TUR/8/02 A/TUR/9/02

Geographical origin Brasil Iraq India Iran Iran Macedonia Saudi Arabia Antalya ,Turkey Van ,Turkey Sivas ,Turkey Ankara, Turkey Afyon, Turkey Kayseri ,Turkey Kastamonu ,Turkey Samsun, Turkey Nigde Turkey Kutahya ,Turkey Ankara,Turkey Izmit, Turkey Malatya Turkey Kırıkkale Turkey Malatya Turkey Manisa Turkey Sivas Turkey Afyon, Turkey, Bolu, Turkey Bingol, Turkey Kars ,Turkey

Date 1955

Reference Knowles et all. (1999)

1964 1977 1987 1996 1996 1999 1973 1980 1985 1989 1991 1992 1995 1996 1997 1997 1998 1998 2000 2000 2001 2001 2001 2001 2001 2002 2002

Knowles N.J. (1999) Tosh, et all.(2000) Marquardt & Adam (1988) Pirbright Pirbright Pirbright Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) Aktas,S., (1998) This work This work This work This work This work This work This work This work This work

Table 2. List of type O viruses used in this study Virus O/GRE/27/96 O/IRQ/30/00 O/IRN/7/94 O/Manisa/TUR/69 O/TUR/10/94 O/TUR/1/96 O/TUR/1/00 O/TUR/2/00 O/TUR/3/01 O/TUR/4/02

Geographical origin Evros,Greece Iraq Iran Manisa,Turkey Nilufer, Bursa, Turkey Kesan, Edirne, Turkey Konya, Turkey Nigde, Turkey Mudurnu, Bolu Sivas, Turkey

Date 1998 2000 1994 1969 1994 1996 2000 2001 2001 2002

Reference Samuel & Knowles (2001) Samuel & Knowles (2001) Samuel & Knowles (2001) Aktas & Samuel (2000) Samuel & Knowles (2001) Samuel & Knowles (2001) This work This work This work This work

94

Table 3. r values obtained against Turkish vaccine strains. A/TUR/6/01 A/TUR/7/01 A/TUR/9/01 A/TUR/1/02 A/TUR/2/02 A/TUR/3/02 A/TUR/4/02 A/TUR/5/02 O/TUR/1/01 O/TUR/2/01 O/TUR/3/01

0.66 0.75 1 >1 0.5 1 0.35 0.75 0.5 0.5 1

References: Aktas, S. (1998). Molecular epidemiology of FMD types O and A in Turkey. PhD Thesis, University of Reading. Felsenstain, J., 1993. PHYLIP- Phylogeny inference package, version 3.5c. University of Washington, Seattle, WA, USA. Knowles, N.J.,Samuel, A.R.(1998). RT-PCR and sequencing protocols for the molecular epidemiology of exotic diseases of animals. IAH, Pirbright Laboratory, Surrey, UK. Marquardt, O. and Adam, K.-H. (1988). Sequences of capsid protein VP1 of two type A footand-mouth disease viruses. Virus Genes, 2: 283-291. Samuel, A.R., Knowles, N.J. and Kitching, R.P. (1988). Serological and biochemical analysis of some recent type A FMDV isolates from the Middle East. Epidemiology and Infection, 101, 577590. Samuel, A.R., Knowles, N.J., Kitching, R.P. and Hafez, S.M. (1997). Molecular analysis of FMD type O viruses isolated in Saudi Arabia between 1983 and 1995. Epidemiology and Infection, 119, 381-389. Thompson ,J.D., Gibson,T.J., Plewniak,F., Jeanmougin,F. and Higgins,D.G. (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 24:4876-4882.

95

A/TUR/3/01 A/TUR/8/02 (

1)

A/TUR/7/01

( 0.75)

A/TUR/4/01

Figure 1. Dendogram depicting relationships between FMDV type A field isolates and the vaccine strains.

A/TUR/9/02 A/TUR/6/01

( 0.66)

A/TUR/1/00 A/TUR/5/01 A/IRN/17/96 A/TUR/2/00 A/TUR/25/98 A/TUR/40/98 A/TUR/12/97 A10/Hol/42 A24/Cr/Bra/55 A/MCD/3/96 A22/IND/17/77 A/TUR/02/80 AMahmatlı A/TUR/12/73 A/TUR/11/97 A/TUR/08/96 A/TUR/3/95 A/SAU/23/99 A/TUR/03/92 0.1

A/TUR/09/91 A/IRN/87 A/TUR/3/89 A/TUR/7/85

O/TUR/2/00 Figure 2. Dendogram depicting relationships between FMDV type O field isolates and the vaccine strain.

O/IRQ/30/00 O/TUR/3/01 (1) O/TUR/1/00

O/TUR/4/02 O/GRE/27/96 O/TUR/1/96 O1/Man/TUR/69 O/IRN/7/94 0.1

O/TUR/10/94

Appendix 13

Modelling Foot-and-Mouth disease: A comparison of epidemiological models François MOUTOU, Benoît DURAND AFSSA, LERPAZ, Epidemiology Unit, BP 67, 94703 Maisons-Alfort cedex, France Tel : 33 1 49 77 13 33, fax : 33 1 43 68 97 62, E mail : f.moutou@alfort.afssa.fr

Introduction Models have been used for some times with the aim of helping in decision making around Foot-and-Mouth Disease (FMD) outbreaks. However, up to very recently, most of these models could mainly be seen as training tools, because Western European and North American countries, as well as Australia and New Zealand, where many of them have been developed, had been free of FMD for many years, or even never experienced the disease like New Zealand. In fact, most of the data used were those collected during the 1967-1968 UK epizootics, under farming conditions certainly different from those prevailing today. More recently, with the sanitary measures –ban on vaccination– implemented over Europe, a new demand arose. However, most of the models were still used either as tools to better understand FMD virus transmission including airborne transmission, to mimic an outbreak, for training purposes, or to compare economical scenarios. The 2001 epizootics, bringing a lot of new data about FMD transmission under today farming conditions, was the occasion for new modelling. In the UK, during 2001, different models were run and published, with conclusions used to control the disease by the authorities. Here we try to present and to compare these models, from what has been published. What is modelling? Modelling is by no means a new tool in epidemiology, even if the development of smaller but much more powerful and easier to use computers -and software- certainly helped a lot. If modelling is aimed at a better understanding of reality and is seen as an instrument in decision making, it more often pinpoints where data are lacking or where understanding of a natural phenomenon is still to be improved. Modelling in epidemiology cannot be seen without a strong connection with field data, even if it should be able to bring help to take decision when not everything is known, a classical situation. FMD is a good example of a disease that can be computed, at least in our west European farming conditions, due to: -fast spreading of the virus through airborne route to four domestic species, -officially good agricultural statistics, -knowledge of animal movements, -the absence, up to now, of wild reservoirs or vectors. During the spring of 2001, different teams made projections, from the beginning of the epizootics, to predict its probable size and duration. A recent synthesis already compared these results (Kao 2002). The present paper will use this reference at large.

103

Modelling FMD in the UK in 2001 Beside the surprise that such an epizootic may represent, it is worth giving of it a good description. In fact, it is only at the end of the year 2001 that such publications were accessible (Gibbens et al. 2001) even if data where already present at the DEFRA. Of course, at the end of February 2001, very few was known. The different teams involved used different approaches, more linked to their own previous modelling experience than to FMD knowledge. The main model categories include deterministic differential equations, micro-simulation, spatial models, and the models could be deterministic or stochastic. In every case, parameters had to be estimated and this proved to be difficult. One of the most classical index is the so called Current Case Reproductive Rate (CRR), which is a transmission parameter indicating how many new outbreaks an existing infected premise (IP) will create. If the value is over 1, the epidemic will increase, if it is below 1, it is decreasing and probably under control. Most of the models are testing their own parameters against this threshold value. Spatial structure of the countryside was of importance, as the distance between farms and between sensitive animals was one of the key to the problem (Morris et al. 2001). However, distance is a difficult parameter to address as it can be seen as a geographical dimension but also as a functional relationship (dairy farms are closer to each other than dairy and beef farms over the same area, as different people, traders, technicians, veterinarians, will visit them). “Moment-closure”, i.e. the density of farms clusters, or “contact matrix”, i.e. distances between pairs of farms, were two sophisticated ways to give value, under many hypotheses, to such distances. The “moment closure” was used by Ferguson et al. (2001). It relies on clusters of farms and on density of farms clusters. The model estimates the probability of virus transmission between farms and farm clusters. The “contact matrix” was used by Keeling et al. (2001): it integrates the distance between farms when estimating the probability of virus spreading should one farm becomes an IP. Each farm could also vary in susceptibility through parameters like: distance to an IP, to a pig farm, to a slaughterhouse, to a road, or to a large farm, and like known commercial links with an IP. One of the main questions asked early during the epidemics, was the culling strategies (and/or the vaccination strategies) to implement, how (delay between suspicion/diagnosis and culling for instance) and why. Such different kind of premises as the following were considered: infected premises (IP), direct contact premises (DC), contiguous premises (CP), premises within 3 km of an IP. The discussion was mainly around a comparison of the results of shorter delays versus longer delays to cull IP animals and/or DC and CP animals. Not only epidemiological considerations were seen, but also economical points of view. This may explain why the no vaccination scheme was preferred. Results Beside the “official” publications, it was also possible to use other information source, through internet, and the following figure is one example (figure 1). It is all working with a logistic model. Other information than just epidemiological or even economical data are added! Such figure, and others, could be found at: http://www1.tpgi.com.au/users/kpduffy/fmd.htm The global shape of the epidemic is clearly seen on this figure. It also compares it with the 1967-1968 epidemic. If the model’s fit is rather good for the beginning of the epidemic, the predicted tail is much shorter, but comparison and interpretation must be cautious. 104

This figure (and this model) is mainly descriptive but recalls when different important decisions were taken.

Figure 1. A simple descriptive model based upon the logistic equation. The comparison of culling scenarios came with the other modelling approaches.

Figure 2. Comparison of the predicted epidemic curves under various control strategies with a deterministic model (Ferguson et al., 2001a)

105

In the case of Ferguson and co-workers (2001a, 2001b), the question is really to look for the control of the epidemic though shorter delays between diagnosis (or clinical suspicion) and culling, i.e. the end of virus excretion by animals (figure 2). Only scenarios with culling of IP and of farms in the surroundings, within a short period of time, will lead to a CRR below 1, i.e. a control of the epidemic. It is however already well known that waiting too many days to cull infected animals will not be a good politic during an outbreak of FMD.

Figure 3. Comparison of the predicted epidemic curves under various control strategies with a stochastic model (Keeling et al., 2001) Black: data (reported daily cases), Pink: 100 runs of the model, Red: mean daily value of the 100 runs

1.0 0.8

SO

0.6

SODC

0.4

SOV2

0.2 0.0 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21

Epidemics duration (weeks)

1 0.8

SO

0.6

SODC

0.4

SOV2

0.2 0 0

40

80 120 160 200 240 280 320 360 400 440 480 520 560 600 640 Epidemics size (outbreaks)

Figure 4. Cumulative distribution of the predicted epidemics size and durations under three control strategies (Durand and Mahul, 2000)

SO: IP cull only, SODC: IP and dangerous contacts cull, SOV: IP cull and ring vaccination from the end of the 1st week

106

Direct costs (mFF)

1 0.8 0.6

SO

0.4

SODC

0.2

SOV2

0 0

100

200

300

400

500

600

700

800

900 1000 1100 1200

Total costs, net of the EU subsidies, borne by the French economy (x1000 mFF) 1 0.8 0.6

SO

0.4

SODC

0.2 0 16.05 16.20 16.35 16.50 16.65 16.80 16.95 17.10 17.25 17.40 17.55

Figure 5. Cumulative distribution of the predicted epidemics costs under three control strategies (Durand and Mahul, 2000)

SO: IP cull only, SODC: IP and dangerous contacts cull, SOV: IP cull and ring vaccination from the end of the 1st week

In fact, all figures 2 and 3 are trying to say the same thing, with different approaches: culling without vaccination is the best scenario to apply. Figures 4 and 5, by comparison, which were published in 2000, are discussing the same item but do not exclude vaccination (Durand and Mahul, 2000; Mahul and Durand, 2000). Here SO means stamping out of IP, SODC: stamping out of IP and of dangerous contacts, and SOV: stamping out of IP and ring vaccination from the end of the first week of the epidemic. The delay of one week is explained by saying that you certainly know a little more after one week, and that it may not be a good strategy to decide and to implement vaccination as soon as the first outbreak is known. These figures compare in terms of epidemic duration and size (figure 4) and in term of cost (figure 5) what will bring each strategy. Figure 4 can be read as the probability (y-axis) that an epidemic duration or size, respectively, will be less than a given value (x-axis). The shortest and smallest epidemics are obtained with the stamping out of IP and of dangerous contact culling policy, when the longest and largest is linked to stamping out of just IP. In terms of costs (figure 5), respectively direct and total, the best scenario is also with the SODC policy, even if for direct costs, SO (stamping out of just IP) is not so different. Discussion To be able to run such models as those published in nearly real time means that a lot of data had already been collected in the field. As the teams were not always the same, between those in the field and those computing a good coordination was necessary. However, from what is known today, the beginning of the epidemics was quite earlier than the time of the discovery of the first case, moment at which time the size, the geographical dispersion of the virus, the actual number of outbreaks and the distribution of the disease, 107

were not known and underestimated. FMD in sheep was not so well known in Europe and the importance of sheep trade, over Europe, was difficult to compute. The situation proved to be quite unique, so these models may be especially devoted to mimic FMD in sheep under conditions closed to February and March 2001 in United Kingdom. In the same time, deciding the shortening of the delays between suspicions and culling is a classical knowledge in case of FMD in our countries. The comparison made by Kao (2002) is clearly a modeller analysis, more than an epidemiological approach. The published papers brought a lot in term of methodology for models, maybe more than just in term of FMD epidemiology. Acknowledgements We thank Kris De Clercq who suggested this contribution, Michelle Rémond who presented it at the meeting, and the Research Group for support. References Durand B. et Mahul O. (2000) ~ An extended state-transition model for foot-and-mouth disease epidemics in France. Preventive Veterinary Medicine, 2000, 47: 121-139. Ferguson NM., Donnelly C.A., Anderson R .M. (2001a) ~ Transmission intensity and impact of control policies on the foot-and-mouth epidemic in Great Britain. Nature, 2001, 413: 542-548. Ferguson NM., Donnelly C.A., Anderson R .M. (2001b) ~ The foot-and-mouth epidemic in Great Britain : pattern of spread and impact of interventions. Science, 2001, 292: 1155-1160. Gibbens J.C., Sharpe C.E., Wilesmith J.W., Mansley L.M., Michalopoulou E., Ryan J.B.M., Hudson M. (2001) ~ Descriptive epidemiology of foot-and-mouth disease epidemic in Great Britain : the five first months. The Veterinary Record, 2001, 149: 729-743. Kao R.R. (2001) ~ Landscape fragmentation and foot-and-mouth disease transmission. The Veterinary Record, 2001, 148: 745-747. Kao R.R. (2002) ~ The role of mathematical modelling on the control of the 2001 FMD epidemic in the UK. Trends in Microbiology, 2002, 10(6): 279-286. Keeling M.J., Woolhouse M.E.J., Shaw D.J., Matthews L., Chase-Topping M., Haydon D.T., Cornell S.J., Kappey J., Wilesmith J., Grenfell B.T. (2001) ~ Dynamics of the foot-and-mouth epidemic : stochastic dispersal in a heterogeneous landscape. Science, 2001, 294: 813-817. Mahul O. et Durand B. (2000) ~ Simulated economic consequences of foot-and-mouth disease epidemics and their public control in France. Preventive Veterinary Medicine, 2000, 47: 2338. Morris R.S., Wilesmith J.W., Stern M.W., Sanson R.L., Stevenson M.A. (2001) ~ Predictive spatial modelling of alternative control strategies for the foot-and-mouth disease epidemic in GreatBritain, 2001. The Veterinary Record, 2001, 149: 137-144.

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Appendix 14

Problems in stamping-out and rendering practice: suggestions for improvements and alternatives Simon J. Barteling Consultant Veterinary Vaccines. address: Nieuwe Keizersgracht 438, 1018 VG, Amsterdam, The Netherlands. E-mail: s.barteling@chello.nl

Summary In general, upon outbreaks of FMD, many efforts are made to contain the disease on outbreak farms. Here, in addition, the principles and rules that have been developed for the highcontainment laboratory, are translated for suspected and outbreak farms, the killing of livestock, the transport of carcasses, and the rendering plant. For the transport of carcasses a (closed) container system is proposed, enabling the lorry and its driver not to become contaminated. It is also proposed to start the rendering process during transport and to operate the rendering plant like a high-containment laboratory (including the same strict rules for its staff). Introduction During the last 30 years a whole set of principles and rules for bio-safety and for highcontainment laboratories have been developed. Many of these principles and rules are already implemented for virus containment on outbreak farms however, one may ask whether more could be done. In this context the transport of carcasses and their rendering are considered as well. Primary containment The main principle of the high-containment laboratory and FMD vaccine production plant is “keep the devil in the bottle” and kill or catch it where it might have escaped. Another principle is that virus cannot escape from rooms where there is no virus. In other words keep the rooms - even though protected by negative air pressure and air-filtration etc. – as virusfree as possible. This will also mean that under normal conditions staff will not become heavily contaminated preventing the associated risks. Wherever “open” manipulations have to be performed e.g. with bottles or tubes containing virus, this should be carried out in a biosafety cabinet. How far is this “primary containment” already practised on suspected and outbreak farms? On the outbreak farm, in general, the diseased animals will excrete the virus in enormous quantities (Sutmoller et al. 2002). The virus behaves like a fine desert dust and probably even worse. On the farm it contaminates all surfaces (outside and inside) including clothes and vehicles. It follows that the most common way of dissemination is not only by infected live animals and contaminated animal products, but also by people, vehicles, and all kind of contaminated materials (fomites) that are leaving infected farms. In general, once FMD is confirmed, the (diseased) animals are killed and the farm is disinfected as soon as possible. For primary containment one could ask oneself whether it would not be better to start (regular) disinfecting the place already when there is suspicion of FMD? Also, is it possible to

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diminish the level of virus aerosols e.g. by regular spraying with the (usual) citric acid solution supplied with acetic acid? Acetic acid is non-toxic and will easily evaporate. Following the principle “where there is no virus it cannot spread” it may make sense if visiting veterinary staff – even when wearing protective clothing - “spray their way” to prevent as much as possible to become contaminated. It could also be good practise in order to make everybody aware of the seriousness of the situation. Changing, and if possible, showering at the gate should of course continue as a decontamination measure. Because cleaning procedures incorporate the risk of making virus aerosols, disinfecting should always precede cleaning, (followed by a final disinfecting round). Laboratory confirmation of suspected cases may take several days, sometimes even weeks. Because vaccination with a potent vaccine may start to protect animals already after 3 or 4 days and stop further dissemination of virus, it may make sense if upon suspicion (of secondary cases) of FMD, all animals on the farm (and contact farms) are immediately vaccinated. For that purpose oil-based vaccines could be available at (local) vaccine banks frozen at -70ºC. One could vaccinate “towards the source” - from the most remote animals towards the animals found with the symptoms (not to forget to change needles regularly and logically). Secondary containment In the laboratory, a second level of containment is provided by the technical facilities of the building: negative air-pressure maintained via HEPA filtration, (heat or chemical) treatment of sewage, airlocks, and (airlock-) showers for the staff. The air treatment is particularly important in places where the formation of virus aerosols cannot be prevented e.g. in the stables. The air-exchange rate will dilute the virus aerosols e.g. an exchange rate of 14 times per hour means that in one hour a million infectious units will be reduced by about a 1000 fold to approximately 1000 units. Often the figures will be even more favourable because air tends to flow from positive to negative pressure or, in other words, from the blowing (the disseminating animals) to the suction point (the filter unit). In the well-ventilated highcontainment stables at Lelystad transmission of disease was only guaranteed if the animals had physical contacts or if the animal caretaker (purposely) acted as intermediary. Very likely, when rooms are well ventilated, virus aerosols are too much diluted to reach the minimum infective dose, the number of infectious units of FMD virus that is required to infect susceptible animals. It is already more than 30 years ago that Sellers (1971) reviewed this topic. He concluded that exposure to relatively high levels of virus does not necessarily result in infection. It is also known that the minimal infective dose of a particular virus strain varies for different animal species and the route of infection. To prevent further spreading of disease, it might help if besides the regular spraying with disinfectant (see above) suspicious farms or farms at risk place small, commercially available (HEPA) filter units in the stables. Anyhow, it seems worthwhile to try to eliminate as much as possible what is considered “uncontrollable” spread by aerosols (Donaldson, 2001). Rules for laboratory staff and visitors on how to behave in and outside the high-containment facilities are, in general, well implemented for the suspicious and infected farms but require constant training and supervision.

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Must all animals on infected premises be killed? In Europe before the discontinuation of vaccination (in 1991) one of the last (type A) outbreaks of FMD occurred in 1987 on a large pig holding (approximately 5.000 pigs) in Northern Italy. Because of limited rendering capacity the authorities decided to vaccinate all the animals in the holding with a potent aluminium hydroxide vaccine emulsified in oil. The pigs in infected pens and “in-contact” pigs were killed and the premises were carefully disinfected and on the farm a sanitary regime was installed to prevent spread of the virus. The other vaccinated pigs were left alive and later went for (normal) slaughter for human consumption. Of these pigs 35 were followed for possible virus reproduction by nasal swabbing at regular intervals. Only from one pig at 7 days post vaccination a minimal amount of virus was isolated probably caused by captured virus aerosol from the environment. Active virus multiplication has not been demonstrated (Panina et al.). A similar approach as described above was successfully followed on another huge pig holding in Northern Italy in 1989 (approximately 10.000 pigs) and in 2001 in South Africa on a very large cattle holding (about 16.000 cattle!) infected with SAT1-type virus. Also in these cases vaccination worked very well and the disease did not reoccur. Whether on infected premises – and on “contact” farms - all susceptible animals must be killed or whether they can be saved by vaccination might be a subject of further considerations. Anyhow, when it is clear that FMD is involved, affected animals (and animals with fever) should be killed as soon as detected (“close the bottle”). If only a few animals are affected and vaccination is starting to protect the others, one could wait and see, leave the farm under quarantine for some time and start to test for carriers which can – for the sake of everyone’s peace of mind - subsequently be eliminated. This approach makes sense for large holdings in particular, not only because of the costs of compensation, but also to prevent associated risks connected with the large scale culling. Killing, transport, and rendering of carcasses Once the infected animals are killed the problem is to transport infectious material from one containment unit (the infected farm) to another (the rendering plant). For that purpose really closed (air-tight) containers/tanks should be used. Ideally, a system should be developed with containers that can be detached from the lorry at the gate of the farm. This will prevent that the lorry (and its driver) becomes contaminated. Before transport, concentrated caustic soda could be added to the safely closed-off content of the tank. This will increase the temperature and will start the rendering process. A provision for rolling the tanks during transport could be added (like for concrete) in order to guarantee freedom of virus - or at least a very low level of surviving virus - upon arrival at the rendering plant. If it cannot be guaranteed that the contents of the containers/tanks are virus-free, the rendering plant should be operated like a high-containment laboratory (including the same strict rules for its staff). The containers/tanks could be dropped outside the rendering plant, using a one way operational system for intake into the plant (via air-locks), disposal of the carcasses in a hall under negative air-pressure, a disinfecting-cleaning-disinfecting procedure to decontaminate the containers/tank and, finally, exclusion of the tanks via another air-lock.

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Concluding remarks During the past thirty years great efforts have been made to increase the safety of FMD highcontainment laboratories and to prevent spreading of FMD from such institutions. However, upon outbreaks, the developed principles and technologies for the high containment laboratory are not yet fully applied for the infected/suspected outbreak farms, neither for the transport of the infected carcasses nor for the rendering plants. Here I have proposed that – in addition to existing practice - principles, methods, and technologies available for the high-containment laboratory be brought further into practice during outbreak situations, proposals that would hopefully contribute in making FMD a more controllable disease. References - Donaldson A.I., Alexandersen S., Sorensen J.H. and Mikkelsen T. (2001) – Relative risk of the uncontrollable (airborne) spread of FMD by different species. Vet. Rec. 148, 602-604 -.Panina G.F., Amadori M., Guadagnini P.F., Massirio I, Melegari M., Pavesi M., & Perini S. (1988). – Il controllo dell’afta epizootica nella specie suina. Selezione Veterinaria, 29 (1 bis), 197-206. - Sellers R.F. (1971). – Quantitative aspects of the spread of foot and mouth disease. Vet. Bull., 41 (6), 431-439 - Sutmoller, P., Barteling, S.J., Sumption, K.J. & Casas Olascoaga, R. 2002/3: “Control of foot-and-mouth disease” In: “Foot-and Mouth Disease”, D.J. Rowlands ed. Elsevier Scientific Publ. Amsterdam (in press). Acknowledgement I thank Dr. Paul Sutmoller for critical remarks, Dr. Franco De Simone for information on the “Italian approach” on large swine holdings in 1987 and 1989, and Mr. Jon Dobson for editorial suggestions.

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Appendix 15

Culling versus vaccination: challenging a dogma in veterinary (FMD) science Simon J. Barteling¹ and Paul Sutmoller² ¹Consultant Veterinary Vaccines. address: Nieuwe Keizersgracht 438, 1018 VG, Amsterdam, The Netherlands. E-mail: s.barteling@chello.nl ²Animal Health Consultant, former chief of Laboratories of the Panamerican Foot and Mouth Disease Center PAHO/WHO. Present address:1502 Largo Road #101, Richmond, Virginia 23233, United States of America E-mail: paulsutmoller@compuserve.com

Summary Outbreaks of FMD can either be controlled by stamping-out or (circle) culling, or by (ring-) vaccination around the outbreak area, or by a combination of the two methods. The pros and cons of the two methods are discussed. A major draw-back – next to many other disadvantages - of the massive circle culling is its contribution to spreading disease. We challenge the dogma that “vaccination against FMD will prevent the symptoms but will not eradicate the disease”. Where outbreaks were controlled by consistent vaccination with a qualified vaccine the disease did not re-occur. Also, there are no documented cases where cattle vaccinated with a qualified vaccine, caused new outbreaks. Therefore, the risks posed by vaccinated carriers must be an acceptable, “close to zero” risk. Certainly, if used in combination with an anti-NSP test, vaccination should become the major tool in controlling outbreaks of FMD, without additional negative consequences for export trade. Introduction The oldest way of controlling outbreaks of FMD is by stamping-out, the killing and disposal of all susceptible livestock on infected farms. Already in 1892 a United Kingdom Act of Parliament stipulated that all susceptible animals must be killed and also, that the farmers would be financially compensated. Although from 1940 onwards vaccines have been developed that protect against the disease, so far the U.K., Ireland, Scandinavian countries, the USA, Canada and some other countries have always stuck to the stamping out policy. Circle culling – invented by epidemiologists/computer modellers – and applied during the 2001 outbreaks, can be considered an extension of the stamping-out approach. Another method of controlling outbreaks is by (ring-) vaccination. However, in 2001 vaccination had considerable drawbacks for export trade unless vaccination was declared “suppressive” and the vaccinated animals were successively killed. Here, we first consider the effectiveness of the two methods in controlling outbreaks. Then we will discuss risks of eventual carriers remaining in the vaccinated population and, what became almost a veterinary dogma that “vaccination against FMD will prevent the symptoms but will not eradicate the disease”. We will also discuss whether it is justified to punish – in terms of export trade - the use of (limited) ring- vaccination for controlling FMD outbreaks.

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Stamping-out Traditionally, in FMD free countries, stamping-out has been the first option to eradicate the disease. As a first line of defense it is often quite successful, at least if the disease has not yet spread too widely and if the density of livestock in the area is relatively low. Also, during the first days of an outbreak a proper vaccine might not be available. The choice of the stampingout option should depend as well on the possibility of tracing dangerous contacts, political will and available resources. In general, stamping-out consists also of the killing and disposal of all susceptible livestock of the outbreak farm and of “contact” farms that are most likely infected, followed by thorough cleaning and disinfection. If the outbreak farm is located at some distance from other farms and without intensive contacts, the slaughter of only infected premises - with surveillance of neighboring farms - might be adequate. In general, however, one must be “ahead” of the disease and, to that end, also slaughter “dangerous contact farms”. However, the latter are difficult to define and decisions can create feelings of arbitrariness and unfairness. In the U.K. and in some other countries in the past the carcasses were burned or buried, however, this creates environmental problems and, therefore, rendering is preferred. If stamping-out of the disease succeeds in a relative short period of time it may be the most economical way of dealing with the outbreak. If the disease is already widespread or occurring in an area with a high-density livestock population it becomes increasingly difficult to bring an outbreak under control and “classical” stamping-out has the following drawbacks: -

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Staff must be available for detecting where the virus came from and where it might have spread in the mean time. Active and intensive surveillance is required in order to be “ahead of the disease” and to detect infection at an early stage. Such surveillance in itself represents a risk of spreading disease. Heavy equipment used in these operations is difficult to decontaminate and might be a source of infection e.g. by contamination of roads when being driven to another job or back home. Large numbers of contractors are involved who are not trained in disease containment. Most of them will be from rural areas and might even live next door to farmers. By having social contact in the farming community they can themselves spread disease. Disposal of cadavers also presents a risk since virus in lesions, excrements and excretions is not rapidly destroyed and might be disseminated by transport of cadavers, by pyres, at burial sites or rendering plants. To our knowledge, neither existing transport systems for carcasses, nor the handling of the carcasses at the rendering plants are bio-secure (Barteling 2002). High speed of operations is needed with logistics that are very much dependent on local circumstances. This can hardly be foreseen in contingency plans and, therefore, in the beginning of outbreaks it will take time before the required speed is reached. This will matter more if during a number of years (e.g. ten years or more) a country had no experience of outbreaks or, like in the U.K. the disease is already widespread when it is detected.

Ring- (circle) culling So-called circle culling around infected premises (I.P.) as has been applied in the UK (and in the Netherlands) can be considered as an extension of usual stamping-out procedures. It has been “invented” by epidemiologists-modellers who make use of computer models on how

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(fast) the disease spreads (Ferguson et al. 2001; Keeling et al. 2001). The aim of the circle is to eliminate incubating infections that may have spread from the outbreak farm(s) and to create a “fire break” zone around the outbreak. The diameter of the circle was based on the analysis of spread of FMD during the outbreak using computer models. However, the calculated distance of spread must include spread due largely to the culling process itself (see above). Also, important parameters such as virus dissemination by the involved species, number of animals involved, important parameters for the farms at risk (see below) and other factors are not incorporated. The advantage is that the decision-making is simple and circle culling reduces much of the need for “tracing and anticipating” and surveillance. When the outbreaks are successfully brought under control, the current (economic) advantage is that the FMD-free OIE status can be obtained shortly after completion of the operation. However, there are major draw-backs with respect to rapid disease control and other elements: - Even more than for “classical” stamping-out a military organization is needed to implement all the logistics for the required speed. - For the creation of a fire-break zone most of the culled farms will not be infected. However, some will be in the (undetected) pre-clinical infection stage. Because of the massive nature of the culling these farms and the equipment used for the hauling and destruction will not get the necessary attention for disinfecting with all the risks of further spread of disease. - Most culled farms within the circle are not infected and do not represent a risk of further spread of the disease and, therefore, are culled unnecessarily. Consequently, one outbreak may cause the unnecessary deaths of large numbers of animals. For example, in The Netherlands there was an outbreak on a calf-fattening farm just outside the main infection zone. Although there was no single indication of virus spread, not even on the farm itself (approximately 500 calves), within the 2 km zone 70,000 animals had to be killed. - The strict application of circle culling poses a threat to zoological collections and valuable (rare) breeding stock as well. - The small risk represented by hobby farms and smallholdings is not taken into account. - Massive killing and destruction of livestock is usually not done with adequate respect for animal welfare and bio-ethical principles. - In contrast to control by vaccination (see below), in the outbreak area (s) the duration of a campaign cannot be predicted. A long drawn-out campaign is very disruptive for the rural society as a whole and includes also sectors like tourism. The rural community may fear the control measures more than the disease, and, because their animals are not protected by vaccination, will live under this fear for several months after the last case. - An enormous serological surveillance exercise is often required to detect residual infection since new cases could easily restart the epidemic at its tail-end, particularly if movement controls are prematurely lifted. - Last but not least, many culls represent a human tragedy and a traumatic experience not only for farmers and their families, but for many veterinarians and others in the rural societies as well. The risk-avoidance behaviour of farmers leads to social isolation and breakdown of the social-economic and trading patterns of rural communities.

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If stamping-out is the method of choice, it should be based on the evaluation of how virus spreads (known risk factors). Certainly, the disease will not spread in a circular manner. The principal routes of virus spread to be considered are by: – animal movements from the farm, during the pre-clinical phase; – estimated virus aerosol production (species and number of animals involved) and weather conditions (temperature, humidity, wind direction) on the days preceding the outbreak (Donaldson et al. 2001); – animal proximity at farm boundaries, e.g. grazing in adjacent fields; – size and animal species of neighbouring holdings e.g. a large cattle holding ( large air sampling volume) represents a greater risk of inhaling a minimal infectious dose” (Sellers 1971) and becoming a new generator of disease than a few sheep or goat of a nearby hobby farmer; – people (veterinarians, inseminators, visiting farmers, cleaning and disinfecting crews, etc.) – vehicles (e.g. animal transport, machinery) contaminating roads that are used by the farmer’s family and associates; Because, in principle, every culling may represent a human and animal welfare tragedy each farm should be judged on its own (risk) parameters. If possible, it would be very helpful if epidemiological factors and associated risks could be incorporated into a computer model to enable rapid risk assessment and decision making for each farm Ring vaccination Instead of killing all susceptible livestock around an I.P. a “fire break zone” can be created by vaccination. The factors determining expected spread (as mentioned above) also should determine the size of the “ring” or area around an IP. In addition, to get “ahead” of the disease one must anticipate the natural spread over the period that is needed to organize the vaccination. In general, this will be a few days plus 4-5 days needed before animals become protected by the induced immunity. In comparison to culling the method is relatively cheap and, therefore larger areas can be vaccinated in a relatively short period of time. The logistics of a vaccination campaign are rather simple. It can be carried out on a large scale by a limited number of (trained) staff under full bio-safety conditions or by farmers and trained farm hands as is common practice in South America. The latter method has the advantage that there will be no risk of cross-contamination between farms caused by the visiting vaccinators. When a potent vaccine is used, outbreaks in the vaccinated zone/ring will usually cease within a week when effective herd immunity is reached. Therefore, ring-vaccinations should be performed without delay and should include all susceptible species. Preferably, the vaccination should be carried out from the outside of the “ring” towards the centre, the I.P. To protect the most endangered farms as soon as possible, vaccination should proceed simultaneously from the I.P. towards the outside. In the immediate vicinity of the outbreak farm, the large (cattle) holdings should be vaccinated first

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because potentially, those are the largest “aerosol collectors” and, therefore, will be most at risk. Ring/emergency vaccinations should be included in any contingency plan: - to stop the disease from spreading; - to avoid all of the above mentioned disadvantages of stamping-out; - to prevent the suffering of animals as much as possible; - to ensure that a few weeks after vaccination life in the affected area can resume its normal course, with minimal socio-economic consequences. Concerns have been raised with regard to human consumption of meat or other products from vaccinated animals. However, there is not any reason to object to human consumption of the meat of vaccinated animals. In Europe meat from vaccinated animals has been consumed for over fifty years, including the meat originating from South America. Ring vaccination followed by slaughter (“suppressive” vaccination) Fear of carriers among vaccinated animals has led to the idea of ”suppressive” vaccination. In that approach, vaccination is used to control the outbreak(s), but all vaccinated animals have to be killed before the FMD-free status can be regained. Vaccination was used in the Netherlands in the main outbreak area to control the 2001 outbreak. In accordance to OIE regulations, the FMD free status was regained 3 months after serological surveillance and after the slaughter of the 160.000 vaccinated animals. As indicated above the period would have been one year if the vaccinated animals were not slaughtered. Vaccination works well to get rid of the disease. However, when vaccination is declared to be “suppressive” several of the problems mentioned for the circle culling remain. An advantage is that the vaccinated animals can be killed over a more extended period. It is interesting to note that, although vaccinated pigs do not become carriers they still must be slaughtered as well! Screening for anti-NSP antibodies In non-vaccinated herds foci of hidden infection can be traced by screening for antibodies against the virus. Vaccination will also raise such antibodies and, therefore might frustrate such tracing; another reason for a primary ban on vaccination. However, virus infection raises antibodies not only against the virus particle but also against non-structural proteins (NSP), the proteins that are needed for virus multiplication. These anti-NSP antibodies are also useful indicators of a past infection and, consequently, of potential carriers. After a single vaccination with a non-purified vaccine, a-NSP antibodies will, in general, not be raised. However, after multiple vaccinations such antibodies might be induced as well. In contrast, vaccines prepared from purified FMD antigens, such as those in the EU vaccine banks, will raise antibodies to the virus particle only. Even upon repeated vaccination no antibodies against non-structural proteins will be raised and past infection can still be traced. Such vaccines, in combination with tests for antibodies against non-structural proteins, will perform like a “marker” vaccine, enabling discrimination between vaccinated animals that are infected (potential carriers) and vaccinated animals that are not infected.

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Although in individual animals the tests to detect a-NSP antibodies are not 100% sensitive, they perform very well if used for screening on a herd basis. If required, testing for the presence of virus e.g. by probang tests or PCR can further reduce the risk of missing an individual animal. Thus, the concern that vaccination blurs the distinction between vaccinated and infected carrier) animals, is no longer founded if purified vaccines are used. Tests to discriminate between potential carriers and vaccinated animals have already widely been used by South American countries to support their claims (at OIE) of freedom of FMD. Dogma The statement that “Vaccination against FMD prevents the symptoms but does not eradicate the disease” has become what one might call a dogma in veterinary science. That dogma was born after it was detected that vaccinated cattle could be carriers as well (Van Bekkum, 1959). Also in that period in many countries (e.g. South-America) the vaccines used were often of poor quality, were sometimes not properly inactivated, and were not always consistently applied. This resulted in “silent”, sub-clinical transmission of FMD and re-occurrence of disease. However, to our knowledge over the past 15 years there are no examples where after consistent vaccination with a qualified vaccine, disease re-occurred. There are, however, many examples that FMD has been eradicated by consistent vaccination: 1. In the sixties The Netherlands suffered from large-scale outbreaks in the non-vaccinated pigs first of type C and later of type O. The virus entered from Germany where general vaccination was not yet applied. The routine Frenkel-type vaccine that was used for cattle did not sufficiently protect the pigs. On the (mixed) outbreak farms the vaccinated cattle were well protected and were left alive, and many of them became carriers (Straver et al. 1970). When the outbreaks were under control, by the application of (expensive) concentrated Frenkel-type vaccine - restrictions on transports and markets were lifted. Also, the vaccination of the pigs (in the outbreak area) was - because of the costs – discontinued. Because of their high turn-over rate, the pig population soon became fully susceptible again. Although at the time there were many mixed farms (with cattle and pigs) no further outbreaks occurred of either “pig-o-phylic” type C or type O strain. 2. The Italian outbreaks of 1985-’86 and of 1987 of type C and type A respectively were controlled by stamping –out of infected premises and additional vaccination. One of the last outbreaks in 1987 occurred in a large pig holding (approximately 5,000 pigs). Because of limited rendering capacity the authorities decided to vaccinate the whole lot with a potent aluminium hydroxide vaccine emulsified in oil. The other vaccinated pigs were left alive and later went for (normal) slaughter for human consumption. Of these pigs 35 were followed for possible virus reproduction by nasal swabbing at regular intervals. Only from one pig at 7 days post vaccination a minimal amount of virus was isolated probably caused by captured virus aerosol from the environment. Active virus multiplication has not been demonstrated (Panina et al.). The same approach was successfully followed in 1989 on another large pig holding. That was the end of types C&A in Europe. 3. Like described above in South Africa in 2000 vaccination was used to protect the nonaffected cattle on a very large cattle holding (about 18.000 cattle) where an outbreak of SAT1 virus occurred. Also there, vaccination worked very well and the SAT1-type FMD did not re-occur (Bruckner et al. 2002). 4. Argentina, large parts of Brazil, Chile, Uruguay, and several other states in the southern cone of South-America became free of FMD by vaccination of the cattle population only and were able to cease vaccination.

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5. After reintroduction of the disease in Argentina, the State of Rio Grande do Sul and Uruguay, the disease was rapidly eradicated, again by vaccination of the cattle population only and even without the slaughter of infected animals (Sutmoller et al., 2002). 6. Israel completely relies on (consistent) vaccination only. During the first 2 years of their life cattle are vaccinated twice a year, after that once a year with a tri-valent vaccine. Sheep are vaccinated once a year only against type O. At the borders sometimes outbreaks of FMD occur in the (nomadic) sheep and sometimes also in the outbreed meat cattle, in the not yet or once vaccinated calves. The infected animals are left alive and in the outbreak area the Israelis simply apply an additional round of vaccination. There are no indications that outbreak strains re-occur. 7. Other examples of recent outbreaks controlled by vaccination without re-occurrence of the disease are: Russia 1995, Albania and Macedonia 1996, South Africa 2000 and 2001, Turkey (Thrace) 2001 (Leforban 2002). Thus it can be concluded that consistent vaccination – no holes in the “vaccination blanket” – with the application of a qualified vaccine can eradicate FMD. Also, that the statement/dogma that “Vaccination against FMD prevents the symptoms, but does not eradicate the disease” does not hold true. There may be an exception for SAT-type viruses. There was circumstantial evidence that vaccinated carriers caused new outbreaks in Zimbabwe (in the late eighties and early nineties) of SAT-type virus (Thomson, 1996). Those outbreaks were on farms in the northern districts while the outbreak strains were related to SAT strains occurring in central Zimbabwe. It may be due to the fact that SAT-type viruses occurring in southern Africa have a special survival mechanism via (wildlife) carriers – buffaloes in particular – that is not known for other FMD sero-types. In addition, one could question whether the vaccines that were used were of undisputed quality and whether it really were the vaccinated carriers which caused the outbreaks. The cattle concerned were moved around from central Zimbabwe to farms in the northern districts and they certainly were not the only animals that were moved around. However, the fact that the strains were related to a strain occurring in central Zimbabwe supported the theory that carriers were involved. However, can introduction of sub-clinically infected animals as a possible cause be excluded? Were all the Lorries that bridged the central and northern districts well disinfected? Were there no other contacts between the central districts and the Northern provinces that may have caused transmission of disease? Thomson (1996) also suggests the possibility that “sexual transmission” - from carrier bulls to cows - might be involved. From the few cases with evidence that carriers introduced FMD into “virgin” herds, bulls were involved as well. (Fogedby, 1961; Sutmoller et al., 2002). Therefore, the possibility of sexual transmission of FMD by carrier bulls might well be object of further studies. Anyhow, the Zimbabwe argument is in our view not sufficiently strong to weaken all the evidence that consistent vaccination can eradicate FMD. Also the Republic of South Africa became free by systematic vaccination and remains free because it is protected by a vaccination zone around the Kruger Park where FMD is endemic in buffalos. Because in all those cases where outbreaks of FMD were controlled by vaccination the vaccinated animals and their products went into the normal consumption circuit without

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causing new outbreaks it can also be concluded that products from vaccinated animals do not represent a risk, or at least an acceptable, “close to zero” risk (Sutmoller & Casas 2002, OIE paper in press). The (possible) presence of vaccinated carrier cattle must also represent an acceptable “close to zero” risk. We base that on the fact that there is no evidence that the large numbers of vaccinated (A, O, and C-type) carriers that in the past must have been around in Europe and in South America, did not hamper the eradication of FMD. In those countries vaccination was no longer needed and could (successfully) be discontinued. However, the assumed risk represented by (vaccinated) carriers puts a heavy penalty on the use of vaccine against FMD in the form of import/export restrictions of animals and animal products, even if for the control of outbreaks only a limited fraction of the cattle population has been vaccinated (pigs don’t become carriers). Therefore, countries with an important export of livestock will omit to use vaccination and will – like in the U.K. – try to control the outbreaks by stamping-out. The question is, whether the presence of some (vaccinated) carriers and the risk they represent justifies regulations that in fact exclude a major and simple method to control outbreaks. So far a proper risk assessment has not been made. Also, the possibility to detect carriers by serological tests against non-structural proteins (see below) has so far not been accepted as a further assurance of an – in our view - already acceptable risk. Conclusions Stamping-out of outbreak and “contact” farms can be used as a first approach to control outbreaks, however, should be based on a proper evaluation of epidemiological and risk factors for each individual contact farm. Massive circle culling will likely contribute to further spread of disease and, therefore will extend the duration of an epidemic. We also have pointed out several other reasons why it cannot be justified as a method to control outbreaks. We have shown that the statement – almost a dogma in veterinary science – that “vaccination against FMD will prevent the symptoms, but will not eradicate the disease” will not hold true. We also gave evidence that the risk of vaccinated carriers causing new outbreaks must be negligible. Anyhow, the current regulations are not based on proper risk assessment also not for the situation after control by stamping-out. In our view both stamping-out and vaccination should have the same consequences for trade, at least, as long as only a limited fraction of the livestock population is vaccinated. If outbreaks are numerous and widespread the risk for trade partners increases anyhow whether they have been controlled by stamping-out or by vaccination. If vaccination can be used without additional punishment, dairy farmers, hobby farmers, and others that have nothing to do with export trade (e.g. zoos) can have their valuable animals protected against a terrible disease. Threatened pigs should be vaccinated anyhow because they might become generators of further spread of disease and, on the other hand, do not become carriers.

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If the above policy is implemented, the ministries of agriculture and livestock industry can select the best options for eradication of the disease that cause the least disruption of social and economic life at the least cost to the community. The demonstration– after an outbreak has been controlled - that the country is free of active FMD and that serological surveys were carried out rapidly and efficiently would demonstrate to the international community the existence of a well-organized veterinary services that takes the eradication of FMD seriously. References - Donaldson A.I., Alexandersen S., Sorensen J.H. and Mikkelsen T. 2001 – Relative risk of the uncontrollable (airborne) spread of FMD by different species. Vet. Rec. 148, 602-604 - Fogedby E: Review of epizootiology and control of foot-and-mouth disease in Europe 19371961. Eur Comm Control of FMD, FAO, Rome, 1963 -. Ferguson N., Donnelly C., & Anderson R. (2001) – The foot-and-mouth epidemic in Great Britain: pattern of spread and impact of interventions. Published online 12; 10.1126/science.1061020 (Science Express Reports) - Keeling MJ, Woolhouse ME, Shaw DJ, Matthews L, Chase-Topping M, Haydon DT, Cornell SJ, Kappey J, Wilesmith J, & Grenfell BT (2001) - Dynamics of the 2001 UK foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape. Science 294 (5543):813-7. -.Leforban, Y. (2002): L’épisode de fièvre aphteuse au Royaume-Uni et en Europe en 2001 : trois importantes questions. Rev. sci. tech. Off. Int. Epiz., 21 (3) in press -.Panina G.F., Amadori M., Guadagnini P.F., Massirio I, Melegari M., Pavesi M., & Perini S. (1988). – Il controllo dell’afta epizootica nella specie suina. Selezione Veterinaria, 29 (1 bis), 197-206. - Sellers R.F. (1971). – Quantitative aspects of the spread of foot-and-mouth disease. Vet. Bull., 41 (6), 431-439. - Straver P.J., Bool P.H., Claessens A.M.J.M., and van Bekkum J.G. (1970): Some properties of carrier strains of foot-and-mouth disease virus. Archiv fúr die gesamte Virusforschung, 29, 113-126. - Sutmoller, P., Barteling, S.J., Sumption, K.J. & Casas Olascoaga, R. 2002/3: “Control of foot-and-mouth disease” In: “Foot-and-Mouth Disease”, D.J. Rowlands ed. Elsevier Scientific Publ. Amsterdam (in press). - Thomson, G. (1996) Role of carrier animals in the transmission of FMD. Rev. sci. tech. Off. Int. Epiz. May 96,1. Acknowledgement We greatly thank Mr. Jon Dobson for editorial comments and Dr Franco De Simone for information on the Italian outbreaks in the eighties.

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Appendix 16

Evaluation of acceptance of alternative FMD eradication strategies in Switzerland 1

Christian Griot1, Lukas Perler2 Institute of Virology and Immunoprophylaxis (IVI), National Reference Laboratory for Exotic Diseases, Swiss Federal Veterinary Office, 3147 Mittelhäusern, Switzerland; 2 Swiss Federal Veterinary Office, 3003 Bern, Switzerland

Foot-and-mouth disease (FMD) continues to be the most important economic threat to the livestock industries throughout the world. The last major outbreaks in Switzerland were recorded in 1965/66, in which more than 1000 farms were affected by FMD virus serotype O. As with other EU countries, Switzerland employed an FMD mass prophylactic vaccination policy until 1990. During this period, approximately 30% of the susceptible livestock population (predominately dairy cattle) was annually vaccinated using a trivalent vaccine. Since 1990, Switzerland no longer applies such a vaccination policy. Instead, an emergency vaccine bank was established, and currently holds approximately 300,000 doses of the four serotypes O, A, SAT-2 and Asia1. Every 5 years the vaccine bank is replaced with a new stock, and if necessary, the serotypes adjusted according to the disease situation. Already during the height of the 2001 UK epidemic several organizations such as the Swiss farmer’s union, animal welfare activists, and breeders’ organizations asked for an immediate introduction of a “mass prophylactic vaccination”. The two major food retailers did not oppose to this proposal. The main rational for this was to “protect the Swiss breeding stock”. In addition, most likely due to the constant media pressure of the outbreaks within the EU, these outbreaks had hit consumer confidence, at a time when it was already reeling from a string of food scares such as BSE, antibiotics residues in food and others. As in the UK, many consumers in Switzerland were horrified in particular by the mass slaughter of animals, the funeral pyres, and the waste of food and protein, respectively. On the issue of vaccination, it was discussed among animal welfare groups and consumers organizations whether this would have been a suitable option for disease control. At this point however it was unclear whether meat and other products from vaccinated animals would have been equally acceptable to the consumers as products from non-vaccinated animals. The Swiss Federal Veterinary Office as well as the IVI updated the public regularly on the relevant issues such as the “facts and figures” of the outbreak, the rationale for using the OIE “stamping out” policy, the measurement taken at the boarders etc. The media as well as the internet (the own websites) were used as the way of distribution. In addition, three “FMD crisis” meetings with the cantonal veterinary services were held. At these meetings new measurements within Switzerland (such as import restrictions with EU countries, restriction of cattle auctions etc) were discussed.

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After the last FMD case on the UK on September 30, 2001 media attention for the disease became negligible. However, there were still several open questions, and therefore it was decided by the Swiss Federal Veterinary Office in collaboration with the IVI to initiate a “round table” discussion to which (i) consumers organizations, (ii) food retailers, (iii) stakeholders such as livestock keepers, (iv) animal welfare groups (v) animal health organizations, and (vi) the Swiss farmers union were invited. The aims of the meeting was to (i) outline the current approach of FMD eradication, (ii) to illustrate disease control measures such as mass culling and the use of emergency vaccination, and (iii) to get an understanding of how consumers organizations would support the marketing of meat and other products derived from vaccinated animals (protective vaccination) without any restrictions (such as special labeling). The round table took place on April 11, 2002. All invited organizations did attend this meeting. The following main conclusions from this “round table” can be summarized as follows: 1. Preventive and control measures: • “Stamping out” of FMD infected herds is accepted to be the basic tool for eradication • Mass prophylactic vaccination is considered (by most organizations) not to be a viable option • Emergency “suppressive vaccination” (vaccinated animals are killed within a certain time period) is rejected by all organizations • Emergency “protective vaccination” (vaccinated animals are used for consumption) is accepted by all organizations • If emergency vaccine is used, there will be an enormous pressure on the farmers outside the designated vaccination zone to vaccinate their animals 2. Marketing of products: • Products derived from FMD vaccinated animals should be marketed through regular (although controlled) channels • There should not be a trade barrier for vaccinated animals since using newly developed tests vaccinated herds can be distinguished from those infected with FMD virus • Consumers should accept products from vaccinated animals 3. Consumers and stakeholder’s knowledge: • Consumers and stakeholders show a lack of understanding for many aspects of the disease. The information provided by the various agencies is too complex. 4. Disease awareness: • Stakeholders disease awareness is only present at an acceptable level if there is an acute risk of disease introduction. • Essential elements of biosecurity are only practiced during the perceived immediate risk of introduction.

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•

Stakeholders need a better understanding of the importance of recording animal movements.

The general feedback by the participants was found to be positive. The media response to the meeting was mediocre; reports were generally seen in the farmer’s press. We strongly believe that these discussions with the stakeholders and the public are urgently needed before the upcoming of a “crisis” in order to gain a higher acceptance of disease control measures in case of an outbreak, not only in case of FMD. It was also shown that these outbreaks have had a major impact on society. Therefore, FMD can no longer be seen just as a purely veterinary and/or agricultural issue; socio-economic factors also need to be considered. Improvement in the methods used for prevention, control and eradication of the disease as well as the evaluation of different strategies in terms of effectiveness will be urgently required in the near future.

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Appendix 17

Studies of quantitative parameters of virus excretion and transmission in pigs and cattle experimentally infected with Foot-and-Mouth disease Virus O UK 2001 Soren Alexandersen*, Melvyn Quan, Ciara Murphy, Jeannette Knight and Zhidong Zhang Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, GU24 ONF, U.K.

SUMMARY Foot-and-mouth disease virus (FMDV) can be spread by a variety of mechanisms and the rate of spread, the incubation period, i.e. the time from receiving an infectious dose of virus until developing clinical signs of disease, as well as the severity of disease depends on a multitude of parameters, including the virus strain, the dose received, the route of introduction, the animal species and the conditions under which the animals are kept. More knowledge in regards to these parameters are urgently needed in order to improve models used to predict spread of disease, especially if such predictions are to be used in practical disease control. Here we extend our previous findings with more detailed studies of virus load, excretion and transmission in pigs and cattle to facilitate use of a viral load framework for assessment of transmission risks. Details of virus load in serum and in oesophageal-pharyngeal (probang) samples are discussed in separate presentations at this meeting. Virus replicated rapidly in inoculated pigs and cattle and when exposed by direct contact the mean incubation period was around 3-4 days for cattle to cattle transmission and 1-3 days for pig to pig transmission depending on the intensity of contact. These differences confirm that a strong correlation exists between challenge dose and length of incubation period. Clinical disease was severe in pigs but relatively mild in inoculated cattle. Interestingly, contact infected cattle appeared to have more severe lesions, especially of the dental pad and gums, than cattle infected by subdermo-lingual inoculation. Viral RNA was recovered in nasal and mouth swabs from inoculated animals soon after they developed a viraemia and probably reflected early virus production and excretion. Viral RNA in nasal swabs and also in mouth swabs from contact animals could be detected several days before they showed other signs of infection and indicates the possibility of detecting exposed animals incubating disease. Viral RNA could also be detected in swab samples after the viraemic phase and may represent background environmental virus that had been trapped in the respiratory tract and mouth or may indicate a somewhat slower clearance/half-life of virus RNA at these sites. However, we can not exclude that this virus may represent an extended low level of virus replication. The pattern of virus RNA levels in pigs was very similar to that in cattle but the amounts of viral RNA recovered were higher.

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INTRODUCTION FMD is most often spread by the movement of infected animals which excrete large amounts of virus. Next in frequency is spread by contaminated animal products, e.g. feeding of milk or unprocessed waste food, a route which is often implicated in international movement of FMD. In such instances introduction of disease is often into pigs. Infection may also be spread by mechanical means, for example via animal contact with virus on the surfaces of transport vehicles, milking machines or on the hands of animal attendants. An additional mechanism is the spread of FMD virus by the wind 5,9,10 . Windborne spread of FMD is most often from pigs as the source to cattle or other ruminants as recipients, because pigs excrete high levels of virus in their breath but are relatively resistant to airborne infection while cattle and other ruminants excrete less virus in their breath but are highly sensitive to infection by the airborne route. More quantitative knowledge in regard to excretion, transmission and disease parameters are urgently needed in order to improve models used to predict spread of disease, especially if such predictions are to be used in practical disease control. METHODS Animals and virus Sixteen Holstein heifers weighing around 150 kg were used. The animals were placed 2 and 2 or 4 and 4 in single rooms in biosecure animal buildings. In experiment 1 four groups of 2 animals were placed in four rooms and all 8 cattle inoculated. Experiment 2 involved two rooms with 4 animals each, two of the animals in each room were selected at random and inoculated while the other two animals in each room were kept as direct contacts throughout the experiment. Inoculation were by subdermo-lingual injection 15,16 of approximately 0.5 ml of virus stock containing 106.9 TCID50 (BTY) of O UK 34/2001 virus (first pig passage). Thirty Landrace cross-bred Large White pigs weighing between 20 and 30 kg were used. In experiment 3 eight pigs were kept in a single room and four randomly selected pigs inoculated in the heel pad as described previously 1,2 with approximately 0.5 ml of virus stock containing 106.9 TCID50 (BTY) of O UK 34/2001 virus (first pig passage). The other four pigs in the same room were kept as direct contacts throughout the experiment. In a previous experiment (not to be described in detail here, but referring to 2 for details) the same O UK 34/2001 virus was used and pigs kept as 4 pairs of a single inoculated and a single contact pig as described below (“one-on-one” experiments). A similar set-up with 4 groups of one inoculated and one contact pig has also previously been done with the C Noville strain (Alexandersen, Zhang and Donaldson, Session of the Research Group of the Standing Technical Committee, European Commission for the Control of Foot-andMouth Disease, Denmark 2001). In experiment 4 six randomly selected pigs were inoculated in the heel pad with approximately 0.5 ml of virus stock containing 105.1 TCID50 (IB-RS-2) of O TAW 9/97 (second pig passage). Two of these inoculated pigs were kept in a single room together with four other pigs kept as direct contacts throughout the experiment. The animal room used for experiment 4 was smaller than the one used for experiment 3, and consequently the space available per pig was similar for these groups. The other 4 inoculated pigs were used in “one-on-one” experiments where a single inoculated and a single contact pig was kept in an individual cubicle in four rooms in order to keep their available area similar to or less (in fact 25% less) than where more

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pigs were kept together in rooms. In the same 4 rooms, two recipient pigs were kept physically separated in a separate cubicle and exposed to aerosol virus emitted over at least a 24 to 48 hour period by the inoculated and contact donor pigs in the other cubicle in the room as described previously for other virus strains 1,2. The recipient pigs were not handled except on the occasions when blood samples were being collected. The animals were examined clinically each day for signs of FMD. Rectal temperatures were recorded daily until 7-10 days after inoculation (dpi). Blood samples (serum tubes and heparin tubes) and nasal and mouth swabs were taken daily or twice daily for the first period after inoculation. The blood samples were immediately transported to the laboratory and serum separated immediately and stored at –80 o C. For RNA extraction, an aliquot of serum was thawed and two volumes of serum added to three volumes of lysis buffer (Roche Lysis Solution) and stored at room temperature until nucleic acid extraction and subsequent analysis. Swabs were taken in duplicate, one swab was placed in 2 ml of maintenance medium and stored at –80o C while the other one was placed in 1 ml of TRIzol (Life Technologies, Paisley, UK) and stored at –80o C. Probang samples were taken from cattle as well, however these data will be presented separately. Measurement of aerosol excretion of FMDV from pigs infected with O TAW 9/97 Samples of the air in the room were collected with a Cyclone sampler on the first and second day of clinical disease. In addition, two pigs with clinical disease were selected on the same days and placed in a 610 litre cabinet 8 and multiple air samples collected with a May sampler as described previously 1. The inside of the cabinet and the walls and ceiling of the room were sprayed with water before commencing the air sampling to ensure that the relative humidity was high and therefore suitable for the survival of airborne virus 8. After measurement, the pigs were returned to their respective boxes. The peak of virus excreted was expressed as the total amount of airborne TCID50 per 90/100 kg pig (i.e. 3 of the approximately 30 kg pigs used per 24 hours as described previously 1,2). Assay for virus The infectivity in the collection fluid from air samplers and in other samples were assayed by inoculation of monolayer cultures of primary bovine thyroid (BTY) cells in roller tubes 20 or for the O TAW 9/97 virus in IB-RS-2 cells. The specificity of the cytopathic effect observed in cell cultures was confirmed by antigen ELISA 12,13,19. Quantitative RT-PCR. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to determine the amount of FMDV RNA in extracts of total nucleic acid from blood and swab samples and from cell cultures. The assay method has been described in detail elsewhere 4,18 (Reid et al, In Press 2002). All extractions involved 0.200 ml of sample and the nucleic acids were finally eluted in a volume of 0.050 ml. All estimations included standard reactions based on in vitro transcribed, molecularly cloned FMDV RNA using samples with a known content of FMDV genome-equivalents and also involved comparison to samples with virus titres determined by virus titration in cell culture. The method is influenced minimally by sample type.

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Assay for antibodies Serum samples were tested by an enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies to FMD virus 11,14 and certain samples verified in virus neutralisation assay (as described in the Manual of Standards for Diagnostic Techniques and Vaccines, l’Office International des Epizooties, OIE, 2000). RESULTS Airborne virus recovery and estimated aerosol excretion and exposure doses The amounts of virus in air samples from the pigs infected with the O Taiwan 1997 virus can be summarized as follows: Airborne excretion of FMDV O Taiwan 1997 was 104.6 TCID50 (IB-RS-2) per 24 hour per 90-100 kg pig at 1 and 2 dpi after inoculation (first and second day of clinical disease). Based on comparable titre comparisons and on comparison of genome numbers per infectious units, as estimated by quantitative real time RT-PCR, this amount of IBRS-2 TCID50s equals a value of around 105.6-6.4 TCID50 in BTY cells for other isolates which grow well in BTYs (Alexandersen and Quan, unpublished observations). Therefore, the amounts of airborne virus emitted by pigs infected with this strain is of a similar magnitude as other type O viruses we have studied 1,2 which seem to fall in the 5.8-6.4 log area. Cyclone samples were taken for 10-20 min from the animal rooms as well, however, the concentration of virus in the air was below the detection limit of the virus isolation assay, albeit that virus RNA could be detected in the samples using RT-PCR due to the higher sensitivity of this technique (data not shown). The low levels detected is due to the limited time of sampling, the dilution of the virus in the air in the animal rooms and due to the ventilation in the room which, albeit being reduced, would cause a reduction of up to 1 log compared to May sampling. However, based on the excretion levels estimated from the May samples, on the RNA levels in the Cyclone samples and on the resulting room concentrations seen in previous experiments 1,2, we expect a concentration of virus in the air of the rooms of 0.1 to 0.25 TCID50 per litre of air. Thus, a 30 kg pig (breathing at about 18 litre per minute) will receive around 103.4-3.8 TCID50 (IB-RS-2) in 24 hours. This amount of exposure equals approximately 104.4-5.6 TCID50 in BTY cells for other strains of virus. Thus, this level of accumulated exposure is in the same range as previous experiments with other strains 1,2. None of the 8 exposed recipient pigs developed clinical disease and only a single pig of the 8 pigs exposed developed a transient antibody response at 14 days post exposure. Clinical signs, virus load in serum and swabs, seroconversion and incubation periods Selected results for experiments 2, 3 and 4 are shown in Figs 1-5. Clinical signs were scored as follows: Lesions on each foot gave one point, lesions in or around the mouth one point and lameness or dullness/reluctance to stand one point. Consequently, each animal could have a maximum score of six. In order to represent these scores graphically, the scores were scaled to fit the range used for other graphs. Virus load in serum is given as number of viral genomes per ml of serum. Comparative titrations on serum and tissue samples indicate that for most isolates one BTY TCID50

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equals roughly 103 genomes, however, for the O Taiwan 1997 virus one IB-RS-2 TCID50 correspond to roughly 104-4.8 genomes. Recent studies indicate that after the peak of virus levels are reached, then up to 101.2-2.0 times more genomes are required to make one infectious unit, probably due to antibodies being present (Quan and Alexandersen, unpublished studies). Some results were confirmed by virus titration in appropriate cell cultures, examples of such data are shown in Fig. 2. It should be noted that preliminary results indicate that for nasal swabs from pigs, the correlation between infectivity and genomes are not nearly as strong as for other sample types indicating that viral genomes in nasal swabs may be either rapidly inactivated or that a substantial amount of the genomes detected is derived from the environment and have a reduced infectivity. These issues will need more research to be resolved. Short summary of results for experiment 1, i.e. 8 cattle inoculated subdermo-lingually with UK 2001 virus and kept in pairs throughout the experiment. High levels of virus RNA was detected already at 24-48 hours after inoculation in both probang samples and in saliva and nasal swabs. The peak of virus in these samples initially followed the levels in serum, however, serum levels declined much faster and virus RNA in serum essentially disappeared as antibodies became detectable at 4 days after inoculation. Virus RNA was consistently detectable for up to 7 days in nasal swabs and consistently for about 5 days in saliva. However, a few saliva samples continued to have low levels of virus RNA up to 18 days after inoculation. Probang samples were consistently positive up to around 8-10 days after inoculation, after that time only two animals continued to be positive (the same animals that were positive on saliva samples at day 18) and in this particular experiment only a single animal was positive at more than 28 days after inoculation. At this time, all other samples were consistently negative. Short summary of results for experiment 2, i.e. 2 rooms each with 2 inoculated and 2 direct contact cattle. The inoculated cattle were given 6.9 logs UK 2001 virus as described above. Selected results can be seen in Fig. 1. An increase in body temperature, vireamia and early lesions were detected in inoculated cattle from around 24 hours after inoculation and in contact cattle around 96-120 hours, giving an incubation period of 3-4 days under these conditions. Vesicular lesions were found on the tongue of inoculated cattle from around 24 hours after inoculation and most inoculated animals also had small vesicles in the interdigital area or along the coronary band on one or two feet. Lesions in contact animals were detected at 96-120 hours and consisted of extensive vesicular lesions of the tongue, dental pad and lips and small to intermediate vesicular lesions in the interdigital area or along the coronary band on two or more feet. Airborne excretion of virus in cattle correlated well with the early phases of clinical disease and virus RNA in nasal and mouth swabs could be detected in inoculated animals soon after viraemia and in contact animals up to 2-3 days before clinical disease was observed. Under these conditions, having both inoculated and contact cattle together, virus RNA could be detected in nasal swabs from all animals for a period of 10 days, significantly longer than detection of viraemia. The pattern of detection of virus and development of clinical disease was relatively uniform, however, development and timing of increased body temperature was somewhat variable (data not shown) and often only lasting 1-2 days and it should be noted that a lack of significantly increased body temperature in individual animals should not be used to exclude the possibility of FMD. Development of antibodies consistently correlated with a significant drop in virus levels

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in serum, however, virus in nasal swabs, and to some extent mouth swabs, could be detected for a longer period than virus RNA in serum. However, at peak levels virus RNA was higher in serum (up to around 108 virus RNA copies per ml, corresponding to roughly 105 TCID50/ml) than in most of the swab samples (generally around 1 log less, probably due to the fact that the swabs, due to the way they were processed before testing, effectively were diluted around 1:10). The peak of virus RNA in mouth swabs from the inoculated animals were as high as in serum, probably due to the extensive lesions on the tongue due to the subdermo-lingual inoculation. Short summary of results for experiment 3, i.e., 4 inoculated and 4 contact pigs kept in a single room. The inoculated pigs were inoculated with UK 2001 virus as described above. Selected results can be seen in Fig. 2. An increase in body temperature, vireamia and early lesions were detected in inoculated pigs from around 24 hours after inoculation and in contact pigs already at 48-72 hours, giving an incubation period of only 24-48 hours under these conditions. Clinical disease was severe and characterised by lameness/reluctance to stand or walk, reduced or no interest in feed and severe vesicular lesions along the coronary bands and heel pad area and sometimes on the tongue and snout. Airborne excretion of virus in pigs, as in cattle, correlated well with the early phases of clinical disease and virus RNA in nasal swabs and in mouth swabs could be detected in inoculated animals soon after viraemia and in contact animals before any clinical disease or a vireamia was observed. Under these conditions, having both inoculated and contact pigs together, virus RNA could be detected in nasal swabs from all animals for the duration examined, i.e. up to 7 days after inoculation, which was after cessation of viraemia. The pattern of detection of virus and development of clinical disease was relatively uniform, however, as for cattle development and timing of increased body temperature was somewhat variable (data not shown), often in the 39-40 C area and only lasted from 1-3 days. Development of antibodies consistently correlated with a significant drop in virus levels in serum, however, virus in nasal and mouth swabs could be detected for a longer period than virus RNA in serum. However, at peak levels virus RNA was higher in serum (up to around 109 virus RNA copies per ml, corresponding to roughly 106 TCID50/ml or around 1 log higher than in the cattle in experiment 1 and 2) than in any of the swab samples. Testing of serum samples for virus infectivity correlated well with the RT-PCR data (Fig. 2), however, RT-PCR can detect samples as positive for a significantly longer period than virus isolation. Short summary of results for experiment 4, i.e., 2 inoculated pigs and 4 contact pigs in one room and 1 inoculated and 1 contact kept in individual cubicles in 4 rooms (one-onone experiments). The rooms used in this experiment was slightly smaller than the one used in experiment 3 and thus the 6 pigs (2 inoculated and 4 contacts) had approximately the same space per pig as the 8 pigs in experiment 3. Due to the cubicle arrangement of the “one-on-one” experiments, these sets of two pigs had approximately 25% less space per pig than the group of 6 pigs or the pigs in experiment 3. Groups of two recipient pigs were kept in separate cubicles (physically separated) together with the “one-on-one” pigs to study the possibility of airborne transmission of the virus. The inoculated pigs received O Taiwan 1997 virus as described above. Even though the recipient pigs would have been exposed to approximately 104.4-5.6 TCID50 (as converted to “BTY” TCID50), a level of accumulated exposure in the same range as previous experiments with other

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strains 1,2, it is worth noting that, even with this highly pig adapted and highly virulent strain, none of the 8 exposed recipient pigs developed clinical disease and only a single pig of the 8 exposed developed a subclinical infection characterised by a transient antibody response at 14 days post exposure that had already dropped at 21 days. The other pig in this cubicle in direct contact with this subclinically infected pig did not develop disease nor antibodies, in agreement with our other studies which suggest that pigs subclinically infected by the airborne route do not transmit the infection to other pigs, even when kept in close contact. Selected results for the inoculated and direct contact pigs can be seen in Fig. 3-5. An increase in body temperature, vireamia and early lesions were detected in inoculated pigs from around 24 hours after inoculation and in contact pigs, as an average, around 72 hours, giving an average incubation period of around 48 hours. The clinical disease and lesions observed was in principle similar to those described above for the UK 2001 virus, albeit with the subjective observation that the vesicles appeared to rupture slightly earlier in case of the O Taiwan 1997 virus. As in the other experiments, elevated temperature did not necessarily correlate well with the development of clinical disease. Virus RNA in nasal swabs could be detected in inoculated animals soon after viraemia and in contact animals before any clinical disease or a vireamia was observed. Under these conditions, having both inoculated and contact pigs together, virus RNA could be detected in nasal swabs from all animals for the duration examined, i.e. up to 9 days after inoculation. The pattern of detection of virus and development of clinical disease was relatively uniform in inoculated pigs (Fig. 3), however, as mentioned earlier the development and timing of increased body temperature was somewhat variable. Development of antibodies consistently correlated with a significant drop in virus levels in serum, however, virus in nasal and mouth swabs could be detected for a longer period than virus RNA in serum. Peak levels of virus RNA was higher in serum (up to around 109 virus RNA copies per ml as in experiment 3) than in any of the swab samples. However, correlation between infectivity and virus genomes was not good on nasal swab samples (data not shown). As mentioned above the average incubation period for these experiments was around 48 hours. However, the spread of disease appeared to be faster in the group with 2 inoculated and 4 contacts than in the 4 groups with “one-on-one”, although space was actually more restricted in the “one-on-one” experiments. Fig. 4 bottom part shows the 4 contact animals exposed to 2 inoculated pigs in an animal room while the top part are the 4 contacts exposed individually to 1 inoculated pigs in each of the 4 individual cubicles. It can be seen that for the bottom panel the incubation period is around 48 hours while for the top panel, i.e. the “one-on one” experiments” the incubation period is slightly longer, around 72 hours. This is also shown in Fig. 5 showing the mean of development of vireamia in inoculated animals (similar in the 2+4 and the one-one-one experiments) and the development of vireamia in the contacts, appearing approximately 24 hours later in the pigs exposed “one-on-one”. It should also be noted, that when exposed “one-on-one”, even with this highly virulent pig-adapted strain, one of the contact pigs (UN76 on Fig. 4) only developed sub-clinical infection characterised by no clinical signs of disease, a low level, transient (detected for one day only) viraemia and a delayed and transient antibody response only detected at 14 days after exposure. This pig had been directly exposed to a pig that developed severe disease (UN84 on Fig. 3) and had received a significant amount of virus as evidenced by detectable virus RNA in nasal swabs (UN76, Fig. 4).

