Open session of the standing technical committee of the EUFMD- 2007

REPORT

CAIRO, EGYPT, 16-19 October 2007

EUROPEAN COMMISSION FOR THE CONTROL OF FOOT-AND-MOUTH DISEASE

Session of the Research Group of the Standing Technical Committee

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EUFMD Research Group - Closed Session 16-18th October Cairo, Egypt

OPENING .......................................................................................................................... 5 ITEM 1 ADOPTION OF THE AGENDA AND ELECTION OF THE CHAIRPERSON ..................... 5 SECTION 1: ITEMS UNDER THE REMIT OF THE INTERNATIONAL SURVEILLANCE AD HOC GROUP.............................................................................................................................. 6 ITEM 2. INTERNATIONAL FMD SURVEILLANCE ................................................................. 6 ITEM 3. TECHNICAL ISSUES ARISING FROM OUTBREAKS IN THE UK IN 2007.................. 8 SECTION II: ITEMS UNDER THE REMIT OF THE LABORATORY STANDARDIZATION AND BIOSAFETY AD HOC GROUP.............................................................................................. 9 ITEM 4. PROMOTION OF MOLECULAR TESTS AS THE FIRST CHOICE METHOD FOR FMD DIAGNOSIS, AND ON REDUCING FALSE NEGATIVE FINDINGS IN TURKEY AND IRAN ...... 9 ON FURTHER DEVELOPMENT OF REAL-TIME RT-PCR (RTRT-PCR) FOR FMD CONFIRMATION IN THIRD COUNTRIES AND IN EUROPE ................................................................................................................... 9 POSITION ON DECENTRALISED TESTING FOR SECONDARY CASES OF FMDV IN NORMALLY FREE REGIONS .............. 9 REDUCING FALSE NEGATIVE RATES IN FMD CONFIRMATION IN IRAN AND TURKEY .......................................10 ITEM 5. FMD LABORATORY BIOCONTAINMENT............................................................... 11 SECTION III: ITEMS UNDER THE REMIT OF THE AD HOC GROUP ON SURVEILLANCE AND SERO-MONITORING ISSUES ........................................................................................... 12 ITEM 6. POST-VACCINATION SURVEILLANCE (PVS) IN FMD FREE COUNTRIES OR POPULATIONS APPROACHING DISEASE FREEDOM ......................................................... 12 6.1 TECHNICAL ISSUES REQUIRING SOLUTIONS IDENTIFIED AT THE PVS WORKSHOPS ..................................12 6.1.1REVIEW OF MAIN CONCLUSIONS OF THE FIRST TWO WORKSHOPS ...................................................12 6.1.2 VIEW POINT ON RESOLUTION OF REMAINING TECHNICAL ISSUES WITH PVS FOLLOWING EMERGENCY VACCINATION ....................................................................................................................13 6.2 PROGRESS WITH USE OF SCENARIO TREES TO OPTIMISE SURVEILLANCE FOR CONFIDENCE IN DISEASE FREEDOM 13 6.3 NSP TEST PERFORMANCE ISSUES.........................................................................................13 ITEM 7 ISSUES ARISING FROM MONITORING OF PREVENTIVE VACCINATION PROGRAMS IN NON-FREE COUNTRIES .............................................................................................. 15 STABILITY OF TYPE O TITRES AFTER VACCINATION (NACI BULUT) .........................................................15 STANDARDISATION OF SERO-MONITORING RESULTS; DRAFT REPORTING FORMAT (CARSTEN POTSZCH) ..............15 MODELLING POPULATION IMMUNITY AFTER VACCINATION CAMPAIGNS : CONCEPT PAPER (ARNAUD LE MENACH, FAO) ..............................................................................................................................15 ITEM 8 FINAL PAPERS –INFORMATION SESSION ........................................................... 16 SECTION IV: OTHER ISSUES........................................................................................... 17 ITEM 9. VACCINATION OF SHEEP: UPDATED GUIDANCE PAPER , PREVENTIVE AND EMERGENCY SITUATIONS............................................................................................... 17 ITEM 10 .GROUP WORK TO DEVELOP CONCLUSIONS, RECOMMENDATIONS AND ACTIONS ............................................................................................................................. 18 ITEM 11. READING OF THE REPORT................................................................................ 18 ITEM 12. OTHER TASKS HELD OVER THE THE 2005-7 WORKPLAN .................................. 18 ITEM 13 PLANNING OF THE OPEN SESSION ................................................................... 18 CLOSING CEREMONY ...................................................................................................... 18

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LIST OF APPENDIXES Page Appendix 1 ………………………………………………………………………………………………………………… 19 List of participants Appendix 2 ………………………………………………………………………………………………………………… 22 Agenda

Appendix 3 ………………………………………………………………………………………………………………… 23 Recent epidemiological events in the European neighbourhood in 2006-07. Keith Sumption

Appendix 4 ………………………………………………………………………………………………………………… 30 Evaluation of the role of sheep and goat in transmission of FMD to Cattle. Vahid Otarod

Appendix 5 ………………………………………………………………………………………………………………… 34 Investigation of molecular epidemiology of FDM virus serotype O and A in Iran in 2006-07. Nazem Shirazi

Appendix 6 ………………………………………………………………………………………………………………… 46 Review of FMD wildlife in Israel. Hagai Yadin, Boris Gelman and Rony King

Appendix 7 ………………………………………………………………………………………………………………… 49 FMD UK. Overview of outbreaks and laboratory involvement. David Patton

Appendix 8 ………………………………………………………………………………………………………………… 55 UK outbreaks 2007: Confirming sequencing results.

Soren Alexandersen

Appendix 9 ………………………………………………………………………………………………………………… 58 Application of real-time RT-PCR for FMD virus.

A. Dekker

Appendix 10 ……………………………………………………………………………………………………………… 60 FMD levels. Summary table of in vivo FMD outputs and laboratory produced levels from live virus handling.

Soren Alexandersen

Appendix 11 ……………………………………………………………………………………………………………… 65 Epizone, EuFMD & EU coordination action FMD-CSF. Kris De Clercq

Appendix 12 ……………………………………………………………………………………………………………… 69 Use of a model to predict expected prevalence of carriers and to design serosurveillance for their detection.

Mark Arnold, John Wilesmith, David Patton, Eoin Ryan, Sarah Cox, Pirbright

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Page Appendix 13 …………………………………………………………………………………………………………………73 Quantitative evaluation of surveillance systems.

Tom Murray

Appendix 14 …………………………………………………………………………………………………………………77 3ABC Elisa in the African context.

F. MAree, B. Blignaut, J. Esterhuysen, L. Heath, W. Vosloo

Appendix 15………………………………………………………………………………………………………………… 81 Use of NSP tests in areas where SAT-type FMD viruses are prevalent: Consultancy report. G. Thomsom

Appendix 16 ……………………………………………………………………………………………………………… 84 FMDV NSP tests, summary of key performances.

Emiliana Brocchi

Appendix 17 …………………………………………………………………………………………………………………87 Comparative evaluation and validation of NSPEs. Donal Sammin.

Appendix 18 …………………………………………………………………………………………………………………89 Problem on stability after the vaccination to type O virus.

Naci Bulut

Appendix 19 …………………………………………………………………………………………………………………92 Standardization of sero-surveillance results?

Draft reporting format?. C. Potzsch

Appendix 20 …………………………………………………………………………………………………………………95 Modelling disease spread under various control strategies. Arnaud Le Menach

Appendix 21 …………………………………………………………………………………………………………………99 Progress report on experimental infections in dromedary and Bactrian camels. S. Alexandersen

Appendix 22 …………………………………………………………………………………………………………………102 Attempt for detection of FMD in wildlife by infra red camera. hagai Yadin.

Appendix 23 …………………………………………………………………………………………………………………104 Vaccination of sheeps; revision of guidelines.

Keith Sumption

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REPORT OPENING A Session of the Research group of the Standing Technical Committee of the EuFMD was held in Cairo, between 16 and 18th October 2007, and jointly hosted by the General Organization of Veterinary Services (GOVS) of the Ministry of Agriculture and land reclamation, Government of Egypt, and by the FAO Regional Office for the Middle-East (FAORNE).

The Session was opened by Mr Ahmed Chikhaoui, on behalf of Mr Albraithen, Regional representative of FAO for the Middle-East, Acting FAO Representative for Egypt, and by Dr Hamed Samaha, Chairman of the GOVS. Eleven of the twelve Committee members were present, plus the ex-officio member for the World Reference Laboratory (Dr Paton). Those present were Kris de Clercq (KdC), Aldo Dekker (AD), Emiliana Brocchi (EB), Bernd Haas (BH), Stefan Zientara (SZ), Hakan Vigre (HV), Donal Sammin (DS), Soren Alexandersen (SA), Naci Bulut (NB), Hagai Yadin (HY) and Georgi Georgiev (GG) .

Invited experts were present from South Africa (Dr Maree), Iran (Drs Otorod and Shirazi).

Observers were present from Australia (Dr Hammond) and Botswana (Dr Thomson). From FAO (EuFMD and FAO AGAH - Animal health Service) were the Secretary, Keith Sumption (KS), Tom Murray (TM), Carsten Potzsch (CP), Francis Geiger (FG), and Arnaud le Menach (FAO-AGAH), and Dr Hassan Aidaros (Manager, FAO/OIE Regional Animal Health Centre, Beirut).

Apologies were received from Mark Bronsvoort, member RG (MB), and Alf Fuessel, DG-SANCO (EC Observer).

The list of participants is found in Appendix 1.

Item 1. Adoption of the Agenda and election of the chairperson The Agenda adopted with minor changes, as given in Appendix 2.

Following the decision of Dr de Clercq to not stand again for the position of Chairman, an election was held, as required by the EuFMD Constitution at the first meeting of the Committee after their election by the previous EuFMD Session (the 37th General Session April 2007).

The Secretary called for nominations from the membership; one nomination was received, of Dr Aldo Dekker, (proposed by Dr de Clercq, seconded by Dr Sammin). There being no other nominations, Dr Dekker was elected as Chairman and indicated his acceptance of the responsibilities. In his response, he proposed a vote of thanks for the leadership and effort of Dr de Clercq, which was enthusiastically supported by all present. Although not in the constitution for the Commission, he asked if Dr de Clercq was prepared to continue as supportive role (ViceChairman),in case of his unavailability, this was accepted.

Chairman of the Group, 2007: Dr Aldo Dekker.

Vice-Chairman, 2007: Dr Kris de Clercq.

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SECTION 1: ITEMS UNDER THE REMIT OF THE INTERNATIONAL SURVEILLANCE AD HOCGROUP Item 2. International FMD surveillance Four papers were presented relating to the FMD situation in the European neighbourhood, excluding the situation in the United Kingdom; an overview of the recent epidemiological events in 2006-7, presented by Keith Sumption (Appendix 3); a paper on the issue of FMD in sheep and goats in Iran (Appendix 4) and on the molecular epidemiology of FMD types O and A in Iran (Appendix 5), and a review of FMD type O epidemic in Israel in 2007 (Appendix 6). Analysis of the situation in Iran had been assisted by geo-referencing epidemiological units and analysis of FMD and vaccination at this level. Capacity for rtRT-PCR has been established at the CVL, Iran, and studies on type A and O molecular epidemiology conducted during a 6 month study tour at the WRL. The regional situation with the A Iran 05 virus genotype had much improved but appeared to be entering a phase where circulation was continuation with low level of outbreaks in Turkey and Iran, possibly preceding extinction in small or larger areas. However, the situation with type O PanAsia II genotype over the past year had been severe, with incursion into most of the ME, and recent spread to central Asian countries. The genotype appeared to have replaced the resident type O in Turkey and now accounted for almost all outbreaks, including those in Thrace in September 07.

Last year type A Iran 05 was main problem, but this year it is O PanAsia type II. It is likely that as population immunity from infection wanes, then replacement by a different serotype occurs.

Vaccination level at less than 20% in cattle is probably not sufficient to change this situation.

Geographic distribution is type A from the East with type O all over.

The 2006-7 type O epidemic in Israel, had as in previous major epidemics, affected wild boar and gazelles which were shown to become infected. Their role in perpetuating domestic outbreaks remains uncertain but given the locations, it is also possible they play a role in transboundary spread, since wild boar may tunnelling under border fences, and possibly aerosols could be sufficient for short range airborne transmission to wild or domestic species.

A collaborative study with DHS (US) and EuFMD support has evaluated the role of thermal imaging for detection of febrile cattle at distance, during the FMD epidemic; this could be achieved up to 30 metres distance.

The situation in Egypt was also discussed, where FMD type A had been confirmed by the local (Abbasia) vaccine research laboratory in samples collected from outbreaks in September 07, and following an apparent break of more than 12 months since previous type A outbreaks. At the time of the Session the virus type had not been confirmed in samples sent to the WRL. Vigorous discussion followed, on: • the issue of vaccine suitability, r-values and their interpretation, and performance of O Manisa vaccine in the field against the O PanAsia II virus; • the apparent drift in FMDV type A from Afghanistan from the A Iran 05 cluster, with reduced match top A22 Iraq and other type A vaccine strains; • there is a paucity of data on the situation in regions outside the ones discussed above, e.g.

South-America; • countries should try to get more information on the quality of the vaccine they use, e.g. by vaccinating small groups (e.g. 10-15 cattle) of animals and collecting approximately 250 ml of serum three weeks post-vaccination to be tested in various laboratories.

Conclusions 1. Access to data is required for risk assessment, therefore sharing of the information with those that require it is important. Although the information obtained is better than in the past, there is still room for improvement on the quality of the data provided to the regional laboratories (data capture) and the data outputs (display).

2. There is a need for a regional ecosystem based (Eurasia, Middle-east and Africa) analysis of virus spread and risk, linking outbreak and laboratory data there might be a role for regional

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3.

4.

5.

6.

7.

animal health centres of OIE/FAO. This would probably need more data, especially on husbandry systems.

Further work is needed to establish the role of sheep and goats in FMD epidemiology in different ecosystems. It is likely small ruminants play a more important role in epidemiology when clinical disease is present in large ruminants. It is probably wise to include smallruminants in emergency vaccinations around outbreaks. Detailed studies in Turkey and Iran are required as the benefit to cost of vaccination of small ruminants is unclear.

Infrared cameras may prove useful to detect clinical disease in extensively kept animals.

The level and duration of protection of commercially available type O vaccines against the O PanAsia II virus strain remains unclear.

More information on antigen quantity in each dose is required to assess the apparent limited protection obtained with the three component type O vaccine used in Israel.

The relative efficacy of monovalent and multivalent vaccines against a particular challenge strain should be further evaluated.

Recommendations 1.

2.

3.

4.

A minimum standard of information should be provided with samples submitted to national and international (WRL and other) reference laboratories.

Guidance to countries is required on the definition of epidemiologically significant events that should be reported; A web-based sequence database for shared access is urgently required, and the ad hoc group should identify how this can be achieved and the suitability of the systems under development for use in risk assessment; A teleconference between the EUFMD secretary, the Research Group Chairman and WRLFMD should immediately follow the issue of WRLFMD quarterly reports, to review findings that may indicate changing level of risk and require follow up actions.

Papers to be prepared by the ad hoc group 1. Minimum standards for information to be provided with samples submitted to national and international (WRL and other) reference laboratories EUFMD member countries and for use in programs supported by EUFMD/EC [Action point: delivery 12/07]; 2. Definition and reporting of epidemiologically significant events: guidance paper, based on criteria developed under the EUFMD/EC/IVO program in Iran [Action point: delivery 12/07]; 3. Paper on the risk of spread from wild boar in the Israeli circumstances (SA and HY); 4. Report on the use of thermal imaging camera for use in extensively kept cattle for the purpose of detection of febrile animals (HY, to be reviewed by DP); 5. Complete the review of duration of immunity after type O vaccination (N Bulut and AD), to include the antibody decline by VNT, and the data should be analysed with linear mixed effect model. (by 12/07).

Further, serum from the trials with ARRIAH vaccine, and or other type O vaccine used in the neighbourhood should be tested by the SAP Institute or RL partners for inclusion in a more complete review (CP, EB and NB).

Recommendations specifically relating to type O control in Turkey The Turkish authorities are recommended to: 1. Undertake an urgent study on increasing biosecurity measures to prevent movement of infection from animal markets in Istanbul to European Thrace [GDPC/EuFMD/EC program]; 2. Monitor immunity in Thrace and Anatolia after autumn vaccination 2007 and subsequent campaigns (SAP Institute/GDPC), and continue NSP sero-monitoring in Thrace with assistance of the ad hoc group of the EuFMD Research Group in design; 3. Study the effect of homologous vaccination on the duration of O Manisa immunity in a large group of animals by vaccinating half of the animals with monovalent type O vaccine; 4. Review the current timing of the vaccination campaigns, to take into account the duration of immunity and the need to achieve maximum immunity before the main periods of risk, particularly the Kurban Bayram festival; 5. Introduce booster vaccination of young animals 6 – 8 weeks after the primary vaccination, at least in regions which are prioritised for FMD control. The priorities should take into consideration the role of young animals in spread between breeding and finishing areas of Turkey;

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6. If the immunity in population is insufficient to achieve the intended purpose a vaccination schedule which results in a better immunity should be introduced.

The CRL should 7. complete a cross-immunity test (EurPh potency test) of O Manisa vs O PanAsiaII challenge, including assessment of the homologous potency based on serology.

The research group should 8. continue work on improving surveillance [including confidence in freedom and optimization of surveillance actions to achieve]; 9. through the ad hoc group on international surveillance, assess the significance of new strains (antigenic and genetic developments, revision of use of r value readout) and advise the EU and vaccine companies on the need for development of new vaccine strains; 10. convene a subgroup with specific interest in the technical support to Turkey, to respond to issues raised by the Turkish authorities through the national FMD task force or by EuFMD or EC; 11. be as a group provided with more information on a regular basis on the status and plans of the EC funded FMD control project in Turkey.

Item 3. Technical Issues arising from outbreaks in the UK in 2007 Two papers were given, an overview of the Pirbright laboratory involvement in surveillance and outbreak investigation, given by David Paton (Appendix 7), by Soren Alexandersen (Appendix 8) on the results of independent full length sequencing of the FMDV genomes as part of the postoutbreak investigation.

Discussion The papers provided detailed insight in the sequence of the outbreak, still is not possible to track the escape and route to the first outbreak from the Pribright Laboratories. All the possible escape mechanism were considered unlikely, e.g. flooding would dilute the virus very much and the first infected premises were upstream, and the pH treatment of the centrifuge waste might have been inadequate, still most of the virus should be inactivated. The incident, however shows that the building concept with different containment buildings on site connected with piping is far from ideal.

The outbreak showed that the assumption that clinical disease will be notified in cattle and pigs is not always true; it depends on the expertise of the owner and the frequency of contact. Notification is reliable in dairy cattle, but less reliable in extensively kept fattening cattle.

In the laboratory lateral flow devices (strip tests) were evaluated and showed valuable in detecting FMD virus infection.

Conclusions 1. The group agrees with conclusions of earlier independent inquiries that the source of the UK 2007 outbreaks was escape of FMDV from the Pirbright site.

2. Far greater attention is needed in contingency plans to the system to be applied to detect infection in extensively kept animals, to ensure early detection (e.g. by requiring a daily inspection by the farmer).

3. Evidence of spread of infection to just outside 10 km surveillance zones raises question of need for larger zones, which could be termed an observation zone, especially given the damage resulting from re-imposition of national standstills.

4. Lateral flow test devices for FMDV detection were demonstrated to be useful to provide rapid diagnosis on farm and in the NRL.

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Recommendations 1. Veterinarians conducting surveillance for clinical FMD must undertake examination of the mouth of each animal under surveillance, following a standard protocol, and be given the facilities to undertake this. If not possible, routine blood testing for viraemia and antibody response must be introduced; 2. Countries should re-evaluate their national FMD contingency plans and allied operational manuals to address the issue of detection of infection in husbandry systems where there is a low level of owner inspection and an expected problem in clinical inspection; 3. The issue of mutual assistance in case of inability of an NRL in Europe to undertake diagnosis, e.g. due to fire, flood or regulatory non-compliance, should be addressed at the CVO level (EUFMD Executive) as well as through development of mutual agreements between NRLs; 4. Further evaluation needed of cost-benefit for preclinical diagnostic tests e.g. large-scale RTPCR on blood or other samples.

Papers to be completed 1. Recommended procedure (standard) for clinical examination and recording of findings during veterinary surveillance visits for detection of clinical FMD (DS: for the report: 1-2 pages, by end October); 2. Draft contract or form of agreement whereby a network of FMD labs in Europe could agree to provide services to NRLs that are temporarily incapacitated; 3. Review paper on the size and purpose of zones established for the purpose of FMD control (protection, surveillance and additional zones) [review at the November Executive; complete

by June 2008].

SECTION II: ITEMS UNDER THE REMIT OF THE LABORATORY STANDARSIZATION AND BIOSAFETY AD HOC GROUP Item 4. Promotion of molecular tests as the first choice method for FMD diagnosis, and on reducing false negative findings in Turkey and Iran On further development of real-time RT-PCR (rtRT-PCR) for FMD confirmation in third countries and in Europe Dr Sumption introduced the item, illustrating the number of countries in the Middle-east and South Asia which now had real-time RT-PCR for HPAI conformation. Several countries had requested FAO to provide training and support to introduce RT-PCR for FMD and other pathogens, mainly to make efficient use of the new capacity but also to address the problem of low sensitivity in other methods (immunocapture ELISA) and biosafety (virus isolation).

Dr Dekker had been requested to provide a guidance paper (Appendix 9) for EuFMD/FAO to assist training and support for laboratories wishing to adopt or evaluate RT-PCR for FMDV diagnosis, including the issue of external quality assurance and harmonisation of test performance.

Discussion The main issues discussed were: sustainability of rtRT-PCR, there being several incidences where tests for HPAI had been established in third countries but per test costs were prohibitively expensive; quality control issues, particularly the use of internal controls to detect possible false negative extractions and amplifications; primer design and risk of false negatives as sequences evolve; and the issue of performance standards versus standardized procedures. Several countries in Europe were involved in decentralizing animal disease confirmation to lower biosecurity labs at sub national level and therefore the need for performance testing of these services within countries was a precedent for a similar international QA for newly established rtRT-PCR capacity in the European neighbourhood.

Position on Decentralized testing for secondary cases of FMDV in normally free regions This issue arose out of the earlier work on pen-side diagnostic tests and their role in FMD diagnosis in relation to laboratory based services normally provided by an NRL in Europe. Donal Sammin provided a summary of key points to be included in the guidance or position paper (Appendix 10).

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Reducing false negative rates in FMD confirmation in Iran and Turkey Keith Sumption summarized the concerns of the Secretariat and Executive Committee that reports of FMD outbreaks in Turkey and Iran in the past year included a high proportion of outbreaks defined on clinical grounds which had not been confirmed by laboratory tests. However, when comparing the results in Turkey with those in other countries the detection rate was not low. Still, the reporting of suspected FMD cases has to be encouraged to keep the percentage of unconfirmed outbreaks at the current level, otherwise it could lead to the range of epidemic events associated with late detection.

The reasons for negative results in suspected cases included late notification, late stage lesions, and treatments of the lesions that would lead to failure to isolate virus, but of course other infections that caused the lesions. Therefore, the protocol for investigation should include active search for early cases, and repeated serology to look for seroconversion in a herd, to reduce the risk of false negative herd investigations. Plans for improving sample collection procedures were in place for introduction into the instructions to field vets in 2008. The situation in Iran was also described by Dr Otorod to be similar.

It was agreed that problems were multiple, involving late notifications, poor sampling, transportation issues and test sensitivity; a review in each country is needed before proposing solutions. It was also agreed that introduction of new test procedures (including pre-clinical diagnosis) offered new opportunities to improve conformation rates.

Conclusions 1. Proficiency testing has shown that between lab variation with RT-PCR (either real time or classical) is lower than with VI and therefore use of the method is encouraged; 2. Several RT-PCR tests have been described, each with its own advantages and disadvantages, therefore it is not possible to prescribe one single RT-PCR test for FMDV; 3. There are limiting factors in the application of real-time RT-PCR in third countries that affect sustainability (e.g. costs of reagents, power supply during process, and contamination control) and therefore countries that elect for using real-time RT-PCR should retain a backup confirmation test; 4. Real-time RT-PCR has a higher analytical sensitivity than conventional RT-PCR for FMD; 5. There is no validated RT-PCR available that can differentiate between serotypes, therefore in countries where more than one type is present or suspected to be circulating it is essential to type the isolate with a another RT-PCR or by virus isolation combined with serological techniques; 6. An internal control may be useful to rule out false negatives; 7. There can be discrepancies between results of RT-PCR and other diagnostic techniques, which can be difficult for authorities, and training in the method must assist the countries to develop a clear process for decision making following RT-PCR results; 8. Quality control procedures are very important for reproducible test performance, e.g. batch control of ingredients; 9. Multiplex testing can be useful for differential diagnosis, but there is often a reduction in sensitivity for a specific agent. Work on internal controls shows that the reduction in sensitivity can be minimised; 10. Test performance of lateral flow devices evaluated in a laboratory setting indicates that they can play a useful role in secondary cases. However, not all serotypes are detected with sufficient sensitivity; one such device did not detect SAT types but detected approximately 86% of the samples containing A, O and Asia-1 serotypes. The other device had the same sensitivity for these serotypes, but also detected some of the SAT types.

Recommendations: 1. Sequences in the target regions should be studied by the WRL and NRLs in endemic regions and the international recommended primers kept under review (yearly update by the CRL/WRL); 2. The international FAO/CRL EQA system involving proficiency panels should be continued and should assist the harmonisation of performance of real-time and classical RT-PCR in the hands of the NRLs; 3. Each NRL using RT-PCR should maintain a back-up protocols to be used to confirm a negative result on samples from strongly suspected FMD cases; 4. Whatever test is applied it should be validated in the laboratory and should achieve a basic level of performance as identified in proficiency tests. The same applies for multiplex tests;

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5. Companies are encouraged to produce standardized kits with primers and probes for FMDV, as currently can be purchased for other diseases such as Avian Influenza, for all possible platforms.

6. Further studies are encouraged to finalize the standardization of serological tests with respect to protection afforded by vaccines against homologous challenge (a study on O Manisa serological responses is part of the FMD Improcon project).

Actions /papers 1. Position paper on the use of RT-PCR for the diagnosis of FMD (AD 1 December 2007) 2. Position paper on the options of decentralized testing (DS 1 December 2007) 3. Analysis of the reasons for low confirmation rates in Turkey and Iran (CP, ZZ and NS by 1 December 2007) and proposed solutions; 4. Sampling instructions for field veterinarians to collect samples with the primary biocontainment at the point of sampling for use in RT-PCR (SZ or BH).

Item 5. FMD laboratory biocontainment Dr Haas introduced this item, as Chairman of the working group (under the Research Group) on FMD laboratory biosecurity. The “Minimum Standards for FMD Laboratories” adopted by the EuFMD Commission in 1993 remained the reference text for the Council Directive on FMD ad therefore the revision of this document fell in the mandate of the EuFMD Commission and inter alia, the research group of the Standing Technical Committee.

A working group had been convened in September 2007 to review the 1993 standards, comprising biosafety officers of six of the licensed laboratories to handle FMDV in Europe for diagnostic, research and vaccine production purposes. In addition the group included a biocontainment expert with expertise in air filtrations, a representative of the WRL (Dr Paton) and the EuFMD Secretary.

The first meeting had agreed that updating of the text was required, for several reasons: technical specifications of components such as air filters had changed; modern biosafety thinking places greater emphasis on documenting the approach taken in assessing and managing risks, and there is generally greater concern on the human element and the need to ensure FMDV risk in considered when laboratories have multiple use, for example where kits and reagents are produced for a range of animal pathogens.

The working group had taken on the updating of sections without proposing major changes to the overall layout of the document, except for introduction of new texts on risk assessment and management, including training.

Discussion The Chairman thanked Dr Haas for his effort, and that of the working group, to progress the updating of the document. Discussion followed the suitability of the OIE requirements for biocontainment for application in endemic areas, where the requirements for handling live FMD do not take into account the status and epidemiologic circumstances, and the impact of escape of endemic strains of FMD is quite different from that in free countries. Members of the group felt that OIE requirements did not differentiate according to risk and therefore it is not surprising that many countries outside of Europe choose to work with FMD virus for diagnostic purposes (and also vaccine production) without fulfilling OIE requirements. The 4 levels used in the OIE code is not in line with the containment levels in human laboratories where the fourth level is for pathogens that can severly harm the person working with the agent and the physical containment requirements are more stringent than at level 3 to protect the person working with the agent. A containment level, in which escape of the agent to people or animals outside the laboratory and is needed when working with FMD, is consistent with a human containment level 3.

The question of the need for EuFMD standards on biocontainment was raised; the Secretary answered that this was really a matter for the European regulatory authorities (at the level of the Council) as the 1993 Standard was referenced in regulations, and until this was changed the EuFMD Commission and inter alia its technical committee were asked to regular review the Standard.

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Conclusions 1. The group agreed on the need to update the 1993 Standards, and to give the responsibility for this to a working group chaired by a member of the Research Group, and accountable to the Chairman for progress in this area.

2. The OIE requirements for laboratories handling live FMD are relevant, but should be reviewed with respect to the numbers of levels included, and consideration should be given to ensure that EuFMD standards are compatible with the OIE requirements, but the EUFMD standards should make clear where risk assessment can enable the safe application of non-live virus techniques in FMD diagnosis, for example in decentralised, secondary laboratories.

3. A working principle in the standard should be that laboratories have responsibility to validate the risk reduction measures they put in place, as part of the risk assessment and risk reduction approach.

4. The working group must consider if the scale of virus production, for example in vaccine production plants, requires a different requirement for risk management than for other situations.

Recommendations 1. The new minimum containments standard should include consensus terms used in the international terminology on biosafety, such as those included in the "Laboratory Biorisk Management Standard".

2. The OIE should be asked to reduce the number of biosafety levels, to be consistent with containment levels in human medicine.

3. The new minimum standard should include strengthened sections on risk assessment and on management.

4. The paper on minimum containment standards should be finalized by the ad hoc working work, and a decision taken on circulation for wider consultation by the Chairman of he research group together with the Chair of the EuFMD Commission and the Secretariat. The entire process of review and consultation should be complete before the Open Session in 2008.

5. The group should refer to the paper the "serology laboratory paper" and the concepts from OIE guidelines Biosafety papers to be completed 1. Revised position paper of minimum requirements for FMD laboratories (BH 31 December 2008) 2. Revised position paper, as an addition or addendum to the document on live virus facilities, to include the requirements for applying non-live FMD techniques in serology and for RT-PCR (BH 31 December 2008).

SECTION III: ITEMS UNDER THE REMIT OF THE AD HOC GROUP ON SURVEILLANCE AND SERO-MONITORNING ISSUES Item 6. Post-vaccination surveillance (PVS) in FMD free countries or populations approaching disease freedom Papers, viewpoints and progress reports on PVS were given by Kris de Clercq, David Paton, Tom Murray, François Maree, Gavin Thomson, Emiliana Brocchi and Donal Sammin.

6.1 Technical issues requiring solutions identified at the PVS workshops 6.1.1Review of main conclusions of the first two workshops Dr Kris de Clercq summarised the main conclusions from the first two simulation exercise workshops (Appendix 11) at which the surveillance approaches after emergency vaccination in relevant European scenario were selected and evaluated by country participants. A final Workshop (week of 22nd October) was planned after which a synthesis of findings and their implications would be needed. It was evident that the workshops had uncovered several areas of remaining difficulty, and a probable need for some changes in the regulatory standards (OIE and Council Directive).

It was agreed by the group that there must be follow on meetings to reach consensus on some points which constrain confidence in application of emergency vaccination/PVS and that the ad hoc

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group of the RG should take this forward with the partners involved in the PVS workshops. The key decision points or technical problems also need to be relayed to the CVO level, for example at the EUFMD executive in order that sufficient time and effort is placed to reach a resolution.

6.1.2 View point on resolution of remaining technical issues with PVS following emergency vaccination Dr David Paton outlined his thinking in a presentation (Appendix 12); in order to better design PVS, a modelling study was conducted to estimate the target prevalence of FMDV infected animals after an epidemics in which EV had been applied. In the model a 10 km ring vaccination was assumed. The model allowed for differences in risks between small herds and large herds, therefore the model predicted little benefit from vaccinating small herds. Whether risk for large holding are truly higher needs to be established, because large holdings often also have a higher standard bio security on the farm. The approach, however very nicely shows how modelling can be used in the planning phase.

6.2 Progress with use of scenario trees to optimise surveillance for confidence in disease freedom Dr Tom Murray, EuFMD, presented a progress report (Appendix 13) on the use of this approach to explore the contribution of sero-surveillance, test performance, and other surveillance activities to an overall level of confidence in freedom from FMD, using the example of Thrace region of Turkey, and using surveillance design for NSP reactors used in 2006 in this region.

The group agreed this was a interesting approach that required to be continued.

Conclusions: 1. NSP workshops have identified need for potential modification to the Council Directive 2003 and/or the OIE Code Annexes, on surveillance after emergency vaccination; 2. There is a need to replace the text and concept of “demonstration of freedom” to “substantiation of freedom from infection” in livestock populations, given the impossibility of the former, in texts with regulatory importance in which the purpose of PVS is indicated; 3. There remains a lack of detail in OIE Code and specific Annexes relating to the requirements for surveillance after emergency vaccination in normally free countries, which affects the decision making of counties in designing and implementing PVS; 4. There is a need to quantify confidence in freedom from infection following surveillance activities after emergency vaccination.

Recommendations 1. Countries performing surveillance should further develop the scenario tree approach as part of the effort to quantify the contribution of each part of an entire surveillance system to confidence in DF; 2. Relating to pigs, the requirement of the Council Directive 2003, currently to test all animals in PVS surveys, should be reviewed, with consideration given to replacement with a 95/5 survey; 3. The EU and OIE should consider making regulations allowing for the slaughter of individual animal slaughter versus herd slaughter, following a singleton or limited number of positive NSP test results. If only the NSP positive animals are slaughtered the risk from such a herd is will be vary low in a vaccinated area. Because all ruminants are tested approximately 85% of the carrier animals will be found and removed; 4. The Research Group should evaluate the literature on disease spread, to determine whether the assumption that small herds contribute less to disease spread is true. If this is true then national Contingency plans should consider and put into place a clear strategy for inclusion or exclusion of small herds in emergency vaccination programs, ahead of any decision to vaccinate, to reduce complications in the later PVS phase.

5. Criteria should be developed to assist countries to substantiate a claim of an effective level of herd immunity in the vaccinated herds and zones.

6.3 NSP test performance issues 6.3.1 Performance of NSP tests for use following SAT type infections

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Two papers were presented for discussion. In the first, Francois Maree (Appendix 14) highlighted the level of diversity in SAT viruses in regions which are utilised in the current NSP tests, with a focus on the 3ABC coding regions. Following experimental infection of non-vaccinated animals, the antibody response as measured by commercially available NSP kits had been of limited duration and with high variability between test systems, although the Ceditest was the most reliable test system currently available. He concluded that there exists a problem for selection and use of NSP test for regions affected by SAT viruses that requires to be resolved.

In the second, Gavin Thomson (Appendix 15), summarized a recent independent review that had been commissioned by the SADC-FMD project, which supported the above view point that currently available NSP test kits had not been shown to have a sufficient performance for use in PVS after SAT infection. He gave an overview of the purpose of NSP test use in areas affected by SAT viruses, particularly the policy issue of how to promote FMD control in populations of cattle in southern Africa that live in close proximity to cape buffalo, and not separated by fences, as increasingly is the trend in the proliferation of trans-frontier national parks across southern Africa.

In his view, a more sensitive test could perhaps be a test based on the 3D protein.

6.3.2 Key performance data for NSP tests; a performance summary for tests available for use in Europe Dr Emiliana Brocchi (Appendix 16) and Dr Donal Sammin (Appendix 17) provided a review of key information on test characteristics and test validation available for recently described NSP antibody tests for FMD. Only the six ELISAs that were compared during the Brescia workshop 2004 (FMD_ImproCon/EuFMD/EC) have achieved a validation level for application in cattle complying with the requirements of the OIE validation template.Comparative performance data of the six NSP tests were made available through a series of publications, with emphasis given to DSp and DSe evaluated for different FMD conditions, the most critical for PVS being the condition of vaccinated and infected animals in the time interval 28-100 days post exposure.

An overview of newly described NSP tests has been provided by EB, with a critical discussion of performance data reported in literature. For most of these tests, estimates of DSp are adequate, in contrast of estimates of DSe that are preliminary and incomplete. Comparability of new DIVA tests for FMD with the validated ones requires availability and testing of comprehensive panels of sera.

Conclusions 1. Insufficient studies have been completed on test performance (DSe and Dsp) with FMD vaccinated and infected sheep; 2. A review is needed to assess the relevance and importance of improving the test performance estimates for vaccinated and infected pigs; 3. There is not sufficient information to demonstrate that there is or is not a problem with use of NSP tests to detect SATs with the Cedi-test or other tests that are comparable to the OIE Index test.

Recommendations: 1. The EU should stimulate in their research programme further development of DIVA tests and /or test combinations suitable as confirmatory test, with diagnostic performance at least comparable to that of best NSP assays currently available; 2. National reference laboratories should study the performance of tests (and test combinations) that are currently available for use by NRLs for detection of SAT infections; 3. The ad hoc group should on a regular (yearly) basis update the “most appropriate validation data ” that indicates the test performance (DSp and DSe) of the commercial anad in-house tests available for Europe; 4. The ad hoc group should also annually review of the reported performance of new generation n NSP tests; 5. The CRL should also use proficiency testing exercises (CRL/Phases studies) to upgrade validation of certain parameters for NSP test used in NRL, such as DSp (through testing of 500 sera from negative populations) and reproducibility (by collating results reported by NRL for the proficiency panel of sera).

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Papers to be completed 1. Review of the performance of NSP tests for detection of SAT infections (Roeder and Thomson – by 1/12); 2. Summary table(s) giving estimates of test performance (DSp and Dse) for the commercially available NSP tests (OIE index test, Ceditest, Svanova, Brescia test) for use in main species (EB and DS, 1/12).

Item 7. Issues arising from monitoring of preventive vaccination programs in non-free countries This issue arises from the program of activities of the EuFMD with non-free countries in the NearEast and South-East Europe. Three papers were presented and discussed; the first, by Naci Bulut, on the problem of inadequate duration of protective antibody titres after type O vaccination in Turkey (Appendix 18); the second, by Carsten Potzsch, on the development of a minimum standard for reporting sero-monitoring activities (Appendix 19), including standards of data collection and expected outputs; the third being a concept paper on development of a model to predict the population immunity in space and time after vaccination campaigns, to assist decision makers to predict the outcome of changing vaccines and vaccination regimes.

Stability of type O titres after vaccination (Naci Bulut, Appendix 18) Dr Bulut showed that the percentage of cattle with LPBE titre higher than 1/100 was diminishing more quickly with type O than with type A or Asia-1. The observed reduction in time was not related to differences in vaccines used. This might indicate that the protective response to type O is more short lived than for the other types, which correlates with the high percentage of O outbreaks in Turkey. The probably lower herd protection to type O is a risk for the evolution of new type O strains. The data should also be analysed in a different way by looking at the titres found.

Nevertheless, the epidemiological situation shows already that new tyoe O strains are evolving and this should be monitored very closely, and perhaps new vaccine strains should be selected to cover better the recent isolates.

Standardization of sero-monitoring results; draft reporting format (Carsten Potzsch, Appendix 19) When sero-monitoring data are made available to neighbouring countries, which is very important to understand the situation in the area, the format of the reports should be standardised. Dr Potzsch developed a draft reporting format which can be used for this purposed.

Modelling population immunity after vaccination campaigns : concept paper (Arnaud le Menach, FAO. Appendix 20) Dr Le Menach showed the outcome of a deterministic SEIR model on the population immunity.

According to this model 30% of the animals protected against FMD infection would be sufficient if movement restrictions are in place (note by AD, this corresponds with a R of approximately 1.4 which has been found after movement restrictions were in the UK. It will be essential to estimate protection against heterologous challenge, because a normal 3 PD50 vaccine will only protect 75%of the animals against homologous challenge. To achieve at least 30% of protection against infection with a heterologous strain a large part of the population has to be vaccinated still).

Conclusions 1. There is a need for harmonization of methods used in sero-monitoring of vaccination in the programs served by the EUFMD Commission to ensure comparability between results of population level surveys and vaccine trials.

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Recommendations 1. The ad hoc group on laboratory standardisation should guide the Secretariat in what should be done, including, as required, the exchange of sera of parallel or repeat testing from the Caucasus, Turkey, Iran and Egypt, to give examples; 2. Continued development of a model to predict the immunity in a population after vaccination campaign over time is strongly supported. If this cannot be continued by FAO staff then a proposal should be made for possible EUFMD/EC/other support, with the aim of development and testing before the Open Session 2008.

Papers to be finalized 1. Finalize paper on type O antibody kinetics induced by different FMD type O vaccines (NB and Aldo Dekker); 2. Finalize paper on standardisation of information collection and information output (Carsten Potzsch).

Item 8. Final papers –information session The final Session involved offered five offered papers; these were subsequently reviewed and conclusions and recommendations reported under relevant ad hoc working group.

8.1 Progress report on experimental infections in dromedary and Bactrian camels (S. Alexandersen, Appendix 21) Dr. Alexandersen showed very eloquent data on studies in which attempts were made to infect dromedary camels.

Conclusion: Dromedary camels have proved refractory to infection with FMDV serotypes O and A, whereas Bactrian camels were successfully infected with serotype A and developed severe lesions on hind feet.

8.2 View point on role of sheep and goats in the epidemiology of FMD in Iran (V. Otarod, Appendix 4) This paper arose from the Iran/EUFMD/EC program. The analysis showed that most outbreaks of FMD were reported in non-vaccinated units. But the absence of laboratory confirmation cannot exclude the some of the disease was caused by other infections than FMD (e.g. PPR or ecthyma contagiosa). The consensus was that assessment of the role of sheep in transmission to cattle could not simply be based on reported incidences in each species but required also sero-monitoring information on age-related exposure.

8.3 Molecular epidemiology of FMD in Iran (N. Shirazi, Appendix 5) This paper reported on progress made during a study tour at the WRL supported by the Iran/EUFMD/EC program. The conclusion was that the typing results need to be further assessed for evidence of local area heterogeneity (“mixing vessels”), local persistence (limited change over time).

8.4 Reports and issues raised in the control of type O FMD in Israel in 2007, including assessment of the use of a thermal imaging device (Hagai Yadin, Appendix 22) FMD is causing epidemics in Israel almost on a yearly basis. The current vaccine contains 3 O type strains, e A type strains and one Asia-1 strain. In 20007 all isolates were type O, and the most likely rout of introduction was from neighbouring countries. Dr Yadin presented several examples of outbreaks and showed that sometimes the number of NSO positive cattle can be very low (1-2 in a herd). In a herd in which there was massive infection in cattle (20% NSP antibody positive) an even higher percentage (80%) of NSP antibody positive sheep were found, although no clinical disease was observed in the sheep.

Because in some previous outbreaks wild boar and gazelles were also affected, a system with a thermal imaging device was tested from a helicopter. The system had been shown to be effective at short distances of approximately 25 meters.

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8.5. Position paper on decentralised testing (Donal Sammin, Appendix 17) The current lateral flow devices are only validated for type A, O and Asia-1. Sensitivity seems similar to the currently used antigen detection ELISA.

SECTION IV: OTHER ISSUES Item 9 Vaccination of sheep: Updated guidance paper, preventive and emergency situations Keith Sumption introduced this Item (Appendix 23). A previous paper on control and eradication of FMD in livestock populations with a high density of sheep had been prepared for the EuFMD Session in 20001. The issues of vaccination of sheep in European countries as part of an emergency response, and in non-free countries as part of routine preventive vaccination, had been raised by CVOs at various meetings including the recent FAO/OIE Roundtable on FMD control in the Middleeast. In countries such as Iran, with 80 million sheep and only 8 million cattle, the question of vaccination also has large resource implications, also the case where emergency vaccination is considered with limited vaccine bank reserves compared to total livestock populations in a control zone.

Since 2000, there had been additional evidence from field and experimental studies on the role of sheep that required consideration. From the UK 2001 it was suggested that sheep acted as ”porters” of FMDV between holdings. This was especially very clear in the period before the national movement ban was in place but the evidence was unclear as to there role in long term persistence of FMDV circulation within an area and of their role as first recipients of infection on a holding (with subsequent spread to cattle). It was clear at a macro-epidemiologic level that FMD could cease to circulate in a country without significant sheep vaccination (types Asia-1 and type A Iran 96 in Turkey, for example) but unclear if type O circulation could cease with only cattle vaccination in such an area. The question of inclusion of sheep in vaccination buffer zones remains important, with sheep included in once yearly vaccination in Thrace region of Turkey but not under the EC funded buffer zone program in the Caucasus.

Discussion The group agreed that the Guidelines needed to be updated and that the issue of vaccination of sheep in endemic countries required specific attention, having major implications for the middleeast.

The lack of data from field studies on the role of sheep remained a problem. The group supported the proposal for specific review of the UK data on the role of sheep and the potential epidemiological impact of vaccination after the movement ban. Detailed serological studies in Iran and Turkey could help resolve issues of the role of sheep and an expert group should develop a proposal for a cost-effective form of study.

Conclusions 1. The Guidelines in the 2000 EUFMD report require updating, and in so doing the authors should consider other relevant texts, particularly requirements stated in the OIE Code.

Recommendations 1. A systematic study on the role of sheep in FMD transmission after the movement ban in UK in 2001, and the possible epidemiologic benefits of vaccination of sheep, should be undertaken [Action: Secretariat] Papers to be prepared 1. Guidelines on the inclusion of vaccination of sheep into control programs in FMD free and nonfree regions 2. The systematic study indicated in Recommendation 1.

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Item 10. Group work to develop conclusions, recommendations and actions The participants divided into three groups, each with a rapporteur.

1. International surveillance group: DP, TM, SA, JH, VO, FG. The group summarized Items 2,3, including FMD information from Iran, Turkey, Israel, plus the study on FMD in camels.

2. Lab standardization and biosafety group AD, EB, BH, HY, NS, DS. Tackled Items 4, 5, and decentralised lab tests.

3. Post-vaccination surveillance and vaccination monitoring group:, SZ, HV, NB, KdC, GG, CP, FM, GT. Tackled Items under Items 7 and 8.

Item 11. Reading of the report The summaries, conclusions and recommendations were collated and a preliminary read-through and amendment was made by the group on day 3.

Item 12. Other tasks held over from the 2005-7 work plan The group discussed items held over from the previous work plan.

1. Type C position paper: no progress, remained an issue. FAO was requested to commission review to be presented to the International Surveillance ad hoc group; 2. Naming of strains (WRL): to be followed up under Laboratory Standardization ad hoc group; 3. Position paper on potency testing in pigs. The Chairman proposed this be completed by the Open Session 2008. As there is no dedicated ad hoc group for vaccine quality assurance, he would oversee this task; 4. Virus inactivation studies: proposals drafted, but not funded. Executive Committee should be informed; 5. Laboratory Contingency Plans: a generic LCP is an output of the Co-ordination Action and will be completed under this project; 6. Replacement of challenge with serological assessment: progress is being made under the Improcon project, to be reported to the Open Session 2008 (progress and implications to be monitored by the Standardization ad hoc group).

Item 13. Planning of the Open Session Venues were discussed; the offer made at the 2006 meeting (Italy and UK) were reconfirmed. The IVO also indicated willingness to host a meeting in Iran.

Dates: end of September 2008 (to enable University accommodation if in UK) to mid October (Italy).

Cost: the group discussed the maximum daily cost for budget purposes, and agreed the total cost per participant must be kept below < 130€ per day.

Decision: both DP and EB would identify options and report back to the Secretariat and Chairman to enable a decision by the 75th Executive (29th November).

Closing ceremony On behalf of all the members of the group, the incoming Chairman thanked the outgoing Chairman (Dr de Clercq) for his outstanding efforts over at least the past 10 years to build a truly collaborative and productive scientific network. This was wholeheartedly endorsed by all present.

The Chairman thanked Dr de Clercq for being willing to say as Vice-Chairman during the Session.

The Secretary thanked all members and participants for their willingness to give time to the international effort on FMD and thanked the Chairman of the GOVS, Dr Samaha, and Deputy Director of the SVSRI Abbasia laboratory, Dr Azab, for their support and hospitality.

Finally he said that the success of the meeting was largely the work of the persons, Nadia Rumich (EUFMD Secretariat) and Yaser Gado, GOVS Liaison officer. Their work was warmly applauded by the group. He also thanked FAO staff in Cairo, particularly M.Mirreh and Toni Ettel for support with arrangements.

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Appendix 1 LIST OF PARTICIPANTS Session of the Research Group of the Standing Technical Committee Cairo – Egypt 16- 19 October 2007

Dr Kris DE CLERCQ (Chairman) Head Department of Virology Section Epizootic Diseases CODA-CERVA-VAR Groeselenberg 99 B-1180 Ukkel, Belgium Tel/Fax: +32-2-3790400 / +32-2-3790666 kris.de.clercq@var.fgov.be Dr Søren ALEXANDERSEN Research Professor Danish Institute for Food and Veterinary Research Department of Virology, Lindholm DK-4771, Kalvehave, Denmark Tel/Fax: +45-72-347833 / +45-72-347883 (direct) or 347901 sax@dfvf.dk Dr Emiliana BROCCHI Head National Reference Laboratory for Vesicular Diseases Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna Via A. Bianchi, 7/9 25124 Brescia, Italy Tel/Fax: +39-30-2290310 / +39-30-2290369 ebrocchi@bs.izs.it Dr Aldo DEKKER Senior Scientist Laboratory Vesicular Diseases Central Institute for Animal Disease Control PO Box 2004, Lelystad 8203 AA, The Netherlands Tel/Fax: +31-320-238603 / +31-320-238668 Aldo.Dekker@wur.nl

Dr Georgi Kirilov GEORGIEV Head of Exotic and Emerging Diseases National Diagnostic and Research Veterinary Medical Institute 1606 Sofia, Bulgaria Tel/Fax: +359-2-8341004 georgivet@yahoo.com Dr Bernd HAAS Head of National FMD Reference Laboratory Friedrich-Loeffler-Institut Federal Research Institute for Animal Health Boddenblick 5 a 17493 Greifswald, Insel Riems, Germany Tel/Fax: +49-(0)3835170 / +49-(0)383517151 bernd.haas@fli.bund.de Dr Stephan ZIENTARA AFSSA – Lerpaz - BP 67 94703 Maisons-Alfort Cedex, France Tel/Fax: +33-1-49-771333 / +33-1-43-689762 s.zientara@afssa.fr Dr Dónal SAMMIN Senior Research Officer Central Veterinary Research Laboratory Daf Laboratories, Backweston, Celbridge Co. Kildare, Ireland Tel/Fax: +353-1-615-7242 / +353-1-615-7253 donal.sammin@agriculture.gov.ie Dr Hagai YADIN Head of Virology Division and FMD Laboratory Kimron Veterinary Institute c/o Ministry of Agriculture PO Box 12 Beit-Dagan 50250, Israel Tel/Fax: +972-3-968-1619 / +972-3-968-1788

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hagaiy@moag.gov.il Dr Naci BULUT FMD Expert Head of the Diagnosis Department FMD Institute Ankara Tel: +90 312 2873600 / Fax: +90 312 2873606 Mobile: +90 533 3571484 nacib@sap.gov.tr Dr Haken Vigre International Epilab Danish institute for Food and Veterinary Research Morkhoj Bygade 19 DK2860 Soborg Denmark hvi@vet.dfu.dk FAO WRL Dr David PATON (ex-officio) Pirbright Laboratory Institute for Animal Health Ash Road, Pirbright, Surrey GU24 0NF, UK Tel/Fax: +44-1483-231012 / +44-1483-232621 david.paton@bbsrc.ac.uk

Prof. Hassan AIDAROS FAO Regional Animal Health Center,Beirut Tel: +2012 2185166 Fax: +202 7607055 haidaros@netscape.net Dr Gavin THOMSON SADC –FAO Project Private Bag 0095 Gaborone Botswana tel: +267 3913357 gthomson@sadc.int Dr Francois MAREE Exotic Diseases Division, OVI, ARC Private Bag X05 Onderstepoort 0110 South Africa Tel: +27 12 5299584/94 Fax: +27 12 5299595/05 mareef@arc.agric.za Dr Ibrahim EL BEN-DARY Govs-Egypt Tel: +2 0 63725533 Ibelbendary@hotmail.com

OBSERVERS Dr Jeffrey HAMMOND CSIRO, AAHL Private Bag 24, Geelong, VIC 3220, Australia Tel/Fax: +61-03-522-75767 / +61-03-522-75555 jef.hammond@csiro.au

Dr Yasser M. GADOU Govs-Egypt Tel: +2 0 05665720 Yaser_Gov@yahoo.com FAO

Dr Vahid OTAROD Iran Veterinary Organization 23, Seyed Asad Abadi St. PO Box 14155-6349, Tehran, Iran Tel/Fax: +98-982-188806407 / +98-982188902712 votarod@hotmail.com

Arnaud LEMENACH EMPRES – FAO Viale delle Terme di Caracalla 00100 Rome, Italy arnaud.lemenach@fao.org

Dr Mohammed Nazem SHIRAZI Iran Veterinary Organization 23, Seyed Asad Abadi St. PO Box 14155-6349, Tehran, Iran Tel/Fax: +98-982-188806407 / +98-982188902712

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EUFMD SECRETARIAT Dr Keith SUMPTION Secretary, EUFMD Animal Health Service Animal Production and Health Division FAO – Viale delle Terme di Caracalla 00100 Rome, Italy Tel/Fax: +39-065705-5528 / +39-065705-5749 keith.sumption@fao.org Dr Tom MURRAY Associate Professional Officer, EUFMD Animal Health Service Animal Production and Health Division FAO – Viale delle Terme di Caracalla 00100 Rome, Italy Tel/Fax: +39-065705-5124 / +39-065705-5749 tom.murray@fao.org Ms Nadia RUMICH EUFMD Secretariat Animal Health Service Animal Production and Health Division FAO – Viale delle Terme di Caracalla 00100 Rome, Italy Tel/Fax: +39-065705-2637 / +39-065705-5749 nadia.rumich@fao.org Dr Francis GEIGER Iran Veterinary Organization 23, Seyed Asad Abadi St. PO Box 14155-6349 Tehran, Iran Tel/Fax: +98-2188957251 / +98-2188902712 francis.geiger@fao.org.

Dr Carsten PÖTZSCH FAO International Coordinator FMD Control and Surveillance Ivedik Cad. No.551 06170 Yenimahalle, Ankara, Turkey 0090 31 23079554 carsten.potzsch@fao.org

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Appendix 2 Agenda of the Research Group Session Day 1 (16th). At FAO-RNE 1 Opening. Adoption of the Agenda, meeting organization 2 International FMD surveillance; recent FMD epidemic events 3 UK outbreak 2007: events, issues arising 3.1. IAH paper (David Paton) 3.2. Lindholm paper on full genome sequencing results ( Soren Alexandersen) 4 FMD confirmation : 4.1. RG position on promotion of molecular tests as the main method/first choice for FMD confirmation. What conditions need to be present for sustainable and harmonised RT-PCR performance? (Aldo Dekker) 4.2. low confirmation rates in endemic regions: problems and solutions; sample collection guidelines 5 FMD lab biosecurity working group (Bernd Haas)

Day 2 (Wedns; at SVSRI Abbasia vaccine production and control centre) 6 Post-vaccination surveillance (PVS); 6.1. technical issues requiring solutions identified at the PVS workshops 6.1.1. Review of main conclusions of the first two workshops (Kris de Clercq) 6.1.2. view point on resolution of remaining technical issues with PVS (David Paton) 6.2. Progress with use of scenario trees to optimise surveillance for confidence in disease freedom (Tom Murray) 6.3. NSP test performance issues 6.3.1. diagnostic sensitivity to SAT infection (Francois Maree, OVI), and purpose of NSP test use in areas affected by SAT viruses (Gavin Thomson, SADC-FMD project) 6.3.2. key performance data for NSP tests; a performance summary for tests available for use in Europe (Emiliana Brocchi and Donal Sammin) 7

Issues arising from monitoring of vaccination programs 7.1. stability of type O titres after vaccination (Naci Bulut) 7.2. standardisation of sero-monitoring results; draft reporting format (Carsten Potszch) 7.3. modelling population immunity: how can we do this? (Arnaud le Menach, FAO)

Discussion and conclusions on issues raised on day 2.

Day 3. Research Group Session at FAO-RNE 8

Final papers –information session 8.1. progress report on experimental infections in dromedary and Bactrian camels (S. Alexandersen) 8.2. View point on role of sheep and goats in the epidemiology of FMD in Iran (V. Otorod) 8.3. Molecular epidemiology of FMD in Iran (N. Shirazi) 8.4. Reports and issues raised in the control of type O FMD in Israel in 2007, including assessment of the use of a thermal imaging device (Hagai Yadin) 8.5. Paper on decentralised laboratory testing (Donal Sammin)

9

Vaccination of sheep: Updated guidance paper , preventive and emergency situations (Keith Sumption)

10 Group work to develop conclusions 11 Reading of the report 12 Planning of the Open Session

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Appendix 3

Thank you Recent epidemic events in the European neighborhood: update

(excludes UK )

EUFMD Standing Technical Committee Session Cairo

member states -support DG-SANCO (Alf Fuessel, Bernard Van Goethem) WRL Pirbright; David Paton, Chris Chisholm, Nigel Ferris (and the typing team) Peter de Leeuw; risk management advice, vaccine bank issues

Keith Sumption Secretary, EUFMD Commission

Veterinary Services of the I.R of Iran – Task Force members Turkey (Dr Pakdil, Huseyin Sungur, Musa Arik, Naci Bulut) Egypt (Dr Samaha, Dr Tawfiq)

Israel; Hagai Yadin

1?

FAO-EMPRES (Giancarlo Ferrari), FAO Ankara, FAO-Egypt FAO procurement service (for all the emergency tenders!)

OIE: Ghazi Yehia, RRME Beirut

VAR, Belgium; Kris de Clercq, Koen Mintiens FLI; Bernd Haas, vaccine trials

FIVE ecosystems have contributed to FMD incursions into European/meditteranean countries in the past 10 years

Colleagues in EUFMD (Tom Murray, Francis Geiger, Carsten Poetsch, Nadia and Laura), FAO Consultants (Zafer Zog, Nick Honhold, E Couacy, JF Valarcher) Giancarlo Ferrari and Mansoor Hussain, GTFS/INT/907/ITA

FMD high risk regions: high weight of infection with antigenic diversity source of emergent viruses

2 3

5

4

Type O (blue), A (green), Asia-1 (orange), SAT2 (purple)

Recent epidemic events – European neighbourhood /Eurasia – 2005 to present

two epidemic waves (A Iran 05, O PanAsia II)

affecting west Eurasia, reaching Thrace (2006,2007, current..)

one exotic FMD incursion (A Egypt 06) plus one “”escape”” (O1 BFS) A Iran 05: epidemic trough, entering endemicity or extinction

Continuing risks

successors emerging ? variant type A viruses to be watched (eg.

Afghanistan 07)

O PanAsia II: epidemic situation in Turkey and Iran continuing, spread into CIS: replacement of endemic genotypes (?)

A Egypt 06: extinct or continuing ?

Asia-1: good situation in west Eurasia, continued spread to east (North Korea in 2007) continuing risk of African types (A, SAT1 and SAT2) from Horn of Africa to near-east type C: risk of laboratory escapes globalisation:

diversified meat and live animal imports southern Africa to Europe: ASF in Georgia, trade between non-free countries (Chinese and Indian exports) official intra-regional trade in middle-east

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Recent FMD incursions – west Eurasia into South -Eastern Europe, threatening Balkan region and Caucasus

1996-2005; type A Iran 96 1999-2002: Asia -1 2005-present: type A Iran 05

2005: before the epidemic, diversity; blue = A Iran 96 and 99; light green= A Iran 05

replacing A Iran 96 in Turkey

2006-present: type O

new PanAsia genotype adding (replacing?) endemic type O in Turkey

Type Type Type Type

FMD type A Iran 05; severe clinical signs in vaccinated cattle (with mortalities in calves and adults) “”FMD – todays real enemy of cattle””

O :98 A96Iran: 2 A99Iran:3 A22(new )2005:15 A22(new)2005:15

FMDV A – New Strain (Irn-05)

Pictures: IVO, Iran

A24/Cruzeiro/BRA/55 [AJ251476] A/K5/80* A/EGY/1/2006 [EF208757] A/K35/80* A/ERI/3/97 A/ERI/3/98 A/SAU/23/86 A/APR/51/05* [EF120400] A/MEU/49/03* [EF120401] A/BHU/27/2003 A/BHU/41/2002 A/BHU/7/2003 OS A/APS/44/05* [EF120402] A/APS/68/05* [EF120403] A/IRN/33/2004 A/IRN/5/2003 A/IRN/9/2003 A/IRQ/2/2002 A/IRQ/100/2002 A/IRQ/99/2002 A/IRQ/24/2002 A/IRQ/59/2002 A/IRQ/33/2002 A/IRQ/60/2002 A/IRQ/107/2002 A/IRQ/108/2002 A/IRN/32/2004 A/IRN/17/2005 A/IRN/1/96 [EF208771] A/TUR/2/2003 A/TUR/14/2002 A/SYR/5/2002 A/TUR/17/2001 A/TUR/4/2003 A/IND/17/77* [AF204108] A22/IRQ/24/64 [AJ251474] A/TAI/118/87* [EF208777] A/TAI/2/97 [EF208778] A/IRN/87 A/SAU/41/91 A/IRN/22/99 [EF208772] A/TUR/5/2003 A/IRN/10/2003 A/PAK/28/2002 A/PAK/9/2003 A/IRN/32/2001 A/IRN/34/2001 A/IRN/2/2002 A/IRN/7/2003 A/IRN/41/2003 A/IRN/7/2004 A/PAK/5/2006 A/IRN/54/2006 A/SAU/15/2005 A/SAU/16/2005 A/IRN/30/2005 A/IRN/27/2005 A/IRN/22/2005 A/IRN/5/2006 A/IRN/40/2005 A/IRN/50/2005 A/IRN/53/2005 A/IRN/36/2005 A/IRN/24/2005 A/IRN/34/2005 A/PAK/1/2006 A/PAK/3/2006 A/IRN/25/2005 A/IRN/4/2005 A/IRN/1/2005 [EF208769] A/IRN/2/2005 A/IRN/7/2005 A/IRN/57/2006 A/IRN/44/2005 A/TUR/8/2005 A/IRN/5/2005 A/IRN/18/2005 A/IRN/14/2005 A/IRN/16/2005 A/IRN/10/2005 A/IRN/13/2005 A/IRN/26/2005 A/IRN/33/2005 A/IRN/43/2005 A/IRN/28/2005 A/IRN/51/2005 A/IRN/54/2005 A/IRN/29/2005 A/IRN/55/2005 A/IRN/42/2005 A/IRN/7/2006 A/TUR/12/2006 A/IRN/38/2005 A/IRN/39/2005 A/TUR/16/2006 A/JOR/3/2006 A/JOR/2/2006 A/JOR/4/2006 A/TUR/11/2005 A/TUR/4/2006 A/TUR/6/2006 A/TUR/20/2006 A/TUR/7/2006 A/TUR/17/2006 A/TUR/8/2006 A/TUR/10/2006 A/TUR/9/2006 A/TUR/14/2006 A/TUR/11/2006 A/TUR/19/2006 A/TUR/18/2006 A/TUR/3/2006 A/TUR/9/2005 A/TUR/1/2006 A/TUR/7/2005 A/TUR/10/2005 A/TUR/12/2005 A/TUR/6/2005 A/TUR/2/2006 A/IRN/31/2005

Irn-96

Turkey 2005-06 Iran 2003-06

Jordan 2006

Irn-99

Pakistan 2006

Saudi Arabia 2005

1%

Unrooted Neighbor-joining tree based on a comparison of the complete VP1 gene. The tree was outgroup-rooted using A24/Cruzeiro/BRA/55. Isolates labelled APR, APS and MEU are from India.