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Virus clearance or decay Assuming that most/all virus RNA detected after peak levels primarily are produced in the period up to the peak then the results can be used to access the speed of virus clearance or decay. This may not be an entirely accurate estimate as some replication may still take place, however, it may provide a useful estimate of the residual levels of virus being present after the peak. More detailed results with regard to the levels in serum and in probang samples will be given in the presentations by Melvyn Quan and Zhidong Zhang, respectively, at this meeting. Using the data from animals inoculated with UK 2001 virus in experiment 2 and 3, calculations of the half-life of virus RNA in nasal swabs from cattle after the peak levels were 7-8 hours in the first phase up to 5 days after inoculation and then 22-24 hours in the period from 5-11 days after inoculation. In the inoculated pigs, the half-life was around 10-12 hours for the period studied, i.e. up to 7 days after inoculation (estimates on the data from experiment 4 of pigs infected with the O Taiwan 1997 virus up to day 9 was of a similar magnitude). Half-life of virus RNA in mouth swabs was of a similar magnitude as for nasal swab samples but appeared to be more variable. Half-life/clearance in serum, after the peak, was much faster with an estimated half-life of around 2-4 hours. These values may represent over-estimates as the inoculated animals are likely to have taken up a significant amount of virus from the incontact animals which replicated virus at a later stage than the inoculated animals. The significance of this may be estimated from the data in experiment 1, where animals were all inoculated and kept two and two in pairs and where the virus in nasal swabs from the cattle only were detected up to day 7 while in experiment 2 it could be detected up to around day 10. Nevertheless, taking the various parameters into consideration the estimates are in a similar range for both cattle and pigs and may suggest that virus RNA may be present in/on animals and in the environment (as the animals essentially are continuous sample devises constantly inhaling large amounts of air) for a considerable amount of time after clinical disease is first detectable. Whether the virus is actually infectious is not known as the level of genome copies at the later time points may be below detection limits of in vitro infectivity assays, especially taking into consideration that antibodies may be present. However, the majority of virus RNA and thus presumable infectivity and infectiousness, is detected in the period starting with early clinical disease and lesions and last for a period of 3-5 days. Before this, a low level of virus RNA can be detected for up to 2-3 days in blood and swabs and low levels are detectable in swabs after the fifth day of disease although antibodies are detectable at this stage. DISCUSSION The experiments described confirmed that cattle and pigs excrete airborne FMDV, as well as virus in nasal fluid and saliva, at maximal levels during the early phases of clinical disease. The maximum amount of airborne virus excreted for the UKG 2001 FMDV in pigs, cattle and sheep has been reported previously 2,4 and corresponds to around 106.1 TCID50 (BTY) in pigs (approximately 90-100 kg) and 104.3 TCID50 in cattle (approximately 150 kg) and sheep (approximately 30 kg) in a 24 hour period. However, in sheep, in contrast to in cattle and pigs, peak excretion in breath is early, i.e. before clinical signs of disease and before significant levels of virus can be detected in serum or in nasal swabs 4. Based on comparative estimates, due to the fact that the O Taiwan 1997 virus grows poorly in BTY cells, the aerosol excretion from pigs infected with the O Taiwan 1997 virus was of a similar magnitude as for the UK 2001 virus, i.e. an estimated maximum of around 105.6-6.4 “TCID50-BTY-equivalents” in a 24 hour period.

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The data presented here suggest that real time RT-PCR provides a powerful tool for detection of FMD virus RNA, i.e. to rapidly and accurately diagnose FMD. In cattle and pigs serum/blood provide a good sample type for diagnosing acute infection, however, the time window is rather narrow. However, the data indicate that swab samples, in particular nasal swabs, provide a useful sample for diagnosing FMD in cattle and pigs on a herd basis, particularly because virus RNA could be found in such samples both before and after development of clinical disease. Virus RNA could even be found for several days in nasal swabs from an exposed pig, although this pig did only develop subclinical infection and only had vireamia for a single day. Consequently, sampling nasal swabs from cattle and pigs appears to have a rather wide diagnostic window and appears to be capable of picking up both clinically and subclinically infected animals and may possibly pick up animals exposed to or close to infected animals, because the respiratory tract in essence are constantly sampling the environment and any virus present will then appear in the nasal swab sample. For sheep the situation is slightly different. For sheep RT-PCR on swabs are also a useful tool 2,4 as mentioned here for cattle and pigs. However, in sheep virus is excreted in the breath of infected animals before any significant amount of virus can be detected in blood or nasal swab samples. Consequently, for very early diagnosis of FMD in sheep it may probably be best to test air samples from suspected sheep. However, as the infection usually spreads relatively slowly in sheep this may not easily be done in practice and possibly an alternative strategy would be to sample and test many sheep using serum samples and nasal swabs. Another potential for early diagnosis in sheep may be mouth swabs as suggested by others 6,7, as it is possible that the virus excreted in breath very early in infected sheep may derive from the superficial epithelia of the mouth or by taking probang samples if virus derives from the pharynx. However, this will require further studies. In the studies described here we primarily quantitated virus load in terms of virus RNA copies. We have previously shown a good correlation between our RT-PCR quantitations of genome numbers and virus infectivity of a number of sample types 3,4, although the ratio of genomes/infectious unit clearly increases when antibodies are present. Moreover, the RT-PCR method can quantitate levels below the detection limits of in vitro infectivity assays. Nevertheless, we consider the levels of virus RNA a useful indicator of potential infectivity. Thus, the time course data and clearance/half-lives reported here may give some guideline to assessment of the level of residual risk of infectivity being present in an exposed or reconvalescent animal at a given time after exposure. Such calculations are naturally only estimates and should not be regarded as exact values. Taking as an example a peak level of 109 genome copies of virus RNA or roughly 106 TCID50 per ml in a sample and a half-life of 24 hours, it would then take approximately 20 days (20 halflives) to reach a level of 1 TCID50 per ml or approximately 30 days to reach a level of only 1 copy of virus RNA per ml of sample. This example is based on the results reported here for nasal and mouth swab samples, virus present elsewhere under conditions more suitable for virus survival, for example the pharynx in ruminants, may be present for a longer period as may, as an example, virus in slurry in winter. Time course studies of virus load, excretion and transmission are of importance in providing quantitative data on which to base veterinary advice or risk analysis in relation to disease control. Similarly, such data can be used to improve models and model assumptions for computer simulation of disease spread and impact of various control options. However, one should bear in mind that many parameters may influence the

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outcome of such studies, including the animal species, the virus strain as well as the specific conditions under which the experiments are performed. Similarly, under field conditions, the parameters will vary widely and it is important to understand the basic quantitative principles governing virus excretion and transmission. Many years of experience in experimental work as well as in the field has indicated that the dose of virus exposure plays an important role in determining the outcome of exposure, i.e. clinical disease, subclinical disease or no detectable effect and in determining the incubation period, i.e. a high dose may result in a short incubation period and a low dose in a long incubation period or perhaps no detectable disease or infection. Investigations during the UK 2001 epidemic (S Alexandersen, R. P. Kitching, and others, unpublished results) have also indicated that the disease initially progressed relatively slowly on infected premises and that the incubation period got shorter as the infection progressed, consistent with a higher level of exposure as the virus is amplified. In the studies presented here, we have generated time course data on virus RNA levels in serum, nasal and mouth swabs from cattle and pigs infected with O UK 2001 virus and from pigs infected with O Taiwan 1997 virus. The levels and patterns of excretion are in principle similar, although pigs appear to amplify virus to a higher level than cattle. No principal differences were apparent for pigs infected with O UK 2001 versus O Taiwan 1997 virus. However, the results indicated that virus is transmitted faster (shorter incubation period) when several animals are kept together (incubation period for the O Taiwan 1997 virus of around 48 hours) as compared to “one-on-one” experiments (incubation period of around 72 hours) where a single inoculated animal is kept together with a single contact animal. This effect was despite the fact that the “one-on-one” animals had less space available than the animals kept as groups. The number of pigs used was not high enough to made the difference in incubation period observed statistically significant (was at the p< 0.15 level using t-test), however our previous experiments documented elsewhere 2 also observed a longer incubation period for the UK 2001 virus in one-on-one experiments (incubation period about 72 hours compared with 24-48 hours in experiment 3 reported here). For comparison it may also be worth mentioning experiments with FMDV type C Noville (excreted at very high levels in the breath of infected pigs) in which one-on-one exposure resulted in an incubation period of 72-96 hours (Alexandersen, Zhang and Donaldson, Session of the Research Group of the Standing Technical Committee, European Commission for the Control of Foot-andMouth Disease, Denmark 2001) and experiments with FMDV O Lausanne where 8 pigs were exposed to 4 inoculated pigs as a group but for only 2 hours (at peak disease of donors) also resulting in an incubation period of 72-96 hours 3. Moreover, the one-on-one experiments with the O Taiwan 1997 virus resulted in one of the direct contacts only developing subclinical infection and in the C Noville experiment two direct contact pigs (exposed one-on-one) only developed subclinical infection. Thus, the data clearly suggest a correlation between the intensity of exposure and incubation period/efficiency of spread and moreover, the data suggest that the actual number of infected animals plays a more important role than the actual density of animals, i.e. the speed and efficiency of spread appeared to follow a pseudo-mass action principle (number of animals) more than a true mass action principle (density dependant) mode of transmission, in contrast to what one may have predicted on theoretical grounds 17,21. Whether this is only the case in pigs and whether it is caused by variability of transmission speed from the individual inoculated animals is currently unknown. In theory, it may be possible that when having several inoculated animals together, transmission primarily takes place from the one first

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excreting significant amounts of virus. However, in the experiments described here the kinetics of virus load in serum and swabs were quite consistent in the individual inoculated pigs. We consider it more likely, that the speed and efficiency of transmission is increased when several pigs are kept together, perhaps because of increased fighting among the individual animals. This would suggest that virus in such cases gain entry through abrasions in the skin, a notion that is reinforced by the fact that the incubation period for pig to pig transmission in experiment 3 (with the UK 2001 virus) was shorter than in experiment 4 (with the O Taiwan 1997 virus) although the O Taiwan virus was detected as fast in inoculated pigs, were excreted at similar levels as the UK virus and albeit that the ventilation in the animal rooms was reduced in experiment 4. Thus, the main reason for this difference, assuming that the virulence of the O Taiwan virus is equivalent to the O UK virus, appears to be that the UK virus experiment (experiment 3) involved 4 inoculated and the O Taiwan virus (experiment 4) only 2 inoculated pigs, while both experiments had 4 contact pigs and the area per pig was similar. Whether a similar effect can be observed for transmission within cattle and sheep is currently unknown. However, the incubation period in the experiment described here (experiment 2) for cattle to cattle transmission was 3-4 days using 2 groups of two inoculated and two contact animals and full ventilation while a previous experiment in sheep also using UK 2001 virus but having six inoculated and 4 contact sheep in the same room and reduced ventilation had an incubation period of only around 2 days. As the sheep experiment achieved a higher airborne concentration of virus than the cattle studies, and considering that increased fighting and increased entry of virus through the skin is considered unlikely for sheep and cattle, we consider it most likely that the reason for the shorter incubation period (faster spread) in the sheep was due to a higher challenge dose of inhaled virus. In any event, the data suggest that efficiency and speed of spread of FMD is highly variable and dependant on many parameters, including the specific conditions under which the animals are kept and the stage of virus amplification in the herd. Thus, transmission efficiency and speed may be very different depending on the specific husbandry of a certain premises or a whole area and may differ over time and space. Under intensive husbandry, animals are likely to be kept inside at a high density and possibly many in direct contact. Thus, in such systems the possibilities for FMD transmission are maximal. However, in less intensive systems stocking density is lower, direct animal contact is low and most animals will spend all/almost all their time outside in the open. In such circumstances transmission is likely to be much slower and less efficient due to a lower direct animal contact and thus a lower dose of virus transmitted. This variability should clearly be kept in mind when giving veterinary advice or doing mathematical modelling on which to base practical disease control. In effect, such control can only meaningfully be achieved by including detailed veterinary expertise and local knowledge. ACKNOWLEDGEMENTS We thank Luke Fitzpatrick, Brian Taylor and Malcolm Turner for their assistance with the handling and management of experimental animals. The research was supported by the Department for Environment, Food and Rural Affairs (DEFRA), UK. REFERENCES 1. Alexandersen, S., I. Brotherhood, and A. I. Donaldson. 2002. Natural aerosol transmission of foot-and-mouth disease virus to pigs: minimal infectious dose for strain O1 Lausanne. Epidemiol. Infect. 128:301-312.

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2. Alexandersen, S. and A. I. Donaldson. 2002. Further studies to quantify the dose of natural aerosols of foot-and- mouth disease virus for pigs. Epidemiol. Infect. 128:313-323. 3. Alexandersen, S., M. B. Oleksiewicz, and A. I. Donaldson. 2001. Early Pathogenesis of Foot-and-Mouth Disease in Pigs Infected by Contact: A Quantitative Time Course Study using TaqMan RT-PCR. J. Gen. Virol. 82:747-755. 4. Alexandersen, S., Z. Zhang, S. M. Reid, G. H. Hutchings, and A. I. Donaldson. 2002. Quantities of infectious virus and viral RNA recovered from sheep and cattle experimentally infected with foot-and-mouth disease virus O UK 2001. J. Gen. Virol. 83:1915-1923. 5. 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. 6. 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. 7. 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:S37-S40. 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. 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. 10. 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. 11. Ferris, N. P. 1987. Development and use of Elisa in the control of foot-and-mouth disease. IAEA-Proceedings 348:65-77. 12. 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. 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. Henderson, W. M. The quantitative study of foot-and-mouth disease virus. A.R.C.report series 8, 1-50. 1949. London, Her Majesty's stationary office. 16. Henderson, W. M. 1952. A comparison of different routes of inoculation of cattle for detection of the virus of foot-and-mouth disease. J. Hyg. (Lond) 50:182-194.

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17. Klinkenberg, D., J. de Bree, H. Laevens, and M. C. de Jong. 2002. Within- and between-pen transmission of Classical Swine Fever Virus: a new method to estimate the basic reproduction ratio from transmission experiments. Epidemiol. Infect. 128:293-299. 18. Reid, S. M., N. P. Ferris, G. H. Hutchings, Z. Zhang, G. J. Belsham, and S. Alexandersen. 2001. Diagnosis of foot-and-mouth disease by real-time fluorogenic PCR assay. Vet. Rec. 149:621-623. 19. Roeder, P. L. and P. M. Le 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. 20. Snowdon, W. A. 1966. Growth of foot-and mouth disease virus in monolayer cultures of calf thyroid cells. Nature 210:1079-1080. 21. Velthuis, A. G., M. C. de Jong, J. de Bree, G. Nodelijk, and M. van Boven . 2002. Quantification of transmission in one-to-one experiments. Epidemiol. Infect. 128:193-204.

FIGURE LEGENDS Fig. 1. Graphs showing the time course of infection in cattle inoculated with O UK 2001 virus or kept in contact with such cattle in experiment 2. Time on the X-axes is given in hours. The mean levels of virus RNA, as genomes per ml, in serum samples and in mouth and nasal swabs are shown together with the titre of antibodies measured in ELISA, the body temperature and the development of lesions. The top panels are inoculated animals and the bottom contact animals. Fig. 2. Graphs showing the time course of infection in pigs inoculated with O UK 2001 virus or kept in contact with such pigs in experiment 3. Time on the X-axes is given in hours. The mean levels of virus RNA, as genomes per ml, in serum samples and in mouth and nasal swabs are shown together with the titre of antibodies measured in ELISA, the body temperature and the development of lesions. The top panels are inoculated animals and the bottom contact animals. Selected data on the levels of infectious virus in serum samples, determined by virus titration in cell culture, are shown in addition. Fig. 3-5. Graphs showing the time course of infection in pigs inoculated with O Taiwan 1997 virus or kept in contact with such pigs in experiment 4. Time on the X-axes is given in hours. The levels of virus RNA, as genomes per ml, in serum samples and in nasal swabs are shown together with the body temperature and the development of lesions. Fig. 3 shows the data for the individual inoculated pigs, the 4 pigs at the top is the four used for the “one-on-one” experiments while the 2 at the bottom was used for the group with 2 inoculated and 4 contact pigs together. Fig. 4 shows the individual data for the contact pigs, the four at the top is the ones used in the “one-on-one” experiments while those at the bottom was from the group with 2 inoculated and 4 contact pigs kept in the same room. Fig. 5 shows the average time course of development of vireamia in inoculated and in contact pigs divided into the group of 2 inoculated plus 4 contacts versus the 4 individual “one-on-one pigs. It can be seen that the development of vireamia is similar among the inoculated pigs, however, the contact pigs exposed “one-on-one” take about 24 hours longer to develop vireamia than the ones exposed as a group.

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Appendix 18

Quantification and duration of Foot-and-Mouth disease virus RNA in bovine esopharyngeal fluid as determined by real-time RT-PCR Zhidong Zhang , Ciara Murphy, Melvyn Quan, Jeanette Knight and Soren Alexandersen Institute for Animal Health, Pirbright, Woking, Surrey, GU24 ONF, U.K.

Abstract: Foot-and-mouth disease virus (FMDV) load was quantitated in experimentally infected cattle in order to improve knowledge about critical features of the initial virus-host interactions which determine disease outcome. A quantitative real-time RT-PCR was used to measure FMDV RNA in oesophageal-pharyngeal fluid (OP-fluid) samples from cattle experimentally infected with type O FMDV by inoculation and contact. The dynamics of FMDV load in OP-fluids exhibited remarkable similarities with viral replication patterns observed in nasal and mouth swab samples for the first week of infection. Viral RNA was recovered at 24 hours post infection (hpi), and rapid viral replication led to peak levels of RNA viral load by 30 hpi to 53 hpi. The extent of virus declined after the peak. Complete clearance of viral RNA occurred in some animals between 7 and 18 dpi. However, viral RNA persisted in OP-fluids at detectable levels beyond 28 dpi in persistently infected cattle (socalled carriers) and was still detectable in some animals at 57 dpi when the experiments were terminated. Viral RNA could not be detected in nasal and mouth swabs from FMDV carriers and non-carriers at any time after 7-18 dpi. An association between the extent of virus replication (growth rate) during acute infection and the outcome of infection (persistence or non-persistence) could not be established. The most significant predictor of the outcome of FMDV infection (persistence or non-persistence) was the extent of viral “clearance” (decay rate) following peak levels. Animals with a slow decay rate became carriers while more rapid clearance of viral RNA was characteristic of non-carriers. Introduction Foot-and-mouth disease virus (FMDV) causes a highly contagious viral disease of domesticated and wild ruminants and pigs. Of considerable importance in the control of FMD is the persistent infection that can occur following clinical or sub-clinical infection in both vaccinated and non-vaccinated ruminants (so-called carriers). There is field evidence to indicate that these carrier animals can precipitate new outbreaks of disease 2. A carrier is defined as an animal from which live virus can be recovered for longer than 28 days after exposure 9,11. Persistent FMDV is eventually eliminated from the carriers, but during the period of persistence, there is considerable variation in the levels of virus recovery from oesophago-pharyngeal fluid (OP-fluid) samples. On the other hand, persistent infection does not occur in all infected ruminants, i.e. only a proportion of infected ruminants become FMDV carriers 2. The mechanism for these phenomena remains to be fully understood although the immune status of the animal prior to contact has been shown not to influence the development of a carrier state. A number of studies on experimentally infected cattle have unequivocally shown the importance of pharyngeal area tissues in FMDV infection and replication during acute disease or persistence 4,5,7,13. During persistence, infectious virus can be isolated from the OP-

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fluid samples2. However, the kinetics of viral RNA load in OP-fluids and its relationship with the outcome of the infection (non-persistence and persistence) remains to be fully elucidated. Intensive and quantitative analysis of FMDV RNA load is made possible by the recent development of sensitive quantitative real-time RT-PCR 1,6,8. In this study the load of FMDV RNA was quantified in OP-fluid samples from cattle experimentally infected with FMDV O during the period of acute and persistent infection. Methods Animals - Twenty standard Compton steers (Holsteins) at 6-10 months of age were used and divided into three groups in this study. Cattle in the first group (Group 1) and the second group (Group 2) were infected with FMDV O UKG34/2001. Eight cattle in Group 1 were infected by intradermolingual inoculation with 106.9 TCID50 and placed in pairs in 4 boxes. The eight cattle in Group 2 were placed 4 and 4 in two boxes, with two cattle in each box infected under the same conditions as the first group. Four cattle in the third group (Group 3) were infected with FMDV O BFS1860 by intradermolingual inoculation of 107 TCID50. After infection, inoculated and direct contact cattle were monitored for clinical signs of disease and the rectal temperatures were recorded daily until 10 days after inoculation. Samples of OPfluid and nasal and mouth swabs were collected from before the start of the experiment (negative controls) and after infection. OP-fluid samples were collected using a probang cup 11 and diluted in an equal volume of Eagle’s-HEPES medium (pH7.2) containing 5% FCS and stored at -80°C until required. The infectivity of OP-fluid samples was determined by inoculation of monolayers of primary bovine thyroid (BTY) cells, essentially as described by Snowdon (1966)10. Nasal and mouth swabs were placed in 1 ml of Trizol (Life Technologies, UK) and stored at -80°C. Nucleic acid purification - 200 µl of samples were mixed with 300 µl of MagNa Pure LC total nucleic acid lysis buffer (Roche, UK). Total RNA was extracted using MagNa Pure LC total nucleic acid isolation kit (Roche, UK) with an automated nucleic acid robotic workstation according to the manufacturer's instruction and stored at -80°C until used. The concentration of the RNA was determined by spectrophotometry. Quantification of viral RNA by real-time quantitative RT-PCR analyses - The level of viral RNA in samples was quantified by a real-time RT-PCR as described previously 3,8. For the generation of standard curves, standard viral RNA was generated from plasmid pT73S containing full-length FMDV (kindly provided by Dr Andrew King, Institute for Animal Health, UK) by in vitro transcription using a commercially available T7 RNA polymerase kit (Ambion, UK) according to the manufacturer's directions. RNA was resuspended in DEPCtreated water and quantified by spectrophotometry. Calculation of the rates of virus growth and decay during infection - Initial viral replication rates were calculated by using the exponential growth rate equation: γ = (lnY1lnY2)/(t1- t 2), where Y1 and Y2 are the virus load values at times t1 and t2, respectively. The virus load doubling time, T2, in hours was calculated by using the equation T2 = (ln2)/γ. Rates of viral decay were calculated by using the decay rate equation (α) = (lnY1-lnY2)/(t1-t2). The viral decay half-life, T1/2, in hours was calculated by using the equation T1/2 = (ln2)/α. Viral decay rates and T1/2 were calculated with the steepest interval of the decay curve following the peak data point.

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Statistical analyses - Statistical analyses were performed on log10-transformed FMDV RNA values, or ln values in the case of viral growth and decay rates. Results Dynamics of viral RNA levels in oesophageal-pharyngeal fluid samples during the acute stage - In order to characterise the early course of viral RNA kinetics in OP-fluids following infection with FMDV, we measured the viral RNA level in OP-fluid samples collected twice a day during the first 2-3 days following inoculation, followed by daily measurements until 7 dpi. At 6 hpi, three of 12 inoculated cattle with FMDV O UKG (Group 1 and Group 2) manifested FMDV RNA, by 24 hpi all inoculated cattle had a quantified level of FMDV RNA ranging from 6.89 to 9.54 log10 copies/ml (average 7.97 ± 0.99; median 7.79) (Fig. 1A and 1B). Viral RNA levels then peaked at 30 or 53 hpi. The maximum level of FMDV RNA varied from 7.52 to 9.54 log10 copies/ml (average 8.55 ± 0.66; median 8.14). Thereafter, FMDV RNA levels decreased by various extents. The contact cattle in Group 2 had a similar kinetic pattern of viral RNA in OP-fluid samples (Fig. 1C). However, Viral RNA levels did not peak (average 8.21 ± 0.74 log10 copies/ml; median 8.06) until 5 days after contact. A similar pattern of FMDV RNA level was also seen in OP-fluids of Group 3 cattle infected with FMDV O BFS (Fig. 1D). The peak levels of FMDV RNA (average 7.95 ± 0.92 log10 copies/ml; median 7.28) was lower than those in cattle infected with FMDV O UKG, but no statistically significant differences were identified (non-parametric test, p > 0.05). The kinetic patterns of viral RNA in the OP-fluid samples exhibited similarities with the dynamics of FMDV RNA observed in nasal and mouth swabs during the first weeks of infection (more detailed results with regard to these will be given in the presentation by Soren Alexandersen at this meeting). The peak levels of viral RNA in OP-fluid samples were not statistically significantly different from those in nasal and mouth swabs (Fig. 2, nonparametric test, p > 0.05). Duration of viral RNA in oesophageal-pharyngeal fluids - Although there is no significant difference in the peak level of viral RNA amongst nasal, mouth swabs and OP-fluid samples, duration of viral RNA in these samples is significantly different. Representative results from Group 1 cattle are shown in Fig. 2. It has been demonstrated that RNA levels in serum declined much faster and become undetectable after 4 dpi when antibodies became detectable3 (more detailed results with regard to these will be given in the presentation by Soren Alexandersen at this meeting). In general, virus RNA in nasal and mouth swabs were detected for a longer period than virus RNA in serum but shorter than that in OP-fluid samples. Virus RNA was consistently detectable for up to 7 days in nasal swabs and consistently for about 5 days in mouth swabs, but 6 out of 12 mouth swabs from inoculated cattle with FMDV O UKG continued to have detectable levels of virus RNA for some days longer and a single animal (a carrier) had virus RNA in the mouth swabs up to 18 days after inoculation. OP-fluid samples were consistently positive up to around 7-10 days after inoculation, after that time 13 out of 20 cattle were still positive and 8 cattle was positive beyond 28 dpi and up to 57 dpi. In Group 1 carrier animals, levels of viral RNA in OP-fluid samples varied from 4.72 log10 copies/ml at 28 dpi to 5.15 log10 copies/ml at 41 dpi. In Group 2 FMDV RNA load varied from 5.54 ± 1.12 log10 copies/ml at 30 dpi to 3.29 ± 0.84 log10 at 44 dpi. Of Group 3 animals, the viral RNA load varied from 5.13 ± 1.65 log10 copies/ml at 30 dpi to 5.15 log10 copies/ml at 39 dpi. All these OP-fluid samples from carriers contained the infectious virus as determined by inoculation onto monolayers of BTY cells. At this time, all other samples were consistently negative.

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The extent of reduction of viral load after peak level in oesophageal-pharyngeal fluids is a critical determinant of the outcome of FMDV infection - To evaluate the possibility that the temporal pattern of OP-fluid viral RNA in the early course of infection is related to the outcome of the infection, cattle were divided into carriers and non-carriers according to the persistent status (persistent and non-persistent) and then the initial virus growth rate and subsequent decay slopes were calculated. Early virus replication in individual animals was assessed by estimation of the mean rate (γ) and standard deviation (SD) of virus growth and the corresponding doubling time, T2 in hours (described in Methods). Growth slopes, in ln units, were calculated by using the initial two quantifiable data points for each animal. As shown in Fig. 3, the mean viral growth rate in carriers (γ = 0.24 ± 0.19; T2 = 2.9 hours) was similar to those in non-carriers (γ = 0.20 ± 0.14; T2 = 3.45 hours). No statistically significant differences were identified (non-parametric test, p > 0.05). To determine whether the extent of reduction of viral load after peak levels in OP-fluid samples is correlated with the outcome of the infection, this phase of the FMDV infection was analysed by calculation of the slopes of early viral decay and virus load half-life in OPfluids, T1/2 in hours (as described in Methods). Decay slopes, in ln units, were calculated from the steepest interval of the decay curve following the peak for each animal. Carriers in Group 1 cattle had a lower decay rate (α = 0.03; T1/2 = 23.1 hours) than that in non-carriers (α = 0.11 ± 0.01; T1/2 = 6.3 hours). A similar phenomenon was also seen in OP-fluid samples in contact cattle of Group 2, in which the carriers had a lower decay rate (α = 0.02 ± 0.01; T1/2 = 34.7 hours) than that in non-carriers (α = 0.08 ± 0.05; T1/2 = 8.7 hours). Moreover, carriers in Group 3 also had a mean decay rate (α = 0.04 ± 0.01; T1/2 = 17.3 hours) which was lower than that in the non-carriers (α = 0.06 ± 0.01; T1/2 = 11.6 hours). Given these data as a whole, the mean decay rate (α = 0.03 ± 0.02; T1/2 = 23.1 hours) in carriers is significantly different from that in the non-carriers (α = 0.09 ± 0.06; T1/2 = 7.7 hours; non-parametric test, p < 0.05) (Fig. 3). These data suggest that the early decay rate of FMDV RNA in OP-fluid samples is a critical determinant of the duration of FMDV persistence. Viral decay rate in nasal and mouth swabs (based on Group 1) was higher than that in OP-fluid samples and no significant difference was identified between carriers and non-carriers (data not shown). Relationship between virus replication during the acute phase and the duration of virus persistence - The animals in this study cleared virus at widely varying times. In order to ascertain whether the characteristics of early viral replication influence the outcome of disease course, the course of acute FMDV infection was divided into three phases: pre-peak, peak and post-peak. The extent of early virus growth might influence the resulting host-virus equilibrium. An association was explored between early viral levels (defined as levels at 24 hours) and peak levels in OP-fluid samples collected from Group 1 animals. There was no correlation among these parameters (r = 0.28, p > 0.05). There also did not appear to be any correlation between early virus growth rates and subsequent events (persistent and nonpersistent). Similar results are also seen in Group 2 and 3 animals. Peak viral load in OP-fluid samples might reflect the extent of cell targets available for FMDV replication. Interestingly, the highest peak viral load was observed in animal UO32, which thereafter exhibited control of virus replication and clearance of the virus after 15 days. No correlation was found between viral peak and subsequent events (persistent and non-persistent) because there is no significant difference in the viral peak levels between carriers (8.38 ± 0.38) and non carriers (8.30 ± 0.33) (non-parametric test, p > 0.05). The post-peak level of viral RNA decline in OP-fluid samples might represent the best measure of host ability to clear virus, as early viral 144

RNA decline in OP-fluid samples was correlated with the duration of virus persistence. The slower the viral RNA declined early after the peak, the longer the virus persisted in OP-fluid samples. Discussion This study has quantitatively analysed the dynamics of FMDV RNA in bovine OP-fluid samples. In order to test the hypothesis that the events occurring shortly after infection dictated subsequent infection outcome (persistent and non-persistent), we performed frequent monitoring of the levels of viral RNA in bovine OP-fluid samples following FMDV O UKG2001 infection. We demonstrated that all OP-fluid samples from carriers which had a quantifiable level of viral RNA as measured by the real-time RT-PCR, were positive in the virus isolation test. Previous studies has also shown that viral RNA copy numbers measured by the real-time RT-CPR correlated well with virus infectivity 1,3. Therefore, the level of viral RNA could be a very useful indicator of the profiles of viral load. Following the peak viral load in OP-fluid samples, the rate and extent of viral decay was significantly different between carrier and non-carrier animals receiving identical inocula. What determines the magnitude of the reduction of RNA load in OP-fluid samples following the peak load is not fully understood. Viral variation seems unlikely to be the cause of these differences. The differential emergence of viral variants with different replicative capacity within the first week of infection seems unlikely among animals that received identical inocula of FMDV O UKG2001. Thus, this observation may simply reflect variations in the kinetics of the host response to infection, i.e. clearance of virus or continuing ability to support replication. We are now investigating the quantitative aspects of early events in the host response to infection in cattle, sheep and pigs to determine whether the observed differences in decay rate is caused by a difference in ability to clear virus or caused by a continuing viral replication. Virus RNA persisted in OP-fluids for a longer period than in other sample types. The reason for this phenomenon remains to be fully understood. Bovine pharyngeal tissue has been identified as a primary site for FMDV infection and replication in vivo 4,5. During persistence, viral RNA was detected in bovine pharyngeal epithelia samples7 and recently localised in epithelial cells of the bovine pharyngeal area 13. These studies of FMDV infection in experimentally infected cattle have unequivocally shown the importance of pharyngeal area tissues in virus infection and replication. The epithelial cells isolated from pharyngeal tissues of FMDV infected cattle shown to persistently harbour FMDV did not show any cytopathic changes for many weeks which further support this theory 12. This leads to the hypothesis that the presence of viral RNA and infectious virus in OP-fluids during persistence is because FMDV may be able to continuously replicate in such a specialised cellular site. Therefore, it may be considered especially important to define those cellular factors and mechanism involved in the regulation of virus expression and clearance in these cells. In fact such knowledge would also be extremely helpful in defining selective approaches to control FMDV in persistently infected animals. This study highlights the role of early events in the establishment of the FMDV infection. The extent of reduction of viral load after the peak is a critical determinant of the outcome of FMDV infection. Elucidation of the mechanisms that account for these observations will provide insight into the pathogenesis of FMD and may have important practical consequences for the development of an effective vaccine for prevention of carriers.

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Acknowledgements We thank Colin Randall, Luke Fitzpatrick, Brian Taylor and Malcolm Turner for their assistance with the handling and management of experimental animals. This work was supported by the Department for Environment, Food and Rural Affairs (DEFRA), UK. References 1. Alexandersen, S., M. B. Oleksiewicz, and A. I. Donaldson. 2001. Early pathogenesis of footand-mouth disease in pigs infected by contact: a quantitative time course study using TaqMan RT-PCR. J. Gen. Virol. 82:747-755. 2. Alexandersen, S., Z. Zhang, and A. Donaldson. 2002. Aspects of the persistence of foot-andmouth disease virus in animals- the carrier problem. Microbes. Infect. 4:1099-1110. 3. Alexandersen, S., Z. Zhang, S. M. Reid, G. H. Hutchings, and A. I. Donaldson. 2002. Quantities of infectious virus and viral RNA recovered from sheep and cattle experimentally infected with foot-and-mouth disease virus O UK 2001. J. Gen. Virol. 83:1915-1923. 4. Burrows, R., J. A. Mann, A. Greig, W. G. Chapman, and D. Goodridge. 1971. The growth and persistence of foot-and-mouth disease virus in the bovine mammary gland. J Hyg. (Lond) 69:307-321. 5. McVicar, J. W. and P. Sutmoller . 1969. The epizootiological importance of foot-and-mouth disease carriers. II. The carrier status of cattle exposed to foot-and-mouth disease following vaccination with an oil adjuvant inactivated virus vaccine. Arch. Gesamte Virusforsch. 26:217224. 6. Oleksiewicz, M. B., A. I. Donaldson, and S. Alexandersen. 2001. Development of a novel real-time RT-PCR assay for quantitation of foot- and-mouth disease virus in diverse porcine tissues. J Virol. Methods 92:23-35. 7. Prato Murphy, M. L., R. F. Meyer, C. Mebus, A. A. Schudel, and M. Rodriguez. 1994. Analysis of sites of foot and mouth disease virus persistence in carrier cattle via the polymerase chain reaction. Arch Virol 136:299-307. 8. Reid, S., N. Ferris, G. Hutchings, Z. Zhang, G. Belsham, and S. Alexandersen. 2002. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay. J. Virol. Methods 105:67-80. 9. Salt, J. S. 1993. The carrier state in foot and mouth disease--an immunological review. Br Vet J 149:207-23. 10. Snowdon, W. A. 1966. Growth of foot-and mouth disease virus in monolayer cultures of calf thyroid cells. Nature 210:1079-1080. 11. Sutmoller, P. and A. Gaggero. 1965. Foot-and mouth diseases carriers. Vet. Rec. 77:968-969. 12. Zhang, Z, G. Hutching, P. Kitching, and S. Alexandersen. 2002. Interferon-gamma cures cells persistently infected with foot-and-mouth disease virus. Arch. Virol. (In press). 13. Zhang, Z and R. P. Kitching. 2001. The localization of persistent foot and mouth disease virus in the epithelial cells of the soft palate and pharynx. J Comp Pathol. 124:89-94.

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Appendix 19

Modelling of viraemia in pigs infected with Foot-and-Mouth disease virus Melvyn Quan1, Louise Matthews2, Ciara M Murphy1, Zhidong Zhang1, Jeanette Knight1, Mark EJ Woolhouse2 and Soren Alexandersen1 1 2

Pirbright Laboratory, Institute for Animal Health, Ash Rd, Woking, Surrey, GU24 0NF, UK. Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK.

Abstract To quantify and predict virus excretion and infectiousness of animals infected with foot-and-mouth disease virus (FMDV), an understanding of the disease dynamics in individual animals is needed. This paper provides a quantitative description of the initial disease process in pigs experimentally infected with FMDV, and describes this process in mathematical terms. Dose-response curves were generated by inoculating groups of pigs with different doses of the FMDV O UKG 34/2001 strain. A significant difference (P = 0.001) between the groups in the length of time to become viraemic was found. No significant differences (P > 0.05) in the in vivo maximum exponential replication rate (r) between the groups were noted. An r of 0.30 was calculated from the experimental data, which equals a virus load doubling time of 2.3 hours. A mathematical model describing viraemia and replication of virus in the epithelium was fitted to the experimental data. Non-specific binding of virus is suggested as a reason for the discrepancy between the data and model. Further experiments are planned to test this hypothesis and improve the fit of the model. Introduction Due to the highly infectious nature of foot-and-mouth disease virus (FMDV) much attention has been focussed on the epidemic nature of the disease. The transmission of the virus depends on - among other factors - species of animal infected, density of animals and amount of virus excreted by individual animals. To quantify and predict virus excretion and infectiousness of animals infected with FMDV, an understanding of the disease dynamics in individual animals is needed. Cottral5 described FMDV viraemia curves in cattle. This paper provides a quantitative description of the disease process in pigs experimentally infected with FMDV, by undertaking a time course study of viral load in blood and describing this process in mathematical terms.

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Materials and Methods Inoculum FMDV O UKG 34/2001 was isolated from a pig at Cheale Meats Abattoir, Brentwood, Essex on 20 February 2001. The virus was passaged once in pigs and an inoculum prepared from epithelial tissue collected from the infected pigs. A 1:10, 1:100 and 1:1 000 dilution series was made with tissue culture medium. The dilutions were made up just before inoculating the pigs. Afterwards, the dilutions were tested by fluorogenic reverse transcription polymerase chain reaction (RT-PCR) and viral titration to confirm quantities and infectivity. Experiment A Twelve female Landrace x Large White pigs, between 20 and 30 kg body weight were placed in a bio-secure animal isolation unit. The pigs were divided into three groups of four pigs, with each group placed in a separate room. A group was inoculated intravenously with 0.5 ml of either a 1:10, 1:100 or 1:1000 dilution. Inoculations started with the group receiving the most dilute inoculum first. The following amounts of FMDV, as determined by viral titration on BTY cells and fluorogenic RT-PCR, were injected into the pigs: • • •

The high inoculation group was inoculated with 105.9 TCID50 or 108.04 FMDV genomes/pig. The medium inoculation group was inoculated with 104.9 TCID50 or 107.04 FMDV genomes/pig. The low inoculation group was inoculated with 103.9 TCID50 or 106.04 FMDV genomes/pig.

Serum samples were collected before the start of the experiment (to serve as controls) and approximately one to five minutes after inoculation. Then, samples were collected every day at 07h00 to 08h00 and 15h00 to 16h00 from the day of inoculation. The frequency of sampling was reduced after four days to once daily/every second day. Serum was collected by venipuncture and collected into plain serum tubes with no additive. The pigs were monitored for clinical signs of disease and temperatures recorded daily. A subjective scoring system was used to evaluate the progression of disease in the pigs. Individual clinical signs were given points (lameness – 1; lesions on one foot – 1, two feet – 2, three feet – 3, four feet – 4; tongue lesions – 1) and then added up. A pig with lesions on any foot was taken to be lame as well. A pig could therefore score a maximum of 6 points. The score was scaled to fit the graphs onto which it was plotted. If clinical disease in a pig was very severe, the pig was humanely euthanased by an overdose injection of pentobarbitone, followed by exsanguination. Only data up to the time the first pig was euthanased was used for analysis.

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Experiment B Experiment B was a repeat of Experiment A, with the exceptions that samples were collected more frequently (every 8 hours) and the distribution of the pigs within the animal isolation unit differed. In the low and medium inoculation group, the pigs were physically separated from each other so that no direct contact could take place. Pigs in the high inoculation group were kept together. Laboratory methods Quantitative measurement of viral genomes in serum and tissues was performed as previously described4,8,9. Viral titrations were performed, as previously described6. Modelling Equations 1 & 2 were used to describe the replication of FMDV in vivo: Equations 1 & 2

dV =γ E − δ V dt dE = δ V + rE − γ E dt where V = viraemia, E = virus in epithelial cells, γ = rate of exit of virus from epithelium, δ = rate of entry of virus into epithelium and r = replication rate. A system of ordinary differential equations were solved by computer, using a 4th order Runge-Kutta algorithm7 programmed in Fortran 95 (FTN95, Salford Software Ltd., UK). Data were represented graphically with Microsoft Excel software. Statistical analysis was performed on Minitab for Windows (Minitab Inc., USA). Results Results for Experiment A are summarised in Figure 1, which represents the logarithmic mean viraemia values for each inoculation group graphically; Table 1, which tabulates the amount of FMDV recovered one to five minutes after intravenous inoculation and Table 2, which lists the maximum exponential replication rate (r) for each animal. Similarly, results for Experiment B are summarised in Figure 2, Table 3 and Table 4. Data from Experiment A and B were combined to look at differences in the start of active viraemia. A Kruskal-Wallis test showed a significant (P = 0.001, H = 13.10) difference between the medians of the low (55.25 hours), medium (39.25 hours) and high (23.50 hours) inoculation groups. The effect of dose on r, irrespective of time, was looked at. A one way ANOVA showed no significant difference (P > 0.05, F = 0.91) in r between the different inoculation groups. The mean r ( r ) of FMDV O UKG 34/2001 was calculated from linear regression of individual viraemia curves ( r = 152

0.30, 95% confidence interval of 0.25 to 0.36), which equals a viral load doubling time of 2.30 hours (95% confidence interval of 1.94 to 2.82). Discussion The layout of pigs in the isolation unit of Experiment A was based on the assumption that the time course for disease progression would be relatively uniform within a group. This was not the case, as is evident by the large standard deviation in Figure 1, especially for the low and medium inoculation groups (the time until first detectable viraemia ranged from 30.0 to 95.5 hours for the low inoculation group of Experiment A, data not shown). The possibility that pigs were infected by other pigs in the group and not by the intravenous inoculation could not be excluded. The experiment was repeated in Experiment B with the layout described above. Pigs are highly resistant to aerosol infection1-4 and are unlikely to be infected by this route when physically separated. As the pigs still had to be handled, the possibility that they could be infected by indirect contact could not be excluded, but the risk was judged to be small and certainly much less than that of Experiment A. The high inoculation group were kept together, as the progress of disease was more uniform, faster and predictable in this group. The time until first detectable viraemia ranged from 31.0 to 119.5 hours for the low inoculation group, which was similar to results obtained from Experiment A. This suggests that the variability seen in the low and medium inoculation groups of Experiment A was not necessarily due to pig to pig transmission, but may have been the result of the dose and route of inoculation. Although different replication rates were noted amongst the inoculation groups in the early stages of the experiments, no significant differences in the r of FMDV between the inoculation groups were found. It appears as if the virus replication rate in different hosts is similar (similar rates were observed with the same strain of virus in cattle hosts, unpublished results), but can differ across virus strains (the replication rate of FMDV 0 UKG 34/2001 appeared to be faster than the O Taiwan 9/97 strain in pigs, even though the latter is a specific pig adapted strain, unpublished results). Although the model reflected the general trends of the experimental data very well, though the degree of variation in virus tires between groups was not closely reproduced at later time points (Figure 3). It is possible that after intravenous inoculation, some virus binds non-specifically to proteins and other cellular components of the circulatory system. Such non-specifically bound virus would be unable to bind to epithelial cells and go on to replicate. This effect would be more noticeable at lower concentrations and may possibly explain the discrepancy in the model and the data. There is some evidence for this when the data from samples taken one to five minutes after inoculation are examined. Although the results presented are based on estimates of the serum volume, a lower percentage of virus was recovered in the low inoculation groups (mean normalised value of 21% for Experiments A & B) than the mid (90%) or high (100%) inoculation groups. The following hypothesis, based on the results above, is suggested: stochastic variation in initiating an infection at low virus concentration is due, in part, to nonspecific binding of virus.

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Formal parameter estimation of γ and δ is planned and it is hoped that this preliminary work will lead to a temporal model of the relationship between viraemia, quantities of virus in epithelium and amount of virus excreted. In turn, this will enable refinement of models used to predict the nature of FMD epidemics. Acknowledgements We thank Luke Fitzpatrick, Brian Taylor, Bev Standing and Malcolm Turner for their assistance with the handling and management of experimental animals. This work was supported by the Department for Environment, Food and Rural Affairs (DEFRA), UK. References 1.

2. 3. 4.

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Alexandersen, S., I. Brotherhood, and I. Donaldson A. 2000. Minimal aerosol infectious dose of the 01 Lausanne strain of foot-and-mouth disease virus for pigs. 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, Rome FAO, Appendix 5, p. 48-57. Alexandersen, S., I. Brotherhood, and A. I. Donaldson. 2002. Natural aerosol transmission of foot-and-mouth disease virus to pigs: minimal infectious dose for strain O1 Lausanne, Epidemiology and Infection 128:301-312. Alexandersen, S., and A. I. Donaldson. 2002. Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs, Epidemiology and Infection 128:313-323. Alexandersen, S., Z. Zhang, S. M. Reid, G. H. Hutchings, and A. I. Donaldson. 2002. Quantities of infectious virus and viral RNA recovered from sheep and cattle experimentally infected with foot-and-mouth disease virus O UK 2001, Journal of General Virology 83:1915-1923. Cottral, G. E., and H. L. Bachrach. 1968. Food-and-mouth disease viremia, Proceedings of the Annual Meeting of the U S Animal Health Association 72:383-99. 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, Journal of Hygiene (London) 68:557-64. Press, W. H., S. A. Teukolsky, W. T. Vetterling, and B. P. Flannery. Integration of ordinary differential equations, Numerical recipes in Fortran 77. The art of scientific computing, 2nd ed, vol. 1 of Fortran Numerical Recipes, Cambridge University Press, p.701-718. Reid, S. M., N. P. Ferris, G. H. Hutchings, and S. Alexandersen. 2001. Evaluation of automated RT-PCR systems to accelerate FMD diagnosis. 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 Island of Moen, Denmark, 12-15 September 2001, Rome FAO, Appendix 25, p. 118-125,. Reid, S. M., N. P. Ferris, G. H. Hutchings, Z. Zhang, G. J. Belsham, and S. Alexandersen. 2001. Diagnosis of foot-and-mouth disease by real-time fluorogenic PCR assay, Veterinary Record 149:621-623.

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Table 1 Experiment A. Amount of FMDV genomes recovered one to five minutes after intravenous inoculation. Animals were not weighed, but were of uniform size and all approximately of the size indicated. Serum volume was calculated as 4.2% of body weight. Values in the second last column were normalised (high inoculation group = 1) in the last column. ID Weight Serum FMDV genomes Mean (log value) Recovered Normalised (kg) volume per animal 1- 5 min of each /inoculated value (ml) after inoculation inoculation group virus (log value) UP50 25 1050 5.34 UP51 25 1050 5.82 UP52 25 1050 6.12 UP53 25 1050 6.04 5.61 0.44 0.26 UP54 25 1050 7.53 UP55 25 1050 8.17 UP56 25 1050 5.34 UP57 25 1050 7.35 6.87 0.81 0.48 UP58 25 1050 8.52 UP59 25 1050 8.30 UP60 25 1050 8.35 UP61 25 1050 8.48 8.19 1.68 1.00

Table 2 Experiment A. The maximum replication rate (r) of FMDV in vivo. An exponential regression equation was fitted through a minimum of three, non-zero points on the steepest part of the exponential growth part of the viraemia curve of individual animals. ‘Period’ indicate the time after inoculation over which the measurement was calculated.

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Table 3 Experiment B. Amount of FMDV genomes recovered one to five minutes after intravenous inoculation. Serum volume was calculated as 4.2% of body weight. Values in the second last column were normalised (high inoculation group = 1) in the last column. ID Weight Serum FMDV genomes Mean (log value) Recovered Normalised (kg) volume per animal 1- 5 min of each /inoculated value (ml) after inoculation inoculation group virus (log value) UQ10 27.0 1134 5.28 UQ11 20.0 840 4.58 UQ12 22.0 924 5.60 UQ13 24.0 1008 6.76 5.53 0.17 0.15 UQ14 22.0 924 8.00 UQ15 19.5 819 7.38 UQ16 27.5 1155 7.53 UQ17 26.0 1092 7.03 7.26 1.41 1.32 UQ18 21.0 882 8.25 UQ19 17.0 714 8.34 UQ20 23.0 966 8.47 UQ21 13.5 567 8.40 8.15 1.08 1.00

Table 4 Experiment B. The maximum replication rate (r) of FMDV in vivo. An exponential regression equation was fitted through a minimum of three, non-zero points on the steepest part of the exponential growth part of the viraemia curve of individual animals. ‘Period’ indicate the time after inoculation over which the measurement was calculated.

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Appendix 20 Expression of inflammatory and antiviral cytokines in pigs experimentally infected with Foot-and-Mouth disease Ciara M. Murphy, Zhidong Zhang, Melvyn Quan, Jeanette Knight and Soren Alexandersen. Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, GU24 ONF, U.K. Abstract The disease produced by foot-and-mouth-disease virus (FMDV) is one of major concern, both from an economical and an animal welfare point of view. Clinical disease is severe, particularly in the case of pigs, which are often affected to a greater degree by FMD than cattle or sheep. The fact that pigs from a clinical point of view become more ill than other susceptible animals lends weight to the possibility that the porcine immune response, in particular the proinflammatory cytokine response, may be significantly elevated since these cytokines, in the process of controlling the infection, can act to make acute clinical disease worse. To explore the cytokine response in pigs in response to FMDV infection, a quantitative real-time RT-PCR was developed and used to measure cytokine mRNA in porcine peripheral blood mononuclear cells (PBMC) following infection of pigs with FMDV O UKG 34/2001 virus. The cytokines studied in this paper include Interferon gamma (IFNγ), Interferon alpha (IFNα), Interferon beta (IFNβ), Interleukin-1 alpha (IL1α) and Tumor necrosis factor alpha (TNFα). The cellular housekeeping gene glyceraldehyde-3-phosphatedehydrogenase (GAPDH) is also measured as an endogenous control. PBMCs were collected from groups of pigs before and during experimental infection with FMDV in order to generate a picture of the cytokine mRNA profiles of healthy animals compared with infected ones. A level of background mRNA expression was noted for each of the cytokines studied. The fluctuations seen in cytokine mRNA expression during the course of FMDV infection will be discussed. Introduction FMDV is an extremely rapidly replicating virus, a feature illustrated by the fact that clinical signs can become visible and quite obvious in pigs and cattle as soon as 24 to 48 hours after infection 1-6. In sheep and goats, the clinical signs are far less obvious and as a result, disease can go unnoticed 5,6,16. Fever, shivering, drooling and lip smacking are all characteristic signs of disease in cattle and lesions are most frequently observed on the tongue and dental pad, along the coronary band of the hoof and in the interdigital cleft 6. Pigs develop similar symptoms to cattle, albeit without the drooling and lip smacking and with the majority of lesions visible on the feet and sometimes around snout and lips 3,6. Lesions may also be observed on the tip and towards the back of the tongue. Clinical disease in pigs is usually very severe, more so than disease in other species. 160

Both humoral and cellular responses are induced as a result of infection with FMDV. However, very little is known about the innate immune response to FMDV 24. The main cells responsible for innate immunity are monocytes, macrophages and neutrophils, which have the ability to phagocytose microbial pathogens. This in turn triggers the cytokine network, resulting in the development of inflammatory responses, followed by specific immune responses 13. Pigs have a larger proportion of γ/δ T cells than other species that are susceptible to FMDV 26,27 and it is possible that these cells may have a role to play in an elevated innate immune response of pigs to FMDV. Cytokines are low molecular weight soluble proteins that are involved in and can regulate the host response to infection, inflammation and immunity, cell growth and cell differentiation. They are primarily secreted by leucocytes but can also be secreted by nearly every nucleated cell type. Acting as chemical communicators between cells, cytokines bind to specific receptors on cell surfaces initiating various signal transduction pathways12,17,21. In order to better understand the immunopathological pathways involved in the inflammatory response to FMDV in pigs, knowledge of the local cytokine profiles is critical. The use of real-time RT-PCR to achieve this provides a rapid, sensitive and quantifiable method of detecting the levels of porcine cytokine mRNA. In addition, this method is capable of detecting the minute quantities of mRNA (only 1% of total cellular RNA) present in cells 23. As a result, this method is becoming increasingly popular for the quantitation of cytokine mRNA 8,10,19,25,28. Materials and Methods Animals: The animals used in these experiments were 24 female Landrace x Large White pigs of 20 – 30kg body weight. Both animal experiments were carried out in biosecure isolation units. Procedure: Experiment 1: Normal control blood samples were taken for a week before inoculation. Three sets of four pigs were inoculated with 3 different doses of FMDV inoculum intravenously starting with the most diluted inoculum first. 0.5 ml of diluted inoculum was inoculated IV into the Vena Cava anterior using a 19G x 1.5” needle and a 2ml syringe. The dose used was (i) 3.9 logs (ii) 4.9 logs and (iii) 5.9 logs. After inoculation each group of pigs (low, medium and high dose) was placed in a separate room. The virus used (final titre 7.2 logs TCID/ml in BTY) was an O UKG 34/2001 inoculum first pig passage. Body temperature and progression of disease of all pigs was recorded. Blood and serum samples were taken in heparin and plain vacutainers respectively. All samples were taken twice daily at 07:30 – 08:30 and 14:30 - 15:30 for the first three days and then daily for a week. Pigs were killed 14 days post inoculation or before if disease was very severe, i.e. pigs with loose hooves, severe secondary infection or severe inability to maintain body temperature. Experiment 2: Normal control blood samples were taken during the four-week period before inoculation. Three sets of four pigs were inoculated with 3 different doses of FMDV 161

inoculum as described previously for Experiment 1. Immediately after inoculation each pig was placed singly into a separate cubicle (no direct contact). The high dose group was kept together in a single box allowing direct contact. The inoculum used was as in Experiment 1. Blood samples for mRNA anlaysis were taken at 07:30-08:30 for the duration of the experiment. Pigs were killed 14 days post inoculation or before if disease was very severe. Isolation of porcine peripheral blood mononuclear cells PBMCs (approx. 1x108cells) were isoloated from 10ml whole heparinated blood using Histopaque® - 1077 (density of 1077 g/dL) and suspended in 400µl of calcium/magnesium free PBS. An aliquot of 120µl of PBMCs, which was equivalent to 3x107 cells, was added to 1680µl of lysis buffer (from Roche mRNA HS kit, as per manufacturers’ recommendations) to a final volume of 1800µl. An aliquot of 600µl from the above 1800µl suspension of PBMCs in lysis buffer contained 1x107 cells – the quantity of cells required for mRNA extraction. The suspensions were vortexed and stored at -80°C until required. Stimulation of porcine peripheral blood mononuclear cells: In order to validate the RT-PCR assay, PBMCs were stimulated and cultured from 0-4 hours, RNA was extracted, reverse-transcribed into cDNA, which was then amplified by real time PCR using different cytokine primer and probe sets. PBMCs were stimulated using lectin, from Phaseolus vulgaris (red kidney bean), at a concentration of 5μg/μl. One Falcon Multiwell™ tissue culture plate, (6 wells, flat bottom with low evaporation lid), was prepared by aliquoting 1ml of RPMI + HEPES media containing lectin, fungizone, penicillin/streptomycin and 10% FBS into each of the wells. One well on each plate contained media only, in order to provide a negative control. Subsequently, 1x106 PBMCs were added to each well and mixed by gentle aspiration. Cells were harvested at the appropriate time and suspended in 1200μl of lysis buffer (from Roche mRNA HS kit). The cells in lysis buffer were vortexed and stored at -80ºC. mRNA extraction and RT-PCR conditions: mRNA isolated from uncultured PBMCs and lectin-activated PBMCs was extracted by the MagNA Pure LC automated instrument (Roche Molecular Biochemicals) by using the mRNA HS isolation extraction kit produced by the same manufacturer. A Jenway Genova MK3 Life Science Analyser was used to measure RNA concentrations. mRNA was then reverse transcribed into cDNA using PE Biosystems TaqMan RT reagents and random primers, as described previously 5,20. Negative controls without RT enzyme were included with every reverse transcription to eliminate the possibility of false positive results due to DNA contamination. Primers and probes for all cytokine target sequences were designed using the computer program Primer Express™ (Applied Biosystems). Quantitative PCR was performed as described previously 3,5,18. Results In order to quantify the results obtained by real-time RT-PCR, standard samples of known concentrations were prepared from vitro-transcribed RNA. These samples were 162

used to construct standard curves for each cytokine mRNA and for the endogenous control, GAPDH. Uniform in vitro expression of porcine GAPDH:

Unstimulated and stimulated PBMCs were tested for the presence of the cellular endogenous control, GAPDH. As predicted, GAPDH levels did not appear to change during cell stimulation, in fact the levels decreased slightly over time. An example of such an experiment is shown in Fig. 1 below.

Figure 1 Expression of GAPDH in lectin-stimulated cells

Cytokine expression by lectin-stimulated porcine PBMCs:

PBMCs were isolated from the blood of two healthy pigs and stimulated with lectin. A relatively low background level of expression of IL-1α and absence of IFNβ was noted in unstimulated PBMCs, Fig. 2 below. For the cytokines IFNγ, IFNα and TNFα a higher level of background mRNA expression was observed. PBMCs stimulated with lectin demonstrated greater levels of expression of all cytokines tested than unstimulated PBMCs, Fig. 2 below.

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diarrhoea and coughing). These cells were then tested for the expression or nonexpression of inflammatory and antiviral cytokine mRNAs, see Fig. 3 below. Over the course of the two uninfected periods, as expected, expression levels of GAPDH mRNA remained relatively stable. In Experiment 1, expression levels of IFNα mRNA were higher than those of the other cytokines, Fig 3 below. The mRNA expression levels of IFNγ and IFNα in Experiment 2 were higher than those of TNFα and IL-1α, but remained relatively stable, Fig 3 below. IFNβ mRNA levels remained negative throughout both animal experiments, so IFNβ was excluded from graphs subsequent to Fig. 3.

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When compared with the patterns of cytokine mRNA expression in uninfected pigs, see Fig. 3 above, it is clear that cytokine mRNA expression was influenced by the presence of the virus.

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The preliminary results from infected pigs in experiment 1 were quite interesting, Fig. 4. This graph depicts the overall average cytokine mRNA expression in the two experiments. There appeared to be a period of cytokine response very early in the infection at 24h PI, with IFNα, TNFα and IL-1α in particular rising above their background levels of expression as illustrated in Fig. 3 above. This increased mRNA expression was followed by a period of possible suppression, which included to a lesser extent the endogenous control GAPDH and TNFα. Experiment 2 also revealed a possible period of suppression of mRNA expression, see Fig. 4. However, in this instance it occurred later, at 72 PI and affected GAPDH to a greater extent. Interestingly, GAPDH mRNA was expressed at a lower level than IL-1α mRNA, which demonstrated a very good average response in this experiment. TNFα mRNA levels were not suppressed as much as the other cytokines at 72h PI, but were then not detectable at 96h PI. It should also be noted that clinical disease occurred later in experiment 2 than in experiment 1, perhaps due to the cubicle arrangements used in experiment 2.