Irn-05

A22 Iraq vaccine

Example of transboundary spread: Development of the A Iran 05 regional epidemic: from Iran in June 2005 to Turkey October 05

The recent type A and type O winter epidemics in Turkey arrows indicate index cases FMD outbreaks in Turkey 2005-2007(March)

FMD outbreaks( reported) since 2005 - June 2006 in cattle population 160

150

Number of FMD reports( Cattle)

140 115

120

180

122 107 93

100

160

86

80 61

59

60 40

26

25

55

59

140 53

52

42

Number

34 26

16

20

Ja n-0 5 Fe b-0 M 5 ar -0 5 A pr0 M 5 ay -0 5 Ju n-0 5 Ju l-0 5 A ug -0 5 Sep -0 5 O ct -0 5 N ov -0 5 D ec -0 5 Ja n-0 6 F eb -0 6 M ar -0 6 A pr -0 M 6 ay -0 6 Ju n-0 6

0

months

120 100

TYPE O

80

TYPE A

60 40

FMD outbreaks in Turkey 2005-2007(March)

MARCH

DECEMBER

FEBRUARY

NO VEMBER

J ANUARY-07

AUGUST

O CTO BER

SEPTEMBER

MAY

JUNE

JULY

APRIL

MARCH

DECEMBER

FEBRUARY

NO VEMBER

J ANUARY-06

MAY

JUNE

JULY

APRIL

AUGUST

TYPE A

60

O CTO BER

TYPE O

80

SEPTEMBER

100

MARCH

120

Month

40

MARCH

FEBRUARY

OCTOBER

DECEMBER

NOVEMBER

JANUARY-07

SEPTEMBER

MAY

JUNE

JULY

AUGUST

APRIL

MARCH

FEBRUARY

OCTOBER

DECEMBER

NOVEMBER

JANUARY-06

SEPTEMBER

MAY

JUNE

JULY

AUGUST

APRIL

0

MARCH

20 FEBRUARY

Number

140

J ANUARY-05

0

160

FEBRUARY

20

180

JANUARY-05

Source: IVO, June 2006 (EUFMD/IVO Regional Workshop, Teheran ) And GDPC, Ankara

Month

24

A Iran 05 epidemic development Source: IVO,

DISTRIBUTION OF TYPE A22 IRAN 2005 OUTBREAKS first 6 months of 2006 (GDPC report to EUFMD)

Non-active (recovered) active outbreak outbreaks occured in last month

Turkey, 2007 report to 30th September

FMD Outbreaks in Turkey Source: FAO National Consultants Report, September 30th 2007

Map 1: Distribution of FMD Type A Outbreaks

Map 2: Distribution of FMD Type O Outbreaks

Type A distribution, June-Sept 07

Outlook for the type A Iran 05 regional epidemic

evidence from Turkey that enter new phase of low incidence, sporadic outbreaks will continue or decline to extinction? how long will this Phase occur? when will the next epidemic wave occur and can it be predicted? should we be concerned about the antigenic and genetic variants of A Iran 05 (e.g. Afghanistan in 2007)? next big thing or just FMDV probing the defences?

25

Example: FMD samples submitted by GTFS project to guide vaccine selection for use in Afghanistan –September 07 Type A viruses from 2007

FMD PanAsia Strain Isolations 2001-2007 (WRL)

r1 Values by VNT

KAZAKHASTAN

A22

MONGOLIA

A Ind 17/82 UZBEKISTAN

A AFG 7/2007

0.33

0.14

A AFG 44/2007

0.32

0.14

KYRGYZSTAN

AZERBAIJAN TURKMENISTAN

TAJIKISTAN CHINA

AFGHANISTAN

ISREAL

SYRIA IRAQ

IRAN

PAKISTAN NEPAL

JORDAN

In the case of neutralisation: r1 = > 0.3. Suggests that there is a close relationship between field isolate and vaccine strain. A potent vaccine containing the vaccine strain is likely to confer protection. r1 = < 0.3. Suggests that the field isolate is so different from the vaccine strain that the vaccine is unlikely to protect.

BHUTAN

BANGLADESH HO NG KONG

SAUDI

UAE

INDIA

MYANMAR

LAOS

ARABIA

OMAN

VIETNAM

THAILAND CAMBODIA

2001

YEMEN

2003 SRI

2004

LANKA

MALAYSIA

2005 2006 2007

Time-line : spread of the new PanAsia type O pandemic in Eurasia, 2006-present Year Month Confirmed (OIE/FAO network – WRL Pirbright and FGI-ARRIAH) 2006 1-06 Pakistan 9 -06 10-06 12-06

Suspected (no samples to network)

Usak Province: index case of the new Type O PanAsia –October 06 (entry point unclear)

Note

Index unknown. FAO project, WRL report

Iran – upsurge Turkey –index case Israel -index Jordan

No OIE report.

Reported by WRL

2007 1 -07 2-07

Northern Iraq Lebanon

4-07 6-07

United Arab Emirates (gazelle) Palestinian Authorities (west bank) Kazakhstan Kyrgyzstan

Total

10

2

Turkey, 2007 report to 30th September Map 1: Distribution of FMD Type A Outbreaks

new Type O PanAsia – also detected in border provinces to Syria (story - wild boar may have intoduced) and Iraq

Out of a clear blue sky..

Map 2: Distribution of FMD Type O Outbreaks

441 type outbreaks in 2007 – most (all?)

PanAsia II genotype steady decline since the January peak of 120 outbreaks only 9 in September one of these was in Thrace region

26

Edirne/Cannakale suspect FMD: October 07 16 calves (2-10 weeks age) moved within Thrace by trader: 2 died 3/10 at IP1, 2 more at IP2. Calves from same group deposited in at least 3 other locations. Classical FMD seen 10th Oct: Source: FAO consultants report to GDPC/FAO

FMD in Egypt – 2006-7

Dangerous contacts – movement of trader with infected group of calves

Index

New introduction or persistent circulation?

senior staff indicated they did not consider officially reported cases to represent the full picture and suspect type A and O circulate at low level mission team observed clinical FMD on a feedlot; unofficial report of type A isolation from samples from this farm; samples submitted to WRL for typing (week of 8th October)

Feedlot, animals 24-30months 71 animals introduced 5th September first cases on 10th clinical signs of FMD seen by mission team on 18th September

Four animals were examined, and vesicular lesions with 2-4 feet affected were seen on all four animals.

Classical tongue lesions were less evident, with healing lesions on the tongue tip in one animal and a possible VL in another.

All animals were in age range 24-30 months.

FMD was diagnosed on clinical grounds,.

.

cases occurred at end of vaccination interval and previous vaccination history not known (assumed vaccinated 6 months)

report of suspected FMD made by Chairman GOVS to FAO/OIE FMD Roundtable (September 07): mission requested to EUFMD mission of Keith Sumption/Hassan Aidaros, 16-20th September 07joint EUFMD

Egypt: suspected FMD outbreaks in 2007

In late August and early September 07, suspect FMD cases had been reported from 3 primary administrative areas (Governorates of Kafersheikh, and Sharkieh in late August , and Berhera in September), near simultaneous appearance of signs (25-27th August in the first two and 10th September (7th? )in Berhera).

The GOVS/DPM teams expressed doubt that the suspect outbreaks were FMD classical tongue lesions were not observed (Kafersheikh and Sharikieh); fever was a main sign, with salivation, ulcers on the dental pad, and mild lameness, affecting about 25% of animals. In this situation differential diagnoses must incude EHD,

FMD infection was not confirmed in samples collected in Sharkieh and Kafersheikh. Vesicular fluids had not been submitted, and materials submitted not described.

Recent Genotypes of FMDV Asia 1

Mission findings

type A and O vaccination, since April 06 type A Egypt 06 : first entry (January 06) of African type A into middle-east; type A vaccine locally prodiuced, introduced April 2006 massive epidemic, last official reported case 6/06. ( type O in April 06)

(WRL + partners , 2006) Date 10th 11 12 13 14 15 16

Group 1 (n=270) New cases 4 37 49 10

Group 2 (n=96) New cases

36 15 10 4

China, Xinjiang Autonomous Region, May 2005

China, Qinghai, Jul 2005 China, Ningxia Mongolia, Dec 2005 Autonomous Region, Aug 2005 Jul 2006 Dec 2005 China, Gansu Jul 2005, Dec 2005 May-Jul 2006

Russia, Chitinskaya, Jan 2006

Russia, Amur, June & Dec 2005 Russia, Khabarovsk, Aug & Dec 2005 Russia, Primorsky, Sept 2005

Tajikistan, 2004 China, Beijing, May 2005 Iran, 2004

China, Hebei, May & June 2005

China

China, Shandong, Apr 2005 & Dec 2005

Afghanistan, 2004

China, Jiangsu, Apr 2005 & Dec 2005 China, Hubei May 2006 China, Hong Kong, group III Feb 2005 group IV

Pakistan, 2002-2005 China, Tibet, Sep 2006 India, 2003-2004

Myanmar, Jul 2005

Vietnam, Nov 2005

Genetic identity not known

group I

group V

group II

group VI

groups I & II

groups II & VI

27

Movement of Asia-1 from west Asia to central asia/China Time –line 2001-4

Unofficial Pakistan

Official (OIE) Endemic

Lab evidence Yes

2001-4

Afghanistan

Yes

Yes

Yes

2003

Tajikistan

Yes ->3 months

December 03

Yes

Yes. 11/03

Yes.

Uzbekistan

Yes. UN news 11/03 Yes. Promed 10/04 FAO mission.

March 05 Yes

No..

FGI-ARRIAH confirmed.

-

Not available.

-

Yes.

Yes

Yes

Yes (April 05 - )

Yes *

Kazakhstan Xinjiang (China)

2005

Hong Kong SAR China –other provinces Russian Federation (Amur) China DPR Korea China

Yes Yes. Yes

Yes. Yes. Yes

Yes *

Yes. *

focus on delivery in four key categories of action in the period 2005-8:

34 member states in Europe governed by member states EC (SANCO) agreement with FAO for support of actions (8 m€ , to 2009) priorities: reducing risk of FMD incursions

Continuing Yes

EUFMD Strategic Plan, 2005-9

Kyrgyzstan

2004

2006 2007

EUFMD Commission

Support to FMD control in “traditional risk areas”threatening south-eastern Europe and Turkey.

Global FMD observation – virus circulation and risk.

Coordination of technical studies to address constraints to policy implementation.

Capacity building across Europe – raising and retaining expertise and competence in the scientific basis of FMD control and in best practises in epidemic management.

Secretariat/FAO was tasked with finding funding and implementing actions:

1: Implementation of an FMDV observation action, supporting European vaccine management through better identification of risk trends and events, including:

Progress

Comments

1.

Limited, but opportunities improving.

Improvements at level of OIE/FAO lab network, and more networking on FMD in some regions. But still no systematic priority system to guide efforts on monitor. Opportunity for FAO/OIE Regional Animal Health Centres to address FMD submission issues. West and East Africa, and west Asia are principle surveillance gaps. Financial resource needed.

Epidemiology expert group needed.

1.

Improved system for monitoring FMD virus strain circulation operational

Technical constraints to preferred European FMD control policies reduced

Some, progress on vaccination to live/ NSP

1.

System for professional development in FMD management/expertise developed

Plan, not implemented, but development of Online training modules started

Plan developed (presented to 72nd Executive) but only module 1 (FMD investigation) module development initiated in 2/07 (Online training resources)

1.

FMD risk surveillance and management programmes operating in target countries

Progress in Iran, also with the Caucasus countries

Targets 2005-8 were in Turkish neighbourhood: Projects or progress in place: Caucasus, Iran, Turkey (EC project 2007-) Gaps: Syria, Iraq “”New”” gaps: Egypt, Ethiopia, sahel, central Asia

1.

FMD incursions/emergencies rapidly controlled, where supported by specific Commission decisions

Good in 2006 (Thrace).

Type A Iran 05 epidemic, incursion 2006 in Thrace: effective control with EC support. Risk management , vaccine supply to Turkey autumn 2006.

Incursion in Thrace 2007: ongoing.

No confirmed FMD incursion in EU countries to date (05-1/07).

Good opportunities to collaborate within Europe. Expertise outside Europe not effectively used. Epidemiology expert group needed. Subgroup on on epidemic modelling required.

More human resources (and facilitation, Use of communication tools) needed to progress working groups /support Chairman

support to developing country veterinary services to collect and submit samples; support to information exchange and networking of FMD Reference Laboratories.

2: Supported actions to address technical constraints identified by the EUFMD Standing Technical Committee, working with the FAO/EC/OIE Co-ordination structure for FMD and CSF laboratories.

3: Implementation of innovative, capacity building action to raise technical competence of key levels of the European epizootic control management.

4: Field programme support:

Category of action (approved: 36th Session)

European neighbourhood pre-accession support to protect Turkey virus intelligence to guide European preparedness increasing recognition that EU is at risk from distant locations

Support the implementation of comprehensive actions for the surveillance and effective response to FMD in the southern Balkans region (Turkey, Greece, Bulgaria); Implementation of a project for early warning of FMD regional risk events, through supported actions with the Islamic Republic of Iran; Implementation of a project for the surveillance and effective response to FMD risk in countries of the South Caucasus (Georgia, Armenia, Azerbaijan); Identification and formulation of project actions to control risk in other countries neighbouring to Turkey, and in other FMD risk situations, as required by the emerging situation.

Progress Report to the 37th Session : April 2007

In conclusion, the European freedom from FMD has been maintained, but was severely threatened from the neighbouring regions by type A and type O regional epidemics in west Asia,

incursions of type A Iran 05 and type O (PanAsia variant) into Iran and Turkey in 2005-6, each resulting in severe, widespread epidemics and which resulted in multiple introductions in Thrace region in 2006 (type A) and 2007 (type A, type O)

The frequency and severity of these events is a major reverse of the favourable epidemiologic situation in 2003-4; type A incursion into Egypt presented a risk situation in early 2006.

European reserves of vaccine have been mobilised in 2006 and 2007 in response to the above threat, for emergency vaccination (Thrace 2006) or strategic application in prevention (Marmara and western Anatolia 2006, Thrace and Marmara, western Anatolia 2007).

The three south Caucasus countries have been assisted with the maintenance of the Buffer Zone : despite the epidemic situation in Turkey and Iran, no FMD outbreaks were reported in the South Caucasus. An emergency reserve as maintained at ARRIAH, Russia, under the EUFMD/EC funding agreement, for mobilisation in case of emergency.

The attention to emergency events, plus lack of funding for active collection of FMD strains circulating in risk areas, has hindered progress on risk assessment and early warning in more distant risk regions to Europe.

28

Progress Report to the 37th Session : April 2007

Regarding the Strategic vision (goal); In 2005 this was defined as: “”- A Europe free of FMD – the FMD disease-free state achieved and maintained in all Europe”.

Progress: FMD freedom in European countries has been maintained in period 2005-7, with exception of Turkey and Israel.

Regarding the Purpose (higher level objectives) : In the 2005-8 plan, it envisaged the member countries, with support from the EUFMD Commission and other partners, working towards the following:

No occurrence of FMD in officially free countries of Europe in the period to 2008.

Effective management of risk of entry through improved access to information of FMDV circulation in source countries and of epidemiologically significant events.

Progress: severe decline in reporting in 2005 and 2006, impacted early warning. Counter measures taken - regional meetings (with OIE, AU-IBAR); more effort in rumour tracking, to promote sample collection, and typing at WRL Pirbright

No occurrence of Asia-1 infection in Turkey over the 4 year period.

Progress: on track – at 2/07. In addition the Republics of Belarus, Serbia and Montenegro, with Kosovo; status of FMD free without vaccination recognised

by OIE in 2006

Progress: on track – to 2/07

Reduction in incidence of other exotic FMD types entering Turkey over this period.

Progress: not achieved, rate of entry and impact increased in period

Progress -ctd

FMD surveillance targets and reporting in Caucasus countries and in Iran (and Iraq, Syria) that meet the requirements of at risk countries for early warning.

Incidence of FMD in Thrace region of Turkey reduced to zero in period ; targets for surveillance, disease investigation and reporting in Thrace region meet need for early and effective control of incursions.

Progress: .surveillance intensity increased in Iran; assistance provided to the Caucasus countries and Syria (on track– to 2/07)

Progress: not achieved. FMD outbreaks in Thrace in 2006, in 2007

Reduction in distribution of type A and type O FMD in Anatolia – defined by increase in the Provinces/area, and in period /time when virus infection is shown not to be present.

Progress: not achieved, reverse

29

Appendix 4

Questions • Which EpiEpi-units should be considered?

Evaluation of the Role of Sheep and Goat in Transmission of FMD to Cattle

• What is the importance of each units (Quantitatively)?

• What is the probability of a unit at a given time to be infected for Cattle and Sheep & Goat FMD? • What is the power of sheep FMDV to be transmitted to cattle?

• What is the effect of Vaccination to protect cattle population of Iran?

Dr. Vahid Otarod

Villages Specifications

Report of outbreaks in GIS • Every EpiEpi-unit should be reported by a code.

– Easy for analysis and comparison.

•

In the Animal Population Structure of I.R.IR, villages are defined defined to be an EpiEpi-unit

•

Villagers keep the animals (cattle , sheep, Goat) in their home

• Every report should consider only one disease

•

Number of animals differ from one village to another

•

Different species of animals may keep separately or together

•

Low level of biosecurity

•

Uneven level of immunity

•

In and Out Movement of animals

•

Common pasture

• Every report should consider only one species of animal.

• For one outbreak of FMD in each species in an EpiEpi-unit , one report should be submitted.

– One EpiEpi-unit may be declared infected two times for two species of animal in a given date.

close contact between animals

Code of Villages reported to be infected for FMD in Cattle 1417054

1504187

1510095

2211029

2601004

2901187

3111040

3209060

3504005

3708010

1001216

1417088

1504197

2004241

2213015

2601015

2901203

3111143

3210077

3504030

3711056

distribution of multi species Infected units (April(April-Sept 2007)

1003143

1501066

1504208

2004308

2213059

2601023

2901248

3111155

3210080

3504088

3904085

1005098

1502026

1505059

2005007

2213060

2601024

2902060

3118264

3210128

3504200

3904243

1008013

1502036

1507034

2012189

2213063

2601025

2902178

3201031

3210161

3504209

3904375

1013012

1502037

1508089

2101194

2213131

2601026

2902179

3201048

3212013

3504214

3905056

1013029

1502039

1508153

2101196

2213136

2601028

2902180

3201170

3212034

3505002

3906018

1014169

1502110

1509004

2101202

2213190

2601029

2902181

3201551

3212091

3505017

3908155

1202005

1502114

1509014

2101268

2213198

2601032

2902216

3201597

3212099

3505018

3908337

1202028

1502152

1509017

2101279

2216065

2601034

2902218

3201753

3213016

3505036

3908374

1203136

1502167

1509021

2101311

2217053

2601037

2902219

3201853

3213038

3505045

3908433

1204074

1502188

1509045

2101345

2219220

2602048

2902220

3201877

3213069

3505047

3908441

1301031

1502189

1509052

2101381

2219245

2603103

2902224

3201899

3214069

3505099

3911069

1301308

1502210

1509053

2104109

2219281

2603129

2902225

3203295

3214162

3505123

3911071

1306014

1502211

1509054

2105109

2301115

2603131

2902226

3204134

3306068

3505124

3912170

500

1306060

1502222

1509057

2106013

2308123

2603157

2902229

3205161

3312014

3505131

3915153

400

1306129

1502229

1509059

2106016

2404007

2604027

2902230

3206009

3313075

3505142

3915219

1402054

1502238

1509068

2106143

2406012

2604033

2902236

3206047

3401025

3505144

3915228

1405082

1502244

1509084

2107025

2408003

2701015

2902265

3206059

3404013

3505164

3916212

200

1407616

1503079

1509090

2107196

2410313

2701016

2903090

3206072

3404060

3505208

3916229

100

1407620

1503093

1509114

2107205

2501084

2701062

2904145

3206077

3501013

3506026

3916285

1407622

1503123

1509115

2107216

2501177

2701071

2904193

3206162

3501064

3506042

3916451

1407624

1503124

1509117

2107219

2504015

2701210

2904225

3206164

3502078

3506065

3917012

1407631

1503128

1509120

2111020

2507097

2701276

2905016

3206207

3502125

3506082

1412004

1504012

1509122

2111024

2509054

2701296

3105109

3208006

3503087

3604028

1417047

1504180

1510026

2111046

2601003

2901160

3111013

3208160

3503164

3608028

Villages Dairy and sheep farms Beef and sheep farms Distribution of infected Epi-units(multi species) in Iran (April-Sept 2007) 600

No. of units

• • •

1001069

EpiEpi-Units of importance

300

0 villages

dairy and sheep farm

beef and sheep farm

3917125

284

30

Code of Villages reported to be infected for FMD in Sheep & Goat 1008007

1502068

1502253

1509053

2107024

2901022

2904225

3210124

3505047

3904226

1013153

1502069

1502254

1509054

2107205

2901030

2904242

3210159

3505098

3904273

1017036

1502070

1502255

1509057

2107209

2901036

2905005

3210161

3505099

3904356

1201009

1502081

1502260

1509059

2108024

2901087

2905006

3213039

3505122

3904374

1202100

1502096

1503093

1509068

2108045

2901137

2905029

3213056

3505123

3905102

1203136

1502101

1504012

1509092

2108061

2901199

2905047

3304021

3505124

3906075

1301224

1502127

1504088

1509110

2108084

2901200

2905082

3306137

3505131

3906076

1301433

1502136

1504185

1509114

2212361

2901203

2905104

3313067

3505147

3908023

1306776

1502143

1504187

1509120

2213136

2901212

2905113

3401046

3505159

3908037

1407619

1502149

1504197

2004226

2216065

2901236

3002054

3406028

3505178

3908065

1414211

1502188

1504208

2004308

2219194

2901260

3003261

3406047

3506015

3908140

1414224

1502189

1505028

2012149

2301128

2902176

3006161

3406054

3506074

3908289

1414225

1502193

1505126

2101138

2404009

2902178

3111149

3406069

3603060

3908441

1417036

1502210

1507014

2101311

2409116

2902179

3118252

3501013

3604028

3908477

1417127

1502215

1507094

2101345

2503164

2902180

3201158

3501061

3607017

3909018

1502013

1502220

1507108

2101355

2601031

2902186

3201569

3501069

3607033

3909048

1502020

1502221

1507129

2102023

2603021

2902228

3201853

3501074

3607036

3911014

1502030

1502222

1508026

2105032

2603092

2902234

3203259

3504030

3607040

3911176

1502035

1502224

1508132

2105066

2603104

2902236

3203337

3504071

3607048

3912108

1502036

1502229

1508156

2105068

2603110

2903120

3206073

3504088

3608010

3913100

1502041

1502230

1509004

2105080

2604008

2903126

3206114

3504177

3608014

3916128

1502062

1502238

1509021

2105109

2604009

2903137

3206147

3504209

3608016

3916129

1502066

1502244

1509024

2107010

2604033

2904193

3206162

3505018

3904201

1502067

1502251

1509028

2107023

2705070

2904223

3210114

3505036

3904203

Code of Villages reported to be Infected for Cattle And Sheep & Goat FMD

1203136

1504187

1509120

2902180

3505018

1502036

1504197

2004308

2902236

3505036

1502188

1504208

2101311

2904193

3505047

1502189

1509004

2101345

2904225

3505099

1502210

1509021

2105109

3201853

3505123

1502222

1509053

2107205

3206162

3505124

1502229

1509054

2213136

3210161

3505131

1502238

1509057

2604033

3501013

3604028

1502244

1509059

2901203

3504030

3908441

1503093

1509068

2902178

3504088

1504012

1509114

2902179

3504209

53 villages

3916257 239

• Probability of finding a village at a given time to be infected for Cattle and Sheep & Goat FMD. P = 0.1 • Population Attributable Risk = 0.04 • Power of Sheep FMDV to be transmitted to cattle = 1.2

No. of units

Risk involved

Villages only cattle infected

231

0.19

Villages only sheep&goat infected

186

0.22

Villages both cattle and sheep&goat infected

53

population attributable risk

0.04

Effect of Vaccination to Protect Cattle Population

31

In 15Inimportant provinces 15 provinces

Vaccination data for 15 provinces

units in which only cattle vaccinated

8738

units in which only sheep&goat vaccinated

1741

units in which cattle, sheep&goat vaccinated

2824 13303

Distribution of vaccination in different units in 15 provinces (April(April-Sept 2007)

units

Number

beef and sheep farm

12

Units

Total Number in Iran

vaccinated

% vaccinated

fattening sheep farm

7

beef and sheep farm

111

12

11

pasture

208

beef farm

1059

91

9

beef farm

91

city

357

141

39

city

141

77

21

Distribution of vaccination in units 12000 10000 8000 Number

Distribution of Vaccinated Units/Total Units

6000 4000

dairy and beef farm

77

2000

dairy and sheep farm

216

dairy farm

1293

p rp um

fa rm

v il la ge

ate w

rm

fa rm

sh ee p

rm fa

fa

da iry

sh ee p

an d

rm

cit y da iry

0

1

0 7

3

20

pasture

1985

208

10

13303

sheep farm

4580

185

4

villages

53564

11053

21

water pump

81

20

25

Total

71959

13303

18

FMD Diagnosis difficulties

• Clinical Differential Diagnosis is not easy

iry

an d

sh

be

ef

fa r

cit d an

da i ry

da

m ee p fa r m da de i ry er fa an fa rm tte d el ni k ng fa sh rm ee p fa rm pa s sh tur e ee p fa rm vi lla ge w at s er pu m p

y

• Mostly confused with other diseases

fa be r m ef fa rm

% of units vaccinated

17

• Mild disease in sheep and goat

ep sh e

11

1293

211

11053

45 40 35 30 25 20 15 10 5 0

nd fa

216

7713

deer and elk farm

185

village

Distribution of vaccinated epiepi-units

be e

366 1931

dairy farm

fattening sheep farm

sheep farm

water pump

da iry

an d

be ef

pa s tu re

be ef fa

be ef

an d sh fa ee tt e p ni fa ng rm sh ee p fa rm

0

dairy and beef farm dairy and sheep farm

– – – – –

PPR Blue tongue Contagious ecthyma Any stomatitis Any lameness

• Most of sheep and goat samples are Negative

32

Visit of a Herd in Zabol (FMD misdiagnosis)

Conclusion • Small number of villages are reported to be infected both for Cattle and Sheep&Goat • Probability of finding a village at a given time to be infected for Cattle and Sheep & Goat FMD is very low • Power of Sheep FMD is not enough to be able to infect cattle • Reports of FMD in sheep is overestimated (field investigations) because of Misdiagnosis.

Misdiagnosis.

• Vaccination coverage for cattle is not enough to cover the role of sheep in transmission of FMD • Last Year Type O PanAsia Epidemie in Iran did not changed the Focal Prevalence of FMD in cattle (two year analysis)

In my opinion Control and Eradication of disease in Endemic areas in Cattle can help Eradication of disease in Sheep and Goat Means that In Endemic areas the effect of Sheep&Goat FMD is very low in spread of disease and there is no need to vaccinate all the population of Sheep and Goat

33

Appendix 5

Investigation of Molecular epidemiology of Foot and Mouth disease virus serotype O and A in Iran in 2006-2007 Nazem Shirazi Iran Veterinary Organisation, Central Veterinary Laboratory, Tehran, Iran.

Summary The aim of the study described in this chapter was to determine the genetic diversity of FMD type O and A viruses isolated from Iran. For this purpose sequences of the complete VP1-coding gene of 116 FMD type O viruses isolated from Iran between 2005 and 2007 were determined. During my visit I was able to determine the sequences of 71 Iranian viruses (67 type O and 4 type A) and 13 others from various Middle Eastern countries. These sequences were also compared to the sequences accumulated at the WRLFMD database at Pirbright to examine the genetic relationship between Iranian type O viruses and viruses obtained from the Middle East and Indian subcontinent. Additionally preliminary work was undertaken to access the possibility of amplification of the complete FMDV genome in a small number of overlapping fragments for complete genome sequencing.

Introduction Foot and mouth disease virus exists in seven antigenically and genetically distinct serotypes and has a single-stranded positive sense RNA genome which undergoes very high level of mutational changes in the nature. During the course of outbreaks, the high rate of mutation in the replicating virus population can lead to the accumulation of genomic changes and eventually to the emergence of immunogenic variants. This poses serious threat to the FMD control campaigns in endemic areas. Nucleotide sequence analysis of the part of the gene coding for VP1 allows direct evaluation of the degree of relationship among FMD viruses (Knowles and Samuel, 2003). It has been found sensitive and precise in revealing the origin and course of epizootics and offers the possibility of preventing further outbreaks. The use of phylogenetic methodology helps in understanding FMDV molecular epidemiology and evolution (Samuel and Knowles, 2001; Knowles and Samuel, 2003).

Recently, Mason et al. (2003), Carrillo et al. (2005) and Cottam et al (2006) have shown the power of complete genome sequencing in molecular epidemiological studies, particularly in farm-to-farm tracing during the UK 2001 outbreak (Cottam et al., 2006).

Materials and methods Viruses and primers The names and origin of the FMDV isolates examined are shown in Table 1. Additionally, I was able to RT-PCR and sequence the following viruses from the Middle East region: O/JOR/5/2006, O/JOR/6/2006, O/JOR/7/2006, O/UAE/1/2007, O/UAE/2/2007, O/ISR/1/2007, O/ISR/3/2007, O/ISR/5/2007, O/ISR/7/2007, O/ISR/9/2007, A/JOR/2/2006, A/JOR/3/2006 and A/JOR/4/2006.

VP1 amplification: Three alternative primer combinations were used for reverse transcription– polymerase chain reaction (RT-PCR) of the VP1-coding region: O-1C244F/NK61, O-1C272F/NK61, and O-1C283F/NK61, which have amplicon sizes of 1,181, 1,153, and 1,142 bp, respectively (Table 2). Forward and reverse primer amounts were 20 and 40 pmol, respectively. Between 4 and 6 internal sequencing primers to ensure coverage of the VP1 region on both DNA strands (Table 2).

Complete genome amplification: Five primer sets (Table 3) were used to attempt to amplify the complete genomes of A/JOR/2/2006 and A/IRN/55/2006, barring the poly(C) tract, in overlapping fragments.

34

RNA extraction and RT-PCR amplification VP1 amplification: Total RNA was extracted from 460 µL of cell culture supernatant using RNeasy kits (Qiagen Ltd., Crawley, West Sussex, UK), according to the manufacturer’s instructions, and resuspended in 50 µL nuclease-free water. This RNA (5 µL) was used as the template in a 1-step RT-PCR (Ready-To-Go RT-PCR Beads; Amersham Pharmacia Biosciences, Chalfont St. Giles, Bucks, UK). The following thermal profile was used: 42°C for 30 min; 94°C for 5 min; 35 cycles of 94°C for 60 s; 60°C for 60 s; and 72°C for 90 s; followed by a final extension of 72°C for 5 min. PCR products were analyzed by electrophoresis on a 1.5% agarose-Tris-borate-EDTA gel containing 0.5 µg/mL ethidium bromide. DNA weight markers (GeneRuler 100 bp DNA Ladder Plus, Ready-ToUse; Fermentas, Inc., Hanover, MD, USA) were run alongside the samples to facilitate product identification and quantification. Post-PCR removal of deoxynucleoside triphosphates and primers was achieved using QIAquick PCR purification kits (QIAGEN), according to the manufacturer’s instructions.

Complete genome amplification: 10 µl of RNA (extracted as above), 4 µl 10 mM primer 5R1 (Table 3), 4 µl 10 mM random hexamers (Promega), 2 µl 10 mM deoxynucleoside triphosphates mix was incubated at 68°C for 3 min and then on ice for 3 min. Eighteen µl of freshly prepared RT mix (4 µl 10X RT buffer (Invitrogen), 8 µl 25 mM MgCl2, 4 µl 0.1 M dithiothreitol, 2 µl RNase OUT (Invitrogen) was added to the sample, followed by 2 µl SuperScript III reverse transcriptase (Invitrogen). The sample was then incubated at 42°C for 4 h, after which the reaction was stopped by incubation at 85°C for 5 min. The cDNA was then cleaned using QIAquick PCR purification kits (QIAGEN), eluting in 30 µl of elution buffer before storage at -20°C. Five overlapping PCR fragments covering the FMDV genome were amplified from each sample by using 47.5 µl of master mix (5 µl 10X buffer, 2 µl MgSO4, 1 µl 10 mM deoxynucleoside triphosphate mix, 1 µl 10 mM forward primer, 1 µl 10 mM reverse primer, 0.2 µl Platinum Taq Hi-Fidelity (Invitrogen), 37.3 µl nuclease-free water) plus 2.5 µl cDNA. The five primer sets consisted are shown in Table 3. Primer sets 1 to 4 were run on a PCR program cycle of initial denaturation at 94°C for 2 min and then 39 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min, ending with incubation at 72°C for 7 min. Primer set 5 was run on a cycle of initial denaturation at 94°C for 2 min and then 39 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 3 min, finishing with incubation at 72°C for 7 min. PCR products were cleaned up using QIAquick PCR purification kits (QIAGEN), eluting in 30 µl in elution buffer.

DNA sequencing PCR amplicons were sequenced by using the DTS Quick Start Kit (Beckman Coulter Inc., Fullerton, CA, USA) according to the manufacturer’s instructions and with the sequencing primers listed in the Table. The sequencing reactions were run on a CEQ8000 Automated Sequencer (Beckman Coulter) according to the manufacturer’s instructions.

Phylogenetic analysis Unrooted Neighbor-joining trees were constructed by using MEGA version 3.1 (Kumar et al., 2004).

The robustness of the tree topology was assessed with 1,000 bootstrap replicates as implemented within the same program.

Results The VP1-coding regions of all the viruses examined were successfully amplified using at least one of the primer sets used. All cDNA amplicons were sequenced on both strands. The relationships between the FMDV O and FMDV A isolates studied are depicted in Figs. 1 and 2, respectively.

Oligonucleotide primers for complete genome amplification were designed based on the sequences of FMDV A and O isolates from the Middle East (A/TUR/2006, PIADC; A/Iran/98, AY593791; A22/Iraq/64, AY593763; and O/Rey/Iran/66, AY593834). Five overlapping PCR’s covered the whole genome, except for the poly(C) tract. These PCR’s were tested on A/IRN/55/2006 and A/JOR/2/2006 with varying success (Fig. 3). The set 4F1/4R1 failed to work on both viruses, while 2F1/2R1 failed on A/IRN/55/2006. However, those that produced amplicons were devoid of nonspecific products and had adequate cDNA to perform sequencing.

35

Discussion The PanAsia strain (Middle East-South Asia topotype) has been the predominant FMDV O virus throughout Asia in recent years. It has been evolving in multiple locations so that many lineages are evident (Fig. 1). One such lineage appears to have originated in Gujarat, India in 2001 (Fig. 1) and is the only PanAsia lineage detect in Iran in 2005-2006 (results for 2007 are in progress) (Fig. 1).

Over the period 2005 to 2007 all FMD A viruses from Iran belonged to the new Irn-05 strain, with the exception of IRN/17/2005, which belonged to the Irn-96 strain (Fig. 2). The Irn-05 strain was also found in Saudi Arabia in 2005, Turkey in 2005-06, Jordan in 2006 and Pakistan in 2006 (Fig. 2). No other type A strains were found in these countries during 2005-2007 suggesting that Irn-05 may have largely supplanted other strains in the region.

Amplification of the complete FMDV A genomes of Middle Eastern isolates in five overlapping fragments should be easily possible with the optimization of primers. A set of sequencing primers will also need to be designed. Alternatively, other methods could be explored; for example, cutting the cDNA into smaller pieces and ligating DNA priming sites onto either end, thus making it possible to sequence all fragments with only two primers.

Acknowledgements It is with great pleasure to pass my gratitude to the staff of the Molecular Characterisation and Diagnostics Group (Jemma Wadsworth, Scott Reid and Donald King) and the Diagnosis of Vesicular Diseases Group (Nigel Ferris and Geoff Hutchings) and to David Paton, Head of the WRLFMD. Also great thanks to my family. Above all, a big thank you to Nick Knowles for making this work a reality.

36

Table 1. The origin of viruses examined during this study and their topotype/strain designations inferred from the VP1 sequence analysis.

Type

WRLFMD Ref. No.

A

IRN/1/2005

A

IRN/2/2005

A

IRN/4/2005

A

IRN/5/2005

A A A A

IRN/7/2005 IRN/10/2005 IRN/13/2005 IRN/14/2005

A

IRN/16/2005

A

IRN/17/2005

A

IRN/18/2005

A

IRN/22/2005

A

IRN/24/2005

A

IRN/25/2005

A

IRN/26/2005

A

IRN/27/2005

A

IRN/28/2005

A

IRN/29/2005

A

IRN/30/2005

A

IRN/31/2005

A

IRN/33/2005

A

IRN/34/2005

A

IRN/36/2005

A

IRN/38/2005

A

IRN/39/2005

A

IRN/40/2005

A

IRN/42/2005

A

IRN/43/2005

A

IRN/44/2005

Location Ghalch-Sadri, Qom, Qom Ghaleh-Sadri, Qom, Qom Shams-Abad, Qom Talkhqab, Markazy Bavanat, Fars Zanjan Ijrod, Zanjan Zanjan GhoyjehTapeh, Zanjan Piranshahr, W. Azarbijan Zanjan Dehloran, Kordestan Brojerd, Lorestan Shadegan, Khozestan Tarom, Zanjan Dehloran, Kordestan Sarab, E. Azarbaijan Sarab, E. Azarbaijan Lafarid, Qom Miyando Ab, W.Azarbaijan Najafabad, Esfahan Shirvan, Brojerd, Lorestan Roniz-Sofla, Estahban, Fars Amir-Abad, Bokan, W. Azarbijan Bokan, W. Azarbijan Arak, Markazi Hallan, Sah Neh, Kerman Shah Marraghea, East Azerbaijan Ghare Ziaodien, West

Species

Date Collected

Sender Ref

Topotype

Strain

Cattle

04/04/2005

84-1

ASIA

Irn-05

Cattle

05/04/2005

84-2

ASIA

Irn-05

Not Known

11/04/2005

84-4

ASIA

Irn-05

Cattle

20/04/2005

84-5

ASIA

Irn-05

Sheep Cattle Cattle Cattle

15/05/2005 28/05/2005 30/05/2005 01/06/2005

84-6 84-9 84-8 84-17

ASIA ASIA ASIA ASIA

Irn-05 Irn-05 Irn-05 Irn-05

Cattle

07/06/2005

84-13

ASIA

Irn-05

Cattle

12/06/2005

84-15

ASIA

Irn-96

Cattle

12/06/2005

84-18

ASIA

Irn-05

Cattle

11/08/2005

84-14

ASIA

Irn-05

Cattle

11/08/2005

84-16

ASIA

Irn-05

Cattle

13/08/2005

84-12

ASIA

Irn-05

Cattle

15/08/2005

84-17

ASIA

Irn-05

Cattle

20/08/2005

84-13

ASIA

Irn-05

Cattle

21/08/2005

84-6

ASIA

Irn-05

Cattle

21/08/2005

84-8

ASIA

Irn-05

Cattle

21/08/2005

84-18

ASIA

Irn-05

Cattle

24/08/2005

84-7

ASIA

Irn-05

Cattle

05/10/2005

184-341 E

ASIA

Irn-05

Cattle

08/10/2005

184-391 L

ASIA

Irn-05

Cattle

08/10/2005

184-421 F

ASIA

Irn-05

Cattle

12/10/2005

184-221WA

ASIA

Irn-05

Cattle

12/10/2005

ASIA

Irn-05

Cattle

12/10/2005

ASIA

Irn-05

Cattle

15/10/2005

184-291 K

ASIA

Irn-05

Cattle

15/10/2005

184-351 EA

ASIA

Irn-05

Cattle

16/10/2005

184-241 WA

ASIA

Irn-05

184-251 WA 184-361M

37

Type

WRLFMD Ref. No.

A

IRN/50/2005

A

IRN/51/2005

A

IRN/53/2005

A

IRN/54/2005

A

IRN/55/2005

A

IRN/5/2006

A

IRN/7/2006

A

IRN/54/2006

A

IRN/57/2006

A

IRN/3/2007

A

IRN/15/2007

O O

IRN/8/2005 IRN/9/2005

O

IRN/12/2005

O O

IRN/20/2005 IRN/21/2005

O

IRN/23/2005

O

IRN/8/2006

O

IRN/9/2006

O

IRN/10/2006

O

IRN/11/2006

O

IRN/12/2006

O

IRN/13/2006

O

IRN/14/2006

O

IRN/15/2006

O

IRN/16/2006

O

IRN/17/2006

Location Azerbaijan Delbarijan, Brojerd, Lorestan Jolfa, East Azerbaijan Haji-Abad, Gume, Gume (Qom?) Damshahr, Gume, Gume (Qom?) Pakdasht, Varamin, Tehran Eslamshar, Teheran Falavarjan, Esfahan Sirk-Mazrdeh, Dahaghan, Isfahan Sirk-Mazrdeh, Dahaghan, Isfahan Amany Pain, Booin Zahra, Gazvin, Central Ardokon, Yazd, Central Kordestan Kordestan Shahr-Babak, Kerman Kazeron, Fars Sepidan, Fars Tekab, W Azarbijan Booinzahra, Qazvin Jafarieh, Qom Dam Shar, Qom Tabas, Yazd Chalous, Mazanderan Miandoab, West Azerbaijan Parsabad, Ardebil Mahabad, West Azerbaijan Komijan, Markarzy Gonbad Kawos, Golestan

Species

Date Collected

Sender Ref

Topotype

Strain

Cattle

23/10/2005

184-401 L

ASIA

Irn-05

Cattle

24/10/2005

184-371 EA

ASIA

Irn-05

Cattle

30/10/2005

184-271 Z

ASIA

Irn-05

Cattle

30/10/2005

184-281G

ASIA

Irn-05

Cattle

09/11/2005

184-381 T

ASIA

Irn-05

Cattle

18/04/2006

Iran-3

ASIA

Irn-05

Cattle

23/04/2006

Iran-8

ASIA

Irn-05

Cattle

00/11/2006

916/A05

ASIA

Irn-05

Cattle

00/11/2006

267/A05

ASIA

Irn-05

Cattle

24/01/2007

IR 71

ASIA

Irn-05

Goats

20/02/2007

IR 66

ASIA

Irn-05

Sheep Cattle

25/05/2005 27/05/2005

84-7 84-14

ME-SA ME-SA

PanAsia PanAsia

Sheep

29/05/2005

84-11

ME-SA

PanAsia

Cattle Cattle

08/08/2005 10/08/2005

84-11 84-9

ME-SA ME-SA

PanAsia PanAsia

Cattle

11/08/2005

84-15

ME-SA

PanAsia

Cattle

29/04/2006

Iran-4

ME-SA

PanAsia

Cattle

09/05/2006

Iran-2

ME-SA

PanAsia

Cattle

09/05/2006

Iran-1

ME-SA

PanAsia

Cattle

12/09/2006

IR 25

ME-SA

PanAsia

Cattle

27/09/2006

IR 20

ME-SA

PanAsia

Cattle

28/09/2006

IR 21

ME-SA

PanAsia

Cattle

01/10/2006

IR 24

ME-SA

PanAsia

Cattle

02/10/2006

IR 22

ME-SA

PanAsia

Cattle

04/10/2006

IR 12

ME-SA

PanAsia

Cattle

04/10/2006

IR 23

ME-SA

PanAsia

38

Type

WRLFMD Ref. No.

O

IRN/18/2006

O

IRN/19/2006

O

IRN/20/2006

O

IRN/21/2006

O

IRN/22/2006

O

IRN/23/2006

O

IRN/24/2006

O

IRN/25/2006

O

IRN/26/2006

O

IRN/27/2006

O

IRN/28/2006

O

IRN/29/2006

O

IRN/30/2006

O

IRN/31/2006

O

IRN/32/2006

O

IRN/33/2006

O

IRN/34/2006

O

IRN/35/2006

O

IRN/37/2006

O

IRN/38/2006

O

IRN/39/2006

O

IRN/42/2006

O

IRN/43/2006

O

IRN/44/2006

Location Shahrood, Semnan Harsin, Kermanshah Jahrom, Fars Farahan, Markazi Bojnord, North Korasan MannehSamalghan, North Khorasan Talesh, Gylan Kerman, Kerman Varamin, Tehran Hamadan, Hamadan Booinzahra, Quzvin Maimoud Abad, Sanandaj, West Azerbaijan Bitoryan, Oshnavieh, West Azerbaijan Qum Sheshlaga Balla, Saveh, Markazi Igdir Sofla, Gonbad, Golestan Shahrkord, Chahr-Mahal Bakhtyar Shahrkord, Chahr-Mahal Bakhtyar Farah, Isfahan Shar Abad Kord, Maneh Samalghan, Norta Khorasan Dizage Fathi, Uromieh, West Azerbaijan Farahan, Markazi Amir Kansy, Meshkinshahr, Ardebil Gazzeran, Jaafarieh, Qum

Species

Date Collected

Sender Ref

Topotype

Strain

Cattle

05/10/2006

IR 19

ME-SA

PanAsia

Cattle

07/10/2006

IR 18

ME-SA

PanAsia

Cattle

09/10/2006

IR 11

ME-SA

PanAsia

Cattle

09/10/2006

IR 13

ME-SA

PanAsia

Cattle

09/10/2006

IR 15

ME-SA

PanAsia

Cattle

09/10/2006

IR 16

ME-SA

PanAsia

Cattle

09/10/2006

IR 26

ME-SA

PanAsia

Cattle

11/10/2006

IR 14

ME-SA

PanAsia

Cattle

15/10/2006

IR 28

ME-SA

PanAsia

Cattle

17/10/2006

IR 17

ME-SA

PanAsia

Cattle

25/10/2006

IR 27

ME-SA

PanAsia

Cattle

00/09/2006

1072

ME-SA

PanAsia

Cattle

02/11/2006

A-36

ME-SA

PanAsia

Cattle

04/11/2006

IR-38

ME-SA

PanAsia

Cattle

07/11/2006

A-48

ME-SA

PanAsia

Sheep

08/11/2006

IR-43

ME-SA

PanAsia

Cattle

11/11/2006

A-49-1

ME-SA

PanAsia

Cattle

11/11/2006

A-49-2

ME-SA

PanAsia

Cattle

19/11/2006

IR-32

ME-SA

PanAsia

Cattle

19/11/2006

A-34

ME-SA

PanAsia

Cattle

20/11/2006

A-35

ME-SA

PanAsia

Cattle

20/11/2006

IR-46

ME-SA

PanAsia

Sheep

21/11/2006

IR-29

ME-SA

PanAsia

Cattle

21/11/2006

A-39

ME-SA

PanAsia

39

O

WRLFMD Ref. No. IRN/45/2006

O

IRN/46/2006

O

IRN/47/2006

O

IRN/48/2006

O

IRN/49/2006

O

IRN/50/2006

O

IRN/51/2006

O

IRN/52/2006

O

IRN/53/2006

O

IRN/55/2006

O

IRN/56/2006

O

IRN/58/2006

O

IRN/59/2006

O

IRN/60/2006

O

IRN/61/2006

O

IRN/64/2006

O

IRN/1/2007

O

IRN/4/2007

O

IRN/5/2007

O

IRN/6/2007

O

IRN/7/2007

O

IRN/8/2007

O

IRN/9/2007

Type

Location Abadeh, Fars Tazehkaind, Uromieh, West Azarbaijan Shahr Ray, Tehran Rezvanshar, Saddogh, Yazd Saddogh, Yazd Shahzand, Markazi Gawmishabad, Dezphol, Khozestan Kaleh Mosalman, Zarinshahr, Isfahan Sirk-Mazrdeh, Dahaghan, Isfahan Sirk-Mazrdeh, Dahaghan, Isfahan Sirk-Mazrdeh, Dahaghan, Isfahan Koh Sohz, Marvdasht, Fars Borastah, Shiraz, Fars Botastah, Shiraz, Fars Binzara, Shiraz, Fars Boraftab, Shiraz, Fars Hamedan, Hamedan, West Chakor Midan, Amlash, Gilan, North Ardogan, Nishabour, Central Khorasan, East Lalyeh, Sari, Mazandaran, North Semnan, Semnan, Central Kadvny, Shiraz, Fars, South Iyrood, Zanjon, Central

Cattle

Date Collected 21/11/2006

Sender Ref IR-51

Cattle

23/11/2006

Cattle

Species

Topotype

Strain

ME-SA

PanAsia

IR-37

ME-SA

PanAsia

25/11/2006

IR-30

ME-SA

PanAsia

Cattle

25/11/2006

IR-40

ME-SA

PanAsia

Cattle

25/11/2006

IR-41

ME-SA

PanAsia

Cattle

25/11/2006

IR-47

ME-SA

PanAsia

Buffalo

25/11/2006

IR-50

ME-SA

PanAsia

Cattle

27/11/2006

IR-31

ME-SA

PanAsia

Cattle

27/11/2006

A-33

ME-SA

PanAsia

Cattle

00/11/2006

1183/A87

ME-SA

PanAsia

Cattle

00/11/2006

967/O

ME-SA

PanAsia

Cattle

05/12/2006

IR-52

ME-SA

PanAsia

Cattle

05/12/2006

IR-55

ME-SA

PanAsia

Cattle

05/12/2006

IR-56

ME-SA

PanAsia

Cattle

06/12/2006

IR-54

ME-SA

PanAsia

Cattle

Not Known

IR-55

ME-SA

PanAsia

Cattle

13/01/2007

IR 69

ip

ip

Cattle

01/02/2007

IR 64

ip

ip

Cattle

01/02/2007

IR 72

ip

ip

Cattle

01/02/2007

IR 73

ip

ip

Cattle

03/02/2007

IR 75

ip

ip

Cattle

07/02/2007

IR 65

ip

ip

Cattle

08/02/2007

IR 62

ip

ip

40

Type

WRLFMD Ref. No.

O

IRN/10/2007

O

IRN/11/2007

O

IRN/13/2007

O

IRN/14/2007

O

IRN/16/2007

O

IRN/17/2007

O

IRN/18/2007

O

IRN/19/2007

O

IRN/20/2007

Not Known

O

IRN/21/2007

Not Known

O

IRN/22/2007

Not Known

O

IRN/23/2007

Not Known

O

IRN/24/2007

Not Known

Location Shirabad, Tabas, Yazd, central Koran Zvrilyeh, Evrdiy, North Khorason, North Deh Kousar, Shazand, Markazi, Central Sarvarabad, Bokan, West Azerbaijan, West Yaparyeh, Qom, Qom, Central Ray, Teheran, Central Eslam Shahr, Tehran, Central Aborash, Modmeh, East Azerbaijan, East

Species

Date Collected

Sender Ref

Topotype

Strain

Goats

10/02/2007

IR 67

ip

ip

Cattle

10/02/2007

IR 68

ip

ip

Cattle/ Sheep/ Goats

12/02/2007

IR 63

ip

ip

Cattle

12/02/2007

IR 74

ip

ip

Cattle

20/02/2007

IR 76

ip

ip

Cattle

25/02/2007

IR 77

ip

ip

Cattle

25/02/2007

IR 78

ip

ip

Cattle

27/02/2007

IR 61

ip

ip

Not Known

575

ip

ip

Not Known

585

ip

ip

Not Known

593

ip

ip

Not Known

597

ip

ip

Not Known

602

ip

ip

Not Known Not Known Not Known Not Known Not Known

ip, in progress (the analysis of these virus sequences was not completed and is currently being finished by WRLFMD staff).

41

Table 2. Oligonucleotide primers used for RT-PCR and cycle sequencing of foot-and-mouth disease viruses.

Primer

Primer sequence (5' to 3')

Location on the FMDV genome

Sense

Gene

Position*

Use

O-1C244F

GCAGCAAAACACATGTCAAACACCTT

+

1C

2469-2494

RT-PCR

O-1C272F

TBGCRGGNCTYGCCCAGTACTAC

+

1C

2497-2519

RT-PCR

O-1C283F

GCCCAGTACTACACACAGTACAG

+

1C

2508-2530

RT-PCR

NK61

GACATGTCCTCCTGCATCTG

-

2B

3630-3649

RT-PCR

NK72

GAAGGGCCCAGGGTTGGACTC

-

2A/2B

3558-3578

sequencing

O-1C499F

TACGCGTACACCGCGTC

+

1C

2724-2740

sequencing

O-1C583F

GACGGYGAYGCICTGGTCGT

+

1C

2808-2827

sequencing

O-1D296F

ACAACACCACCAACCCAAC

+

1D

3181-3199

sequencing

O-1D628R

GTTGGGTTGGTGGTGTTGT

-

1D

3181-3199

sequencing

* position on the genome of O1/Kaufbeuren/FRG/66 (EMBL/GenBank accession no.

X00871).

Table 3. Primer sequences used for complete genome amplification.

Primer

Primer sequence (5' to 3')

Sense

Length

Location on the FMDV genome*

1F1

TTGAAAGGGGGCGCTAGGGTYTCA

Forward

24

0001-0024

1R1

GGGTGAAAGGCGGRCTYCGGGT

Reverse

22

0351-0372

2F1

CCCCCTAAGTTACCACCGTC

Forward

20

0441-0460

2R1

GTTRATRATRCTNCCAGTGTTGCCTG

Reverse

26

1805-1830

3F1

CAYGCTGGYATYTTCYTGAAAGGACA

Forward

26

1570-1595

3R1

CCIGTGGCCAATTCCTCAAACGC

Reverse

23

4135-4157

4F1

GTCATTGACCTCATGCAAACMCA

Forward

23

3481-3503

4R1

GTCTCTTGCGAGTCTCGCGGATC

Reverse

23

5661-5683

5F1

CCWCARCCRCCCCTCCAGAACGT

Forward

23

5338-5360

5R1

GGCGGCCGCTTTTTTTTTTTTTTT

Reverse

24

8252-8266

*, position on the genome of A/Iran/98 (AY593791).

42

No. of Taxa : 149 Data File : n:\evd\meg\db\fmdv\o\IRN2006G.meg Data Title : FMDV O Iran 2006 Data Type : Nucleotide (Coding) Analysis : Phylogeny reconstruction Tree Inference : ============================== Method : Neighbor-Joining Phylogeny Test and options : Bootstrap (500 replicates; seed=1234) Include Sites : ============================== Gaps/Missing Data : Pairwise Deletion Codon Positions : 1st+2nd+3rd+Noncoding Substitution Model : ============================== Model : Nucleotide: Kimura 2-parameter Substitutions to Include : d: Transitions + Transversions Pattern among Lineages : Same (Homogeneous) Rates among sites : Uniform rates No. of Sites : 639 No Of Bootstrap Reps = 500 Only bootstrap values of 70% and above are shown

98

O/IRN/60/2006 O/IRN/64/2006 O/UAE/1/2007 98 O/UAE/2/2007 O/IRN/59/2006 O/IRN/47/2006 O/IRN/58/2006 O/IRN/27/2006 O/IRN/24/2006 O/IRN/16/2006 O/IRN/30/2006 O/IRN/19/2006 O/IRN/44/2006 O/IRN/28/2006 O/IRN/42/2006 O/IRN/14/2006 O/IRN/20/2006 O/IRN/34/2006 O/IRN/23/2006 O/IRN/12/2006 O/IRN/13/2006 O/IRN/46/2006 O/IRN/8/2006 O/IRN/9/2006 O/IRN/10/2006 70 O/IRN/21/2006 O/IRN/26/2006 O/IRN/61/2006 O/IRN/18/2006 O/IRN/31/2006 O/IRN/17/2006 O/IRN/38/2006 O/IRN/39/2006 O/IRN/51/2006 O/JOR/5/2006 O/ISR/5/2007 98 O/ISR/7/2007 O/ISR/9/2007 78 O/ISR/1/2007 O/ISR/3/2007 O/JOR/6/2006 O/JOR/7/2006 O/IRN/35/2006 O/IRN/32/2006 O/IRN/37/2006 O/IRN/33/2006 O/IRN/55/2006 O/IRN/15/2006 O/IRN/48/2006 75 O/IRN/49/2006 O/IRN/52/2006 O/IRN/45/2006 O/IRN/29/2006 O/IRN/43/2006 O/IRN/11/2006 O/IRN/50/2006 O/IRN/53/2006 O/IRN/56/2006 O/IRN/25/2006 O/PAK/12/2006 87 O/PAK/14/2006 O/PAK/11/2006 O/PAK/15/2006 O/PAK/4/2006 O/PAK/9/2006 O/PAK/6/2006 O/PAK/16/2006 O/PAK/8/2006 O/PAK/10/2006 O/IRN/22/2006

PanAsia

O/NEP/4/2003 (DQ165059) O/BHU/48/2003 O/BHU/49/2003 (DQ164867) O/NEP/5/2003 (DQ165060) O/NEP/6/2003 (DQ165061) O/NEP/2/2003

93

76 85

87

97

9. West Azerbaijan 2. Qom 3. Markazi 27. Golestan 19. Fars 24. Esfahān 15. Khuzestan 16. Chaharmahal and Bakhtiari 1. Tehran 23. Yazd 6. Ardebil 28. North Khorasan 12. Kermanshah 11. Hamedan 5. Gilan 4. Qazvin 26. Mazanderan 25. Semnan 10. Kurdistan

98

99

76

91

O/IRN/8/2005 O/IRN/21/2005 O/IRN/9/2005 O/IRN/23/2005 O/IRN/12/2005

O/PAK/9/2005 O/PAK/13/2005 O/BHU/33/2004 (DQ165046) O/BHU/39/2004 (DQ164870) O/BHU/30/2004 (DQ165045) 93 O/BHU/26/2004 (DQ165043) 75 O/BHU/28/2004 (DQ165044) O/MAY/6/2003 (DQ165058) O/MAY/6/2005 O/MAY/3/2005 99 O/MAY/4/2005 O/MAY/7/2005 O/IND/136/01* (IVRI) O/IND/151/01* (IVRI) 99 O/IND/155/01* (IVRI) O/IRN/1/2000 (DQ164892) O/TUR/5/2000 (DQ164983) O/IRQ/30/2000 (AJ303499 DQ165057) 76 O/PAK/1/2005 83 O/PAK/15/2005 O/PAK/19/2005 O/IRN/67/2001 (DQ164897) O/PAK/14/2005 O/IND/34/00* (IVRI) 99 O/IRN/2/2003 (DQ165048) O/IRN/16/2003 (DQ165052) 98 O/KUW/2/2006 87 99 O/KUW/3/2006 O/SAU/8/2005 O/SAU/14/2005 O/IRN/8/2004 (DQ165054) 97 O/TUR/2/2001 (DQ164985) O/TUR/12/2002 (DQ164988) O/AFG/16/2003 (DQ165035) O/IRN/9/99 (AJ318838) O/IRN/24/99 (AJ318839) O/SAR/1/2000 (AJ318860) 99 O/UKG/12/2001 (AJ311724) 99 O/IRN/6/2004 (DQ165053) O/ISR/2/2004 (DQ164900) 92 O/ISR/1/2005 97 O/ISR/2/2005 O/Snir/ISR/05* (DQ372716) 83 O/ISR/1/2006 99 O/ISR/2/2006 O/TUR/1/2005 O/TUR/5/2005 O/TUR/2/2005 O/TUR/3/2005 O/TUR/4/2005 O/TUR/5/2002 (DQ164987) O/PAK/1/2003 (DQ165065) O/IRN/15/2004 (DQ165055) 99 O/PAK/15/2002 (DQ165062) O/IRN/6/2003 (DQ165050) 97 O/IRN/20/2004 (DQ165056) O/IND/53/79 (AF292107) O/PAK/17/2003 (DQ165069) O/IND/R2/75* (AF204276) O1/Manisa/TUR/69 (AJ251477) O/ISR/2/88 (DQ164899) 99 O/MOR/1/91 O/MAY/1/2002 (DQ164923) O/TAI/189/87* (TRRL) O/HKN/6/83 O/HKN/9/2005 84 O/PHI/5/95 (DQ164946) O1/BFS 1860/UK/67

Irn2001

ME-SA 81

SEA Cathay Euro-SA

85

99

0.02

Figure 1. Mid-point rooted Neighbor-joining tree showing the relationships between the foot-andmouth disease virus serotype O isolates studied.

43

No. of Taxa : 120 Data File : n:\evd\meg\db\fmdv\a\IRN2007A.meg Data Title : FMDV A (IRN2007A.LST) Data Type : Nucleotide (Coding) Analysis : Phylogeny reconstruction Tree Inference : ============================== Method : Neighbor-Joining Phylogeny Test and options : Bootstrap (1000 replicates; seed=88958) Include Sites : ============================== Gaps/Missing Data : Pairwise Deletion Codon Positions : 1st+2nd+3rd+Noncoding Substitution Model : ============================== Model : Nucleotide: Kimura 2-parameter Substitutions to Include : d: Transitions + Transversions Pattern among Lineages : Same (Homogeneous) Rates among sites : Uniform rates No. of Sites : 645 No Of Bootstrap Reps = 1000 Only bootstrap values of 70% and above are shown

9. West Azerbaijan 2. Qom 3. Markazi 27. Golestan 19. Fars 24. Esf ahān 15. Khuzestan 16. Chaharmahal and Bakhtiari 1. Tehran 23. Yazd 6. Ardebil 28. North Khorasan 12. Kermanshah 11. Hamedan 5. Gilan 4. Qazvin 26. Mazanderan 25. Semnan 10. Kurdistan 7. Zanjan 8. East Azerbaijan 14. Lorestan 21. Sistan and Baluchistan

100

98

99 100

A/TUR/6/2005 A/TUR/2/2006 A/TUR/12/2005 A/IRN/31/2005 A/TUR/7/2005 A/TUR/3/2006 A/TUR/9/2005 A/TUR/18/2006 A/TUR/1/2006 A/TUR/10/2005 A/TUR/11/2006 74 A/TUR/19/2006 A/TUR/9/2006 A/TUR/14/2006 A/TUR/7/2006 A/TUR/17/2006 A/TUR/8/2006 A/TUR/10/2006 A/TUR/6/2006 A/TUR/20/2006 A/IRN/38/2005 A/IRN/39/2005 A/TUR/11/2005 A/TUR/4/2006 A/TUR/16/2006 A/JOR/4/2006 A/JOR/2/2006 99 A/JOR/3/2006 A/IRN/42/2005 A/IRN/7/2006 A/TUR/12/2006 A/IRN/51/2005 70 A/IRN/54/2005 A/IRN/28/2005 A/IRN/29/2005 A/IRN/55/2005 A/IRN/43/2005 A/IRN/18/2005 A/IRN/14/2005 A/IRN/10/2005 A/IRN/16/2005 A/IRN/13/2005 A/IRN/26/2005 A/IRN/33/2005 A/IRN/5/2005 A/IRN/2/2005 89 A/IRN/4/2005 A/IRN/1/2005 (EF208769) A/IRN/25/2005 A/PAK/1/2006 A/PAK/3/2006 100 A/IRN/7/2005 A/IRN/57/2006 A/IRN/44/2005 94 A/TUR/8/2005 72 A/SAU/15/2005 98 A/SAU/16/2005 A/IRN/54/2006 A/IRN/24/2005 86 A/IRN/34/2005 A/IRN/36/2005 A/IRN/53/2005 A/IRN/27/2005 A/IRN/5/2006 A/IRN/22/2005 A/IRN/30/2005 A/TUR/324/2007* (FMDI-Ankara) A/IRN/40/2005 A/IRN/50/2005 89 A/IRN/41/2003 A/IRN/7/2004 A/IRN/3/2007 A/PAK/5/2006 A/IRN/15/2007 A/IRN/2/2002 A/IRN/7/2003 100 A/IRN/32/2001 A/IRN/34/2001 100 A/IRN/10/2003 A/PAK/28/2002 A/PAK/9/2003 100 A/IRN/87 A/SAU/41/91 A/IRN/22/99 (EF208772) A/TUR/5/2003 100 A/TAI/118/87* (EF208777) A/TAI/2/97 (EF208778)

Irn-05

Irn-99

100

ASIA

A/IND/17/77*

93

74

A22/IRQ/24/64 (AJ251474) A/BHU/41/2002 A/BHU/7/2003 A/MEU/49/03* (India) (EF120401)

100

A/BHU/27/2003 88 79 100

A/APR/51/05* (India) (EF120400) A/APS/44/05* (India) (EF120402) A/APS/68/05* (India) (EF120403)

A/SAU/23/86

100

EURO-SA 98

AFRICA

73 95

A/TUR/17/2001 A/TUR/4/2003 A/SYR/5/2002 100 A/TUR/14/2002 A/TUR/2/2003 A/IRN/1/96 (EF208771) A/IRN/32/2004 97 A/IRN/17/2005 A/IRN/5/2003 97 A/IRN/9/2003 100 A/IRN/33/2004 A/IRQ/2/2002 A/IRQ/107/2002 A/IRQ/24/2002 A/IRQ/59/2002 A/IRQ/33/2002 A/IRQ/60/2002 A24/Cruzeiro/BRA/55 (AJ251476) A/K5/80* A/EGY/1/2006 (EF208757) A/K35/80* A/ERI/3/97 A/ERI/3/98 100

Irn-96

0.02

Figure 2. Mid-point rooted Neighbor-joining tree showing the relationships between the foot-andmouth disease virus serotype A isolates studied.

44

M

A/JOR/2/2006 1 2 3 4 5

6

1

2

A/IRN/55/2006 3 4 5 6

M

Fig. 3. Agarose gel of amplicons generated from RTPCRs using primers spanning the complete FMDV genome for A/JOR/2/2006 and A/IRN/55/2006.

Primers were: 1) 1F1/1R1; 2) 2F1/2R1; 3) 3F1/3R1; 4) 4F1/4R1; 5) 5F1/5R1; 6) negative control; M) DNA markers (3000, 2000, 1500, 1200, 1031, 900, 800, 700, 600, 500, 400, 300, 200 & 100 bp)

45

Appendix 6

Review of FMD in wildlife in Israel

Hagai Yadin, Boris Gelman and Rony King

Year

Samples

Sn Pos.

11

2

1988

19

1

1989

4

0

1990

-

-

1991

1

0

1992

33

12

Virus Isol.

17

2

1994

117

23

1995

388

37

2

Sum

590

77(13%)

8

Wild boars FMD Sero. Pos. Per District 1987 - 1995 East. Upp. Galilee

23/276 42/179 8.3

23.5

Wes. Up. Galilee

West.

Galilee

Golan heights

Issachar Heights

Total

Nr. Sending's

120

59

179

Nr. Blood samples

1261

443

1704

92 (7.3%)

32

124

(7.2%)

(7.2%)

Survey of wildlife for FMD Virus isolation (carriers) 1986-1995 Gazelles

Wild Boars

Nr. Sending’s

155

47

Nr. OP samples

1131

47

Nr. FMDV Pos.