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Average Cytokine Expression in Low, Medium and High Dose Groups (Exp. 1)

The low dose group of 4 pigs appeared to demonstrate a similar pattern of expression for the IFNα, TNFα, IL-1α in Fig. 5 below. There was an increase in expression at 24h PI, followed by a rapid decline, which extended to 48h PI. IFNγ demonstrated an earlier initial increase in expression 6h PI, which was also followed by a rapid decline up to 48h PI. There then followed an increase in the expression of all four cytokines, with IFNγ showing the greatest initial increase of 5 logs at 54h PI. IFNα, TNFα and IL-1α then increased further at 78h PI. Interestingly, both of the peaks of IFNγ occurred earlier than the peaks of the other three cytokines. The medium dose group of 4 pigs did not demonstrate the same pattern of expression as the low dose group in Fig. 5 below. There did not appear to be an early response at 24h PI, as there was in the case of the low dose group. However, there was a decline in expression of IFNα, IFNγ, and TNFα mRNAs at 48h PI. IL-1α mRNA expression did not decline at this point, but was maintained. By 54h PI an increase in the expression of all cytokines occurred with IFNγ demonstrating the greatest degree of response once again. IFNγ again responded well in the high dose group of 4 pigs, Fig. 5 below, with a large increase in expression 6h PI that was maintained until 48h PI when it decreased. By 54h PI, IFNγ expression was elevated again. There was a poor response in the case of IFNα, but TNFα in particular and IL-1α demonstrated peaks at 24h and 72h PI. Low Dose Group Average Cytokine Expression 9 8 7

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Average Cytokine Expression in Low, Medium and High Dose Groups (Exp. 2):

It is difficult to comment on the patterns of expression of the cytokines in experiment 2, Fig. 6 below, because of the behaviour of GAPDH, which closely follows the patterns of cytokine mRNA expression, with the exception of TNFα. We think it is likely that the FMD infection induces a complex pattern of mRNA induction and degradation leading to a very complex picture that not only involves inducible cytokine mRNAs but perhaps other cellular constituents that may include GAPDH and other RNAs. Another less likely explanation could be that no “real” cytokine response has been detected and that the results are merely illustrating a combination of the quality of the PBMCs isolated from each blood sample and the quality of the RNA extracted from those PBMCs. There is evidence against this, as it appeared that there was a genuine response in the case of IL-1α in all three groups of pigs. The background level of expression determined for IL1α was less than 2 log copies per 1.0E+7 cells, see Fig. 3. In contrast, the levels of expression during this experiment were as high as 7 log copies per 1.0E+7 cells at 48h PI, an increase in expression of 5 logs. IL-1α and TNFα mRNA levels were both elevated at 48h PI in the low dose group of pigs, this was followed by the possible period of suppression that occurred at 72h PI in experiment 2, 24 hours later than the decrease in experiment 1. However, TNFα mRNA levels did not decrease as much as the other cytokines at 72h PI, but were not detectable at 96h PI. The medium dose group of pigs exhibited a similar pattern of cytokine mRNA expression with regard to suppression at 72h PI. However, there did not seem to be an equally good response from IL-1α, which peaked at 48h PI at 5.5 log copies per 1.0E+7 cells compared with 7.0 log copies per 1.0E+7 cells in the low dose group. The decline in mRNA expression at 72h PI was not as severe for the medium dose group of pigs, with all mRNAs remaining within the detectable range. The high dose group of pigs again demonstrated a similar pattern of cytokine expression. In this case the decline at 72h PI was even less pronounced and was followed by an increase in the expression of all mRNAs but TNFα at 96h PI.

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Discussion The aim of this study was to gain a deeper understanding into the pathobiology of FMDV infection in pigs and into the pathogenesis of the disease through the quantitation of the mRNA levels of a variety of antiviral and proinflammatory cytokines. The very severe nature of FMD infection in pigs leads us to believe that the porcine immune system is particularly activated in order to eliminate the virus, and that this activation may cause some of the general clinical signs observed in pigs. By studying cytokine expression, an insight into the innate immune response to FMDV can be gained. In the present preliminary study, cytokines were quantitated by real-time RT-PCR, an increasingly popular method for the detection of cytokine mRNA 8,9,11,15,19,25. The assay utilised in this study was validated by determining the magnitude of induction of cytokine mRNAs by activation of porcine PBMCs. This work demonstrated that the assay could effectively be applied to the detection of cytokine mRNA fluctuations in porcine PBMCs. The housekeeping gene, GAPDH was used as an endogenous control to demonstrate that cellular mRNA was being efficiently extracted, reverse transcribed into cDNA and amplified by real-time PCR. GAPDH expression remained relatively constant in the in vitro PBMC experimental system, however there was evidence of a degree of fluctuation of GAPDH in the in vivo system. In our in vivo system average GAPDH levels were determined to be approximately 106 copies of GAPDH per 107 cells (the number of cells from which mRNA was extracted). A fluctuation of GAPDH mRNA expression of almost 2 logs was seen in these uninfected pigs, Fig. 3. However, the observed fluctuations of cytokine and GAPDH mRNA levels were much greater in FMDV infected pigs, Fig. 4. The effect of FMDV infection on GAPDH was mainly observed as a severe decrease in expression, Fig. 4. There is evidence that GAPDH transcription can be inducible in some systems 7,10. Nevertheless, we believe that it is more likely that the suppression effect seen on GAPDH and the cytokines studied was as a direct result of FMDV infection or possibly as a result of the effects of fever and other host processes induced by the virus. Neither of the two animal experiments carried out revealed a positive result for the expression of IFNβ. However, IFNβ was expressed (albeit at a relatively low level, see Fig. 2) by the lectin-stimulated PBMCs. The likely reason for this is the cells being assayed in this study; IFNβ is predominantly a product of fibroblasts 22, not PBMCs as were sampled here. The expression of IFNβ by lectin-stimulated PBMCs may be the result of their over-stimulation to the degree that more signalling pathways were activated than occurs during FMDV infection. The overall average cytokine expression for the two animal experiments revealed a similar pattern in both cases, see Fig. 4 above. In experiment 1 an initial peak of cytokine mRNA expression was seen from as early as 6h to 24h PI. This was followed, by a decline in mRNA expression at 48h PI. Expression appeared to recover by 54h PI, it is possible that the translation of early expressed cytokine mRNA into protein communicators led to a second round of cytokine mRNA expression. Experiment 2 (with animals in groups 1 and 2 housed individually) revealed a slightly different pattern of cytokine mRNA expression The first peak of cytokine mRNA expression was not seen before 48h PI, but was followed by a rapid decline at 72h PI, similar to the decline observed in experiment 1 at 48h PI. Levels of mRNA expression in experiment 2 did not begin to recover until 96h PI. As previously mentioned, this may be an effect of the cubicle arrangements in the second experiment, where pigs were 170

housed individually and developed disease later than pigs in experiment 1. Cytokine mRNA expression appeared to recover more rapidly in the case of experiment 1, this may be due to higher levels of virus in the environment, or possibly because the pigs in experiment 2 had experienced unrelated health problems in the period immediately before FMD infection. The high dose group of pigs in experiment 2 were housed as a group like those in experiment 1, but still exhibited a later peak of cytokine mRNA expression, see Fig. 6. However, in comparison with the other two groups in experiment 2, the recovery of cytokine mRNA expression in the high dose group occurred earlier and was elevated by 96h PI, see Fig. 6. FMD infection appears to induce a very complex picture of mRNA stimulation and degradation. If mRNA is indeed degraded at certain points in the disease process, this may be due to a direct effect of the virus. However, we think it is more likely that this is due to the effects of cytokines released as a result of infection. For instance through the induction of intra-cellular RNase-L by interferons, which produces extensive cleavage of both host and viral mRNA leading to the inhibition of protein synthesis 14. More work is required both at the mRNA and the protein level so that virus and host effects during infection can be elucidated. Reference List 1. Alexandersen, S., I. Brotherhood, and A. I. Donaldson. 2002. Natural aerosol transmission of foot-and-mouth disease virus to pigs: minimal infectious dose for strain O1 Lausanne. Epidemiol. Infect. 128:301-312. 2. Alexandersen, S. and A. I. Donaldson. 2002. Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs. Epidemiol. Infect. 128:313323. 3. Alexandersen, S., M. B. Oleksiewicz, and A. I. Donaldson. 2001. The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative timecourse study using TaqMan RT-PCR. J. Gen. Virol. 82:747-755. 4. Alexandersen, S., Z. Zhang, and A. Donaldson. 2002. Aspects of the persistence of foot-and-mouth disease virus in animals-the carrier problem. Microbes. Infect. 4:1099. 5. Alexandersen, S., Z. Zhang, S. M. Reid, G. H. Hutchings, and A. I. Donaldson. 2002. Quantities of infectious virus and viral RNA recovered from sheep and cattle experimentally infected with foot-and-mouth disease virus O UK 2001. J Gen. Virol. 83:1915-1923. 6. Bachrach, H. L. 1968. Foot-and-mouth disease. Annu Rev Microbiol 22:201-244. 7. Barroso, I., B. Benito, C. Garci-Jimenez, A. Hernandez, M. J. Obregon, and P. Santisteban. 1999. Norepinephrine, tri-iodothyronine and insulin upregulate glyceraldehyde-3-phosphate dehydrogenase mRNA during Brown adipocyte differentiation. Eur. J. Endocrinol. 141:169-179. 8. Blaschke, V., K. Reich, S. Blaschke, S. Zipprich, and C. Neumann. 2000. Rapid quantitation of proinflammatory and chemoattractant cytokine expression in small tissue samples and monocyte-derived dendritic cells: validation of a new real-time RTPCR technology. J. Immunol. Methods 246:79-90.

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9. Brieland, J. K., C. Jackson, S. Hurst, D. Loebenberg, T. Muchamuel, R. Debets, R. Kastelein, T. Churakova, J. Abrams, R. Hare, and A. O'Garra. 2000. Immunomodulatory role of endogenous interleukin-18 in gamma interferon-mediated resolution of replicative Legionella pneumophila lung infection. Infect. Immun. 68:65676573. 10. Bustin, S. A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25:169-193. 11. Casteels, K. M., C. Mathieu, M. Waer, D. Valckx, L. Overbergh, J. M. Laureys, and R. Bouillon. 1998. Prevention of type I diabetes in nonobese diabetic mice by late intervention with nonhypercalcemic analogs of 1,25-dihydroxyvitamin D3 in combination with a short induction course of cyclosporin A. Endocrinology 139:95-102. 12. Goodbourn, S., L. Didcock, and R. E. Randall. 2000. Interferons: cell signalling, immune modulation, antiviral responses and virus countermeasures. Journal of General Virology 81:2341-2364. 13. Janeway, C. A., Jr. and R. Medzhitov. 2002. Innate immune recognition. Annu Rev Immunol. 20:197-216. 14. Le Page, C., P. Genin, M. G. Baines, and J. Hiscott. 2000. Interferon activation and innate immunity. Rev Immunogenet. 2:374-386. 15. Liu, Z., K. Geboes, S. Colpaert, L. Overbergh, C. Mathieu, H. Heremans, M. de Boer, L. Boon, G. D'Haens, P. Rutgeerts, and J. L. Ceuppens. 2000. Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions. J. Immunol. 164:6005-6014. 16. Nandi, S., B.S.Negi, and A.K.Sen. 1999. Epidemiology of foot and mouth disease in sheep and goats. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases 20:1-10. 17. O'Neill, L. A. 2000. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci. STKE. 2000:RE1. 18. Oleksiewicz, M. B., A. I. Donaldson, and S. Alexandersen. 2001. Development of a novel real-time RT-PCR assay for quantitation of foot- and-mouth disease virus in diverse porcine tissues. J. Virol. Methods 92:23-35. 19. Overbergh, L., D. Valckx, M. Waer, and C. Mathieu. 1999. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11:305-312. 20. Reid, S., N. Ferris, G. Hutchings, Z. Zhang, G. Belsham, and S. Alexandersen. 2002. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay J Virol. Methods 105:67. 21. Schutze, S., K. Wiegmann, T. Machleidt, and M. Kronke. 1995. TNF-induced activation of NF-kappa B. Immunobiology 193:193-203.

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22. Sen, G. C. and P. Lengyel. 1992. The interferon system. A bird's eye view of its biochemistry. J. Biol. Chem. 267:5017-5020. 23. Sirlin J.L. 1972. Molecular Species of RNA, p. 51-82. Biology of RNA. Academic Press, London. 24. Sobrino, F., M. Saiz, M. A. Jimenez-Clavero, J. I. Nunez, M. F. Rosas, E. Baranowski, and V. Ley. 2001. Foot-and-mouth disease virus: a long known virus, but a current threat. Vet. Res. 32:1-30. 25. Stordeur, P., L. F. Poulin, L. Craciun, L. Zhou, L. Schandene, A. de Lavareille, S. Goriely, and M. Goldman. 2002. Cytokine mRNA quantification by real-time PCR. J. Immunol. Methods 259:55-64. 26. Takamatsu, H. H., M. S. Denyer, and T. E. Wileman. 2002. A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells. Vet. Immunol. Immunopathol. 87:223-224. 27. Yang, H. and R. M. Parkhouse. 2000. Characterization of the porcine gammadelta Tcell receptor structure and cellular distribution by monoclonal antibody PPT27 Immunology 99:504-509. 28. Yin, J. L., N. A. Shackel, A. Zekry, P. H. McGuinness, C. Richards, K. V. Putten, G. W. McCaughan, J. M. Eris, and G. A. Bishop. 2001. Real-time reverse transcriptasepolymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol. Cell Biol. 79:213-221.

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Appendix 21

Foot-and-Mouth disease virus transmission between individually housed calves A. Dekker, A. Bouma and M.C.M. de Jong

Introduction Foot-and-Mouth disease (FMD) virus was most likely introduced in the Netherlands by calves imported from Ireland via an FMDV-contaminated resting point in Mayenne, France. The calves were sent to a mixed veal-calf/dairy-goat farm and housed individually (Figure 1). The first clinical signs of FMD were reported in the goats, three weeks after arrival of the calves (index case 2001/03). At culling of the farm, serum samples were taken from all calves (74) and 100 goats on that farm. From the 74 calves 4 were positive for antibodies against FMDV, whereas 87 of the 100 goats sampled were serological positive. The serological positive calves were not found in an isolated part of the stable (Figure 1). Based on the information given in Figure 1, it seemed that these calves were able to have contact with other calves. 23 calves 25 calves 2+

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Since only a small percentage of the calves was serologically positive, it was questioned whether the hypothesised route via Mayenne was likely or even possible. After all, FMD is known to be very contagious and the spread of FMD virus among calves appeared to be only limited. The calves on the index farm (NET/2001/03) were initially housed individually. As for every contagious disease, the contact structure between infectious and susceptible animals is important and in part determines whether an infection will spread. In theory, infection of animals housed in a row will stop spreading, if transmission relies on direct contact between animals, because the chance of transmission will always be smaller than 1. However, we had no information on the reduction of transmission of FMD virus if such a housing system is used. In a previous challenge experiment using tethered calves no transmission from infected calves (10 vaccinated and 2 controls) to 5 non-immune contact calves was observed when the 174

calves were culled 7 days after infection (personal observation). This indicated that transmission between calves that are individually fed does not happen very easily. In the current experiments we try to estimate the transmission rate of FMD virus can in individually housed calves without direct and with direct contact. Materials and methods Animal experiments Conventionally reared calves of 4 to 9 weeks of age were randomly allocated to the experimental groups. In experiments 1-4, one inoculated calf was housed at 1 metre distance from the contact calf. In experiments 5 and 6, the inoculated calf was housed between two contact calves (Photo 1). The animals arrived at "High Containment" facilities 5 - 7 days before the start of the experiment. At day 0, the experiment started with inoculation of one of the calves in each group was intranasally inoculated with 1.5 ml of FMD virus in each nostril. The other animal(s) in each group were in-contact calves. The body temperature off all calves was recorded daily. After inoculation heparinised blood samples were taken from all calves daily until seven days, buccal swabs using cotton gauze and a forceps until 14 days after inoculation, and serum samples weekly. Animal caretakers first took care of the contact calves, and changed shoes and clothing before taking care of the infected calves. Calves had their own feeding and drinking bucket. In experiment 1and 2 all calves were culled 30 days after inoculation, in experiment 2 and 3 the calves were culled 43 days after inoculation and in the last two experiment 71 days after inoculation.

Photo 1: Housing of the calves in the experiments with direct contact.

Virus Samples from outbreak NET/2001/01 and NET/2001/03 were grown in the tongue of FMD serological negative calf. After approximately 25 hours all vesicles were collected and used to prepare the challenge virus, which was stored at -70 °C. The challenge virus was titrated on cattle tongue before use, and a titre of 105.9 cattle ID50 was found. In the experiments the virus was diluted 1500 times and 1.5 ml was inoculated in each nostril, resulting in approximately 1500 cattle ID50 per calf. After inoculation samples of the freshly prepared inoculum as well as the inoculum used in the stable were titrated on secondary porcine kidney cells as well as on secondary lamb kidney cells.

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Virus neutralisation test The virus neutralisation test was performed as described previously (Dekker and Terpstra, 1996) using O1Manisa virus and secondary porcine kidney cells. Results Titration of the inoculum on secondary pig and lamb kidney cells resulted in a dose of 6800 PFU and 9800 PFU per calf, respectively. In 5 out of the 6 inoculated animals clinical signs of foot-and-mouth disease were observed, with a marked rise in body temperature for one or two days between 5 and 7 days after inoculation. Vesicles were observed for the first time 6 to 9 days after infection, in some animals reddening of the dental pad was seen one or two days earlier. Lameness was not observed, but due to the housing system (Photo 1) also unlikely to be observed easily. In all inoculated animals a four-fold increase in neutralisation titre was observed between 7 and 11 days after inoculation. In one of the inoculated calves no vesicles or lesions were detected, but the animal had fever for one day and became serological positive after 15 days. In the inoculated calves the peak in antibody titre was detected between 10 and 27 days after inoculation. Antibody titres declined towards the end of the experiment, but stayed in all inoculated animals well above the cut-off defined by the FAO cut-off reference serum. None of the contact calves showed a rise in body temperature, or any vesicles or lesion. All contact calves remained serologically negative. Table 1: Clinical and serological observations. Maximum rectal temp (°C)

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Day first Maximum four-fold VNT titre rise (10log) antibodies 8 2.4 <0.3 15 1.05 <0.3 7 >2.4 <0.3 7 >2.4 <0.3 <0.3 7 >2.4 <0.3 <0.3 11 >2.4 <0.3

Discussion The purpose of this study was to determine the transmission rate of FMD virus among calves that had only limited contact. In neither of the experiments virus transmission was observed, therefore, the transmission rate could not be estimated. These results indicate that the occurrence of minor outbreaks, as observed on two veal-calf herds during the Dutch FMD epidemic in 2001, were possible.

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Although it is known that FMD virus can spread by air it has recently been established that the virus dose required to infect pigs with a recent European isolate is much higher than previously was determined for other strains (Alexandersen and Donaldson, 2002). New data on the minimal infectious dose of these isolates for cattle and sheep are not available. In the 2001 FMD outbreak in the Netherlands two outbreak farms were found in which infected calves were found in the same stable as non-infected calves. On these outbreak-farms a housing system similar to the one in this study was used. These field data suggested that airborne transmission, although well described previously, did not occur in these situations. As mentioned before, when an infection spreads only via direct contact, theoretically an infection chain ends when animals are housed in a row and have only direct contact to their neighbours. Only large outbreaks are seen in calves on a row when a calf can also infect calves further away via indirect contact, for example by air. We, therefore, started the experiment with a distance of 1 metre between animals. However, none of the 4 contact calves at 1 metre distance did contract the infection. Subsequently we tried to quantify the transmission when direct contact was possible (Photo 1). Also in this situation transmission did not occur, despite the clinical signs in both inoculated animals. Because none of the contact calves got infected it is not possible to make a quantitative estimation of the transmission rate. The results of these experiments do question the role of airborne transmission in FMD infection of cattle. In the housing system applied in these experiments calves have their own feeding and drinking bucket, which is cleaned daily. Whereas in traditional housing systems calves used to be fed by drinking out of the same through or by sharing the drinking buckets. Also in housing systems for dairy cattle sharing drinking and feeding places is common. In the outbreaks in the Netherlands there was no indication that the virus was not able to spread in dairy farms or goat farms. Additionally it is also very unlikely that young cattle are less susceptible for FMD virus than older cattle. Therefore the most likely conclusion is that the contact structure of the housing system applied in these experiments is the major cause of the reduction in transmission. This would indicate that airborne transmission between cattle is a very unlikely route. Sharing feeding, drinking, milking machine and e.g. injection needles probably causes transmission in other housing systems. Because ingestion of FMD virus is not known to be a very efficient route of infection, small wounds in the mouth or on teats are more likely the port of entry. In these experiments each inoculated calf received 1500 times the dose that in 50% of the cases produces vesicles on cattle tongue. In cell culture, however, the calculated dose was even 4.5 to 6.5 times higher, which is common when comparing sensitivity of cell culture to the sensitivity of cattle. One of the inoculated calves did not show clinical signs. Based on the four-fold increase of antibodies the virus dose used did result in infection of all inoculated calves. Antibodies in the subclinical infected animal appeared later and the maximum titre was lower (table 1). The titre, however, stayed above the titre (0.55 10log) defined by the FAO cut-off serum (Moonen et al., 2000). Extrapolation and generalisation of these results from these experiments to the field situation, indicating a very low risk of infection if young cattle are involved, is dangerous. In our containment facilities the direction of the air was from head to tail, and the complete air content of the stable is refreshed approximately 7 times per hour. Moreover, there was no contact between the animals via animal caretakers or drinking buckets. In conclusion, the experimental data are in agreement with the field observations and suggest that the occurrence of the minor outbreaks as observed on the two veal-calf herds during the Dutch FMD epidemic in 2001 were possible.

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References Alexandersens, S., Donaldson, A.I. 2002. Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs. Epidemiology and infection 128 (2); 313-323. Dekker, A., Terpstra, C. 1996. Prevalence of foot-and-mouth disease antibodies in dairy herds in the Netherlands, four years after vaccination. Research in Veterinary science 61; 89-91. 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. Research in veterinary science 43; 339-346. Moonen, P., Miedema, G.K.W., van Hemert-Kluitenberg, F., Chenard, G., Dekker. A. 2000. Comparison of FMDV neutralisation tests using three different cell lines: validation of the new FAO reference sera. Session of the research group of the European commission for the control of foot-and-mouth disease, Borovets, Bulgaria, 5 to 8 September 2000.

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Appendix 22

Comparisons of the Complete Genomes of Asian, African and European Isolates of a Recent Foot-and-Mouth Disease Virus Type O Pandemic Strain (PanAsia) P.W. Mason1, J.M. Pacheco1, Q.-Z. Zhao1,2 and N.J. Knowles3 1

U.S. Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Greenport, NY 11944, USA. 2 Lanzhou Veterinary Research Institute, Lanzhou, Gansu, PR China 3 Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.

Abstract To further analyze the relationships between foot-and-mouth disease type O viruses belonging to the PanAsia strain complete genomes were amplified using long-range PCR techniques, and the PCR products were sequenced revealing the sequences for the entire genomes of five PanAsia isolates as well as an animal-passaged derivative of one of them. These genomes were compared to two other PanAsia genomes, and these analyses revealed that all portions of the genomes of these isolates are highly conserved and provided confirmation of the close relationship between the viruses responsible for South African and UK outbreaks. Introduction During the last 12 years a strain of foot-and-mouth disease (FMD) virus serotype O, named PanAsia, has spread from India throughout southern Asia and the Middle East (Knowles et al., 2000). During 2000, this virus strain caused outbreaks in the Republic of Korea, Japan, Russia (Primorsky Territory), Mongolia and South Africa (KwaZulu-Natal Province), areas that last experienced FMD outbreaks in 1934, 1908, 1964, 1974 and 1957, respectively. In February 2001, the PanAsia virus strain spread to the United Kingdom where, in just over seven months, it caused outbreaks on 2,030 farms. From the UK it quickly spread to the Republic of Ireland, France and the Netherlands. Previous studies that utilized reverse transcription-polymerase chain reaction (RT-PCR) to sequence the VP1-coding region of the RNA genomes of approximately 30 PanAsia isolates demonstrated that the UK virus was most closely related to the virus from South Africa (approx. 99.7% nucleotide identity) (Knowles et al., 2001b). Phylogenetic analyses of these VP1 sequences and others held in the WRLFMD database showed that the UK and South African viruses were more closely related to PanAsia virus isolates from the Far East than to those occurring in the Middle East or the Indian sub-continent (N.J. Knowles, unpublished data). To further elucidate the relationships between certain virus isolates belong to this strain, a long-range RT-PCR technique (Tellier et al., 1996) was developed to allow the complete genomes to be amplified and sequenced. The resulting sequence data was subjected to a number of analyses.

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Materials and Methods Viruses For the six viruses sequenced in this work, passages (p) of viruses prior to sequencing were performed in pigs (PIG), cattle (BOV), primary bovine thyroid cells (BTY), IB-RS-2 porcine kidney cell line (RS), baby hamster kidney cell line 21, clone 13 (BHK), and secondary porcine kidney cells (PK). O/TAW/2/99 is a bovine probang isolate from Kinmen Island Prefecture, Taiwan Province of China, BHKp3, BTYp1, BHKp2. O/TAW/2/99bov, is an experimental bovine-derived derivative of O/TAW/2/99 passaged an additional two times in pigs prior to its inoculation into a heifer. O/Tibet/CHA/99 is a bovine isolate from Tibet, People’s Republic of China (reported to OIE, May 31, 1999; OIE volume 12, no. 21, June 4, 1999), BOVp1, BHKp3; the sequence data reported here are similar to those recently deposited in GenBank for this isolate (accession no. AF506822). O/SKR/2000 is a bovine isolate from early May of 2000, from the Chungju county of the Kyunggi province, Republic of Korea, BHKp1. SAR/19/2000 is a bovine isolate from KwaZulu-Natal, Republic of South Africa, PKp2, RSp1, BHKp1. O/UKG/35/2001 is a porcine isolate from Cumbria, United Kingdom, PIGp2. O/JPN/2000 designates the Japanese isolate, whose sequence data were provided by T. Kanno (T. Kanno et al., Virus Genes, in press). O/SKR/2000gb is used to designate the data from a 2000 South Korean isolate deposited in Genbank (accession no. AF377945). RT-PCR and sequencing Sequence data were obtained from RNA isolated from the indicated source using TriZOL (Life Technologies, Gaithersburg, MD), reverse-transcribed with Superscript II polymerase (Life Technologies), and amplified using the polymerase chain reaction (PCR) using Herculase polymerase (Stratagene, Lo Jolla, CA, USA) and the indicated primers (see Table 1). Following amplification, the cDNA fragments were purified from acrylamide gels by elution (S-fragment amplicons) or agarose gels with Qiagen Resin (Qiagen, Valencia, CA), and sequenced using selected primers (Table 1) and asymmetric amplification with Big-dye terminators (ABI, Foster City, CA) followed by resolution on an ABI 3700 sequencer. Genome scanning analyses A program written by on of the authors (NJK) was used to compare the complete genome sequence of O/UKG/35/2001 to those of 15 FMD viruses representing five of the seven serotypes. The percentage nucleotide identities were calculated for a sliding window of 300 nucleotides stepping at 15 nucleotide intervals. The resultant values were plotted on a graph. Phylogenetic analyses Distance matrices were calculated using with the program Clustal X (Thompson et al., 1997). The phylogenetic tree was constructed using a Neighbor-joining algorithm (Saitou & Nei, 1987) implemented in the program Clustal X and drawn using the program TreeView 1.6 (Page, 1996). Confidence limits were calculated by the Bootstrap re-sampling method (1000 replicates) (Efron et al., 1996) as implemented within Clustal X.

180

Results and Discussion Sequencing strategy and sequence data acquisition: Typically RT-PCR analyses are compromised by the fact that the sequence data at the ends of the products are fixed by the primer sequences, and therefore the precise genome sequence are replaced with the primer sequences, which may be divergent. In the case of 3' polyadenylated RNAs (such as the FMDV genome), RT and amplification with oligo(T)-containing primers insures that the only genetic information lost is the number of adenosine (A) residues in the poly(A) tract (which is, in any case, variable between genomes within an isolate). In the case of the 5' end of the genome, the problem of loss of sequence data upon amplification can be overcome by using terminal transferase to add a homopolymer to the 3' end of RT-produced single-strand cDNA, and then amplifying through the 5' end of the cDNA utilizing a homopolymer complementary to the added bases and an antisense primer (Frohman et al., 1988). In the case of FMDV, sequencing of its genome by RT-PCR methodology is further complicated by the presence of a long polypyrimidine tract (of variable length) near the 5' end of the genome, that contains predominantly cytosine (C) residues. Using oligonucleotide primers that border this region, it is possible to reverse-transcribe cDNAs, and amplify them using PCR, although the poly(C) tracts are truncated by successive rounds of PCR. Using these methods, we developed a fulllength, authentic sequence data for a 1999 Taiwan isolate of the PanAsia topotype (O/TAW/2/99). This sequence reflects the authentic genome in all positions except the number of A residues in the poly(A) tract, and the number of C residues in the poly(C) tract, as well as the possible small number of U and A residues that could be present in this poly(C) tract. Several nucleotides (positions 454, 460, and 461) are reported as C/U mixtures, consistent with the expected quasispecies nature of the genome. All other positions could be assigned to a single nucleotide, but among these there were undoubtedly mixtures at multiple positions. Similar methods (with the exception of RACE) were utilized to determine the sequences of five other viruses (O/TAW/2/99bov; O/Tibet/CHA/99; O/SKR/2000; O/SAR/19/2000; O/UKG/35/2001). Since terminal transferase tailing was not employed, the first 19 bases of the S-fragment for these five sequences are dictated by the primer, although it should be noted that these bases are highly conserved among all seven serotypes of FMDV (Harris, 1980), so the 19 bases reported in Fig. 1a are highly likely to reflect the authentic genome sequences. In addition, primer-determined sequences account for the last base of the Sfragment for O/TAW/2/99bov, O/Tibet/CHA/99, O/SKR/2000, and O/SAR/19/2000, and the last 10 bases of the S-fragment of O/UKG/35/2001. In addition, for all viruses except the O/TAW/2/99 and O/Tibet/CHA/99, the first 22 bases of the L-fragment of the genome are from primer-encoded sequences, although, once again, due to conservation among viruses, these are also likely to be identical to the authentic genome, given the high degree of conservation observed elsewhere. Among these five genomes, unambiguous assignments (with the caveats listed above) could be made for all but one base in the O/SAR/19/2000 sequence (an A/G mix at 2182) and one base in the O/SKR/2000 sequence (a C/G mix at position 4009). Interestingly, our O/SKR/2000 sequence data showed numerous differences with a recently deposited sequence (GenBank accession no. AF377945) of the L-fragment of O/SKR/2000 (2.76% different; see below). 5' and 3' untranslated regions (UTR): The 5' and 3'UTRs of the FMDV genome contains a number of distinct elements that have been identified based on their predicted secondary structure and, in some cases, biological functions. As expected, the vast majority of the sequence differences are in regions between the elements, or do not affect the predicted 181

secondary structure (data not shown). Of interest is the finding that the 3’UTR of the SKR sequence deposited in GenBank has five deletions that were not present in the genomes of the seven other PanAsia isolates (see also concluding remarks). Polyprotein: As with the above analyses of the UTRs, the analyses of the predicted polyprotein sequences of the eight isolates revealed a high level of identity. In all of the coding regions the conservation at the protein level is greater than the nucleotide level, indicating that silent codon changes were greatly preferred (data not shown). The numbers of amino acid differences in each mature polypeptide when comparing O/UKG/35/2001 with the two most closely related isolates, O/SAR/19/2000 and O/JPN/2000, are shown in Table 2. Of interest is the fact that there is not a single deletion or insertion within the polyprotein, although the sequence of O/SKR/2000gb contains a deletion closely followed by an insertion in the coding region for VP2 (1B), which is most likely explained by a sequencing error (producing two new codons. Complete genome analyses: The complete genome sequences of the eight PanAsia isolates were aligned with those of other FMD viruses using Clustal X. A program written by one of us (NJK) was then used to compare O/UKG/35/2001 to each of the others employing a "scanning window"; the percentage nucleotide sequence identity measured over 300 bases between pairs of viruses is plotted on the Y-axis as the window of comparison was moved in 15 nucleotide steps. The divisions on the ordinate represent the passage by the comparisonwindow of 300 nucleotides starting with positions 1-300, and terminating at approximately 7947-8247. Fig. 1a shows all the type O viruses compared to O/UKG/35/2001. All the PanAsia virus sequences are very closely related across the whole genome with O/SAR/19/2000 being the most closely related and O/SKR/2000gb the most different. The other two type O viruses are clearly distinct. Fig. 1b shows a scanning window analysis comparing O/UKG/35/2001 to viruses of other serotypes. This analysis reveals the clear differences among all the coding regions, with the greatest differences detected in the region coding for the outer capsid polypeptides. Interestingly, complete genome analyses revealed that only minor changes in the genome of FMDV can dramatically alter virulence in animals, presumably by altering functional changes through mechanisms that are not readily apparent. Specifically, we found that there were only a small number of differences in sequences of a virus recovered from a probang sample (O/TAW/2/99) that was avirulent in cattle and poorly infectious in pigs (JMP and PWM, unpublished data) and a derivative obtained by two passages in pigs (once by inoculation, and a second time by contact) and a single passage in a cow that presented a severe disease (O/TAW/2/99bov; JMP and PWM, unpublished data). These differences consisted of eight nucleotides (five amino acids) in the polyprotein-encoding region, one nucleotide in the pseudoknot region, one nucleotide in the IRES region of the 5' UTR and no changes detected in the S-fragment or 3' UTR regions. Using the complete genome sequence data, the relationships between the eight PanAsia viruses were determined using phylogenetic algorithms (Fig. 2). In general, the year of isolation correlates well with genetic relationship, and these analyses reveal that O/UKG/35/2001 is most closely related to O/SAR/19/2000, consistent with earlier analyses with data from the VP1-encoding region of the genome (Knowles et al., 2001b). Although powerful, these analyses can only be used to determine that the UK virus is the closest relative of the SAR virus. They cannot be used to imply the mechanism of introduction into the United Kingdom. Thus, these data are consistent with either a common source of the 182

outbreaks in these two regions or indicate that South Africa is the source of the strain that caused the outbreak in the UK. Concluding comments: Taken together, analyses of the complete genome sequence data reveals a remarkable conservation among the PanAsia virus isolates, which appear to be much more stable than other type O viruses circulating in Asia at during the same time period (Knowles et al., 2001a; Samuel & Knowles, 2001). Not only are the PanAsia genomes devoid of specific hot-spots for change, the scanning analyses reveal that there have not been any recent intra- or intertypic recombination of the PanAsia viruses. These data are particularly interesting in terms of the fact that the PanAsia viruses have been so effective in their spread across Asia, and appear to have, in some areas, replaced enzootic strains of FMDV O (N.J. Knowles and A.R. Samuel, unpublished observations). Comparisons of the sequence data of O/SKR/2000, an isolate from early May, 2000 in the Kyunggi province of South Korea revealed a large number of differences (2.76%) with a recently deposited sequence of the L-fragment of another O/SKR/2000 sequence (GenBank accession no. AF377945). This large number of differences strongly suggests that multiple genetic lineages of FMDV were responsible for the outbreaks reported in South Korea in 2000, a particularly intriguing hypothesis given the reported re-introduction of FMD in 2002. Acknowledgements We would like to thank Toru Kanno, National Institute for Animal Health, National Institute of Animal Health, Department of Exotic Disease, Kodaira, Tokyo, Japan for providing prepublication sequence data on O/JPN/2000. We also thank Juan Lubroth, FADDL, PIADC, Greenport, NY, USA for supplying O/SKR/2000, and Wilna Vosloo, OVI, Onderstepoort, South Africa, for supplying O/SAR/19/2000 and Nigel Ferris, IAH-Pirbright, UK for supplying O/UKG/35/2001. The valuable technical assistance of Jason Gregory is acknowledged. This work was partially supported by a grant from the National Research Initiative Competitive Grants program of USDA/CSREES (Grant #99-35204-7949), the Agricultural Research Service of the USDA (CRIS Project #1940-32000-035-00D), and the UK Department of the Environment, Food, and Rural Affairs (DEFRA) Grant no. 2919. References Efron, B., Halloran, E. & Holmes, S. (1996). Bootstrap confidence levels for phylogenetic trees. Proc Natl Acad Sci U S A 93, 13429-34. Frohman, M. A., Dush, M. K. & Martin, G. R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A 85, 8998-9002. Harris, T. J. (1980). Comparison of the nucleotide sequence at the 5' end of RNAs from nine aphthoviruses, including representatives of the Seven serotypes. J Virol 36, 659-664. Knowles, N.J., Samuel, A.R., Davies, P.R., Kitching, R.P., Venkataramanan, R., Kanno, T., Scherbakov, A.V., Drygin, V.V., Zhao, Q. Z. and Xie, Q.-G. (2000). Emergence of a pandemic strain of foot-and-mouth disease virus serotype O. 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, Borovets, Bulgaria, 5-8 September, 2000. Rome: FAO, Appendix 1: 20-31.

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Knowles, N. J., Davies, P. R., Henry, T., O'Donnell, V., Pacheco, J. M. & Mason, P. W. (2001a). Emergence in Asia of Foot-and-Mouth Disease Viruses with Altered Host Range: Characterization of Alterations in the 3A Protein. J Virol 75, 1551-1556. Knowles, N. J., Samuel, A. R., Davies, P. R., Kitching, R. P. & Donaldson, A. I. (2001b). Outbreak of foot-and-mouth disease virus serotype O in the UK caused by a pandemic strain. Vet Rec 148, 258-9. Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357-8. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406-425. Samuel, A. R. & Knowles, N. J. (2001). Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes). J Gen Virol 82, 609-21. Tellier, R., Bukh, J., Emerson, S. U. & Purcell, R. H. (1996). Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon. Proc Natl Acad Sci U S A 93, 4370-3. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-82.

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Table 1. Oligonucleotide primers used for RT-PCR and sequencing. Number P614 P599 P55 P606

purposea/senseb PCR/sense SEQ/sense SEQ/antisense PCR/antisense

P609 P600 P166 P4 P603 P548 P607e P536 P538 P539 P540 P541 P498f P617 P514 P500g P618 P534 P502 P503 P512 P515 P367 P517 P530 P15

PCR/antisense PCR/sense SEQ/sense SEQ/antisense PCR/sense SEQ/antisense PCR/sense SEQ/sense SEQ/sense SEQ/sense SEQ/sense SEQ/sense PCR/sense PCR/sense SEQ/sense PCR/antisense SEQ/sense SEQ/sense SEQ/sense SEQ/antisense SEQ/sense SEQ/sense SEQ/sense SEQ/sense SEQ/antisense PCR/antisense

a b c d e f g

sequencec tatcccgggttcTTGAAAGGGGGCGTTAGGG CACYYTCCGCCTACTTGGTCGT GCCCAAGACAGCGCTAACG cgtgcctaGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGTGAAAGGTGG tatcctaGGGGGGGGGGGGGGGGT CCAAGTTTTTACCGCCTTTCCCGG GGAGGCCTTGTACAAACACGATC CCTCTAGACCAGGAAAGACC TGGCCGCGGGAACTCCTCCTTG CCTGAAGGGCATCCTTAGCC ACCTCCRACGGGTGGTACGC YRACGGGTGGTACGCGAT GGRGTSACKTACGGGTAC CTMCCRACTGACCACAAAGG YAAGGTSTATGCCAACAT ATYAAYCTSCACTTCATGTT AATTACACATGGCAAGGCCGACGG CCATCAAAGCCACTCGGG CCAACCCCGGGCCTTTCTTCTTCTCTGACG GACATGTCCTCCTGCATCTG TGATTCTTGCCATCCGCG AGCGTGTTTGAAGAGCGG CAGATATCAATTCCTTCCCAAAAGGCT cccggatccaTATGGTCCTTCAGCTTGTGG gagagatctggacatatgCCGGTCCTCTAGAGCGTCAAA ACCCCCGTCGTCGGCGTGATCAA TGCTGCTGACTACGCGTC GTGATGGCCTCGAAGACCCTCG GCAGGTAAAGTGATCTGTAGCTTGG ggcggccgcTTTTTTTTTTTTTTT

positiond S (1-19) S (208-229) S (227-245) S (359-378) S (368-378) PK (377-400) cre (606-628) IRES (679-698) IRES (785-806) IRES (937-956) L (1554-1573) L (1559-1576) VP2 (2040-2057) VP2 (2196-2215) VP2 (2540-2257) VP3 (2916-2935) VP3 (3167-3190) VP1 (3763-3780) 2B (3940-3969) 2B 4008-4027) 2C (4477-4494) 2C (4635-4654) 3A (5364-5390) 3A (5814–5833) 3B (5836-5856) 3C (6375-6397) 3D (6908-6925) 3D (7881-7902) 3D (8034-8058) poly(A)

PCR indicates primers used for PCR (also RT, in the case of antisense primers). SEQ indicates primers used for sequencing only. Not all primers were utilized for all genomes. Primer’s sense on the genome. sequence of the oligonucleotide, 5’ to 3’, standard abbreviations, lowercase bases do not match genomes but were needed for cDNA manipulations for other purposes. Functional element followed by nucleotide numbers, in parentheses) for the PanAsia genome numbered as shown in Fig.1. Also known as L-463F (Samuel & Knowles, 2001). Also known as O-1C564 (Samuel & Knowles, 2001). Also known as NK61 (Samuel & Knowles, 2001).

185

Table 2. Number of amino acid substitutions in the polyprotein of O/UKG/35/2001 compared to O/SAR/19/2000 and O/JPN/2000.

L

O/UKG/35/2001 vs O/SAR/19/2000 2

O/UKG/35/2001 vs O/JPN/2000 3

VP4

0

0

VP2

0

2

VP3

0

1

VP1

1

1

2A

0

1

2B

0

0

2C

0

5

3A

1

1

3B x 3

1

1

3C

1

0

3D

1

3

186

100 90 80 70 60 50

O/TAW/2/99

O/TAW/2/99bov

O/Tibet/CHA/99

O/SKR/2000

O/SKR/2000gb

O/JPN/2000

O/SAR/19/2000

O/Yunlin/TAW/97

O1/Kaufbeuren/FRG/66

Fig. 1a. Genome scanning comparisons of genomic sequence data. The origins of the sequences not derived from this work are as follows: O1/Kaufbeuren/FRG/66 [M35873, X00871]; O/Yunlin/TAW/97 [AF308157]; O/SKR/2000gb [AF377945]. The S-fragment of O/SKR/2000gb was not available.

7920

7590

7260

6930

6600

6270

5940

5610

5280

4950

4620

4290

3960

3630

3300

2970

2640

2310

1980

1650

1320

990

660

330

1

40

100 90 80 70 60 50

A12/UK/119/32

A22/Azerbaijan/USSR/65

C1/Santa Pau/SPA/70

C3/ARG/85

Asia1/IND/63/72

SAT2/KEN/3/57

7920

7590

7260

6930

6600

6270

5940

5610

5280

4950

4620

4290

3960

3630

3300

2970

2640

2310

1980

1650

1320

990

660

330

1

40

Fig. 1b. Genome scanning comparisons of genomic sequence data. The origins of the sequences not derived from this work are as follows: A12/UK/119/32 [L11360; M10975]; A22/Azerbaijan/USSR/65 [X74811, X74812]; C1/Santa Pau/SPA/70 [AJ133357]; C3/ARG/85 [AJ007347]; Asia1/IND/63/72 [X83209, Y17973, AF227965, Y09949, AF207524, AF088224, AF207525, AF207526, AF088223, AF207520]; SAT2/KEN/3/57 [AJ251473]. The S-fragment of SAT2/KEN/3/57 was not available. Similarly, the 3’ UTR was not available for Asia1/IND/63/72.

SAT2/KEN/3/57 Asia1/IND/63/72 289

A12/UK/119/32

992

A22/Azerbaijan/USSR/65

698

C3/ARG/85

1000

C1/Santa Pau/SPA/70 O/Yunlin/TAW/97

1000

1000

O1/Kaufbeuren/FRG/66

O1/Campos/BRA/58

423

O/SKR/2000gb O/SKR/2000

1000

1000

O/JPN/2000 1000

O/SAR/19/2000

1000 5%

995

O/UKG/35/2001

O/Tibet/CHA/99 775

O/TAW/2/99

1000

O/TAW/2/99bov

Fig. 2. Neighbor-joining tree (outgroup-rooted on SAT2/KEN/3/57) showing relationships among the PanAsia viruses and other FMDVstrains for which complete (or nearly complete) genome sequence data is available. Relationships were based on a comparison of the complete genome see Methods). Database accession numbers for the sequences not reported in this study are shown in the legend to Fig.1, except for O1/Campos/BRA/58 [AJ320488].

Appendix 23

Characterisation of monoclonal antibodies against foot-and-mouth disease vaccine strain C1 Oberbayern and their reactivity with field isolates N. Aggarwal*, S. J. Cox, R. J. Statham, P. V. Barnett International Vaccine Bank for FMD, Institute for Animal Health, Pirbright Laboratory Pirbright, Surrey GU24 0NF, U.K.

Introduction The 2001 epidemic of foot-and-mouth disease (FMD) in the U.K. and mainland Europe was a timely reminder of the devastating consequences of this disease. Although the epidemic was brought under control by slaughter of all infected/in-contact animals and traditional zoosanitary measures, vaccination was considered as an option, at-least in the early stages of the epidemic. The International Vaccine Bank (IVB) for FMD at Pirbright holds quantities of seven strains of inactivated FMD virus (FMDV) antigen over liquid Nitrogen, ready for immediate formulation into vaccine if required. These will protect against the viruses of serotypes that are most likely to threaten the livestock of the UK or other IVB member countries (i.e. serotypes A, O, C and Asia 1). The antigenicity of FMDV can change because of frequent mutations in it's genome, and thus evade the immunity provided by vaccines. It is therefore necessary to monitor the field isolates for their relatedness to vaccine strains to ensure that currently available vaccine will provide protection against any new isolate. Currently, an ELISA based on the use of polyclonal hyperimmune serum is used by the WRL for FMD for the antigenic profiling of field isolates. However, the inherent variation in the properties of polyclonal serum batches complicates the interpretation of results. An antigen profiling ELISA, based on monoclonal antibodies (MAbs) has also been described (Samuel et. al., 1991). We aim to refine this approach in order to have a more reliable ELISA that could ultimately alleviate the need for in-vivo testing of selected vaccine strains. As a first step towards this goal, we have started production and characterisation of monoclonal antibodies to cover the entire antigenic spectrum of vaccine strains held by the IVB. In this report results are presented of studies that have been done with the C1 Oberbayern vaccine strain of FMDV. Materials and Methods Cells and Viruses All the strains of FMDV were procured from WRL for FMD at Pirbright and were grown either in BHK-21 or IB-RS2 cells. Monoclonal antibody production Six-week old BALB/c mice were immunised by the sub-cutaneous route with 10 µg of C1 Oberbayern antigen that was received from the IVB. Immunisation was repeated at day 21 and 35 after first inoculation. Three days after the last immunisation, spleens were aseptically 190

removed and fusions performed essentially as described by Aggarwal and Holmes (2000). The resulting hybridomas were screened by an antigen capture ELISA (see below) and cloned twice by limiting dilution before amplification. Isotypes of the MAbs were determined using a commercial kit (Sigma). Virus neutralisation test MAbs were tested for their ability to neutralise the homologous virus in a microneutralisation test according to the method of Golding et al., 1976. Western Blotting This was essentially done as described by Towbin et. al. (1979) with some modifications. C1 Oberbayern was grown in BHK-21 cells, inactivated with binary ethylenimine and purified by the sucrose density gradient method. Varying concentrations of virus were mixed with Laemmli sample buffer (Bio Rad) and disrupted by boiling at 100°C for 5 mins. Polypeptides were separated by 14% Tris-Tricine gels and the reactivity of the MAbs with FMDV polypeptides detected immunologically. ELISA a) Hybridomas were screened using an antigen capture ELISA as described previously (Aktas and Samuel, 2000). For this, sucrose density gradient purified C1 Oberbayern antigen was used at a concentration of 1µg/ml. b) A similar ELISA was used to determine the reactivity of MAbs with trypsin treated virus. For this, 100 µg of sucrose density gradient purified C1 Oberbayern antigen was treated with 50 µl of trypsin (stock solution 2 mg/ml) as described by Crowther et. al., 1993. c) An antigen profiling ELISA, as described by Aktas and Samuel, 2000, was used to determine the reactivity of five field isolates with each of the MAbs. Results Five MAbs were produced from two fusions and characterised. Isotypes of these MAbs are shown in Table 1. All of these MAbs except E11.A9 reacted with polypeptides in Western blotting (Table 1) indicating that they recognised a linear epitope. Conversely, E11.A9 recognised a confirmational epitope. Two MAbs, E11.A9 and E2.B4 were shown to be neutralizing. E2.B4, A4.D12 and C2.A3 recognised trypsin sensitive epitopes (Table 1). Field isolate reactivity patterns against the five MAbs are shown in Figure 1. Discussion The antigen profiling ELISA results indicate that the five MAbs recognise four different epitopes. MAbs A4.D12 and C2.A3 appear to recognise an identical epitope where as the rest of the MAbs recognise individual epitopes. We are currently defining the critical residues for these MAbs. Antigen profiling studies suggest that all the five field isolates tested share some epitopes with the vaccine strain C1 Oberbayern. However, none of the field isolates tested showed an identical reactivity as the vaccine strain. In any outbreak of FMD, if vaccination is to be implemented as a control policy, one main issue is to find a vaccine strain that is antigenically similar to the field isolate in question. Our observations in this report and those of various previous workers (Barteling et. al., 1986; Brocchi et. al., 1998; Samuel et. al., 2000) indicate that it is possible to measure antigenic relatedness between vaccine strains and field isolates in an ELISA based on MAbs. The panel of C MAbs used in this study was very limited but it is hoped that when complete panels of MAbs against this and other vaccine strains are available, it will be possible to define the 191

antigenically important homologous epitopes between field and vaccine strains. However, the significance of each antigenic epitope in relation to protection against disease still remains to be determined. Once more complete information is available, it will help to define epitopes which must be matched to identify vaccine strain that will offer greatest protection against a field isolate. Given the scarcity of such data at present the use of MAb based antigen profiling ELISAs for vaccine strain selection as a replacement for more traditional methods, reducing the use of animals, is a distant prospect. The importance of continually producing more MAbs and delineating the significance of each epitope in protection against disease and analysing such information in conjunction with data generated from polyclonal based serological tests is essential to make further progress. Given the amount of work required to produce and characterise MAbs, it would be highly desirable for different research groups to co-operate in these studies to speed up the work and to avoid duplication of the efforts. Acknowledgment We would like to thank Dr. N. Ferris (IAH, Pirbright) for providing rabbit and guinea pig sera used in capture ELISAs. References

Aggarwal, N. and Holmes, M.A. (2000) “Production and characterization of two monoclonal antibodies reactive with equine IgG Fc receptors.” Vet. Immunol. Immunopathol. 73:63-71 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), 744-753 Barteling, S.J., Boerke, J. and Woortmeyer, R. (1986) "Use of MAbs for surface scanning of different field and vaccine strains of A-type FMDV new approach of subtype identification?" Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm. Control of FMD, Madrid, Spain. 32-34 Brocchi, E., Gamba, D., Bugnetti, M. and De Simone, F. (1998) "Monoclonal antibody profiling of recent FMD type A field viruses and comparison with reference virus strains." Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm. Control of FMD, Pirbright, U.K. 56-61 Crowther, J.R., Farias, S., Carpenter, W.C. and Samuel, A.R. (1993) "Identification of a fifth neutralizable site on type O FMD virus following characterization of single and quintuple monoclonal antibody escape mutants." J. Gen. Virol. 74: 1547-1553 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." Research in Veterinary Sciences 20: 142-147 Samuel, A.R., Knowles, N.J., Samuel, G.D. and Crowther, J.R. (1991) "Evaluation of a trapping ELISA for the differentiation of FMDV using MAbs." Biologicals 19: 299-310 Samuel, A.R., Turner, L.S. and Knowles, N.J. (2000) "Antigenic and genetic characterisation of FMD type O viruses from the far East." Rpt. Sess. Res. Grp. Stand. Tech. Comm. Eur. Comm. Control of FMD, Borovets, Bulgaria. 58-66 Towbin, H., Staehelin, T. and Gordon, J. (1979) "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA. 76: 4350-4354.

192

Table 1: Characteristics of C1 Oberbayern MAbs MAbs

Isotype

Reactivity in

Neutralising

Trypsin

Western blota

activityb

sensitive

E11.A9

IgG2a

-

+

No

A4.D12

IgG1

+

-

Yes

D7.G2

IgG1

+

-

No

C2.A3

IgG2a

+

-

Yes

E2.B4

IgG2a

+

+

Yes

a – No reactivity; + reactivity b – No activity; + activity

C1 Ober C Phil 9/94 C Phil 4/90 C Nep 1/94 C Bhu 2/91 C Ban 1/92

E11.A9

A4.D12

C2.A3

D7.G2

E2.B4

< 10 % reactivity 10-50 % reactivity 50 – 100 % reactivity Figure 1: Reactivity of vaccine strain C1 Oberbayern and field isolates with MAbs raised against IVB C1 Oberbayern vaccine strain

193

Appendix 24

Monoclonal Antibodies against FMDV type Asia 1: preliminary characterization and potential use in diagnostic assays Grazioli S., Fallacara F. and Brocchi E. Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy

Introduction Foot-and-Mouth Disease (FMD) is economically the most important disease of domestic livestock. Outbreaks with devastating economic consequences still occur; the 2001 FMD crisis in the UK was a traumatic experience. The most serious source of the risk of entry of FMD is illegal import of animals or animal products from countries where FMD is endemic. This aspect, associated with increasing immigration from third countries, induces the need that European countries are prepared to recognise and control any of the FMD viruses currently circulating in the world. FMD monitoring systems should ensure that outbreaks are detected at an early stage and that subclinical persistence of infection is promptly recognized. To satisfy these requirements there is the need for updated and new diagnostic tools, which cover the complexity of the FMD diagnostic task. The most significant innovations of the last decade in FMD diagnosis have been the introduction of RT-PCR for sensitive detection of FMDV presence in biological samples and the serological distinction between vaccinated and infected animals, based on the detection of antibodies to viral non structural proteins. Minor advances have been reported concerning reagents and methods for the detection and characterization of FMDV antigens and antibodies. These assays are traditionally performed using polyclonal immune sera, although it has been proven that immunoassays for many infectious diseases, including FMD, can greatly benefit from the use of monoclonal antibodies (MAbs). Furthermore, MAbs represent valuable tools for studying the antigenicity of viruses; this application is particularly important for FMD viruses, which show frequent variability and may thus evade immunity provided by vaccines. Selection of appropriate vaccine strains, that considering their antigenic profile are expected to induce protection against circulating field viruses, is crucial in FMD control based on vaccination strategy. Several reports described the antigenic structure of FMDV and compared the antigenic profile of FMDV isolates using MAbs. Also in diagnostic field, MAbs-based ELISAs for both antigen and antibody detection are being used in our laboratory for almost 20 years on a routine basis (Brocchi et al., 1986, Brocchi et al., 1990). However, most MAbs to FMDV used to these purposes were raised against old reference strains of type O, A, C, which are no more representative of viruses currently circulating. Then, one main objective of our laboratory is updating the MAbs collection, taking into consideration either other FMDV types or isolates showing significant antigenic variation. Within this programme, we have produced a new panel of MAbs, raised towards FMDV type Asia 1. Here we describe their partial characterization and a preliminary evaluation of their potential as diagnostic reagents in ELISA assays for both antigen and antibodies detection.

194

Materials and Methods Viruses FMDV isolates used were received from the World Reference Laboratory, Pirbright, UK; they are listed in table 1. Viruses were propagated in IBRS-2 cells monolayers and harvested when cytopathic effect was maximum. The strain used for mice immunisation was inactivated with binary ethylenimine and purified by ultracentrifugation through a 25% (w/w) sucrose cushion. Monoclonal antibodies (MAbs) Two Balb/C mice were immunised with 2-3 doses respectively of 20 µg each of purified FMDV Asia 1, strain Nepal 29/97, at intervals of one month. Cells fusion and cloning of positive hybridomas were performed following procedures standardised in our laboratory (Brocchi et al. 1993). Selection was based on results of a trapping ELISA against the homologous virus strain. Ascitic fluids of selected hybridomas were induced in mice. MAbs were purified from ascites by ammonium sulphate precipitation and conjugated with peroxidase using a modification of the method described by Tjissen (1985). Isotype of the MAbs was determined using a commercial ELISA (Boehringer Mannheim isotyping kit). Trapping ELISA The assay used for both screening hybridomas and antigen characterisation was a trapping ELISA (Samuel et al., 1991). Essentially, each MAb was reacted with pre-titrated concentrations of viruses (supernatant of infected cells) which had been trapped using a polyclonal rabbit Asia 1 antiserum. End-point titres of MAbs were determined as the reciprocal dilution that gave optical density readings higher than 0.2, while the reactivity of mutants and field isolates with each MAb was expressed as a percentage of the corresponding reaction with the parental strain, assumed to be 100%. Sandwich ELISA MAbs-based sandwich ELISA for antigen detection has been carried out as described previously (Brocchi et al., 1993). Solid phase competitive ELISA The principle and the procedure of the MAbs-based competitive ELISA, firstly described for the quantification of antibodies specific to the European subtypes O1, A5 and C1 of FMDV (Brocchi et al., 1990), and later applied to other infectious diseases (Brocchi et al., 1993, Brocchi et al., 1995) was adapted for the detection of FMDV type Asia 1 specific antibodies. The MAb selected for this application was 5E10, used either as antigen catching or as competing, peroxidase-conjugated antibody. Virus Neutralization (VN) test VN test was carried out in microplates against 100 TCID50 of the homologous FMDV Asia 1 and IBRS-2 as substrate. The final dilution required to neutralise 50% of the inoculated cultures was calculated. MAb neutralization-resistant (MAR) mutants The selection of mutants resistant to neutralization by MAbs was carried out as described previously (Borrego et al. 2002).

195

Western Blot analysis Hybridoma supernatants were assayed against the purified homologous FMDV, resolved by SDSpolyacrilamide gel electrophoresis 12% and transferred to nitrocellulose filters, following standard procedures (Harlow & Lane, 1988, Towbin et al., 1979). Results and Discussion Characterization of monoclonal antibodies Twenty-four MAbs were raised against FMDV type Asia 1, strain Nepal 29/97, in two fusion experiments. Table 1 outlines results of a preliminary characterisation, including the following details: immunoglobulin class, ability to neutralize viral infectivity, identification of linear epitopes in viral proteins, specificity with respect to the homologous type Asia 1 and the heterologous types O, A and C, profile of reactivity with field isolates; analysis of these results lead to the classification of the MAbs in 6 separate groups. Concerning immunoglobulin class, the MAbs panel include 21 IgG1, 1 IgG2a and 2 IgM. Results of VN test revealed that 10 MAbs neutralized viral infectivity, showing variable neutralising titres, whilst 24 MAbs were not neutralising. According the different pattern of reactivity shown, the 14 non-neutralizing MAbs could be clustered in three separate subgroups: group 4, including two type-specific MAbs, group 5, including 11 MAbs with different degree of cross-reactivity with two or even three heterologous FMDV types examined, and group 6 including one single MAb, directed against a linear epitope in VP2 (as demonstrated by reactivity in Western Blot), trypsin-sensitive (not shown) and shared by the three heterologous FMDV types examined. In contrast, all the neutralizing MAbs were type-specific; end-point titres of hybridoma supernatants in trapping ELISA did not necessarily correlate with VN titres, since several factors may influence this aspect, like antibody concentration, antibody affinity or mechanism of neutralisation. Results of Western blot test indicated that three of the neutralising MAbs react with VP1; as virus trypsin treatment prevented this reactivity, the target site is likely the linear trypsin-sensitive sequence corresponding to the G-H loop (site A) in VP1. A further and accurate way to distinguish neutralizing MAbs and to map the relevant sites is the selection and the characterisation of MAb neutralization resistant mutants. So far, 3 of the neutralizing MAbs were used to select escape mutants. The pattern of reactivity of each mutant with the 10 neutralizing MAbs in a trapping ELISA proved evidence that these MAbs identify at least 3 different antigenic sites (figure 1 and table 1). One site, already distinguishable on the basis of its location in a linear sequence of VP1, was defined by the 3 MAbs 5C12, 4E10, 4F10: mutations induced in any of the relevant escape mutants annulled the reaction of the three MAbs, but did not alter the reactivity of the other seven MAbs. Another independent site includes three additional MAbs: in fact, the mutant resistant to MAb 5E10 lost reactivity not only with 5E10 itself, but also with two further MAbs, 3C6 and 1F10, while maintaining full reactivity with other MAbs. Finally, the remaining four MAbs were not affected by mutations in the two previous sites: this reactivity indicates that they correspond to a distinct site.

196

Figure 1. ELISA reactivity of FMDV Asia 1 MAR mutants with the panel of neutralizing MAbs Antigenic sites defined by neutralising MAbs

4D8

5G4

2G1

2C3

Site 3 1F10

3C6

5E10

Site 2 4F10

4E10

5C12

Site 1

MAR mutants

5C12-a 5C12-b 5C12-c 5C12-d 4F10-a 4F10-b 5E10-a parental

MAR mutants are named according to the MAb used to select them. Reactivity is expressed as percentage of binding with respect to the parental strain, FMDV Asia Nepal 29/97, assumed to be 100%: white cells, no reactivity; light grey, 25% reactivity; black, 100% reactivity.

The level of conservation of the sites identified by the MAbs was analysed by testing their reactivity in a trapping ELISA with 11 isolates representative of a thirty-year period, being the oldest virus of this panel originated in 1973 (Asia 1 Turkey 15/73) and the most recent one in the year 2000 (Asia 1 Greece 1/2000). The antigenic profiles (table 1) show that non-neutralizing MAbs displayed a broad reactivity with the isolates; the antigenic stability of the relevant sites was expected, mainly concerning sites 5 and 6, which were common even to heterologous FMDV types. Also sites 1 and 2 involved in neutralisation were proven to be conserved in the majority of isolates, whilst the neutralising site 3 was shown to be almost exclusive for the homologous strain, being mutated in all other isolates. With respect to this particular panel of MAbs, FMDV Asia 1 isolates showed limited antigenic variation, with the exception of one isolate, Cambodia 3/93, which was not recognised by any of the MAbs specific for the Asia type. Evaluation of MAbs performances in diagnostic assays Preliminary studies to investigate the potential of the characterised MAbs as diagnostic reagents were undertaken.

197

Figure 2. Sandwich ELISA for antigen detection: results obtained with representative pairs of catching and conjugated MAbs catch. Mab - 4F10 type specific conj. Mab - 5F10 cross reactive

2,5 2 1,5

Asia 1 Asia 1 12S O A C

a)

1 0,5

3 Optical Density

Optical Density

3

0

catch. Mab - 2A4 cross reactive conj. Mab - 5F10 cross reactive

2,5 2 1,5 1

b)

0,5 0

1/5

1/15

1/45

1/135

1/405 1/1215

1/5

1/15

Antigen dilution

catch. Mab - 4F10 type specific conj. Mab - 5F10 cross reactive

2,5 2 1,5

Asia 1 Asia 1 12S O A C

c)

1 0,5

3

0 1/5

1/15

1/45

1/135

1/45

1/135

1/405 1/1215

Antigen dilution

Optical Density

Optical Density

3

Asia 1 Asia 1 12S O A C

catch. Mab - 2A4 cross reactive conj. Mab - 5E10 type specific

2,5 2 1,5 1

d)

Asia 1 Asia 1 12S O A C

0,5 0

1/405 1/1215

1/5

Antigen dilution

1/15

1/45

1/135

1/405 1/1215

Antigen dilution

Titration curves of supernatants of FMDV infected cultures Asia 1 : strain Nepal 29/97 homologous to MAbs, infectious titre 107.9 TCID/ml Asia 1 12S : same as before, treated at 56°C for 30 min. O : O1 Switzerland 1965; A : A5 Italy 1962; C : C1 Noville

For antigen detection, performances of a sandwich ELISA carried out with several combinations of different MAbs, used as catching and conjugated antibody, were evaluated using supernatants of infected cells as source of antigen: titration curves for either the homologous virus type, Asia1, and the heterologous types O, A and C, were performed; the reactivity with 12 S viral particles, obtained by heat treatment (56°C, 30 min.) was analysed also. A strong signal was observed with most MAbs, with a detectability limit of 105 TCID50/ml, expected from immunoassays which do not benefit from a preliminary amplification step (figure 2). In particular, when combinations of two type-specific MAbs were used as catching and conjugated antibody, only the homologous type was detected (figure 2a), whilst combinations of two crossreactive MAbs did recognise the homologous as well as the heterologous types with similar profile and sensitivity (figure 2b). This result offers the possibility of choosing a universal assay for FMDV detection or a type-specific one, depending on requirements. Furthermore, figures 2c and 2d report antigen titration curves obtained by combinations of one type-specific with one cross-reactive MAb: in both cases the reactivity was specific for the homologous type only: then, virus typing can be achieved by combining a universal catcher with a type-specific conjugated MAb or vice versa. This result further extends the possibility to modulate and adapt antigen detection assays to different needs.

198

All MAbs pairs reacted with 12S particles, showing a slightly lower signal in the case of neutralizing MAbs and slightly higher reactivity when non-neutralizing MAbs were used.

percentage inhibition

100

Neg

Cut off

Weak

Strong

75 50

Figure 3. Evaluation of a monoclonal antibody-based competitive ELISA (SPCE) for the assessment of antibody to FMDV type Asia 1 (catching and conjugated MAb 5E10) A) Results obtained with PHASE XVII sera (candidate reference sera)

25 0 1/10

1/30

1/90

serum dilution

1/270

B) Frequency distribution of percentage inhibition observed with negative sera (n° 107 cattle, n° 74 pigs)

For antibody assessment, a solid phase competitive ELISA was designed, determining the capability of sera to block the binding of a specific MAb to the virus. The virus was preliminarily captured onto microplates wells by the catching MAb 5E10, that allowed the use of a non-purified preparation as source of virus. Competition between test sera and the peroxidase-conjugated MAb 5E10 was then evaluated. Epitopes target of neutralizing, type specific MAbs are expected to be more immunogenic, then the selection of 5E10, satisfying these requirements, as competing antibody should direct the detection of specific sets of serum antibodies. Sensitivity and specificity of this experimental assay were preliminary evaluated by testing a panel of field negative sera from cattle and pigs and the Asia 1 specific sera of phase XVII exercise, aimed at selecting candidate reference sera, and including a cut-off, a weak positive and a strong positive serum, together with a negative one. Results, obtained using pre-determined optimal concentration 199

of antigen and competing MAb, are shown in figure 3, expressed as percentage inhibition produced by tested sera. In particular, figure 3A shows titration curves of the 4 candidate reference sera: the 4 sera were distinguishable each other and gave, at the screening dilution 1/10, percentage inhibition coherently distributed along the whole scale: the negative serum presented inhibition close to 0%, the cut-off, weak and strong positive sera scored inhibition of 35%, 65% and 92 % respectively. Figure 3B shows frequency distribution of percentage inhibition produced by negative sera at the screening dilution 1/10: pig sera displayed a normal distribution, characterised by a restricted percentage range (0-40%), with mode in the class 11-20% and none out of 74 samples examined over 40%; differently, cattle sera showed a wider distribution, extended up to 80% inhibition in exceptional cases, although maintaining the mode in the class 11-20%. Selecting 60% inhibition as threshold, the test was able to detect weak and strong positive reference sera, but missed the cut-off serum; with regard to specificity, the assay presented approximately 3% of false-positive results (3 out of 107) in the cattle population but none in the pig species. Although assay standardisation is still in course, the SPCE for the detection of Asia 1 specific antibodies proved to work in principle. Several assay parameters will be evaluated to improve specificity and sensitivity, in particular: modulation of antigen/antibody concentration, more stringent buffer conditions and the assessment of other MAbs and competitive designs, including washing steps and changing from a solid phase to a liquid phase competitive ELISA. Conclusion Within the programme of developing/updating MAbs suited for the study and the diagnosis of recent FMDV isolates, we have produced a panel of 24 MAbs against FMDV type Asia 1. Their preliminary characterisation demonstrated that 10 type specific MAbs identify at least 3 different antigenic sites involved in neutralisation, two of them conserved in different isolates and the remaining restricted to the homologous strain; 14 non-neutralizing MAbs showed high antigenic stability among Asia 1 isolates and some of them presented different degrees of heterotypic reactivity also. Studies to investigate the potential of these MAbs as diagnostic tools lead to the development of prototype assays for both antigen and antibody detection. In particular, sandwich ELISAs for either the specific detection of Asia 1 FMD virus or a universal detection of FMD viruses have been developed, showing a detection limit of 105 TCID50/ml. A SPCE for the specific detection of Asia 1 antibody is under evaluation: the assessment of few positive and negative sera provided promising results, although assay standardization is not yet finalized and performances may be improved. Acknowledgments We wish to thank Dr D. Gamba for the work in MAbs production, Dr M. Bugnetti for growing viruses, Dr. B. Haas for providing rabbit sera used in capture ELISAs and Dr . F. De Simone for his valuable advice. Work was supported by national grant IZSLER 07/98 from National Ministry of Health and by Concerted Action grant FAIR CT98 4032 from the European Union.