3 (0.26%)

6

1993

Golan Heights

survey 1986 - 1993

FMD SN Positive

Wild Boars – FMD survey 1987 - 1995 1987

Gazelles - FMD serological

Jordan valley

Sum.

1/26

6/31

5/79

77/591

3.8

19.3

6.3

13% Pos.

8

Is there any dependence between FMD outbreaks in domestic farms / wildlife Year

No. Outbreaks

% Pos Gazelles

% Pos. Boars

1985

25

-

1986

0

6

1987

12

7.5

1988

8

7.6

18 (2/11)

1989

20

10.5

5.2 (1/19)

1990

8

17.2

-

1991

2

6.2

0

1992

1

2.8

36 (12/33)

1993

3

3.6

12 (2/17)

1994

26

7.4

20 (23/117)

1995

5

9.5 (37/388)

46

No. of FMD Outbreaks in relation to Sero positive Wildlife 40

FMD Outbreak in Gazelles 2007

% Pos. Gaz. % Pos. Boar No. Outbreaks

30 20 10

Years

1995

1993

1991

1989

1987

1985

0

FAO World Reference Laboratory for Foot and Mouth Disease* REPORT FOR FEBRUARY 2007 Country WRL for FMD Animal Date of Collection Sample Identification VI/ELISA RT-PCR Final report ISR 1/2007 ISR 2/2007 ISR 3/2007 ISR 4/2007 ISR 5/2007 ISR 6/2007 ISR 7/2007 ISR 8/2007 ISR 9/2007 ISR 10/2007

Goat Goat Cattle Cattle Cattle Cattle Cattle Cattle Cattle Cattle

01.01.07 01.01.07 03.01.07 03.01.07 24.01.07 24.01.07 26.01.07 26.01.07 27.01.07 27.01.07

UNITED ARAB UAE 1/2007 Gazelle EMIRATES UAE 2/2007 Gazelle

O Positive O O Positive O O Positive O O Positive O O Positive O O Positive O O Positive O O Positive O O Positive O O Positive O

10.01.07 10.01.07

O Positive O O Positive O

FMD outbreak in wild life 22/03 Gazelles 50-120

1300 gazelles

died

8 virus isolation type o

47

Appendix 7

2007: FMD Report Cases (~205)

FMD 2007 UK Overview of outbreaks and laboratory involvement

David Paton Pirbright Laboratory

Phase 1: August/Normandy • 3 Aug: IP1 (3 locations) • 6 Aug: IP2 (3 locations) • 3 contact herds culled • 24 Aug: PZs lifted • 8 Sep: SZ lifted • Origin: contamination from Pirbright site

Index Case – IP1b

Index Case – IP1b (a)

Map 1: Normandy site

(b)

A – stream running through fields Cattle here on Sat

B – cattle drooling (c) Stream

Cattle here on Thurs/ Fri

6 sheep

C – Recent 3 day tongue lesion

Scale bar: 100m

(e) Kill box

2 sows, 7 piglets

(d)

(f)

D – Older 8 to 9 day old tongue lesion E and F – 5 to 6 day interdigital foot lesions

49

Report on FMDV O UK 2007 VP1 sequences Software: MEGA 3.1 No. of Taxa : 35 Data File : n:\evd\meg\db\fmdv\o\UKG2007c.meg Data Title : O UK 2007 Data Type : Nucleotide (Coding) Analysis : Phylogeny reconstruction Tree Inference : ============================== Method : Neighbor-Joining Phylogeny Test and options : Bootstrap (1000 replicates; seed=69221) Include Sites : ============================== Gaps/Missing Data : Pairwise Deletion Codon Positions : 1st+2nd+3rd+Noncoding Substitution Model : ============================== Model : Nucleotide: Kimura 2-parameter Substitutions to Include : d: Transitions + Transversions Pattern among Lineages : Same (Homogeneous) Rates among sites : Uniform rates No. of Sites : 639 No Of Bootstrap Reps = 1000 Only bootstrap values of 70% and above are shown

100

EURO-SA

ME-SA

100

O/UKG/11/2007 O/UKG/94/2007 O/UKG/150/2007 O/UKG/7/2007 O/UKG/93/2007 O/UKG/144/2007 O/UKG/91/2007 O/UKG/124/2007 O/UKG/9/2007 93 O/UKG/126/2007 O/UKG/95/2007 O/UKG/92/2007 O1/BFS 1860/UK/67 (AY593816) O1/BFS 1860/UK/67 (AY593815) O1/BFS 1860/UK/67 (J02185) O1/BFS 1860/UK/67 (E00225) 73 70 O1/Wettmar/FRG/1/88 75 O1/Burgw edel/87 O1/Burdorf /FRG/2/88 96 O/Zusmarshausen/FRG/84 82 O1/Kaufbeuren/FRG/66 (X00871) O1/Lausanne/SWI/65 O1/Brugge/BEL/63 (AY593817) O1/Campos/BRA/58 (AY593818) O1/Campos/BRA/58 (AY593819) O1/Campos/58 (M95781) O1/Campos/58 (K01201) 81 O/Campos/BRA/58 (PBEP B99-04 CV epi trit O/Campos/BRA/58 BHK8 (15-03-90) O/Campos/BRA/58 (BK3 05-11-2004) O6/UK/1/24 (AY593829) O1/Manisa/TUR/69 (AJ251477) O/SAR/1/2000 (AJ318860) O/UKG/12/2001 (AJ311724) 100 O/UKG/35/2001 (AJ539141) 70

Pirbright site: shared IAH / Merial Facility

0.02

Possible sources of FMD O1 BFS virus at Pirbright • IAH users (<10 ml) – – – –

Two different stocks of virus in use July 12th , 16th , 17th , 18th , 23rd , 24th , 25th One close to original 1967 isolate One derived from Wellcome vaccine strain

• Merial (12,000 L) – – – –

Merial vaccine strain Large vaccine batches started July 17th & 19th Centrifuge steps July 19th and 23rd Discharge of citric acid treated cell slurry July 22nd and 25th

Possible transmission routes for FMD virus from Pirbright • Aerosolised virus transported by wind • Water borne virus • Fomites accidentally transported by persons or vehicles • Fomites spread by animals mechanically • Intermediate animal hosts that became infected • Deliberate introduction

Wind conditions favouring spread from Pirbright 15th, 19th, 20th and 23rd July

South Farnborough - Wind

Source of air sampled by cattle on IP1b on 23rd July

350

250 200 150 100

05/08/00

04/08/00

03/08/00

02/08/00

01/08/00

31/07/00

30/07/00

29/07/00

28/07/00

27/07/00

26/07/00

25/07/00

24/07/00

23/07/00

22/07/00

21/07/00

20/07/00

19/07/00

18/07/00

17/07/00

16/07/00

0

15/07/00

50 14/07/00

Direction

300

50

Precipitation recorded at the Institute for Animal Health

Drain Runs from Merial and IAH

A total of 63.6mm were recorded at midnight on 21 July (data refers to the previous 24 hour period).

Pilot digs on 25th July Animal UK262726300944 UK262726400952 UK262726700934 UK262496200273 UK262726500939 UK262858600875 UK262726400959 UK263516100443 UK262483302411 UK262726300958 UK261466400546 UK262483702422 UK262496500269 UK262148500119 UK262148700114 UK261466300524 UK262148300117 UK261466200586 UK262483202410 UK262496600270 UK262709400470 UK262496200266 UK261466100529 UK263516300445 UK261466200530 No eartag black limousin UK262483402426 UK262858100877 UK26146400518 UK262858400915 UK261466500519 UK261466600527 UK261466400553 UK261466600548 UK261466200544 UK261466300545 UK261466200551 UK261466600520

Oldest lesion 3 4 >5 5 5 6 4 6 >7 6 6 >7 >7 >6 6 >7 6 >7 >7 7 >5 7 7 7 6 >7 >7 10 >6 6 >7 >7 >7 >7 >6 7 >7

CEDI-NS 32 51 51 36 25 45 41 49 46 54 46 46 49 41 31 48 48 66 46 48 42 47 51 51 60 52?

53 42 46 50 46 54 47 52 74 58 62 61

WRL SPCE 32 42 46 57 42 61 44 70 57 57 67 76 52 61 43 82 73 74 71 81 74 74 83 84 86 78 78 70 94 71 94 88 75 81 96 90 93 90

Cedi-O 18 32 15 27 29 48 35 49 35 48 46 46 28 40 63 63 60 62 51 65 46 55 74 69 77 59 61 47 79 37 90 96 71 88 89 77 77 79

PCR POS POS POS POS POS POS POS inc POS POS POS inc inc POS ─ inc POS inc POS POS POS ─ POS inc POS ─ POS POS inc inc ─ ─ ─ ─ ─ ─ ─ ─

overall age (d) 3 3 4 5 5 5 5 5 5 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 >6 >7 >7 >7 >7 >7 >7 >7 >7

Effect of lesion age and incubation period on time of infection Estimating lesion ages on IP1b

Likely times of lab escape of virus • • • • • • •

16 -7

17 -6

18 -5

July 19 -4

20 -3

21 -2

22 -1

23 0

24 1

25 2

26 3

27 4

28 5

29 6

30 7

31 8

August 1 2 9 10

3 11

4 12

12 13 14 15 16 -14 -13 -12 -11 -10

17 -9

18 -8

July 19 -7

20 -6

21 -5

22 -4

23 -3

24 -2

25 -1

26 0

27 1

28 2

29 3

30 4

31 5

August 1 2 6 7

3 8

4 9

14 15 16 17 18 -14 -13 -12 -11 -10

July 19 -9

20 -8

21 -7

22 -6

23 -5

24 -4

25 -3

26 -2

27 -1

28 0

29 1

30 2

31 3

August 1 2 4 5

3 6

4 7

9 10 11 12 13 -14 -13 -12 -11 -10

14 -9

15 -8

NB Could be further lag between escape of virus and exposure of cattle

Normandy Outbreaks

20th July: flooding 23rd July: best airborne spread day 22nd July: 1st centrifuge waste discharged 25th July: 2nd centrifuge waste discharged 20th July: 4 lorries via Westwood Lane 25th July: 2 lorries via Westwood Lane 23rd July: Infection day for IP1 assuming 3 day incubation and 9 day oldest lesion

51

Area at risk to airborne spread of FMD from IP1

Risk of airborne spread from IP1

Phase 2: September/Egham

IP2c: pre-clinical diagnosis

•

IP3

• • • • • • •

Confirmed 12 Sep 2007 281 cattle 8 pigs 8 locations Clinical findings 2 locations positive in lab Culling completed 16 Sep

•

IP4

• • • • • •

Confirmed 15 Sep 54 cattle (location B) 743 pigs (location A) Clinical findings in cattle only Laboratory –cattle positive, pigs negative Culling completed 16 Sep

WINDSOR

5 km IP6 IP6b

IP8 EGHAM

IP3b IP3c IP7

IP4 IP4b IP5

•No clinical signs at slaughter

•

IP6

• • • • • •

Confirmed 21 September 34 cattle 2 locations 2 out of 32 cattle at 1 location with 2-4 day lesions 2 virus +ve All seronegative

WOKING

•19 of 58 animals RT-PCR positive blood samples •11 of 58 animals virus isolation positive blood samples

PIRBRIGHT SITE

IP2a IP2b IP1a IP1b

GUILDFORD

IP2b IP2c

•

IP7

• • • • • • •

Confirmed 24 September 16 cattle Single location 14 with acute signs 1-4 day lesions 15 virus +ve 2 seropositive (with 4 day lesions)

•21 of 58 positive in one or other test •Indicates near simultaneous infection of multiple animals •First use of real-time preclinical diagnosis for FMD in field?

•

IP5

•

IP8

• •

Detected 16 September. No acute signs.

17 out of 22 cattle with 2-3 week old lesions. All seropositive, virus negative.

12 out of 16 sheep seropositive; 10 with old lesions. 2 pigs –no lesions; seronegative, virus negative.

Confirmed 17 September. Single location.

• • • •

Confirmed 29 September 134 cattle, 16 sheep 4 locations (3 PZ, 1 SZ, just outside PZ) 54 cattle at infected site. 8 with lesions, estimated maximum 3-4 days.

Other sites no signs, initial serology negative

• •

•

Bovington IPA ongoing

Wiggins Raynen

Berryman Crown Estates

IPA culled

Regan

August and September control zones, with 3km PZ and 10km SZ

Cook Salmon Whitty Robertson

Wallace

52

Genealogy and infection profiles A

IAH2

IP6b

AY593815

IP1b(2) MAH

IAH1

IP3c

IP1b(1)

IP8

IP3b IP4b IP2b

IP7

IP2c IP5

B IP1b(2) IP1b(1) IP2c IP2b

IP5 IP4b IP3c IP3b IP6b IP7

30-Sep-07

23-Sep-07

16-Sep-07

02-Sep-07

09-Sep-07

26-Aug-07

19-Aug-07

12-Aug-07

29-Jul-07

05-Aug-07

22-Jul-07

15-Jul-07

IP8

Date

IAH-P Laboratory Activities Aug-Oct 2007 • • • • • • • • •

Visited 18 holdings Laboratory diagnostic tests on 54 report cases Serology on ~ 19,000 blood samples Preclinical RT-PCR screening of ~1,500 samples Web-based access for Defra to lab results Sequencing of outbreak and related viruses Meteorological analyses Consultant advice to Defra Information to inquiry teams

Lab tests carried out • • • • • •

Ag ELISA & rtRT-PCR main virological tests Used virus isolation selectively Evaluated LFDs in lab and on farm SPCE-O & Cedi-O for serological screening Cedi-NS and VNT for confirmation Probang testing on three holdings

53

Conclusions • Pirbright site the likely source of the August outbreaks in Surrey • IAH or Merial - ?

• Drains as source - possible • Lorries as means of delivery - ?

• Second phase of outbreaks in September is derived from August cases not reintroduction (but other views) • Sequence phylogeny recreates transmission pathways and predicts missing links that seem to have been found

Risk and Control Zones

Discussion points

Conclusions (2) • How did virus get from Normandy to Egham?

• Cattle main affected species as in 1967 • Classical lesions evident but poorly observed • Airborne transmission?

• Preclinical diagnosis used for first time • Rapid LFD tests evaluated

• • • • • •

Cattle as indicator species?

Size of control zone?

Use of vaccination?

Virus survival in soil?

Role of wildlife and vectors?

Contingency planning for non-availability of national lab?

Acknowledgements • Nigel Ferris, Geoff Hutchings • Don King, Scott Reid, Katja Ebert • Yanmin Li, Kate Swabey, Ginnette Wilsden, Pip Hamblin, Phil Keel, Bob Statham • Nick Knowles, Eleanor Cottam, Jemma Wadsworth • Annette Hendry, Juliet Dukes, Julie Stirling, • Chris Chisholm, John Bashiruddin • Chris Oura • Eoin Ryan, Nick Juleff, Bartek Bankowski, Bryan Charleston • Many other scientific and non-scientific staff from IAH and elsewhere • Field and headquarters staff at Defra

54

Appendix 8

Conclusions •

UK outbreaks 2007: Confirming sequencing results •

Professor Soren Alexandersen Department of Virology, National Veterinary Institute Danish Technical University Lindholm, Denmark Spread of the PanAsia lineage of the O1 BFS 1967 Middle East-South Asian (ME-SA) topotype of FMDV-O Significant spread up to about 60 km away. Mainly in cattle.

• •

First appearance 1990 1993 1994 1995 1996 1997 1998 1999 2000 2001

IAH N.J. Knowles, 15 October 2001

Solid colour = PanAsian strain present Hashed colour = Type O present, PanAsian strain suspected

Rapid sequencing of samples received from the UK resulting in a thorough report of the 4 sequences of 7600 nucleotides each within a one week period.

It was later revealed to us that the results closely reflected those determined

by IAH internally.

We concluded, that the outbreak isolate, Isolate B, originated with high probability from isolate A (Merial) or isolate C (IAH Immunol) while an origin from strain D (IAH disinfection testing) is unlikely. We can not conclude with high certainty whether the origin of isolate B is in fact isolate A or Isolate C, however, based on the number of differences, isolate A (Merial) may be slightly more likely than isolate C, but this can not be assessed with any statistical significance as there is only a single difference between isolate A and C. According to the available evidence and information, the virus is most likely to have escaped from the Pirbright site most likely through faulty piping combined with the movement of constructors and soil etc from the grounds The Pirbright site sewage system appeared to have been approved by defra, however, the EU regulations clearly demand that the entire effluent system is within containment (and having at least double piping with sensors if going outside containment).

Recommendations •

It is strongly recommended that all laboratories and vaccine plants in the EU working with FMDV is approved according to the overall EU regulations instead of only being treated by local, national and historical regulations.

2

October 2007

FMD UK 2007 The identity of the outbreak virus (O1 BFS 1967) suggested that it may have escaped from the containment facilities at either IAH-Pirbright Laboratory OR the commercial Merial vaccine production facility at the same site.

FMD UK 2007 continued

Lindholm was asked to very rapidly produce and analyse consensus sequences from 4 FMDV isolates already sequenced at IAH. This was accomplished by sequencing of the L-fragment, containing the full coding sequence, around 7600 nucleotides from 4 isolates in less than a week. This was accompanied by a thorough analysis of the sequences and a full report was given to the UK authorities within 8 days.

Seriousness of the UK 2007 epidemic, just a small escape or more like a real epidemic?

Per 5 October 2007: • 2 IPs plus 6-7 IPs = 8-9 IPs However, • 2IPs, + 2 PCR positive, + 3 Infection negated, one disappeared = 8 premises for period one • 5-7 IPs. + 1 SOS, + 5 DC, + 11 infection negated = 22-24 premises • Total up to 32 premises involved, quite different from the official number of 8 IPs.

The work started in the morning of Tuesday 13 August where four FMD virus samples from the UK came to Lindholm.

Three of the samples where from the two laboratories, IAH-Pirbright Laboratory and Merial, one was from an infected cattle at the initial infected premises (IP 1).

RNA was extracted, reverse transcribed into cDNA, amplified by PCR in a way that generate overlapping fragments and also inactivates any infectious virus. Each isolate is amplified with multiple overlapping primer sets and then gel-purified before being sequenced in both directions - resulting in a total of around 136 sequence reactions.

Sequence reactions were then submitted to the Vet-DTU sequencing facility in Copenhagen which ran the reactions on their automated equipment. (Similar sequencing reactions were also shipped to a commercial, large-scale sequencing facility in order to maximise the likelihood of a fast and high quality result).

Raw sequence data where immediately made available to Lindholm over a shared server drive.

The highest quality sequence data are usually found in the L segment - approximately 7600 nucleotides out of the total viral genome of 8200-8500 nucleotides. Overlapping nucleotide segments were subsequently assembled into (almost) full length sequence data using standard computer software.

After this the various virus samples could then be compared at the nucleotide level.

On Tuesday 21 August (after 1 week) a report with the final results and conclusions was submitted to the UK authorities.

55

Airborne disease transmission Over land

Overview of FMDV genome

O1 BFS 1967

Significant spread up to about 60 km away.

Mainly in cattle.

•Picornaviridae family •Aphthovirus genus

Cell culture passaged virus likely to be heperan sulphate binding and to be of significantly reduced in vivo excretion and virulence until adaptation to animals

P1-2A 5' UTR CCn

Lab Lb 1A 1B

1C

P2 2A 1D 2B

2C

P3 3A3B 3C

3D

3' UTR AAA

IRES

L or Large fragment

con_all_compat Strain C

IAH immunology O1 BFS strain 1.00

COMPARISONS Strain A

1.00

Merial O1 BFS for making vaccine

Strain B

OUTBREAK VIRUS IP1

Strain D

Strain used for desinfection testing

O1 Manissa from GenBank AY593823xxxxx

Number of differences among the 4 isolates

A. Discrepancies between the sequences Isolate B

Number of nt differences in the consensus nucleotide sequences (around 7600 nt) D C B

A 8 1 5

D __ 9 11

C __ __ 6

Number of amino acid differences in the CDS (ORF of 2332 aa) A D C D 4 __ __ C 1 5 __ B 1 3 2

A = Merial C = IAH Immunol.

Isolate D

Isolate A

Isolate C

CDS position [nt]

SYN/NONSYN AAProtein change*

G G C A G C T C T T

G G A 88 NONSYN E-K Leader (29) A G G 1091 NONSYN C-Y VP2 (78) T T T 1284 SYN VP2 A G G 1679 NONSYN H-R VP3 (56) A G G 1691 NONSYN G-D VP3 (60) T T T 2340 SYN VP1 C C C 4242 SYN 2C A C C 4399 NONSYN L-I 3A (42) C T T 4506 SYN 3A C T T 5928 SYN 3D * Amino acid changes are given using the one letter code and the first aa is that of isolate B while the second is for isolates having a different aa in this position.

A = Merial C = IAH Immunol.

B = Outbreak strain D = Desinfection strain

VP3(56) R is heperan sulphate binding Do do do VP56(56) H not heperan sulphate binding Do do do

Glycine (G) at VP3 (60) is typical for a highly cell culture-passaged and aspartic acid (D) for a low passaged O strain or directly from field material. Together this indicate that the outbreak strain (B) has lost the ability to bind strongly to heperan sulphate but that is still is not fully adapted to in vivo conditions (have not yet an aspartic acid at VP3 position 60).

56

Conclusion The outbreak isolate, Isolate B, originated with high probability from isolate A (Merial) or isolate C (IAH Immunol) while an origin from strain D (IAH disinfection testing) is unlikely.

We can not conclude with high certainty whether the origin of isolate B is in fact isolate A or Isolate C, however, based on the number of differences, isolate A (Merial) may be slightly more likely than isolate C, but this can not be assessed with any statistical significance as there is only a single difference between isolate A and C.

How did the virus escape what are the lessons to be learned

Requirements for facilities working on live FMDV (S. Alexandersen, version 14 November 2005) OIE requirements for Group 4 Animal Pathogens in blue and EU requirements for FMDV in red - some requirements may not be explicitly stated in the EU regulations but has been inferred by the author.

Yes

1. Not next to known fire hazard 2. Workplace separated from other activities

Yes Yes

3. Personnel access limited

Yes Yes

4. Protected against entry/exit of rodents and insects

Yes Yes (and birds)

5. Liquid effluent must be sterilised

Yes and monitored Yes 100C for 1 hour or equivalent, controlled and recorded Alternatively, >pH 12 for 10 hours, controlled and recorded

5b. Entire effluent system within containment

Yes

5c. Devices to prevent backflow into supply lines and to have automatically filled liquid U-seals in sewage system

Yes

6. Isolated by airlock. Continuous internal airflow

Yes Yes

As an example of this, the Danish FMD lab at Lindholm Island was in the early 1990-ties asked to install a boiler system directly below the FMD labs At the Pirbright site apparently only defra regulations were fulfilled and the EUregulations not enforced The lesson is to have everyone follow the common regulations and not treat labs differently

Acknowledgement CO-AUTHORS: Joern Klein, Preben Normann, Thomas Bruun Rasmussen and Soren Alexandersen We also thank Tina Pedersen for her efforts in transport of samples and Lars Mølbak, Kirsten Vestergaard, Mette Boye and Lasse Dam Rasmussen from the Vet-DTU Section of Molecular Microbiology in Copenhagen for preparing the final sequencing reactions and for running these on their automated sequencing equipment.

Thanks to the HSE and Spratt investigations.

57

Appendix 9

Why molecular techniques for FMD diagnosis

Application of real-time RT-PCR for foot-and-mouth disease virus

Presentation based on IAEA meeting in Vienna December 2005

A. Dekker

Issues that should be addressed

RT-PCR tests available (old data)

Primer selection: how can labs now what is the recommended primer for any region?

Sample handling: on site into lysis buffer, or at a biocontainment facility?

Kits: are the main extraction methods and reagents available in kit form to reduce risk of errors, simplify scaling up?

Differential diagnosis Standards External QA

Fit for purpose

Status

Test needs

Tests

1. Countries/regions free of FMDV without vaccination

Detection of FMD in first sample.

Antigen capture ELISA confirmed by VI, or RTPCR. Antigen capture ELISA or RT-PCR or VI

2. Countries/regions free of FMDV with vaccination

Detection of FMDV or evidence of virus, including the ability to detect virus in persistently infected animals Serological tests to detect evidence of virus replication

VI (confirmed by antigen capture ELISA) or RT-PCR

3. Country/region regaining freedom of FMDV circulation using emergency vaccination

Ability to detect virus in persistently infected animals Serological tests to detect evidence of virus replication

VI or RT-PCR. (ELISA as confirmation of VI) Antibodies against NSP

4. Country/region regaining freedom of FMDV circulation using culling infected farms

Ability to detect circulating virus

VI or RT-PCR. (ELISA as confirmation of VI) Antibodies against SP and/or NSP (ELISA)

Detection of virus in tracing from Index case.

Evidence of circulating virus from serology

5. FMDV infected countries/regions All conditions

RT-PCR techniques available (e.g. HPAI laboratories) RT-PCR can be standardised RT-PCR proven to be reliable

FORMAT Oem et al (2005) Lomakina et al., (2004) Barlic-magnja et al., (2004) Rasmussen et al., (2003) Moonen et al., (2003) Clavijo et al., (2003) Saiz et al., (2003) Callahan et al., (2002) Reid et al., (2002)

Real-time conventional RT-PCR hybridisation Real-time Real-time conventional conventional Real-time Real-time

TARGET 2B 3D VP1 and 3D 3D 3D 3D 3D 3D 5’UTR

Serotypes detected all all all all all O, A, C all all all

Primer selection

5’ UTR

Antibodies against NSP (ELISA)

Highly validated Used in outbreak situation Variable genome region

3D

Well validated More conserved genome region (but still strains exist that might be missed)

VI Antigen capture ELISA RT-PCR Antibodies against FMD Antibodies against NSP

58

Sample handling

Depends on situation in country

Ideally two samples

With RNA stabiliser for Molecular detection With glycerol buffer (or dry if time to lab is short)

Kits

All laboratories use kits

RNA isolation

RT-PCR

Differential diagnosis

Clinical diagnosis is important

No laboratory tests for diseases not in the clinical differential diagnosis Reduce possibility for false positive results

Multiplex testing vs multiple tests

Standards

Each laboratory should use standards for batch control Try to develop international standards

Quiagen, Roche or others

Differential diagnosis PROS - MULTIPLEXING Lots of information from one assay.

Cheaper to run one assay than 10 individual assays.

More information in a shorter time than if all assays were run individually.

Can accommodate multiple loci for the same agent in nucleic acid detection assays.

One-step diagnosis.

Valuable as a screening tool.

MULTIPLEXING - CONS More complex sample matrices to consider and validate.

One does not always want to test for all of the diseases included in a particular multiplex assay.

High equipment costs and relatively high reagent cost compared to traditional techniques.

Requires a strong technical background to trouble shoot problems

External QA

Trizol for RNA stablisation Roche or Quiagen kits for RNA isolation

All laboratories had ISO accreditation Proficiency tests show higher concordance between RT-PCR tests compared to VI Need of testing new isolates in various laboratories

Appendix 10

59

TENTATIVE Conclusions •

•

FMDV levels: Summary table of in vivo FMDV outputs and laboratory produced levels from live virus handling – A DIFFICULT TASK WITH MANY UNCERTAINTIES

•

•

Professor Soren Alexandersen Department of Virology, National Veterinary Institute Danish Technical University Lindholm, Denmark

There is still a lack of knowledge regarding quantitative levels and amounts of FMDV excreted during animal infection, laboratory manipulation and vaccine production.

Also a lack of knowledge on how excretion is translated into risk and level of transmission by various routes, distances and time.

Pigs are the highest risk for causing airborne transmission and it may be likely, or speculated, that pigs are in general the highest risk of causing transmission followed by cattle and then sheep.

Laboratory work appear to be of relative minor risk compared to work with infected livestock, however, large-scale vaccine production may produce very large FMDV levels and it is currently unknown whether these levels and total amounts can be effectively controlled, contained and removed within containment.

Recommendations •

Further targeted studies should be conducted aimed at quantitating and understanding excretion by various routes from infected livestock, laboratories and large-scale vaccine production AND to characterise how such levels correspond to transmission to other animals and herds by the major routes.

2

October 2007

Summary Table: Peak excretion compiled and partly assumed by Alexandersen, S. (Mainly from Alexandersen et al., 2003).

Cattle Conc 0.1-0.2/l Max 1-2/l

Amount 4.3 5.3

Nasal fluid/ (Culture fluid)

5-5.6 C 7.1-8.3

? ?

5

?

6

?

Saliva

6.5 C 7.5-8.8

12 13-14

6.5

11

6

10

OP-fluid

5.5-7 C 8.3

11-12.5 13.5

5.5-7

?

?

?

Faeces

1.5 C 2-3.3

6.2 6.7-8

2

5

?

?

Urine

2.5 C,A 4.9

6.5 9

?

?

?

?

Preputial sw.

Semen

4.5 5-6

?

6-7

? ?

? ?

? ?

? ?

Milk Serum

6-6.6 5-5.5 C 6-7.8

10-11 10 10,5-12.3

5

9

5-7.2 7

9-11 11

Lesion epi.

8-9

10

8-9

9

9-10

10-11

Air

Sheep Conc Amount 1-2/l 4.3

Pigs Conc 20/liter C Nov ↑

Amount 6.1 7.6-8.6

Lab work Conc Amount <0.004/l < 2

Vaccine prod Conc Amount ? ?

8

9?

11

15

Summary estimates of relative excretion and potential infectiousness of individual animals freely after Alexandersen, S. Based on the notion that peak concentration of FMDV in cattle and in sheep appears to be similar and the peak concentration in pigs appears to be approximately 10-100 times higher.

Thus, if cattle is ~ 500 kg, sheep ~ 50 kg and pigs on average 80100 kg, then relative excretion may likely be: Sheep: 1 Cattle: 10 In the breath, for most strains, one pig Pig: 100 excrete as much as 60 cattle or sheep, but for C Noville one pig can excrete as much as 300-3000 cattle or sheep

Assuming 100 litre saliva, 20-40 kg faeces, 10-20 litre urine, 30 litre serum (if bled out), and 10-100 g lesion material excreted on peak day of excretion Referenced in: Alexandersen, S., Z. Zhang, A. I. Donaldson, and A. J. Garland. 2003.

Mean concentrations of FMDV in the secretions and excretions of cattle before and

Table 1.

From: Alexandersen, S., Z. Zhang, A. I. Donaldson, and A. J. Garland. 2003. The Pathogenesis and Diagnosis of Foot-and-Mouth Disease.

J. Comp Pathol. 129:1-36

after the first appearance of macroscopical lesions resulting from indirect contact with infected pigs

Selected estimated minimum doses* for various species and routes of exposure Sampl e

Virus type

Results (bovine thyroid cells ID50/ml or g) on stated days after first appearance of macroscopical lesions -4

Species

Inhalation

Intradermal

Intramuscular

Nasal instillation

Oral

Cattle

10

100

104

104-105

105-106

Sheep

10

100

104

104-105

105-106

Pigs

>800

100

104

Unknown

104-105

-1

0

1

O

-

-

-3 0.2

-2 3.2

4.9

5.3

2.6

-

2 -

3 -

4 -

5

C

-

-

-

3.9

5.9

6.1

0.4

-

-

-

-

-

O

-

0.3

2.8

5.6

6.1

5.5

4.1

4.3

2.6

1.9

0.9

1.3

2.5

-

Ser

PhF C

-

2.2

4.7

6.8

7.1

6.3

5.5

5.1

3.0

2.1

2.4

O

-

0.3

1.6

4.7

6.5

6.4

5.4

3.6

1.8

0.5

-

-

C

-

1.4

5.3

7.0

7.5

6.7

6.3

4.4

3.0

1.5

0.3

-

OS

*

The estimated minimum doses are those reported to cause clinical disease. It is emphasized that these are not absolute values but represent estimates based on different experiments that are not necessarily directly comparable. It is possible that even smaller doses might produce infection if large numbers of animals were exposed. Doses are given as TCID50 (bovine thyroid tissue culture 50% dose endpoint estimates). For further information see the text and associated references. It should be noted that for intradermal and intramuscular inoculation doses from 5-10 fold lower are cited in the literature, but without details of the assay systems used (Sellers, 1971).