References Borrego B, Carra E, Garcia-Ranea JA and Brocchi E. (2002). Characterization of neutralization sites on the circulating variant of Swine Vesicular Disease virus: a new site is shared by swine vesicular disease and the related Coxsackie B5 virus. J Gen Virology. 83: 35-44

200

Brocchi E, Capucci L, De Simone F, Panina GF (1986). Potential of monoclonal antibodies (MAbs) for FMD diagnosis and characterization of the isolates. Report of Sess. Res. Gr. St. Tech. Committee of the Europ. Comm. Control FMD, Italy, Rome, 1986, p 30-31. Brocchi E., De Simone F., Bugnetti M., Gamba D., Capucci L. (1990) Application of a monoclonal antibody-based competition ELISA to the measurement of anti-FMDV antibodies in animal sera. Report of Sess. Res. Gr. St. Tech. Committee of the Europ. Comm. Control FMD, Lindholm, Denmark, June 24-25 1990, Appendix 14. Brocchi E, Gamba D, Poumarat F, Martel JL, De Simone F. (1993). Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies. Rev Sci Tech Off Int Epizoot. 12: 559-570. Brocchi E, Berlinzani A, Gamba D, De Simone F. (1995). Development of two novel monoclonal antibodybased ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus. J Virol Methods. 52: 155-167 . Brocchi E, Gamba D, Bugnetti M, De Simone F (1998). Monoclonal antibodies profiling of FMD viruses type A currently used for FMD control. Report of the Sess. Res. Gr. St. Tech. Committee of the Europ. Comm. Control FMD, UK, 14-18 Sept. 1998, p 56-61. Harlow E. & Lane D. (1988). Immunoblotting protocols. In Antibodies: a Laboratory manual, p 479-510. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Samuel, A.R., Knowles, N.J., Samuel, G.D. and Crowther, J.R. (1991) "Evaluation of a trapping ELISA for the differentiation of FMDV using MAbs." Biologicals 19: 299-310. Tjissen P. (1985). Preparation of enzyme-antibody or other enzyme-macromolecule conjugates. In Laboratory techniques in biochemistry and molecular biology. Practice and therapy of enzyme immunoassays (R.H. Burdon & P.H. van Knippenberg, eds). Elsevier, Amsterdam, 221-277.

201

Table 1 – Pattern of reactivity of 24 monoclonal antibodies raised against FMDV type Asia 1, strain Nepal 29/97

-

IgG1

5C12

> 32

>20480

VP1

625

-

-

IgG1

5E10

24

nd

-

125

-

IgG1

3C6

3

1920

-

125

IgG1

*2° 1F10

> 32

nd

nd

125

IgG1

2C3

12

nd

-

IgG1

2G1

24

nd

IgM

5G4

6

IgG2a

4D8

1

IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

4G6 *2° 3D8 4B1 4B2 2F7 2G8 *2° 2B11 3G3 2A4 3B6 5H5 4G2 *2° 3H12

-

IgG1

125 5F10 VP2 *2° : MAb originated from the second fusion;

homologous

Cam 3/93

-

Nepal 58/88

-

Tur 15/73

125

Kuwait 2/81

VP1

Gre 1/2000

>20480

SAU 39/94

> 32

Iran 58/99

4F10

Pak 3/98

IgM

Pak 2/98

-

India 10/82

-

Cam 9/80

-

(homologous)

125

Nepal 29/97

C1

VP1

Ag site

A5

1920

Noville

O1

12

Italy 1962

Western Blot

4E10

Swtz.1965

asc.fluid

IgM

Asia (titre)

MAb

Ig class NEUTRALISING MAbs NON NEUTRALISING MAbs

percentage reactivity (ELISA) with 11 FMDV isolates - type Asia 1

ELISA Trapping

culture supernatant

VNT titre

100 100 100 100 100 100 100 100 100 70 100

0

100 100 100 100 100 100 100 100 100 70 100

0

-

100

20

0

-

-

100 100 50

20

0

-

-

-

100 100 100 100 100 100 100 100 75 100 40

0

-

-

-

100

625

-

-

-

100

50

10

10

10

10

10

10

10

10

10

5

-

625

-

-

-

100

50

0

0

0

0

0

0

0

0

0

0

nd

-

125

-

-

-

100

0

50

50

35

20

0

0

0

0

0

0

nd

-

5

-

-

-

100

0

0

0

0

0

0

0

0

0

0

0

125 3000 125 625 2000 625 625 2000 625 625 25 2 125

+/+/(+) (+) (+) + + + + + +

(+) (+) (+) + + + + + +

+/+/+ + + + + +

+

+

-

-

nd

nd

nd nd

-

nd

-

-

-

nd

nd

nd

nd

-

nd

nd

nd nd

202

1

2

3

75 100 100 100 100 100 100 100 25 50

50

50

50

35

not done

40

50

not done

100 100 100 100 100 100 100 100 100 100 100

4

100

not done

0

not done

100 100 100 100 100 100 100 100 100 100 100 100

5a

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

5b

100 100 100 100 100 100 100 100 100 100 100 100 100

5

not done

not done

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

5c

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

not done

not done

100 100 100 100 100 100 100 100 100 100 100 100 + 6 (+) : reduced signal with respect to the homologous type

Appendix 25

Diagnosis of Foot-and-Mouth disease virus by automated RT-PCR Scott M. Reid, Nigel P. Ferris, Geoffrey H. Hutchings and Soren Alexandersen Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom

Abstract Automated 5' nuclease probe-based (TaqMan®) reverse transcription polymerase chain reaction (RT-PCR) procedures using a MagNA Pure LC were evaluated for foot-and-mouth (FMD) virus diagnosis during the United Kingdom (UK) 2001 epidemic. Epithelial suspensions (ES), serum or whole blood, milk and oesophageal-pharyngeal fluid (“probang”) samples submitted to the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright, were tested as well as supernatant fluids following inoculation of cell cultures with ES. New programmes have been inserted into the software of the MagNA Pure LC to improve the extraction of nucleic acids from test samples and controls and to increase the speed, flexibility, capacity and reproducibility of the RT and PCR procedures without harming the assay sensitivity. Like the initial programmes for automation, the new programmes enabled definitive diagnostic results to be achieved on first passage cell culture supernatant fluids but a 96-well PCR assay can now be performed by 2 people within an extended working day. Our results indicate that our realtime automated RT-PCR could be recommended instead of virus isolation during an outbreak to accelerate FMD diagnosis. The positive-negative acceptance criteria for the testing of probangs by automated RT-PCR is under consideration. Preliminary results from experimentally infected animals show that the virus can be detected in probangs but different assay acceptance criteria to that based on the testing of ES and cell culture supernatant fluids will have to be used. 1. Introduction If laboratory investigations are to play a part in FMD diagnosis following the control policies introduced by the UK government during the 2001 epidemic then the time to perform tests will have to be considerably accelerated. In the WRL for FMD, Pirbright, an evaluation is being made of the application of automated RT-PCR technology for the rapid and accurate diagnosis of the disease. Automated RT-PCR procedures for the diagnosis of FMD virus using tissue epithelium, serum or whole blood, milk and probang samples were initially evaluated on samples submitted to the WRL for FMD during the UK 2001 epidemic and from animals experimentally infected with the FMD virus serotype O UK 2001. A primer/probe set designed for the intended detection of all 7 serotypes of FMD virus was used (Reid et al., 2002) and the results of this evaluation were directly compared to the routine diagnostic tests of ELISA and virus isolation in cell culture (Reid et al., 2001a; 2001b). Automated programmes increased the speed and capacity of the RTPCR assays by enabling larger panels of clinical samples (together with positive and negative controls) to be tested simultaneously by RT-PCR. In an extended working day, one operator 203

could realistically obtain results from 64 test (and control) samples by performing 2 runs of a 32well RT-PCR assay. The Pirbright Laboratory have further developed and evaluated automated RT-PCR procedures for FMD virus diagnosis by creating new MagNA Pure LC programmes in attempts to improve the efficiency of the extraction of total nucleic acid from test and control samples and to increase the speed, flexibility and reproducibility of the automated procedures for RT and PCR without affecting the sensitivity achieved by the initial programmes. 96-well automated assays would greatly increase the diagnostic capacity and consistency of RT-PCR and could easily be performed on a routine basis. The results from assays involving new automated programmes for the nucleic acid extraction, RT and PCR steps and their comparison with the routine diagnostic procedures of ELISA and virus isolation in cell culture are presented in this study. 2. Materials and methods 2.1. Sample preparation, ELISA and virus isolation ES and other suspensions of samples submitted for FMD virus diagnosis from the UK 2001 FMD epidemic were prepared, tested by ELISA and inoculated onto primary calf thyroid cell cultures (Ferris and Dawson, 1988). IB-RS-2 cells were also used for virus isolation on some sample submissions. ES was similarly prepared and tested from overseas sample submissions. Serum or whole blood ("blood") and probang samples from the epidemic were similarly inoculated onto primary calf thyroid cell cultures. Samples showing a cytopathic effect (CPE) were harvested and the FMD virus specificity of the supernatant fluids was confirmed by ELISA. Supernatant fluids of a selection of cell cultures not showing a recognisable CPE on first and second passage following inoculation with ES or other suspensions were collected. 2.2. Total nucleic acid extraction Prior to the extraction procedure, 0.2 ml of ES was added to 1 ml TRIzol® Reagent (Life Technologies, UK). Blood and probang samples were added to an equal volume of lysis/binding buffer (Roche, UK) and each sample mixed for 10-15 sec. Cell culture supernatant fluids were either added to an equal volume of lysis/binding buffer or 0.2 ml added to 1 ml of TRIzol® Reagent. Samples were placed in batches of 32 inside a MagNA Pure LC (Roche, UK) programmed to extract total nucleic acid to a final elution volume of 0.1 ml. A new total nucleic acid extraction programme was also used to provide a final elution volume of 0.05 ml and therefore achieve a greater concentration of extracted nucleic acid. 2.3. Reverse transcription The MagNA Pure LC was programmed to mix 6 µl of each nucleic acid with 9 µl RT mix in batches of 32 samples. For the 96-well RT-PCR assay (called the ‘fast RT-PCR protocol’), this RT process was carried out on 3 consecutive sets of 32 nucleic acids in a single 96-well plate and the RT of 96 samples was completed by placing the plate in a PTC-100TM thermal cycler (MJ Research, Inc.) and incubating successively at 48oC for 45 min, 95oC for 5 min and 20oC for at least 20 min. A faster RT programme was later created to achieve the same function but which could mix 32 samples of nucleic acid with RT mix in a shorter time period of around 5 min. The 204

slower RT programme was only used for 96-well assays (‘fast RT-PCR protocol’) and the newer faster one has mainly been used for 32-well assays. 2.3. PCR amplification by 5' nuclease probe-based reaction Redundant primers and a fluorogenic 5' nuclease probe were as described previously (Reid et al., 2001a; 2001b; 2002). RT products were added to the PCR mix by an automated process in which the MagNA Pure LC was programmed to add 7 µl cDNA from batches of either 32 or 96 samples (as in the 96-well ‘fast RT-PCR protocol’) to 18 µl PCR mix containing 0.9 pmol/µl each of the forward and reverse primers and 0.3 pmol/µl of probe. This was followed by PCR amplification in a GeneAmp® 5700 Sequence Detection System (Applied Biosystems) using the amplification programme described previously (Reid et al., 2001a; 2001b; 2002). A quantitative value was assigned to each PCR reaction and positive/negative cut-off criteria was applied which included a ‘borderline’ region. Samples falling within this region require retesting by the PCR to determine their status. 2.4. Reproducibility of automated RT-PCR The intra-assay reproducibility of the 96-well ‘fast RT-PCR protocol’ was monitored by incorporating FMD-positive and FMD-negative control samples within each set of 32 samples. Intra-assay reproducibility of the same automated RT-PCR procedure was also assessed by testing a single cell culture grown virus preparation in every well of the 96-well plate. Inter-assay reproducibility of this protocol was determined by testing a batch of 32 samples in more than one assay run. 3. Results 3.1. Automated 96-well ‘fast RT-PCR protocol’ The results of the 96-well ‘fast RT-PCR protocol’, ELISA and virus isolation on ES, other suspensions, blood and probang samples are summarised in Table 1 (section A). RT-PCR detected more positive sample submissions than ELISA and was positive on more ES than virus isolation. Blood samples positive by virus isolation were generally also positive by RT-PCR but only one probang was positive by virus isolation. This probang was borderline by the RT-PCR as were 2 other probangs submitted from other premises but which were negative by virus isolation. Table 2 (section A) compares the results of the ‘fast RT-PCR protocol’ with virus isolation on cell culture supernatant fluids inoculated with ES or other suspensions. Fifteen supernatant fluids produced a CPE on first passage cell culture and were positive by RT-PCR while 17 supernatant fluids which did not produce a CPE after first (and then second) passage (and therefore not tested by ELISA) were negative by RT-PCR.

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3.2. 32-well RT-PCR protocol with alternative programmes for nucleic acid extraction and reverse transcription The results from a 32-well RT-PCR using the alternative programme for extraction of total nucleic acid from test and control samples to a final elution volume of 0.05 ml followed by the more rapid programme for automated RT on ES and cell culture supernatant fluids are shown in Table 1 (section B) and Table 2 (section B) respectively. The RT-PCR had an equivalent sensitivity to virus isolation and a higher sensitivity than ELISA for detection of FMD virus in ES. Four supernatant fluids produced a CPE on first passage cell culture and were RT-PCR positive. Fifty one supernatant fluids which did not produce a CPE after first and second cell culture passage (not tested by ELISA) were negative by RT-PCR. 3.3. Reproducibility of automated RT-PCR The standard deviation was low when the cell culture grown virus preparation was tested in all 96 wells by the ‘fast RT-PCR protocol’ and the inter-assay reproducibility of this procedure was also good based on the re-testing of the batch of 32 samples and the re-testing of other selected samples (data not shown). 4. Discussion Automated 5' nuclease probe-based RT-PCR can provide FMD diagnostic results more rapidly than conventional RT-PCR or non-automated 5' nuclease probe-based RT-PCR methods but further development of the methodology has increased test capacity and consistency to achieve 96 results in a single plate by one person within 2 typical working days or by 2 people in around 12 hours. The sensitivity of this 96-well assay (‘fast RT-PCR protocol’) compared well with virus isolation (the so-called gold standard) for the testing of ES and blood while the more recentlydeveloped 32-well RT-PCR protocol had an equivalent sensitivity to virus isolation on ES. Both RT-PCR methods detected FMD viral RNA in positive cell culture supernatant fluids on first passage (showing a recognisable CPE) and were also clearly negative on first passage cell cultures not showing a CPE and which did not produce a CPE on second passage so that definitive diagnostic results could be achieved by RT-PCR on first passage cell cultures alone. The described automated RT-PCR could be recommended instead of virus isolation during an FMD outbreak to achieve diagnostic results within a much smaller time-scale. More samples will be tested to confirm that passaging of samples in cell culture may not be necessary for the issue of laboratory results although it would still be useful to isolate samples in cell culture for confirmation of a first outbreak in a previously FMD-free country. The improved nucleic acid extraction and RT programmes used in the 32-well RT-PCR can be applied for testing batches of 96 (or 64) test/control samples in a single plate to provide diagnostic results in an even shorter time-scale than that of the 96-well ‘fast RT-PCR protocol’ (total assay time cut from 12 to 10-11 hours). This new 96-well RT-PCR assay will have to be evaluated on more ES and cell culture supernatant fluids but it has already achieved promising results on probangs taken from experimentally infected animals even though the positive/negative acceptance criteria has not been firmly established (S. M. Reid et al., unpublished results). This is likely to differ from that currently used for the testing of ES and cell culture supernatant fluids. 206

Acknowledgements The authors thank Sylvia Grierson (Veterinary Laboratories Agency, Addlestone) and Antoinette Cherubini (Central Veterinary Research Laboratory, Abbotstown, Dublin) for their help. This work was supported financially by the Department for Environment, Food & Rural Affairs (DEFRA), UK. References Reid, S. M., Ferris, N. P., Hutchings, G. H., Zhang, Z., Belsham, G. J., Alexandersen, S., 2002. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay. J. Virol. Methods 105, 67-80. Reid, S. M., Ferris, N. P., Hutchings, G. H., Zhang, Z., Belsham, G. J., Alexandersen, S., 2001a. Diagnosis of foot-and-mouth disease by real-time fluorogenic PCR assay. Vet. Record 149, 621-623. Reid, S. M., Ferris, N. P., Hutchings, G. H., Alexandersen, S., 2001b. Evaluation of automated RT-PCR systems to accelerate FMD diagnosis. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Island of Moen, Denmark, 12-15 September, 2001. Rome: FAO 2001 Appendix 25, pp. 118-125. Ferris, N. P., 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.

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Table 1. Comparison of the 96-well ‘fast RT-PCR protocol’ (section A) and the new automated 32-well RT-PCR protocol (section B) with ELISA and virus isolation for the testing of epithelial suspensions (ES), other suspensions, blood and probang samples

Test procedure

Type of material and the number of samples positive (FMD virus), negative (NVDa) or borderline ES

A

B

a

FMD

NVD

Probangs

FMD

NVD

Borderline

Borderline

FMD

NVD

ELISA

104

104

8

Virus isolation

124

92

0

26

226

0

1

159

RT-PCR

134

80

2

27

220

5

0

157

ELISA

13

17

0

Virus isolation

14

16

0

RT-PCR

14

15

1

Otherc Borderline

FMD

NVD

Borderline

0

3

0

0

0

3

0

3

0

3

0

NVD, no virus detected.

b Blood c

Bloodb

and probang samples are not tested directly by ELISA but inoculated onto primary calf thyroid cell culture. ELISA then used to test supernatant fluids from cell cultures showing a CPE.

Suspensions prepared from “bone marrow” samples.

208

Table 2. The results of the 96-well ‘fast RT-PCR protocol’ (section A) and the newer automated 32-well RT-PCR protocol (section B) on cell cultures inoculated with epithelial suspensions (ES)

Test procedure

Ratio of number of samples positive (FMD virus) or NVDa in passages of cell culture following inoculation with ES First passage (0-48 hr)

A

B

a

Positive

NVD

Positive

NVD

Virus isolation

15/15

0/17

NT b

0/17

RT-PCR

15/15

0/17

NT

NT

Virus isolation

4/4

0/51

NT

0/51

RT-PCR

4/4

0/51

NT

NT

NVD, no virus detected.

b NT,

Second passage (48-72 hr)

not tested.

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Appendix 26

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 Scott M. Reid, Nigel P. Ferris and Geoffrey H. Hutchings Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK

Abstract Results compiled from the reverse transcription polymerase chain reaction (RT-PCR) collaborative study under the European Union (EU) Concerted Action Group Project PL 98-4032 involving the laboratories in Pirbright, Brussels, Valdeolmos, Brescia, Tübingen, Lelystad and Lindholm were presented in order to evaluate the sensitivity and specificity of the locally employed RT-PCR procedures. All 7 laboratories were supplied with 40 unlabelled epithelial suspensions (ES) of foot-and-mouth disease (FMD) virus positive clinical material (10 each of the serotypes O, A, C and Asia 1) and 5 of the laboratories supplied with the supernatant fluids (cc) following the inoculation of the ES in cell culture. At the Pirbright Laboratory virus was detected (but not serotyped) in 36 of the 40 ES and in 39/40 cc by conventional (not nested and non-quantitative) RT-PCR and in 36/40 ES by a prototype immunocapture method. Similar sensitivity with universal primer sets was achieved in RT-PCR assays on the ES and cc in Brescia but 39/40 ES were detected by a novel RT-PCR in Valdeolmos. A nested PCR protocol in Tübingen also detected 39/40 ES but a positive result on all 40 ES samples was obtained by quantitative RT-PCR for the universal detection of FMD virus (TaqMan® assay at Pirbright, LightCycler protocol at Lelystad and SYBR® Green assay at Lindholm). Serotype-specific primers were used only in Pirbright and Brussels but a more thorough comparison of the sensitivity and specificity of the RT-PCR procedures can be obtained only after all of the experimental details employed by the laboratories (particularly amplification programmes, cut-off criteria and the use of positive and negative controls) are considered in greater detail. 1. Introduction RT-PCR procedures are becoming more important for diagnosis of FMD virus and three studies by Reid et al. (1998; 1999; 2000a) at the OIE/FAO World Reference Laboratory for Foot-andMouth Disease (WRL for FMD), Pirbright, reported the use of conventional RT-PCR formats involving universal or serotype-specific primer sets for the testing of ES and cc prepared from large panels of positive FMD virus specimens of all 7 serotypes. Since the beginning of the United Kingdom (UK) 2001 FMD epidemic, new RT-PCR methodologies for FMD virus diagnosis have been developed and evaluated at the WRL for FMD to supplement the routine diagnostic procedures of ELISA and virus isolation in cell culture (Ferris and Dawson, 1988) and which have involved non-automated and automated fluorogenic assays (Reid et al., 2001a; Reid et al., 2001b; Reid et al., 2002a; Reid et al., 2002b, in press). Results currently suggest that a definitive diagnostic result might be achieved by RT-PCR at the end of one cell culture passage 210

(Reid et al., 2002b, in press). This would obviate the necessity of passaging samples in cell culture so that laboratory results could be issued much faster. At the first annual European Union (EU) Concerted Action PL 98-4032 meeting during 5-7 May 1999 at the Federal Research Centre for Virus Diseases of Animals [BFAV] in Tübingen, a collaborative study was organised between the Veterinary and Agrochemical Research Centre (VARC) in Brussels, BFAV Tübingen and the WRL for FMD, Pirbright, to provide an assessment of the sensitivity and specificity of the RTPCR procedures employed by these laboratories. The RT-PCR protocols used by the EU laboratories are not standardised and inter-laboratory variation has not been investigated so the study could suggest improvements in the diagnosis of FMD virus by RT-PCR. At the EU Concerted Action meeting in Pirbright the following year, the Center of INIA (CISA-INIA) in Valdeolmos (Madrid) and the Istituto Zooprofilattico Sperimentale in Brescia joined the study and the number of participating laboratories rose to 7 at the next meeting in Brussels in 2001 when the laboratories of CIDC-Lelystad (Central Institute for Animal Disease Control Lelystad) and the Danish Veterinary Institute for Virus Research in Lindholm requested to participate. This study will investigate the FMD diagnostic potential within the EU of some of the more recently introduced RT-PCR methodologies such as quantitative (Taqman® and SYBR® Green) RT-PCR as well as previously described conventional or nested RT-PCR formats such as those of Vangrysperre and De Clercq (1996) and Moss and Haas (1999). Some of the results obtained by the WRL for FMD were reported previously (Reid et al., 2000b). 2. Materials and methods 2.1. Preparation of the unlabelled samples for the blind trial between the collaborating laboratories The 10 ES each of the FMD virus serotypes O, A, C and Asia 1 and the cc resulting from the propagation of the ES in cell culture were prepared and aliquotted at the WRL for FMD and distributed (unlabelled) to the laboratories of BFAV Tübingen, VARC in Brussels, Center of INIA (CISA-INIA) in Valdeolmos and to the Istituto Zooprofilattico Sperimentale in Brescia (as well as to the WRL for FMD) as previously described (Reid et al., 2000b). The 40 ES but not the cc were also sent to the laboratories in Lelystad and Lindholm. 2.2. Summary of the RT-PCR methods used in each laboratory 2.2.1. WRL for FMD, Pirbright Conventional RT-PCR Total RNA was extracted from the trial samples, uninfected negative control ES and cc and subjected to RT as described previously (Reid et al., 2000b). The universal O/A/C/Asia 1 primer set (1F/1R) designed for the intended diagnosis of all 7 FMD virus serotypes was used as it was 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., 2000a). The specific primers P33/P38/P87-P92/P40/P74P77 designed for the diagnosis of the serotypes O, A, C and Asia 1 (Vangrysperre and De Clercq, 1996) and evaluated in RT-PCR procedures by Reid et al. (1999; 2000a) were also used. The RT

211

product from each sample was tested with the 1F/1R primer pair and with the cocktail of the specific primers P33/P38/P87-P92/P40/P74-P77 in RT-PCR procedures as described previously (Reid et al., 2000b). Immunocapture (antigen capture) RT-PCR Each ES was tested in a prototype immunocapture RT-PCR method for FMD virus diagnosis involving PCR amplification with the 1F/1R primer set as described previously (Reid et al., 2000b; 2000c). Ten ES samples were tested by another prototype immunocapture protocol involving 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) in separate PCR amplifications as described previously (Reid et al., 2000b; 2000c). Quantitative (fluorogenic or TaqMan®) RT-PCR A forward primer, reverse primer and probe were designed from conserved sequences of the FMD viral genome for use in a quantitative PCR machine. All ES and cc were tested in a TaqMan® RT-PCR using a GeneAmp® 5700 Sequence Detection System (Applied Biosystems, UK) to give a positive or negative result for FMD virus in each sample based on quantitative values (Reid et al., 2002a). 2.2.2. VARC, Brussels Twenty eight samples each of the ES and cc were tested by RT-PCR using serotype-specific primers designed in-house from the 1D region of the virus genome (Vangrysperre and De Clercq, 1996). 2.2.3. CISA-INIA, Valdeolmos All ES and cc were tested by the novel RT-PCR protocol of Sáiz et al. (2001) using the primer set A1 designed from the 3D1/3D2 region of the genome for the detection of the FMD virus serotypes O, A, C and Asia 1 as a group (Rodríguez et al., 1992; Núñez et al., 1998) and the primer set U (Sáiz et al., 2001) for the universal diagnosis of all 7 serotypes of FMD virus. 2.2.4. Istituto Zooprofilattico Sperimentale, Brescia The cc was re-passaged in order to produce larger volumes for testing. Thirty nine of the 40 ES and all 40 re-passaged cc were tested by an RT-PCR using a primer set F17/F21 which is the primer set A1 (Rodríguez, et al., 1992; Núñez et al., 1998) and which was used in this study by the laboratory of CISA-INIA in Valdeolmos. Thirty nine of the 40 ES and 16 cc were also tested by an RT-PCR with a primer set LD2/LR2 (Lomakina, unpublished) designed from the 3D region of the FMD virus genome for the intended universal detection of all 7 serotypes. 2.2.5. BFAV Tübingen All ES and cc were tested by nested PCR in combination with a plaque test as described by Moss and Haas (1999) with the slight modification of using TRIzol ® Reagent for RNA extraction. 212

2.2.6. Danish Veterinary Institute for Virus Research, Lindholm RNA was purified from 90 µl of each ES sample by a QIAamp® Viral RNA Mini Spin Kit (QIAGEN) and RT performed on 5 µl of the extracted RNA by a RETROscript First Strand Synthesis Kit (Ambion). PCR amplification was carried out by SYBR® Green assay on 5 µl cDNA using the universal O/A/C/Asia 1 primer set (1F/1R) of Reid et al. (2000a) in a 7700 Sequence Detection System (Applied Biosystems). The 1F/1R primer set was used by the WRL for FMD in conventional and immunocapture RT-PCR methodologies as previously described (2.2.1.). Further testing of the samples will be performed using a QuantiTect SYBR® Green RTPCR Kit (QIAGEN). 2.2.7. CIDC-Lelystad The LightCycler RT-PCR protocol of Moonen et al. (2001) using labelled probes was used to test the 40 ES samples. 3. Results 3.1. WRL for FMD, Pirbright RT-PCR results obtained on the 40 ES and cc samples obtained by each laboratory are shown in Table 1. At the WRL for FMD, one sample was not detected by conventional RT-PCR with the primer set 1F/1R either as an ES or cc but all other cc samples were detected as were 36 of the 40 ES samples including all of the serotype O and Asia 1 viruses. This mirrored the results from a previous evaluation with this primer set in conventional RT-PCR (Reid et al., 2000a) 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 cc of type Asia 1 viruses using both thermocycler programmes but were less successful for detection of the other serotypes which again mirrored previous results (Reid et al., 1999). The specific primers did however detect the serotype C virus which was negative with the primer set 1F/1R. Annealing temperature significantly influenced the performance of the specific primers for detection of serotype A strains as the performance of the primers dropped when this was 59oC rather than 58oC (Reid et al., 2000b). The performance of the 1F/1R primer set on the 40 ES samples in the immunocapture RT-PCR were identical to those achieved by the same primers in the conventional RT-PCR (Reid et al., 2000b). The results of the immunocapture RT-PCR on the 10 ES samples with the specific primers were also similar to those achieved by the conventional RT-PCR but the serotype A strains were not detected (Reid et al., 2000b, data not presented here). All 40 samples of ES and cc were positive for FMD virus by the TaqMan® RT-PCR. 3.2. VARC, Brussels The specific primers correctly serotyped some of the trial samples but a selection of the ES and cc produced results corresponding to more than one serotype rather than to the single FMD virus serotype. The results did indicate that FMD virus was present in 26 of the 28 ES and/or cc samples tested.

213

3.3. CISA-INIA, Valdeolmos Primer set A1 detected 34/40 ES and 37/40 cc samples. Three viruses were negative on both ES and cc. However, the primer set U detected 39/40 ES and all 40 cc. The ES of one serotype C virus was negative with both primer sets. 3.4. Istituto Zooprofilattico Sperimentale, Brescia RT-PCR with primer set F17/F21 detected 35/39 ES and 39/40 cc viruses (compared to 34/40 ES and 37/40 cc detected using the same primer set at CISA-INIA) and all 40 virus strains were detected either as an ES and/or cc. The primer set LD2/LR2 detected 33/39 ES and 15/16 cc in an RT-PCR. 3.5. BFAV Tübingen Nested PCR detected 39 out of 40 ES, of which 30 were already positive after first amplification. All 40 cc were detected by nested PCR and of these, 36 were positive after first amplification. The ES sample testing negative by nested PCR was positive in the other laboratories. 3.6. CIDC-Lelystad and Danish Veterinary Institute for Virus Research, Lindholm All 40 samples of ES were positive by RT-PCR at Lelystad and Lindholm by the LightCycler and SYBR® Green assays respectively. 4. Discussion The laboratories of the WRL for FMD, CISA-INIA, Istituto Zooprofilattico Sperimentale, BFAV Tübingen, CIDC-Lelystad and Lindholm correctly achieved a positive diagnostic result for FMD virus on either the ES or cc (and in most cases on both ES and cc) of all 40 trial samples by their RT-PCR procedures although positive results were not achieved solely on the ES of all samples by the RT-PCR procedures at CISA-INIA, Istituto Zooprofilattico Sperimentale and BFAV Tübingen. The conventional RT-PCR at the WRL for FMD also failed to detect FMD virus in all ES and had an equivalent sensitivity to a prototype immunocapture assay, which may have a potential role in 'on-site' diagnosis of FMD by RT-PCR, with the universal primer set 1F/1R. Nested and quantitative methodologies were used for universal FMD virus diagnosis. Nested PCR at BFAV Tübingen was positive on all samples except one ES but primary diagnosis of FMD virus in all 40 ES was achieved by the quantitative TaqMan®, LightCycler and SYBR® Green RT-PCR assays at the WRL for FMD, Lelystad and Lindholm respectively. The TaqMan® assay was also positive on all 40 cc. This highlighted the FMD diagnostic potential of these assays given that the test results can be obtained within a few hours of sample receipt and in a much shorter time-scale than virus isolation in cell culture. These quantitative RT-PCR formats also avoid the necessity of analysing PCR products by gel electrophoresis, which is laborious, time-consuming and relatively insensitive. Interestingly, the primer set 1F/1R was used in the SYBR® Green RT-PCR assay (Lindholm) and in conventional and immunocapture RT-PCR formats at the WRL for FMD and while all 40 ES samples were detected by the SYBR® Green assay, 36/40 ES were detected by the other methodologies. This may have been due to the 214

positioning of the positive to negative cut-off in the respective assays. To achieve a more substantial evaluation of the sensitivity and specificity of the described RT-PCR procedures it will be necessary to compare aspects like this and other key features of the assays such as the amplification programme and the role played by positive and negative control samples in the validation of the test. It would also be interesting to investigate the cause of negative results. The primer and primer/probe sets used in the study were predominantly designed for the universal diagnosis of FMD virus in clinical samples rather than for serotype-specific diagnosis. Such an approach is clearly useful in a circumstance similar to that of the UK 2001 epidemic in order to establish the presence of FMD virus in clinical samples so that control measures could be implemented quickly. However, serotype-specific, semi-specific or strain-specific primer (or primer/probe) sets could also extend the scope of the RT-PCR formats by detecting emerging FMD virus strains. Serotype-specific primers were used only in non-quantitative RT-PCR formats at the WRL for FMD and in Brussels and while more positive results were achieved at the WRL for FMD with the universal primer set in conventional RT-PCR, a positive result was produced with serotype-specific primers on the cc of a type C strain which was negative with the universal primer set (sample no. 19). This study was a true blind trial in that the samples were tested ‘unseen’ in the laboratories but it was known beforehand that the ES and cc of all 40 samples were positive for FMD virus. A bias could therefore be imparted to the testing approach as more tests can be performed on any given sample in order to ‘achieve’ the positive result. Such a luxury would not normally be available in the diagnostic laboratory. Furthermore, it would have been interesting to have included samples which did not contain FMD virus. This would have provided a greater test of the specificity of the diagnostic RT-PCR methodologies. Acknowledgements The authors thank Kris De Clercq (VARC, Brussels), Esther Blanco (CISA-INIA), Emiliana Brocchi (Istituto Zooprofilattico Sperimentale), Bernd Haas (BFAV Tübingen), Aldo Dekker (CIDC-Lelystad), Karin de Stricker (Danish Veterinary Institute, Lindholm) and their co-workers for participating in this study and Soren Alexandersen (Institute for Animal Health, Pirbright) for the design of the primers and probe and for technical assistance with the TaqMan® RT-PCR. The RT-PCR assays carried out at the WRL for FMD, Pirbright, were supported financially by the Department for Environment, Food & Rural Affairs (DEFRA), UK. References Reid, S. M., Forsyth, M. A., Hutchings, G. H., 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., 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., Knowles, N. J., 2000a. Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J. Virol. Methods 89, 167-176.

215

Ferris, N. P., 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. Reid, S. M., Ferris, N. P., Hutchings, G. H., Zhang, Z., Belsham, G. J., Alexandersen, S., 2001a. Diagnosis of foot-and-mouth disease by real-time fluorogenic PCR assay. Vet. Record 149, 621-623. Reid, S. M., Ferris, N. P., Hutchings, G. H., Alexandersen, S., 2001b. Evaluation of automated RT-PCR systems to accelerate FMD diagnosis. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Island of Moen, Denmark, 12-15 September, 2001. Rome: FAO 2001 Appendix 25, pp. 118-125. Reid, S. M., Ferris, N. P., Hutchings, G. H., Zhang, Z., Belsham, G. J., Alexandersen, S., 2002a. Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay. J. Virol. Methods 105, 67-80. Reid, S. M., Grierson, S. S., Ferris, N. P., Hutchings, G. H., Alexandersen, S., 2002b. Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of FMD virus. J. Virol. Methods, in press. Vangrysperre, W., 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 genomically and/or symptomatically related viruses. Arch. Virol. 141, 331-344. Moss, A., 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., Hutchings, G. H., Ferris, N. P., 2000b. 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. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Borovets, Bulgaria, 5-8 September, 2000. Rome: FAO 2000 Appendix 21, pp. 181-187. Reid, S. M., Hutchings, G. H., Ferris, N. P., 2000c. The development of an antigen capture reverse transcription polymerase chain reaction method for foot-and-mouth disease virus antigen detection. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Footand-Mouth Disease, Borovets, Bulgaria, 5-8 September, 2000. Rome: FAO 2000 Appendix 19, pp. 167172. Sáiz, M., de la Morena, D. B., Blanco, E., Núñez, J. I., Fernández, R., Sánchez-Vizcaíno, J. M., 2001. A novel method for detection of FMDV from culture and clinical samples by RT-PCR and restriction enzyme analysis. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, Island of Moen, Denmark, 12-15 September, 2001. Rome: FAO 2001 Appendix 26, pp. 126. Rodríguez, A., Martínez-Salas, E., Dopazo, J., Dávila, M., Sáiz, J. C., Sobrino, F., 1992. Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus. Virology 189, 363-367. Núñez, J. I., Blanco, E., Hernandez, T., Gómez-Tejedor, C., Martín, M. J., Dopazo, J., Sobrino, F., 1998. A RT-PCR assay for the differential diagnosis of vesicular diseases of swine. J. Virol. Methods 72, 227235.

216

Moonen, P., Boonstra, J., Hakze-van der Honing, R., Boonstra-Leendertse, C., Jacobs, L., Dekker, A., 2001. Validation of a LightCycler based RT-PCR for the detection of foot-and-mouth disease. Report of the Session of the Standing Technical Committee of the European Commission for the Control of Footand-Mouth Disease, Island of Moen, Denmark, 12-15 September, 2001. Rome: FAO 2001 Appendix 27, pp. 127.

217

Table 1. RT-PCR results obtained on epithelial suspensions (ES) and virus preparations in cell culture (cc) following inoculation with ES WRL for FMD, Pirbrighta

No. of sample and FMD virus serotype O/A/C/Asia 1

O/A/C/Asia 1

(1F/1R) (all)

(specific)

ES

cc

ES

cc

1 A

+

+

A

A

2 Asia 1

+

+

Asia 1

3 O

+

+

4 Asia 1

+

5 C

VARC, Brussels b

TaqMan® (all)

O/A/C/Asia 1

A1 (O/A/ C/Asia1)

(specific)

Istituto Zooprolilattico, Bresciad

U (all)

F17-F21 (3D)

BFAV Tübingene

LD2-LR2 (3D)

PCR

nested PCR

CIDC-Lelystadf

Lindholmg

Labelled probes

O/A/C Asia 1 (1F/1R) (all)

ES

cc

ES

Cc

ES

cc

ES

cc

ES

cc

ES

cc

ES

cc

ES

ES

+

A

A

+

+

+

+

+

+

+

NTh

+

+

+

+

+

+

Asia 1

+

-

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

O

O

+

O

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

+

Asia 1

Asia 1

+

Asia 1

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

-

+

-

-

+

-

O

-

+

+

+

+

+

+

+

-

-

+

+

+

+

6 A

+

+

A

A

+

-

-

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

7 Asia 1

+

+

Asia 1

Asia 1

+

O, A, Asia 1

O, A, C, Asia 1

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

8 A

+

+

-

-

+

A

A

-

+

+

+

-

+

-

+

+

+

+

+

+

+

9 C

+

+

C

C

+

-

C

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

10 O

+

+

-

-

+

O

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

11 A

+

+

-

-

+

O

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

12 C

+

+

-

C

+

-

A, C

+

+

+

+

NT

+

NT

NT

-

+

+

+

+

+

13 A

+

+

-

-

+

-

A

+

+

+

+

+

+

-

-

-

+

+

+

+

+

14 Asia 1

+

+

Asia 1

Asia 1

+

-

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

15 O

+

+

-

O

+

O

O

+

+

+

+

+

+

-

+

+

+

+

+

+

+

16 A

-

+

-

-

+

-

A

-

-

+

+

+

+

+

+

+

+

+

+

+

+

17 Asia 1

+

+

Asia 1

Asia 1

+

O

O

+

+

+

+

+

+

+

NT

+

-

+

+

+

+

18 C

+

+

-

-

+

-

C

+

+

+

+

+

+

+

+

-

+

+

+

+

+

19 C

-

-

-

C

+

C

C

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

20 A

+

+

-

+

-

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

21 Asia 1

+

+

Asia 1

+

O, Asia 1

O, A, C, Asia 1

+

+

+

+

+

+

+

NT

-

+

+

+

+

+

A Asia 1

ES and cc

CISA-INIA, Valdeolmosc

NT

218

+

+

O

-

+

O

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

+

+

Asia 1

Asia 1

+

O, Asia 1

O, Asia 1

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

24 A

+

+

-

-

+

O, A

O, A

+

+

+

+

+

+

+

+

-

+

-

+

+

+

25 C

+

+

-

-

+

C

C

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

26 O

+

+

O

-

+

O

O

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

27 C

+

+

C

C

+

C

C

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

28 A

+

+

-

-

+

-

-

-

-

+

+

-

+

-

+

+

+

+

+

+

+

29 O

+

+

O

O

+

NT

NT

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

30 Asia 1

+

+

-

-

+

NT

NT

+

+

+

+

+

+

+

NT

-

+

+

+

+

+

31 O

+

+

-

-

+

NT

NT

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

32 Asia 1

+

+

Asia 1

Asia 1

+

NT

NT

+

+

+

+

+

+

+

NT

+

+

+

+

+

+

33 A

+

+

A

A

+

NT

NT

+

+

+

+

+

+

+

+

+

+

+

+

+

+

34 O

+

+

-

-

+

NT

NT

+

+

+

+

+

+

+

+

+

-

+

+

+

+

35 C

+

+

-

-

+

NT

NT

+

+

+

+

+

+

+

+

+

+

+

+

+

+

36 O

+

+

-

-

+

NT

NT

+

+

+

+

-

+

-

+

+

+

+

+

+

+

37 C

-

+

-

C

+

NT

NT

-

+

-

+

-

+

-

+

-

+

+

+

+

+

38 Asia 1

+

+

Asia 1

Asia 1

+

NT

NT

+

+

+

+

+

+

+

+

+

+

+

+

+

+

39 O

+

+

O

O

+

NT

NT

-

-

+

+

+

- (?)

+

+

-

-

+

+

+

+

40 C

+

+

-

-

+

NT

NT

+

+

+

+

+

+

+

+

-

+

+

+

+

+

22 O 23 Asia 1

a Conventional and immunocapture RT-PCR procedures using the universal O/A/C/Asia 1(1F/1R) primer set for detection of all 7 serotypes. Conventional RT-PCR also performed with the specific primer sets used in Brussels for detection of the serotypes O, A, C and Asia 1. The conventional and immunocapture RT-PCRs produced identical results on the ES of all 40 samples with the 1F/1R primer set. The TaqMan® assay used a universal primer set for the intended detection of all 7 serotypes. b RT-PCR procedure using specific primers for the detection of individual serotypes O, A, C and Asia 1. c Novel RT-PCR procedure using primer set A1 for the detection of serotypes O, A, C and Asia 1 as a group and the primer set U for the universal detection of all 7 serotypes. d RT-PCR procedure using primer set F17/F21 (same as primer set A1 used in Valdeolmos) and primer set LD2/LR2 for the universal detection of all 7 serotypes. e PCR (first amplification) followed by nested PCR. f LightCycler RT-PCR using labelled probes. g SYBR® Green assay using the 1F/1R primer set as used by the WRL for FMD, Pirbright, in conventional and immunocapture RT-PCR. h NT, not tested. +, positive result. -, negative result

219

Appendix 27

Diagnosis of A22 Mahmatlı and O1 Manisa FMD viruses from field samples: Development of a latex agglutination test kit Nilgün Özdural*, Aysel Candaş , Melahat Cengiz *Sap Institute, PK 714,Ulus, 06044 Ankara, TURKEY

Abstract Foot-and-mouth disease (FMD) is a severe highly contagious and acute disease of swine and cloven-hoofed animals. Among other diagnosis test methods, complement fixation test (CFT) and ELISA are widely use for FMD diagnosis. Recently, Latex Agglutination Test (LAT) has found an ever increasing application in the diagnosis of some types of bacterial, viral and parasitical infections. In this research a new LAT kit was developed as an alternative method to the other diagnosis tests. First two different size mono-disperse polystyrene latex particles were prepared with a modified emulsion polymerisation of styrene monomer. Subsequently these latex particles were coated with two different rabbit anti FMD IgG, which were against A22 Mahmatlı and O1 Manisa FMD viruses. In this research, the virus detection was successfully achieved. Due to high sensitivity of LAT, prepared in this work, special attention should be given to the time of the observation of agglutination. Prolong time, results some cross-reactions. Keywords: Rapid diagnosis, Foot-mouth disease, Latex agglutination test 1. Introduction Rapid confirmatory diagnosis of FMD is necessary to initiate procedures to control the spread of disease and to reach the source of an outbreak. For this purpose laboratory based test are necessary to detect and differentiate the FMD from vesicular viruses and also to differentiate FMD virus types. State owned SAP Institute (Ankara,Turkey), with a prime mission of preparation of FMD vaccine and FMD diagnosis, receives field samples for FMD diagnosis. All field samples are routinely tested and serotyped by CFT, ELISA combined with virus isolation in cell culture. The shipment of the epithelium or vesicular fluid from the scene of the suspected outbreak plays a vital role on the precision of diagnosis. Delays and unproper shipment conditions in the transportation of samples to the laboratory might cause degradation of virus antigen. In this laboratory, the positive specimens are diagnosed by CFT and ELISA, the remaining of field samples being typed after virus amplification in cell culture. The virus amplification procedure may require up to 4 days additionally. Virus isolation also requires laboratory cell culture facilities which can be difficult and expensive to maintain. RT-PCR procedure while extremely useful as a diagnostic technique along with ELISA and virus isolation (Reid et al., 1998, 1999), require skilled personnel, contamination-free laboratory space and specialized equipment. ______________________________________________________________

* Corresponding author: Tel.: 00-90-312-287 36 00/157; fax: 00-90-312 287 36 06 e-mail : nilgunozdural@hotmail.com

220

FMD is one the most contagious animal virus disease of cloven-hoofed livestock and its most dramatic effects can be seen in FMD-free countries which have a well developed export-trade. In these conditions, the population of non-immun animals provides the ideal conditions for rapid spread of disease after introduction. The early reporting disease, followed rapid diagnosis in the laboratory, are very important to prevent spread of disease. A confirmatory diagnosis at the point of suspected outbreak of FMD would be very useful for faster diagnosis. The faster diagnosis would increase disease awareness and would be improve epidemiological information. 1. 2. 3.

4.

The methods of diagnosis might generally be given as following: Clinical diagnosis Serological diagnosis Virological diagnosis a. Virus isolation b. İmmunological method (CFT, antigen capture ELISA) c. Nücleic acid recognition method (Nücleic acid hybridization assay, RT-PCR, Nested PCR) Field tests These tests mainly rely on agglutination assays. Consequently, serotyping is easy to perform with these assays.

An example such a test is Stapylococus aureus naturally bound to FMD virus guinea-pig antibody via the protein A (Montassier et al., 1994) and this test was successfully applied on field samples in Turkey and display high sensitivity for oesophago-pharyngeal samples (Ünver et al, 2000) Another one is coloured latex particles coupled to a monoclonal antibody in a chromatographic strip test. Favorable results were obtained in a laboratory-based study and this test also showed higher sensitivity than the ELISA for epithelial suspensions and nasal swabs (Reid et al., 2001) The principle of LAT kit developed in this work is based on latex agglutination. Uniform latex particles (ULPs) have an intriguing history. The Dow Chemical Company had been manufacturer of polystyrene, including latex used to formulate paints. In 1947, lot no. 3584 was manufactured among others, and a Dow chemist sent a sample of it to a colleague at the University of Michigan. He wanted to get a picture of it using the electron microscope. The electron micrograph showed spherical particles of astonishing and unexpected uniformity. This finding became even more significant because no other lot of latex had particles of similar uniform diameter. By 1956 the technology was complete, including the method of building larger diameter particles from smaller ones. Latex Agglutination Tests or Latex Immunoassays both start with latex particles. These are tiny plastic spheres suspended in water, which have same very special properties for test development. First there are ‘billions and billions’ of particles in a single millilitre of latex. The light scattering from all these particles causes latex to look milky.

221

Second, they are all perfect spheres of the same size (uniform diameter). They all have the same surface properties (uniform surface chemistry). Therefore, they all behave the same and do things together. They can be uniformly coated with antibodies (or other proteins) due to hydrophobic interaction of portions of the protein with the polystyrene surface of the particles. After coating the particles are said to be ‘sensitized’ (they still look milky). If a drop of antibody coated particles is mixed with a drop of sample (urine, serum, etc) containing antigen, then the latex will become ‘agglutinated’; the milky latex looks ‘curdled’. The sub-microscopic particles aggregate to form large visible clumps, resembling chunks of chalk rolling around on a plate. This is principle of all latex agglutination tests. Simple latex agglutination slide tests are performed on a glass or paper slide to give a yes/no answer. A simple slide test does not require a fancy instrument it does not even require electricity. Latex test are inexpensive as compared with the other techniques (Bangs, 1988). The research of our group on rapid diagnosis test of FMD dates back to the year 1991. The development of the method was carried out in three stages during the year 1991-1996 at the laboratories of SAP Institute. The first stage comprises the preparation of uniform latex particles a modification of well known emulsion polymerisation process were elaborated, where styrene monomer was polymerised. After the preparation of latex particles, they are coated with rabbit anti FMD IgG, which were against A22 Mahmatlı and O1 Manisa FMD viruses. The latex particles are coated with antibody via van der Waals forces and they are store in 2 mL sized capped tubes, to be used test reactive. During the first stage of this research, some preliminary agglutination tests were also carried out. The detailed description of latex preparation and coating procedures are given elsewhere. (Ozdural et al., 1994). The second stage of this research was mainly focused on differentiation of types of FMD virus. A22 Mahmatlı and O1 Manisa FMD viruses were taken as test viruses so as to demonstrate the type detection (Ozdural et al., 1994). The logical extension and the third stage of this work was the comparison of the results of this newly developed latex agglutination technique with well established CFT technique (Ozdural et al., 1996). 2. Material and methods 2.1. Preparation of latex particles A modified emulsion polymerisation technique is used for polymerisation of styrene monomer (Petkim A.S., Turkey). Long chain water-soluble polymers were added to the emulsion polymerisation medium. So as to obtain mono-sized latex particles. Detailed description of the procedure is given elsewhere (Ozdural et al., 1994). 2.2. Coating (sensitising) of latex particles The rabbit anti FMD IgG, which were against A22 Mahmatlı and O1 Manisa FMD viruses were obtained from Pirbrigth Laboratory (UK). Lowry protein determination technique is 222

used for determining the IgG concentration (Peterson, 1983). The optimum amount of coated IgG were found by trying different initial IgG concentrations. 2.3. Latex agglutination Test The agglutination procedure is carried out on a black coloured analysis plate with a three detection spot. 2.3.1. Detection of agglutination a)

b) c) d)

The concentration of IgG coated latex (test reactive) were as follows: Latex concentration (D= about 1µm) 5.68 mg latex/1 mL latex suspension IgG concentration at coated latex suspension 0.95 mg IgG/1mL latex suspension 10 µL of test reactive placed on one of the detection spot of the analysis plate 10 µL of field sample (prepared for CFT) dropped on to the test reactive and mix for 15-20 second Tilt the analysis plate carefully a couple of times and look for agglutination If the result is ambiguous control the test results by using control reagent (BSA solution 0.1% by weight).

2.3.2. Differentiating the FDA virus types For this purpose all three detection spots of the analysis plate are used. a) b)

c)

10 µL of field sample (prepared for CFT) placed on all three detection spots of the analysis plate - Drop 10 µL of BSA solution (0.1% by weigth) to the first detection spot of analysis plate - Drop 10 µL of test reagent prepared against O1 Manisa FMD virus to the second detection spot of analysis plate - Drop 10 µL of test reagent prepared against A22 Mahmatlı FMD virus to the third detection spot of analysis plate Tilt the analysis plate carefully a couple of times and look for agglutinations 3. Results and Discussion 106 field samples were simultaneously cross-tested with using both CFT and LAT (developed in this work) techniques. Table 1. summarises the results.

223

Table 1. Cross Test Results of Field Samples CFT ___________________________________

LAT __________________________________

Number of samples

A22 positive

O1 positive

Number of samples

A22 positive

O1 positive

95

-

+

94

-

+

4

+

-

10

+

-

7 _____ 106

-

-

2 _____ 106

-

-

Table 1 illustrated the close agreement between CFT and LAT (developed in this work) results. Thus one might conclude that the LAT is good candidate for being routinely used in the field for FMD virus diagnosis. Furthermore, the LAT technique does not require a fancy and complicated instrument and it is cost effective as compared with the other techniques. Due to the high sensitivity of LAT, prepared in this work, special attention should be given to the time of observation of agglutination. Prolonged time, results cross-reactions. Alternatively cross-reactions can be minimized by diluting the sample, so as to generate specific protocols for different types of FMD virus diagnosis. References Bangs, L.B., 1988. Latex agglutination tests. Amer. Clin. Lab. News, 7, 20-26 Montassier, H.J., Araujo, J.P., Pinto, A.A., 1994. Rapid co-agglutination test for the detection and typing of foot-and-mouth disease. J. Virol. Methods 50, 29-42 Ozdural, N., Yigit, A., Çelik, N., 1994. Sap virusu tayinine yönelik lateks aglutinasyon test kiti geliştirilmesi. 1. Ulusal Veteriner Mikrobiyoloji Kongre özet kitapcığı, Ankara, Turkiye, 61 Ozdural, N., Candas, A., Cengiz, M.,1996. Diagnosis of samples came from fields with polystyrene latex particles coated with antibodies against O1 Manisa and A22 Mahmatlı FMD virus. 1. International Veterinary Microbiology Congress Abstract, İstanbul, Turkey, 47 Reid, S.M., Ferris, N.P., Brüning, A., Hutchings, G.H., Kowalska, Z., Akerblam, L., 2001. Development of a rapid chromatographic strip test for the pen-side detection of foot-and-mouth disease virus antigen. J. Virol. Methods 96,189-202 Peterson, G.L. 1983. Determination of total protein. Methods Enzymol. 91, 95-121 Reid, S.M., Fortsyth, M.A., Hutchings, G.H., Ferris, N.P.,1998. Comparison of reverse transcription polymerase chain reaction, enzyme linked immunusorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease. J. Virol. Methods 70, 213-217

224

Reid, S.M., Hutchings, G.H., Ferris, N.P., 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 Ünver G., Alkan F., 2000. Serotyping of FMDV with coagglutination test as an alternative to CFT and ELISA. Report of the session of the FAO research group of the standing technical committee of the European Commission for the control of foot-and-mouth disease. Borovets, Bulgaria, 152-158.

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Appendix 28

FAO Collaborative Study Phase XVII: Standardisation of FMD Antibody Detection D J Paton, R M Armstrong, L S Turner, P A Hamblin, M Corteyn, D Gibson, J Anderson Institute for Animal Health, Ash Road, Pirbright, Woking Surrey GU24 0NF

Introduction The aims of the current study were agreed at the FAO EU FMD meeting in Borovets, 2000. The previously agreed reference sera to O Manisa, A22 and C Noville were to be adopted by O.I.E. as official standards. New reference sera were to be prepared against O SKR, A Iran 96 and Asia 1 Shamir. These should be distributed for evaluation, along with a panel of coded proficiency test sera. New primary sera were prepared at the start of 2001, but the FMD epidemic severely delayed subsequent progress. During 2002, the candidate reference sera were distributed and this report is mainly concerned with their evaluation. No proficiency testing with a panel of coded sera was performed. Materials and Methods Candidate reference sera for each of the FMD strains O SKR, A Iran 96 and Asia 1 Shamir were derived by diluting strong positive original sera in a standard, pooled negative serum (Table 1). Strong positive original sera were obtained from cattle after either intradermolingual inoculation (O SKR) or vaccination, including a 21 day boost (A Iran 96 and Asia 1 Shamir). Participants (Table 2) were requested to examine the sera by both ELISA and virus neutralisation test (VNT). In the case of ELISA, participants were asked to use both the liquid phase blocking ELISA (LPBE) and the solid phase competition ELISA (SPCE) as well as other available tests, including those for non-structural (NS) antibodies. All laboratories received a protocol for a version of the SPCE modified from that of Mackay et al. (2001), along with the necessary reagents for a serotype O version of the test. Results Results were available from all but one of the laboratories that had been sent samples and these are summarized in Tables 3-6. For the standard tests, the cut-offs used at Pirbright have been applied for interpretation of the results, although it is appreciated that other interpretations may be in use. Some results are not tabulated, since they were not directly comparable due to differences in the way the tests were performed or in which the results were expressed. The negative serum was found negative in all tests, in all labs.

226

The serotype O and A VNT results were strain dependent and use of a non-homologous subtype of virus, such as O Manisa instead of O SKR, or A22 instead of A Iran 96, generally reduced the antibody titre (Table 3). For the serotype O sera, the strong positive, weak positive and cut-off sera had mean arithmetic titres of 1:193, 1:37 and 1:15 respectively from the three laboratories who tested it using the homologous virus. Equivalent mean homologous titres were 1:661, 1:148 and 1:57 for the serotype A and 1:88, 1:22 and 1:8 for the serotype Asia 1 sera respectively. The results of LPBE testing are summarised in Table 4, except for those of lab 2, which reported results for serotypes O and A as percentage inhibitions at a single dilution. In this case, the strong positive sera were scored positive, as was the weak positive O SKR serum. The weak positive A Iran 96 serum and both cut-off sera were scored negative in lab 2. For the other LPBE results, all but one laboratory had the correct result with each of the strong positive sera, whereas the weak positive and cut-off sera gave inconsistent qualitative results (Table 4). There were also wide fluctuations in the quantitative results. For example, two labs found all of the type A sera positive with much higher titres than the others. The variability was not obviously related to the subtype used as antigen and for generation of the polyclonal rabbit and guinea-pig reagents. For example, most labs used O Manisa reagents for serotype O, but there were nevertheless, considerable quantitative and qualitative differences in results. Eight laboratories provided results from a serotype O specific SPCE test, after using the original or modified method of Mackay et al. (2001), which have cut-offs of 30% and 60% respectively (Table 5). One lab evaluated a new ELISA kit from Lelystad and another used a SPBE. Some labs also reported results for prototype SPCE based on type A specific or type Asia 1 specific reagents (Table 5). The strong positive sera were mostly found positive, but the weak positive sera sometimes gave results just below the cut off level. The cut-off sera were mostly scored negative. More sera were scored positive using the original Mackay method and cut-off of 30%, than using the modified method and the 60% cut-off. Some labs also tested for cross-serotype reactivity in VNT and structural protein ELISAs (data not shown). Results from Lab 1 for VNT and lab 2 for LPBE, indicated no or negligible cross-serotype reactivity by VNT and LPBE respectively. SPCE results from Pirbright and lab 2 showed weak cross-reactivity, with the A Iran 96 strong positive serum scoring positive or near-positive in the test using O Manisa reagents. The CEDI serotype O test evaluated by lab 4 scored all of the heterologous strong positive sera as positive, and also gave borderline reactions with some of the weak positive heterologous sera. In different NS tests, the sera obtained from vaccinated animals (serotypes A and Asia 1) were consistently negative. All NS tests scored the strong positive O SKR serum as positive. In-house NS tests gave a positive result with the weak positive O SKR serum, whilst it was scored positive, weak positive or negative by the Chekit-FMD-3ABC ELISA (Bommeli) in the three labs that evaluated this kit. Only the in-house blocking 3ABC ELISA of lab 8 scored the O SKR cut-off serum as positive.

227

Discussion It was not always clear how closely laboratories had followed the methods described in the OIE Manual of Diagnostic Tests. For example, a range of unusual serum dilutions was used to calculate end-point titres. A further point of ambiguity was how end-point titres had been calculated from serum dilutions, and whether initial or final serum dilutions had been used for this purpose. This needs to be standardised and it is recommended that final dilutions are used, as described in the OIE Diagnostic Manual. As expected, consistent results were obtained for the negative and the strong positive sera. However, the weak positive sera scored positive or inconclusive in VN tests with homologous viruses. The cut-off sera scored inconclusive or negative in the equivalent tests. Similar, though variable, results were obtained with ELISA, indicating that these sera are too weak for use as reference standards. There was an indication of reduced sensitivity if a heterologous strain of virus was used in the VNT, for example using O Manisa in place of O SKR, or A22 in place of A Iran 96. In the case of the A Iran 96 weak positive serum the mean arithmetic titre fell from 1:148 with the homologous virus to 1:13 with A22. The effect of test strain selection on the LPBE and SPCE was difficult to evaluate from these results, although in the case of LPBE, use of A22 instead of A Iran 96 did appear to impair sensitivity. The most inconsistent results were obtained with the A Iran 96 sera. In some labs, the titres obtained for VNT and LPBE were much higher than in others, such that even the cut-off serum was sometimes clearly positive. The O SKR sera should be suitable reference standards for NS serology once strengthened. The definitions for the weak and cut-off sera should be improved and explicitly related to the purpose for which testing is performed. It is suggested that the weak positive serum should represent a minimum standard for detection in any test used for herd-based serosurveillance. It should be primarily designed by reference to the virus neutralization test (VNT) using homologous virus. The cut-off for the VNT when screening on a herd basis is 1:45. Therefore the weak positive serum should have a mean VNT value of 1:64. The cut-off serum should represent a minimum standard for detection in any test used for individual animal certification. It should be primarily designed by reference to the virus neutralization test (VNT) using homologous virus. The cut-off for the VNT when screening on an individual animal basis is 1:16. Therefore the cut-off serum should have a mean VNT value of 1:32. Conclusion More explicit instructions should accompany ring test reagents, to improve standardisation of approaches and interpretations. It is difficult to produce weak positive and cut-off sera that behave similarly in different tests. Homologous VNT should be the gold standard, but preferably, a range of sera from different animals should be available in order to select standards with the broadest range of acceptable qualities.

228

Initial results with the SPCE and NS tests are promising and need to be further evaluated. It is proposed that the weak and cut-off positive sera need to be made stronger, so that the weak positives are consistently positive by most labs by VNT, and the cut-off sera are consistently inconclusive positive (i.e. not negative). Acknowledgements Thanks are due to all of the labs and their staff, who participated in this collaborative exercise. References Mackay DKJ, Naci Bulut A, Rendle T, Davidson F, Ferris NP. (2001). A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus. Journal of Virological Methods 97, 33-48.

229

Table 1. Dilutions of strong positive original sera to make reference sera Original sera

Dilution of original sera to make: Strong +ve serum Weak +ve serum

Cut-off serum

O SKR (34 days after infection)

1:2

1:10

1:42

A Iran 96 (39 days after 1st vaccination)

1:2

1:24

1:90

Asia 1 Shamir (47 days after 1st vaccination)

1:2

1:18

1:80

Table 2. Participants Dr Roland Silber, Vienna, Austria Dr Karl Sorensen, Lindholm, Denmark Dr Emiliana Brocchi, Brescia, Italy Dr Luis Romero, Madrid, Spain Dr Naci Bulut, Ankara, Turkey Dr Kris de Clercq, Ukkel, Belgium Dr Bernd Haas, Tuebingen, Germany Dr Aldo Dekker, Lelystad, The Netherlands Dr Barabara Thuer, Mittelhausern, Switzerland

230

Table 3: Summary of Virus Neutralisation Test results

PIRBRIGHT LAB 1 LAB 2 LAB 3 LAB 4 LAB 5 LAB 6 LAB 8 LAB 9

O SKR Mean reciprocal titres obtained for reference sera Strong +ve Serum Weak +ve Serum Cut-off Serum Log10 Arithmetic Log10 Arithmetic Log10 Arithmetic 2.2 159 1.67 47 1.27 19 2.1 128 1.65 45 1.2 16 1.9-2.2 80-160 1.3-1.6 20-40 1 10 2.2 159 1.2 16 neg neg 1.95 90 1.2 16 neg neg 1.73 54 1 10 neg neg 2.41 260 1.66 46 1.2 16 1.65 45 1.35 23 0.6 4 2.5 342 2 90 1.3 18

PIRBRIGHT LAB 1 LAB 1 LAB 2 LAB 3 LAB 4 LAB 5 LAB 6 LAB 8 LAB 9

A Iran 96 Mean reciprocal titres obtained for reference sera Strong +ve Serum Weak +ve Serum Cut-off Serum Log10 Arithmetic Log10 Arithmetic Log10 Arithmetic 2.18 151 1.43 27 1.12 13 2.4 256 1.35 24 1.05 12 >3.0 >1024 2.1 128 1.65 48 1.9-2.2 80-160 1.0-1.3 10-20 neg neg 3.45 2818 2.11 129 1.71 51 1.65 45 neg neg neg neg 1.35 22 1.08 12 0.78 6 2.71 512 2.26 182 1.81 65 2.25 181 0.9 8 neg neg 3 959 2.4 257 2 101

PIRBRIGHT LAB 1 LAB 2 LAB 3 LAB 4 LAB 5 LAB 6 LAB 8 LAB 9

Asia 1 Shamir Mean reciprocal titres obtained for reference sera Virus used Strong +ve Serum Weak +ve Serum Cut-off Serum Log10 Arithmetic Log10 Arithmetic Log10 Arithmetic 2.12 132 1.6 40 1 10 Asia 1 Shamir 1.65 48 1.2 16 0.9 8 Asia 1 Shamir nd nd nd nd nd nd nd 2.2 159 1.2 16 neg neg Asia 1 1.65 45 neg neg neg neg Asia 1 Shamir 2.1, 1.88 128, 75 1.43, 1.2 27, 16 0.9, 0.9 8, 8 Asia 1 Shamir 2.56 363 1.81 65 1.2 16 Asia 1 2.1 128 1.35 23 0.6 4 Asia 1 Shamir 2.4 255 1.7 53 1.2 17 Asia 1 Nep29/97

Key to interpretation used at Pirbright and applied to all results >=1.65 (>=45) is positive 1.2-1.5 (16-32) is incocnclusive

<1.2 (16) is negative

Virus used O SKR O Manisa O O SKR O Manisa O Lausanne O SKR O Manisa O UK 31/01 Virus used A Iran 96 A22 A Iran 96 A A Iran 97 A 22 Iraq A22 A Iran A Iraq A Iran 96

Table 4: Summary of Liquid Phase Blocking ELISA results

PIRBRIGHT LAB 3 LAB 5 LAB 6 LAB 8** LAB 9*

O SKR Mean reciprocal titres obtained for reference sera Strong +ve serum Weak +ve serum Cut-off serum Reagents used Log10 Arithmetic Log10 Arithmetic Log10 Arithmetic 2.88 759 2.25 178 1.38 24 O SKR 2.93 851 2.16 145 1.54 35 O Manisa 10 5 5 O Lausanne 2.89 776 2.31 204 1.82 66 O Manisa 1.49 31 0.95 9 neg neg O Manisa 1.96 92 1.40 25 1.23 17 O Manisa

PIRBRIGHT LAB 3 LAB 5 LAB 6 LAB 8** LAB 9

A Iran 96 Mean reciprocal titres obtained for reference sera Reagents used Strong +ve Serum Weak +ve Serum Cut-off Serum Log10 Arithmetic Log10 Arithmetic Log10 Arithmetic >3.45 >2818 2.8 631 2.15 141 A Iran 96 3.9 7943 2.81 646 2.28 191 A Iran 97 160 20 5 A22 2.71 513 1.71 51 neg neg A22 2.47 300 1.36 23 0.85 7 A 3.25 1789 1.88 75 1.32 21 A Iran 96

PIRBRIGHT LAB 3 LAB 6 LAB 8** LAB 9**

Asia 1 Shamir Mean reciprocal titres obtained for reference sera Reagents used Strong +ve serum Weak +ve serum Cut-off serum Log10 Arithmetic Log10 Arithmetic Log10 Arithmetic 3.08 1202 2.45 282 1.73 54 As 1 Sham 3.4 2512 2.74 550 2.05 112 As 1 Sham Asia 1 2.55 355 1.78 60 neg neg >2.7 512 2.28 191 1.64 44 Asia 1 2.1 128 1.5 32 1.1 13 Nep 29/97

Key to interpretation used at Pirbright and applied to all results >=1.95 (>=90) is positive 1.65-1.95 (45-89) is inconclusive

* Monoclonal antibodies used as capture and conjugate reagents ** Solid phase blocking ELISA format used

<1.65 (45) is negative

Table 5: Summary of results from Solid Phase Competition ELISA

PIRBRIGHT LAB 1 LAB 2 LAB 4 LAB 5 LAB 6 LAB 8 LAB 9 LAB 1* LAB 3* Blocking tests LAB 4 CEDI

O SKR Mean percentage inhibition values obtained for reference sera Strong +ve Weak +ve Cut-off serum serum serum Reagents used 91 64 10 O Manisa 87 61 33 O Manisa 54 40 12 O Manisa 79 47 13 O Manisa 90 73 35 O Manisa 83 54 25 O Manisa 88 53 14 O Manisa 52 24 7 O Manisa 61 29 6 O Manisa 85 61 35 O Manisa 93

73

53

O Manisa

PIRBRIGHT LAB 1* LAB 1* LAB 3*

A Iran 96 Mean percentage inhibition values obtained for reference sera Strong +ve Weak +ve Cut-off Reagents used serum serum serum 89 45 12 A Iran 96 85 42 24 A22 85 71 48 A Iran 96 95 86 71 A Iran 97

PIRBRIGHT LAB 1* LAB 3*

Asia 1 Shamir Mean percentage inhibition values obtained for reference sera Strong +ve Weak +ve Cut-off serum serum serum Reagents used 93 78 42 Asia 1 shamir 77 54 35 Asia 1 shamir 90 88 71 Asia 1

* These results are for original Mackay et al (2001) method, with purified antigen and 30% cut-off, wheras others use modified method including cruder vaccinal antigen and reagents distributed for this exercise with working cut-off of 60% Positive results shown shaded

Table 6: Summary of results from non-structural antibody ELISAs Strong +ve serum

O SKR Weak +ve serum

Cut-off serum

Indirect 3ABC ELISA (In-house test, Pirbright) positive positive doubtful Blocking 3ABC ELISA (In-house test, Lab 8) 276 59 12 3ABC trapping ELISA (In-house test, Lab 9) 0.75 0.25 negative Chekit-FMD-3ABC (Bommeli Kit, Lab 9 results) positive doubtful negative Chekit-FMD-3ABC (Bommeli Kit, Pirbright results) positive negative negative Chekit-FMD-3ABC (Bommeli Kit, Lab 5 results) positive positive negative Results for all reference sera from vaccinated animals (A Iran 96 and Asia 1 Shamir) were negative

Appendix 29

Specific detection of antibodies against FMD by engineered β-galactosidase enzymatic sensors Esther Blanco (1), Jordi X.Feliu (2), Francisco Sobrino (1,3) and Antonio Villaverde (4) (1) (2) (3) (4)

CISA-INIA. Valdeolmos 28130, Madrid.Spain Biokit.Barcelona.Spain CBMSO, UAM. Madrid.Spain UAB. Bellatera, Barcelona.Spain

SUMMARY The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. In this work we have explored the antibody responsiveness of newly engineered β-galactosidases accommodating up to three insertions per monomer of an antigenic FMDV peptide, that were much more immunoreactive than their parental proteins. The results obtained analyzing sera from immune animals (infected or vaccinated), as well as specific monoclonal antibodies, by the FMDV biosensor prototypes, point out the utility of these devices for simple diagnosis of FMD in an easy and fast assay. INTRODUCTION In a general context, a biosensor is constituted by a sensing element coupled with, or integrated within a physicochemical transducer. In these analytical devices the presence of a target ligand induces changes on the property of the sensor surface, which are then converted to a macroscopic signal or electronic information. Whereas the recognition component is a biological species with binding properties, the transducer element can be non-biological. Both elements have to be sufficiently close together to allow a good transmission of the alterations promoted by the analyte upon binding. Proteins possessing an intrinsic signal transduction capacity, such as enzymes, are good candidates to be used as transducer elements. Up to now, several enzymes with easy detectable activity, like green fluorescent protein or β-galactosidase have been adapted upon specific engineering, to detection of different ligands, including ions, enzyme inhibitors and antibodies. Escherichia coli β-galactosidase is made up of four identical subunits, and catalyses the hydrolysis of lactose into galactose and glucose as well as different galactopyranoside analogue substrates that render coloured products. β-galactosidase has been employed as a carrier protein for bioproduction of heterologous polypeptides, since fusions at both ends and insertions of small peptides in solven-exposed surfaces keep its native structure, stability and enzymatic activity. Employing this principle, several recombinant β-galactosidases, accommodating one copy per monomer of a 27-mer antigenic peptide from the VP1 capsid protein of FMDV serotype C, were constructed. 235

The activity of the resulting enzymes is stimulated by antibodies directed against the viral peptide up to a 200% factor. Interestingly, the new FMD biosensor prototypes, displaying a higher number of peptide copies per monomer are much more immunoreactive than those with only one copy, reaching activation factors of about 400%. MATERIAL AND METHODS The figure 1 shows the different recombinant β-galactosidase proteins displaying one, two or three copies of the FMD antigenic peptide, named GH23, that corresponds to residues 134-156 from VP1 capsid protein of FMDV isolate C-S8c1 (Spain 70). The peptide was inserted at amino acid positions 279, 795 and a fusion to the C-terminus of β-galactosidase. The engineered enzymes were purified by affinity chromatography and dialysed against Z buffer. The modulation assay was performed as described the figure 2, and the serum used included the monoclonal antibodies 4C4, SD6 and 3E5, that recognise different B-cell epitopes located in the GH23 peptide, and sera collected from experimentally vaccinated and infected pigs, cattle and guinea-pigs (1). RESULTS The reactivity of the chimeras displaying two of three copies to GH23 peptide with FMDV-directed mAbs was higher than that of single insertion mutants. In addition, the multiple peptide insertion affected the properties of the enzymes by reducing their specific activity compared to the parental, pseudo-wild type protein LACZ (2), a promising feature regarding the possible regulable nature of these enzymes (3). As expected, the activity of most of these enzymes can be modulated upon incubation with sera from immune guinea-pigs (Figure 3). Despite an important variability in the activity increase of the new proteins to antibodies, improved activation factors have been observed. In particular, protein HT7278CA, that displays 12 copies of the viral peptide per enzyme molecule, shows an activity increase close to 400% upon incubation with either anti-peptide monoclonal antibodies or sera from immune guinea pigs. The analysis of the enzymatic activity at different antibody amounts demonstrated that the extent of activity increase depends on antibody concentration (Figure 4). Both the improved activation factors and the reduced incubation times prompted us to investigate the effectiveness of these new sensors with sera from immune animals. Therefore, we tested several sera from swine and cattle that were either vaccinated or infected with FMDV. Swine infected or vaccinated with FMDV C-S8c1, the virus serotype C /subtype C1) from which the peptide inserted in β-galactosidase had been selected, are those that gave the highest activation factors relative to a non-immune serum.

236

Sera from cattle immunised with a trivalent vaccine including viruses of serotypes A, O and C (this one of subtype C3 but not C1) or infected with a C3 virus only induced modest activations factors. No differences were observed between sera from heterotypically infected animals (serotype A and O) and non-immune serum. DISCUSSION The results obtained with these new generation of β-galactosidase biosensors, indicated that the enzymatic assay is potentially useful for detection of anti-FMDV antibodies in different hosts, without the requirement of species-specific antibodies for their detection. Therefore the proteins characterised here could be the basis of a new and fast diagnosis assay, which would require further and detailed verification with an extended number of sera. The utility of this kind of biosensors including antigenic peptides from non-structural proteins could be especially interesting in order to develop a new assay capable to discriminate between infected and vaccinated animals. Therefore, we are now working in the constructions of new biosensors displaying different antigenic peptides from nonstructural 3B. In addition, the different activation factor obtained depending on the virus serotype would allow determining antigenic distances between virus strains, which could represent a powerful tool in epidemiological studies but also in the analysis of architectural features of continuous B-cell epitopes. REFERENCES (1) J.X.Feliu, N.Ferrer-Miralles, E.Blanco, D.Cazorla, F.Sobrino and A.Villaverde. Biochim.Biophys.Acta Apr 29; 1596 (2): 212-24 (2002). (2) J.X.Feliu, X.Carbonell, A.Villaverde, FEBS Lett. 474 (2000) 87-92. (3) N.Ferrer-Miralles, J.X.Feliu, A.Villaverde. Biochem.Biophys.Res.Commun. 275 (2000) 360-364. (4) A.Benito, M.Vidal, A.Villaverde. J.Biotechnol. 29 (1993) 299-306.

237

Figure 1

A) Inserted sequence (GH23 peptide)

Virus FMDV (C-S8c1)

134

TTYTASARGDLAHLTTTHARHLP156

B) 795

279

C

-C

LACZ

-C -C -C

M278VP1 JX795A JX8CA

-C -C

HD78CA HD72CA

-C

HD7872A

-C HT7278CA • •

Amino acid sequence of the viral peptide from FMDV inserted in β-galactosidase Schematic representation of the different recombinant β-galactosidase proteins with the FMDV insertion sites.

238

Figure 2

ASSAY 1.Recombinant protein (2 pmol) 2.Sera problem

1 hour incubation at 28ºC 3.ONPG (2-nitrophenyl-β-D-galactopyranoside)

20 minutes incubation 4..Na2CO3 1M (stop the reaction)

Results = A414 antibodies – A414 in absence of antibodies

239

400 350

(%)

β-Galactosidase activity

Figure 3

SD6 4C4 3E5 Guinea-pig

300 250 200 150 100 50 0

JX8CA

HD7872A JX795A HD78CA HT7278CALACZ HD72CA M278VP1

One

Two

Three

Insertions Modulation of enzymatic activity of the recombinant proteins using anti-FMDV VP1 monoclonal antibodies SD6, 4C4, 3E5 and immune guinea pig sera.

240

Figure 4

400

HD72CA HD7272A HT7278CA M278VP1 JX795A

3E5

SD6

Sera

400 300

200

200

100

100

β-Galactosidase activity (%)

300

10-2

10-1

100

10-1

100

Antibody concentration (ng/µl)

101

10-4

10-3

10-2

Serum dilution

Effect of the antibody concentration in the enzyme activation with different monoclonal antibodies and immune guinea pig sera.

241

Appendix 30

Modification and further validation by different laboratories of a FMDV type O specific ELISA for detection of antibodies in all susceptible species L. Jacobs 1) ,G. Chènard2) , T. Jelsma 1) and K. de Clercq 3). 1)

Cedi-Diagnostics B.V. The Netherlands ID-Lelystad, the Netherlands 3) CODA, Belgium 2)

Summary A quick and simple Mab based ELISA for the detection of antibodies directed against type O of FMDV was developed by ID-Lelystad. The ELISA was specially developed to be used on a semi-automatic pipetting system. Specificity and sensitivity of the ELISA was previously described by G. Chènard et al. (2001). To decrease the variation between test results when used manually and to increase the specificity some slight modifications were applied. Additionally the modified Ceditest® FMDV type O ELISA was taken in production and send to different laboratories for validation. The modified Ceditest® FMDV type O was compared with the original test using the same sera, if available. Additionally the test was validated during a workshop organised by the FAO-EUFMD commission in Sofia, Bulgaria, by the CODA-CERVA-VAR in Belgium and by the world reference laboratory of FMDV in Pirbright, England. Also the Ceditest type O ELISA was used by the BFAV Tűbingen/Riems, Germany and by the State Veterinary Institute, Lindholm in Denmark. Test procedure and modifications Original test Plates coated with antigen Add 40 μl buffer + 10μl serum and 10 μl serum Incubate 1 hour at room temperature Add 50 μl conjugate Incubate 1 hour at room temperature Wash the plates Add 100μl TMB Stop the reaction after 15 minutes

Modified test Plates coated with antigen Add 90 μl buffer (+5% horse serum ) Incubate 1 hour at room temperature Wash the plates Add 100μl conjugate Incubate 1 hour at room temperature Wash the plates Add 100μl TMB Stop the reaction after 15 minutes

Incubation time of the modified Ceditest® FMDV type O is the same, only one extra washing step is included to increase the specificity and to decrease test variation. Calculation and interpretation of the results of the modified Ceditest® FMDV type O %inhibition = 100-((OD monster-OD blank)/OD max-OD blank)*100) Positive >= 50% Negative < 50% 242

Comparison of the sensitivity and specificity after modification Table 1: Sensitivity and specificity of the Ceditest® FMDV type O before and after modification Species Sensitivity Before

Sensitivity After modification Bovine 96.9 (n=548) 98.2 (n=214) Porcine 90.8 (n=392) 85.7 (n=105) Ovine 96.2 (n=474) 98.8 (n=82) Average 94.6(n=1177) 95 (n=401)

Specificity Before

Specificity After modification 95.5(n=1544) 98.9 (n=1315) 99 (n=610) 99.7 (n=295) 94.1 (n=414) 99.8 (n=751) 96.2(n=2782) 99.5 (n=2391)

Note: The virus neutralisation test was the golden standard Sera used for validation are described in detail by G. Chėnard et al., 2001. To be short: Cattle were vaccinated/infected with: O1 Manisa, O1 Hong Kong. O1 Italy, O1 Weerseloo, O1 Algarije Pigs with:O1 Italy, O1 Taiwan, O1 BFS Sheep with:O1 Marokko

Validation performed by other laboratories Validation done during a regional workshop on FMD organised by the FAO-EUFMD commission in Sofia, Bulgaria from 18-22 March 2002 (data provided by K. de Clercq) During this workshop 3 ELISA’s, the Checkit-FMD-3ABC, the Ceditest® FMDV type O and the SPC-ELISA with the reagents and protocols of WRL in Pirbright were practised and discussed. Sera used during the workshop: • Sera from cattle in Turkey (n=107) vaccinated with a trivalent vaccine ( O1 Manisa, A22 and Asia 1) • Sera from an outbreak in Bulgaria (n=27) • Sera from FAO phase 16 of which 2 originated from type O vaccinated animals • Sera from type O infected cattle from Greece (n=3) • Sera from negative cattle from Greece (n=15) • Sera from negative sheep from Greece (n=9) Results Of the sera from Turkey 100 scored positive in the Ceditest Four sera that were negative in the Ceditest were also negative in the SPC-ELISA (Pirbright). Two sera that were negative in the Ceditest were positive in the SPC-ELISA and one serum was not tested in the SPCELISA.

243

From the Bulgarian outbreak all animals scored positive. Additionally the FAO sera and the type O infected cattle from Greece scored positive. Sera from negative cattle and sheep from Greece scored negative. Overall comment: “the Ceditest ® FMDV type O was repeatable among participants and showed to be very robust” Validation performed by CODA-CERVA-VAR, Belgium, February 2002 (data provided by K. De Clercq) Sera used for the validation • Sequential sera of two calves vaccinated with O1 Manisa starting from day 3 post vaccination until day 63 post vaccination • Sera from negative sheep (n=80). • Sera from negative pigs (n=32). • Sera from negative cattle (n=152) • To determine the test variation a mixture of serum of three vaccinated bovids (O1, A5 and C1) were used undiluted and in different dilutions. Results • Specificity in pigs and sheep was 100%. Specificity in cattle was 99.6%. • Considering the sensitivity both calves tested positive from day 3 until the end of the sampling period. • Test variation (see table 2) Table 2: Test variation Dilution Mean % inhibition Undiluted 94.9 1:3 83.9 1:10 60.8 1:20 46.9 1:30 36.7 1:40 30.8

Standard deviation 1 2.2 3.3 2.4 2.7 2.6

Validation performed by World Reference Laboratory of FMD in Pirbright, England, April 2002 (data provided by D. Paton) Sera used for the validation • Sequential sera from 1 sheep inoculated with FMDV type O1 Greece • Field sera from a flock of sheep suspected with FMD during the 2001 outbreak (n=80) • Sequential sera from cattle vaccinated with O1 Manisa (n=60) • Sequential sera from pigs vaccinated with O1 Manisa (n=36)

244

Results

Table 3: Serial bleed from 1 sheep inoculated with FMDV type O1 Greece 94 Days post PI SPC-ELISA with O1 Manisa PI Ceditest ® FMDV infection as antigen type O 0 0 0 1 0 1 2 0 0 3 0 9 4 0 8 5 43 42 6 58 57 8 66 78 9 76 80 10 85 82 12 90 95 14 88 98 16 88 90 20 88 95 23 88 96 Bold is positive Italic is inconclusive Sheep field sera Of a flock of suspected sheep 80 field sera were collected and tested in the virus neutralisation test (VNT), the SPC-ELISA and in the Ceditest® FMDV type O. In the VNT a titre of >45 is considered to be positive and of <11 is considered to be negative. Titres in between are inconclusive. In the SPC-ELISA, PI >60% are considered positive, PI <50% are considered negative and in between inconclusive. In the Ceditest® FMDV type O, PI >=50% are positive and <50% are negative. Of the 80 sheep, 68 tested negative in the as well the Ceditest as in the VNT, 10 sheep tested positive in both assays. Two sheep tested inconclusive in the VNT of which one sheep tested positive and one tested negative in the Ceditest. All sheep that tested positive in the Ceditest tested also positive in the SPC-ELISA. Three sheep that tested inconclusive in the SPC-ELISA tested negative in the VNT and in the Ceditest. One sheep that tested positive in the SPC-ELISA tested negative in the Ceditest and in the VNT.

245

Table 4: Cattle vaccinated with O1 Manisa Animal Days post VNT PI Ceditest® FMDV vaccination titre type O 1 0 Neg 0 21 1/64 91 2 0 Neg -2 21 1/64 61 3 0 Neg 14 21 1/90 84 4 0 Neg 7 21 1/22 32 5 0 Neg 10 21 1/512 87 6 0 Neg 9 21 1/64 62 7 0 Neg 1 21 1/32 34 8 0 Neg -1 21 1/22 47 9 0 Neg 0 21 1/22 58 10 0 Neg 20 21 1/128 85 11 0 Neg 4 21 1/32 43 12 0 Neg 2 21 1/22 46 13 0 Neg 17 21 1/32 59 14 0 Neg -1 21 1/22 48 15 0 Neg -1 21 1/32 57 Bold is positive Italic is inconclusive

All sera positive in the VNT (titre>45) tested positive in the Ceditest® FMDV type O. Of the 8 sera that were inconclusive in the VNT (titres between 11-45) 3 tested positive and 5 negative in the Ceditest® FMDV type O.

246

Table 5: Pigs vaccinated with O1 Manisa Pig

Days post vaccination 1 0 28 2 0 28 3 0 28 4 0 28 5 0 28 6 0 28 7 0 28 8 0 28 9 0 28 10 0 28 11 0 28 12 0 28 Bold is positive Italic is inconclusive

VNT titre Neg Neg Neg Neg Neg Neg Neg 1/16 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg neg

PI SPC-ELISA O1 Manisa as antigen 6 59 6 64 7 62 17 90 14 40 16 83 15 33 10 54 9 69 29 54 16 10 18 15

PI Ceditest®FMDV type O 0 50 -2 56 -1 58 -1 95 -3 43 -4 70 -1 36 -3 35 -2 78 -1 51 1 0 0 -2

In pigs the VNT seems to be very insensitive test when compared to both the SPC-ELISA and the Ceditest® FMDV type O. All sera that tested positive in the SPC-ELISA tested positive in the Ceditest® FMDV type O. Of the 3 inconclusive sera in the SPC-ELISA, 2 tested positive and 1 tested negative in the Ceditest® FMDV type O.

247

Appendix 31

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 BERND HAAS* and KARL JOHAN SORENSEN# *Federal Research Centre for Virus Diseases of Animals (FRCVDA) D-72076 Tübingen, Germany # Danish Veterinary Institute DK-4771 Kalvehave, Lindholm

A study was performed in order to validate ELISAs for the differentiation of FMDV-infected cattle from those that had merely been vaccinated. Sets of sera from naive and from vaccinated animals as well as from cattle that had been vaccinated and subsequently infected were tested for antibodies to nonstructural proteins (NSPs) of FMDV in a commercial test, the Chekit FMD-3ABC bo-ov (Bommeli/Intervet) and a competitive ELISA developed at the Danish Veterinary Institute. Probang samples from infected animals were examined by plaque test and by RT-nPCR in order to determine whether the animals had become virus carriers. With sera from naive or vaccinated, but non-infected animals the specificity of both tests exceeded 99%. In 21 out of 22 bovines, that had been challenged after vaccination, antibodies to FMD polyprotein 3ABC could be detected. The percentage of vaccinated and infected cattle scored positive by NSP serology depended on the test used and the time after infection. The competitive ELISA showed a higher sensitivity than the Chekit ELISA. However, when the cut off value of the Chekit ELISA was set at 10% instead of 20%, the sensitivity of this test could be increased significantly with only a slight loss of specificity. It became evident, that the seroconversion to NSPs in some animals was delayed significantly in comparison to seroconversion to structural proteins as well as in comparison to seroconversion to NSPs in non-vaccinated, infected animals. In both ELISAs the sensitivity exceeded 80%, but had to be seen in relation to time after infection. In contrast to that of the competitive NSP ELISA, the sensitivity of the Chekit test decreased after about four months p.inf.. It can be concluded that the 3ABC-ELISA will be a useful tool in FMD control and eradication programmes facilitating the detection of virus circulation in FMD-vaccinated populations, because on a herd basis, vaccinated and infected cattle can be differentiated from those that had merely been vaccinated. 20 out of 22 vaccinated and infected animals became carriers, proving once again that even high potency vaccines can not prevent the development of a carrier state after massive challenge. Keywords: foot-and-mouth disease, cattle, vaccination, diagnosis, non-structural proteins, 3ABCELISA

248

Material and Methods Sera. In the frame of a validation trial sera of naive German cattle were tested in the CHEKIT ELISA by German regional laboratories; results of 593 sera were reported to the FRCVA until early August 2002 and included into this study. 414 sera of cattle vaccinated experimentally with a commercial vaccine (Bayer AG) were tested at the FRCVDA. 990 sera of naive Danish cattle as well as sera of 960 sheep and 1056 pigs were tested in the competitive ELISA at the Danish Veterinary Institute. 134 samples of sera from cattle vaccinated with commercial FMD vaccines and challenged 21 or, in case of the A24 trial, 28 days post vaccination, with homologous FMD virus, were collected following potency trials that, in principle, had been performed according to the method described in the European Pharmacopoeia. Only one vaccinated animal used for the study had not been protected. The cattle were sequentially bled for up to 22 weeks post infection (w.p.i.). The sera were tested in the Chekit FMD-3ABC bo-ov at the FRCVDA and in the competitive ELISA at the Danish Veterinary Institute. Detection of the carrier state A cell suspension plaque Test (PT) was used to test probang samples for FMD virus. BHK21-CT cells, a special clone of BHK21 cells selected for optimal sensitivity to FMDV, were used for the cell suspension plaque test as previously described (Moss and Haas, 1999). Probang samples were kept at – 70°C until testing and 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 examined. A reverse transcription nested polymerase chain reaction (RT-nPCR) was used as an additional tool to determine whether an animal was a virus carrier. The PCR was carried out as previously described (Moss and Haas, 1999) with some modifications. Probang aliquots examined by RT-nPCR were mixed with extraction buffer immediately after thawing (Trizol, GibcoBRL). Primers were derived from the genome of strain O1 Kaufbeuren, spanning from N-terminus of polymerase 3D to Cterminus of 3A. The PCR resulted in a product of approximately 900 bp. One µl of this product was amplified in a nested PCR. All samples producing a DNA band of the expected size of about 900bp or 580bp, respectively, were considered positive. ELISAs The presence of 3ABC protein was examined using two ELISA - the commercially available CHEKIT FMD-3ABC bo-ov, Dr. Bommeli AG, Switzerland, and a competitive ELISA developed at the Danish Veterinary Institute, Lindholm. The Chekit FMD-3ABC bo-ov was performed according to the manufactures instructions. Briefly, dilutions of the sera were incubated in microtiter plates pre-coated with recombinant FMDV 3ABC viral antigens. Antibody specific for 3ABC bound to the antigen and is recognized by adding a antiIgG-peroxidase conjugate. After washing and adding of a chromogen, the degree of colour which develops in the well was proportional to the amount of antibody specific for 3ABC present in the sample. Results were expressed as % by comparing the optical density (OD) of samples to the OD recorded for the positive control. The manufacturer considers values below 20% as negative, values between 20 and 30 % as ambiguous and values above 30% as positive. However, also the feasibility of a lower cut off value for the doubtful range of 10% was investigated.

249

The competitive NSP ELISA was performed as described previously (Sorensen et al., 1998a) with modifications. Briefly, microtiter plates were prepared by capturing 3ABC protein produced in the Baculovirus expression system with a monoclonal antibody (MabD5) coated on the plates. Dilutions of the sera were incubated and competing antibody bound to 3ABC was recognised by adding the peroxidase conjugated MabD5 directed against the protein. After washing and addition of chromogen (TMB), the colour development was measured and the results expressed as ODp values (percentage of average negative control value). Results Detection of the Carrier state In case of the A24 and O BFS experiments (9 animals), samples were taken weekly and examined in both the plaque test and RT-nPCR. Detailed results are shown in table 1. In all 9 animals virus could be recovered beyond day 28 p. inf. Most virus titres were quite low. Only in two animals, No 9437 and 9259 occasionally virus titres of more than 50 PFU/100µl were recorded. The intermittent nature of virus excretion could be seen especially in case of animals 22129 and 83 793. While PCR results in general were in good agreement with virus isolation results, there were contracting results on some samples of low virus content. The carrier state for all animals included in the study is indicated in tables 3 and 4 in the column 3. 20 out of 22 vaccinated and infected animals became carriers. Cut off and specificity Not all German regional laboratories involved in a validation trial for the Chekit FMD-3ABC bo-ov test had reported their results until early August 2002, but on the basis of available data on 593 cattle sampled for screening purposes unrelated to FMD, the high specificity claimed by the manufacturer could be confirmed. For the calculation of specificity, results in the “doubtful” range were counted as positive, because all doubtful or positive sera would be retested by EITB and true positives can be expected to be confirmed. Using a cut off of 20% only 1 out of 593 sera reacted positively (99,8% specificity) and at a cut off of 10% 3 further sera were scored as “doubtful” (99,3% specificity). Also of 414 sera from animals vaccinated with a commercial vaccine, three gave doubtful result (99,3% specificity). The specificity of the competitive NSP ELISA was determined with sera collected at slaughterhouses in Denmark. A normal distribution of ODp values was obtained with serum samples from 990 cattle, 960 sheep and 1056 pigs (figure 1). The mean ODp values, SD and specificity at a cut off value of 50 (Positive result with ODp value < 50) are shown in table 2. Table 2. Results obtained with sera from naive animals Cattle Sheep Pigs number of sera (n) 990 960 1056 Mean ODp values 82 84 98 Standard deviation (SD) 17 14 13 Specificity % at cut off 50 99.4 99.6 99.8 Sensitivity In table 3 the results of testing sera from 22 vaccinated and subsequently challenged animals in the Chekit FMD-3ABC bo-ov are shown. The ELISA results at different times after infection are given

250

as percentage of net OD of the positive control. Light shading of the boxes indicate a doubtful result of between 10 and 20%. Dark shading indicates a positive result. In table 4 the results of testing sera from 22 vaccinated and subsequently challenged animals in the competitive NSP ELISA are shown. The ELISA results at different times after infection are given as Odp values. Dark shading of the boxes indicates positive result. In figure 2 the results from tables 3 and 4 are summarized by grouping all sera taken during a twoweeks period into one group (only one serum per animal was included into the same group) and calculating the percentage of animals recognized as positive by the ELISAs in that group. The competitive ELISA showed a higher sensitivity than the Chekit ELISA. With a cut of 10% instead of 20%, the sensitivity of the later test could be increased significantly. Seroconversion to NSPs in some animals (6845, 62438, 9430, 36259) was delayed significantly in comparison to seroconversion to structural proteins as well as in comparison to seroconversion to NSPs in nonvaccinated, infected animals, which can be expected during the first (Hamblin 1986) or the second week (Sorenson 1998, Mackay, 1998) after infection, respectively. In both ELISAs the sensitivity exceeded 80%, but had to be seen in relation to time after infection. Discussion In both the competitive ELISA and the Chekit ELISA with the modified cut off value, the percentage of seropositive animals rose from about 65% one month after infection to more than 80% within a few weeks. In contrast to that of the competitive NSP ELISA, the sensitivity of the Chekit ELISA showed a clear decrease after about four months p.inf.. Unfortunately, the competitive NSP ELISA is currently not available as a commercial test kit for large scale usage. With the original cut off, the sensitivity of the Chekit ELISA reached only about 60% after 3 months and quickly declined. However, the specificity of the Chekit FMD-3ABC bo-ov test for cattle was only slightly reduced to 99.3% by applying a lower cut off for the doubtful range of 10% instead of 20%, while the sensitivity was markedly improved. One carrier animal (91368) showed only a very brief and weak seroreaction to NSPs in the competitive ELISA. In the Chekit test, even at a cut off of 10%, four animals, of which 3 were carriers, showed no or almost no reactivity. The specificity of the competitive ELISA with sera from naive animals clearly exceeded 99% for all species tested. Similar results were obtained with vaccinated animals (data not shown); however, experiments are still ongoing. Several authors studied NSP antibody response in vaccinated cattle exposed to infection to determine whether or not animals which are protected by vaccination seroconvert to NSPs following exposure to FMD virus (Berger et al., 1990, Diego et al., 1997, Haas, 1997, Mackay et al. 1998, a, b, c). Brocchi et al. (1998) showed that the great majority of cattle which were vaccinated as part of vaccine potency trials seroconverted to 3ABC following challenge. Since well documented sera from field outbreaks are difficult to obtain, also we used sera from vaccine potency trials. In our experiment 21 out of 22 cattle that had been vaccinated and subsequently infected reacted positively in 3ABC serology. Both Mackay et al. (1998b) and Sorensen et al. (1998b) examined sets of sera collected from vaccinated cattle following challenge in which the carrier status of the animals was known. Some vaccinated animals which became persistently infected following challenge did not seroconvert to NS proteins. Although the majority of vaccinated animals responded to NSPs, the response in some animals was delayed, weak or absent.

251

On the basis of our results and that of others (Bergmann et al. 1998, Brocchi et al. 1998, Mackay et al. 1998a, b and c, Sorensen et al. 1998a and b) we conclude that with NSP serology virus replication involving more than a few animals would be detected by whole herd testing with 3ABC serology. Because infection can be traced in vaccinated herds, in future hopefully it will no longer be necessary to slaughter all vaccinated animals as it was practiced in the Netherlands during the FMD eradication campaign in 2001. Whereas our results showed again that absolute differentiation of infection from vaccination is not possible by serological means alone, it has to be considered that also virus isolation and PCR do not solve this problem under field conditions. Whilst the isolation of FMDV in orogharyngeal scrapings collected from convalescent cattle („probang test“ ) is reliable for the detection of FMDV in clinical samples collected during the acute stage of FMD, the low virus titre and the intermittent nature of virus recovery at later stages and the low sample throughput considerably reduce its value for the detection of carriers in the field. Whereas transmission from carriers to susceptible animals seems to be a very rare event, this hazard is responsible for the long duration of trade restrictions after an outbreak. In order to gain acceptance for “protective” emergency vaccinations the question has to be answered what restrictions will be imposed on the vaccinated animals and their products and on the non-vaccinated part of the national herd. This will certainly depend on the level of confidence with which vaccinated herds can be classified as “non infected”, which depends on the size of infected clusters and the sensitivity of the NSP tests. The size of infected clusters after emergency vaccination is not known. “Vaccinating into an outbreak” or disease introduction briefly after vaccination would probably lead to a relatively high prevalence of infected animals and several animals would seroconvert to NSPs. However, if in a well protected herd one or a few animals get into contact with FMDV, e.g. by a person violating biosecurity rules, this would create different situation. It is currently impossible to say how many of such animals would eventually become virus carriers. Allowing some animals that had been used for vaccine potency trials to survive for some time in high security stables probably does not mimic the field situation after an outbreak perfectly. In vaccine trials animals are usually infected (day 21 or 28 p.vacc.) into the tongue instead by contact and the prevalence of carriers of about 90% recorded here may appear higher than would be expected in the field. However, since there are few well documented post outbreak sera from the field in Europe, an estimate of the sensitivity of NSP tests in vaccinated European herds currently has to be based mainly on sera from vaccine trials. To change that, some well controlled “experimental outbreaks” in high security facilities will have to be funded in the future. In the meantime we should try to gain support for emergency vaccinations complemented by NSP serology and restrictions for the vaccinated animals that, while facilitating to keep them alive and use their products, still allow to quickly restore confidence of trading partners in the FMD-free status of the non-vaccinated part of the animal population. References 1. Berger H-G., Straub O.C., Ahl R., Tesar M., Marquardt O.: Identification of the foot-and-mouth disease virus replication in vaccinated cattle by antibodies to non-structural virus proteins. Vaccine 1990, 8, 213-216. 2. Bergmann I.E., Auge de Mellor P., Neitzert E., Beck E., Gomes I.: (1993) Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens. Am. J. Vet. Res. 1993, 54, 825-831. 3. Brocchi E., De Diego M.I., Berlinzani A., Gamba D., De Simone F.: Diagnostic potential of Mabbased ELISAs for antibodies to non-structural proteins of foot-and-mouth disease virus to

252

4. 5. 6. 7. 8. 9. 10. 11.

12.

differentiate infection from vaccination. Proceedings of the Final Meeting of Concerted Action CT93 0909, Vet. Q. 1998, 20, 20-24. De Diego M., Brocchi E., Mackay D., De Simone F.: The non-structural polyprotein 3ABC of footand-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Arch. Virol. 1997, 142, 2021-2033. Haas B.: Possible role of 3ABC-ELISA in screening of cattle after an outbreak. 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, Brasov, Romania, 23-27 September 1997, 52-54 Hamblin C., Barnett I.T.R., Crowther, J.R.: A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. II. Application. J. Immun. Meth. 1986, 93, 123-129. Mackay D.K.J., Forsyth M.A., Davies P.R., Berlinzani A., Belsham G.J., Flint M., 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, 446-459. Mackay D.K.J.: Differentiating infection from vaccination in foot-and-mouth disease. Proceedings of the Final Meeting of Concerted Action CT93 0909. Vet. Q 1998, 20, 2-5. Mackay D.K.J., Forsyth M.A., Davies P.R., Salt J.S.: Antibody to nonstructural proteins of foot-andmouth disease virus in vaccinated animals exposed to infection. Proceedings of the Final Meeting of Concerted Action CT93 0909. Vet. Q 1998, 20, 9-11. Moss, A. and Haas, B.: Comparison of the plaque test and reverse transcription nested PCR for the detection of FMDV in nasal swabs and probang samples; J. of virological Methods, 1999, 80, 59 - 67 Sorensen K.J., Madsen G.K., Madsen E.S., Salt J.S., Nqindi J., Mackay D.K.J.: Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the nonstructural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus. Arch. Virol. 1998, 143, 1461-1476 Sorensen K.J., Hansen, C.M., Madsen E.S., Madsen, K.G.: Blocking ELISAs using the FMDV nonstructural proteins 3D, 3AB, and 3ABC produced in the Baculovirus expression system, Proceedings of the Final Meeting of Concerted Action CT93 0909. Vet. Q 1998, 20, 17-20.

253

Table 1 Animal Weeks p.inf. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

22 129

PT Neg 7.8 16 0.6 8.8 9.3 3 Neg Neg Neg Neg Neg Neg Neg 0.1 Neg Neg 0.3 Neg Neg

36 259

PCR PT Pos n.d. Pos n.d. Pos Neg Pos >58 Pos 51 Pos 22 Neg 44 Neg 11 Neg 5 Neg 2.4 Neg 0.3 Neg 20 Neg 3.8 Neg Neg Pos 1.8 Neg n.d. Neg n.d. Neg n.d. Neg n.d. Neg n.d.

PCR PT n.d. Neg n.d. Neg Neg Neg Pos Neg Neg Neg Pos 0.3 Pos n.d. Pos n.d. Pos n.d. Pos n.d. Neg n.d. Neg n.d. Pos n.d. Neg n.d. Pos n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

###

###

###

PCR PT Neg 8.5 Neg 17 Pos >140 Neg >85 n.d. 23 Neg 32 n.d. 11 n.d. 47 n.d. 27 n.d. 15 n.d. 8.5 n.d. Neg n.d. 1.6 n.d. 4.9 n.d. 2.8 n.d. 0.3 n.d. 2.5 n.d. 1.6 n.d. 1 n.d. Neg

PCR PT Pos 0.5 Pos 8 Pos 2.5 Pos 5.3 Pos 4.8 Pos 5.4 Pos 8.6 Neg 1.6 Pos 0.1 Pos Neg Pos 1.6 Pos 0.3 Pos 8 Pos 0.6 Pos 0.3 Pos 2.6 Pos 0.3 Pos 3.1 Pos 0.9 Neg 4.4

PCR PT Pos 0.5 Pos 8 Neg 1.3 Pos 4 Pos Neg Pos 0.8 Pos Neg Neg 0.5 Pos Neg Pos 1.4 Pos 0.9 Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg

83 793

88 813

PCR PT Pos 1 Pos 0.8 Pos 8.8 Pos 1.8 Pos 1.5 Pos 0.3 Pos Neg Pos Neg Pos Neg Pos Neg Neg Neg Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

80 835

PCR PT Pos Neg Neg 1.1 Pos 0.1 Pos 7.5 Pos 8.8 Pos 0.4 Neg Neg Pos 0.1 Neg 0.4 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Neg Neg Neg Pos Neg Neg Neg

62 941

PCR PT Pos Neg Neg 6.5 Pos 1.4 Pos 4.3 Pos 3 Pos 2 Pos 1.6 Pos 3.9 Pos 3 Pos 7.3 Pos Neg Pos 1.3 Pos 1.5 Pos 2.6 Pos 5.5 Neg 0.9 Neg 0.1 Neg 0.9 Neg 0.1 Neg 0.1

PCR Pos Pos Neg Pos Pos Pos Pos Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Results of cell suspension plaque test and RT-nPCR obtained with probang samples Plaque test titres are given as PFU per 100 µl

Fig. 1 40

35

30

Frequency %

25

20

15

10

5

0 1-9

Cattle

10-19

Sheep

20-29

30-39

40-49

50-59

60-69

70-79

80-89

90-99 100-109 110-119 120-129 130-139 >139

ODp values

Pigs

Distribution of ODp values obtained with sera of naive animals in the competitive NSP ELISA n = 990 cattle, 960 sheep and 1056 pigs

Fig. 2 100 90 80 70

%

60

Chekit 20% Chekit 10%

50

Competitive

40 30 20 10 0 3-4

5-6

7-8

9-10

11-12 13-14 15-16 17-18 19-20 21-22

weeks p. inf.

Percentage of vaccinated and infected cattle recognized by NSP ELISAs Chekit 10% and 20%, respectively, indicate the lower cut off value used to classify sera as doubtful Sera classified as "doubtful" were considered positive for the calculation of specificity

Table 3 Protection

Carrier

Animal

Dose weeks p. inf.

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

87919 Yes

Yes

O Man

0,25

63

53.4

38.8

30

91367 Yes

Yes

O Man

1

2.3

8.3

7.8

5.6

35.8 8.4

91368 Yes

Yes

O Man

1

3.2

0.6

2.1

3.3

4.8

6257 Yes

No

O Man

1

6.5

7.3

7

4.5

6845 Yes

Yes

O Man

1

9

9.9

10.4

15.3

20614 Yes

Yes

A 22

0,25

64.7

63.9

61.6

38167 Yes

Yes

A 22

1

23.5

28.8

13.8

38168 Yes

Yes

A 22

1

181.3

153.4

107.1

38170 Yes

Yes

A 22

1

19.7

19.8

18.8

38175 Yes

Yes

A 22

0,25

10

16.5

11.3

49341 Yes

No

A 22

0,25

28.9

34

47.5

32.6

50.6

25.2

4.9

18

49343 Yes

Yes

A 22

1

62438 Yes

Yes

A 22

0,25

22129 Yes

Yes

A 24

1

9430 Yes

Yes

A 24

0,25

9437 Yes

Yes

A 24

1

9440 Yes

Yes

A 24

0,25

16.7

19.4

44.1

3.5

3.8

10.4

19.2

25.9

16.6

15.4

12.1

7.8

5.8

6.8

32.6

19.8

16.6

11.7

12.8

13.9

11.2

9.7

9.9

6.5

116.2

46.1

42.9

27.7

25.2

22.8

13.6

16

15

15.6

83793 No

Yes

A 24

0,0625

60.1

52.7

28.2

17

12.3

13

13.5

12

8.1

6.3

88813 Yes

Yes

A 24

1

30.1

39

33.4

58

36.2

37

28.7

32

23.4

32.6

80835 Yes

Yes

A 24

1

62941 Yes

Yes

A 24

0,25

36259 Yes

Yes

O BFS

1

11

14.8

20.1

17.5

19.2

26.8

13.6

18.8

13.4

13.1

15.1

36.4

46.9

41.6

33

28

25.3

21.3

21.8

15

6.3

2.3

183.7

178

48.6

52.1

38.3

Reactivity in the Bommeli Chekit-FMD-3ABC ELISA (Percentage of net OD of positive control)

Table 4 Protection

Carrier

Animal

Dose weeks p. inf.

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

87919 Yes

Yes

O Man

0,25

10

10

6

7

91367 Yes

Yes

O Man

1

40

30

14

20

17

91368 Yes

Yes

O Man

1

60

63

58

48

53

6

6257 Yes

No

O Man

1

70

80

73

66

6845 Yes

Yes

O Man

1

44

21

16

14

20614 Yes

Yes

A 22

0,25

10

5

6

38167 Yes

Yes

A 22

1

27

7

6

38168 Yes

Yes

A 22

1

7

6

6

38170 Yes

Yes

A 22

1

11

6

9

38175 Yes

Yes

A 22

0,25

44

22

20

49341 Yes

No

A 22

0,25

11

7

7

49343 Yes

Yes

A 22

1

12

7

7

62438 Yes

Yes

A 22

0,25

32

9

6

22129 Yes

Yes

A 24

1

59

24

12

9430 Yes

Yes

A 24

0,25

95

86

30

8

8

6

7

5

6

7

9437 Yes

Yes

A 24

1

10

10

17

9

6

6

6

5

6

7

9440 Yes

Yes

A 24

0,25

23

24

11

10

6

6

6

4

5

4

83793 No

Yes

A 24

0,0625

18

12

9

8

7

11

13

14

12

11

88813 Yes

Yes

A 24

1

25

12

8

7

6

5

6

6

7

7

80835 Yes

Yes

A 24

1

42

31

23

19

17

13

15

13

17

16

62941 Yes

Yes

A 24

0,25

51

23

6

6

9

6

5

4

4

4

36259 Yes

Yes

O BFS

1

40

41

6

Reactivity in the competitive NSP ELISA (Percentage of control value)

4

5

4

4

Figure 2

Percentage of vaccinated and infected cattle recognized by NSP ELISAs 100 90 80 70

%

60

Chekit 20% Chekit 10% Competitive

50 40 30 20 10 0

3-4

5-6

7-8

9-10

11-12

13-14

weeks p. inf.

15-16

17-18

19-20

21-22

Appendix 32

Detection of IgG against the Foot-and-Mouth Disease Virus (FMDV) nonstructural polyprotein 3ABC in cattle and sheep R.M.Armstrong, Z.Zhang, N.Aggarwal, P.A.Hamblin and S.J.Cox BBSRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF

Summary Results from in-house assays to detect cattle or sheep IgG against 3ABC were compared with those from a commercially available test kit in naïve or infected animals, and those first vaccinated then challenged with foot-and-mouth disease virus (FMDV) type O. Bovine IgG1 could be detected 7 days post infection or challenge (dpi or dpc) using the in-house method (3 days after neutralising antibodies) but only after 10 days using the kit. Ovine IgG1+2 was detected by the in-house method as early as 10-12 dpc (an average 4 days after neutralising responses) and 2.5 days before the commercial method. When two steers were exposed to aerosols from donor pigs infected with O UKG 2001, one appeared uninfected while the other reacted in the in-house assay against 3ABC after 12 days. In a similar experiment using donor sheep, it was not until 28 dpc that both steers became positive. With the in-house test, sheep left in contact for 11 days with donors of the same species reacted against 3ABC 12 dpc but those exposed to aerosol only after 20 days. Eight vaccinated then challenged cattle were positive using the in-house test at 10 dpc as opposed to only three, which all showed clinical signs, with the test kit. In sheep given a low potency vaccine that protected against disease there was good agreement between postchallenge virus replication and reactions with the in-house 3ABC assay by which four animals were positive but only one reacted in the commercial assay. In the unvaccinated but challenged control group, the kit showed responses three days later than the in-house test. Amongst 380 ovine field samples, sensitivity was 77% among VNT positive sera with the inhouse assay but only 13% using the commercial product.

Introduction If FMD is suspected to have been transmitted to species in which infection is frequently subclinical such as sheep, widespread serological surveillance may be required in order to identify animals which have been exposed to the virus and developed antibodies. Several classes of immuno-globulins such as IgG may be produced, including those that neutralise, depending on the time which has elapsed since infection (Mulcahy et al., 1990; McCullough et al., 1992; Salt et al., 1996). The virus neutralisation test (VNT; OIE Manual of Standards, 2000) is considered definitive in determining the antibody status of livestock, whether they are suspected of having been infected or for certification prior to international trade. However, it can only detect immunoglobulins against the capsid or structural proteins of the virus. If animals subjected to serosurvey are unvaccinated, previous infection is readily detected by tests for structural antibodies. However, if vaccination is introduced at any stage to control the spread of FMD, 257

positive reactors may have developed antibodies in response to either infection and/or immunisation. In the case of animals traded across international borders, there may be uncertainty over both their vaccination and infection status. Therefore, vaccination may have considerable implications for the strategy involved in serological diagnosis. Taken together with the fact that the process of identification of individual stock can be difficult in some species (e.g. sheep) which may not remain tagged for long periods, it becomes imperative to distinguish between such antibodies. It is also important to recognise that vaccinated animals may also become infected, and cattle and sheep which recover from infection, whether or not they have also been immunised, may become carriers (Burrows, 1966; Sutmoller and Gaggero, 1965) and thus be a potential source of new infection. In addition to capsid proteins, certain non-structural (NS) ones are also produced during the process of infection by FMD virus and against which immuno-globulins may be formed. Though produced by viral replication, and hence initially present in all virus harvests, NS proteins are later removed during the purification process used in the formulation of modern vaccines and it is unlikely these would elicit such antibodies. Several ELISAs have been developed to differentiate infection from vaccination by the detection of antibodies against a number of distinct NS proteins (De Diego et al., 1997; Mackay et al., 1998; Sorenson et al., 1998) expressed in either E. coli or Baculovirus. Perhaps the most reliable single NS indicator is the poly-protein 3ABC which appears to produce conclusive evidence of previous infection (Mackay et al., 1998), whether or not the animals have also been vaccinated. Antibodies against 3ABC have been detected up to 395 days post infection in both cattle and sheep (Sorenson et al., 1998). This paper compares the results obtained with sera collected from naïve, infected (including field sera) and vaccinated then infected cattle and sheep in two experimental assays and a commercial kit that detects the IgG class of antibody against 3ABC.

Materials and Methods Sera submitted for import/export purposes from healthy cattle and sheep in the UK and other European countries prior to the UK outbreak of FMD in 2001 were used to represent naïve populations in the in-house assays. Some of these sera had given inconclusive antibody reactions but were shown to be negative on re-sampling. Samples were also obtained from cattle and sheep used in the transmission experiments described later (Aggarwal et al., 2002, Cox et al., in preparation) and were used to compare performance of the assays below. In addition, 380 field sera (from a total submission of 815) collected from UK sheep as part of the sero-surveillance activities during the 2001 disease outbreak of FMDV type O were used to assess sensitivity. Poly-protein 3ABC expressed in E. coli (pMF21; Flint, 1995) was produced by the method of De Diego, et al., (1997) then 200µg used to immunise rabbits at four sites sub-cutaneously. Animals were boosted with the same dose after 28 days (P.Davies, personal communication). Sera were tested by checkerboard titration in the in-house indirect sandwich ELISA for activity (see protocol below; all volumes 50µls): Indirect Sandwich ELISA 1) F96 Maxisorp Nunc Immuno plates were coated with an optimal dilution of rabbit anti3ABC serum overnight at room temperature (RT) and stored at +4°C for up to two weeks. 258

2) Before use, plates were washed three times with phosphate buffered saline (PBS; pH 7.27.6). Each was divided into sections of three columns, the first of which received buffer only (PBS + 0.05% Tween 20 (PBST) + 3% commercial unsweetened, organic soya milk + 1% normal horse serum) while an optimal dilution of 3ABC in buffer was added to the next two columns. Plates were incubated at 37°C for 1 hour. Meanwhile, buffer + 1% E.Coli sonicate was used to make a 1/200 dilution (Mackay et al., 1998) of each test serum. Under these conditions an OD of 2.2-2.4 should be obtained from highest reacting 21 day post infection sera (dpi), one of which (or dilution of) was selected that gave approximately 1.5 OD at 492nm. This was further diluted in normal serum of the same species (used as negative control) to provide a weak, borderline (M.Forsyth, personal communication) positive which should produce a consistent weak/strong positive ratio (w/s). 3) Plates were re-washed then the test and control serum dilutions added to three wells of a row (i.e. to two wells containing 3ABC and one without) and incubated as above. 4) Plates were washed again then an optimal dilution of mouse anti-bovine IgG1 (Serotec, UK; MCA 627) or anti-goat/sheep IgG1+2 (Sigma, UK; G-2904) added in buffer and incubated at 37°C for 1 hour. 5) After re-washing, an optimal dilution of anti-mouse immunoglobulins/HRP conjugate (Dako, Denmark; P0260) was added and incubated as above. 6) The plates were washed again to include a thorough handwash which flooded the plate to remove excess conjugate. The reaction was then developed by addition of the chromogen, 5.05mM ortho-phenylene-diamine dihydrochloride (Sigma, UK; P8412) and substrate, 4.4mM (0.015%) H2O2 in phosphate-citrate buffer (Sigma, UK; P4809). Colour was allowed to develop at RT for 15 minutes and the reaction stopped by addition of 1.25M H2SO4. Optical density was measured at 492nm. 7) Results for test and control sera were processed as follows: The mean of the two wells with 3ABC minus that containing only buffer was calculated. Then for each plate, the ratio of the test sample OD and weak positive control was divided by that obtained for the strong positive, resulting in the test/positive or t/p ratio. This corrected for plate to plate and between test variation. Test sera giving t/p ratios higher than or equal to that of the weak positive control on the same plate were considered positive. Occasionally, control sera were added twice and the median calculated. Commercial Test Kit The FMD-3ABC bo-ov Chekit (Bommeli Diagnostics, Switzerland) was used to detect IgG in cattle and sheep. Colour was allowed to develop at RT until approximately 0.8 OD at 405nm was obtained for the 1/100 dilution of the positive control (which was titrated to include 1/200 and 1/400 in every test) and 0.1 for the negative. Results were expressed as a percentage of the kit positive control using a macro program (D.Rebeski, Bommeli, personal communication). The appropriate species of control sera used for the experimental assays were always included. Sera were considered ambiguous if the 1/100 dilution gave 20-30% “percent positive” (pp) and unequivocally positive if >30%. Virus Neutralisation Tests (VNT), Solid Phase Competition (SPC) ELISA, Probang Sampling/Virus Isolation and Real Time Polymerase Chain Reaction (RT-PCR) Neutralisation tests were carried out as prescribed in the OIE Manual of Standards (2000) while the competition ELISA was performed according to Paiba et al., (in preparation). Probangs were collected and tested for the presence of live FMDV by the method of Ferris and Dawson (1988). RT-PCR was carried out as described by Reid et al., (2001). 259

Results Negative sera T/P ratios found in the in-house assays amongst naïve UK and other European cattle and sheep are presented in Fig 1. A “doubtful” range was defined with lower cut-offs of 0.04 and 0.09 respectively (corresponding to 98% specificity in both species). Upper cut-offs were determined by the w+/s+ ratio, which in bovines ranged from 0.14-0.21 while in sheep, 0.180.26 was acceptable. Cattle inoculated with O1BFS 1860 (UK, 1967) Three steers inoculated intra-dermolingually all showed clinical signs at 2 dpi and demonstrated live virus in probang samples at 3-5 dpi. UH 28 and 29 became carriers while UH 27 did not. All were VNT positive at 4 dpi. Two animals reacted positively in the inhouse assay against 3ABC at 7 dpi, the other at 10 dpi (Fig 2) while the commercial kit produced affirmative results in all three cattle only at 10 dpi. Sheep exposed to aerosols from pigs infected with O UKG 2001 Of three animals, UI 69 and 70 showed clinical signs at 8 dpc, and UI 71 at 6 dpc (Aggarwal et al., 2002). UI 69 became positive by VNT at 10 dpc, UI 70 at 6 dpc, and UI 71 after 8 days. IgG against 3ABC was detected in UI 69 after 19 days in both 3ABC tests. However, UI 70 was positive at 12 dpc using the in-house method but only after 19 days with the kit, and UI 71 reacted after 10 and 12 days respectively (Fig 3). Real time PCR revealed viral RNA in a probang from UI 69 at 8 weeks post challenge (results not shown). Cattle exposed to aerosols from pigs and sheep infected with O UKG 2001 Of two animals exposed to pig aerosols, one appeared not have been infected while the other, UI 62 was clinically positive and reacted in the VNT at 6 dpc. UI 64 and 65, exposed to sheep aerosols showed signs of FMD only after 17 and 14 days (Aggarwal et al., 2002) and were both VNT positive at 20 dpc. UI 62 was positive in the in-house assay against 3ABC after 12 days but not until 19 days using the commercial test. Cattle exposed to infected sheep all reacted at 28 dpc in both tests except UI 64 which gave only ambiguous results in the kit (Fig 4). Sheep exposed to aerosols from, or in contact with, sheep infected with O UKG 2001 Three animals (UI 72-4) left in-contact with sheep previously infected, developed clinical FMD faster (after 8-12 days) than the two (UI 66 and 67) which only received aerosols (Aggarwal et al., 2002) though most became VNT positive after 16 days. The in house test showed 3ABC antibodies as early as 12 dpc amongst animals in-contact but after 20 days if only exposed to aerosol. The kit revealed IgG as early as 16 dpc in-contact but only the beginning of a response in the aerosol group at 28 dpc (Fig 5). UI 72 and 73 were viral RNA positive after 8 weeks (results not shown).

260

O1 Cattle Vaccine Potency Test Three groups of five steers were given different dilutions of a commercial vaccine, and there were two unvaccinated controls. Eleven of those immunised had developed a neutralising antibody response at 21 days post vaccination (dpv), though only seven showed protective levels i.e. approximately log10 1.4-5 (P.Barnett, personal communication). Animals were challenged at this point with 105 TCID-50 administered by tongue inoculation. Only four cattle were kept beyond 10 dpc – amongst these, live virus was isolated from probangs at 28 dpc in all, clinical signs being observed in UM 64 and 72 but not in UM 60 or 69. Overall, eight were positive in the in-house 3ABC assay at 10 dpc but only three using the commercial assay (all having shown disease), though another two were very nearly so (Table 1). Two reactors in the in-house assay (UM 63 and 68) gave t/p ratios of 0.45 and 0.83 but were negative using the commercial test. O1 Sheep Vaccine Potency Test: A group of seven animals were immunised with an experimental low potency vaccine which gave protection from clinical disease and reduced but did not eliminate virus replication. Together with seven unvaccinated controls, all were challenged after 14 days by aerosol exposure from infected pigs. VNT responses had developed in only three of those vaccinated by 10 dpv and were slightly changed at 4 dpc. All controls except one showed virus replication at 2 dpc (results not shown), all were positive using the in-house 3ABC test by 11 dpc but with the commercial assay, they generally reacted only at 14 dpc. In the vaccinated group, five sheep were probang positive, and four reacted against 3ABC in the in-house test but only one using the kit. Sensitivity of the latter could be enhanced by using the in-house ovine weak positive as the diagnostic indicator, which generally fell in the 10-15% percent positive range. Sheep field sera from UK 2001 FMD outbreak Of 380 sheep sera collected as part of the sero-surveillance activities during the UK FMDV type O epidemic and screened using the SPC ELISA, , those giving ≥50% inhibition (70; 18%) were re-tested using the VNT when 30 were positive (8%; Table 3) according to the criterion in the OIE Manual of Standards (2000). Of the VNT reactors , 23 (77%) were positive using the in-house assay but only 4 (13%) with the commercial test. Discussion In addition to robustness and reliability, an important requirement of any veterinary diagnostic test kit is high specificity since litigation may be initiated if false positives are encountered and animals are slaughtered unnecessarily. The lack of a confirmatory test is also a reason for requiring a high specificity. However, specificity should not be achieved at the expense of sensitivity; rather a balance should be arrived at, best befitting the product’s intended use and, taking into account other assays or strategies which may be used. One major use of tests to detect antibodies against the non-structural proteins of FMD, including any resulting commercial product, is in conjunction with those for measuring structural responses in sero-surveillance of livestock in which overt symptoms may be mild or absent altogether, such as sheep. This may be required following vaccination during or after an outbreak in otherwise FMD-free countries. During the epidemic of FMD in the UK in 2001, millions of sheep sera were tested for structural antibodies using the solid-phase competition 261

ELISA (Paiba et al., in preparation), positives being confirmed by VNT. As no vaccination took place, non-structural tests were not required. However, in countries where immunisation is practised in response to a disease outbreak, not only animals from herds proven infected during the epidemic may be slaughtered but also, at a later stage, those which have been vaccinated. Any use of such prophylaxis on a large scale could have profound implications for serology, especially as the UK outbreak saw a watershed in attitudes towards mass slaughter of livestock. It is also very important to distinguish between immunisation and infection in animals that have crossed international borders illegally or whose history of exposure to FMDV antigen is otherwise uncertain. The in-house method used to detect cattle or sheep IgG, of which there are two main subclasses, against the non-structural polyprotein 3ABC of FMD proved considerably more sensitive than the commercial assay. IgG1 could be detected experimentally 3 days later than the virus neutralising response but 3 days earlier than by the test kit in inoculated cattle. In sheep, where the moment of infection was less well defined, detection of IgG1+2 with the inhouse assay was, on average, 4 days later than a positive VNT result, but 2.5 days earlier than with the commercial product. The detection of waning antibodies was also poor – three sheep infected by pig aerosol of O UKG 2001 were still positive experimentally after 214, 79 and 44 days, whereas using the commercial test, affirmative diagnoses were achieved only up to 176, 44 and 25 days respectively (not shown in results). In vaccinated then challenged animals, eight cattle were found positive by the in-house method but only three which showed clinical signs reacted in the kit. Moreover, two found unequivocally positive in the in-house assay tested negative with the kit. In sheep given a low potency experimental vaccine which gave protection against disease but did not entirely prevent virus replication, there was good agreement between isolation of live virus and the appearance of 3ABC antibodies using the in-house test, but only one sheep was positive with the commercial assay. Amongst controls, the experimental test detected IgG 11 dpc while the kit only revealed antibodies in most animals at 14 dpc. The manufacturers claim the Chekit FMD-3ABC bo-ov assay has an underlying specificity of 99.95 and 100% for cattle and sheep respectively. However the in-house indirect sandwich ELISA, which appears to possess nearly 100% specificity with bovine sera is still considerably more sensitive than its commercial counterpart. This may partly be due to its targeting of the IgG1 isotype, which is generally produced in higher quantities than IgG2 in cattle infected with virulent strains or those vaccinated then challenged (Mulcahy et al., 1990). However, the in-house ovine assay for IgG1+2 – the isotypes detected by the commercial product – also appeared considerably more sensitive than the kit. Sensitivity could be enhanced somewhat in the commercial test by basing diagnosis upon the performance of the ovine weak positive control which was demonstrated to be close to borderline. This generally fell in the 10-15% percent positive range, but even re-setting the cut-off to this level still resulted in poor sensitivity compared to the in-house assay. Assuming the recommended serum dilution of 1/100 is the most appropriate as regards diagnostic potential, the problem may well lie with the conjugate, the nature of which is unknown. However, if problems associated with sensitivity can be overcome, the kit’s inherent robustness and high reproducibility will make it a powerful diagnostic tool.

262

References

Aggarwal, N., Zhang, Z., Cox, S., Statham, R., Alexandersen, S., Kitching, R.P. and Barnett, P.V. (2002) Experimental studies with foot-and-mouth disease virus, strain O, responsible for the 2001 epidemic in the United Kingdom. Vaccine, 20, 2508-2515. Burrows, R. (1966) Studies on the carrier state of cattle exposed to foot-and-mouth disease virus. J. Hyg. 64, 81-90 De Diego, M., Brocchi, E., Mackay, D. and De Simone, F. (1997) The use of the non-structural polyprotein 3ABC of FMD virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Archives of Virology 142, 2021-2033. Ferris, N.P. and Dawson, M. (1988) Routine allocation of enzyme linked immunosrbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases. Vet. Microbiol. 8, 249-56. Flint, M. A study of foot-and-mouth disease virus polyprotein processing. PhD Thesis, University of Reading, 1995 Mackay, D.K.J., Forsyth, M.A., Davies, P.R., Berlinzani, A. Belsham, G.J., Flint, M. and Ryan, M.D. (1998) Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant non-structural proteins in ELISA. Vaccine 16, No 5, 446-459 McCullough, K.C., de Simone, F, Brocchi, E., Capucci, L., Crowther, J.R. and Khim, U. (1992) Protective immune response against foot-and-mouth disease. J.Virol, 66, 1835-40. 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, Vol 8, 249-256 Office International des Epizooties (OIE) Manual of Standards for Diagnostic Tests and Vaccines, 2000. Paiba, G.A., Anderson, J., Paton, D., Soldan, A., Alexandersen, S., Corteyn, M., Wilson, J., Turner, L., Hamblin, P., Mackay, D.J. and Donaldson, A. Validation of a foot and mouth disease antibody solid-phase competition ELISA (SPCELISA) In preparation Reid, S., Ferris, N., Hutchings, G., Zhang, Z., Belsham, G. and Alexandersen, S. (2001) Diagnosis of foot-and-mouth disease by real time fluorogenic polymerase chain reaction assay. Vet.Rec. 149, 6213. Salt, J.S., Mulcahy, G. and Kitching, R.P. (1996) Isotype-specific antibody responses to foot-andmouth disease virus in sera and secretions of ‘carrier’ and ‘non-carrier’ cattle Epidemiol. Infect. 117, 349-360 Sorenson, K.J., Madsen, K.G., Madsen, E.S., Salt, J.S., Nqindi, J. and Mackay, D.K.J. (1998) Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovvirus Arch. Virol. 143, 1461-1476. Sutmoller, P. and Gaggero, A. (1965) Foot-and-mouth disease carriers. Vet. Rec. 77, 968-9.