O

-

-

0.7

3.3

5.6

5.6

4.5

3.1

1.2

0.1

-

-

C

-

0.9

3.9

5.5

7.1

6.5

5.7

2.7

1.0

0.4

-

-

O

-

-

0.2

3.3

4.4

5.4

5.3

4.5

3.3

1.8

-

-

C

-

-

0.6

2.0

3.6

5.0

3.3

0.9

-

-

-

-

O

-

-

-

0.5

2.4

2.0

1.9

1.4

1.5

-

-

-

NS

LS

U

60

Airborne disease transmission Over land

Over the sea

VIRUS AEROSOLS FROM INFECTED ANIMALS • Airborne virus in droplets and droplet nuclei excreted in the breath First from the upper and later from the lower respiratory tract • > 6 micron in diameter: ~ 35% 3 to 6 micron in diameter: ~ 40% 1 to 3 micron in diameter: ~ 25% • Stable at a relative humidity above 55-60%, a temperature below 330C Relatively stable to UV light (indirect effect of drying and temperature)

Peak amount of airborne virus emitted in 24 hours by an

Concentration in the air of rooms with affected animals

infected pig (90-100 kg), sheep (30-40 kg) or heifer

Pigs - Up to 2.570 TCID50/liter

Strain of virus

Amount (log TCID50a/24 h)

Pig

O UKG 34/2001

6.1 (day 1-2 of clinical disease)

Pig

Other type O’s,

5.8-6.4 (day 1-2 of clinical disease)

Day -2

e.g. O TAW 97, BFS etc.

Adjacent rooms or dead animals 0.04-0.25 TCID50/liter

Pig

Type C Noville 1/73

7.6-8.6 (day 1-2 of clinical disease)

Heifer

O UKG 34/2001

4.3 (day 1-2 of clinical disease)

O UKG 34/2001

4.3 (1-2 days BEFORE clinical disease)

Labs working on Sheep FMDV - < 0.004 TCID50/liter air a

Generation of aerosols during vaccine production is possible, but levels are unknown

FMDV excretion in aerosol from pigs, cattle and sheep *Measured for FMDV O UKG 2001. In logs TCID50s.

(around 200 kg).

Cattle or sheep Concentrations up to 0.2-0.7 SPECIES TCID50/liter air

Species

TCID50 = bovine thyroid tissue culture 50% dose end-point.

Minimal dose*

Inhalation rate

Threshold plume

TCID50**

M3 / 24 hours***

conc. to infect

Cattle

10

150

0.07

Pigs

>800

50

>16

Sheep

10

15

0.7

Day 0

Day 1

Day 2

Day 3

Pigs

1.8

6.1*

6.1*

4.7

2.9

Cattle

1.8

4.3*

4.2

4.1*

2.4

3.4/4.3*

4.3/<3.0*

1.5

Sheep

1.7

Day -1

2.9 /4.3*

Day 4

TCID50 /M3

Effect of species and number of animals excreting virus on the

Heddon-On-The-Wall FMD 4

predicted risk for airborne spread to different species downwind.

Airborne excretion and animals with disease Excretion airborne

Have or have had disease

Acute disease (5 days)

Distance downwind at which species are at risk Species excreting virus

Cattle

Sheep

Pigs

400

1000 infected animals (Millions)

Excretion in TCIDs (x 6 logs)

500

300

200

Modellers simplification

Pigs

6-30-100 km

2-10-30 km

<0.2 km

Cattle

0.7km

0.2 km

<0.1 km

Sheep

0.7km

0.2 km

<0.1 km

100

100 infected animals 0 1

4

7

10

13 Date in February

16

19

22

25

Pigs

2-10-30 km

0.4-2-6 km

<0.1km

Cattle

0.2 km

<0.1 km

<0.1 km

Sheep

0.2 km

<0.1 km

<0.1 km

61

WHAT WE NEED TO KNOW AND UNDERSTAND ABOUT AIRBORNE FMDV

SOURCE OF VIRUS IN ANIMALS

•Amounts/distribution of infectious virus in single particles

• Vesicular lesions and skin; • Breath; milk; faeces; semen, all secretions and excretions; all tissues during acute phase • Bone marrow and lymph nodes in carcass meat. Drop in pH after death in cattle inactivates

•Probability of infection in relation to virus concentration and time of exposure

virus in skeletal muscles

• Change in pH in pig meat is variable not been documented for sheep meat

•Need to define these parameters in order to develop a simulation model predicting probability - not plus/minus

• PERSISTENCE OF VIRUS IN PHARYNX AFTER RECOVERY

Measuring FMDV infectivity and RNA RNA to infectivity ratio in air samples ranges from 101.7- 4.1 (2.93+/-0.85) with the lower ratios in the smaller droplets

The FMDV receptor (integrin αvβ6) expression in bovine tongue Tropism & Persistence Regulated expression & integrin activation Cellular signaling

The ratio is ~ 102-3 for serum Tongue, pig 2 days pi labelled with antibodies to FMDV 3ABC Bovine inoculated - mean Pig inoculated - mean

Pig inoculated - mean V i r aemi a

Mean viraemia

O UK 2001 pigs

Virus in breath

Temperature

Clinical signs

10

41 41

8

40

10 8 6 4 2 0 0

Anti body

V i r us i sol ati on

1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 24 48 72 96 120 144 168 192 216 240 264

cattle

NB virus in breath is in log TCID50/hour slopes predicted

38 2

37 37

0

V i r aemi a

24

48

72

96

120

144

168

192

216

240

1.E+05 1.E+04

6 4

1.E+03 1.E+02

2 0 0

1.E+01 1.E+00 24 48 72 96 120 144 168 192 216 240 264

264

Pig contacts - mean

Mean viraemia

Virus in breath

Temperature

Pig inoculated - mean ___ Nasal ___ Mouth swabs

Clinical signs 10

10

41

8 6

8 6

41

4

40

2

40

0 0

24 48 72 96 120 144 168 192 216 240 264

39 Pig contacts - mean

39 4

38

2 0 24

48

72

96

120

144

168

192

216

240

264

41 40

6

NB virus in breath is in log TCID50/hour

4

2

37 37

0

36 0

24

48

72

96

120

144

168

192

216

240

6

37

4

Temperature

41 41

8

40 40

6

48

72

96 120 144 168 192 216 240 264

1.E+00 24 48 72 96 120 144 168 192 216 240 264

Bovine contacts - mean V i r eami a

A nt i body

10 8

1.E+05 1.E+04

6 4

1.E+03 1.E+02

2 0 0

1.E+01 1.E+00 24 48 72 96 120 144 168 192 216 240 264

M ean bov contact

Nasal swabs

Mouth swabs

10 8 6 4 2 0 0

48

96

144

192

240

288

336

39

slopes predicted

39

Bovine contacts - mean

38

37 37

24

0

Clinical signs

10

0 0

1.E+01

0

Bovine inoculated - mean Virus in breath

38

0

1.E+03 1.E+02

2

Bovine contacts - mean Mean viraemia

2

2

1.E+04

6 4

264

4 8

37

1.E+05

8

38 38

A nt i body

10

39

10

38

36 0

V i r aemi a

41

39

Vi r us i s ol ati on

10 8

36 0

A nti body

Bovine inoculated - mean

Clinical signs

40

Log genomes estimated by RT-PCR

Log genomes estimated by RT-PCR

39 38

Temperature

Pig contacts - mean

39

4

Virus in breath

10

8

40 6

Mean viraemia

O UK 2001

36 0

24

48

72

96

120

144

168

192

216

240

264

M ean bo v cont act

Nasal swabs

M out h s wabs

10 8 6 4 2 0 0

48

96

144

192

240

288

336

62

RT-PCR quantitation of FMDV RNA in nasal swabs from sheep, Group averages +/- S.D.

Quantitative aspects

Average inoculated

• Species differences

30

Average contact

Fever and clinical disease inoculated

Contacts

Porcine

Bovine

FMDV genomes (log)/ml serum

10

105 TCID/ml

20

Aerosol inoculated

and air

Aerosol contact

Six “inoculated” sheep were inoculated with FMDV O UK 2001 in the coronary band and four “contact” sheep kept in the same room.

Ventilation reduced to 3-5 air changes/ hour to resemble intensive, indoor conditions.

8 3

10 TCID/hour/sheep

6

102 TCID/ml

10

4 1 TCID/ml

2 0 0

0 0

12

24

36 48 Hours

60

7

Viraemia

72

14

21

28

Days

Virus stability

RT-PCR quantitation on nasal and rectal swab

Dependant on pH, temperature, humidity AND initial concentration

Sheep inoculated with FMDV O UKG 2001

pH stability

30

• Average rectal swab 105 TCID/ml

20 (50-Ct)

Average nasal swab Average nasal swab contacts

102 TCID/ml

10 Average rectal swab contacts 1 TCID/ml

0 0

7

14

21

28

Days

Heat stability •

72C for 15 sec give a 104-105-fold reduction in infectivity 80-90C for 30 sec give a 105.4-106.0-fold reduction >= 132C for at least 1 sec give a 105-106-fold reduction It should be realised that virus within e.g. the cellular fraction may be more heat resistant Sterilisation (Fc/F0 >= 3, i.e. at least 121C at the coldest point for 3 min) likely to inactivate FMDV by a factor of at least 1012

• More stable in cool conditions than in warm

The exact rate of inactivation is depending on many parameters e.g. organic material and ionic strength etc. Furthermore, the inactivation of FMDV is biphasic and a small fraction of virus may survive if inactivation measures are minimal rather than optimal A guiding rule of thumb for FMDV is: Half-life, or under optimal conditions the 10fold reduction time, equals roughly – 12 hours at pH 6.5 or 10 1 min at pH 6 or 11 1 sec at pH 5 or 12

Example: • If peak level of 109 genomes of virus RNA (roughly 106 TCID50) per ml in a sample & A half-life of 24 hours: • It would take approximately 20 days to fall to 1 TCID50 per ml or approximately 30 days to 1 copy of virus RNA per ml

• Drying may reduce infectivity by a factor of 101102-fold but surviving FMDV is often very stable

63

Fig. 5

- mainly inhalation - inhalation or ingestion

•

• SPREAD OF FMD • CONTROLLABLE SPREAD – movement of infected animals – movement of animal products e.g. meat, milk, offal – vehicles, milk tankers, people (stockmen) etc

8 6 4 2 0 0

24

48

72

96

120

144

Hours

Box 1-4 = 1:1 (IP 72h)

Box 5 = 2:4 (IP 48h)

Logarithmic mean of the three inoc groups High Mean

Mid Mean

Low Mean

10 8 6 4 2 0 0

24

Hours

• UNCONTROLLABLE SPREAD – spread by the carriage of virus on the wind or by wildlife

Mean box1-4 vs Mean box5 Inoc box1-4 Inoc box5 Contact box1-4 Contact box5 10

Temperature (°°C)

• All species - through cuts and abrasions in skin and mucosae

Field and experimental experience suggest that the efficiency of spread, length of the incubation period and severity of clinical signs of FMD is highly dependent on husbandry and local conditions

PIGS - FMDV O Taiwan 1997 FMDV genomes (log)/ml serum

• Ruminants Pigs

FACT

1 molecule: calculated incubation period of 13 days and 22 hours

48

72

96

120

144

168

V0=100000

V0=10000

V0=1000

Low

Medium

High

192

1.E+09 Virus genomes/ml serum

• ROUTES OF INFECTION

1.E+08 1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00 0

24

48

72

96

120

144

Time (hours)

Pig exp. 2 – One-on-one exposure, 1+1, 2 hours at 1-4 pi plus group (4+4) at 4 days pi

5

7 9

THEREFORE

10

5 5 4 Subclinical – viraemia day 5-11

• The number of animals kept together in direct contact and the timing and length of contact influence the incubation period, the efficiency of spread and the risk of subclinical, persistent or abortive infection • Spread of FMD may be rapid and efficient under certain (intensive) conditions. Such conditions are often found when kept indoor in large groups or for example in connection with transport, markets, lambing, shearing etc • Under extensive conditions, often outdoor, or where animals are individually confined the spread of FMD may be slow and inefficient

Ackowledgement I thank all former and current colleagues for helpful discussions and collaboration on FMD

Appendix 11

64

3 FMD-NSP workshops: EPIZONE, EUFMD (FAO) & EU COORDINATION ACTION FMD-CSF

Workshop on the design and interpretation of post Foot-and-Mouth Disease (FMD)vaccination serosurveillance

by NSP tests

REPORT

Workshop 1: January 2007, for dense cattle countries (Western Europe) Workshop 2: June 2007, for Balkan and Mediterranean countries Workshop 3: October 2007, for Central European, Nordic and Baltic countries

Dr. Kris De Clercq

FMD-NSP workshop objectives (1)

Europe:

FMD-free without vaccination When FMD outbreak: possibility of emergency vaccinate-to live-policy followed by post-vaccination serosurveillance to return to FMDfree without vaccination

FMD-NSP workshop objectives (2) Workshop objectives: 1. design and implement a survey to substantiate free from infection with a certain degree of confidence after performance of vaccination; 2. guidance to interpretation on the follow-up on seropositive animals/herds/flocks; 3. guidance to use of laboratory test results in decision-making; 4. identification of the resources (laboratory, veterinarians) required.

FMD-NSP workshop objectives (3) Make participants familiar with: NSP tests available, other relevant tests and their use Computer tools for calculation of required number of samples to be taken The legislation relevant for NSP testing The detection of carrier animals: relevant potential control measures

FMD-NSP workshop participants (1)

A team from 1 country preferably consists of 1 epidemiologist, 1 lab expert, 1 crisis manager, the CVO. Formation of working groups consisting of teams coming from 2 countries (in different meeting rooms, each equipped with PC, MS Office, internet connection, printer, beamer, telephone, flip chart)

65

FMD-NSP workshop scenario’s (2)

FMD-NSP workshop scenario’s (1)

Working groups provided with:

Scenario 1: limited number of outbreaks/only bovine vaccinated, Scenario 2: limited number of outbreaks/all animals (bovine, pigs, sheep, goats) vaccinated Scenario 3: large number of outbreaks/only bovine vaccinated.

Scenario 4: limited number of outbreaks/only bovine and pigs vaccinated Scenario 5:limited number of outbreaks, one in a very peculiar and big pig holding/only bovine and pigs vaccinated Scenario 6: larger number of outbreaks in a mountainous area/all susceptible animals vaccinated

• surface of the different zones, • composition of the various clusters/stratification by radius, • composition of the various clusters/stratification by size.

• spatial information

FMD-NSP workshop tasks for working groups

FMD-NSP workshop scenario’s (3) size

number of cattle holdings

total number of cattle

average number of cattle

1 to 4

54

125

2

5 to 10

37

266

7

11 to 25

20

327

16

26 to 50

10

373

37

51 to 100

7

438

63

101 to 250

17

2.521

148

251 to 500

4

1.340

335

501 to 1000

2

1.466

733

0

0

-

151

6.856

1001 to

FMD-NSP workshop results

Background information of epizootic Characteristics of outbreak clusters (general composition, size and composition) Information about the different scenario’s (culling, vaccination, …)

Tables:

make the best possible survey design (not necessarily design proposed by OIE Guidelines or EU Directives), to follow-up the seropositive herds/animals/flocks and use of lab results in decision-making identify the necessary resources for their own scenario.

FMD-NSP workshop conclusions and recommendations (1) C.: Despite different working groups/countries clear degree of similarity between approaches

Presentation of results by groups in plenary session + discussion

C.: Vaccination-to-live → survey (NSP testing) → substantiate free from infection = realistic and achievable option in FMD control. But: stamping out remains part of control policy

66

FMD-NSP workshop conclusions and recommendations (2) C.: NSP assays:not sensitive enough (cf. carriers)

→ combine clinical, serological and epidemiological investigations (cluster analysis) to conclude on infection status of vaccinated herds.

R.: Conclusions on infection status of herds after FMD outbreaks in a vaccinated population: based only on a survey system incl. at least clinical, serological and epidemiological investigations.

R.: Performance characteristics of survey system should be determined wrt. different species

FMD-NSP workshop conclusions and recommendations (3) C.: Prove freedom from infection for vaccinated animals is impossible ↔ substantiate freedom from virus circulation in vaccinated animals ↔ freedom from infection in nonvaccinated animals; R.: The term ‘demonstrate absence of infection’ should be replaced by ‘substantiate absence of infection’;

FMD-NSP workshop conclusions and recommendations (5)

FMD-NSP workshop conclusions and recommendations (4) C.:

EC Dir. (2003/85/EC) for FMD control – 2 surveys: a)

b)

For detecting presence of FMD virus (Article 56 combination of clinical, epidemiological and serological investigations, high overall system sensitivity); to substantiate freedom from infection (Article 61) high specificity - might include a 2nd serosurveillance;

C.:

Test all animals in vaccinated population (cf. EC Dir. 2003/85/EC) not achievable in areas with dense vaccinated pig population or within big vaccinated pig herds.

R.:

Consider amendment of the Directive in order to allow a within-herd sampling scheme based on a 5% prevalence with 95% confidence for vaccinated pigs;

C.:

Herds with seroreactors: follow-up by serology based on NSP assays (welldefined performance characteristics).

C.:

If specificity of serological test system were known: consider seroreactor rates above Herd Cut Point only ↔ EC Dir. 2003/85/EC

R.:

Contingency plans: include a clear flow chart for follow-up of seropositive herds (minimum requirements of App. 3.8.7. of TAHC)

FMD-NSP workshop conclusions and recommendations (6) C.: I. II.

R.:

R.:

Detection of virus circulation by clinical surveillance + paired serology: test all ruminants When virus circulation: herd slaughter When carriers: slaughter of reactor animals only After FMD outbreak: Test all (large?) ruminants to substantiate freedom from infection in a vaccinated population.

Evidence of virus circulation → declaration of an outbreak In case of carriers: seek consensus on the slaughter of reactor animals only.

Change in outbreak definition in OIE guidelines and EC Dir. for carriers.

FMD-NSP workshop conclusions and recommendations (7) C.:

Vaccination of small herds remains controversial.

2 options were discussed: a)

b)

R.:

non-vaccination policy - integration as sentinels vaccination policy: they contribute in achieving the necessary level for population protection and because of political reasons.

The vaccination of small herds should be further discussed.

67

FMD-NSP workshop conclusions and recommendations (general) R.:

Explore relative confidence attainable with “herd based” and “individual” certification for different herd sizes and prevalences

FMD-NSP workshop remarks

R.:

To refine the application of NSP tests, more work could be done in predicting the expected prevalence of infection within and amongst vaccinated herds.

R.:

Functional FMD expert groups should be created in every country.

Acknowledgments (2)

Acknowledgments (1) Financial support: • • • • •

Change of OIE code: containment of infection region is possible, country status remains unaffected.

Training for set-up of information system for field data is necessary for some countries.

Countries with large population of backyard animals: problems in collection, control and management of disease information.

Scenarios assume perfect movement control. This is not a real life situation!

Probang testing will not prove absence of virus circulation → more problems than solutions to substantiate freedom from infection.

Introduction of negative animals as sentinels in a vaccinated pig or cattle herd is of limited value due to low transmission rate.

For several countries vaccination will not be the main option, especially if there are a lot of small herds.

The organisation of a workshop on ‘vaccination: how and when, after or in face of an outbreak’ should be considered.

FAO EUFMD TAIEX EPIZONE WP 4.3 EU Coordination Action FMD-CSF Veterinary and Agrochemical Research Centre Belgium

Logistic support: • Belgian Food Agency • Veterinary Administration of the Republic of Slovenia • Veterinary and Agrochemical Research Centre Belgium • Taiex

Jorgen Westergaard, Olga Zorko, Philippe Houdart, Pierre Kerkhofs, Koen Mintiens, Camilla Brasch-Andersen, Wim Pelgrim, Breda Hrovatin, Jedrt Maurer-Wernig, Aleksandra Hari, John Wilesmith, Dónal Sammin, Paul Van Hoeyweghen, John Bashiruddin, Ase Uttenthal, Preben Willeberg, Alf Füssel, Peter De Leeuw, Keith Sumption, Tom Murray, Gideon Bruckner, Lea Knopf, Isabel Minguez, David Paton, Aldo Dekker, Matthias Greiner, Yves Leforban.

NSP tests - Gaps OIE NSP ad hoc Group: - Sufficient data for - cattle - DSp naïve sheep & pigs and +/- non-vac infected sheep & pigs - Data lacking for - vac + vac/inf sheep (+ carriers) - experimentally vac + inf pigs - subclinically inf pig problem

68

Appendix 12

Outline of approach

Use of a model to predict expected prevalence of carriers and to design serosurveillance for their detection

• Simulation of epidemic with/without vaccination • Include simple model of the probability of farm being a carrier • Model post epidemic testing using NSP and record status of each herd • Examine sensitivity and specificity of NSP testing at herd level

Mark Arnold – VLA John Wilesmith – Defra David Paton, Eoin Ryan, Sarah Cox – IAH Pirbright

4/13/2009

Transmission model

2

Transmission vs distance from IP

• Transmission depends on – Number of animals in IP – Distance from IP – Number of animals in susceptible farm

4/13/2009

3

4/13/2009

Susceptibility to infection postvaccination

Animal number Infectiousness/susceptibility

Animal (and farm) susceptibility reduced according to days between exposure and vaccination

0.13 0.13 Susceptibility= 36 × ( N CATTLE ) + ( N SHEEP )

Infectiousness=

4/13/2009

4

1.4 × ( N CATTLE ) 0.13 + ( N SHEEP )0.13

5

4/13/2009

6

69

Probability of carriers in vaccinated farms

Likelihood of disease post vaccination

Estimate initial infs

Infectiousness assumed to correlate with probability of disease

Calculate clinicals

Determine if IP

Estimates from 2001 epidemic stil into ke Efficacy of vaccinationm equation or simulation Likelihood of disease vs days post vaccinationexperiment <5 days post vaccination OR >0 clinicals – dairy; >1 clinical -suckler

IF NOT AN IP

Calculate if carriers 4/13/2009

7

50% probability for each infected

4/13/2009

8

Carrier probability vs herd size and no of clinicals for detection

Number of initial infections Small dependence on number of cattle: Mean infs= 4.1+0.005Ncattle

4/13/2009

9

4/13/2009

Rate of vaccination

Other assumptions

• Commences 7 days after initial IP identified • 50 vaccination teams, vaccinating up to 250 animals per day • Clinical inspection of sheep and pigs also performed • 10k vaccination ring around each IP, nearest farms vaccinated first 4/13/2009

10

• Time to detection for infected herds taken from 2001 epidemic • All animals in each IP are culled, time to slaughter taken from 2001 epidemic

11

4/13/2009

12

70

Post epidemic testing

Outline of simulations

• Commences 30 days after last IP • Cattle tested on all vaccinated farms with Cedi screen/Cedi retest/Svanova confirm • Sensitivity=67%, specificity=99.99%.

• Sufficient animals tested to detect 5% prevalence with 95% probability • Have also looked at testing all animals

• Population at risk=Devon • 5 initial infections • 200 runs of each of the following scenarios: – No vaccination – Vaccinate farms>50 cattle, <10k from IP – Vaccinate all cattle farms < 10k from IP • 44% of herds with cattle have <50 in Devon 4/13/2009

13

4/13/2009

Epidemic size distribution – 200 runs

14

Mean no of IPs/day Vaccinating small farms – 66 vaccinated farms/IP, 71% reduction in cases Vaccinating farms >50 cattle – 35 vaccinated farms/IP, 68% reduction in cases

4/13/2009

15

4/13/2009

Distribution of carrier farms by herd size

Within herd prevalence Farms with many initial infs->IPs Farms infected late after vaccination have few initial infs Therefore carriers occur at low prevalence

Reducing number of farms as herd size increases But higher incidence of those farms

4/13/2009

16

17

4/13/2009

18

71

Sensitivity of testing (at farm level) Strategy Vacc >50 only Vacc all

Specificity of testing (at farm level)

True positives 335

Detected

Sensitivity

118

35%

325

140

43%

Vacc >50 only Vacc all

4/13/2009

19

Effect of changing key parameters

4/13/2009

Vaccinate herds>50 cattle, Devon Mean initial infections=5% of herd size 2 clinicals to be an IP 3 clinicals to be an IP Vaccine effect 50% slower Cumbria Cheshire 25% of infected cattle ->carriers 75% of infected cattle ->carriers Mean initial infections=5% of herd size, 2% of herd clinical for detection

False positives 1,562

99.32%

397,000

1,890

99.52%

Mean within – Percentage of herd prevalence vaccinated of carriers farms that are carriers 1.18 0.16

Specificity of testing at herd level (%)

Sensitivity of testing at herdlevel (%)

99.3

39.6

1.30

0.14

99.5

49.5

1.36

0.17

99.3

44.6

1.42

0.19

99.3

46.0

1.28

0.11

99.3

43.5

1.21 1.26 1.05

0.13 0.17 0.12

99.3 99.3 99.3

44.8 42.6 38.0

1.33

0.18

99.3

50.2

1.51

0.18

99.4

46.7

4/13/2009

20

Main results (1) • Little benefit for vaccinating small herds in terms of total cases • Carrier farms distributed across all herd sizes – fewer large herds but higher incidence of large herds • Small number of carriers in positive herds means low herd-level sensitivity (with 5% design prevalence) – therefore need to test all animals in vaccinated herds 21

4/13/2009

22

Conclusions

Main results (2)

• If do vaccination well expect few carriers that are difficult to detect • As, if not more important than NSP testing will be

• Vaccinating small herds – Smaller average herd size of positives – Higher herd level sensitivity – larger proportion of animals tested – Higher herd level specificity – But far more vaccinated farms to test

• Same conclusions from results of NSP testing in Cumbria 4/13/2009

Specificity

True negatives 233,000

• 98.4% specificity if all animals tested (for vacc>50)

• 75% sensitivity if all animals tested (for vacc>50)

Parameter change

Strategy

23

– Demonstrating effectiveness of vaccination programme – Detection of clinical cases

• Virus circulation 4/13/2009

24

72

Appendix 13

Presentation

Quantitative Evaluation of Surveillance Systems

FAOFAO-EUFMD

Securing Freedom From FMD The Surveillance Component

Simplified

example of Scenario Tree methodology to quantitatively evaluate Targeted random survey Issues relating to estimation of prior probability of FMD presence Application to Thrace Potential beneficial outcomes from the use of this method 1

FAOFAO-EUFMD

2

Questions

FAOFAO-EUFMD

3

Given that surveillance systems show no positive results. How confident are we that this region is free from FMD (NPV)?

– What is the probability that FMD is present prior to survey (prior) – After combining the survey results with the prior and presuming that all survey evidence suggests the absence of FMD, how confident are we that this is a true reflection of the situation.

– Given that all results are negative, then what is the probability of error and that disease is actually present. (Posterior) (Probability of Disease Presence) FAO4 FAO-EUFMD

Examples:Targeted Random Serosurveillance 152

Villages surveyed

64

Juvenile animals surveyed per Village

Diagnostic

FAOFAO-EUFMD

5

FAOFAO-EUFMD

Test Sensitivity eg 0.81

Data For Illustrative Purposes Only

6

73

Scenario Tree

Quantitative evaluation of TRSS Question: If the detection of one NSP positive animal in a village represents FMD presence and all animals test negative, then how confident are we that FMD is absent.

FAOFAO-EUFMD

7

FAOFAO-EUFMD

Adjusting Minimum Expected Prevalence

Formulae Probability any sample for an infected herd tests positive

Pupos=EPI* Seu Pupos=EPI*Seu

=Effective probability infected*Unit Test Sensitivity

Herd Sensitivity

HSe=1 HSe=1--(1(1-Pupos)^n

n is the number of animals sampled within the herd

Surveillance System SSCSe= SSCSe= 11-(1(1component (HSe*EPIh))^nh (HSe*EPIh))^nh sensitivity

Negative Predictive Value

The

nh is the number of herds sampled EPIh is the effective probability that the herd is infected

NPV=(1NPV=(1-prior)/(1prior)/(1(prior*SSCSe )) (prior*SSCSe))

Posterior probability Posterior=1Posterior=1-NPV of FMD

FAOEUFMD FAO of -FMD

8

Data Illustrative Only

9

Effective probability of Infection EPI Used to account for the fact that minimum expected prevalence would vary from region to region and between animal groups.

The relative risks are adjusted (Adjusted Risk AR) to account for variation between groups while not effecting the regional minimum design prevalence EPI (for each group) = AR X Design Prevalence

minimum probability that any animal within a village is the design within herd prevalence (Pu (Pu)) But risk varies between strata and this serosero-survey targets Juveniles Calculate the minimum “effective probability” that any Juvenile animal within an infected herd is infected.

FAOFAO-EUFMD

10

Effective Probability of Infection

FAOFAO-EUFMD

11

ARNeo =

1 JRRj + ARRa + Neo

ARj= RRj X ARc ARA= RRAX Arc Effective probability infected (EPI) = AR XPU

FAOFAO-EUFMD

J = Proportion Juveniles In population A = proportion of Adults in population Neo = proportion of Neonates in population RRj=Relative Risk Juveniles to Neonates RRa=Relative Risk Adults to Neonates

12

74

PstarU Test Sensitivity sampled/herd Herds tested Probability That any one test from an infected Herd is positive Herd Sensitivity SSC sensitivity

0.05 0.8 64 152 0.0005556

0.97 0.77

NPV Posterior

0.82 0.18

RRisk 5 3 1

Juvenile/Neonate Adult/Neonate Neonate

rrjuv rrad rrneo

Adjusted Risk 1.39 0.83 0.28 0.28

Calculation of Adjusted Risk Neonate

Propneo propjuv propad

FAOFAO-EUFMD

EPI 0.069 0.042 0.014

arjuv arad arneo

Unlikely that all tests are negative, as few tests are 100% specific Use of Confirmatory tests added to scenario tree

0.1 0.4 0.5

13

Data Illustrative Only

Can use scenario Tree to evaluate the effect of variations in testing parametres

FAOFAO-EUFMD

Data Illustrative Only

14

Combining Surveillance System Components

Screening Test Specificity

The

Posterior Probability for NSP serosero- surveillance is used as the Prior Probability when calculating Negative probability of the overall surveillance system and so on.

Inputs can be used in the form of distributions to account for uncertainty

A

highly specific Screening test (e.g.

NSP tests) leads to a greater negative predictive value

FAOFAO-EUFMD

15

FAOFAO-EUFMD

16

Prior probability of FMD presence

First Quantitative Evaluation – – – –

Expert Opinion Literature Risk Assessments Levels of various risk factors Periods of increased marketing (e.g. kurban) kurban) Increased incidence of FMD in Anatolia New Strains in Anatolia Vaccination Coverage

Subsequent Evaluations – Uncertainty associated with estimation of prior probability decreases – Prior is based on posterior probability from a previous incursion and the probability of a new incursion in the intervening time period

FAOFAO-EUFMD

17

FAOFAO-EUFMD

18

75

Application To Thrace/Turkey

Application to Thrace Turkey

Turkish Veterinarians collect and model current available data carry out quantitative evaluation Workshop of Turkish and International experts to analyse results and assumptions The construction of a framework for the progressive improvement of surveillance systems Repeat annually

Identify the surveillance options available Quantify these within an overall surveillance system Identify risk factors for new incursions of FMD Quantify how these risk factors effect the chance of new incursions into Thrace.

1.

2.

3.

4.

FAOFAO-EUFMD

19

FAOFAO- EUFMD

Sources of Data/Information/Knowledge

Potential Surveillance Options Targeted

Random Serosurveillance Clinical Surveillance at Markets SeroSero-surveillance at Markets Veterinary farm inspections Compulsory notification Outbreak investigations Other veterinary activities

Turkey – – – – – – – –

– eg Tb and Brucellosis testing FAOFAO-EUFMD

20

21

Markets GDPC Farmers Local Veterinary Offices SeroSero-surveys Consultants Veterinary Clinics SAP Institute

Primary Research Literature Reviews Research Group members EUFMD Reports EUFMD Meeting Reports Risk Assessments ????

FAOFAO-EUFMD

22

Potential Beneficial Outcomes

Effective surveillance is a critical component in FMD Control Structured and progressive approach to surveillance and control in Turkey Potential for comparison with other regions and monitoring progress from year to year.

Provide Direction for further research Provides a platform for the practical application of latest research work Is useful for evaluating individual surveillance system components A surveillance system that provides a high level of confidence in FMD Freedom is one that provides for early detection of new FMD Incursions This could work could progress into Eastern Turkey

FAOFAO-EUFMD

23

FAOFAO- EUFMD

24

76

Appendix 14

Introduction

3ABC ELISA in the African context

Difficult differentiate between vaccinated and infected animals.

FMD free status requires proof.

FMDV vaccines: SemiSemi-purified antigen depleted of most nonnon-structural proteins (NSP).

Host immune response is directed mainly towards the structural proteins proteins (SP).

F.F. Maree, Maree, B. Blignaut, Blignaut, J.J. Esterhuysen, Esterhuysen, L. Heath, W. Vosloo Transboundary Animal Diseases ARC

Presence of antibodies (Ab (Ab)) to the NSP’s – indicates animals naturally infected with live replicating FMDV.

Serological tests available: The 3ABC ELISA detects antibodies against the NSP, like 3ABC polypeptide. polypeptide.

ELISA and VNT tests measure antibodies against the structural proteins proteins

Assessment of commercial 3ABC ELISAs

Gene relationship of 3C coding region West African SAT2

A variety of 3ABC ELISA kits are commercially available. based on the NSPs of the European types (A, O and C)

45% nucleotide variation

Significant variation and poor correlation were observed between the sensitivities of the different kits.

Positive results were observed for only a few weeks post infection. infection.

Ugandan SAT strains

32% amino acid variation Euro-Asian types A, O and C Southern African SAT types

ELISA for structural proteins

LPBE

SEQUENT IAL SERA 3.5 3.0

titer

2.5 2.0 1.5 1.0

East African SAT1 & 2 and West African SAT1

133

119

91

105

77

63

49

35

21

13

9

11

7

5

3

P re

0.0

1

0.5

days post infection KNP 196/91/1

KNP 19/89/2

KNP 10/90/3

0.02

Cut-off

Assessment of commercial 3ABC ELISAs

Assessment of commercial 3ABC ELISAs

Cattle infected with SAT1/KNP/196/91 SAT1/KNP/196/91

Cattle infected with SAT2/KNP/19/89 SAT2/KNP/19/89

200

90

180 160

Cedi Svanovir Bommeli Checkit UBI

140 120 100

80 70

Cedi Svanovir Checkit UBI

60 50 40

80 60

30

40 20

20 10

0

Days post-infection

12 6

98

11 2

84

70

56

42

28

14

12

8

6

4

10

PRE

2

0

406

371

336

301

266

231

196

161

126

91

56

21

5

10

0

0

PRE

Days post-infection

77

Assessment of commercial 3ABC ELISAs

Assessment of commercial 3ABC ELISAs

Cattle infected with SAT3/KNP/10/90 SAT3/KNP/10/90

SAT1/KNP/196/91 SAT1/KNP/196/91

120

Cedi Svanovir Checkit UBI

100 80

Bommeli Checkit

SAT3/KNP/10/90 SAT3/KNP/10/90

SvanoSvanovir

UBI

Cedi

SvanoSvanovir

UBI

Cedi

SvanoSvanovir

UBI

0.76

0.74

0.91

0.15

0.56

0.78

0.38

0.31

0.19

0.82

0.65

0.19

0.19

0.48

0.73

Cedi

60

SAT2/KNP/19/89 SAT2/KNP/19/89

Cedi

SvanoSvanovir

0.66

0.49

0.19

40

> 0.6 = Good

Kappa

14 7

12 6

84

Days post-infection

10 5

63

42

21

9

3

12

0.4-0.6 = Medium < 0.4 = Weak

6

0

0

20

Gene relationship of 3C coding region

60

SAT2/ZIM/17/91 /14/90 SAT2/ZIM 6 2/93 /4/9 ZAM/ AM SAT1/ T3/Z /83 1 SA /7 /6 3/02 /ZIM SA /5 2 OZ/ /M S AT a t1 T1 s SA

30

sat1/

40

27% variable aa

98

84

70

56

42

28

14

12

8

10

6

4

9

2

C4Tierra

A29Peru/6

N/7/83 SAT2/SE

0

14% variable aa

9 C a/6 is AB a n re-3 M u O1 esh G O1 67 ros/ a se O1C efa ACan

C1O C 1N berbayern /60 ov ille/ 65

SAT2/SE N/5/75

3A

SAT2/GHA/8/91

32% variable aa overall

Days post-infection

Alignment of the 3ABC polyprotein

ay/98

ntina/69 C5Arge C3Rese nde/55 AArgen tina/00 O6Pir br ig A1 Ba ht/65 ye OK rn E /Bav Ty N 19 aria 1-3 pe AB A1 C 76 -3 AB C

EuroEuro-Asian types A, O and C

0.02

PRE

OTaiw an/97

AUrugu

Southern African

0

SAT1/N SAT IG/5/81 1/SU SA D/3 T2 sat1 /76 /7 IS /R W RL A/2 /4/6 /0 2 1

SAT2 /UGA S /2/02 SA AT3 /U G T1 A/2 /U G /9 7 A/1 /9 7

4 AN G/4 /7 SA T2/

4 1/7 AI/ /Z /75 T2 /15 SA NIG 00 T1/ U/6/ SA /S A T2 21 SA nyake -3 /3/57 sat2 SAT2/KEN

20 10

East and West African

R/00 O1SK

SA T3 /B S A EC T1 /1 /N /6 sat1 AM 5 /3 /3 6SW SWA107/9 8 A/4 SAT3/ZIM/ 0/61 /49 5/91 sat1/1BECH 8 sat1/BOT/1/6 srhod sat1-4 ch ya be -3 n sat3 ke -3 t3 a s

Cedi Svanovir Checkit UBI

50

SA SA T2 /K SAT2 T1 /K N P /S AR NP/1 /1 9 /16 96 /8 SAT2/M OZ/4/8 /83 /9 19 SAT1/SAR/9/81 3

sat2-2

6% variable aa

70

sat1-20 6 HOD/5/6 /90 sat1/R P/10 /KN SAT3 7/99 /76 a s N/3 L/3 -2 /T A M A t3 sa T3/ SA

T1 SA

Cattle infected with SAT2/UGA/2/02 SAT2/UGA/2/02 80

sat2-1rhod SA T2/R HO/1/ SAT1 48 S A SA /KEN/5/98 T2 /K T1/T AN EN /1/9 /8/9 9 9

SAT1/U GA/3/9 9

Assessment of commercial 3ABC ELISAs

NeighbourNeighbour-joining tree

Structure of FMDV 3C protease

3B

SAT1, 2, 3

mAb binding site 2

“Back” view of 3C

3C

SAT1, 2, 3

Hypervariable regions Mab epitopes

mAb binding site 1

mAb binding site 3 Hypervariable regions

Deduced amino acid sequence alignment of the non-structural 3ABC polypeptide of FMDV SAT1, 2 and 3 strains and European A, O and C types. Binding sites of Mabs used in commercial kits overlap with hyper variable regions, with up to 30% amino acid variation.

Most of the variation maps opposite to the active site of the 3C protease.

78

3B amino acid alignment

3A amino acid alignment

O1 Manisa 3B 3B1

3B2

1

11

21

3B3

1

11

Southern Africa SATs

C-terminal of 3A

21

81

1

11

21

91

101

111

121

131

141

KRQQMVDDAV NDYIEKANIT TDDKTLDEAE KNPLETSGAS TIGFRERTLP GHKASDDVST EPAKPVEDRP

GPYAGPLERQKPLRVKTKLPQQE GPYAGPMDRQKPLKVRARAPVVKE GPYEGPVKKPVALKVKAKNLIVTE T

M

Q

KLQPRP KA

K

RA

R

V

VR LEK Q

RLKSKL

R

A

D LL

A

L TRAP

T P

TM

E

S

M

K E

EEVL DEHTARSG A

Q

E

G RK Q VVDKP GVV KK KEG GQAPRKD KT

V

Q RH FQGTA SIS H

T

N

KTGTS

aa variation in alignment with 34 SAT isolates

AETAE-R GATGEEE

H Y

P A

Q

Q E

Q

T

variable aa positions in SATs

46.5% variable amino acids

Mab epitopes

R L K TS N

50% variable amino acids

Hydrophilicity profile of 3ABC

Cloning of truncated 3ABC

Hopp and Woods Scale Mean Hydrophilicity profile

S-tag

T7 promotor 1

20

Mean Hydrophilicity

15

tr3ABC 10

Induction

ptr3ABC

5 0 -5

His-tag

-10 -15 53 amino acids

74 amino acids

amino acid residue

N 3B

3C

Truncated 3ABC

Expression of truncated 3ABC in E. coli

TrNC clones TrC clones 2

3

Poly-His tag

Solubility and purification of rec 3ABC

Western blot using anti-SAT2 sera

SDS PAGE gel non rec un 1

His His His His

N

C 3A

4

1

3

6

8

9

8

TrNC clones 1 2

TrNC clones TrC clones un

9

1

2

3

4

1

3

6

8

9

8

unin

9

ind

unin ind

insoluble

soluble

75 50 50 37 37

50 25

25

37

Time induced: 5mM IPTG

3h

24h

3h

24h

Time induced:

3h

24h

3h

24h 25

BOT/4/06-specific polyclonal serum from infected animals.

79

ELISA detection of truncated 3ABC

ELISA detection of purified tr3ABC

2.5

2.5

His

2

OD450nm values

3ABTrC

OD450nm values

2

1.5

1

0.5

1.5

1

0.5

0

0 1

2

3

4

5

2-fold dilution

6

TrC

7

TrNC

8

2

3

4

5

2-fold dilution

6

TrAC

7

TrC

The antigen was captured onto poly-H tagged mAb plates.

Plates coated with truncated 3ABC protein.

BOT/4/06/2 infected cattle sera (PS).

BOT/4/06/2 infected cattle sera (PS).

Negative bovine sera (NBS).

Negative bovine sera (NBS).

Summary

1

Negative

Acknowledgements and Collaborators

Significant variation and poor support were observed between the different NSP kits.

32% amino acid variation in a complete alignment of 3C.

4545-50% variation in 3B and CC-terminal of 3A.

8 amino acid deletion in CC-terminal of 3A in an SAT2 isolate from Senegal.

Hypervariable regions overlap MAb binding sites.

A truncated, soluble form of a SAT2 3ABC polypeptide was successfully successfully expressed in E. coli. coli.

8

Negative

TADs: TADs:

Belinda Blignaut

Jan Esterhuysen

Dr. L. Heath

Dr. J. van Heerden

Collaborators:

Sonja Maree

Biochemistry, OVI

Prof. J. Theron

Microbiology, UP

Dr. E. Rieder

PIADC

80

Appendix 15

Outline of presentation

Closed Session of the EU FMD Research Group Use of NSP tests in areas where SATtype FMD viruses are prevalent: Consultancy report

The problem Consultancy report – Dr Peter Roeder, Taurus Animal Health, UK: Conclusions Recommendations Why NSP testing is so important in the wider context of FMD management in southern Africa

Gavin Thomson, SADC FMD Project, Botswana

SADC FMD Project

SADC FMD Project

The problem

Roeder report

An NSP test, or set of NSP tests, is needed for surveillance & management of FMD in regions of Africa where SAT-type viruses are prevalent Initial appraisal of available test kits/systems raised questions about the sensitivity of available tests (& also the ‘purity’ of antigens included in current FMD vaccines produced in the region) The SADC FMD Project therefore appointed an independent evaluator to provide a formal opinion as to the suitability of the presently available NSP tests in areas where SAT-type viruses are prevalent

Conclusion Despite paucity of information, data exists to support the view that currently available NSP tests do not work well with SAT viruses, especially SAT2 & SAT3 There is a need to develop NSP tests which are reliable to use within the SADC environment & this merits support

SADC FMD Project

Roeder report Recommendations Targeted action is needed to produce appropriate diagnostic reagents & apply them in robust test formats Essentially what is needed is to extend what has been done for the Eurasian FMDV lineage to the SAT lineage – product development Collaborative action is necessary – lead by SADC reference laboratories, leading international labs working in the NSP field, some African national labs & international agencies (FAO & OIE)

SADC FMD Project

Roeder report Recommendations (cont.)

Following elements are necessary: • Intensification of studies on phylogeny & antigenic characterisation of SAT viruses, including NSPs • Development of an assay (more likely assays) capable of detecting, with high sensitivity, the circulation of SAT viruses in livestock populations, irrespective of vaccination status, i.e. detection of SAT antibody ‘footprints’ • Exploration of the use of anti-3D assays for the detection of virus circulation in free-living wildlife populations

81

SADC FMD Project

Why are TFCAs an issue in relation to FMD?

EU-funded (4 year / >€12 million) project aimed at increasing capacity to manage FMD in the SADC Region generally & to assist three countries specifically – Malawi, Mozambique & Zimbabwe

Because: TFCAs will increase the number & distribution of wildlife reservoirs;

In this context, SAT viruses & wildlife assume special significance & the issue of wildlife is escalating

They straddle the borders of 2-5 countries – the ideal is that TFCAs should be linked by corridors

Particular problem relates to the transfrontier conservation area (TFCA or ‘Peace Parks’) concept because it is in conflict with international norms associated with management of transboundary animal diseases (TADs) generally & FMD specifically

FMD & Limpopo NP as an

illustration of the TADs problem

The TFCA movement has major political, financial & popular backing & is a fact of life!

Great Limpopo Transfrontier Park (GLTP)/GLTFCA

Background • SAT viruses are maintained in free-living situations by African buffalo (Syncerus caffer) • But the buffalo show no physical sign of infection • These viruses are sometimes transmitted from buffalo to domestic animals, cattle particularly, by close contact • Once cattle are infected they can propagate the viruses independently of buffalo • Therefore a major effort has traditionally been made in SADC Region to keep buffalo & cattle apart as a means of preventing FMD occurring in livestock (additional to vaccination of cattle) • Infection in cattle trade sanctions & knock-on effects

GLTFCA

A village on the banks of the Limpopo River adjacent to the LNP (GLTFP) – there are many like this

The Limpopo River is no barrier!

This area not surveyed due to aircraft problems

82

Results of an aerial count in the Shingwedzi River Basin portion of Limpopo National Park, Mozambique (Whyte & Swanepoel, 2006) SPECIES Buffalo

COUNT 225

SPECIES

Bushbuck

1

Roan

Bushpig

8

Sable

Elephants

630

COUNT

Ostrich

36 6 62

Steenbuck

12

Giraffe

23

Warthog

48

Grey Duiker

56

Waterbuck

86

Ground Hornbill

50

White rhino

16

Impala

496

Wildebeest

358

Kudu

273

Zebra

325

7

Cattle

3142

257

Goats

527

Lichtenstein's Hartebeest Nyala

The situation & its implications?

• About 25 000 people – who own unknown perhaps 7-8 000 head of cattle – are living within borders of the Limpopo NP; their relocation unlikely to occur soon!

• Therefore the Limpopo NP is at high risk of initiating an outbreak of FMD • Movement of cattle out of Limpopo NP happens on a regular basis & is difficult to prevent threatens the whole of southern Mozambique • The only tools we have at our disposal for managing FMD in this situation is surveillance & vaccination. In this context effective NSP tests are indispensable • But it’s complicated by livestock policy defining future development

Satellite view of southern Mozambique Proposed beef export zone The juxta-positioning of the GLTP & the beef export zone is incompatible with the international rules, i.e. those laid down by OIE. Thus confusion reigns – huge problem for rural development!

16

TFCAs / livestock policies are often incompatible with international norms!

• This is an illustration of the type of problem that is evident in a number of SADC countries • TFCA concept, in general, is not compatible with norms of international animal health control • However, there are new possibilities based on concepts such as ‘compartmentalisation’ & ‘commodity-based trade’ which provide an opportunity • Future development – irrespective of the approach adopted – will depend on the availability of effective NSP tests able to identify SAT virus infections in vaccinated populations • This issue is not unimportant in safe-guarding Europe from future FMD threats

83

Appendix 16

OIE VALIDATION TEMPLATE FMDV NSP tests summary of key performances

Check list 3 3.1

Objective Create a reference source of key performance data of NSP tests (OIE index test and tests available in Europe) Users: epidemiologists, lab staff, tests selection, …

Possible Model : OIE validation template

4

Calibration

4.1

3.2

Repeatability

3.3

Analytical specificity

3.4

Analytical sensitivity

Validation - Stage II Reference Animals

4.1.1

Negative reference animals

4.1.2

Positive reference animals

4.1.3 Validation – Stage III

4.2

5.1

Laboratory selection

4.3

5.2

Evaluation panel

5

5.3

Update information when required

Validation – Stage I

6

Performance Estimates

4.3.1

Reproducibility

Laboratories

6.2

Test applications

6.3

International reference reagents

6.4

Inter-laboratory testing programmes

6.5

International recognition

Dx Se and Sp estimates with defined reference animals

4.3.2

Dx Se and Sp estimates without defined reference animals

4.3.3

Agreement between tests

Validation – Stage IV

6.1

Experimental animals Threshold determination

SPECIES Cattle – sheep/goats - pigs

IZSLER

IZSLER

BRESCIA

BRESCIA

OUTPUTS OF BRESCIA WORKSHOP MILESTONE → Evaluation of NSP tests available in Europe and comparison with the OIE index test

Brescia International Workshop 2004 Most accredited data

Publications Vaccine 2006 → comparative evaluation – tests performances EUFMD 2006 → ROC/LR analyses Vaccine 2007 → estimates of Dse and DSp by Bayesian appr.

Vaccine 2006 → use of NSP tests after emergency vaccination

Parallel testing with 6 NSP tests Same samples, different sources Single laboratory – different operators Transparent results

Workshops Brussels 2007 → design and interpretation of postvaccination surveillance by NSP tests (simulation exercise) IZSLER

IZSLER

BRESCIA

BRESCIA

OIE VALIDATION TEMPLATE Check list 3

OIE VALIDATION TEMPLATE

Validation – Stage I

4

Validation - Stage II

3.1

Calibration

4.1

Reference Animals

3.2

Repeatability

3.3

Analytical specificity

3.4

Analytical sensitivity

Performance data relevant to Stage I and II for the NSPtests available in Europe and for the OIE index test can be found in the papers originated from Brescia WS Complete for cattle

4.1.1

Negative reference animals

4.1.2

Positive reference animals

4.1.3

Experimental animals

4.2 4.3

Progress in stage III Two evaluation panels prepared

5

Threshold determination

5.1 Laboratory selection

Performance Estimates

4.3.1

Dx Se and Sp estimates with defined reference animals

4.3.2

Dx Se and Sp estimates without defined reference animals

4.3.3

5.2 Evaluation panel

Agreement between tests

Preliminary for other species Presented in “tables format” in several occasions

Validation – Stage III

Question: find the format to summarise/present lots of data

5.3 Reproducibility

-

IAH (JVDI 2007): 36 cattle sera Panaftosa (submitted): 34 cattle

Reproducibility - index test: data available - commercial tests: maybe deducible from Phase XIX, Phase XX results ? (several labs tested the proficiency panels with available NSP tests)

IZSLER

IZSLER

BRESCIA

BRESCIA

84

Field studies in cattle detection of infection/viral circulation in vaccinated populations

OIE VALIDATION TEMPLATE Progress in stage IV

6

Validation – Stage IV

Applications in several field cases

6.1

Laboratories

6.2

Test applications

6.3

International reference reagents

6.4

Inter-laboratory testing programmes

6.5

International recognition

• Zimbabwe (SAT 1 & SAT 2 → 6 NSP tests • Israel (type O) → 6 NSP tests • Cameroon (O, A SAT2) → 3 tests (OIE Index, Danish, Chekit)

International standards sera described

• South Caucasus → Brescia test, 2005-2007 survey • Taiwan (VetMic 2006) → UBI & Ceditest)

IZSLER

2004: stratified survey in pluri-vaccinated pop in area FMD-free with vaccination since 2002 1% + by UBI, 4% + by Ceditest No vaccine interference according exp trials IZSLER

BRESCIA

BRESCIA

Field studies in pigs

Recent developments in NSP assays

the 3 commercial NSP tests available for pigs (Ceditest, UBI, Chekit)

• Hong Kong 2005 → Donal • Taiwan (J Vir Met 2004, Vet Mic 2007) • Kinetic and duration studies, up to 400 d.p. outb.

– Ab drop more rapidly than in cattle (non carrier status in pigs?), 30 → 60% pos at 25 weeks post outb.

– Sensitivity → UBI ≥ Ceditest > Chekit

• Diagnostic specificity – naive → 100% – repeatedly vaccinated 65 → 84% in 1997-98 years 84 → 100% in 2004 (improved assays or purified vaccines?)

UBI less specific UBI for screening (3-5% FP), Ceditest for confirmation (0.03% FP)

Indirect ELISA format Source

Antigen

ELISA format Detector Ab

Vet Mic 2007 China

3ABC (E.coli) coated

Vaccine 2006 Argentina

3ABC (E.coli) MAb-trapped

J.Vet Sci 2005 Korea

2C peptide (15 aa) coated

Indirect

α-species Ig-HRP

JV Diagn Inv 2006 Japan/Uk

2B peptide (13 aa, KLH carrier) coated

Indirect

α-bovine Ig-HRP

Vaccine 2007 India

3A+3B peptides (49 aa) chimeric thimerovirus VLP self-assembled in E.coli, coated

Indirect

α-species Ig-HRP

Indirect

α-species Ig-HRP

Indirect

α-bovine Ig-HRP

(α-3A Mab-trapping

IZSLER BRESCIA

Recent developments in NSP assays competitive ELISA format ELISA format

competitorAb

Biotinilated 3ABC (E.coli) Trapped by streptav

competitive

α-3ABC guinea pig serum + α-guinea pig Ig-HRP

3AB histag (E.coli) coated

competitive

α-3B MAb-HRP

J Imm M 2007 Australia

3ABC (E.coli) coated

competitive

Chicken α-3B rec Ab (by phage libraries of scFv)

J Imm M 2007 Canada

3D histag (E.coli) coated

competitive

α-3D MAb + α-mouse Ig-HRP

Source

Antigen

JVM 2004 Canada JVM 2007 Korea

Simultaneous testing against 4 Ag → potential confirmatory test Only preliminary feasibility studies in cattle, sensitivity similar to Indirect ELISAs Vaccine 2006 Canada

3ABC+3A+3B+3D (E,coli, histag)

Multiplex Luminex assay

Biotinilated α-bovine Ig + streptavidine R-PE conjugate

Performances of recently described NSP assays Source Vet Mic 2007 China

Antigen 3ABC (E.coli) coated

ELISA format Detector Ab Indirect

α-species Ig-HRP

DSp → evaluated : N and V cattle/pigs/sheep (2606 in total) Kinetic/Duration → evaluated (different species, Infected) Diagnostic Sensitivity Some exp animals, I, different species (confirmation of cut-off) Few field samples Type O / Asia 1 (not all infected animals react POS) Comparison with Cedi (98.05% concordant) and UBI (93.2% concord) IZSLER BRESCIA

85

Performances of recently described NSP assays Source Vaccine 2006 Argentina

Antigen

ELISA format Detector Ab

3ABC (E.coli) MAb-trapped

Indirect (α-3A Mab-trapping

α-bovine Ig-HRP

Performances of recently described NSP assays Source J.Vet Sci 2005 Korea

Antigen

ELISA format Detector Ab

2C peptide (15 aa) coated

Indirect

α-species Ig-HRP

Kinetic/Duration → evaluated

2C peptide recognised by cattle, not by pigs Suited only for cattle Not “normalized” results → epression by OD values

Diagnostic Sensitivity

DSp → evaluated : 467 N and 768 V cattle

DSp → evaluated : N and V cattle (1626) →

99.6%

Only cattle, types O and A Few, not well defined Infected cattle → 96-97% Extensive use for post outbreaks surveillance (A Arg 2001), >100,000 samples, detection of virus circulation in vaccinated population) IZSLER

→ > 99%

Kinetic → confirmation of observations by other studies Diagnostic Sensitivity Few nfected cattle, type O (O/SRK 2000) → similar to Chekit

BRESCIA

Performances of recently described NSP assays

Performances of recently described NSP assays Source

Source JV Diagn Inv 2006 Japan/Uk

Antigen

ELISA format Detector Ab

2B peptide

Indirect

(13 aa, KLH carrier) coated

α-bovine Ig-HRP

IZSLER BRESCIA

Vaccine 2007 India

Antigen

ELISA format Detector Ab

3A+3B peptides (49 aa) chimeric thimerovirus VLP self-assembled in E.coli, coated

Indirect

α-species Ig-HRP

Not “normalized” results → expression by OD values Studies performed only in cattle

DSp → preliminary analysis : 190 sera to define cut-off

DSp → preliminary evaluation : > 300 sera to define cut-off

Kinetic/Duration → evaluated in different species (seroconv ≥ 10 dpi, not all animals seronvert: incongruency between figs and text)

Kinetic/Duration → evaluated

Diagnostic Sensitivity

Diagnostic Sensitivity Field samples Zimbabwe, comparison with other NSP tests Promising test, screening or confirmatory, new target antigen

IZSLER

Several exp and field sera (different species, including buffalos), but poorly defined Comparison with Ceditest IZSLER

BRESCIA

BRESCIA

Performances of recently described NSP assays

Performances of recently described NSP assays

Source

Antigen

ELISA format

Detector Ab

Source

Antigen

ELISA format

Detector Ab

JVM 2004 Canada

Biotinilated 3ABC (E.coli) Trapped by streptav

competitive

α-3ABC guinea pig serum + α-guinea pig Ig-HRP

JVM 2007 Korea

3AB histag (E.coli) coated

competitive

α-3B MAb-HRP

DSp → extensively evaluated (3098 N + 313 V field sera, three species) 99.97% (only 1 bovine serum positive)

DSp → ≈ 100% (1780 Naive, three species) Kinetic → evaluated in≠ species (seroconv 7 - 10 dpi, 4 FMDV types)

Kinetic → evaluated in≠ species Diagnostic Sensitivity → not evaluated

Diagnostic Sensitivity → not evaluated (only 19 cattle, 56 dpi,type O

IZSLER

IZSLER

BRESCIA

BRESCIA

Performances of recently described NSP assays Source J Imm M 2007 Australia

Antigen 3ABC (E.coli) coated

ELISA format

Detector Ab

competitive

Chicken α-3B rec Ab (by phage libraries of scFv)

Rec Antigen and rec Antibody (recombinant single chain variable fragments, scFv, selected from phage libraries generated from chickens immunised with 3ABC, E.coli expression)

Performances of recently described NSP assays Source J Imm M 2007 Canada

Antigen

ELISA format

3D histag (E.coli) coated

competitive

Detector Ab α-3D MAb + α-mouse Ig-HRP

Pepscanning of 3D with infected cattle sera → MAb produced against peptide 4, best reactor

Validation not started

DSp and DSe not evaluated Feasibility studies performed with few I cattle → 80% detected

Only feasibility studies with few N and I samples

Kinetic evaluated in cattle and sheep Pigs do not react (do not recognise peptide 4) IZSLER

IZSLER

BRESCIA

BRESCIA

86

Appendix 17

Introduction Comparative evaluation and validation of NSPEs Dónal Sammin, CVRL-DAF, Ireland

Studies • FMD-ImproCon multinational workshop; IZS-Brescia, May 2004; cattle (± sheep/pigs); Brocchi et al., 2006 [App 01] + Dekker et al. [App 08] • EUFMD/WRL field study; Zimbabwe, April 2004; cattle, SAT-type infection; Sammin et al., VR, 2007 • EUFMD/WRL field study; HK-SAR, March 2005; (+ experimental study, IRL, 2006); pigs; Paton et al.

• 2003/85/EC: vaccination to live and post-vaccination surveillance • DIVA testing => use of NSP-based tests • OIE-approved method ex. Panaftosa • 5 other NSPEs in Europe • Evaluate comparative performance • Validate for purpose

FMD_ImproCon WS; IZS, Brescia; May 2004 Brocchi et al., 2006 [App 01] MATERIALS & METHODS • 3551 sera [BE, DE, DK, ISR, IT, NL, TK, UK] • 2579 (67%) bovine; 703 ovine; 269 pig • Different vaccination and infection status • All sera tested x6 NSPE in parallel • Panaftosa, IZS-B, Ceditest, Svanovir, Chekit and UBI ELISAs

• Proposed field and exptl studies on sheep, 2007/8

WORKSHOP; IZS, Brescia; May 2004 MATERIALS & METHODS: BOVINE SERA

• Experimental (n = 1037 sera) 425 V+ I62 [54] V- I+ 21 [17] C+ 550 [285] V+ I+ 225 [67] C+ • Field (n = 1542 sera) 672 V- I867 V+ I± (ISR/ZIM)

WORKSHOP; IZS, Brescia; May 2004 MATERIALS & METHODS: NSP ELISAs ELISA

Antigen

Format

“Grey zone”

Panaftosa

3ABC

INDIRECT; coated

YES

IZS-Brescia

3ABC

INDIRECT; trapping

YES

Ceditest FMDV-NS

3ABC

BLOCKING; trapping

NO

SVANOVIR FMDV 3ABC-Ab ELISA

3ABC

INDIRECT; coated

NO

CHEKIT-FMD-3ABC

3ABC

INDIRECT; coated

YES

UBI FMDV NS ELISA

3B

INDIRECT; coated

NO

87

WORKSHOP; IZS, Brescia; May 2004 RESULTS: diagnostic specificities

• 1100 bovine sera [675 V- I- & 425 V+ I-] • 97.2% - 98.5% on first screening test • 98.3% - 99.7% on retesting positives

WORKSHOP; IZS, Brescia; May 2004 RESULTS: detection rates • CATTLE V- I+ (n = 54) & V+ I+ (n = 285) • Sub-categories of V+ I+ cattle: • • • •

(i) infection demonstrated (n = 164); (ii) carrier status demonstrated (n = 67); (iii) not carriers (n = 26); (iv) no evidence of infection (n = 17)

• 7-14, 15-27, 28-100 and >100 days p.i.

• Subcategories (i) and (ii), 14-27dpi: Panaftosa, IZS-B and Ceditest NSPEs >60% seropositive • Carrier detection rate, 28-100dpi; Panaftosa 93.9%, IZS-B 86.4% and Ceditest 86.4%

EUFMD/WRL field study; Zimbabwe • • • • •

Objective: evaluate NSPEs for SAT-type FMD 403 cattle; 6 herds; April-May 2004 SAT1/SAT2, 5 herds, 1-5 months pi 12 - 35% of herds were “carriers” (probangs; VI & PCR) Overall seroprevalence of 56% - 75% with NSPEs; 81% with SPCE and 91% with VNT • Carrier detection rate of 70% - 90% • Sammin et al., Veterinary Record (2007) 160: 647-654

Still to do…

• Publish paper on HK & IRL pig studies • Publish paper on LRs and ROC analysis of results from Brescia WS • Perform field and experimental studies on sheep during late 2007/early 2008

EUFMD/WRL field study; HK-SAR & exptl study on vaccinated pigs, IRL Objective: evaluate 3 x NSPEs for use in pigs Ceditest, UBI and Chekit ELISAs Field study • 405 pigs; 4 vaccinated herds; Feb-March 2005 • Type O infection, 2 herds, 1-2 months pi • All 3 NSPEs detected infection in pigs; different dSE and dSP • Cedi was both sensitive and specific; UBI was less specific and Chekit was less sensitive Experimental study • 99 pigs; vaccination x2; sampling 28 dpv • dSP: 100% for Cedi and UBI; 98% for Chekit

CONCLUSIONS • Multiple sources of data • Data differs in reliability and level of detail • Most reliable data is from independent comparative evaluation studies • Significant difficulty for manufacturers to evaluate/validate new generation NSP tests.

RECOMMENDATIONS • Publish a critical review (meta-analysis) of ALL available data (from published and nonpublished sources) • Provide guidelines on minimum requirements for evaluation/validation of NSP tests for DIVA purposes.

Appendix 18

88

PROBLEM ON STABILITY AFTER THE VACCINATION TO TYPE O VIRUS (NOTIFYING A TECHNICAL PROBLEM)

Naci BULUT, VMD Head of the Diagnosis Department FMD (Sap) Institute, Ankara, Turkey

Introduction

Turkey is endemic with Foot and Mouth Disease (FMD) since his disease history which has been resulted enormous economic loses and damage on animal health.

The virus sero-types caused the disease have been changed years to years. As a summary of the circulating the sero-types in the country are as follow:

Type O:

Type A:

It is the main domestic type of the country which is responsible for many outbreaks burst in the country.

Although it was standstill occasionally, because of dominating the other sero-types outbreaks, the disease due to the type O has been continued its effects and emerged frequently a new outbreaks by maintaining circulating of the live virus in the environment or introduced from the outside of the country.

After the O 1 Manisa genetic lineage was prevailed in many years, O Pan-Asia sub-strain has been introduced since end of the 2006 which was caused outbreaks picks during the Kurban Festival, winter and spring time of this year.

Although it has been declined the outbreaks with this substrain, it has been still circulated all over the country and affected damages on animal health by occurring occasional outbreaks.

Type AsiaAsia-1:

It was the exotic type for the country which has been introduced three times up to 2001. not recorded any occurrence since 2001.

This sero-type has been occurred occasionally in country.

Type A 22 Mahmatli, same with A22 Iraq, has been appeared and caused outbreaks infrequently and in limited area since 1996.

Afterward this time, last in a decade, three different types, introducing from the outside of the country, type A Iran 96, A Iran 99 and A Iran 05 respectively the last one was constituted big outbreaks event during the 2006 all over the country and now finally seems in peace occurred in limited area a few outbreaks.

Dynamics of the spreading the virus:

dynamics of the spread is depend on the situation in which region, time of the year and type of the outbreaks, it can be summarized as follow:

89

Summarize of dynamics of virus spread Emerging a new virus strain from the outside of the country

Insufficient animal movement and market control

Kurban Festival movement

Sequentially occurrence of the outbreaks

Maintenance of carrier state

Poor disease awareness& puplic awareness

Inadequate disinfections

It can be questionable that one sero-type emerges to country can be controlled easily by same measures, conversed good immune response, however the other one can maintenance which is not reacted good antibody conversion and, even if, durability of the immune level declines in a short periods.