263

TABLE 3: EXAMINATION OF 380 SHEEP FIELD SERA FROM A TOTAL SUBMISSION OF 815 DURING UK 2001 EPIDEMIC OF FMDV TYPE O

ALL INITIALLY SCREENED BY COMPETITIVE ELISA - THOSE WITH ≥50% INHIBITION (70) THEN TESTED BY VIRUS NEUTRALISATION TEST (VNT) POSITIVE BY VNT (≥1/45) INCONCLUSIVE (1/16-1/32)

= =

30 10

IN-HOUSE ASSAY FOR SHEEP IgG AGAINST 3ABC OF VNT POSITIVES

=

23 (77%) POSITIVE + 4 (13%) DOUBTFUL

OF VNT INCONCLUSIVES

=

5 (50%) POSITIVE + 2 (20%) DOUBTFUL

OF VNT POSITIVES

=

4 (13%) POSITIVE + 4 (13%) AMBIGUOUS

OF VNT INCONCLUSIVES

=

0 POSITIVE + 0 AMBIGUOUS

BOMMELI 3ABC CHEKIT

271

Fig 1:

Frequency distributions of naïve UK and other European cattle & sheep showing start of doubtful range and acceptable values for weak to strong positive ratio (w+/s+). Samples with ratio equal to or greater than weak control are considered positive. CATTLE 100 90 80

Frequency

70 60 50 40 30 20 10 0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0.22

0.24

0.26

Test/positive ratio

SHEEP 100

90

80

Frequency

70

60

50

40

30

20

10

0

0.03

0.06

0.09

0.12

0.15

0.18

0.21

Test/positive ratio

0.24

0.27

0.3

0.33

Fig 2:

Detection of IgG against 3ABC in cattle inoculated with O1 BFS 1860. Animals all showed clinical signs at 2 dpi and were VNT +v at 4 dpi

Fig 3:

Experimental assay 1.8

1.4

1.4

UH 27 +v at 7dpi

1.2

UH 28 +v at 10dpi

1.2 Test/positive ratio

Test/positive ratio

1.6

UH 29 +v at 7dpi

1 0.8 0.6 0.4 0.2 0

0

4

7

10

16

26

0.8 0.6 0.4 0.2 0

Days post infection Commercial kit

300 250 200

UH 27 +v at 10dpi

120

UH 28 +v at 10dpi

150

UH 29 +v at 10dpi

100 50 0

0

4

7

10

Days post infection

16

26

% of positive control

% of positive control

1

100 80 60 40 20 0

Detection of IgG against 3ABC in sheep infected with O UKG 2001 by pig aerosol. Clinical signs appeared in UI 69 & 70 at 8 dpc, UI 71 at 6 dpc. UI 69 was VNT +v at 10 dpc, UI 70 at 6 dpc & UI 71 at 8 dpc Experimental assay 4 2 1

UI 69 +v at 19dpi

8

UI 70 +v at 12dpi

6

UI 71 +v at 10dpi

4 2 0

0

4

6

8

10

12

19

25

12

19

25

Days post challenge

Commercial kit 0 0 0

UI 69 +v at 19dpi

0

UI 70 +v at 19dpi UI 71 +v at 12dpi

0 0 0

0

4

6

8

10

Days post challenge

Fig 4:

Detection of IgG against 3ABC in cattle exposed to O UKG 2001 by aerosol from infected pigs (UI 62 & 63) or sheep (UI 64 & 65) Animal UI 62 UI 63 UI 64 UI 65

Clinical Signs 6 dpc none 17 dpc 14 dpc

Exptl 3ABC + 12 dpc never 28 dpc 28 dpc

Kit 3ABC + 19 dpc never amb 28 28 dpc

Experimental assay

0.8 0.7 Test/positive ratio

VNT + 6 dpc never 20 dpc 20 dpc

UI 62

0.6

UI 63

0.5

UI 64

0.4

UI 65

0.3 0.2 0.1 0

0

4

6

8

10

Days post challenge

12

19/20

25/28

Fig 5:

Detection of IgG against 3ABC in sheep exposed to O UKG 2001 by aerosol (UI 66 & 67) or through direct contact (UI 72-74) with sheep already showing clinical signs of infection Clinical Animal Signs UI 66 none UI 67 13 dpc UI 72 12 dpc UI 73 12 dpc UI 74 8 dpc

VNT + 16 dpc 16 dpc 16 dpc 16 dpc 10 dpc

Exptl 3ABC + 20 dpc 20 dpc 16 dpc 20 dpc 12 dpc

Kit 3ABC + never >28 dpc 16 dpc 20 dpc 16 dpc

Test/positive ratio

Experimental assay 1.4 1.2 1 0.8 0.6 0.4 0.2 0

UI 66 UI 67 UI 72 UI 73 UI 74

0

4

6

7

8

10

12

16

20

28

16

20

28

Days post challenge Commercial kit

% of positive control

160 140

UI 66

120 100

UI 67

80

UI 72

60

UI 73

40

UI 74

20 0

0

4

6

7

8

10

12

Days post challenge

Table 1: Comparison of 3ABC results from O1 Manisa vaccine potency test in cattle. Groups given varying doses then challenged after 21 days Experimental assay Vaccine Animal 21 DPV 21 DPV 10 DPC 51 DPC 65 DPC 78 DPC 91 DPC group number 3ABC VNT 3ABC 3ABC 3ABC 3ABC 3ABC

1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 Non-vacc Non-vacc

UM 58 UM 59 UM 60 UM 61 UM 62 UM 63 UM 64 UM 65 UM 66 UM 67 UM 68 UM 69 UM 70 UM 71 UM 72 UM 73 UM 74

neg neg neg neg neg neg neg neg neg neg 0.05 neg 0.12 neg neg neg neg

2.55 neg 1.35 0.34 1.65 0.05 neg 1.2 2.1 neg 1.2 0.83 neg 1.02 neg neg 1.35 neg 2.25 neg neg 0.45 1.2 neg 1.5 0.14 1.5 0.42 1.5 0.38 neg no sample neg 0.54

no sample no sample 0.09 no sample no sample no sample 0.72 no sample no sample no sample no sample 0.09 no sample no sample 0.35 no sample no sample

no sample no sample 0.09 no sample no sample no sample 0.58 no sample no sample no sample no sample 0.08 no sample no sample 0.37 no sample no sample

no sample no sample 0.08 no sample no sample no sample 0.39 no sample no sample no sample no sample 0.08 no sample no sample 0.49 no sample no sample

no sample no sample 0.05 no sample no sample no sample 0.37 no sample no sample no sample no sample 0.05 no sample no sample 0.32 no sample no sample

Commercial kit Group group

1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 Non-vacc Non-vacc

Animal 21 DPV 21 DPV 10 DPC 51 DPC 65 DPC 78 DPC 91 DPC number 3ABC VNT 3ABC 3ABC 3ABC 3ABC 3ABC

UM 58 UM 59 UM 60 UM 61 UM 62 UM 63 UM 64 UM 65 UM 66 UM 67 UM 68 UM 69 UM 70 UM 71 UM 72 UM 73 UM 74

Key:

neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg

2.55 neg 1.35 29.5 1.65 neg neg 105.1 2.1 neg 1.2 neg neg 66.9 neg neg 1.35 neg 2.25 neg neg neg 1.2 neg 1.5 neg 1.5 20.2 1.5 40.4 neg no sample neg 29.2

= =

no sample no sample neg no sample no sample no sample 65.5 no sample no sample no sample no sample neg no sample no sample 72.8 no sample no sample

Doubtful/Ambiguous Positive

no sample no sample neg no sample no sample no sample 43 no sample no sample no sample no sample neg no sample no sample 66.6 no sample no sample

no sample no sample neg no sample no sample no sample 45.7 no sample no sample no sample no sample neg no sample no sample 64.2 no sample no sample

no sample no sample neg no sample no sample no sample 36.5 no sample no sample no sample no sample neg no sample no sample 74.7 no sample no sample

Table 2: Comparison of 3ABC results from control sheep and those receiving an experimental low potency O1 vaccine & challenged after 14 days

Experimental assay Sheep UO87 UO88 UO89 UO90 UO91 UO92 UO93 UO94 UO95 UO96 UO97 UO98 UO99 UP0

Sheep UO87 UO88 UO89 UO90 UO91 UO92 UO93 UO94 UO95 UO96 UO97 UO98 UO99 UP0

Group Vacc

Unvacc

Group Vacc

Unvacc

VNT 10 dpv neg neg 1.5 1.65 neg 1.2 neg neg neg neg neg neg neg neg

7dpc

9dpc

11dpc 0.11

0.1

no sam

0.1

0.37

VNT 4 dpc neg neg

VNT 7dpc no sam no sam

neg 1.5 neg 1.35 1.35 neg neg neg neg neg neg neg

neg no sam no sam no sam 1.8 2.1 2.25 2.25 2.1 2.1 no sam 2.25

0.55 0.92 0.34 0.47 0.7 0.6 0.23 1.04

14dpc 0.25

22dpc 0.23 0.27

28dpc 0.13 0.14

35dpc 0.21 0.1

42dpc 0.15 0.14

0.1

0.18

0.18

no sam

0.28

1.12 1.37 0.57 no sam 1.23 1 0.54 1.2

0.91 1.35 0.55 0.92 1.11 0.94 0.55 1

0.6 1.17 0.47 0.85 1.05 0.68 no sam 1.14

0.6 1.1 0.4 0.62 1.18 0.79 0.92 1.14

0.4 1.1 0.29 0.55 0.99 0.57 0.81 1.02

14dpc

22dpc

28dpc

35dpc

42dpc

19.7

19.7

Commercial kit 9dpc

11dpc

20.4

12.5

14 13.8 17.6 13.3

17.6

47.2 71.7 50.9 no sam 48.3 12.2 34.7 19.1 39.7 77.1 Key:

= no sam = = = =

39 23.3 90 78 46.5 39.6 41.5 34.7 86.4 83 38.1 30.6 29 no sam 84.7 92.4

15.7 83.6 35.8 30 85.7 39.5 73.3 74.8

68.6 28.9 23.1 60.5 25.9 80.1 74

Negative No sample taken Doubtful/Ambiguous Positive (Pirbright weak +v) Positive

Appendix 33

A comparison of two 3ABC ELISA’s in an African cattle population with endemic multiple serotype Foot-and-Mouth disease B.M.deC. Bronsvoort1, K.J. Sørensen5, J. Anderson3, A. Corteyn3, V.N. Tanya2, R.P.Kitching4, K.L. Morgan1 1

University of Liverpool, Dept. Veterinary Clinical Sciences and Animal Husbandry, Leahurst, Neston, Wirral, CH64 7TE, UK. 2 Institute of Agricultural Research for Development, Regional Centre of Wakwa, B.P. 65, Ngaoundere, Cameroon. 3 Institute of Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK. 4 National Centre for Foreign Animal Disease Canadian Food Inspection Agency Winnipeg, Manitoba, Canada. 5 Danish Veterinary Institute, Virology Department, Lindholm, DK4771 Kalvehave, Denmark.

Keywords: 3ABC, CHEKIT, competitive ELISA, ROC Abstract The development of a foot-and-mouth disease virus (FMDV) serological test which is quick and easy to use, which can identify all 7 serotypes and which can differentiate vaccinated from convalescing or potential carriers would be a major advance in the epidemiological tool kit for FMDV. The polyprotein 3ABC has recently been proposed as such an antigen and a number of diagnostic tests are being developed. This paper compares the performance of the 3ABC FMD CHEKIT bov-ov ELISA (Bommeli) and a competitive 3ABC ELISA developed in Denmark, in an African cattle population in an endemic FMDV region of Cameroon with multiple serotypes. The test parameters (sensitivity, specificity and predictive value) were examined over a range of test cut-off’s. The results indicate that both tests lack sensitivity though the CHEKIT-ELISA is particularly low at the recommended cut-off. Their performances at different cut-off’s and how they might perform at the herd-level are discussed. Introduction The non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) have received considerable attention in recent years with the search for improved serological tests for FMDV 1-5. The virus neutralisation test (VNT) 6 and liquid phase blocking ELISA (LPBE) 7-9 are currently the recommended tests by the OIE. However, these tests require each serotype to be tested separately, are time consuming to perform, require virus containment facilities and cannot differentiate vaccinated from convalescing animals. The development of a single, quick to use test that covered all 7 serotypes as well as differentiating vaccinated from convalescing animals would be a major advance in the epidemiological tool kit for FMDV. The NSP’s are only expressed in animals with replicating virus and therefore only animals that have been infected with live virus should develop antibodies to these 272

proteins 1,3,10,11. The currently used inactivated vaccines have been purified to remove cellular proteins and NSP’s and should not induce antibodies to these proteins. However, in practice vaccinated animals do produce antibodies to some of the NSP’s such as 3D 5 and antibodies to others such as 2C were found to rapidly fall below detectable levels 12,13 . The polyproteins 3ABC and 3AB appear to be the most promising protein as diagnostic antigens 4,14-17. Recent studies of FMDV in Morocco and in Taiwan using 3ABC or 3AB ELISA’s are encouraging 18-20. If these tests are to be useful it is essential that they are evaluated in a range of populations since the diagnostic sensitivity (Se) and specificity (Sp) are population parameters that describe test performance for a given reference population 21 and previous studies have shown that when applied in tropical veterinary medicine, many diagnostic tests do not perform as well as expected 22. In addition, the overall test performance can be evaluated and the cut-off optimised for field use. The following paper describes a comparison of the 3ABC FMD CHEKIT bov-ov ELISA 1 (CHEKIT-ELISA) and a 3ABC competitive ELISA (C-ELISA) developed in Denmark. This work forms part of a larger study of the epidemiology of FMDV in the Adamawa Province of Cameroon 23. Sera from a population based sample of the cattle population of the Adamawa Province, an endemic FMDV region of Cameroon with multiple serotypes, was used to evaluate the two tests. The tests were compared to the VNT which was considered to be the ‘gold standard’. The Se (the conditional probability that an animal is test positive given it is diseased) and Sp (the conditional probability that the animal is test negative given that it is not diseased) of the two tests were estimated and the overall test performances compared using receiver operating characteristics (ROC). The test parameters are then discussed in terms of their implications when applied at the herd level. Materials and Methods Study Population A full description of the study area, the livestock population and the study design is given elsewhere 23. In brief, the study area was the five administrative Divisions of the 64,000km2 Adamawa Province of Cameroon. A sample frame of the cattle herds in the area was constructed from the rinderpest vaccination lists held by the local Veterinary Centres. A cross-sectional study design was used with a stratified (by Division), twostage (by Centre and Herd) random sampling strategy. The program ‘Survey Toolbox’ 2, calculated a two-stage sample of 54 centres and 3 herds per centre, allowing for a 10% non-response rate. A questionnaire was administered to each herdsman and 5 juvenile (824 month old) and 5 adult (>24 months old) cattle were cast, examined for lesions and a serum and oropharyngeal fluid or probang sample (OP) taken from each animal. Five randomly selected animals gives a 95% probability of detecting at least one positive animal in a herd of 70 if the test is 100% sensitive and specific. The herds were not believed to have been vaccinated since no vaccine was available in the country and no 1 2

Bommeli Diagnostics, Stationsstrasse 12, CH-3097, Liebefeld, Bern, Switzerland. http://www.ausvet.com.au/surveillance/toolbox.htm

273

herdsman reported using vaccine. The study was conducted between April and November 2000 which encompasses the rainy season when herds are close to their home areas. Tissue culture of OP samples The OP samples were inoculated onto bovine thyroid cell monolayers following the OIE/WRL protocol. The cultures were incubated at 370C for up to 72 hours and examined for any signs of cytopathic effect (cpe). Cultures with positive cpe were then typed using the WRL sandwich antigen ELISA 24,25. Virus Neutralisation Test (VNT) VNT’s were performed following OIE/WRL protocol 6. The serum samples were tested against FMDV type O Manisa, type A (P59/2000-VBM/153/09) and type SAT2 (P26/2000 – FDL/74/10) respectively. The 100 TCID50 end point was estimated for each sample 26 and the standard OIE cut-off of ≥1/10-1.56 (1/45) used. 3ABC CHEKIT ELISA The CHEKIT-ELISA was used according to the manufacturers instructions. Briefly, the serum was diluted 1/100 and added in duplicate to the wells of a 96 well microtitre plate pre-coated with the vector expressed viral 3ABC antigen. Antibodies specific to 3ABC were bound to the antigen forming an antigen/antibody complex on the plate surface. Unbound antibody was washed away. A horseradish peroxidase labeled guinea pig antibovine IgG conjugate was added which bound to any antibody/antigen complexes. Unbound conjugate was removed by washing and the chromagen substrate added. The degree of colour that developed was proportional to the amount of antibody complexed on the plate surface and read at 405nm with a spectrophotometer. The final reading for the sample was calculated as follows using the means of the pairs of samples and the median of the 4 positive and negative controls on each plate: value% =

ODsample − ODneg OD pos − ODneg

X 100

The manufacturers recommend interpretation is an %OD of less than 20% is negative, 20-30% is ambiguous and greater than 30% is positive. FMD-3ABC Competitive ELISA The C-ELISA was performed as described previously 15 with modifications. Microtiter plates were prepared by capturing 3ABC protein produced in the Baculovirus expression system with a monoclonal antibody (MabD5) coated on the plates. Dilutions of the sera were added followed immediately by the competing antibody, horseradish peroxidase conjugated MabD5. After washing and addition of chromagen substrate (TMB H2O2) the colour development was measured and the results expressed as percentage of negative control (ODp) values. Analysis The results of the 3 VNT’s were combined such that an animal that was positive for one or more serotypes was classed as positive on the combined VNT (cVNT). The cVNT and 274

ELISA results were first compared at the animal level in 2x2 tables. The Se and Sp of each test was calculated using a range of cut-offs from 10-30% for the CHEKIT-ELISA and ≤50% for the C-ELISA. The test characteristics were further investigated using the ROC calculated by using AccuROC 3. The herd-level sensitivity (HSe) (the conditional probability that a herd is test positive given that it is diseased) and specificity (HSp) (the conditional probability that a herd is test negative given that the herd is not diseased) of the two tests were examined over a range of possible within-herd prevalences using the Herdacc 4 software in a theoretical herd of 100 animals using a cut point of one positive animal. Results The virus isolation results from the probang samples indicated that there were at least 3 serotypes of virus actively circulating in the population and therefore VNT’s were carried out for serotypes O, A and SAT2 only. The prevalence of antibodies to these 3 serotypes are given in Table 1 below. Table 1. Seroprevalence of serotypes O, A and SAT2 in the Adamawa Province of Cameroon. Test Juvenile Adult SAT2 42.9 (±4.7) 67.0 (±4.3) A 12.4 (±4.4) 55.8 (±7.1) O 5.1 (±2.7) 15.0 (±3.9) The results of the C-ELISA (Table 2) and CHEKIT-ELISA (Table 3) were then compared with the cVNT results using 2x2 tables and the Se, Sp and predictive values estimated.

Table 2. The Se and Sp of the C-ELISA at 50% compared to combined VNT tests for O, A, and SAT2. 50% cut-off cVNT + cVNTPredictive Value C-ELISA + 647 48 93.1+ % C-ELISA 260 422 61.9- % Se=71.3% Sp=89.8%

3 4

AccuROC v2.4, www.accumetric.com Herdacc v3.0 David Jordan, University of Guelph, Guelph, ON, Canada, N1G 2W1.

275

Table 3. The Se and Sp of the 3ABC at 30% (a), 20% (b), and 10% (c) compared to combined VNT tests for O, A, and SAT2 for cattle in Cameroon. (a) 30% cut-off cVNT + cVNTPredictive Value CHEKIT + 212 10 + 95.5% CHEKIT 695 460 - 39.8% Se=23.4% Sp=97.9% (b) 20% cut-off CHEKIT + CHEKIT (c) 10% cut-off CHEKIT + CHEKIT -

cVNT + 314 593 Se=34.6%

cVNT22 448 Sp=95.3%

Predictive Value + 93.5% - 43.0%

cVNT + 537 370 Se=59.2%

cVNT74 396 Sp=84.3%

Predictive Value + 87.9% - 51.7%

The results show the C-ELISA to have a low sensitivity and specificity at the recommended cut-off. In comparison, the sensitivity of the CHEKIT-ELISA is extremely low at the recommended cut-off of 30% though the specificity is very high. The test cutoff’s were examined further using the ROC shown in Figure 1 below. From the curves it is clear that the C-ELISA performs better overall though the cut-off of 50% may not be optimal. The CHEKIT-ELISA though not performing as well overall is not optimised at a cut-off of 30%. Lowering the cut-off to 15% would greatly increase the sensitivity of the test without too much loss of specificity. The area under the two curves were compared using a non-parametric method 27.

276

Figure 1. ROC for 3ABC CHEKIT-ELISA and C-ELISA The estimates of the HSe and HSp of the C-ELISA and the CHEKIT-ELISA when applied to a theoretical herd of 100 animals are given in Tables 4a and 4b below. A range of herd-level prevalences were investigated from 1 to 30% for each of the tests. For the C-ELISA the low Sp is rapidly compounded with the need to increase the sample size to increase the HSe. Therefore using a cut-off of 45 or 40% would increase the Sp and overcome some of the imperfections of the test. The CHEKIT-ELISA by having a high Sp is less prone to the rapid decline in HSp when applied to the herd. However, because the Se is also very low many more animals have to be sampled depending on the underlying herd prevalence.

277

Table 4. The herd-level sensitivity and specificity of the C-ELISA (a) and CHEKIT-ELISA (b) (a) Assumptions: Cut-point = 1, Sensitivity = 0.713, Specificity = 0.898. Sampling without replacement. Herd size = 100. ______________________________________________________________ Sample HSPEC HSENS: size: 0 1 3 5 10 15 30 (10) (11) (12) (13) (16) (19) (29) ______________________________________________________________ 1 0.900 0.110 0.120 0.130 0.160 0.190 0.290 11 0.294 0.742 0.774 0.802 0.869 0.914 0.982 21 0.083 0.936 0.951 0.963 0.984 0.993 1.000 31 0.020 0.987 0.992 0.995 0.999 1.000 1.000 41 0.004 0.998 0.999 0.999 1.000 1.000 1.000 ______________________________________________________________ (b) Assumptions: Cut-point = 1, Sensitivity = 0.234, Specificity = 0.979. Sampling without replacement. Herd size = 100. ______________________________________________________________ Sample HSPEC HSENS: size: 0 1 3 5 10 15 30 (2) (2) (3) (3) (4) (5) (8) ______________________________________________________________ 1 0.980 0.020 0.030 0.030 0.040 0.050 0.080 11 0.791 0.209 0.298 0.298 0.377 0.449 0.620 21 0.622 0.378 0.511 0.511 0.617 0.701 0.860 31 0.474 0.526 0.676 0.676 0.780 0.851 0.955 41 0.346 0.654 0.799 0.799 0.884 0.934 0.988 _______________________________________________________________

aColumns

bNumbers

are HSPEC/HSENS followed by no. of infected animals in the herd. in brackets are the predicted no. of test positives in the herd

Discussion The results of this analysis indicate that at the individual animal level the C-ELISA performs better than the CHEKIT-ELISA in an Africa cattle population, though neither test performs particularly well. From a practical point of view there are advantages to using a competitive ELISA in that there is only one conjugate needed for all species. The additional use of a Mab as the competitor should make the standardisation of the test between laboratories much easier. As mentioned in the introduction, Se and Sp are not fixed values and will vary between sub-populations and between populations conditional on the distribution of influential covariates (eg. age, stage of disease). Therefore, it may be useful to calculate the stratum specific Se and Sp where possible or use logistic regression models that include the covariates in order to obtain estimates, as these may

278

have greater diagnostic utility and may be applied to other populations anticipated that such analyses will be carried out.

21,28

. It is

It was not unexpected that the Se and Sp were lower than reported in previous evaluations15. This is most likely due to there being cross reactions from other pathogens/antigens which animals are exposed to in the tropics, different breeds (Bos indicus), and management systems, the multiple serotypes of FMDV that the animals were exposed to and the much wider range of disease states in a natural population21. In addition the use of the combined VNT results as a ‘gold standard’ has not been previously used and the estimates should therefore be interpreted with a degree of caution. Although the tests are less than perfect they still may be useful depending on the circumstances in which they are used. When applying a test to a herd it is important to realise that the HSe and HSp are dependent on, the individual test Se and Sp, the number of animals tested, the true prevalence in infected herds, the herd cut-off value used to classify herds as positive and the variation in Se, Sp and prevalence among herds 29. In particular, if a test is interpreted at the herd level, as long as the individual test Sp is very high, lower individual Se’s can be overcome to some degree. So although these results can not be directly applied to the European setting the basic implications still apply. There are clearly a number of senarios where a NSP test could be useful. For example, in endemic areas where vaccination is started as a control measure, being able to monitor the levels of sub-clinical disease would help decision making. Using the right cut-offs these tests could be used. However, as the prevalence falls, both tests but in particular the CHEKIT-ELISA will have increasing problems with low HSe unless most of the herd are sampled. Another proposed use has been at borders. The main problem with this will be that the test would need to be interpreted at the individual animal level unless all the animals were from the same source. For Europe, which is driving much of this research, the motivation is to be able to screen vaccinated herds after an outbreak and identify herds with sub-clinical infections that could become ‘carriers’. Recent changes in OIE regulations will allow countries to resume trading after 6 months instead of 12 months if they vaccinate and can then screen and demonstrate freedom from disease. This change assumes that there is a test that will distinguish vaccinated from sub-clinically infected/convalescing animals. However, it is unclear what the prevalence of such animals would be in a herd though it is likely to be low in which case both these tests, even with adjusted cut-off’s, may struggle to achieve acceptable HSe and HSp.

279

APPENDIX Apparent prevalence AP True prevalence Sample size per herd n

TP

AP=(1-Sp)+(Se+Sp-1)*TP Hse=1-(1-AP)n HSp=Spn Acknowledgments The authors would like to thank Dr. Charles Nfon and Mr. Hamman Saidou Mustaffa for their considerable efforts in trying conditions in the field. In addition we thank P. Hamblin and D. Gibson for working on the VNT and ELISA. Mark Bronsvoort is a Wellcome Trust Research Training Fellow in Clinical Tropical Epidemiology. References 1.

Berger HG, Straub OC, Ahl R, Tesar M, Marquardt O. Identification of foot-and-mouth disease virus replication in vaccinated cattle by antibodies to non-structural virus proteins. Vaccine 1990;8(3):213-6.

2.

Sorensen KJ, Madekurozwa RL, Dawe P. Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA. Veterinary Microbiology 1992;32(3-4):25365.

3.

Lubroth J, Brown F. Identification of native foot-and-mouth disease virus non-structural protein 2C as a serological indicator to differentiate infected from vaccinated livestock. Research in Veterinary Science 1995;59(1):70-8.

4.

De Diego M, Brocchi E, Mackay D, 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. Archives of Virology 1997;142(10):2021-33.

5.

Sørensen KJ, Hansen CM, Madsen ES, Madsen KG. Blocking ELISAs using the FMDV nonstructural proteins 3D, 3AB, and 3ABC produced in the Baculovirus expression system. Veterinary Quarterly 1998;20:S17-S20.

6.

Golding SM, Hedger RS, Talbot P. Radial immuno-diffusion and serum nestralisation techniques for the assay of antibodies to swine vesicular disease. Research in Veterinary Science 1976;20(2):142-147.

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Hamblin C, Barnett ITR, Crowther JR. A new enzyme-linked-immunosorbent-assay (elisa) for the detection of antibodies against foot-and-mouth-disease virus .2. Application. Journal of Immunological Methods 1986;93(1):123-129.

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8.

Hamblin C, Barnett ITR, Hedger RS. A New Enzyme-Linked-Immunosorbent-Assay (Elisa) For the Detection of Antibodies Against Foot-and-Mouth-Disease Virus .1. Development and Method of Elisa. Journal of Immunological Methods 1986;93(1):115121.

9.

Hamblin C, Kitching RP, Donaldson AI, Crowther JR, Barnett ITR. Enzyme-Linked Immunosorbent-Assay (Elisa) For the Detection of Antibodies Against Foot-and-MouthDisease Virus .3. Evaluation of Antibodies After Infection and Vaccination. Epidemiology and Infection 1987;99(3):733-744.

10.

Bergmann IE, de Mello PA, Neitzert E, Beck E, Gomes I. Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens. American Journal of Veterinary Research 1993;54(6):825-31.

11.

Pinto AA, Garland JM. Immune response to virus-infection-associated (VIA) antigen in cattle repeatedly vaccinated with foot-and-mouth disease virus inactivated by formalin or acetylethyleneimine. Journal of Hygiene, Cambridge 1979;82:41-50.

12.

Foster M, Cook A, Cedillo L, Parkhouse RM. Serological and cellular immune responses to non-structural proteins in animals infected with FMDV. Veterinary Quarterly 1998;20 Suppl 2:S28-30.

13.

Mezencio JM, Babcock GD, Meyer RF, Lubroth J, Salt JS, Newman JF, Brown F. Differentiating foot-and-mouth disease virus-infected from vaccinated animals with baculovirus-expressed specific proteins. Veterinary Quarterly 1998;20 Suppl 2:S11-3.

14.

Mackay DKJ, Forsyth MA, Davies PR, Berlinzani A, Belsham GJ, Flint M, Ryan MD. Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA. Vaccine 1998;16(5):446-459.

15.

Sørensen KJ, Madsen KG, Madsen ES, Salt JS, Nqindi J, Mackay DKJ. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus. Archives of Virology 1998;143(8):1461-1476.

16.

Shen F, Chen PD, Walfield AM, Ye J, House J, Brown F, Wang CY. Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA. Vaccine 1999;17(23-24):3039-3049.

17.

Bergmann IE, Malirat V, Neitzert E, Beck E, Panizzutti N, Sanchez C, Falczuk A. Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with an enzyme-linked immunoelectrotransfer blot assay. Archives of Virology 2000;145(3):473-489.

18.

Blanco E, Romero LJ, El Harrach M, Sanchez-Vizcaino JM. Serological evidence of FMD subclinical infection in sheep population during the 1999 epidemic in Morocco. Veterinary Microbiology 2002;85(1):13-21.

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19.

Chung WB, Sørensen KJ, Liao PC, Yang PC, Jong MH. Differentaiting foot-and-mouth disease virus infected pigs from vaccinated pigs by blocking ELISA using non structural protein 3AB as antigen and its application to an eradication program. Journal of Clinical Microbiology 2002;40:2843-2848.

20.

Huang CC, Lee F, Tu WJ, Lee SH, Huang TS, Lin YL, Jong MH, Lin SY. Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus. Veterinary Microbiology 2002;84(4):317-326.

21.

Greiner M, Gardner IA. Epidemiologic issues in the validation of veterinary diagnostic tests. Preventive Veterinary Medicine 2000;45(1-2):3-22.

22.

Greiner M, Kumar S, Kyeswa C. Evaluation and comparison of antibody ELISA's for serodiagnosis of bovine trypanosomosis. Veterinary Parasitology 1997;73:197-205.

23.

Bronsvoort BMd, Tanya VN, Kitching RP, Nfon C, Saidou HM, Morgan KL. Foot-andmouth disease and livestock husbandry practices in the Adamawa Province of Cameroon. Tropic Animal Health and Production 2002;in press.

24.

Roeder PL, Le Blanc Smith PM. 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 1987;43(2):225-32.

25.

Ferris NP, Dawson M. 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 1988;16(3):201-209.

26.

Karber G. Beitrag zur kollectiven Behandlung pharmakologischer Reikenversuche. Arch. exp. path Pharmak. 1931;162:480-483.

27.

DeLong ER, DeLong DM, Clarke-Pearson DL. Comparing the areas under two or more correlated receiver operating characteristic curves: A non-parametric approach. Biometrics 1988;44:837-845.

28.

Coughlin SS, Trock B, CCriqui H, Pickle LW, Browner D, Tefft MC. The logistic modelling of sensitivity, specificity and predictive value of a diagnostic test. Journal of Clinical Epidemiology 1992;45:1-7.

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Christensen J, Gardner IA. Herd-level interpretation of test results for epidemiologic studies of animal diseases. Preventive Veterinary Medicine 2000;45(1-2):83-106.

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Appendix 34

Recently generated data with the CHEKIT-FMD-3ABC ELISA kit and methods to monitor the operational performance of a 3ABC ELISA L. Schalch1, D.E. Rebeski1, H. Samaras1, G. Lozano1, B. Thuer2, C. Schelp1 1 Bommeli Diagnostics, Stationsstr. 12, 3097 Liebefeld-Bern, CH 2 Institut für Viruskrankheiten und Immunprophylaxe (IVI), Nationales Tierseuchenreferenzlabor, 3147 Mittelhäusern, CH

Summary The CHEKIT-FMD-3ABC ELISA is a commercially available test kit for the detection of antibodies to the non-structural (NS) proteins of FMD virus. The assay has been launched during November 2001 and is still undergoing extensive validations in numerous laboratories. In this paper recently generated data are presented. Additionally, a full section is dedicated to data for the operational performance monitoring under manual usage and under fully automated conditions. Introduction Foot and mouth disease (FMD) is the most contagious disease of mammals and has a great potential for causing severe economic loss in susceptible cloven-hoofed animals. Diagnosis of FMD is made by the demonstration of FMD viral antigen or nucleic acid in samples of tissue or fluid. Detection of specific humoral antibody can also be used for diagnosis, especially in wildlife. Up to now, the humoral antibody detection required the absence of any history of vaccination, as it was not possible to differentiate a serological response to natural infection from that due to vaccination. The tests generally used are virus neutralisation (VN) and ELISA (Hamblin et al. 1986, Hamblin et al. 1987 and Van Maanen et al. 1990). These are also the prescribed tests for trade. The VN test is serotype specific, requires cell culture facilities and takes 2-3 days to provide results. The conventional ELISA is also serotype specific, sensitive and quantitative, and has the advantage that it is quicker to perform, less variable, and is not dependent on tissue culture systems. Low titre false-positive reactions can be expected in a small proportion of the sera in either test. An approach combining screening by ELISA and confirming the positives by the VN test minimises the occurrence of false-positive results. The detection of antibody to the nonstructural (NS) proteins of FMD virus has been used to identify past or present infection with any of the seven serotypes of the virus, whether or not the animal has also been vaccinated. The test can be used on a herd basis to detect FMD virus infection in vaccinated and unvaccinated populations (De Diego et al. 1997, Brocchi et al. 1998, Dekker et al. 1998, Malirat et al. 1998). In this paper data generated with the commercially available CHEKIT-FMD-3ABC ELISA during the most recent field studies are presented. An additional part is dedicated 283

to the operational performance monitoring using charting methods. Comparative data generated by hand or with a fully automated ELISA processor are presented. Material and methods 1.

CHEKIT-FMD-3ABC

The CHEKIT-FMD-3ABC bo-ov and po enzyme immunoassay (ELISA) kit provides a rapid, simple, sensitive and specific method for detecting antibodies against the pathogen responsible for Foot-and-Mouth-Disease (FMDV) in serum or plasma samples of bovine, porcine and ovine origin. This test allows to discriminate between samples from infected (3ABC positive) and vaccinated (3ABC negative) animals. The assay was performed according to the instructions provided by the manufacturer. 2.

Samples sets

2.1

Sera from non infected and not vaccinated, from vaccinated and from challenged cattle A set of 18 samples (6 negative control animals, 6 vaccinated animals, and 5 challenged animals) collected during one vaccination and challenge trial were analysed. 2.2 Sheep vaccination and challenge trial 17 sheeps were vaccinated and challenged. Samples were collected on day 0, 7, 14, 21 and 28 days after vaccination and 6 days after challenge 2.3 Bovine serum samples collected 21 days after vaccination 414 bovines were vaccinated and sampled 21 days later. 2.4 Multiple vaccination effect on test specificity, cattle 5 bovines were vaccinated with 4-fold standard dose on days 0, 28 and 56. Starting from day 0 the animals were sampled every 14 days until day 140 after vaccination. 2.5 Multiple vaccination effect on test specificity, swine 5 swine were vaccinated with 4-fold standard dose on weeks 0, 4, 8, 12 and 16. Starting from day 0 the animals were sampled every second week until week 18 after vaccination. 2.6 Negative bovine field samples collected in different European countries Bovine samples collected in several European countries were analysed in order to assess the test specificity. 948 German, 813 Austrian, 184 Swiss, 92 British and 33 Swedish samples were tested. 2.7 Negative ovine field samples collected in different European countries Ovine samples collected in several European countries were analysed in order to assess the test specificity. 92 German, 276 Swiss and 184 British samples were tested.

284

2.8 Negative porcine field samples collected in different European countries Porcine samples collected in several European countries were analysed in order to assess the test specificity. 534 Danish, 240 Swiss and 255 French samples were tested. 2.9 204 bovine serum and heparin- and EDTA plasma samples were tested 204 bovine were sampled in Germany using dry tubes (serum), heparin and EDTA (plasma) tubes. 2.10 Samples from experimental infection trials Several sample sets collected during different experimental infection trials were tested. 2.11 Laboratory proficiency testing Randomly collected routine serum and plasma sample were analysed under field conditions in 6 independent laboratories in Germany. 2.12 Samples used for the operational performance monitoring For the operational performance monitoring 4 different samples and/or controls were used. The first control was the conjugate control in other words an empty microtiter plate well. As negative and as strong positive serum the negative and the positive control respectively provided in the test kit was used. The weak positive control was obtained by preparing a 2 fold dilution of the strong positive control with the negative serum. Table 1: CHEKIT-FMD-3ABC data generated with negative samples with samples from Vaccinated and with samples from infected bovine

Designation Negative control Positive control 100 106 108 97 98 99 42 47 40 25 76 108 109 110 111 114 116

Status

PV PV PV PV PV PV PI PI PI PI PI NI NI NI NI NI NI

Sera Serotype

A A C O O O A A O SAT-1 SAT-2

CHEKIT-FMD-3ABC OD % 0.089 1.283 0.097 0.122 0.087 0.105 0.121 0.090 4.000 2.341 2.298 1.502 1.004 0.094 0.092 0.083 0.083 0.091 0.099

PV: post vaccination PI: post infection NI: no infection

285

0 100 1 3 0 1 3 0 327 189 185 118 77 0 0 0 -1 0 1

Results The first data described have been generated with bovine samples. In table 1. the CHEKIT-FMD-3ABC results generated with samples from non vaccinated non infected, from infected and from vaccinated animals are shown. The vaccination and the infection were performed with various FMD virus serotypes (A, C, O, SAT-1 and SAT-2). The results confirm a good specificity with samples from negative and negative but vaccinated animals. On the other hand, the results generated with samples from animals infected with different serotypes show the strain independency of the assay. The second data set describe results generated during a sheep vaccination experiment. During this experiment 17 sheep were vaccinated and sampled every 7 days until 28 days after the vaccination. On day 28 the animals were challenged and samples were collected 6 days post infection. All samples were analysed with the CHEKIT-FMD-3ABC and with the VNT, whereas the VNT was made with the homologous virus strain Lamb C1 Detmold. With the ELISA all samples prior to challenge were scored negative. With VNT the seroconversion following vaccination is observed (see table 2). Six days after infection the samples show very high titers in the VNT. With the ELISA 3 out of 13 analyzed samples showed an increased reactivity, whereas only one sample was scored ambiguous. (2) Results Chekit-FMD-3ABC Vaccination experiment #94.117 Sheep

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0dpv

-1 1 -1 -1 1 0 0 0 2 0 1 0 0 0 1 1 3

7dpv

5 5 2 2 0 1 2 2 2 3 -1 0 2 1 1 0 2

14dpv

3 1 1 1 -1 0 1 1 1 1 3 0 -1 0 1 1

Results VNT (Lamb C1Detmold)

21dpv

28dpv

6dpi

-1 0 2 1

2 2 4 1

1 0 2 1 1 3 3

2 0 2 1 0 2 1

1 7 4 1 4 17 10 24 4 6 4 8 3

0dpv -

7dpv 0.45 0.45 0.9 -

14dpv 0.6 1.65 1.05 0.6 1.05 0.45 1.05 1.05 1.65 1.35 0.3 0.6 -

21dpv 0.75 1.2 0.9 1.8 1.2 0.6 1.05 1.05 1.35 1.35 1.5 0.75 -

28dpv 6dpi 1.5 1.8 1.5 >=2.55 0.9 2.4 2.25 2.4 1.65 2.4 0.75 >=2.55 0.75 >=2.55 ns 1.35 ns 1.35 >=2.55 1.5 >=2.55 >=2.55 1.05 >=2.55 >=2.55 0.75 >=2.55 >=2.55 ns ns: no serum

Table 2: Sheep vaccination trial with FMF serotype Lamb C1 Detmold. 17 sheeps were vaccinated with a Lamb C1 Detmold serotype based FMD vaccine. On the left hand side the CHEKIT-FMD-3ABC results are shown. On the right hand side the VNT titers generated with the homologous FMD serotype are shown.

In order to confirm that even multiple vaccinations was not affecting the diagnostic principle with NS proteins, 5 bovine were vaccinated (O1 Manisa) 3 times (day 0, 28 and 56) with a 4-fold vaccine dose. From each animal, serum, heparin and EDTA plasma samples were collected every second week until week 20 after vaccination. All samples were analysed with the CHEKIT-FMD-3ABC. None of the animals showed an increased reaction in the ELISA (see figure B below), confirming the absence of NS proteins in a vaccine produced according to the standard procedure. This experiment additionally 286

confirms that any sample type tested (serum, heparin or EDTA plasma) did not affect the test performance. All samples were also analysed in a conventional ELISA using inactivated whole virus (O serotype). With these results the sero conversion after vaccination can be documented (see Figure 1.).

anti-SP-O (%)

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dpv

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40 30

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20

heparin EDTA

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dpv

Figure 1. Multi-vaccination trial with 5 bovine. Five bovine were vaccinated on day 0, 28 and 56 with a 4 fold normal dose FMD O Manisa vaccine. Serum, heparin and EDTA plasma samples were collected every 14 days until day 140. (A) all samples were analysed with an ELISA based on inactivated O1 Manisa FMD virus. (B) All samples were also analysed with the CHEKIT-FMD-3ABC.

A similar experiment as described above was performed with swine. Five swine were vaccinated (O1 Manisa) 5 times (day 0, 4, 8, 12 and 16) with a 4-fold standard vaccine dose. Samples were collected every second week until 18 weeks after vaccination. In the CHEKIT-FMD-3ABC all samples were scored negative (see Figure 2).

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anti-3ABC (%)

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Cut-off < 20 %

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18

weeks post infection Figure 2. Multi-vaccination trial with 5 swine. Five swine were vaccinated on day 0, 4, 8, 12 and 16 with a 4 fold normal dose FMD O Manisa vaccine. Serum, heparin and EDTA plasma samples were collected every 14 days until day 140. All samples were analysed with the CHEKIT-FMD-3ABC.

In order to further assess the test specificity different samples sets collected in different European countries were analysed. In the figure below the frequency distribution obtained for each sample set is shown. Except one that was scored ambiguous, all samples were clearly negative in the CHEKIt-FMD-3ABC (see Figure 3). 700 616

cut-off

Fig. 1A. Distribution of negative bovine samples (n = 2070)

497

Switzerland, n = 184 Great Britain, n = 92

446

500

Germany, n = 948 Austria, n = 813

Sweden, n = 33

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0 <0

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5 6 1

48

49 44

100

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40-50 50-60 % classes

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80-90

90-100

>100

Figure 3. Frequency distribution of negative bovine samples. The CHEKIT-FMD-3ABC results of FMD negative bovine samples from Germany, Austria, Switzerland, Great Britain and Sweden are represented

A similar specificity trial as described above was run with ovine samples. 552 ovine samples collected in different European countries (Germany, Switzerland and Great Britain) were analysed with the CHEKIT-FMD-3ABC ELISA. All samples resulted negative indicating a specificity of 100% (see Figure 4).

288

180

171

cut-off

157

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Fig. 1B. Distribution of negative ovine samples (n = 552) Germany, n = 92

Number of samples

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Switzerland, n = 276 Great Britain, n = 184

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specificity: 100.00%

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Figure 4. Frequency distribution of negative ovine samples. The CHEKIT-FMD-3ABC results of FMD negative bovine samples from Germany, Switzerland and Great Britain are represented.

Another specificity trial was run with swine samples. For this study 1029 swine samples collected in several European countries (Denmark, France and Switzerland) were analysed the CHEKIT-FMD-3ABC. With this sample type, one was scored positive and two ambiguous. All other samples were scored negative (see Figure 5). Due to the fact that in numerous field laboratories different sample types (serum, heparin or EDTA plasma are sent in for analysis, the CHEKIT-FMD-3ABC characteristics were evaluated under this aspect. For this reason, 204 bovine samples were collected at the same time in two different farms with dry (serum), heparin (plasma) and EDTA tubes. An additional variable was that in one farm the animals were not vaccinated against Bovine Herpes Virus 1 (BHV1, n =89) whereas in the second farm the animals were BHV1 vaccinated with a gE marker vaccine (n = 105).

289

400

cut-off

Fig. 1C. Distribution of negative porcine samples (n = 1029)

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Denmark, n = 534 France, n = 255 Switzerland, n = 240 224

250 200

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Percent positivity

Figure 5. Frequency distribution of negative swine samples. The CHEKIT-FMD-3ABC results of FMD negative swine samples from Denmark, France and Switzerland are represented.

30

+

25

?

20 15

-

10 5 0 Serum

Heparin

EDTA

Figure 6. Box- and whisker plot of FMD negative bovine samples tested with the CHEKIT-FMD-3ABC. 204 sample collected in dry (serum), heparin (plasma) and EDTA (plasma) tubes were analysed.

290

All samples were analysed and the results compared. The results are represented as boxand whisker plot in Figure 6. Except one animal which gave an ambiguous result independently of the sample type all animals were scored negative. The analytical test sensitivity was further investigated by analysing samples collected during various experimental infections of cattle, sheep and swine. For all species the FMD virus serotype A24 was used. These long term experiments were carried out in order to investigate the FMD specific antibody kinetic over a longer time. All the experiments were conducted at the "Institut für Viruskrankheiten und Immunprophylaxe (IVI), Nationales Tierseuchenreferenzlabor", in Mittelhäusern, Switzerland. For all three species it is shown, that the NS protein specific antibody sero conversion can be detected with the CHEKIT-FMD-3ABC as early as 8 to 10 days after infection. For all animals an NS protein antibody titer decrease is observed after a certain time. In this experiment for bovine the antibody started to decrease after 28 to 50 days. Two animals out of 5 remained positive during the observation period, whereas the others became negative 133 and 218 days after infection (see Figure 7). 270 250

Bovine005 Bovine 003 Bovine 769 Bovine 772 Bovine 773

230 210

CHEKIT-FMD-3ABC (%)

190 170 150 130 110 90 70 50

cut off

30

+ ?

10

-

-10

0

4

7

9

11 14 21 30 43 98 126 133 155 161 166 182 190 212 232 240 268 294 303 331 364

dpi

Figure 7. 3ABC specific antibody kinetic following experimental infection. 5 bovine (003, 005, 769, 772 and 883) were experimentally infected and sampled at different time points after infection (dpi). All samples have been analysed with the CHEKIT-FMD-3ABC.

291

For the two sheep used in this study the sero conversation after infection was observed between 7 and 10 days after infection. For one animal all samples were scored positive during the whole experiment. For the second animal a rapid antibody titer decrease expressed in lower %-values in the CHEKIT-FMD-3ABC was observed between day 126 and 155 after infection (see Figure 8). 230 210

Sheep 1 Sheep 2

CHEKIT-FMD-3ABC (%)

190 170 150 130 110 90 70 50

cut off +

30

?

10

-

-10

0

4

7

9

11 14 21 30 43 98 126 133 155 161 166 182 190 212 232 240 268 294 303 331 364 391

dpi

Figure 8. 3ABC specific antibody kinetic following experimental infection. 2 sheep (1 and 2) were experimentally infected and sampled at different time points after infection (dpi). All samples have been analysed with the CHEKIT-FMD-3ABC.

For swine the sero conversion was observed between 8 and 10 days after infection. For all animals except one the FMD NS protein specific antibodies measured with the CHEKIT-FMD-3ABC persisted throughout the whole observation period of the experiment. For one animal the antibody titer started to decrease between day 21 and 70 after infection and the last sample taken on day 301 after infection gave even a negative results (see Figure 9).

292

290 270

Pig 65 Pig 67 Pig 68 Pig 69

250 230

CHEKIT-FMD-3ABC (%)

210 190 170 150 130 110 90 70 50

cut off +

30

?

10

-

-10

0

4

7

9

11

14

21

30

43

98

126 133 155 161 166 182 190 212 232 240 268 294

dpi

Figure 9. 3ABC specific antibody kinetic following experimental infection. 4 swine (65, 67, 68 and 69) were experimentally infected and sampled at different time points after infection (dpi). All samples have been analysed with the CHEKIT-FMD-3ABC.

In order to test the laboratory proficiency and also to introduce the assay in the routine laboratory, a field trial was organised in six official laboratories in Germany in collaboration with the FMD national reference centre in Tübingen. For this trial, various randomly collected routine samples collected from cattle, sheep, goat and swine were analysed in the different laboratories. Table XX summarizes the results which were generated. Due to the fact that the CHEKIT-FMD-3ABC has been designed according to the overall CHEKIT parameter range, which is widely used in these laboratories, the assay was well accepted and easily introduced. In total 1849 samples have been analysed. Except for one laboratory, which could not validate one plate according to the manufacturer’s guidelines, no major problems were encountered for the test introduction. As only negative samples were analysed it was only possible to assess the diagnostic assay specificity. Nine out of the 1849 tested samples were scored ambiguous. Considering all 9 samples as false positive an overall test specificity of 99.51% has been calculated. Two out of these 9 samples were cattle samples and 7 were swine samples (for details see Table 3).

293

Laboratory Species #1 #2

Cattle Sheep, goat Swine Cattle Swine

#3 #4 #5 #6

Cattle Sheep Swine Cattle Sheep Swine Cattle Swine Cattle Sheep Swine

Sample Matrix Mean SD Mean + 3SD Max. DSp 92 92 92 92 92 92 92 42 40 22 22 159 22 276 180 231 27 184

Plasma Plasma Plasma Serum Plasma Serum Plasma Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum

-1 -2 -7 -2 0 1 1 2 4 1 0 -1 16 -1 -1 2 1 -1

2 2 3 2 3 3 3 1 2 3 2 3 4 2 2 3 4 5

6 5 2 4 8 10 10 6 10 10 7 7 29 6 6 12 12 14

4 13 6 2 13 11 12 4 9 8 6 8 25 13 12 34 19 33

Remarks 100 100 100 100 100 100 100 100 100 100 100 100 77 100 100 99 100 99

1 plate rejected

5 doubtful samples Swine samples re-tested at BD 2 doubtful samples 2 doubtful samples

Table 3. Results generated during a field trial in Germany are summarised. In 6 independent German laboratories randomly collected samples from various species (bovine, ovine, caprine and swine) were analysed with the CHEKIT-FMD-3ABC. 3ABC. For each animal species and for each laboratory the mean %-value (mean), the standard deviation (SD), the mean + 3x SD, the maximal value (Max.) and the calculated diagnostic specificity (DSp) are shown. In the last column some comments provided by the respective laboratory has been included.

Nowadays the ELISA technology is widely applied in veterinary diagnostics. With the availability of standardized test kits and with the routine use of the technique in nearly all veterinary diagnostic laboratories, it became a very reliable diagnostic tool. Although it is a very robust method, many steps during the assay procedure can dramatically influence the test performances. Therefore, every laboratory should implement quality-monitoring procedures in order to generate reliable results. Besides defining standard laboratory procedures or participating to so-called ring trials organized by national and international reference laboratories, one technique to guarantee reliability is to use internal quality control methods in combination with charting methods. Charting methods can be implemented in a very pragmatic way by including one reference sample on each microtiter plate and to monitor the respective result between assays over time. A more sophisticated charting method has been described by Rebeski et al. 2001. For this method, it is necessary to know the control data range for internal quality control (IQC) samples, which have to be defined during the assay development phase. Once these limits have been set, the user can simply compare his own results generated with the IQC samples with the range set by the assay developer. A very convenient way to analyse such data has proved to be the Shewart-like charting (Shewart, 1931) technique. In order to analyse the operational performance for the CHEKIT-FMD-3ABC two approaches have been chosen. First it was decided to run the assays manually as performed by a majority of all users. Second the same experiment was run by using a fully automated ELISA processor (Tecan Genesis, Tecan Schweiz AG, Seestrasse 103, 8708 Männerdorf, Switzerland). By using both approaches it was possible to assess the test performance as such and to get an idea about the influence of the human variability on the test characteristics.

294

To run the assay four controls consisting of a conjugate control (CC), a strong positive (C++) a low positive (C+, 1 to 2 dilution of C++) and a negative (C-) serum sample were used. For the manual processing every control was distributed as four sets of quadruplicate on each plate. For the automated processing every control was distributed as six sets of quadruplicate on each plate. For both procedures a total number of 20 microtiter plates were analysed on different days. For the manual and the automated ELISA processing the complete data charting methods on the operators level was performed. The charts are meant to visualize the agreement of the true operational work performed locally with the expected performance determined by the assay developer to control and monitor the plate to plate and day to day and trend performance. Shewart-Like control detailed (D) and summary daily data (SDD) charts are generated to plot the daily distribution of the IQCs. The chart shows the number of plates along the x-axis against the actual absorbance values or the PP values (y-axis), respectively. In addition the UCL-LCL ranges representing the overall mean OD and PP values + 3 standard deviations (STDEV) are indicated. The daily IQC results from single plates (mean OD and PP values + 2 STDEV) and an overall mean + 2 STDEV derived from all plates on one occasion is plotted. The intra-laboratory analysis of the variation of the IQC replicates within and between plates is referred to as assay repeatability. Detailed daily precision (DDPre) charts plot percent coefficient of variation (CV%) which is a measure of relative dispersion of IQC replicates based on the STDEV. The CV% is calculated by the STDEV of four PP replicates divided by the respective mean for single plates. For this study, the UCL is set as CV = 10%, which was empirically determined and recommended for evaluation of standardised ELISA’s (Jacobson, 1998). The detailed daily proficiency (DDPro) charts plot the intra-laboratory assay proficiency computing the ratio of antibody binding to non-binding (B/B0) of the median PP of C+ divided by C- from each plate. For the calculation, the median of four IQC replicates rather than the mean is chosen to approach the true value rather than the value more biased by dispersion of four replicates. Also, the small difference of antibody activity of C+ compared to Cwas considered more suitable to indicate reduced assay proficiency than the higher ratio C++ divided by C-. The overall mean of B divided by B0 ratio + 2 STDEV derived from all plates on one occasion is also plotted. The tentative UCL-LCL range is determined from the overall mean value C+ divided by C- binding ratios + 3 STDEV which is obtained from the data described before. Figure 10 shows all the data which were generated during the full automation procedure. During this experiment a plate was always analysed by hand (Plate_Hand) and the results were compared to the automated procedure (Plate_RPMA25). Except on one day at which the C++ was higher than the UCL, all data were within the UCL-LCL.

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A

Detailed and Summary Daily Data (D & SDD) Chart #1 Plots of optical density (OD) values (C++ AVG ODs ± 2 STDEV). Horizontal lines represent tentative upper and lower control limits (AVG ODs ± 3 STDEV).

2.5 2.4 2.3 2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

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Detailed and Summary Daily Data (D & SDD) Chart #2 Plots of optical density (OD) values (C++ AVG ODs ± 2 STDEV). Horizontal lines represent tentative upper and lower control limits (AVG ODs ± 3 STDEV).

2.5 2.4 2.3 2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

C

Detailed and Summary Daily Data (D & SDD) Chart #1 Plots of percent positivity (PP) values (C++, C+, C- and Cc AVG PPs ± 2 STDEV). Horizontal lines represent tentative upper and lower control limits (AVG PPs ± 3 STDEV).

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120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

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Plots of percent coefficient of variation (CV %) of percent positivity (PP) values (C++, C+, C- and Cc). Horizontal line represents tentative upper control limit of CV = 10 %.

Detailed Daily Precision (DDPre) Chart #1

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F

Plots of percent coefficient of variation (CV %) of percent positivity (PP) values (C++, C+, C- and Cc). Horizontal line represents tentative upper control limit of CV = 10 %.

Detailed Daily Precision (DDPre) Chart #2

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Detailed Daily Proficiency (DDPro) Chart #1

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SD020822

0

Figure 10. (A & B) Illustration of a daily single and summary plate OD values plotted on a D and SDD chart (C++ mean ODs + 2 STDEV). Horizontal lines represent tentative UCL and LCL (overall mean ODs + 3 STDEV) as determined during this study. (C & D) Illustration of a daily single and summary plate PP values plotted on a detailed and SDD chart during this study (C++, C+, C- and CC mean PPs + 2 STDEV). Horizontal lines represent tentative UCL and LCL (overall mean PPs + 3 STDEV) as determined during this study. (E) Illustration of CV% of PP values plotted on DDPre chart during this study. The horizontal line represents the upper the tentative upper control limit of CV = 10%. (G & H) Illustration of C+/Cbinding ratios plotted from median PP values of four replicates on a daily detailed proficiency chart during this study. The daily overall mean B/B0 ratio + 2 STDEV is also recorded. Horizontal lines represent tentative UCL and LCL (overall mean + 3 STDEV) as determined during this study.

The same proficiency analysis was performed by hand and by using the TECAN instrument. The results are compared in Table 4. What is observed is that the ranges defined by the STDEVs are slightly reduced when using a fully automated system.

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BD-FMD_UCL-LCL

TECAN-FMD_UCL-LCL

AVG STD AVG + 1 STD AVG - 1 STD AVG + 2 STDs AVG - 2 STDs AVG + 3 STDs AVG - 3 STDs

Cc (ODs) 0.063 0.008 0.071 0.055 0.078 0.048 0.086 0.040

C++ (ODs) 0.836 0.153 0.990 0.683 1.143 0.530 1.296 0.377

C+ (ODs) 0.387 0.066 0.453 0.321 0.518 0.255 0.584 0.190

C- (ODs) 0.061 0.008 0.069 0.054 0.077 0.046 0.085 0.038

AVG STD AVG + 1 STD AVG - 1 STD AVG + 2 STDs AVG - 2 STDs AVG + 3 STDs AVG - 3 STDs

Cc (ODs) 0.093 0.011 0.103 0.082 0.114 0.071 0.124 0.061

C++ (ODs) 1.688 0.081 1.769 1.607 1.850 1.525 1.932 1.444

C+ (ODs) 0.900 0.044 0.944 0.856 0.988 0.811 1.033 0.767

C- (ODs) 0.094 0.011 0.105 0.082 0.116 0.071 0.127 0.060

AVG STD AVG + 1 STD AVG - 1 STD AVG + 2 STDs AVG - 2 STDs AVG + 3 STDs AVG - 3 STDs

Cc (PPs) 8 2 10 6 11 4 13 3

C++ (PPs) 100 2 102 98 104 97 105 95

C+ (PPs) 47 4 51 42 55 38 59 34

C- (PPs) 8 2 9 6 11 4 13 2

AVG STD AVG + 1 STD AVG - 1 STD AVG + 2 STDs AVG - 2 STDs AVG + 3 STDs AVG - 3 STDs

Cc (PPs) 5 1 6 5 7 4 7 4

C++ (PPs) 100 1 101 99 102 98 103 97

C+ (PPs) 53 2 55 51 57 49 59 47

C- (PPs) 6 1 6 5 7 4 8 3

AVG STD AVG + 1 STD AVG - 1 STD AVG + 2 STDs AVG - 2 STDs AVG + 3 STDs AVG - 3 STDs

B/B0 (PPs) 6 1 8 5 9 4 10 2

AVG STD AVG + 1 STD AVG - 1 STD AVG + 2 STDs AVG - 2 STDs AVG + 3 STDs AVG - 3 STDs

B/B0 (PPs) 10 1 11 9 12 7 13 6

Percent proportion of CV % values < 10% (n=120) Cc C++ C+ C45.00 95.00 91.67 55.00

Percent proportion of CV % values < 10% (n=120) Cc C++ C+ C86.67 99.17 98.33 75.00

Max CV% value Cc 31

Max CV% value Cc 18

C++ 33

C+ 16

C69

C++ 19

C+ 11

C20

Table 4. Summarized data generated during a operational performance testing of the CHEKIT-FMD-3ABC by hand (BD FMD UCL-LCL) and with a full automated ELISA processor TECAN Genesis RPM (TECAN-FMD UCL-LCL). For both approaches the overall mean (AVG) for the optical densities (OD), the percentage values (PP) and the binding ratio (B/B0 = C+PP/C-PP) are shown.

Discussion The data presented in the first part of this chapter confirm what has already been observed during previous evaluations performed with the CHEKIT-FMD-3ABC. Overall the specificity reaches an acceptable level for a reliable diagnostic. As far as the diagnostic sensitivity is concerned, the results confirm that most probably NS protein based FMD diagnostics are suitable for a herd diagnostic and that the CHEKIT-FMD3ABC is a potential candidate, commercially available meeting these requirements. In the second part the assay robustness is documented. With this data it is shown that the CHEKIT-FMD-3ABC is a valuable and reliable tool for the routine FMD diagnostic under field conditions.

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Acknowledgment We would like to express our gratitude to all institutions and colleagues who helped to generate the data presented in this work. Special thanks shall go to the team of the IVI in Mittelhäusern in Switzerland, which put at disposal the laboratory facility and the precious samples collected during experimental infection trials. We are gratefull to Dr. Haas from the German FMD reference laboratory and all German laboratories having coordinated and run the field trial in Germany. Literature

Brocchi E., De Diego M.I., Berlinzani A., Gamba D. And De Simone F. (1998) Diagnostic potential of MAB-based ELISAs for antibodies to non structural proteins of Foot and Mouth Disease Virus to differentiate infection from vaccination. Vet. Quart. Vol 20 suppl. 2. De Diego M., Brocchi E., Mackay D., and De Simone F. (1997) The non-structural polyprotein 3ABC of Foot and Mouth Disease Virus as a diagnostic antigen in ELISA to diffrentiate infected from vaccinated cattle. Arch. Virol. 142. Dekker A. and Gijsen E. (1998) The possible use of native Foot and Mouth disease nonstructual protein 3A in a serological screening test. Vet. Quart. Vol 20 suppl. 2. Hamblin C., Barnett I.T.R. & 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. Hamblin C., Kitching R.P., Donaldson A.I., Crowther J.R. & Barnett I.T.R. (1987). Enzymelinked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. 3. Evaluation of antibodies after infection and vaccination. Epidemiol. Infect., 99, 733-744 Jacobson R.H. (1998) Validation of serological assays for diagnosis of infectious diseases. Rev. Sci. Tech. Off. Int. Epiz. 17. Malirat V., Neitzert E., Bergmann I.E., Maradei E. and Beck (1998) Detection of cattle exposed to Foot and Mouth Disease Virus by means of an indirect ELISA test using bioengineered nonstructural polyprotein 3ABC. Vet. Quart. Vol 20 suppl. 2. Rebeski D.RE., Winger E.M., Ouma J.O., Kong Pages S., Büscher P., Sanogo Y., Dwinger R.H. and Crowther J.R. (2001). Charting methods to monitor operational performance of ELISA method for the detection of antibodies against trypanosomes. Vet. Parasitol. 96. Shewart W.A. (1931). Economic control of quality of manufactured product. Van Nostrand, New York (republished by ASQC Quality press, Milwaukee, WI, 1980). Van Maanen C. (1990). A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot and mouth disease types A, O and C. 1. Method and characteristics. Vet. Microbiol., 24, 171-178.

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Appendix 35

Synthetic peptide-based serosurveillance and vaccine system for FMD Scott Liu1, Tseng Yuan Chang1, Alan M. Walfield1, Mei Lun Zhang1, Kenneth K. Sokoll1, Shih Ping Chen2, Ming Chang Li2, Yeou Liang Lin3, Ming Hwa Jong3, David Yang2, Nancy Chyr4, and Chang Yi Wang1,4 1

United Biomedical Inc., 25 Davids Drive, Hauppauge NY 11788, U.S.A. Animal Technology Institute Taiwan (ATIT), Chunan, Miaoli, Taiwan. 3 National Institute of Animal Health Taiwan, Tanshui, Taipei, Taiwan. 4 United Biomedical Inc. Asia, Shijr City, Taipei, Taiwan. 2

Introduction We have designed a synthetic peptide-based system for the control of FMD, having synthetic peptide-based NS immunoassays and peptide-based marker vaccines. The UBI® system for FMD has two indirect ELISAs for screening animals based on 3B nonstructural protein (NS) synthetic antigen, for detection of animals that may be carrying infectious virus and for differentiation of exposed from vaccinated animals (the UBI® FMDV NS EIA SWINE and CATTLE) [1,2]. The addition to the UBI® system of two peptide-based confirmatory tests, a 3B NS blocking ELISA (the UBI®FMDV NS 3B Neutralization Assay) and a 3A NS indirect ELISA (the UBI® NS 3A EIA) is reported here. We report on the initial validation studies of these tests and on a large-scale field trial showing the overall performance of the UBI® NS immunosurveillance system that includes the 3B screening test and the two confirmatory immunoassays. The other half of the UBI peptide-based system uses VP1 G-H loop peptide immunogens in marker vaccines against FMDV. The G-H loop peptide has been optimized for crossreactivity to FMDV and for immunogenicity by the inclusion of cyclic constraint for relevant conformation, by extending the critical G-H loop site with adjoining VP1 sequences for additional Th epitopes and more authentic conformation, and by linkage to a promiscuous UBITh® T helper epitope. The extrinsic T cell help provided by the UBITh® site overcomes a lack of responsiveness to the G-H loop domain in many swine and cattle that is due to genetic restriction. The incorporation of consensus residues into the hypervariable positions of the G-H loop domain provides for broad cross-reactivities across strains. A novel consensus O immunogen (Fig. 1) was formulated into acceptable water-in-oil vaccine formulations and successfully protected swine from infection by heterologous FMDV O1 Taiwan [3]. UBI vaccines serve as marker vaccines because of the absolute absence of NS antigens, and so are complementary when used together with the UBI NS peptide immunoassays. Furthermore, peptide-vaccinated animals can be readily identified by use of a UBI serotype-specific VP1 ELISA [2]. In the present communication we report on further efforts to develop our swine vaccine, including a dose response, a vaccine stability and a vaccine duration study. And, we present a preliminary formulation study directed towards application of the peptide-based vaccine to cattle. 303

Materials and Methods Peptide synthesis The peptide antigens for immunoassays and the peptide immunogens for vaccines were produced by synthesis on a solid-phase support using an Applied Biosystems Peptide Synthesizer Model 430A, and Fmoc protection for the α-NH2 terminus and side chain protecting groups of trifunctional amino acids. Peptides having combinatorial library Th were prepared by providing a mixture of the desired amino acids at the specified positions. Completed peptides were cleaved from the solid support and side chain protecting groups removed by 90% trifluoroacetic acid. Synthetic peptide preparations, except for the library immunogens, were characterized for correct composition by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry using a PerSeptive Biosystems/Vestec LaserTec Benchtop 11 Mass Spectrometer, and by Reverse Phase HPLC. The library immunogen for the vaccine was characterized by size exclusion chromatography to a specification that requires 90% of the integrated area to exceed a mass threshold limit value, and by Edman degradation for N-terminal amino acid analysis. Liquid phase cyclization of the peptide immunogens was accomplished by dissolving the peptides in water at 0.8 mg/ml, pH 3, adding DMSO to 1% (v/v) and adjusting to pH 7.5 with NH4OH. The solution was incubated at ambient temperature in air and checked daily for 3 days by colorimetric assay using Ellman’s reagent until disulfide bond formation was at least 90% complete. Sera A total of 14 positive sera (Table 1) from animals infected with various FMDV serotypes were obtained from the United States Department of Agriculture (USDA) and used in the study. To test the specificity of the confirmatory tests, 1612 normal swine sera and 1200 ruminant (800 cattle, 200 sheep, and 200 goats) sera, originally collected from US slaughterhouses, were purchased from Covance and Valley Biomedical and used in the study. In addition, 10,169 field sera samples from pigs vaccinated with commercial FMDV O vaccines, which were collected from pig farms in Taiwan in a nationwide serological survey in 2001, were also tested with the confirmatory tests. ELISAs All ELISA assays, including the NS EIA confirmatory tests, were performed as described in the Direction Inserts of the test kits. Briefly, 100 μl of diluted samples are added to the microtiter wells of the UBI® FMDV NS assay and allowed to react at 37˚C for I hour. The sample wells are then washed six times with UBI® Wash Buffer. A standardized preparation of HRP-conjugated ImmunoPure® Protein A/G (Pierce Chemical Co., Rockford IL, USA) is added to each well and incubated for 30 minutes at 37˚C. The wells are again washed six times and then reacted with 3,3’,5,5’-tetramethylbenzidine (TMB) for 15 minutes. Reactions are stopped by the addition of 1.0 M H2SO4 and the A450 determined. For the FMDV NS 3B Neutralization Assay, serum samples were individually prediluted in duplicate with Specimen Diluent and with NS 3B Neutralization Buffer, before adding to NS Reaction Microplate coated with the NS 3B peptide [1]. The NS 3B Neutralization Buffer is equivalent in composition to the Specimen Diluent except that the 3B peptide was added to the buffer at a final concentration of 50 g/ml. The rest of the assay procedure was identical to that of the 304

UBI FMDV NS EIAs. The neutralization or inhibition rate of the NS 3B Neutralization Buffer to each serum sample was calculated by the following formula: % NEUTRALIZATION RATE (NR) =

(A450 of SD) − (A450 of NB) (A450 of SD)

x 100%

Where “A450 of SD” is the absorbance of sample mixed with the Specimen Diluent; “A450 of NB” is the absorbance of sample mixed with the NS 3B Neutralization Buffer. Based on preliminary studies, the Cutoff for the NS 3B Neutralization Assay was set at 60% Neutralization Rate (NR); i.e. sera giving NR values equal or greater than 60% are considered to be true positive, those giving NR values less than 60% are considered to be false positive. The procedure of the FMDV NS 3A EIA was identical to that of the FMDV NS (3B) EIA. Neutralizing Antibody Assay The quantitative N.A. assay for antibodies that neutralize FMDV is performed with BHK-21 cells in flat-bottomed microtiter plates. The test is an equal volume test in 50 µl. Starting from a 1:4 dilution, sera are diluted two-fold across the plate. Diluted sera are mixed with equal volumes of FMDV O1 Taiwan (200 TCID50/50 µl) and incubated at one hour at 37˚C. Cells at 1 x 106 cells/mL are added in medium containing 10% bovine serum. Microscopic examination is feasible after 48 hours. Titers are expressed as the reciprocal of the final dilution of serum at the 50% endpoint. NS Enzyme-linked Immunoassays (EIAs) background False-positive samples can impose a serious problem for immunoassays for detection of FMD. Because non-specific reactivity and cross reactivity can both give false-positive results in enzyme-linked immunoassays (EIAs), no EIA test can give a specificity of 100.0%. The UBI® FMDV NS EIAs are no exception to the rule. Despite their excellent specificities in detecting antibodies to the non-structural (NS) protein 3B in normal and vaccinated swine and cattle samples, which ranges from 97.5% to 99.3%, false positives still occur among samples from non-infected animals [1,2]. False-positive test results can be ominous uncertainties: 1. In the early phase of an outbreak, before official confirmation of FMDV infection, uncertainties to the accuracy of a positive test result can be devastating, as any delays to the final confirmation of FMD outbreak will lead to wider spread of the disease. 2. After the first case of FMD has been confirmed in an outbreak, false-positive test results can be severely damaging to farmers and economies. Quick detection is critical to effectively control this extremely contagious disease, and in many cases no time will be allowed for laborious confirmation tests. Consequently, all suspicious animals will be slaughtered. Farms and agricultural areas can be wrongly classified

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as “infection zones” or “infection centers”, and emergency response resources will be wasted and local economies will be severely affected by this classification. There are several OIE-recognized FMD diagnostic methods that can be used for confirmation of the NS EIA results. However, for the following reasons, those tests described in the OIE Manual of Standards for Diagnostic Tests and Vaccines (O.I.E. 2000) cannot be readily conducted or completed within a short period of time (a few hours). 1. Two non-immunological virus identification methods are described in the OIE Manual, virus isolation by tissue culture and reverse-transcription polymerase chain reaction (RT-PCR). Both methods require tissue samples isolated from the epithelial cells of the suspected animals. However, in many cases, it is very inconvenient, timeconsuming, or impossible (due to termination) to isolate epithelial samples from suspected animals. It often requires veterinarians to go back to the farms that the sera samples were originally collected to obtain the epithelium samples of the suspected animals. Moreover, the tissue culture procedures for viral amplification and detection requires at least 1-2 days. This 1-2 days delay will be very costly in a real FMD outbreak situation, particularly in the early stage of an outbreak when no infection has been confirmed and no measures have been put into place to prevent or control its spread. RT-PCR is a highly specialized technique that requires unusual care and skill for accurate results. 2. There are also several enzyme-linked immunosorbent assays (ELISAs) described in the OIE Manual that can be used to confirm the NS EIA results. However, these serological methods use either antisera to the whole viral particle or to a major structural component of the virus (the 146S antigen), which can only detect FMDV infection in cases of high systemic viremia. Therefore, these methods are often not very sensitive. In addition, because polyclonal antisera against many viral proteins are used as the capturing and detecting antibodies, the specificity of these assays tends not to be very good. 3. The viral neutralization (VN) test is also a method for detection of antibodies to FMDV. This method requires growing cells by tissue culture, and therefore often takes at least 1-2 days. Furthermore, the sensitivity of this method is often not good enough because that in many cases FMDV infections do not trigger high titers of neutralizing antibodies, and that the virus serotype used in the VN test may not be of sufficient serological cross-reactivity to the infecting virus for high sensitivity. In addition, sera from vaccinated, but not infected, animals also contain neutralizing antibodies, which can complicate the interpretation of the result of the VN test. 4. The Western blot method for detection of anti-NS antibodies is laborious and time consuming, and requires a high degree of skill and experience, so cannot be considered an easy, fast, and convenient confirmation method.

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Therefore, there is a need for an FMD confirmatory test that is sensitive and specific, easy to perform, and quick to finish and generate results. This confirmatory test, once developed will be extremely useful for confirmation of positive test results of FMDV NS EIAs. Fast and reliable confirmation of the NS EIA diagnostic results will be critical to FMD management and control, especially in the early phase of an outbreak and in the later eradication phases. We have developed two confirmatory tests that are designed to rapidly confirm positive results by UBI® FMDV NS EIAs. The UBI® NS confirmatory tests, which consist of the UBI® FMDV NS 3B Neutralization Assay and UBI® FMDV NS 3A EIA, use the same indirect ELISA format that allows the assays to be easily performed and finished within two hours. The UBI® FMDV NS 3B Neutralization Assay (NA) is a blocking ELISA that has been designed to confirm whether a positive seroreactivity identified by a UBI® FMDV NS EIA results from the specific reaction of serum antibodies to the NS 3B peptide. A 3B seroreactivity specifically directed to FMDV will be markedly reduced by mixing the serum with NS 3B Neutralization Buffer containing the soluble NS 3B peptide. In contrast, a non-specific 3B seroreactivity will be largely unaffected by mixing the sample with NS 3B Neutralization Buffer. During the course of the 3B NA, each specimen is diluted in duplicate; in one well, the specimen is mixed and diluted with the Specimen Diluent of the UBI® FMDV NS EIA (SWINE) or (CATTLE), and in the other well, it is mixed with the NS 3B Neutralization Buffer. The NS 3B Neutralization Buffer is basically the Specimen Diluent plus the 3B peptide. The diluted samples are then added to the Reaction Microplate wells. FMDV NS 3B-specific antibodies, if present, bind with the liquid-phase 3B peptide antigen during mixing with the Neutralization Buffer, and most of them are no longer available for binding to the solid-phase 3B peptide coated on the Reaction Microplate. Nonspecific antibodies do not bind to the solution-phase peptide antigen after mixing with the Neutralization Buffer, and remain capable of binding to the Reaction Microplate surface. Any specimen showing a reduction of A450nm of certain percentage or greater in the FMDV NS EIA following mixing with 3B Neutralization Buffer will be considered seropositive (i.e., contains 3B-specific antibodies). Any specimen showing a reduction of the A450nm of less than that percentage will be considered negative for antibodies to FMDV. The UBI® FMDV NS 3A EIA employs for its sensitizing agent a 3A non-structural (NS) protein peptide that contains antigenic determinants taken from the immunoreactive domains of the NS 3A protein [1]. NS 3A is the first protein of the NS 3ABC polyprotein which has been most often used as the solid phase antigen in NS EIAs developed elsewhere. The NS 3A EIA had been tested by UBI® during initial research on selection of an NS antigen for development of a commercial NS EIA test. It was found in our previous study that the NS 3A polypeptide imparts the assay with excellent sensitivity that is as good as the NS 3B EIA [1]. However, in those studies on vaccinee samples, the FMDV NS 3A EIA had poorer specificity than that of the NS 3B EIA. That

307

finding was the basis for choosing NS 3B as the solid phase antigen for UBI® FMDV NS EIAs. The 3A peptide antigen may nevertheless be used for a confirmatory test. Having a 3A antigen as the solid-phase immunosorbent provides for an EIA that is serologically independent to the 3B-based UBI FMDV NS EIA. Thus, the FMDV NS 3A EIA (CATTLE) or (SWINE) provides an assay that is very similar to the UBI® FMDV NS EIA (CATTLE) or (SWINE) in principle and procedure, but capable of independent confirmation of the repeatably reactive samples identified by the 3B-based NS EIAs. We describe in this report the results of initial validation studies of the UBI® NS EIA confirmatory tests. EIA Results Validation of FMDV NS confirmatory tests with FMDV positive sera The confirmatory tests were tested against a panel of FMDV-infected animal sera obtained from the USDA (Table 1). It was found that both tests correctly identified all sera samples in the panel as positive. The NR of each sample was greater than 80% in the NS 3B Neutralization Assay, and the Signal/Cutoff ratio of each sample in NS 3A EIA was greater than 1.0 (Tables 2 and 3). Validation of FMDV NS confirmatory tests with normal animal sera The 1612 normal swine samples from the U.S. were first screened with UBI® FMDV NS (3B) EIA (SWINE). A total of 11 repeatably reactive samples were identified. These presumably false positive samples were then tested with FMDV NS 3B Neutralization Assay. Nine of the 11 repeatably reactive samples were identified as false positive by the Neutralization Assay. The remaining 2 positive samples were both identified as negative after testing with the FMDV NS 3A EIA (SWINE) (Table 4). Thus, a specificity of 99.87 % (1610/1612) was achieved when only the NS 3B NA was performed, and a combined specificity of 100.00% was achieved when both confirmatory tests were applied. For the 1200 normal ruminant samples from the U.S., a total of 9 repeatably reactive samples were identified by UBI® FMDV NS (3B) EIA (CATTLE). These 9 false positive samples were tested by both NS 3B Neutralization Assay and FMDV NS 3A EIA (CATTLE). Two out of the 9 samples were identified as false-positive by the Neutralization Assay, and 6 out of the remaining 7 samples were found non-reactive by NS 3A EIA (CATTLE) (Table 5). Thus, a specificity of 99.42 % (1193/1200) was achieved when only the NS 3B Neutralization Assay was performed, and a combined specificity of 99.92% (1199/1200) was achieved when both confirmatory tests were applied. Validation of FMDV NS confirmatory tests with sera from vaccinated swine The samples tested were from a recent Taiwanese survey in which a total of 10,169 swine samples were collected from 85 farms, with 120 animals (60 sows and 60 growth pigs) randomly screened per farm. All animals had received at least two doses of FMDV O vaccines. The infection status of these animals was unknown since FMD is endemic in

308

Taiwan. From the serological screening by the UBI® FMDV NS EIA (SWINE), a total of 413 repeatably reactive (RR) samples were identified. Of the 413 RR samples, 330 were found to be false positive, i.e., the reactivities were not specific to the NS 3B peptide antigen, when tested by the FMDV NS 3B Neutralization Assay. The remaining 83 samples were subjected to the FMDV NS 3A EIA (SWINE), and 58 of them remained positive after the second confirmatory test. To analyze the true infection status of these 58 triple-positive samples, a herd analysis was applied. FMDV is highly contagious and thus multiple infections within a herd would be expected. Therefore, it was assumed that for herds where multiple triple-positive samples were identified, they are most likely to be true positives. Conversely, if a triple-positive sample was from a farm where only a single triple-positive sample was identified, it is likely that this sample was false positive. By these criteria, 50 out of the 58 triple-positive samples were found to be true positive, and the other 8 to be false positive. Thus, based on this analysis, the specificity on vaccinee sera for UBI® FMDV NS EIA (SWINE) plus the NS 3B NA was 99.18 % (10036/10119); the specificity for UBI® FMDV NS EIA (SWINE) plus both the confirmatory tests was 99.92% (10111/10119) (Table 6). Vaccine results Swine For the vaccine dose response and stability trial shown in Table 7, the synthetic FMDV VP1 immunogen shown in Fig. 1 was dissolved into water and formulated into water-inoil vaccines at the doses shown in the Table, with Seppic Montanide ISA 50v as the oil phase. The final volumes of peptide vaccines and placebo were kept constant at 1.0 ml, the commercial vaccine was in 2.0 ml. Pigs were immunized on weeks 0 and 4 by intramuscular injection and challenged with FMDV O1 Taiwan on week 8 by injection of 104.5 TCID50 of pig-passaged virus. Swine were monitored for clinical signs of FMD. These included body temperatures elevated to ≥40º for three successive days, lameness and vesicular lesions on the snout and coronary bands of the legs. Groups 1-3 were a dose response experiment using a four-fold range from 3.12 to 50 µg. The vaccine was protective at all doses, so we were unable to estimate a PD50. The neutralizing antibody titers by day of challenge were correlated to dose. Group 3, the group given the lowest dose, also had the lowest neutralizing responses. Group 4 was given 50 µg doses of freshly prepared peptide vaccine. Groups 5 and 6 received comparable doses that had been aged for 6 months and one year, respectively, to evaluate vaccine stability. The vaccine remained fully stable and effective for at least six months as shown by the protected animals, while retaining significant potency for one year. The placebo controls all experienced disease, while the positive control group given a commercial FMDV O1 Taiwan vaccine was fully protected. Table 8 shows the results of a vaccine duration study. At the start of the study, pigs were given a single 25 µg I.M. dose of the FMDV O consensus immunogen in ISA 50v. At monthly intervals, groups of five were challenged by an FMDV PanAsia isolate. The duration of protective immunity lasted for at least six months.

309

Cattle A synthetic serotype O peptide immunogen was prepared as water-in-oil vaccine formulations in ISA 50v, at a dose of 200 µg (Table 9). One group of three cattle was given that emulsion, which is equivalent to a higher dose of the vaccine that was fully protective in swine. A second group was given the emulsion augmented by a proprietary peptide stabilizer. These synthetic peptide vaccines were given I.M. on days 0 and 21. A control group was given placebo (ISA 50v only). The animals were challenged intradermolingually with 104 BID50 of a PanAsia O virus on day 42. The neutralizing antibody titers at day 35 were predictive of protection for the groups given either of the two vaccine formulations. However, only the group that received the vaccine formulation with peptide stabilizer was protected. The augmented formulation was needed to achieve protection in cattle. The neutralizing antibody titers in both swine and cattle did not relate to protective immune responses. N.A. titers were low in the low dose group (Group 3) of swine who nevertheless were protected (Table 7), and high and predictive of protection in the cattle given the unaugmented peptide emulsion vaccine who nevertheless were not protected (Table 9). A lack of correlation between neutralizing antibodies and protective immunity suggests that other correlates of protection are involved. Discussion The peptide-based FMDV NS confirmatory tests provide a system that allows rapid confirmation of positive results of UBI® FMDV NS EIAs. In addition, because these confirmatory tests can screen out false-positive samples, they also allow end users to lower the Cutoffs of the UBI® FMDV NS EIAs, to raise assay sensitivity without severely degrading overall specificity. Although lowering the assay Cutoff for higher sensitivity will inevitably create more false positive results on the screening tests, the majority of those samples will be identified as false positive by the confirmatory tests. In a recent study designed to test whether false positive samples created by lowered assay Cutoffs can indeed be eliminated by the confirmatory tests, we have confirmed the prediction that the confirmatory tests can improve the specificity of UBI® FMDV NS EIAs even though the Cutoffs of the assays were significantly reduced (results not shown). Thus, while the lowered Cutoffs increased the assay sensitivity, the subsequent application of the NS confirmatory tests more than compensated by increasing overall assay specificity. The combination of lowering assay Cutoffs and using confirmatory tests to sort out the positive samples allow net gains in both sensitivity and specificity. An effective synthetic peptide-based vaccine for FMD has been developed for pigs and is now being developed for cattle. The UBI vaccine formulations are 1) effective without adverse local reactions, 2) they have stability beyond six months (the peptide antigens themselves are stable for years), 3) the consensus antigen approach provides for activity against a broad range of viruses in a given serotype, and 4) the peptide vaccines can be economically produced and sold at acceptable cost because of the small peptide doses. Moreover, as peptide-based vaccines, they have the unique advantages of absolute

310

biosafety in both manufacturing and use, high flexibility to quickly confront newly emerging subtypes because new peptide antigens can be rapidly manufactured. As subunit vaccines they have the advantage of being antigenic marker vaccines. The peptide-based vaccine presented here reproducibly elicited protective immune responses in natural host species. The antigen was designed to accurately mimic the critical VP1 G-H loop epitope for cross-reactivity to FMDV. That it does so was demonstrated by the capability of the vaccine to produce neutralizing antibodies. However, protective immunity here was not well-correlated to neutralizing antibody titers, suggesting involvement of other correlates of immunity. The antigen was designed for enhanced immunogenicity by the addition of promiscuous T cell help and immunogenicity was further enhanced by oil adjuvant and a peptide stabilizer. We believe that these additional vaccine components contributed to other critical factors such as cytokine responses for innate immunity, and for altering the balance between Th1 and Th2 immune pathways. These issues are presently being investigated. A peptide-based system for differential enzyme-linked immunoassays in combination with synthetic peptide vaccines is an integrated system for FMD control. The chemically defined immunogen of UBI vaccines are site-specific antigenic markers whose effects can be readily detected by the use of UBI® VP1 EIAs, useful for monitoring the effectiveness of vaccination campaigns. And, the VP1-specific responses to the peptide vaccines can be readily distinguished from the immune response to virus by the UBI® FMDV NS EIAs. Literature Cited 1 2 3

Shen, F., Chen, P.D., Walfield, A.M. et al. Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA. Vaccine 1999, 17, 3039-3049. Wang, C.Y., Chang, T.Y., Walfield, A.M. et al. Synthetic peptide-based vaccine and diagnostic system for effective control of FMD. Biologicals 2001, 29, 221-228. Wang, C.Y., Chang, T.Y., Walfield, A.M. et al. Effective synthetic peptide vaccine for foot-and-mouth disease in swine. Vaccine 2002, 20(19-20), 2603-2610.

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Figure 1 ISISEIKGVIVHKIETILF-εK-VYNGNCKYGENAVTNVRGDLQVLAQKAARCLPTSFNYGAIK T RT TR UBITh®1 FMDV O consensus immunogen for swine and cattle. T cell help is provided by the combinatorial library UBITh®1 site shown, linked through an ε-lysine to the consensus 0 VP1 G-H loop site [3]

312

Table 1. Serum panel for initial validation studies of FMDV NS Confirmatory Tests ________________________________________________________

Serum Panel FMDV Type/Strain ____________________________________________________ GPa-R00023

A/5 Westerwald

GP-R00059

A/22 Turkey

GP-R00071

A/27 Columbia 67

GP-R00079

O/1 Campos

GP-R00100

C/3 Resende

GP-R00101

C/4 Tierra del Fuego

GP-R00105

Asia 1/1 Pakistan

GP-R00116

SAT1/4 S. Rho 58

GP-R00124

SAT2/1 Rho 48

Bov-R00132

A24

Bov-R01053b

A/5 Westerwald O/1 Kaufbeuren SAT2/1 Rho 48

Bov-005-35c

A24

Sw-067-28d

A24

Bov-001

A24

________________________________________________________ a Guinea

pig sera.

b Steer was

sequentially inoculated by the indicated strains on

days 1, 29, and 84, respectively. c Bovine dSwine

serum collected at day 35 post infection.

serum collected at day 28 post infection.

313

Table 2. Validation of FMDV NS 3B Neutralization Assay with FMDV Positive Sera Control Buffer

Neutralization Buffer

0 µg/mL

50 µg/mL

Peptide (NS 3B) Conc Sample ID

Abs

Ratio

Abs

Ratio

% Neutralization Rate

1. GP-R00023

2.478

8.73

0.154

0.54

93.8%

2. GP-R00059

2.896

10.20

0.403

1.42

86.1%

3. GP-R00071

2.900

10.22

0.307

1.08

89.4%

4. GP-R00079

2.687

9.47

0.333

1.17

87.6%

5. GP-R00100

2.019

7.11

0.099

0.35

95.1%

6. GP-R00101

2.638

9.29

0.201

0.71

92.4%

7. GP-R00105

2.853

10.05

0.246

0.87

91.4%

8. GP-R00116

0.720

2.54

0.075

0.26

89.6%

9. GP-R00124

2.121

7.47

0.167

0.59

92.1%

10. Bov-R00132

2.342

8.25

0.225

0.79

90.4%

11. Bov-R01053

0.861

3.03

0.048

0.17

94.4%

12. Bov-005-35

1.489

5.25

0.145

0.51

90.3%

13. Sw-067-028

1.847

6.51

0.206

0.73

88.8%

14. Bov-001

0.781

2.75

0.072

0.25

90.8%

314

Table 3. Validation of FMDV NS 3A EIA with FMDV Positive Sera Control Buffer

Neutralization Buffer

0 µg/mL

50 µg/mL

Peptide (NS 3A) Conc Sample ID

Abs

S/C Ratio

Abs

S/C Ratio

% Neutralization Rate

1. GP-R00023

2.122

7.48

0.201

0.71

90.5%

2. GP-R00059

2.543

8.96

0.701

2.47

72.4%

3. GP-R00071

2.211

7.79

0.398

1.40

82.09.4%

4. GP-R00079

1.878

6.62

0.181

0.64

90.4%

5. GP-R00100

1.792

6.31

0.157

0.55

91.2%

6. GP-R00101

2.099

7.40

0.270

0.95

87.1%

7. GP-R00105

2.486

8.76

0.193

0.68

92.2%

8. GP-R00116

0.329

1.16

0.069

0.24

79.0%

9. GP-R00124

0.984

3.47

0.129

0.45

86.9%

10. Bov-R00132

0.675

2.38

0.116

0.41

82.8%

11. Bov-R01053

0.407

1.43

0.051

0.18

87.5%

12. Bov-005-35

1.767

6.23

0.195

0.69

89.0%

13. Sw-067-028

2.210

7.79

0.269

0.95

87.8%

14. Bov-001

1.432

5.05

0.222

0.78

84.5%

315

Table 4. Validation of FMDV NS Confirmatory Tests on Normal Swine Samples Cutoff

NS 3B EIA

NS 3B Neutralization Assay

Sample ID

Abs

S/C Ratio

Abs

S/C Ratio

NRC

0.083

0.31

0.083

RC

1.151

4.35

1.151

Cutoff COVANCE Lot#1 141 184 424

0.256

% NR

NS 3A EIA Abs

S/C Ratio

0.31

0.075

0.15

4.35

1.198

2.33

0.256

0.514

0.522 1.070 0.304

2.09 4.04 1.15

0.079 0.986 0.081

0.30 3.72 0.31

87.5% 7.9% 73.4%

COVANCE Lot#2 75 351 387 494 498

0.612 0.331 0.311 0.395 0.316

2.31 1.25 1.17 1.49 1.19

0.644 0.428 0.289 0.424 0.312

2.43 1.62 1.09 1.60 1.18

-5.2% -29.3% 7.1% -7.3% 1.3%

Valley Biomedical 31 130 131

0.329 0.317 0.689

1.24 1.20 2.60

0.246 0.476 0.665

0.93 1.80 2.51

25.2% -50.2% 3.5%

316

0.172

0.33

0.093

0.18

Table 5. Validation of FMDV NS Confirmatory Tests on Normal Ruminant Samples FMDV-NS ( 3B ) Control Sample ID NRC NS (3B) RC Cutoff Repeat Reactive COVANCE Bovine – 085 Goat – 012 Valley Cattle sera 207 235 291 317 323 404 425

FMDV-NS ( 3A )

Neutralization Buffer

Control

Abs 0.086 1.334 0.307

S/C Ratio 0.28 4.35

Abs 0.086 1.334 0.307

S/C Ratio 0.28 4.35

% NR

Abs

S/C Ratio

0.457 0.894

1.49 2.91

0.058 0.054

0.19 0.18

87.3% 94.0%

0.078 0.203

0.24 0.52

1.138 0.352 0.321 1.031 0.497 0.942 0.199

3.71 1.15 1.05 3.36 1.62 3.07 0.65

0.059 0.046 0.046 0.074 0.584 0.052 0.410

0.19 0.15 0.15 0.24 1.90 0.17 1.34

94.8% 86.9% 85.7% 92.8% -17.5% 94.5% -106.0%

0.074 0.059 0.063 0.077 0.183 1.122 0.192

0.20 0.16 0.20 0.24 0.58 3.75 0.64

317

Table 6. Specificity of UBI® FMDV Confirmatory Tests on Swine and Ruminant Samples Specificity Test

Normal Ruminant

Normal Swine

Vaccinated Swine

NS 3B EIA

99.2% (1191/1200)

99.3% (1601/1612)

>95.9% (9756/10169)

NS 3B EIA + 3B Neutralization Assay

99.4% (1193/1200)

99.9% (1610/1612)

99.18% (10036/10119)

NS 3B EIA + 3B Neutralization Assay + NS 3A EIA

99.9% (1199/1200)

100.0% (1612/1612)

99.92% (10111/10119)

318

Table 7. Vaccine dose and stability studies in swine.

Group

1 2

3

4 5 6 7 8

Tag #

401 403 404 405 406 407 408 409 410 411 412 413 415 416 438 417 418 419 423 425 439 426 427 428 434 435 436 456 464 468

Dose

50 μg/ 1.0 mL, fresh

12.5 μg/ 1.0 mL, fresh

3.12 μg/ 1.0 mL, fresh

50 μg/ 1.0 mL, fresh 50 μg/ 1.0 mL, aged 6 months 50 μg/ 1.0 mL, aged 12 months Commercial FMD O1 Taiwan Vaccine, 2.0 mL Placebo

319

N.A. Titer 8 wk 45 181 11 25 11 32 64 8 11 8 6 4 3 3 3 6 25 32 32 32 25 11 32 3 512 256 724 25 3 3

Protection

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

Table 8. Duration study of single dose vaccine Month Post-injection 1 2 3 4 5 6

UBI vaccine No. infected/n 0/5 0/5 0/5 0/5 0/5 0/5

Placebo No. infected/n 5/5 5/5 5/5 5/5 5/5 5/5

Table 9. Formulation study for cattle

Group

Tag #

1

7 8 9 10 11 12 18 19 20

2 3

Formulation 200 μg/ISA 50v 200 μg/stabilizer/ISA 50v Placebo

320

N.A. Titer 5 wk 724 256 64 256 128 512 3 3 8

Protection

+ + + + -

Appendix 36

Regulation of FMD vaccines within the European Union K De Clercq 1 and D K J Mackay 2

Introduction The EUFMD European Pharmacopoeia Working Group made a proposal for revision of the FMD vaccine monograph (Amadori, 1999; De Clercq, 2000; De Clercq 2001) and presented its reports to Group 15V of the European Pharmacopoeia (Ph. Eur.). EUFMD informed the Immunological Working Party of the Committee for Veterinary Medicinal Products (CVMP) of the European Agency for the Evaluation of Medical Products (EMEA) of its activities and proposed a discussion forum including all key players: EMEA, EU, EUFMD, OIE, Ph. Eur. and the FMD vaccine producers. The IWP welcomed this initiative and proposed the CVMP to set up an ad hoc group. In regulatory terms, foot-and-mouth disease (FMD) vaccines are often seen by their manufacturers, and to some extent by their users, as a ‘special case’ due to the special nature of the disease against which they provide protection. From a legal and regulatory perspective, however, FMD vaccines, like all vaccines, are immunological veterinary medicinal products and are therefore subject to the requirements of the veterinary pharmaceutical Directive 2001/82/EC. This directive requires that all veterinary medicines that are placed on the market within the European Union must be authorised by means of a marketing authorisation and lays down the minimum requirements in terms of quality, safety and efficacy that medicines must meet in order to obtain an authorisation. The directive provides an exemption from the requirement for an authorisation when a product is to be used in the event of ‘serious disease epidemic’ provided that there is no authorised medicine for use against the disease concerned and provided that the European Commission is informed of the detailed conditions of use. The term ‘serious disease epidemic’ is not further defined but clearly applies to outbreaks of FMD. The European Commission itself currently utilises this exemption to allow use without an authorisation of vaccines prepared using antigens maintained in the strategic antigen reserves of the EU FMD antigen Bank. The recent epidemic of FMD in the United Kingdom, the current Commission reviews of both pharmaceutical and FMD legislation, and a greater perception that future control strategies might involve a policy of ‘vaccination to live’ have all contributed to the setting up by the Committee for Veterinary Medicinal Products (CVMP) of an ad hoc group to prepare guidelines on the requirements for FMD vaccines. The CVMP ad hoc group comprises members of the Immunologicals Working Party of the CVMP, of the Research Group of the European Commission for the Control of FMD of the FAO, and of the OIE. Invited as observers are representatives from DG Enterprise and DG SANCO of the European Commission and from the European manufacturers of FMD vaccines. The group has met on several occasions and is currently finalising the first draft of the ‘Position paper on requirements for vaccines VAR, Belgium Corresponding author; Veterinary Medicines Directorate, Woodham Lane, Addlestone, Surrey KT15 3LS, United Kingdom. D.mackay@vmd.defra.gsi.gov.uk

1 2

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against foot-and-mouth disease’ which will go out for consultation during 2002. The document proposes practical means whereby applicants for marketing authorisations for FMD can meet the requirements of EU legislation. The group has consulted widely with other international organisations to ensure that the draft proposal have wide acceptance. As part of the consultation process it will be necessary for the relevant EU authorities to consider whether or not changes are needed to EU pharmaceutical legislation to cope with the particular technical issues raised by FMD vaccines. Ultimately the standards set will be those that will apply in the EU and authorities in other regions will have to consider to what extent they may wish to apply them in their own areas. Within the EU, directive 2001/82/EC requires that any product placed on the market must meet the requirements of the relevant monograph of the European Pharmacopoeia where one exists for the type of product concerned. In the case of FMD vaccines, there is a monograph laying down the minimum requirements for inactivated FMD vaccines for ruminants. Group 15V of the European Pharmacopoeia have published a revision proposal for this monograph and are currently working to develop a monograph for FMD vaccines for pigs. The CVMP ad hoc group has taken account of the revision proposal in preparing the draft guideline and have put forward a number of proposed amendments to the revision proposal for consideration by Group 15V. During the consultation processes for both documents care will be taken to ensure that they remain compatible and complementary. This paper describes the approach taken in the draft guideline to those features that make FMD vaccines a ‘special case’ in terms of authorisation. It is important to emphasise that the approach outlined in this document is the outcome of scientific assessment by the ad hoc group of the regulatory challenges presented. Whether or not these proposals are ultimately adopted will depend on the outcome of the process of scientific and legal consultation through which the guideline will now progress. FMD vaccines as a special case From a regulatory perspective, FMD vaccines represent a special case due to the number and antigenic diversity of strains that might be used alone or in combination within the context of an authorisation. EU Pharmaceutical legislation is based on the regulatory model that a vaccine comprises a fixed formulation of ingredients including defined amounts, or limits, of one or more antigens. Any change in terms of addition, substitution or removal of an antigen requires a variation resulting in a new authorisation (known as a ‘line extension’). The guideline proposes that a slightly amended definition is required in the case of FMD vaccines and that an FMD vaccine should be defined as a formulation of ingredients including defined amounts (limits) of one or more antigens that varies only in the number and types of antigen present. The maximum permitted number and amount of antigens shall be specified in the authorisation in relation to the safety studies presented. This definition is intended to allow an authorisation to cover the potentially large number of different strains of FMD virus which may be included in an authorised product in any combination without each separate combination requiring an individual authorisation. The issue of whether or not this proposed definition is acceptable within current legislation will require further consideration during the consultation process.

322

The second factor that makes FMD vaccines a special case is the requirement to ensure that there is a mechanism whereby antigens produced from new master seed viruses can be rapidly incorporated into the authorised product. This need will arise in situations where there is an incursion of a ‘new’ strain of FMD virus against which established, and therefore previously authorised, vaccine strains do not provide protection. The current approval process for a line extension to include a new strain can take up to 210 days which is not an appropriate time frame for response to an FMD outbreak. The guideline therefore considers a number of mechanisms by which this time frame might be reduced. Quality Requirements The guideline is underpinned by the assumption that the method of production and the nature of the antigens are identical for all strains of FMD virus to be included in the authorisation and that the strains differ only in their antigenic characteristics. In this way it is possible to consider that vaccine strains, and the antigens produced from them, are themselves individually authorised once they have been shown to meet the quality requirements of the authorisation. In the case of new strains, there may not be sufficient time available to complete the full range of tests for freedom from extraneous agents according to the relevant CVMP guidelines before there is a need to incorporate the antigen into a vaccine for emergency use. In this case is may be necessary to seek a provisional authorisation based on a risk assessment for freedom from extraneous agents until such time as full testing can be completed. This assessment will take into account the risks of contamination at source in relation to the species and country of origin. The guideline specifies that master seed viruses should be treated with an organic solvent to inactivate enveloped viruses and should be inactivated with a first order inactivant. Consideration must also be given to reducing at source any possible risk of contamination with agents responsible for transmissible spongiform encephalopathies. An area of particular current interest is that of identifying infected animals, whether or not they have also been vaccinated, by means of measuring antibody to one or more of the non-structural (NS) proteins of FMD virus. Manufacturers may therefore wish to provide potential customers with information on whether or not their vaccine induces antibody to NS proteins. Modern FMD vaccines contain purified preparations of virions from which tissue culture components, including NS proteins, have been removed to a lesser or greater extent through the production process of the antigen. There is currently insufficient information on the immunogenicity of the various NS proteins to set levels below which FMD vaccines can be considered to be ‘free’. What is important is the ability of any residual, contaminating NS protein to induce an antibody response in the target species that is sufficient to interfere with a diagnostic test. The guideline therefore proposes that manufacturers should look for antibody to defined NS proteins in the sera of cattle (or other species for which the vaccine is indicated) that have been repeatedly immunised with vaccines containing the maximum amount and number of FMD antigens permitted under the authorisation. The guideline does not attempt to prescribe which antigen(s) should be studied nor which test should be used, but requires that the test used is fully validated. The manufacturing process must include a purification step to remove NS protein contamination and manufacturers can support their claim by demonstrating, using suitable immunochemical methods, that their antigen preparations are free from 323

defined NS protein(s) or contain only low levels. There is no EU legislation covering the evaluation or authorisation of diagnostic tests. This, together with the fact that ‘vaccinated’ and ‘infected’ states are not mutually exclusive in FMD, means that claims to ‘differentiate’ infection from vaccination or for ‘marker’ vaccines are inappropriate and should not be included on the summary of product characteristics (SPC) for FMD vaccines authorised in the EU. Claims that vaccines do not interfere with the detection of infected animals by means of NS antibody tests would be acceptable. There is currently no consensus on which NS protein is the most reliable marker of infection and the tests available have been validated to different levels. The claims made in relation to NS proteins must clearly reflect the studies presented and should be limited to stating which NS protein has been studied and by which test. In this way, potential customers can make informed decisions on an appropriate choice of a vaccine and a companion diagnostic test to be used when choosing to follow a policy of ‘vaccination to live’. Safety Requirements The current revision proposal of the European Pharmacopoeia monograph replaces the intradermolingual safety test with a conventional double dose safety test in the most susceptible category of animals using each recommended route of administration. This test is therefore suitable for vaccines adjuvanted with either aluminium hydroxide or oil. The guideline proposes that the safety of an FMD vaccine can be demonstrated by conducting this double dose safety trial using a vaccine containing the maximum amount and number of antigens. Provided that the results of this test are acceptable, then the safety of lesser amounts or combinations of antigens would not need to be individually demonstrated before authorisation. Likewise, when inclusion is sought for a new strain into an existing authorisation, safety studies should be performed with vaccines formulated to contain the maximum proposed amount of the new antigen. The new antigen shall be considered satisfactory in terms of safety provided that the local and systemic reactions seen with the new strain are no more severe than those seen for the established, authorised strains. Efficacy requirements The guideline considers in detail the question of efficacy of FMD vaccines in relation to potency. The European Pharmacopoeia monograph for FMD vaccines establishes a minimum potency of 3 PD50 for an FMD vaccine to be placed on the market in the EU and such vaccines are termed ‘standard potency’ in the draft guideline. Published data suggests that there are benefits to be gained in emergency circumstances from the use of higher potency vaccines. Vaccines with higher antigen payloads have been shown to induce protective immunity more rapidly and, possibly, to increase the ‘breadth’ of the immune response in terms of antigenic cover. The guideline proposes that vaccines with a potency of ≥ 6 PD50 could be classified as ‘higher potency’ vaccines. However, there is currently insufficient evidence to define such vaccines as ‘Emergency’ vaccine, as the extent to which standard potency vaccines can induce the same level of protection as higher potency vaccines has yet to be fully evaluated. The guideline highlights the balance that must be drawn between increasing the potency of a vaccine and decreasing the number of doses that can be prepared from a given antigen stock but considers that the final choice of potency should rest with customer

324

and should be chosen in relation to the epidemiological situation in which the vaccine will be used. The definitive test for the ability of an FMD vaccine to protect against disease (termed immunogenicity) is established by means of the Ph. Eur. challenge potency test in cattle. The most difficult issue addressed by the guideline is the use of alternative, serological tests to demonstrate the immunogenicity of FMD vaccines. The guideline takes into account the considerable amount of published evidence showing that a good correlation exists between serum antibody titre and protection against FMD in cattle. However, the ‘pass level’ titre above which cattle may be considered protected depends on a large number of factors including the serotype of FMD virus used for vaccination and challenge, the serological test used to measure antibody and the conditions under which challenge occurs. The European Pharmacopoeia General Text 5.2.7 states that, challenge studies should be conducted using a challenge strain that is different from the strain used for vaccination. However, in the case of FMD, the guideline proposes that there should be an exemption from this requirement and that challenge should be conducted with a strain homologous to the vaccine strain, provided that it induces the required level of clinical signs in the challenged, unvaccinated controls. The results of a challenge study with a homologous strain provide information on the quality of the antigen and the vaccine in terms of their ability to induce protection against the most relevant antigenic strain. From the customer’s perspective however, it may be necessary to conduct additional trials to establish if the vaccine strain also protects against field strains relevant to their particular situation. In order for a new strain to be considered for inclusion within an authorisation the manufacturer should conduct full potency challenge tests in cattle with a satisfactory result demonstrating that a vaccine blended with a defined amount of antigen has a potency of at least 3 PD50. The revision proposal for the European Pharmacopoeia monograph proposes that subsequent batches can be released on the basis of a serological test in cattle demonstrating that the antibody levels induced by a test batch are not significantly less than those induced by at least three batches shown to be potent by challenge in cattle. The proposal also requires that the serological test used is fully validated and that a satisfactory correlation has been shown between the results of the serological test and protection in cattle. The guideline proposes conditions that should apply in order to reduce the amount of testing required to establish a suitable correlation between the two tests and a pass level for a particular strain. The test to be used shall be the virus neutralisation test using as antigen the vaccine strain for which a serological pass level is being sought. The divided dose challenge study shall show a graded response and at least some animals vaccinated with lower doses shall not be protected. Provided these criteria are met only a minimal number of challenge tests, possibly as few as one, should be required to establish a titre corresponding to the minimum ‘pass level’ associated with a vaccine having a potency of at least 3 PD50. This pass level can then subsequently be used for all vaccines containing the same or greater amounts of the given antigen, irrespective of the other antigens with which the particular vaccine under test is formulated. If the proposal to accept the use of serological alternatives to potency testing in cattle is accepted, there will be a greater need for standardised reference sera for FMD serology. The European Department for the Quality of Medicines of the European 325

Pharmacopoeia has recently undertaken to coordinate a project to produce, evaluate, store and distribute such sera in relation to potency testing of FMD vaccines. Manufacturers may, of course, use serological tests other than the virus neutralisation test but in this situation the test must be fully validated and the correlation between titre and protection established for each antigen and each combination of antigens included within the authorisation. In terms of the other efficacy parameters of FMD vaccines, the claims made shall reflect the data presented in terms of duration of immunity and reduction of infection, mortality and/or clinical signs. Immunogenicity shall be demonstrated for each species for which an indication is sought. In general, for strains which are virulent in all FMD-susceptible species, batch potency shall be demonstrated in cattle. However, for strains which have a particular virulence and tropism for a species other than cattle e.g. pigs, batch potency shall be demonstrated in the most relevant species. The ad hoc group does not consider there is a need to develop a specific monograph for FMD vaccines for sheep and awaits the outcome of the work of Group 15V on the development of a monograph for FMD vaccines for pigs. Conclusions In general, the majority of the requirements that apply to all immunological veterinary medicinal products apply equally well to FMD vaccines. There are however some unique features of the disease and vaccines used against it that require a different approach to fulfilling the requirements of the relevant legislation if authorisation of FMD vaccines is going to become routine within the EU rather than an exception. Recent policy changes in terms of how FMD outbreaks should be controlled make vaccination more likely and recent experience suggests that the 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 hoping to place FMD vaccines on the market within the EU. References Amadori M. (1999 Proposal for amendments of the FMD Monograph of the European Pharmacopoeia. Report of the Session of the FAO Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, 29 September - 01 October, Maisons-Alfort, France, p. 88-93 (AGA: EUFMD/RG/99). De Clercq K. (2000). Proposal for revision of the FMD Monograph in the European Pharmacopoeia. Report of the Session of the FAO Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, 5-8 September, Borovets, Bulgaria, p. 198-205 (AGA: EUFMD/RG/00). De Clercq K. (2001). Revision of the European Pharmacopoeia monograph for FMD vaccine and European guidelines on requirements for FMD vaccine production. Report of the Session of the FAO Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease, 12-15 September, Island of Moen, Denmark, p. 174. (AGA: EUFMD/RG/01).

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Appendix 37

FMD vaccines: PFMD Vaccines Potency Testing in the Target Species Lukas Bruckner, Christian Griot Institute of Virology and Immunoprophylaxis (IVI), National Reference Laboratory for Exotic Diseases, Swiss Federal Veterinary Office, CH-3147 Mittelhäusern, Switzerland

A batch of FMD vaccine has been tested for potency in cattle. The sample was prepared from the Swiss antigen bank for emergency use. FMD serotype A Iran 96 antigen was emulgated in an oily adjuvant. Briefly, the potency test was performed as described in the monograph of the European Pharmacopoeia (Ph.Eur.) “Foot-and-Mouth-Disease Vaccine (inactivated) for Ruminants”. In contrast to the requirement of the Ph.Eur. the challenge was performed with a heterologous virus strain, A Iran 99. All cattle vaccinated with either a double, a single or 1/3 of a recommended dose were protected from FMD. In contrast, non-vaccinated control animals showed typical FMD lesions on all 4 feet. The experiment showed, that despite the antigenic difference, a vaccine prepared from FMD virus serotype A Iran 96 was capable to induce within 21 days after vaccination, protection from virulent challenge with FMD serotype A Iran 99.

327

Appendix 38

Potency control of FMD vaccine in the field between 2001-2002 Gülhan AYNAGÖZ, A.Naci BULUT and Aydın COŞKUNER Şap Institute PK 714 06044 Ankara - Turkey

In this paper the results of the herd immunity and challenge tests carried out with the FMD vaccine produced in Şap Institute is presented. Vaccination is the most important control and the eradication strategy for animal virus diseases. A variety of serological tests were developed and evaluated by many scientists in order to find out a better alternative for the challenge method for vaccine potency testing and assessed the correlation of the results with protection. These are herd immunity level, virus neutralisation test, plaque reduction test, the metabolic inhibition test, mouse protection test, different forms of ELISA using polyclonal or monoclonal antibodies. The main aim of this study is to improve an alternative method for potency test instead of challenge. Because challenge method is very high cost and diffucult to find antibody free animals and using 3 cattle are not reliable. In Ankara Şap Institute, for FMD potency testing, 3 antibody free cattle are vaccinated with undiluted vaccine and cattle as a control are used for every batch. Sera are collected at 14th and 21st days after vaccination. The challenge is done by the intra dermolingual inoculation of 1x104 ID50 cattle adapted homologous FMDV on 21st day. Cattle are observed 7 days for clinical signs of FMD and after this period animals are slaughtered and the mouth and the foot are checked for FMD lesions. The foot and the mouth lesions should be observed at least three feet on the control cattle. The mouth lesions may be observed on vaccinated animals, though there should be no lesion on the feet. Sera are tested against FMD virus by SNT and ELISA. In addition to safety and potency tests in the laboratory, starting from the 2001, the vaccines are regularly being tested in the field. European Pharmacopoeia cannot be followed because of the high cost and difficulty of the availability of antibody free animals and animal welfare. The first recorded attempt to produce an international standard for defining potency of FMD vaccines for cattle was made in the recommendations of the XIII Conference of the Permanent Commission of the OIE on Foot-and-Mouth Disease in 1972. In the field control; every vaccine series (trivalent vaccine) have been controlled in private or state farms. The animals are bled before vaccination and then tested for FMD antibodies in the laboratory by LPB ELISA. Animals with low antibody titre are chosen for the test. For this purpose 16 monovalent vaccine series have been controlled up to now. The team from Şap Institute went to vaccinate cattle which have got low FMD antibodies in provinces

328

and villages where FMD outbreak was not seen. Sera were collected at 21st or 28th days post vaccination and tested by LPB ELISA. The results of the antibody titres and of challenge are shown in Table 1-2. FMD vaccine batches will be continued to control for herd immunity levels next year. If there is a correlation between challenge test and herd immunity levels . According to that result in the field control test should be considered to replace the challenge test for FMD potency testing. This would provide several advantages such as, reduced cost and time, reliability, reproducibility, addressing animal welfare, vaccines of several types could be tested simultaneously in animals housed together.

329

Table 1: Antibody titre and challenge results for monovalent vaccine which were produced in 2001 Number of series

Type of vaccine

01/07

* **

Asia 1

Number of vaccinated animals 75

Protective antibody levels % * 85.06

Results of Challenge ** 0/3

01/08

A96-Iran

93

85.82

0/3

01/09

A96-Iran

67

85.2

0/3

01/10

A96-Iran

23

100

0/3

01/14

Asia 1

91

100

0/3

01/15

Asia 1

290

94.53

0/3

01/17

O1

156

82.2

0/3

01/18

A96-Iran

140

97.5

0/3

21 days after vaccination Protected animals/The number of vaccinated cattle

Table 2: Antibody titre and challenge results for monovalent vaccine which were produced in 2002 Number of series

Type of vaccine

02/01

* **

Asia-1

Number of vaccinated animals 70

Protective antibody levels % * 98.66

Results of Challenge ** 0/3

02/02

Asia-1

114

100

0/3

02/03

A96-Iran

193

88.05

0/3

02/04

O1

108

99

0/3

02/05

O1

95

87.05

0/3

02/06

O1

165

87.12

0/3

02/07

O1

67

97.15

0/3

02/08

O1

157

86.63

0/3

21 days after vaccination Protected animals/Number of vaccinated cattle 330

Appendix 39

Monoclonal antibody based detection of 3A containing non-structural proteins in Al (OH)3 adjuvanted Foot-and-Mouth disease vaccines Fuat Özyörük, Ünal Parlak, Gülhan Aynagöz, Hidayet Bozoğlu Foot-and-Mouth Disease Institute, Ankara-Turkey

Abstract To detect 3A containing non-structural proteins [3A, 3AB and 3ABC (3A-NSPs)] in foot and mouth disease (FMD) vaccines, and to find out how they are eliminated are required. This study proposes a standard method based on enhanced detection of 3A-NSPs in Al(OH)3 adjuvanted FMD vaccines by an amplified Western blot assay (AWB). Limit of detection of AWB was found 11.5 ng/lane using anti-3A monoclonal antibody (MAb) 2C2 and recombinant 3ABC. Cellular debris (CD) of five baby hamster kidney cells (BHK-21) infected or non-infected with different type of FMDV, and elution fluids acquired from fifteen batches of FMD vaccines were analyzed for their reactivity with MAb 2C2 by AWB. All infected CD were reactive with Mab 2C2 at the molecular weight (MW) of 28 kDa identical to hypothetical MW of NSP-3AB, whereas none of the FMD vaccines tested had bands reactive with Mab 2C2. The results indicated that NSP-3AB was sedimented together with BHK-21CD, and FMD vaccines tested did not contain 3A-NSPs more than 12.3 ng/lane in the context of detection limit. Introduction Purity of the FMD vaccine is mainly associated with effective separation of virus in supernatant from CD (1, 4). It is important that vaccine be free from cellular proteins as well as non-structural viral proteins (NSP). Presence of NSPs in FMD vaccines would result in ambiguity whether or not vaccinated animals have FMD history, as NSP antibody response is the most reliable way to differentiate infected/convalescing from vaccinated animals in the field to facilitate prevention of new outbreaks. Many publication have revealed that anti-NSP 3ABC, 3AB, 3A, 2C responses in animals are specific indication of FMD history other than vaccination (2, 3, 8-11, 14). However these findings cannot justify that estabilshment of NSP detection procedures in FMD vaccine plants is unnecessary; because at what stage of the production, how, and how much of these NSPs are eliminated are not known except for 2C. In one study, absence of anti-2C antibodies from the sera of vaccinated animals, due to association of 2C with cellular debris that is separated from the virus harvest during vaccine production, have been reported (8). Present study proposed to establish a standard method based on enhanced detection of 3A-NSPs in FMD vaccines and CD of baby hamster kidney BHK-21 by an anti-3A monoclonal antibody. The results indicated that NSP-3AB was sedimented together with BHK-21 CD, and FMD vaccines examined did not contain 3ANSPs more than 12.3 ng/ml.

331

Material and methods Cell cultures and viruses A continuous cell line, BHK-21 Cl-13 cells, (provided from Animal Cell Culture Laboratory of FMD Institute, Ankara) were used for propagation of FMD vaccine strains currently used in Turkey; A Aydin 96, O1 Manisa and Asia-1. After development of cytopathic effect, cells were harvested by freezing and thawing three times. Alternatively, virus-cell suspensions in the vaccine production were homogenized with 2% percent chloroform at 4 ºC for 1 h followed by sedimentation over night. Clarification was improved by the treatment of diatoma earth and filters with 15, 5, 1.5 and 0.6 μm pore size. 1-2 ml of frozen-thawed cell virus suspension or chloroform treated cell sediment was centrifuged at 5000 rpm for 10 min.. Supernatant was discarded prior to pellet was washed with PBS by vortexing. The last two steps were repeated three times in sequence, and preparations were tested by AWB as described below. MAb and recombinant protein IgG1 MAb 2C2 recognizing a linear 3A epitope on FMDV was used to detect 3A-NSPs in vaccines and CDs (3). A recombinant protein, the 3ABC polypeptide of FMDV O1 Kaufbeuren, was used to figure out detection limit of AWB assay (3). Recombinant 3ABC concentration was determined by the combination of bicinchoninic acid assay (Micro BCATM; Pierce), sodium dodecyl sulphate polyacrylamide gel electrophoresis and Integrated Optical Density analysis (GelPro 3.1; Media Cybernetics). Production of FMD vaccines adsorbed to aluminium hydroxide gel and preparation of elution fluids acquired from vaccines Al(OH)3 adjuvanted FMD vaccines were produced according to manufacturer’s manual in the context of international standarts (7), and elution fluids containing viral proteins were acquired from aluminium hydroxide gel while concentrated 62 fold, as described previously (5). They were tested by liquid phase blocking ELISA in order to confirm the presence of viral proteins (6). AWB assay Recombinant 3ABC, BHK-21 CD and elutes of FMD vaccines were evaluated for their reactivities with anti-3A MAb 2C2 by AWB. Samples were heated at 100ºC for 3 min in 0.03 M tris (pH 6.8) containing 2% SDS, 10% glycerol 0.01% bromphenol blue, and 100 mM dithiothreitol and loaded 30 μl/lane. Then they were subjected to electrophoresis in 4 to 20% SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane (Pierce, Rockford, IL, USA) at 150 V for 1 h with a Mini TransBlot apparatus (Bio-Rad, Hercules, CA, USA). Membrane was blocked with blocking buffer (SuperBlock®; Pierce), reacted with anti-3A MAb 2C2 and secondary goat anti-mouse horseradish peroxidase conjugate(HRP) (Pierce). Binding of HRP conjugates were evaluated with a chemiluminescence reagent (SuperSignal® West Pico, Pierce) developed on X-ray film (X-Omat; Kodak, N.Y., USA). Results and discussion Detection limit of AWB assay To establish a standard assay, recombinant 3ABC with known concentrations was tested in AWB using anti-3A MAb 2C2. As expected, MAb 2C2 was reactive with recombinant 3ABC and bands were visible up to 11.5 ng/lane (Fig. 1. lane 6). This detection limit (11.5 ng/lane)

332

was found extremely low compared to amount (0.003 ng/lane) stated by previous studies as well as manufacturer of the kit (12, 15). Low sensitivity in AWB remained to be determined. 1

2

3

4

5

6

78 kDa ►

FIG. 1. AWB of anti-3A MAb 2C2 with recombinant 3ABC. Lanes 1-6 were loaded with decreasing amount (ng/lane) of recombinant 3ABC; 390, 185, 92.5, 46.2, 23.1 and 11.5 respectively.

AWB of anti-3A MAb 2C2 with BHK-21 CDs Previous studies (2, 3, 8-11, 14) revealing the association of NSP-2C with CD, and lack of antibody response against 3A, 3AB, and 3ABC in vaccinated herds let us to hypothesize that 3A-NSPs may as well be captured by CD during clarification. To test this hypothesis, FMD infected or non-infected BHK-21 CDs were analyzed whether they had 3A-NSPs. All examined CDs infected with FMDV had bands at 25-28 kDa reactive with anti-3A MAb 2C2 (Fig. 2. lane 1-4) while no band was observed with non-infected BHK-21 CD (Fig. 2. lane 5). Molecular weights of reactive bands were identical with the hypothetical location of native 3AB (Fig. 2. lane 6). Inferior bands were probably degradation products of 3AB but the possibility of 3A presence among those bands should not be disregarded. On the other hand absence of 3ABC band (corresponding to 48 kDa) reactive with MAb 2C2 could be explained by the fact that this polypeptide is neither translation nor cleavage product in picornaviridae (13). 1

2

3

4

5

6

60 39 28 18.8

FIG. 2. AWB of anti-3A MAb 2C2 with infected or non-infected BHK-21 CDs. All lanes were loaded with

BHK-21 CDs either infected with an FMD vaccine strain (lanes1-4) [A Aydin 98, O1 Manisa, Asia-1, and Asia-1 (from production)] or non-infected (lane 5). Theoretical separation of 3ABC (48.1 kDa), 3AB (25.1 kDa) and 3A (17.3 kDa) according to their calculated molecular weights was shown in lane 6. Numbers to the left of each panel are in kilodaltons

AWB of anti-3A MAb 2C2 with elution fluids acquired from FMD vaccines To determine if there was residual contamination of 3A-NSPs in the detection limit of AWB, elution fluids of FMD vaccines were analyzed in AWB using MAb 2C2. None of the tested FMD vaccines had bands corresponding to the hypothetical locations of 3A-NSPs (fig. 3. lane 1-15). These data indicated NSP 3AB were eliminated during clarification and not leaked through the FMD vaccines tested. If 3A-NSPs were exist but not determined due to out of detection limit, amounts of 3A-NSPs would not be more than 12.3 ng/ml in vaccines, taking to account of loading volume and detection limit. Even this amount seems very low to induce anti-3ABC

333

antibody response; it is not known how much 3ABC required for antibody response detectable with 3ABC-ELISA kits.

1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16

60 39 28 18.8

FIG. 3. AWB of anti-3A MAb 2C2 with elutes acquired from FMDV vaccines. Type O vaccines (serial numbers: 14, 4, 5, 6, 7, 8 and 9) are in lanes 2, 4, 6, 7, 10, 13 and 14. Type A vaccines (serial numbers: 3, 11 and 10) are in lanes 5, 9 and 11. Type Asia-1 vaccines (serial numbers: 13, 15, 12, 2, and 1) are in lanes 1, 3, 8, 12 and 15. BHK-21 CD infected with type A virus as a positive control is in lane 16.

Acknowledgement We thank Dr. E. Brocchi and Dr. F. De Simone for evaluating data, and providing MAb 2C2 and recombinant 3ABC protein. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Barteling, S. J., and J. Vreeswijk. 1991. Developments in foot-and-mouth disease vaccines. Vaccine. 9:75-88. Brocchi, E., M. I. De Diego, A. Berlinzani, D. Gamba, and F. De Simone. 1998. Diagnostic potential of Mab-based ELISAs for antibodies to non- structural proteins of foot-and-mouth disease virus to differentiate infection from vaccination. Vet Q. 20:S20-4. De Diego, M., E. Brocchi, D. Mackay, and F. De Simone. 1997. 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. 142:2021-33. Doel, T. R. 1999. Optimisation of the immune response to foot-and-mouth disease vaccines. Vaccine. 17:1767-71. Doel, T. R., and R. F. Staple. 1982. The elution of foot-and-mouth disease virus from vaccines adjuvanted with aluminium hydroxide and with saponin. J Biol Stand. 10:185-95. 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. 93:115-21. International Office of Epizootics. 1996. Manual of standards for diagnostic tests and vaccines : lists A and B diseases of mammals, birds and bees, 3rd ed. Office international des epizooties, Paris, France. Lubroth, J., M. J. Grubman, T. G. Burrage, J. F. Newman, and F. Brown. 1996. Absence of protein 2C from clarified foot-and-mouth disease virus vaccines provides the basis for distinguishing convalescent from vaccinated animals. Vaccine. 14:419-27. Lubroth, J., A. Lopez, A. K. Ramalho, R. F. Meyer, F. Brown, and G. C. Darsie. 1998. Cattle response to foot-and-mouth disease virus nonstructural proteins as antigens within vaccines produced using different concentrations. Vet Q. 20:S13-7.

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10. 11. 12. 13.

14.

15.

Mackay, D. K., M. A. Forsyth, P. R. Davies, A. Berlinzani, G. J. Belsham, M. Flint, and M. D. Ryan. 1998. Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA. Vaccine. 16:446-59. Malirat, V., E. Neitzert, I. E. Bergmann, E. Maradei, and E. Beck. 1998. Detection of cattle exposed to foot-and-mouth disease virus by means of an indirect ELISA test using bioengineered nonstructural polyprotein 3ABC. Vet Q. 20:S24-6. Mattson, D. L., and T. G. Bellehumeur. 1996. Comparison of three chemiluminescent horseradish peroxidase substrates for immunoblotting. Anal Biochem. 240:306-8. Rueckert, R. R. 1996. Picornaviridae: the viruses and their replication, p. 609-654. In B. N. Fields and D. M. Knipe and P. M. Howley and R. M. Chanock and J. L. Melnick and T. P. Monath and B. Roizman and S. E. Straus (ed.), Fields Virology. Lippincott-Raven, Philadelphia, PA. Sorensen, K. J., K. G. Madsen, E. S. Madsen, J. S. Salt, J. Nqindi, and D. K. Mackay. 1998. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus. Arch Virol. 143:1461-76. Walker, G. R., K. D. Feather, P. D. Davis, and K. K. Hines. 1995. SuperSignal® CL-HRP: A new enhanced chemiluminescent substrate for the development of the horseradish perxide label in Western blotting applications. Journal of NIH Research. 7:76.

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Appendix 40

Foot-and-Mouth disease in Argentina: development of vaccines for emergency, control and eradication of the disease Smitsaart, E.1, Mattion, N.1, Mazzuca,G.2, Robiolo, B.3, Maradei, E.2, Filippi, J.1, Sadir, A.4, Falczuk, A2, La Torre, J.3, Pedemonte, A.2 , D´aloia, R.2, Periolo, O.3, Cadenazzi, G.2, Palma, E.4 & Bellinzoni, R1 1-Biogenesis S.A. Ruta Panamericana km. 38,2. (B1619IEA) Garín 2-SENASA Fleming 1653 (1640) Martínez 3-CEVAN- CONICET. Serrano 669 (1414) Buenos Aires 4- INTA-CICV y A. CC 77 (1708) Morón. Buenos Aires, Argentina E-mail: esmitsaart@biogenesis.com.ar

Abstract In May 2000, six years after the last outbreak, Argentina was declared by OIE “FMD-free without vaccination”. In July 2000, first outbreak of FMD was reported and the disease spread through the main livestock area. Initial control measures included slaughter, ring vaccination and restriction of animal movements. Later on the control programme consisted mainly in mass vaccination of cattle population and a continuous sanitary surveillance. FMDV type O was responsible of outbreaks in 2000 and type A circulated in 2000 to January 2002 (last report). This paper describes the development steps and challenge studies to assess the proper dose and vaccine composition to generate potent vaccines to face the outbreak. Relatedness of the new isolates with several type A vaccine strains was established by serum neutralization assays. Herd immunity levels in vaccinated cattle under vaccination programme were assessed by liquid-phase blocking sandwich ELISA on farms with no detection of viral activity. No detectable antibodies to non structural proteins were induced by the developed vaccines as was clearly established by several available non structural protein detection tests. The Argentinean FMD Vaccine and Antigen Bank played a major role in the development and scale up of vaccines that induced high protection levels in the cattle population. More than 187 million doses of multivalent vaccines were manufactured and delivered since the end of 2000 to May 2002. In co-ordinated activities the governmental institutions (National Service for Agrifood Health and Quality, SENASA, Center for Animal Virology, CEVAN, National Institute of Agricultural Technology, INTA) and the private industry (the Argentinean FMD Antigen and Vaccine Bank) along with farmer associations were able to control the outbreak using emergency and mass vaccination. Introduction The Argentinean National Foot-and-Mouth disease (FMD) control programme 1990-1997 led to the control and eradication of the disease, reaching official status recognition by the OIE as “FMD-free country where vaccination is practised” in 1997 and without vaccination in May 2000. 336

In July 2000, 14 months since vaccination was stopped, FMD outbreaks reappeared, reaching a maximum of outbreaks in May 2001. FMD affected primarily regions of major livestock concentration. The disease has mainly affected cattle (99%), concerning mostly animals younger than 2 years. FMDV types O and A were confirmed by National Reference Laboratory and further characterization of the isolates indicated that the type O strains were related to previous O type isolates from South America. In addition, molecular analysis indicated that type A isolates belonged to two different lineages genetically distant from each other (Piccone at al., 2002). Initially, control measures including slaughter, ring vaccination and restriction of animal movements were taken by National Health Authorities (SENASA). Emergency vaccination was implemented firstly with available trivalent vaccines (O1 Campos, A24 Cruzeiro and C3 Indaial) and bivalent vaccines (O1 Campos, A24 Cruzeiro). In April 2001, a systematic vaccination campaign was started including vaccination twice a year, in conjunction with control measures such as epidemiological surveillance, strategic emergency vaccination and restriction of animal movement. Regarding vaccine reserves, the Argentinean FMD Vaccine and Antigen Bank was created in 2000 upon a national tender. It holds cryopreserved FMDV concentrated antigens able to be formulated into final vaccine, and ready-to-use multivalent vaccine. It also has operational capacity to scale up and formulate vaccine from “fresh” antigen in case of spread of the disease. The Vaccine and Antigen Bank played a major role in the control of the recent disease outbreak in Argentina. The present report describes the development and performance of vaccines used in the emergency and in the routine FMD vaccination campaign initiated in April 2001 in Argentina. Materials and methods FMDV strains FMDV vaccine strains included in the Argentinean Vaccine and Antigen Bank were provided by the National Reference Laboratory of SENASA. In addition, epithelial tissue from cattle infected with selected field strains A Arg 2000 and A Arg 2001 were also provided by the same Laboratory. FMDV strains have been characterized by Complement Fixation Test (Alonso et al, 1992), by monoclonal antibody profiling (Seki et al, in preparation) and by nucleotide sequencing (König et al, 2001), results that have been reported in separate communications (Piccone et al, 2002 ; König et al, 2002). Serological characterization by serum neutralization test (SNT) Microserum neutralization test was carried out according to the method of Rweyemamu et al. (1977) using baby hamster kidney (BHK21 clone 13) cell monolayers. Bovine sera from vaccinated cattle taken 30 days after vaccination with oil adjuvanted vaccines against A24 Cruzeiro, A Arg 2000, A Arg 2001, A 81 Arg 87 or A Arg 79 were used. The two last ones were kindly provided by Dr N. Fondevila (INTA, Argentina). 337

Test was carried out as a two dimensional dose-response test and serum titre was determined as the highest dilution of serum that neutralizes exactly 100 TCID50 of virus and calculated by the Reed-and Muench method (1938). The relationship between strains was calculated by the ratio: r = serum titre against heterologous virus /serum titre against homologous virus (Rweyemamu et al.,1977). Vaccines and experimental design FMDV strains were propagated in BHK21 cell suspension cultures and inactivated with binary ethyleneimine (BEI). Antigens were concentrated and partially purified with polyethylene glycol 6000. Vaccines were prepared as water-in-oil emulsions. Aqueous phase (40%) was composed of concentrated antigen from the corresponding FMDV vaccine strains, to which 3 mg/dose of saponin was added. Oil phase was composed of 90% Marcol 52® (Exxon, France) and 10% Montanide 888® (Seppic, France). The vaccine dose was 2 ml except for O-A24-C3 vaccine in which a 3 ml dose was used (Table 1a, Trial 1). Immediately after the reintroduction of FMDV, bivalent (O / A) and trivalent (O / A /C) vaccines that were available already formulated were used. Antigenic characterization and results of protection studies in cattle led to the incorporation to the bivalent O1 Campos/A24 Cruzeiro formulation of the new field strains recently adapted to large scale production, firstly A Arg 2000, and later on A Arg 2001 (Table 1a, Trials 1 and 3). In order to optimize the quantitative formula to be used in vaccines for mass vaccination, dose-response studies using decreasing 140S antigen content of the new A strains were performed (Table 1b, Trials 2 and 4). Animals One hundred and sixty eight (158) Hereford breed cattle aged 18-24 months free of FMD antibodies were used. They belonged to the FMD free zone in Argentina, the Patagonia region. Twenty two (22) Hereford X Aberdeen Angus breed cattle aged 18-24 months free of FMD antibodies originated from Buenos Aires province were also used (O-A24-A2000 vaccine, Table 1b, Trial 3) Animals were kept during the whole experiment in controlled pens. Challenge studies Challenge was performed at 30 (Trials 1, 2 and 3) or 90 days post-vaccination (dpv) (Trial 4) by inoculation of 10.000 suckling mouse letal dose 50% (SMLD50%, Trials 1, 2 and 3) or 10.000 bovine infectious dose 50% (BID50%, Trial 4) by the intradermolingual route (Table 1a, 1b). Two unvaccinated cattle were included in each trial as controls. One week after challenge, cattle were examined for clinical signs of FMD. Cattle were considered unprotected when typical FMD lesions developed at least in one foot.

338

Detection of antibodies (Ab) to structural FMDV proteins in cattle sera The Ab titres against each one of the viral strains present in the vaccine were examined by Liquid-Phase Blocking Sandwich ELISA (LPBE) (Robiolo et al, 1995) on sera collected at day 0 and at different intervals after vaccination. Statistical differences between groups were estimated by analysis of variance. Detection of antibodies against FMDV non structural proteins (NSP) in cattle sera Antibodies to NSP were assessed on post vaccination sera of vaccinated cattle groups collected at 30 dpv . Ab to 3ABC NSP were determined in SENASA by using ELISA 3ABC and EITB (Bergmann et al., 2000), and in CEVAN by ELISA 3ABC (Robiolo et al, 2002). Cattle sera of Trial 4 (Table 1b) were submitted to INTA to determine Ab to 3ABC using the ELISA kit From Pirbright/Brescia (De Diego et al., 1997) and to NSP by a Peptide ELISA kit (United Biomedical Inc, Shen et al, 1999). Assessment of herd immunity by a sero-epidemiological survey in cattle under the FMD vaccination programme The LPBE was applied to monitor the FMD immunity status in vaccinated cattle belonging to the vaccination area (North of 42º parallel) in December 2001 following statistical sampling (designed by SENASA). The serum samples from cattle 1-2 years of age and older than 2 years were collected after spring vaccination by SENASA. First screening of the samples (N=8810) was performed by ELISA 3ABC-EITB (Bergmann et al., 2000). Serum samples from farms scoring negative results by ELISA 3ABC-EITB were then tested by LPBE (Robiolo et al., 1995). Details of the number of samples assayed by LPBE of each age category are described in Table 4. The expected protection percentage (EPP) for each FMDV type was previously calculated by comparing LBPE Ab titres on sera collected at 60 days post vaccination with results of protection challenge tests in cattle by applying the logistic regression fitting. LPBE titres above log10=1.8 for O and A FMDV types, were associated with EPP above 80% (SENASA, 1996).

339

Table 1a: Cross protection studies. Vaccines and experimental design Trial Challenge Nº strain 1

A Arg 2000

3

A Arg 2001

Vaccine

Vaccine strains1

O

6

MV A24 MVA2000 O-A24-C3 O-A24-C3 rv*

A24 A2000 O-A24-C3 O-A24-C3

33 33

MV A24 MV A2000 MV A2001 O-A24-A2000 O-A24-A2000 rv*

A24 A2000 A2001 O-A24-A2000 O-A24-A2000

15.8 15.8

140S content2 Vaccination A24 A2000 A2001 (dpv)3

Nº of animals4

Challenge (dpv)5

10 17 17

10 -

-

day 0 day 0 day 0 0 and 113 dpv

10 10 10 10

30 30 141 141

10 6.5 6.5

10 2.7 2.7

10 -

day 0 day 0 day 0 day 0 0 and 21 dpv

10 10 10 10 10

30 30 30 120 141

Vaccination

Nº of

Challenge

(dpv)3

animals4

(dpv)5

Table 1b: Dose-response studies. Vaccines and experimental design Trial Challenge Nº

strain

2

A Arg 2000

4

A Arg 2001

Vaccine 1 2 3 4 5 6

Vaccine strains1

O-A24- A2000 O-A24- A2000 O-A24- A2000 O-A24-A2000-A2001 O-A24-A2000-A2001 O-A24-A2000-A2001

140S content2 O

5 2.5 1.25 5 8 4.4

A24 5 2.5 1.25 6 5 3.2

A2000 A2001 3 1.5 0.75 0,75 3 1.4

6 3 1.5

day 0 day 0 day 0 day 0 day 0 day 0

10 10 10 16 14 16

30 30 30 90 90 90

1: FMDV strains: A24= A24 Cruzeiro Br/55, O= O1 Campos, C3=C3 Indaial, A2000=A Arg 2000, A2001=A Arg 2001. 2: µg of 140S per vaccine dose. 3: 2 ml /dose except for O1 Campos, A24 Cruzeiro, C3 Indaial polyvalent vaccine in which a 3 ml/dose was used. dpv: days post vaccination 4: Number of animals/vaccinated group. Additionally, two unvaccinated control cattle for each trial were used 5: Challenge dose for Trials 1, 2 and 3: 10000 SMLD50% of A Arg 2000 or A Arg 2001 FMDV strains. Challenge dose for Trial 4: 10000 BID50% of A Arg 2001 FMDV strain. Route: intradermolingual. dpv: days post- primary vaccination. 6: MV Monovalent vaccines *rv: revaccinated groups

340

Results FMDV strains FMDV strains A Arg 2000 (isolated from Gral Villegas, Buenos Aires Province, 2000) and A Arg 2001 (isolated from Trenque Lauquen, Buenos Aires Province, 2001) were adapted to large-scale production and conditions of virus propagation optimized. In addition, inactivation kinetics of virus suspensions of each FMDV strain with BEI was established. In process controls such as identity and purity of antigen of the new strains had been standardized. Immunogenic properties of the A Arg 2000 and A Arg 2001 antigens were tested as described below (Trial 1, MV A2000 and 3, MV A2001). Serological characterization by serum neutralization test (SNT) Serological relationships between A Arg 2000 and A Arg 2001 field strains using A24 Cruzeiro, A Arg 2000, A Arg 79 and A Arg 87 post vaccination cattle sera are shown in Table 2. The r-values obtained are indicative of low levels of cross protection. Challenge studies and Ab response after vaccination Results of protection, LPBE Ab titres after vaccination and non-structural protein Ab results are shown in Table 3a and 3b. Trial 1: Cattle vaccinated with MV A2000 vaccine induced higher percentage of protection against A Arg 2000 FMDV strain than cattle given one dose of vaccines including the heterologous A24 Cruzeiro strain (MV A24 Cruzeiro and the polyvalent O-A24-C3 vaccine). In contrast, cattle receiving two doses of polyvalent O-A24-C3 vaccine showed protection as high as that achieved by MVA2000 vaccine. High levels of Ab against A24 Cruzeiro by LPBE were detected at 30 dpv in all groups. The mean group Ab responses against A Arg 2000 were high in all vaccinated groups, with the exception of MV A24 vaccinated group which showed mean Ab titre of log10 =1.65, in agreement with the lowest level of protection achieved (Table 3a). Trial 2 : High levels of protection were induced by polyvalent O-A24-A2000 vaccines containing decreasing concentration of 140S antigen (3, 1.5 or 0.75 µg 140S of A Arg 2000 FMDV strain) revealing the high efficacy of the polyvalent vaccine formulations including A Arg 2000 FMDV strain in inducing resistance to virus challenge. LPBE Ab titres to A24 and A2000 increased significantly (P<0.05) with 140S antigen content in each vaccine (Table 3b). Trial 3: Cattle vaccinated with MV A2001 vaccine induced higher percentage of protection against A Arg 2001 FMDV strain than cattle given one dose of vaccines including A24 Cruzeiro Br 55, A Arg 2000 or both. In contrast, cattle receiving two doses of polyvalent vaccine lacking homologous A2001 showed protection as high as that achieved by one dose of MV A2001 vaccine. As in Trial 1, two doses of polyvalent vaccines lacking homologous FMDV strain in vaccine composition, induced strong immunity and protection. The lowest group mean Ab titres were detected in cattle vaccinated with MV A2000, in association with the failure of protection against A2001 challenge. However, the immune response achieved by the same vaccine in animals of Trial 1 was higher than that found in this trial (Table 3a).

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Trial 4: When A Arg 2001 strain was included in polyvalent formulations, a minimum of 6 µg of 140S of A Arg 2001 was necessary to induce above 80% of protection against homologous challenge after a single dose. LPBE Ab titres to A2000 and A2001 increased significantly (P<0.01) with 140S concentration of A Arg 2001 in each vaccine (Table 3b).

Table 2: Serological characterization by serum neutralization test

Cattle serum A24 Cruzeiro Br55 A Arg 2000 A Arg 79 A 81 Arg 87

r values* A2000 A2001 0.20 1 0.11 0.38

0.15 0.25 0.11 0.32

N** 4 4 1 1

* r value is a measure of relationship between virus strains and is given by r= serum titre against heterologous strain/ serum titre against homologous strain **Number of serum tested in duplicate

342

Table 3a: Cross protection studies: protection against challenge, LPBE Ab titres and non structural protein Ab results Trial Nº

Challenge

1

strain A Arg 2000

Vaccine

3

A Arg 2001

MV A24 MV A2000 O-A24-C3 O-A24-C3 rv* MV A24 MV A2000 MV A2001 O-A24-A2000 O-A24-A2000 rv*

Percentage of Protection 30 90 60 100 30 0 80 60 90

LPBE Aba log10 /SDb A24

A2000

A2001

2.4 /0.27 2.25 /0.25 2.53/0.11 >2.7/0 2.20/0.42 1.66/0.17 1.96/0.5 2.82/0.22 2.92/0.31

1.65/0.22 2.21/0.27 2.26/0.28 2.63/0.13 1.84/0.59 1.66/0.34 2.27/0.49 2.52/0.28 2.87/0.32

ND ND ND ND 1.8/0.56 1.39/0.35 2.94/0.26 2.13/0.21 2.35/0.30

NSP Abc +ve/total 30 dpv 0/10d 0/10 d 0/10 d 0/10 d 0/10 d 0/10 d 0/10 d 0/10 d,e 0/10 d,e

Table 3b: Dose-response studies: protection against challenge, LPBE Ab titres and non structural protein Ab results Trial number

Challenge

2

strain A Arg 2000

4

A Arg 2001

Vaccines 1 2 3 4 5 6

Vaccine Percentage of strains Protection O-A24-A2000 100 O-A24-A2000 90 O-A24-A2000 100 O-A24-A2000-2001 81 O-A24-A2000-2001 57 O-A24-A2000-2001 31

LPBE Aba log10 /SDb A24

A2000

A2001

2.04/0.3 1.74/0.21 1.63/0.22 2.41/0.38 2.39/0.53 2.24/0.37

2.22/0.4 1.95/0.39 1.75/0.24 2.33/0.5 2.1/0.49 1.79/0.38

ND ND ND 2.48/0.59 2.18/0.38 1.94/0.34

NSP Abc +ve/total 30 dpv 0/10 d 0/10 d 0/10 d 0/16 d,e 0/14 d,e 0/16 d,f,g

a- Detection of Ab by liquid-phase blocking sandwich ELISA against A24 Cruzeiro Br 55, A Arg 2000 and A Arg 2001 in cattle sera collected at day of challenge as stated in Tables 1a-1b. b-Standard deviation. c- Non structural protein Ab determinations. d- ELISA 3ABC (Robiolo et al, 2002), e-ELISA 3ABC- EITB (Bergmann et al, 2000), f- 3ABC ELISA Brescia-Pirbright (De Diego et al, 1997). g- Peptide ELISA (United Biomedical, Shen et al, 1999). *revaccinated group, second dose was administrated at 30 dpv (O-A24-C3 vaccine) or at 21 dpv (O-A24-A2000 vaccine)

343

Assessment of herd immunity by LPBE in sera from cattle under the FMD vaccination programme Cattle 1- 2 years of age evidenced high immunity levels against all FMDV strains included in the vaccine revealing satisfactory vaccine coverage in the age category of higher disease risk. The EPP for A Arg 2000 and A Arg 2001 FMDV strains were calculated by using the logistic regression fitting established previously for the A type FMDV vaccine strains. Specific comparisons of LPBE Ab titres and protection against A Arg 2001 are being undertaken in order to validate a correlation between both variables for this particular FMDV strain. Table 4: Percentages of cattle with high-expected protection percentage (EPP>80%) according to age during 2001 serological survey in Argentina. Percentages of cattle with EPP >80% against FMDV strains*

Age category** 12-24 m >24 m

N

O1C

A24 Cruzeiro

A 2000

A2001

4972 1243

91 % 91 %

94 % 93 %

87 % 89 %

83 % 87 %

* LPBE titres were assessed individually against each FMDV strain contained in the vaccine, on cattle sera from farms showing negative results by ELISA 3ABC-EITB (Bergmann et al., 2000). LPBE titres above log10= 1.80 were associated with EPP >80% (SENASA; 1996) ** age in months

Discussion and conclusions After the first outbreaks reported in 2000 in cattle in the North East of Argentina, the disease spread to several provinces, except the Patagonian region, primarily through movements of cattle. FMDV type O circulated in 2000 and type A circulated in 2000 to January 2002. The National Reference Laboratory, SENASA, received routinely tissue samples from suspected cases of vesicular disease. Primary diagnosis was done by ELISA and complement fixation test and through serial passages in tissue culture or suckling mice. Further characterization was performed by monoclonal Ab profiling and nucleotide sequencing, results that were reported in detail separately. Briefly, the sequencing data indicated that the type O FMDV isolates were related to South American isolates (König et al, 2002). On the other hand, the type A virus strains that circulated during 2000 and 2001, named A Arg 2000 and A Arg 2001, belonged to two different lineages (Piccone et al., 2002). Type O isolates were antigenically related to the O1 strains isolated in the region (data not shown). This is probably the reason of the quick disappearance of outbreaks caused by this virus following vaccination with vaccines containing O1 Campos. For A types, the relationships between field strains and the vaccine strains studied indicated low levels of antigenic relatedness with “r” values lower than 0.4. Further studies are ongoing to establish the statistical significance of the neutralising Ab data presented here and to investigate relationships of these strains with other vaccine strains.

344

The results obtained in challenge studies indicated that protection against A Arg 2000 and A Arg 2001 after primary vaccination was induced by vaccines containing homologous strains. However, vaccines containing heterologous type A strains achieved satisfactory levels of protection only after revaccination (Trials 1 and 3, Table 3a). In order to induce high protection in singly vaccinated animals, SENASA decided to include the new strains into the vaccine, in a first stage A2000 and later on A2001. To optimize the quantitative formula to be used in vaccines for mass vaccination, doseresponse studies using decreasing 140S antigen content of the new A strains were performed. Trials 2 and 4 showed that protection against A Arg 2001 required vaccines with higher homologous 140S content than those which induced protection against A Arg 2000 (Table 3b). In these studies, we did not assess the influence of other type A strains present in polyvalent vaccines, which may possibly had played a role in crossprotection, as it was observed by others (Barnett et al., 2001). As shown for the vaccines used in this study, formulations containing more than 6 µg of A Arg 2001 were needed to induce above 80% protection. It should be noted that for oil vaccines, virus challenge at 30 dpv is more severe than that at 90 dpv, the latter challenge method had been adopted by Sanitary Authorities to control the potency of oil vaccines able to induce long duration of immunity. Higher Ab titers against A Arg 2001 were found in vaccinated groups with higher protection percentage against A Arg 2001 challenge (Trials 3 and 4). The mean group Ab titres to A2001 related with protection in this study were also shown to be higher than those for other strains (Robiolo et al, 1995). More data are being collected in order to establish a correlation between Ab titre and protection against challenge for this FMDV strain. It is also desirable that immunogens to be used in prophylactic campaigns do not interfere with the assessment of viral activity in the vaccinated cattle population. In this regard, the developed vaccines did not induce Ab against non structural proteins as assayed by different detection methods. The assessment of the herd immunity to FMD along with the detection of viral activity has been shown to be reliable indirect indicators of the progress of the FMD control campaigns and to diagnose areas under risk (Smitsaart et al., 1998, Fondevila et al., 1997). In our study, herd immunity was above 80% assuring satisfactory protection levels in the cattle population (Table 4). It should be noted that in this study only Ab due to vaccination had been considered, as cattle sera from farms with positive reactors to non structural proteins were not included. In addition, the vaccine coverage and vaccine efficacy were demonstrated by the dramatic decrease in the number of outbreaks since September 2001 after the second round of vaccination of 55 million cattle (Figure). The success of the control of FMD in Argentina has been the result of the co-ordinated activities of the different local institutions such as SENASA, INTA, CEVAN, farmer associations, and the Argentinean FMD Vaccine and Antigen Bank, on topics such as characterisation of virus strains, epidemiological studies, vaccine development and vaccine performance. The existence in Argentina of a FMD Vaccine and Antigen Bank played a major role in the control of the recent disease outbreaks. It provided not only the know how to adapt

345

field strains to manufacturing conditions, but also the scale up of the production was rapidly accomplished due to the fact that the manufacturing capacity of the facility was available, and readily increased to provide all the doses needed for the immediate and subsequent campaigns. Soon after SENASA authorities decided to start the mass vaccination programme, the Vaccine Bank increased its production capacity up to 14 million doses per month of tetravalent vaccine by the end of 2001. In this regard, more than 187 million doses have been delivered from October 2000 to May 2002, supplied by a single plant. At the same time, all the reagents and procedures necessary for inprocess controls were generated. Moreover, the Quality Assurance System (ISO 9001) allowed proper internal and external controls of critical steps in the vaccine production process. In terms of safety, there were minimal reports of secondary adverse reactions to vaccination. On the other hand, the vaccines were manufactured using only local animal products. This aspect guaranteed minimal risk concerning bovine spongyform encephalopaties, since Argentina, according to geographical risk assessment, is considered as highly unlikely to present a BSE risk (Category I, Scientific Steering Committee-EU). In summary, safe vaccines that induced protection against field strains were readily developed and scaled up, which allowed rapid control of FMD, giving satisfactory herd immunity for the cattle population along with decreasing of FMD outbreaks, particularly since the introduction of A Arg 2001 strain in the vaccine formulation. Acknowledgements The authors wish to thank the SENASA's Epidemiology Consultant Committee for providing epidemiological data. References Alonso, A. Martins, M., Gomes, M.P.D. et al. (1992). Foot-and-mouth disease virus typing by complement fixation and enzyme-linked immunosorbent assay using monovalent and polyvalent antisera. J. Vet. Diagn. Invest. 4: 249-253. Barnett, P.V., Samuel, A.R., Statham, R.J. (2001) The suitability of the “emergency” foot-andmouth disease antigens held by the International Vaccine Bank within a global context. Vaccine 19:2107-2117 Bergmann, I., Malirat, V., Neitzert, E., Beck, E., Panizzutti, N., Sanchez, C., Falczuk, A. (2000) Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with an enzymelinked immunoelectrotransfer blot assay. Arch Virol, 145 (3) 473-89. De Diego, M., Brocchi, E., Mackay, D.K. and De Simone, F. (1997) The non-structural polyprotein 3ABC of FMDV as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Arch Virol; 142: 2021-2033 Fondevila, N., O´Donnell, V., Duffy, S., León, E., Smitsaart, E.& Schudel, A. Seroepidemiological indicators for evaluating foot-and-mouth disease control campaigns. (1997) Rev.sci.tech.Off int. Epiz 16 (3)784-792.

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König, G.A., Gomez, E., Seki, C., Mazzuca, G., La Torre, J., Maradei, E., Sadir, A., Palma, E., Piccone, M.E. (2002) Caracterización Molecular del Virus de la Fiebre Aftosa (VFA) de serotipo O circulante en Argentina en el año 2000. VII Congreso Argentino de Virología, Buenos Aires, Argentina. 23-25/09/2002. Piccone M.E., König G., Seki C., Mazzuca G., Robiolo B., Maradei E., Periolo O., Sadir A, Falczuk A., La Torre J. & Palma. E.L. (2002) Genetic analysis of type A foot-and-mouth disease virus isolated in Argentina during the 2000-2001 outbreak. EUROPICTamerica2002. Sea Crest, USA. 13-19/05/2002. Rweyemamu, M.M., Pay, T.W.F. & Parker, M.J. (1977) Serological differentiation of foot-andmouth disease virus strains in relation to selection of suitable vaccine viruses. Dev.Biol.Stand. 35:205-214 Robiolo, B., Grigera, P.R., Periolo, O. H., Seki, C., Bianchi,T., Maradei, E. and La Torre, J. L. (1995) Assessment of foot-and-mouth disease vaccine potency by liquid-phase blocking ELISA: a proposal for an alternative to the challenge procedure in Argentina. Vaccine 13: 1346-1352 Robiolo, B. Morvillo, V. Scodeller, E., Seki, C., Grigera, P., Maradei, E., Periolo, O. & J.L LaTorre (2002). ELISA 3-ABC-Monoclonal: un método para diferenciar animales vacunados de infectados en Fiebre Aftosa. VII Congreso Argentino de Virología, Buenos Aires, Argentina. 23-25/09/2002. Seki, C., Periolo, O., Soares Campos, G., Grigera, P., Remorini, P.L., Capozzo, A., Falzuc, A., Maradei, E. and La Torre, J. Monoclonal antibodies developed against South American Aphtovirus strains. Application in the quality control of commercial vaccines and in the characterization of field isolates. In preparation. SENASA. Resolución 219/95, Boletín Oficial N° 28125, Disposición Oficial N° 03/96. Shen, F., Chen, P.D., Waldfield, A.M., Ye,J., House, J., Brown,F. et al. (1999) Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA. Vaccine, 17: 3039-3049 Smitsaart, E., Zanelli, M., Rivera, I., Fondevila, N., Compaired, D., Maradei, E., Bianchi, T., O'Donnell, V. & Schudel, A.A. (1998) Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines. Prev. Vet. Journal. 33: 283-296.

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Figure: number of outbreaks notified in Argentina in the 2000-2002 period. Source: SENASA

FMD outbreaks 2000-2002 700 600

Frequency

500 400 300 200 100 0 outbreaks

Jul- Aug- Sep- Oct- Nov- Dec- Jan- Feb- Mar- Apr- May- Jun00 00 00 00 00 00 01 01 01 01 01 01 4

38

18

16

11

37

65

203

247

359

605

540

Jul- Aug- Sep- Oct- Nov- Dec- Jan- Feb- Mar- Apr- May- Jun- Jul01 01 01 01 01 01 02 02 02 02 02 02 02 324

68

17

1

348

6

1

1

0

0

0

0

0

0

349

Appendix 41

Peparation of synthetic peptide FMD vaccine with newly developed antigen- polymere conjugates be used as immonogen and vaccine in veterinary medicine S. I. Deliloglu Gürhan1, M. Mustafaev Akdeste2, Z. Mustafaeva Akdeste2, G. Aynagöz3, G. Unver4, N. Unal3, N. Celik3 E.U. Bioengineering Dep., 35100 Bornova IZMIR- TURKEY, 2TUBITAK MAM GMBAE, Gebze KOCAELI- TURKEY, 3Foot and Mouth Disease Institute, PO Box 714, 06044 ANKARA- TURKEY, 4 Veterinary Control and Research Institute, 35100 Bornova IZMIR- TURKEY 1

ABSTRACT Preparation of newly developed immunogens and vaccine preparation by conjugation of synthetic polymers and peptide antigens of 40-60 and 135-160 amino acid sequences of immunogenic VP1 capsid protein of "A" type FMDV which causes epidemics in Turkey was the aim of this project. Thus, by the modification of the immunogenisity of the antigens, development of the new FMD vaccines, diagnostic reagents, pharmaceuticals and biotechnological preparations was considered. The peptide chains were prepared by Fmoc-solid- phase synthesis chemistry. Electrostatic polycomplexes (EC) of bovine serum albumin- polypeptide conjugates (BSA.P) with polycations-copolymers of 4- vinyl- N- ethyl pyridine and 4- vinyl- N- cetylpyridine bromides and Cu2+- induced ternary polycomplexes with anionic copolymers of acrylic acid with Nisopropyl- acryl amide and N- vinyl- pyrrolidone (PE) were used. A protein- peptide and synthetic polyelectrolyte (PE) - peptide conjugates consist of the samples of the different domains of foot-and-mouth disease virus (FMDV) VP1 which covalently bound to bovine serum albumin (BSA), polyacrilic acid (PAA), copolymers of acrylic acid (AA) with Nisopropylacrylamide (NIPAAm) (CP1) and N- vinylpyrolidone (CP2). For preparation of the conjugation systems Na- (2- ethylhexsyl) sulphosuccinate and octane were used as surface active material and regular organic phase respectively. Primarily four different synthetic peptide vaccines prepared as described above were tested in mice for their antibody stimulating efficiency and in guinea-pigs for protection values against homolog challenge virus strain. The vaccine (VAC2) demonstrated minimum side effects in guinea-pigs and highest protection against the challenge virus was selected for vaccination of cattle. Peptide specific and intact virus specific antibody responses were demonstrated by enzyme- linked immunosorbant assay (ELISA) and neutralisation test (NT) with the 14 and 21 day post vaccination sera of the cattle vaccinated with (VAC2). The results indicated that VAC2 is able to protect guinea-pigs against "A" type FMDV challenge but not induce sufficient specific antibody against neither the synthetic peptides nor to intact virus in cattle. However, some of the animals responded to the synthetic vaccine but the antibody level estimated with both LPB ELISA and NT was not in acceptable level for protection. Only one cattle performed VAC2 induced immune response just below the protection limit (NI=1.2) even 14 days post vaccination. It is clear that some quantitative and 349

qualitative modifications such as addition of T-cell epitope sequences or increasing the quantity of the peptide in the vaccine formula will aid to induce immune response in cattle. In the author’s knowledge, this work is the first synthetic peptide vaccine trial in Turkey. INTRODUCTION The suggestion using synthetic peptides as foot-and-mouth disease (FMD) vaccine was made after the determination of the high neutralisation titre with one segment of VP1 about 20 years ago. In parallel with the developments in technology, at present, synthesis of some amino acid domains of VP1 can be realised rapidly and in a correct form [20]. Generally, the target is preparation of vaccine or diagnostic material for FMD. The protection effect of synthesised biocojugates as a mixture in oil emulsions were detected in the immunisation of the animals [9]. In these studies the polypeptides synthesised as antigen were primarily coupled to a protein carrier then this conjugate was mixed with an oil adjuvant (e.g.: VP1-protein + IFA). A number of researches are being carried out to develop synthetic peptide vaccines against viral pathogens including FMDV. Most of them are able to protect laboratory animals such as mice, rabbits and guinea pigs. However, there is no such vaccine which can be used in the field for the farm animals yet [6, 8, 17, 19, 25, 28]. The first synthetic peptide vaccine against an animal disease which fully protects the target animal is for the parvovirus infection of dogs [14]. Recently, edible synthetic peptide vaccines prepared by the expression of VP1 of FMDV to transgenic plants are developed [7, 23]. In fact, synthetic peptides are not strong antigens. They need to be conjugated to a suitable adjuvant. Most of the vaccine development studies are focussed on the preparation of a good effective adjuvant [18]. The adjuvant is very important as well as inoculation route, dose and formulation of the antigen. If a suitable adjuvant was selected, controlled release of the antigen and, as a result of it, long lasting immunity with single inoculation can be achieved [16]. The 200-213 and 140-160 amino acid residues of VP1 protein conjugated with key hole limpet haemocyanine could stimulate neutralising antibody response and protect the guinea pigs against FMDV challenge [5, 10]. Higher peptide mass (5mg) and boosting the cattle could stimulate antibody response in cattle. However, complete protection against intact virus was not reported [24]. Although the hopeful results were reported with modified vaccines, biosynthetic vaccines and synthetic peptide vaccines none of them can compete with the conventional inactivated vaccines yet. Additionally, researches on the modified vaccines lost their importance after the prohibition of EC countries the importation of the meat from the countries where modified intact virus vaccines are applied [13]. Recently, a synthetic peptide vaccine against O type FMDV effective in swine was reported [30]. Some others are also carrying out the field trials [12].

350

In addition to the receptor sites for protein antibodies, the unbound peptides can interact with helper T cell receptors and Ia antigens. Starting from this fact it was hypothesized that the protection problem of cattle could be solved by the addition of an extra T cell epitope into the vaccine formulation [11]. Another encouraging technique is utilisation of the non immunogenic synthetic polyelectrolyte (PE) as antigen carriers [21]. In the present study, a novel approach to a totally synthetic vaccine, which consists of FMDV VP1 peptides (40-60 and 135-160 residues), prepared by chemical synthesis and nonimmunogenic membrane active polyelectrolyte (PE) is reported. MATERIAL AND METHODS Peptide Synthesis: 40- 60 and 135- 160 amino acid residues of "A" Aydin 98 (A Iran 96) serotype of FMDV were synthesized by Merrifield solid-phase technique with an automatic synthesizer (Millipore’ s Automated Peptide Synthesizer, USA) [23, 26]. Those peptides were purified and characterized with chromatographic, spectroscopic and fluorimetric analysis. The experiments were repeated with the same sequences synthesized by SIGMA GENO§YS (USA). P1

40-60 (21 mere)

Val-Lys-Ile-Asn-Asn-Thr-Ser-Pro-Thr-His-Val-Ile-Asp-Leu-Met-Gln-Thr-His-Gln-His-Gly

P3

135-160(26 mere)

Lys-Tyr-Ser-Ala-Thr-Gly-Glu-Arg-Thr-Arg-Gly-Asp-Leu-Gly-Ala-Leu-Ala-Ala-Arg-Val-Ala-Thr-Gln-LeuPro-Ala

Table 1. : Synthesised amino acid sequences of A Aydın98 FMDV strain [1].

Polyelectrolyte Sythesis: Cationic polyelectrolytes (PE) are the copolymers of 4-vinyl-Nethylpyridine (PEVP) and 4-vinyl-N-cethylpyridine (PECVP). PE was obtained by quaternization of narrow fractions of poly-4-vinylpyridine (Pn = 103) with ethyl and cethylbromides by the method previously desribed [15]. The anionic PEs are polyacrylic acid (PAA), copolymers of acrylic acid (AA) with N-isopropylacrylamide (NIPAAm) (CP1) and N-vinylpyrolidone (VP) (CP2). CP1 was prepared by the radical copolymerization of AA with NIPAAm in the presence of 2-oxoglutaric acid as a UV initiator by irradiation of UV light (365 nm) under nitrogen at room temperature. CP2 was obtained by the radical copolymerization of AA with VP in a methanol solution in presence of a radical initiatorazodinitrile-bis-isobutiric acid at 60 0C. The copolymers obtained were purified by three precipitations from methanol into ether. Polyelectrolyte-peptide and protein-peptide conjugate synthesis: To carry out PE-peptide (PE.Pep) and BSA-peptide (BSA.Pep) conjugation reactions, surfactant stabilized micro emulsion of water in organic solvents, i.e. hydrated reversed micelle (HRM) systems (BSA – Bovine Serum Albumin Mw = 70000, pI = 4.9, from Sigma Chemical Co., St.Louis, MO) was used. Using the HRM technique, a series of protein-peptide and PE-peptide conjugates were prepared including FMDV VP1 epitopes of different regions which were covalently bounded to BSA, CP1 and CP2. Polyelectrolyte complex synthesis: To prepare a PE-BSA.Pep electrostatic complexes, various concentrations of the BSA.Pep conjugate solutions were added to PEVP (or PECVP), dissolved in phosphate buffer (PBS), pH 7.2. To produce the PE-Cu2+ complex, the 351

CuSO4.5H2O (pH 4) solution was added to PE, dissolved in PBS. The desired pH values were adjusted with 1 M NaOH. The ternary PE- Cu2+-protein complexes were, in turn, prepared by adding BSA.Pep conjugate solution to the PE- Cu2+ solution. Both of the amino acid sequences (P1 and P3) coupled to 4 different polymeric complexes (CP1, CP2, PECVP and CP-Cu2+-BSA). Thus, 4 different vaccine compositions were prepared (VAC1, VAC2, VAC3 and VAC4). VAC1 VAC2 VAC3 VAC4

(CP1-P1) + (CP1-P3) (CP2-P1) + (CP2-P3) (PECVP-BSA.P1) + (PECVP-BSA.P3) (CP-Cu2+-BSA.P1) + (CP-Cu2+- BSA.P3)

Table 2: Composition of the synthetic peptide vaccines.

Side Effects in Guinea pigs: 4 animals for each vaccine were inoculated s.c. (2ml/ animal). The local and general reactions were detected and recorded during the 1 month of inspection period. Side Effects in Cattle: VAC2 which passed both guinea pig tests was inoculated to 13 cattle. The local and systemic adverse reactions were inspected clinically for 7 days. Immunisation Studies: Vaccine Doses: Synthetic peptide quantities of vaccines which used in immunisation and potency trials are summarized in Table 3. Animal Mouse Guinea-pig Cattle

P1 (µg/dose) 50 500 1500

P3 (µg/dose) 50 500 1500

P1 + P3 (µg/dose) 100 1000 3000

Vaccine dose (ml) 0.2 2 2

Table 3: Final synthetic peptide concentration of one vaccine dose for animals.

Mouse: 8 week old Balb/c mice were immunized intra venous (i.v.) with 4 vaccine candidates. Synthetic peptide combination (P1+P3) was used as negative control. Animals were bled at weekly intervals and antibody titres were estimated with indirect ELISA. Cattle: 10 cattle (in a fattening farm near Ankara) were inoculated with VAC2 s.c. Animals were bled before vaccination (on the day of vaccination), 14th and 21st days pv.. Antibody response against both synthetic peptides and intact virus were tested with indirect ELISA and LPB ELISA respectively. Development of neutralizing antibodies against whole virus was also detected with NT with BHK cells. Protection Test: Guinea-pig protection test was applied as described by Barnett, CARABİN 2002 AND Wotzler et al 2002. [3, 31]. Groups of four animals were immunized with twofold dilutions of the 4 vaccine candidates and an aluminium hydroxide-saponine adjuvant vaccine prepared with the inactivated A98 virus as control. Immunization was done subcutaneously 2ml vaccine/ guinea-pig where the final synthetic peptide concentration in each dilution was 1 mg, 0.5mg, 0.25mg and 0.125mg. 21 days post vaccination animals were challenged with 400 guinea-pig ID50 "A" 98 virus/ animal. A group of four unvaccinated guinea-pigs was also infected with the challenge virus as control. 352

RESULTS AND DISCUSSION Vaccine Site Reactions in Guinea-pigs: Four vaccine formulations were tested in guineapigs. There was abnormal reaction in the animals vaccinated with VAC1 and VAC3. A local hyperaemia in the inoculation zone was detected for 1-2 days in the animals after vaccination with VAC2. Nevertheless, severe local and systemic reactions appeared just a few minutes after inoculation of VAC4. In coordinated pace, loss of appetite for 1 day, apses with large hyperaemia and large swelling zone in the inoculation area. This severe side effects with VAC4 were attributed its Cu+2 content of the polymer. Vaccine Site Reactions in Cattle: All of the cattle were inspected for 1 week post vaccination. Mild reactions were detected such as increase in body temperature of two animals (39.1-40.1) for 1 or 2 days and a small lump with 30-40 mm Ø insensitive to pressure lasting for 7 days maximum. Immunogenicity: Mice: The mice immunised intravenously with adjuvant-free polypeptide were not responded to the antigen. Whereas, primary peptide specific immune response increased in the first 7 days and the titres were steady up to 23rd day p.v. in the animals vaccinated intravenously with PE-polypeptide conjugates (Table 4). Since peptides are known as poor immunogens, unresponsiveness to the peptides without adjuvant was expected. The highest antibody level was detected in the sera of the mice inoculated with VAC2 14 days post vaccination and starting from the 21. day pv peptide specific antibody titre decreased gradually. In fact, the characteristics of antibody development and the level of immune response was not dependant with the preparation method of the biopolymer systems and the structure of the polymer carriers. Potency in Guinea-pigs: Guinea-pig potency test is still an acceptable and reliable method in determination of the potency of the FMD vaccines [4]. Starting from that point, 4 PEpolypeptide conjugates (VAC1, VAC2, VAC3, and VAC4) which developed the immunogenic activity in the mice were selected to be used in guinea-pig potency test. The conventional vaccine, with Al (OH)3 adjuvant, inactivated virus which contains 8.9µg 146S antigen was protected all of the animals. Guinea-pigs vaccinated with VAC2 which contains 1mg synthetic peptide/dose was also protected the entire animal. The protection ratio in the animals vaccinated with the same dose (1mg) of peptide conjugated with different polymers (VAC3 and VAC4) was ¾. However, VAC1 developed weak protection. The importance of the adjuvant in the potency of the inactivated or subunit vaccines is a well known reality [11, 20]. For that reason, recently most of the vaccine development studies are targeted to find out more effective adjuvant with minimum side effects. Also in the present study, guinea-pigs vaccinated with the same quantity of synthetic peptides conjugated with different PE’s were protected against the same quantity of the virus in different levels (Table 4). VAC2 was selected as primary candidate for the further experiments because of its higher protective capacity and lower toxicity in lab animals. Vaccine Trial with Cattle: 15-18 months old 10 cattle were vaccinated subcutaneously with VAC2 which contain 3mg synthetic peptide / doses. Animals were bled 14 and 21 days post vaccination. Antibody response to homolog intact virus was evaluated with both LPB-ELISA 353

and NT in BHK21 cell line. Although there was a mild increase in the titres of 8 animals out of 10, none of them passed over the acceptable levels. Since, NI 0.9-1.3 considered uncertain. Only one cattle (ear tag No.99) could reach up to that level with NI 1.2 in 14th day p.v. However, to be honest, this animal was probably primed before vaccination (NI 0.3 at day 0). In the present study vaccination of the guinea-pigs with 1mg of PE- conjugated synthetic peptides (40-60 and 135-160 amino acid. residues of VP1) developed complete protection. Antibody response in mice with 100µg of the same peptide vaccine conferred the effectiveness of preparation. Nevertheless, 2 ml vaccine with 2 mg synthetic peptide was not sufficient to develop antibody response against homolog virus. Antoni et al (1988) showed that the cattle with high antibody titre at 21st day p.v. against synthetic peptide vaccine developed generalised lesions after challenge with 10.000 ID50 of the homolog virus [2]. Contrary, in some experiments some of the cattle with insufficient antibody titre could be protected after challenge [27]. It is clear that there is some other factors play important role in the protection mechanism of the animals. This can be cellular immunity or other type immunological responses [3]. For this study increasing the quantity of the synthetic peptide per dose could also be a solution to the problem. Tam et al (1989) showed that sufficient protection in cattle could be achieved with 5mg synthetic peptide [24]. Another alternative is preparation of new vaccine combinations with some additional amino acid residues. Volpina el al (1999) declared that besides mice, guinea-pigs and rabbits also sheep and cattle responded to the vaccination with synthetic peptide vaccines contain 170-188 amino acid residues of VP1 protein of A type FMDV [29]. The last solution but not the least is to make some modifications in synthetic polymer composition and coupling mechanisms.

Figure 1: Peptide specific antibody formation dynamics in the mice immunised with biopolymer systems combined by two polypeptides (40-60 and 135-160 sequences) containing PE-peptide conjugates, electrostatic and ion coordination bonds (Cu2+). (1■) VAC1, (2●) VAC2, (3▲) VAC3, (4▼) VAC4, (5♦) P1+P3.

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TESTS Mice

Peptide specific Ab response (indirect ELISA)

Guinea-pigs

Side effects

Potency Side effects Virus specific Ab response (NT&ELISA) Potency *number of protected guinea-pigs /number of challenged ND- not done Cattle

RESULTS VAC1 VAC2 VAC3 VAC4 VAC1 VAC2 VAC3 VAC4 V AC1 VAC2 VAC3 VAC4 VAC2

– + + ND no negligible (1-2 days) no sever – (0/4)* ≤2ml (≤1mg) + (4/4)* 2ml (1mg) + (3/4)* 2ml (1mg) + (3/4)* 2ml (1mg) no ± ND

Table 4: Summary evaluation of the control tests applied to the synthetic peptide vaccines.

REFERENCES 1. Aktaş S., Molecular epidemiology of foot and mouth disease types O and A in Turkey. Thesis R7871, Ph.D. Thesis, University of Reading, (1998). 2. Antoni,F.,Soos, T, Varga, J., Mecs, I., Penke, B., Horvath, K., Bognar, K., Tubloy, S. Efficacy of experimental synthetic peptide vaccines against foot and mouth disease, Magyar Allatovosok Lapja, 43(9), 561-6, (1987). 3. Barnett, P. V., Carabin, H. A review of emergency foot-and-mouth disease (FMD) vaccine, Vaccine, 20, 1505-1514, (2002). 4. Barnett, P. V., Statham, R. J. Stratified and cryogenically stored (SACS) vaccines, a new concept in emergency foot-and-mouth disease vaccine formulation and storage, Vaccine, 20, 2060-2064, (2002). 5. Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M., Sutcliffe, J. G., Lerner, R. A. et al. Protection aganist foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence, Nature (London), 298-30, (1982). 6. Brown, F.: In Immunochemistry of viruses, eds. Regenmortel and Nevrath, p.265, Elsevier Sci. Pub., Amsterdam, The Netherlands (1985). 7. Carillo, C. et al Protective immune response to foot and mouth disease virus with VP1 expressed in transgenic plants, J. Virol.,72(2), 1688-90, (1998). 8. Chinsangaram. J. et al Antibody response in mice inoculated with DNA expressing foot and mouth disease virus capsid proteins, J.Virol., 72(5), 4454-7, (1998). 9. Deen, C., Claasen, E., Gerritse, K., Zegers, D.N. and Boersma, J.A.W. A novel carbodiimide coupling method for synthetic peptides enhanced anti-peptide antibody responses, J. Immunol. Methods., 129 119-25, (1990). 10. Dimarchi, R., Brooke, G., Gale, C., Cracknell, V., Doel, T. and Mowat, N. Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science, 232, 639, (1986). 11. Francis, M. J., Hostings, G. Z., Syred, A. D., McGinn, B., Brown, F. and Rowlands, D. J. Overcoming genetically controlled non-responsiveness to uncoupled FMDV peptide using “foreign” helper T-cell determinants. Nature (London), 330, 168-70, (1987). 12. http://www.unitedbiomedical.com 13. Kobbs-Conrad, S., Gerdon, A. and Kaumaya, P.T.P. Multivalent B- and T-cell epitope vaccine design. In Proceedings of the Twelfth American Peptide Symposium. J.Smith and J.Rivier, eds, ESCOM, Leiden, (1991). 14. Langeveld, JP et al First peptide vaccine providing protection against viral infection in target animal: studies of canine parvovirus in dogs. J.Virol., 68(7), 4506-13, (1994).

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15. Laporte, J., Grusclaude, J., Wantygem, J., Bernard, S. And Rouze, P. Neutralisation en culture cellulaire du pouvoir infectieux du virus de la fievre aphteuse par des serums provenant de porcs immunises a laide d’une proteine virale purifiee. C. R. Acad. Sci. Ser. D. Sci. Nat., 276, 3399, (1973). 16. Lofthouse, S. A., Kajihara, M., Nagahara, S., Nash, A., Barcham, G. J., Sedgmen, B., Brandon, M. R., Sano, A. Injectable silicone implants as vaccine delivery vehicles. Vaccine, 20, 1725-32, (2002). 17. McCullough, K.C., De Simone, F., Brocci, E., Capucci, L., Crowther, J.R. and Kihm, U., Protective immune response against foot and mouth disease, J.Virol.66(4), 1835-40, (1992). 18. Mustafaev, MI.., Norimov, AS., Petrov,RV.:In Synthetic modulators, 1992 eds. R.Petrov, Nauka, Mocow, 250-370. Vaccines: New-Generation Immunological Adjuvants, 1994, eds. G.Gregoriadis, A.C.Allison and G. Poste, Plenum Press, New York. 19. Nalin, D. R., Evidence based vaccinology, Vaccine, 20, 1624-30, (2002). 20. Oldstone, MB, Discriminated selection among viral peptides with the appropriate anchor residues: implications for the size of the cytotoxic T-lymphocyte repertoir and control of viral infection, J.Virol., 69(12), 7423-9, (1995). 21. Oxenius, A CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines J.Virol.,73(5), 4120-6, (1999). 22. Petrov, R.V., Mustafaev, M.I., Norimov, A.Sh. Physico-Chemical Criteria for the Construction of Artificial Immunomodulators and Immunogens on the Basis of Polyelectrolyte Complexes. Vol.4. pp. 1-113, Harwood Acad. Publ. GmbH, UK. (1992). 23. Santos, M.J.S., Wigdorovitz, A., Trono, K., Ríos, R.D., Franzone, P.M., Gil, F., Moreno, J., Carillo, C., Escribano, J. M., Borca, M.V. A novel methodology to develop a FMDV peptidebased vaccine in transgenic plants. Vaccine, 20, 1141-7, (2002). 24. Synthetic Peptides. Ed. Gregory, A., Grant USA, 283-9, (1992). 25. Tam, J.P. Multiple antigenic peptide system: A novel design for synthetic peptide vaccines and immunoassay. In Synthetic Peptides; Approaches to Biological Problems. J.P. Tam and E.T. Kaiser, eds. Alan R. Liss, Inc., New York, pp. 3-18. (1989). 26. Taboga, O et al A large-scale evaluation of peptide vaccines against foot and mouth disease: lack of solid protection in cattle and isolation of escape mutants J.Virol., 71(4), 2606-14, (1997). 27. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., Weigele, M. Fluorescamine: A reagent for assay of amino acids, peptides, proteins and primary amines in the Pico mol range.Science, 178, (1972). 28. Van Lierop, M.J., Wagenaaar, J.A.,Van Noort,J.M. and Hensen, E.J. Sequences derived from the highly antigenic VP1 region 140-160 of FMDV do not prime for bovine T-cell response against intact virus, J. Virology, 69(7), 4511-4, (July 1995). 29. Vasantha, S., Antony, A. And Lal, S.M. Liposome encapsulated subunit (VP1) and virion vaccines against foot and mouth disease. Acta virol., 31, 109-15,(1987). 30. Volpina, O.M., Surovoy, A.Y., Zhmak, M.N., Kuprianova, M.A., Koroev, D.O., Chepurkin, A.V., Toloknov, A.S., Ivanov, V.T. A peptide construct containing B-cell and T-cell epitopes from the FMD viral VP1 protein induces efficient antiviral protection. Vaccine, 17, 577-84, (1999). 31. Wang, CY, Chang, TY, Walfield, AM, Ye, J, Shen, M, Chen, SP, Li, MC, Lin, YL, Jong, MH, Yang, PC, Chyr, N, Kramer, E, Brown, F, Effective synthetic peptide vaccine for footand-mouth disease in swine Vaccine 20, 2603-2610, (2002). 32. Wutzler, P., Sauerbrei, A., Klöcking, R., Brögmann, B., Reimer, K., Virucidal activity and cytotoxicity of the liposomal formulation of povidone-iodine, Antiviral Research, 54, 89-97, (2002).

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ACKNOWLEDGEMENT This work was carried out in Foot & Mouth Disease Institute, Ankara and Institute for Genetic Engineering & Biotechnology of TUBITAK Marmara Research Center, Gebze Kocaeli. The authors are grateful to Prof. Dr. E. Bermek who founded the collaboration between the organizations, to the General Directorate of Agricutural Research of Ministry of Agriculture and Rural Affairs (TAGEM), Turkish Scientific and Technical Research Organisation (TUBITAK) and Ege University Science and Technology (EBILTEM) for their financial support, to Dr. H. Zengin for his moral support, Dr. A. Naci Bulut for his excellent technical help in ELISA applications and to Dr. I. Türküz for his technical help in vaccination and bleeding of the cattle on the farm.

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Appendix 42

Proposals for Completion of Phase XVII and for Phase XVIII The available results of the Phase XVII study were discussed at the EUFMD Research Group Meeting in Izmir (16-20 Sept 2002). It was agreed that Dr Paton and Dr De Clercq would prepare proposals for the completion of Phase XVII and for the new Phase XVIII project. 1. Phase XVII 1.1. Background The candidate reference sera prepared and evaluated in Phases XV and XVI have been adopted by OIE. They represent three strains of FMD, O Manisa, A22 and C Noville. Under Phase XVII, candidate reference sera have been prepared and evaluated for three new FMD virus strains, namely, O South Korea (O SKR), A Iran 96 and Asia 1 Shamir. There has been uncertainty over the exact definition of the weak positive and cut-off reference sera. According to OIE guidelines, a weak positive should be weak, but consistently positive and cut-off sera are not defined. The following is now proposed: FMD weak positive serum. This is the weak positive serum for use in herd-based serosurveillance. It should be primarily designed by reference to the virus neutralization test (VNT) using homologous virus. The cut-off for the VNT when screening on a herd basis is 1:45. Therefore the weak positive serum should have a mean VNT value of 1:64. FMD cut-off serum. This is the weak positive serum for use in individual animal certification. It should be primarily designed by reference to the virus neutralization test (VNT) using homologous virus. The cut-off for the VNT when screening on an individual animal basis is 1:16. Therefore the weak positive serum should have a mean VNT value of 1:32. Based on the above criteria, the weak and cut-off sera appear to be too weak, and it is proposed to strengthen the new panel for O SKR, A Iran 96 and Asia 1 Shamir. 1.2. Workplan This will require a six months extension to the project, until the end of June 2003. October 2002 Phase XVII results to be circulated to the participating laboratories. Proposals for completion of Phase XVII and for new Phase XVIII to be prepared. December 2002 New candidate reference sera for O SKR, A Iran 96 and Asia 1 Shamir to be sent to the same 9 laboratories that conducted the evaluation in August 2002. These sera must first be BEI inactivated and adjusted so that the weak positive and cut-off sera are stronger. Inocuity testing of the heat treated (56ºC for 30 minutes) and BEI inactivated sera can proceed after the sera have been distributed. March 2003 Evaluation reports concerning the reference sera sent out in December, to be returned to the WRL by mid-March. 358

June 2003 Submit documentation in support of new reference sera to O SKR, A Iran 96 and Asia 1 Shamir to OIE. 2. Phase XVIII 2.1. Background The main aims of this study will be: • • • • •

Introduction of solid phase competition ELISA. Preparation of secondary standards based on reference sera to O, A and Asia 1. An analysis of what exactly is done in this respect by participating laboratories. Use of callibrated tests to examine negative serum populations from own region. Use of callibrated tests to examine a proficiency test panel. Standardisation of internal quality control procedures.

For VNT, participating laboratories should as a minimum, use the homologous virus in their test system. As a working positive control, a titration of a strong positive may be used, so long as the acceptable result is established by comparative tests using the appropriate reference sera. For liquid and solid phase ELISA, O1 Manisa reagents are recommended for serotype O, Asia 1 Shamir for serotype Asia 1 and either A 22 or A Iran 96 for serotype A (latest results need to be analysed before coming to a decision on this). To ensure that the necessary reagents are made available, it will be necessary to send out a plan to participants as soon as possible and to start preparing the documentation (import permits, etc) well in advance of the start date. 2.2. Workplan This will run from January 2003 to December 2004. December 2002 Send consultation letter out on proposals, request plans for preparation of secondary standards and initiate preparation of licences required for reagent transfers, including all possible viruses i.e. O Manisa, O SKR, A22, A Iran 96 and Asia 1 Shamir. Arrange receipt of bulk Asia 1 and A Iran 99 sera from Drs Yadin and Griot. July 2003 Send out reference sera and proficiency panel, along with reagents including detailed protocols for all steps. May 2004 Results to be reported back to WRL for collation and analysis. September 2004 Presentation of results at Research Group meeting.

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Appendix 43

LIST OF PARTICIPANTS Dr Christian Griot Director Institute of Virology and Immunoprophylaxis CH-3147 Mittelhaeusern Switzerland Tel/fax : 41-031-8489211 / 8489222 e-mail : christian.griot@ivi.admin.ch

Members of the Research Group: Dr Kris De Clercq (Chairman) Department of Virology Section Epizootic Diseases CODA-CERVA-VAR Groeselenberg 99 B-1180 Ukkel Belgium Tel/fax : 32-2-3790400 / 32-2-3790401 e-mail : kris.de.clercq@var.fgov.be

Dr Bernd Haas Head of FMD Diagnostic Laboratory Federal Research Centre for Virus Diseases of Animals Paul Ehrilich Strasse 28 D-72076 Tübingen Germany Tel/fax : 49-7071-9670 / 49-7071-967305/905 e-mail : bernd.haas@tue.bfav.de

Dr Aldo Dekker Senior Scientist Laboratory Vesicular Diseases Central Institute for Animal Disease Control P.O. Box 2004 B203 AA The Netherlands Tel/fax : 31-320-238858 / 31-320-238668 e-mail : a.dekker@id.wag-ur.nl

Dr Per Have Danish Veterinary Institute Department of Virology Lindholm DK 4771 Kalvehave Denmark Tel/fax : 45-55869728 / 55869700 e-mail : ph@vetinst.dk

Dr Franco De Simone Head, Centro Nazionale di Referenza per Le Malattie Vescicolari Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Via A. Bianchi, 9 25124-Brescia Italy Tel/fax : 39-30-2290310 / 39-30-2290310 e-mail : fdesimone@bs.izs.it

Dr Vilmos Pálfi Head, Diagnostic Department Central Veterinary Institute 1149 Budapest, Tábornok Utca 2 Hungary Tel/fax : 36-1-2527533 / 36-1-2226069 e-mail : palfi@oai.hu

Dr Alex Donaldson International Veterinary Consultant 290 London Road Guildford, Surrey GU4 7LB UK Tel: 44-1483-567385 e-mail : donaldson.a@aol.com

Dr Hagai Yadin Head of Virology Division and FMD Laboratory Kimron Veterinary Institute c/o Ministry of Agriculture P.O. Box 12, Beit-Dagan 50250 Israel Tel/fax : 972-3-9681619 / 972-3-9681788 e-mail : hagaiy@moag.gov.il

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Dr Michelle Rémond AFSSA LERPAZ 22 rue Pierre Curie – BP 67 94703 Maisons Alfort Cedex France Tel/fax : 33-1-49771317 / 33-1-43689762 e-mail : m.remond@alfort.afssa.fr

Dr N. Ünal Head of Cell & Virus Bank FMD Institute / SAP Institute PK 714 06044 Ankara Turkey Tel/fax : 90-312-2873600 e-mail : nilayunal@hotmail.com

Dr Françoise Lebreton AFSSA LERPAZ 22 rue Pierre Curie – BP 67 94703 Maisons Alfort Cedex France Tel/fax : 33-1-49771317 / 33-1-43689762 e-mail : f.lebreton@alfort.afssa.fr

Observers from member countries : Dr Roland Silber Head of HSL Department Österr. Agentur für Gesundheit und Ernährungssicherheit Robert Kochgasse 17 A-2340 Mödling Austria Tel/fax : 43-1-8021212-10 / 43-1-8021212-11 e-mail : mks.silber@batsb.at

Dr Anja Laabs Veterinarian, Doctoral candidate Free University of Berlin 13355 Berlin Germany Tel/fax : 49-30-20936028 / 49-30-20936067 e-mail : alaabsqk@hotmail.com

Dr Georgi Georgiev National Veterinary Service 15 P Slaveikov Blvd Sofia Bulgaria Tel/fax : 359-2-9441514 / 359-2-9525306 e-mail : georgivet@yahoo.com

Dr Helen Hondrokonki FMD Institute of Athens Neapoleos 25 Ag. Paraskevi Athens 15310 Greece Tel/fax: 30-10-6007016 / 30-10-6082085 e-mail: fmdi@otenet.gr

Dr Ivan Holko State Veterinary Institute National Reference Laboratory of FMD and Vesicular Diseases Sídlištní 136/24 16500 Prague 6 Czech Republic e-mail : ivan.holko@atlas.cz

Dr Helen Reboutzakou FMD Institute of Athens Neapoleos 25 Ag. Paraskevi Athens 15310 Greece Tel/fax: 30-10-6007016 / 30-10-6082085 e-mail: fmdi@otenet.gr

Dr Karl Johan Sørensen Danish Veterinary Institute Department of Virology Lindholm 4771 - Kalvehave Denmark Tel/fax : 45-558-69531 / 45-558-69700 e-mail : kjs@vetinst.dk

Dr Patrick J. O’Reilly Head of Virology Central Veterinary Research Laboratory Abbotstown Castleknock Dublin 15, Ireland Tel/fax : 353-1-6072623 / 353-1-6072663 e-mail : patrick.oreilly@agriculture.gov.ie

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Dr Simon J. Barteling Consultant Veterinary Vaccines Simbar Consultancy Nieuwe Keizersgracht 438 1018 VG Amsterdam The Netherlands Tel/fax : 31-20-6207688 e-mail : s.barteling@chello.nl

Dr Mihai Danes Senior Researcher I.N.M.V. Pasteur Institute Calea Giulesti nr. 333 Bucharest, Sector VI Romania Tel/fax : 40-1-2206929 / 40-1-2206907 e-mail : pasteur@fx.ro

Dr Phaedra Eblé Scientist Central Institute for Animal Disease Control (CIDC-Lelystad) Section Notifiable and Exotic Viral Diseases P.O. Box 2004 8203 AA Lelystad The Netherlands Tel/fax : 31-320-238800 / 31-320-238668 e-mail : p.l.eble@id.wag-ur.nl

Dr Tadej Malovrh Veterinary Faculty Gerbiceva 60 Ljubljana Slovenia Tel/fax : 386-1-4779352 / 386-1-4779768 e-mail : tadej.malovrh@vf.uni-lj.si Dr Esther Blanco Scientific Researcher Centro de Investigación en Sanidad Animal (CISA-INIA) Carretera de Algete a El Casar de Talamanca s/n Valdeolmos, 20130 Madrid Spain Tel/fax : 34-91-6202300 / 34-91-6202247 e-mail : blanco@inia.es

Dr Dick Smit Head Development & Troubleshooting FMD vaccine Production ID-Lelystad P.O. Box 65 8200 AB Lelystad The Netherlands Tel/fax : 31-320-238667 / 31-320-238668 e-mail : d.i.smit@id.wag-ur.nl

Dr Hüseyin Sungur Director General General Directorate of Protection and Control Ministry of Agriculture & Rural Affairs Akay Cad. No. 3 Bakanliklar, Ankara Turkey Tel/fax : 90-312-4174176 / 90-312-4188005 e-mail : vet_service@kkgm.gov.tr

Dr Hanny Swam ID-Lelystad Intervet International PO Box 35 5830 AA - Boxmeer The Netherlands Tel/fax : 31-485-585389 / 31-485-587707 e-mail : hanny.swam@intervet.com

Dr M. Zafer Zög Senior Veterinarian (Liaison Officer) General Directorate of Protection and Control Ministry of Agriculture & Rural Affairs Akay Cad. No. 3 Bakanliklar, Ankara Turkey Tel/fax : 90-312-4174176 / 90-312-4178209 e-mail : zaferz@kkgm.gov.tr

Dr Liesbeth Jacobs Project Manager Cedi-Diagnostics BV P.O. Box 2271 8203 AG Helystad The Netherlands Tel/fax: 31-320-238426 /31-320-214379 e-mail: c.e.jacobs@id.dlo.nl

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Dr Naci Bulut Chief of the Serology Laboratory Diagnosis Department Tel/fax : 90-312-2873600-181 / 2873606 e-mail : nacibulut@hotmail.com

Dr Musa Arik Head of Animal Health Department General Directorate of Protection and Control Ministry of Agriculture & Rural Affairs Akay Cad. No. 3 Bakanliklar, Ankara Turkey Tel/fax : 90-312-4182436 / 90-312-4178209 e-mail : musaa@kkgm.gov.tr

Dr Sükran Yilmaz Biologist Cell & Virus Bank Department Tel/fax: 90-312-2873600 / 90-312-2873606 e-mail: sukranyilmaz@hotmail.com

Dr Ismet D. Gürhan Department of Bioengineering Ege University Faculty of Engineering 35100 Boronova - Izmir Turkey Tel/fax : 90-232-3884955 / 90-232-3884000 e-mail : ismetgurhan@hotmail.com

Dr Fuat Özyörük Chief, Typing Laboratory of FMD Tel/fax : 90-312-2873600 / 90-312-2873606 e-mail : fuat@vetmed.wsu.edu Other staff of the SAP Institute:

Prof. Dr Ersin Istanbulluoglu Local Consultant Angora Evleri Turk Mavisi Sokak. A6-1 Beysukent Ankara Turkey Tel/fax: 90-312-2251320 / 90-312-2251320 e-mail: eistan@superonline.com

Dr Hüseyin Zengin, Director Dr. Gülhan Aynagöz Dr Nedret Çelik Dr Abdulnaci Bulut Dr Ünal Parlak Dr Hidayet Bozoğlu Dr Can Çokçalişkan Dr Nilgün Özdural Dr Ümit Şengül Dr Aydin Çoşkuner Dr Mehmet Karakaya Dr Ergül Uzunlu Dr Taibe Arsoy Dr Tuncer Türkoğlu

FMD Institute / SAP Institute PK 714 06044 Ankara, Turkey Dr Sinan Aktas Deputy Director Head of Diagnosis Department Tel/fax : 90-312-2873600-201 / 2873606 e-mail : aktass@hotmail.com

Dr Gareth Davies Veterinary Consultant Zinnia Kettlewell Hill Woking GU21 4JJ Surrey UK Tel/fax : 44-1483-772042 e-mail : garethdavies@intonet.co.uk

Dr Şeref Akyüz Chief of the Cell Culture Laboratory of The Production Department Tel/fax : 90-312-2873600-244 / 2873606 e-mail : serefakyz@hotmail.com

Dr Tim Doel Site Manager, Merial Animal Health Limited Ash Road, Pirbright Woking GU24 0NF Surrey UK Tel/fax : 44-1483-238111 / 44-1483-238102 e-mail : tim.doel@merial.com

Dr Gülhan Aynagöz Head of Control Department Tel/fax : 90-312-2873600-234 / 2873606 e-mail : gulhanaynagoz@hotmail.com

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Dr Ahmed Daoud Biogenesis El Sekka, El Beida Abbassia, Postal Code 11381 P.O. Box 131 Cairo Egypt Tel/fax : 2-02-4821009 / 2-02-6858321 e-mail : svri@idsc.gov.eg

Dr Mark Bronsvoort University of Liverpool Leahurst Neston CH64 0TT UK Tel/fax : 44-0151-7946027 / 44-0151-7946028 e-mail : bmdecb@liverpool.ac.uk Prof. Kenton Morgan Professor of Epidemiology University of Liverpool Leahurst, Neston CH64 0TT UK Tel/fax : 44-0151-7946027 / 44-0151-7946028 e-mail : K.L.Morgan@liverpool.ac.uk

Dr Hossam Fawzy El Sekka, El Beida Abbassia, Postal Code 11381 P.O. Box 131 Cairo Egypt Tel/fax : 2-02-4821009 / 2-02-6858321 e-mail : svri@idsc.gov.eg

Dr Michael Thrusfield Department of Veterinary Clinical Studies University of Edinburgh Royal (Dick) School of Veterinary Studies Easter Bush Veterinary Centre Roslin, Midlothian EH25 9RG UK Tel/fax: 44-131-6506223 / 44-131-6506588 e-mail : m.thrusfield@ed.ac.uk Other observers :

Dr Kenichi Sakamoto Chief of Diagnosis Laboratory National Institute of Animal Health 6-20-1 Josuihoncho Kodaira 187-0022 Japan Tel/fax : 81-423-211441 / 81-423-255122 e-mail : skenichi@affrc.go.jp

Dr Eliana Nora Smitsaart Biogenesis Ruta Panamericana km. 38.2 Garin (B 1619 IEA) Provincia de Buenos Aires Garín Argentina Tel/fax : 54-3327-448355 / 54-3327-448324 e-mail : esmitsaart@biogenesis.com.ar

Dr Kazuo Yoshida Chief of Epizootiology Laboratory National Institute of Animal Health 6-20-1 Josuihoncho Kodaira 187-0022 Japan Tel/fax : 81-423-211441 / 81-423-255122 e-mail : yoshidak@affrc.go.jp

Dr Alfonso Clavijo Head, Foot-and-Mouth Disease Unit National Centre for Foreign Animal Diseases 1015 Arlington St, Suite T2300 Winnipeg, Manitoba R3E 3P6 Canada Tel/fax : 204-7892047 / 204-7892038 e-mail : aclavijo@inspection.gc.ca

WRL : Dr David Paton Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-231012 / 44-1483-232621 e-mail : david.paton@bbsrc.ac.uk

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Dr Melvyn Quan Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-232441 / 44-1483-232448 e-mail : melvyn.quan@bbsrc.ac.uk

Prof. Soren Alexandersen Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-231025 / 44-1483-232448 e-mail : soren.alexandersen@bbsrc.ac.uk Mr Scott Reid Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-232441 / 44-1483-232621 e-mail : scott.reid@bbsrc.ac.uk

Dr Robert Armstrong Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-231441 / 44-1483-232448 e-mail : robert.armstrong@bbsrc.ac.uk Dr Ciara Murphy Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-231441 / 44-1483-232448 e-mail : ciara.murphy@bbsrc.ac.uk

Dr Nigel Ferris Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-232441 / 44-1483-232448 e-mail : nigel.ferris@bbsrc.ac.uk

EU/EC : Dr Zhidong Zhang Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-232441 / 44-1483-232448 e-mail : zhidong.zhang@bbsrc.ac.uk

Dr Alf-Eckbert Füssel DG SANCO/E2 Animal Health, Welfare and Zootechnics Rue Froissart, 101, 3/64 B-1049 Brussels Belgium Tel/fax : 32-2-2950870 / 32-2-2953144 e-mail : alf-eckbert.fuessel@cec.eu.int

Dr Neeraj Aggarwal Pirbright Laboratory Institute for Animal Health Ash Road Pirbright, Surrey GU24 0NF UK Tel/fax : 44-1483-231161 / 44-1483-236430 e-mail : neeraj.aggarwal@bbsrc.ac.uk

Professor Reinhard Ahl Chairman of the Scientific Sub-Committee for Animal Health of the EU-Commission Paul Ehrlich Strasse 28 D-72075 Tübingen Germany e-mail : RFH.Ahl@t-online.de

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Merial SAS Dr Philippe Dubourget Adjoint, Direction Grandes Prophylaxies Merial 29 Avenue Tony Garnier BP 1723 69348 Lyon Cedex 07 France Tel/fax : 33-4-72723072 / 33-4-72723181 e-mail : philippe.dubourget@merial.com

IZS Dr Emiliana Brocchi Reparto Biotecnologie Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna Via Bianchi 9 – 25124 Brescia Italy Tel/fax : 39-30-2290310 / 30-30-2290369 e-mail : ebrocchi@bs.izs.it

Bayer – AG Dr. Joachim Grunmach Bayer AG, D-51368 Leverkusen Bayer Health Care Animal Health Operations Quality Assurance Osterather Str. 1a D-50739 Cologne Tel/fax: +49 221 1778 270 / +49 221 1778 299 E-mail: joachim.grunmach.jg@bayer-ag.de

OIE Dr Alejandro A. Schudel Head of the OIE Scientific Technical Department OIE 12, rue de Prony – 75017 Paris France Tel/fax: 33-1-44151882 / 33-1-42670987 e-mail: a.schudel@oie.int

Bommeli Diagnostics Dr Lucas Schalch General Manager Stationsstr. 12 CH-Liebefeld Bern Switzerland Tel/: 41-319706267 e-mail : lucas.schalch@intervet.com

AOAD Dr Akel Mansour Director, Projects Department Arab Organization for Agricultural Development 7 Amarat Street P.O. Box 474 11111 - Khartoum Sudan Tel/fax : 249-11-472176 / 249-11-471402 e-mail : aoad@sudannet.net

United Biomedical, Inc. Dr Scott Liu Director of Regulatory Affairs Hauppauge New York, USA Tel/fax : 1-631-2732828 / 1-631-2731717 e-mail : sliu@unitedbiomedical.com

Dr Elsayed Elowni Arab Organization for Agricultural Development 7 Amarat Street P.O. Box 474 11111 – Khartoum Sudan Tel/fax : 249-11-472176 / 249-11-471402 e-mail : aoad@sudannet.net

Dr Alan Walfield Director, Patent & Licensing Affairs 25 Davids Drive Hauppauge New York, USA Tel/fax : 1-631-2732828x1119 / 1-6312731717 e-mail : awalfield@unitedbiomedical.com

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FAO Animal Production and Health Division FAO Viale delle Terme di Caracalla Rome, Italy Fax no : 39-065705-5749 Secretariat Dr Keith Sumption Secretary, EUFMD Animal Health Service Tel : 39-065705-5528 e-mail : keith.sumption@fao.org Ms Egiziana Fragiotta Administrative Clerk, EUFMD Animal Health Service Tel : 39-065705-2637 e-mail : egiziana.fragiotta@fao.org

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