According to disease experiences of Turkey within the last decade, situation of outbreaks of type A and O can be given an interesting example for this dilemma.

&control measures Patchy and poor vaccination coverage

Summarize of results on the differences of between two types protection level within the last several years

TWO PHENOMENA That has been observed; During the type A Iran 05 outbreaks in 2006:

Data showing protection rate determined within the last several years in the country were assorted within three groups: Group 1: It was represented results of field sera collected from cattle and sheep belong to state farms.

Since those farms are well structured, they implement regular vaccination and always monitor protection rate of their population before/after vaccination.

These figures were generated from 3280 sera from 15 farms which represented around 16000 sized populations

-seed virus was adapted easily to cell cultures and prepared a good potent vaccine a short periods -this vaccine gave an effective immune response -this acquired protection level was prevailed for a long period, 7-10 months

On the contrary, O1 Manisa vaccine antigen for O Pan-Asia sub strain outbreaks were exposed different characteristics:

-cell culture adaptation and harvesting the virus culture are crucial, -although the amount of type O antigen containing within the vaccine is 2/3 times more which is conversed weak immune response, -and it is easily declined the protection level within the 2 or 3 months

The answers of those phenomena that might be found can be solved the problems.

FIRST GROUP RESULTS TYPE O ANTIBODY DURATION Vaccine1/H1

100

Vaccine1/H2

80

Vaccine1/H3

60

Vaccine2/Ha

40

Vaccine2/Hb

20

Vaccine2/Hc

0

Vaccine2/Hd

1st Month

3rd Month

MONTHS

TYPE A ANTIBODY DURATION

Vaccine2/He Vaccine3/H#

120 PERCENTAGE O F A N T IB O D Y L E V E L

P E RCE NTAG E O F A N T IB O D Y L E V E L

120

SECOND GROUP

Vaccine1/H1

100

Vaccine1/H2

80

Vaccine1/H3

60

Vaccine2/Ha

40

Vaccine2/Hb

20

Vaccine2/Hc Vaccine2/Hd

0 1st Month

3rd Month

MONTHS

Vaccine2/He Vaccine3/H#

It has been conducted regularly serosurvey in Thrace region for two main objective one of them is to monitor vaccination effectiveness.

This group data were generated from those studies

90

THIRD GROUP

SECOND GROUPS RESULTS

P E RCE NTAG E O F AB LEVEL

SEROSURVEY RESULTS FOR TYPE O

TYPE A 100

80

1998

60

2000

40

2001

20

2003 2006

0 1ST MONTH

2ND MONTH

AB LE V E L

100

80

1998

60

2000

2001

40

2003

20

2006

0

4TH MONTH

1ST MONTH

2ND MONTH

4TH MONTH

PERIODS

PERIODS

Evaluation those results

THIRD GROUP RESULTS

PERCENTAGE OF Ab LEVEL

v accine trial for e ffective ness in importe d cattle population

100 90 80 70 60 50 40 30 20 10 0

vac-1 vac-2

type A

1st month

type O

type A

2nd month

type O

type A

type O

4th month

vac-7 vac-8 vac-9

type A

5th month

TIME PERIOD

Situation of O Pan- Asia sub strain, can O1 Manisa be covered to this new isolates or we need a new vaccine strain?

Those three different situations are indicated that: seroconversion to type O vaccine was weak and acquired antibody level can be declined in a short period as well.

In this circumstance, it can be raised a question that does damage of integrity of antigen take form this weak immune response, which is concluded that this strain became as dominant strain and continue circulating in the field?

However each stage of vaccine preparation relating to antigen integrity has been checked by several virological and molecular based tests, such as SG for 146S and antigenic characterization (R value, Mab Profiling) and finally confirmed antigen is used for formulation.

As a conclusion, we need a new vaccine strain for type O which to be give strong and durable protection for the control of the disease!.

Attempt preparation O Pan-Asia vaccine strain

Vaccine suitability

vac-3 vac-4 vac-5 vac-6 type O

In this year to evaluate durability of protection rate and vaccine effectiveness, it was carried out a pilot study at a big farm contained imported naive young cattle.

It has been implemented three times vaccination: 0 day, 1st month and 4th mont and collected sequentially sera as postvaccination

According to R value results (by WRL, VNT), O1 Manisa vaccine strain was cover to O Pan-Asia isolates by multiple vaccination by potency vaccine.

However, according to field observation, vaccination with O1 Manisa strain was reacted poor seroconversion, although vaccination was implemented multiple (3 times within the 3 months) and also vaccinated young animals were affected by the infection.

Difficulties on adaptation of O Pan-Asia sub strain as seed virus for vaccine production According to current lab results, attempt that preparation vaccine strain from O PanAsia field isolates has been not achieved yet, because of adaptation problem:

-difficult and long cell culture adaptation

- very small plaque characteristic of antigen and low infective titres A/Kastamonu/1104/2006 A/Kastamonu/1354/2006 A/Corum/385/2007 A/Samsun/1025/2006 A/Samsun/685/2006 A/Samsun/474/2006 A/Samsun/798/2007 A/Samsun/718/2007 98 A/Samsun/884/2007 A/Kastamonu/794/2007 A/Kastamonu/860/2007 A/Elazig/542/2005 50 0 A/Elazig/548/2005 23 A/Ordu/665/2006 34 A/Igdir/1050/2006 A/Kastamonu/337/2006 0 6 A/Bolu/658/2006 A/Isparta/370/2007 8 80 A/Konya/1372/2006 A/Bolu/565/2005 72 A/Bingol/571/2006 71 0 A/Diyarbakir/1088/2006 14 A/Bingol/1096/2006 A/Corum/532/2007 52 A/Samsun/739/2006 12 A/Samsun/741/2006 A/Samsun/241/2006 25 A/Samsun/537/2006 0 A/Igdir/979/2006 A/Erzurum/203/2006 2 A/ Nevsehir/331/2006 39 A/Kars/957/2006 52 A/Balikesir/330/2006 A/Ardahan/347/2006 A/Ankara/546/2005 A/Konya/562/2005 A/Tekirdag/194/2006 0 A/Tekirdag/270/2006 A/ Ankara/969/2006 A/Bolu/552/2005 A/Konya/700/2006 65 A/Mersin/560/2005 A/Gaziantep/569/2005 A/IRN/7/2006 A/Konya/1029/2006 A/Kayseri/1024/2006 A/Kirklareli/175/2006 A/Bilecik/564/2005 0 A/Kirklareli/161/2006 A/Tunceli/1091/2006 A/Ankara/29/2006 A/Adana/1048/2006 66 A/Kastamonu/1107/2006 A/ Afyon/583/2006 A/IRN/29/2005 A/IRN/13/2005 35 A/IRN/16/2005 35 A/IRN/10/2005 20 A/Cankiri/399/2007 A/Aydin/1400/2006 A/Canakkale/1012/2006 0 A/Eskisehir/1115/2006 21 A/Bursa/1250/2006 00 A/Samsun/738/2006 97 A/Afyon/1374/06 0 A/Usak/1401/2006 A/Tokat/48/2007 99 0 A/Malatya/57/2007 A/Edirne/6/2007 1 A/Sivas/894/2007 A/Erzurum/859/2007 6 A/IRN/7/2004 13 85 A/IRN/41/2003 100 A/IRN/26/2003 44 A/Diyarbakir/664/2006 48 A/Kars/726/2006 0 A/Kutahya/570/2005 A/Erzincan/1084/2006 2 70 A/Mus/1443/2006 44 A/Mus/544/2007 12 A/Kars/764/2006 60 A/Kastamonu/208/2007 20 84 A/ Amasya/1433/2006 1 A/Sinop/1071/2006 A/Cankiri/696/2007 18 69 33

Şekil 2: 2005-2007 yıllarında Türkiye’de ve bölgede seyreden A Iran 2005 tipi şap viruslarının moleküler epidemiyolojisi

Şekil 1: 2006-2007 yıllarında Türkiye’de ve bölgede seyreden O tipi şap viruslarının moleküler epidemiyolojisi

14

5 33

O/Mus/947/2007 O/Mus/951/2007

64 52

O/Mus/927/2007 O/Aksaray/916/2007 O/Usak/933/2007 O/Aydin/879/2007 41 O/Sirnak/778/2007 98 O/Zonguldak/780/2007 O/Tokat/886/2007 47 O/Mugla/13/2007 39 O/Diyarbakir/1481/2006 35 68 O/Elazig/740/2007 O/Batman/620/2007 80 57 O/Sirnak/285/2007 58 O/Eskisehir/639/2007 O/Nigde/908/2007 84 O/Erzurum/310/2007 O/Erzurum/309/2007 42 O/Karabuk/1472/2006 16 O/Sivas/773/2007 O/Erzincan/5/2007 O/Sirnak/628/2007 1 7 O/Gaziantep/654/2007

130 0

12

No. of Taxa : 79 Data File : C:\Molep\AylikRaporlar\2007\Eylul 2007\EylulO.meg Data Title : : EylulO.aln Data Type : Nucleotide (Coding) Analysis : Phylogeny reconstruction Tree Inference : ============================== ->Method : Neighbor-Joining ->Phylogeny Test and options : Bootstrap (1000 replicates; seed=64238) Include Sites : ============================== ->Gaps/Missing Data : Complete Deletion ->Codon Positions : 1st+2nd+3rd+Noncoding Substitution Model : ============================== ->Model : Nucleotide: Maximum Composite Likelihood ->Substitutions to Include : d: Transitions + Transversions ->Pattern among Lineages : Same (Homogeneous) ->Rates among sites : Uniform rates No. of Sites : 362 No Of Bootstrap Reps = 1000

65

O/Balikesir/955/07 O/Kirklareli/967/2007

25

O/Konya/623/2007 O/Osmaniye/876/2007 O/PAK/14/2006 O/PAK/4/2006 O/IRN/9/2006 O/IRN/10/2006

57 45

32

O/Kayseri/954/2007 O/Konya/961/2007

61 23

O/Bursa/758/2007 O/Bursa/913/2007 O/Kutahya/671/2007 O/Sirnak/883/2007

12 15 70 85

O/Samsun/938/2007

O/Sakarya/784/2007 O/Bursa/723/2007 O/Afyon/923/2007 O/Istanbul/731/2007 O/Konya/120/2007 O/Erzurum/595/2007 O/Istanbul/325/2007 O/Kayseri/928/2007 O/Afyon/404/2007 65 O/Kutahya/688/2007 13 58 O/Konya/83/2007 O/Kirklareli/357/2007 58

67

No. of Taxa : 113 Data File : C:\Molep\AylikRaporlar\2007\Eylul 2007\EylulA.meg Data Title : : EylulA.aln Data Type : Nucleotide (Coding) Analysis : Phylogeny reconstruction Tree Inference : ============================== ->Method : Neighbor-Joining ->Phylogeny Test and options : Bootstrap (1000 replicates; seed=64238) Include Sites : ============================== ->Gaps/Missing Data : Complete Deletion ->Codon Positions : 1st+2nd+3rd+Noncoding Substitution Model : ============================== ->Model : Nucleotide: Kimura 2-parameter ->Substitutions to Include : d: Transitions + Transversions ->Pattern among Lineages : Same (Homogeneous) ->Rates among sites : Uniform rates No. of Sites : 428 No Of Bootstrap Reps = 1000

66

97

O/Sakarya/288/2007

O/Usak/1423/2006 O/Erzurum/328/2007 O/Erzurum/584/2007 O/Erzurum/553/2007 O/Erzurum/583/2007 O/Corum/458/2007 O/Erzurum/581/2007 O/Erzurum/765/2007 O/Bolu/882/2007 O/Ardahan/600/2007 25 O/Erzurum/450/2007 47 O/NEP/4/2003 O/BHU/41/2003 47 O/BHU/28/2004 39 O/MAY/6/2003 25 O/NEP/6/2003 35 O/NEP/5/2003 43 O/BHU/33/2004 70 O/Tibet/CHA/99 78 O/JPN/2000 26 O/Konya/512/1999 O/Duzce/207/2005 38 O/Ankara/250/2001 O/Denizli/TUR/441/11/03 50 O/KMaras/1127/2006 99 O/Osmaniye/473/2006 O1/Manisa/Turkey/69 O1/FRG/Kaufbeuren66 20 61

69

62

56

30

17

14 99

2

A/Ardahan/937/2007 A/Ardahan/958/2007 A/Ardahan/925/2007

A/Ardahan/950/2007 A/IRN/1/2005 A/IRN/4/2005 A/Denizli/1068/2006 A/Diyarbakir/553/2005 A/IRN/22/2005 A/IRN/5/2006 A/Aydin/1451/2006 A/Usak/467/2007 4 25 A/ Van/1269/2006 A/Hakkari/1266/2006 25 69 A/Van/1416/2006 39 A/ Hakkari/1240/2006 17 43 A/Bolu/324/2007 51 A/Hakkari/520/2007 100 A/PAK/1/2006 99 A/PAK/3/2006 A/IRN/53/2005 A/PAK/5/2006 A/IRN/6/2002 A/IRN/7/2003 100 64 A/IRN/2/2002 A/IRN/34/2001 100 A/IRN/32/2001 A10Holl/NET/1942 76

2

60

18

74

0. 02

97

0.02

91

Appendix 19

… steps of serosurveillance

Standardization of sero-surveillance results?

Planning/ survey design

Draft reporting format?

Carsten Pötzsch Ankara, Turkey

FAO/EUFMD EC/DG-SANCO

Turkey Description: • Regular serosurveillance in Trace since 2001(SP, NSP) • In Anatolia 2003 (NSP, SP) and 2004 (NSP, SP; research) • Survey design: epidem. valid • Aims: - vacc. success - risk assessments, temp. & spatial patterns Output: • Adjustment of control policies • Follow-up actions

Syria Description: • Driven by availability of diagnostic resources; no routine ss. (1000 samples in 2004/05 with SP tests; 2006 some follow-up of clinical suspect cases with one CEDI kit) • Survey design: not clear • Aims: not clearly formulated (to determine vaccine coverage) Output: (no ss.)

Field survey/ sample collection

FMD field situation

Aims of FMD ss. include: • Early warning/ risk assess.

• Description of temporal and spatial patterns of FMD • Success of vaccination campaigns • FMD freedom

Diagnostics

Reporting

Follow-up actions

Data analysis/ results

Data management

Iran Discription: • Ca. 20,000 samples in provinces in 2007 (SP, NSP) • Survey design: epidem. valid • Aims: epidem. understanding Output: • Start of national ss.

Armenia Description: • ca. 4000 post- vaccination samples, 2x p. year since 2004 • Survey design: epidem. valid • Aim: vaccination success Output: • Routine ss., yet not part of national disease control strategy • Analysis: percentages on district level • No follow-up action

92

Transcaucasus FAO FMD project

Distribution of % NSP antibodies (district level)

Description: • Postvaccination serosurveillance (SP and NSP) in the buffer zone 3x since 2003 • Prevaccination and market ss. in 2006 • Postvaccination ss. in BZ & whole countries, incl. market ss. planned in 2007

2005

• Survey design: epidem. valid • Aims: - vacc. success - risk assessm./ temp. & spatial patterns 2003

2006/07, up to 1 yr age

… steps of serosurveillance

Transcaucasus (FAO FMD project)

Planning/ survey design

Output: • • • • • •

FMD field situation

Reporting

Problems

Assessment of vaccination success Risk assessments Follow-up investigations Epidemiological understanding Policy advise Regional information gathering and sharing

Problems II

Field survey/ sample collection

- In every country specific situation and problems - no universal and straightforward solution

Follow-up actions

Data analysis/ results

Diagnostics

Data management

Standardized reports?

• FMD is – often not of high priority for vet. services – mostly controlled by vaccination • FMD ss. is not prioritized in these countries

• Reporting should be based on standardized national formats ( Draft reporting format ------> national )

• Limited advantage of FMD ss. to countries • Limited resources for ss.

• Output parameters should be standardized

93

Draft guidelines for obtaining good quality serosurveillance results as a prerequisite for the standardization of reporting I Planning/ survey design II Field survey/ sample collection III Diagnostics IV Data management V Data analysis/ results VI Reporting

pt. II. Field study/ sample collection The following data/ information should be collected (minimum requirement): • Sample No. • (Country, Region) • District • Village/market • (Spatial reference, compatible to the GIS used ) • Owner • Animal identification: number, name, markings • Sex • Age • Date of last vaccination • Vaccination serotypes • Date of last disease • FMD serotype •

Description of the epidemiological situation:

pt. V. Data analysis/ results pt. IV. Data management

• Data should be entered electronically, in spreadsheet or database format.

What were the objectives of the study, which are the questions to answer?

Analysis of test results based on following variables and categories: • • • • • •

Spatial: all administrative levels Temporal (if applicable) Age (or age groups) sex, breed, species: (if applicable and meaningful) vaccination (e.g. yes/no, type and/or no. of vaccinations received, time since vaccination) disease (e.g. yes/no, time since disease)

Results are only applicable to the population of interest and with the given statistical power; to other population only with care, epidemiological knowledge and common sense.

Use of data and reports • Reported data should be linked to geo-reference data • can then be incorporated into GIS Presentation of data, e.g.: • Vaccination maps • Combined with mapping of outbreaks and suspicions • Time series maps

Conclusions • Good surveillance practices required for quality data/reports • Reporting needs stimulation and feedback • Management of international reporting and of ss. databases is a large task that needs long term commitment There is big potential: – to improve regional and national disease surveillance (e.g. problem with immunity levels, clusters of NSP Ab) – to increase trust in the preventive measures across boundaries – to improve epidemiological understanding of FMD and in general – to focus training needs

94

Appendix 20

Forecasting the spread of a disease Foot and mouth in a new environment

EUFMD Research Group

Modelling spatial and temporal transmission of footand-mouth disease in France: identification of high-risk areas, Le Menach A, Legrand J, Grais RF, Viboud C, Valleron AJ, Flahault A, Vet Res. 2005

Modelling disease spread under various control strategies Modelling vaccination coverage under various vaccination scenarios Le Menach Arnaud, FAO EUFMD, Cairo, 17/10/2007

Introduction

Modeling disease spread

• What would be the consequences of foot and mouth reintroduction in France and the impact of various control strategies?

• Mathematical models – Keeling et al. (Science 2001) : deterministic approach – Ferguson et al. (Science 2001): stochastic approach

• Disease Characteristics – Highly contagious disease and highly resistant virus – Transmission : direct or indirect contact over long distance – Foot and mouth disease epidemic in UK in 2001

• Farm based stochastic model adapted to French agricultural structure and derived from Keeling et al study.

• More than 2000 cases, 6 millions animals culled • Disease spread before the detection • Cattle and sheep mainly affected

– Species heterogeneity – Spatial heterogeneity : distance between farms – National movement restriction (no long distance spread)

State transition

Farm-based stochastic Model

With control strategies Minimum control

• Farm passes into the following states :

Pe i = 1 − exp( −[Su k N i ][ Trk ∑ N jK (d ij )]) j

S

Pei

4 days E

1 to 3 days

5 days I1

15%

I2

R

• Probability that a susceptible farm (i) be infected by contagious farm (j) is : – Suk and Trk : susceptibility and transmisssibility parameters – Ni et Nj : number of livestock in each farm – K(dij) : weighting factor depending upon distance

2 days S’ E’ I1’

95

Simulation methods

An epidemic simulation in France

• Monte-Carlo simulation •

200 simulations for 365 days length mean

• Simulation of farms location based on assumed town boundaries (square) • Simulation oh the number of livestock in each farm

• Location and number of index cases

Number of cases

• Farms structure (280 000 farms, 20 millions heads of cattle and 7 millions heads of sheep, source Agreste)

• Randomly chosen according to farms density • 50 or 20 index cases time (days)

• Simulation of control strategies

• An epidemic issue from 50 index cases :

• Only animals of infected farms are culled • Animals around an infected farm are culled or vaccinated

– Simulated cumulative cases 16 354 [8 610 ; 20 858] – Median length 358 days – Epidemic peak 87 cases at day 122

• R0 values for each farm

R0 values

Control strategies comparison • R0 values map – High risk areas

Reference One day Ring vaccination

Number of cases

Ring cull

Ring vaccination and cull

• Pays-de-la-Loire • Midi-Pyrénées • Auvergne and Limousin

R0=5+ R0=4

• County risk – Haute-vienne R0=1,77 [1,73 ; 1,8] – Hautes-Pyrénées R0=0,35 [0,33 ; 0,37]

R0=3 R0=2 R0=1

– 65 % decrease in the number of cumulative cases

Discussion • Models would provide useful tools to help decision makers • Impact of neighborhood culling – No silent period and no long distance contact taken into account – Implementation difficulty – high level of spatial resolution

Predicting vaccination coverage in time and space Achievement Predicting coverage rates to identify risk areas of low coverage, and periods in the year when this will occur under various vaccination scenario

• Data quality – both situation with no exact data – high influence of clusterisation

• Future perspective – highlighting contact matrix – Application to other farming context

Immunity and vaccination • Serological and mucosal immune responses after vaccination and infection with FMD in pigs, Eblé et al., Vaccine, 2006

Modeling disease spread and vaccination • Modeling vaccination strategies against foot and mouth disease Keeling et al. Nature, 2003.

• Farm based model evaluating

• 3 groups of pigs – Non vaccinated, vaccinated and vaccinated with 4 fold the dose – Half of the pigs were infected with FMD virus

• Results – Vaccination reduce virus excretion – Vaccination inducing IgA may induce more protective response – Display immunity response up to 116 days after injection (vaccine)

– National Prophylactic vaccination campaign in advance of an outbreak – Reactive vaccination during an outbreak – Preventive vaccination (based on epidemiological heterogeneity of the transmission)

• Main results – In case of movement restriction, immunization of more than 30% of the animals leads to R<1 – Reactive vaccination successful in combination with culling taken into account delay between vaccination and protection (promotion of preventive vaccination)

96

Methodology

Vaccination coverage prediction problematic • Objective – Temporal-spatial map of immunity rates at population level following FMD vaccination campaigns.

• Heterogeneity – Assuming a certain vaccination coverage percentage, how would evolve the overall coverage in time and space depending on the following constraints

• Compartmental model in time and space describing the population dynamic and the immune history – Discrete (or continuous time): data driven – Spatial resolution: administrative unit

• Compartments – Species: 3 groups (cows, pigs, sheep and goats) – Age: 2 groups (young and adult) – Immunity: 3 groups (no immunity, 1st vaccination immunity, 2nd vaccination immunity)

• Heterogeneity in individual vaccination response • Heterogeneity in population dynamics (slaughtering, exportation, importation, new born...)

• Heterogeneity in age and response to vaccine • Heterogeneity in immune history (no vaccination ever received, immunity acquired through vaccine or through the disease...)

– 18 compartments (isolated by group of 6 compartments driven by species type)

Population dynamic and demographic data

Population dynamic and demographic data

Initial Population P0

• Resident animal R – – – – –

Initial Population P0 Imported animals A Exported animals E New born animals B Slaughtered animals S

Population

Importation Exportation

Number of livestock

Slaughter New Born

• At time t+1, Rt+1=P0t+At t+1+Bt

t+1-

Et

t+1-St

t+1 Time

Population evolution in a same compartment

Immunity Dynamics

As,a,i

Immunity

Es,a,i Prob (Vac +)

Previously Immune

Ps,a,i

Evaluation of vaccination coverage in time

Ps,a,i

Ss,a,i

Vs,a,i

Prob (Vac -)

Bs,a,i

Naïve Young Naïve Adult

Population dynamic T

Vaccination Campaign T+1

T+2

Time

Immune evolution in a same compartment Adult cows Vaccinated Adult cows Non Vaccinated Updated demographic data

Deterministic compartmental model • Constraint

Adult cows vaccinated 2nd

– Static evolution and no consideration of biological variation – An entire compartment share the same immunity status

Adult cows vaccinated 1st Adult cows non Vaccinated

Need to set up a threshold to decide when a compartment is not considered as vaccinated

• Advantages – Ease of computer implementation T

97

Individual stochastic simulation • History of each animal is tracked with associated probability of being eliminated, vaccinated, created etc..

Outputs : vaccination coverage at time t VC = ∑ (Ps, a, i (t) × Is, a, i(t) ) • 100% animals vaccinated s , a ,i

100%

• Advantages – High precision and flexibility in the evaluation – Continuous evolution

60% 50%

• 80% adult cows vaccinated for the first time • 20% young cows vaccinated for the first time At t: VC effective=100%

• Constraint

VC real = 20% × 50% + 80% × 60% = 58%

– Difficulty in implementation t

Necessary Data (1) • Population dynamic – Detailed Census by lower administrative unit • Number of sheep, goat, cows and pigs for each age group

– Trade • Number of animals exported from the region for each category per unit of time • Number of animals imported in the region for each category and each per unit of time

– Number of birth for each species per unit of time – Number of slaughtered/deceased animals for each category per unit of time

Conclusion • Few research papers on forecasting vaccination coverage – Pubmed research – Virology papers – Modeling disease spread under various control measures

• Data driven – Availability of data will influence methodology to be used – Influence the time steps (continuous to discrete) – Influence of the spatial level of resolution

• Individual or compartmental models

Necessary Data (2) • Immunity – Vaccination coverage and date of the last campaign – Type of vaccine used – Immunity curves for each category per unit of time and for each previous immunity status – Immunity threshold: under which value (percentage) an animal is not considered as vaccinated

Discussion • Relevance of all categories (age, focusing on only cattle vaccination...)?

• Availability of data?

• Other key parameters not taken into account?

– Immunity and population dynamics evaluated simultaneously

98

Appendix 21

Conclusions Camelus dromedarius and Camelus bactrianus – differences in susceptibility to experimental infection with FMDV Prof. Soren Alexandersen and Dr. Magdalena Larska Department of Virology, National Veterinary Institute Danish Technical University Lindholm, Denmark &

Dr. Ulli Wernery, CVRL, Dubai, UAE

•

We conclude that dromedaries (Camelus dromedarius) are of very low susceptibility to infection with FMDV serotype O and A

•

Although they may sometimes become infected at low level after direct inoculation, they appear not to transmit infection even by close direct contact and dromedary camels are unlikely to play any significant role in the natural epidemiology of FMD

•

Dromedary camels appear to only mount a limited antibody response to FMDV and no antibodies to NSP were detected!

•

Experiments with 2 Bactrian camels inoculated with FMDV serotype A indicated that these are rather susceptible, replicate the virus to considerable titers and develop severe lesions of the hind feet, only.

Although the Bactrians replicated the virus and developed rather high levels of antibodies, also against NSP, the experiment did not indicate any spread to other susceptible animals at the site and moreover, none of the bactrians carried the virus for more than a few weeks and did thus not become carriers.

Recommendations •

It is strongly recommended that the dromedary and the Bactrian camel are seperately evaluated when assessing the susceptibility to FMDV 2

October 2007

Camelids classification

Habitat of C. dromedarius and C. bactrianus

• • •

Class Mammalia Order Artiodactyla Suborder: Tylopoda - Camelidae Old World Camels (Camelini): Dromedary and Bactrian camel New World Camels (Lameli): llama, alpaca, guanaco, vicuña

•

Suborder Ruminantia (cattle, sheep, goats, water buffalo, giraffe, deer, bison)

•

Suborder Suiformes (pigs, hippopotamuses, peccaries)

Camels can be milked, ridden, loaded with baggage, eaten, harnessed to a plough or wagon, traded for goods or wives, exhibited in a zoo or turned into sandals and camel hair coats ...(Bulliet, 1975)

Differences between camelids and ruminants •toenails and soft padded feet •3 compartments in stomach (C1-3) •small and elliptical red blood cells •IgG2 and IgG3 lack light chains (HCAb)

Cattle, sheep, goats and pigs African & water buffalo Kudu, impala, warthog, deer

Experiment

I

II

Virus

O UAE 542-99 (origin Dubai, Arabian gazelles, 5p. BHK 21)

O UAE 542-99 (vesicular epithelium from heifer)

Animals

2 dromedaries

2 heifers (positive control)

5 dromedaries (2 previously inoculated)

4 sheep 5 dromedaries (contacts)

2 sheep (positive control)

Clinical signs

No

Yes

No

No

Yes

Viraemia

No

Yes

1/5

No

Yes

No

Yes

No

Yes

Virus in probangs and mouth swabs

No

Yes

PB 1/5 (6 DPI in RT-PCR) MS 4/5 (1-3 DPI)

Seroconversion in camels

No

Yes

3 (at 10-14 and 6-10 DPI)

Experiment 1 at CVRL, Dubai

FMD: WIDE HOST RANGE

Previous experiments

Dromedary Camels????

Camel Experiment 2

Daily Temperature of FMD treated and control contact camels

Camel 26 (Control) Camel 27 (Control) Camel 28 (Control) Camel 29 (Control) Camel 31 (Control) Camel 30 (Treated) Camel 32 (Treated) Camel 33 (Treated) Camel 34 (Treated) Camel 35 (Treated)

39,0 38,5 38,0

Temperature

37,5 37,0 36,5 36,0 35,5 35,0 34,5 34,0 -10

-5

0

5

10

15

20

25

30

Days PI

99

Experimental infection III

Log10 virus or RNA per ml

Camel Exp. 2 Viraemia 9 8 7 6 5 4 3 2 1 0

532v 537v 34v 532r 537r

2 inoculated sheep (SCI), previously vaccinated ♂♀

34r Dect. lim. V 0

1

2

3

4

5

6

10

14 21 28

Dect. lim. R

Days

2 inoculated Bactrian camels (BCI B3♂ B3♂, B2♀ B2♀)

Camel Exp. 2 Antibodies 700

532ae

600

537ae

500

34ae

400

30ae

300

33ae

200

532av

100

537av

0

34av 0

1

2

3

4

5 Days

6

10

14

21

28

30av

Titre

Titre

Camel Exp. 2 Antibodies 45 40 35 30 25 20 15 10 5 0

8 inoculated dromedary camels (DCI) 5 contact dromedaries (DCC) ♂

4 sentinel sheep as contacts (SCC) ♀

34ae 30ae 33ae 34av 30av 33av 0

1

2

3

4

5

6 10 14 21 28

Dect. lim. (50%)

Days

33av

Sheep got NSP antibodies from day 6-10 while NO NSP antibodies were detectable in camels

Inoculation and sampling

Inoculation of Bactrian camels under the skin of the tongue – beware the bull is “all teeth”

Camels inoculated subepidermolingually with 107.0 TCID50 of FMDV type A SAU 22/92 inoculum in a volume of 0.25 ml each Sheep were infected by intradermal inoculation into the coronary band of a forefoot with same dose of FMDV type A SAU 22/92 as the camels

Mouth swabs

The Vet in the “lions” mouth

Is it a carnivore or just a Bactrian camel

When teeth and hands come close together

Close contact after inoculation, and then a long sleep

100

Clinical signs -Bactrians

Clinical signs - camels

Temperature of Bactrian camels inoculated with A SAU 22/92 ( BCI)

41,0 40,5 40,0 Temperature

39,5 39,0 38,5 38,0 37,5 37,0 36,5 36,0 0

1

2

3

4

7

10

14

21

28

DPI

DCI OB5 DCI 658 DCI 31 DCI 184 DCI 76 DCI 77 DCI 254 DCI 231 DCC 183 DCC 180 DCC 264 DCC 175 DCC 244 BCI B2 BCI B3

• average temp. in dromedaries = 37.8 (SD 0.6) • Dromedary camels inoculated and contacts – NO clinical signs of FMD, NO lesions • Contact sheep - NO clinical signs of FMD, NO lesions

41.0 40.0 Temperatur e

Temperature of Dromedary and Bactrian camels inoculated with A SAU 22/92 (DCI and BCI) and controls (DCC)

Average temp. = 37.4°C SD = 0.83

39.0 38.0 37.0 36.0 0

1

2

3

4

7

BCI B2

lesion on the hind feet – 9 DPI BCI B2

• • •

10

14

21

28

DPI

BCI B3

Bactrians showed no signs of FMD until 7 DPI On 7 DPI both B2 and B3 developed lameness of the hind feet with fluid coming out of lesions in and around the foot pads Most severe lameness of hind legs, pain and lesions on day 10 PID. The B2 camel lost the epidermis of the sole of the foot pad

The animals gradually improved, lesions healed and they regained health up to 21 DPI •Bactrian B2 had completely shed the skin of the footpad sole and new tissue replaced it.

lameness, depression 9-14 DPI

Dromedaries: • No virus detected in the sera, probang samples nor in mouth swabs • No antibodies

Conclusions

Conclusions •

Bactrian camels were found to be susceptible to experimental infection with FMDV type A SAU 22/92

•

Bactrians showed moderate to severe clinical signs although the onset of the clinical signs of FMD was late comparing to susceptible animals. Both of the camels had lesions only on the hind feet.

•

They developed viraemia and seroconverted (significant level of Ab against FMDV up to 130 days p.i.).

•

The presence of FMDV in probangs only transient – no carriers.

•

No transmission of FMDV from Bactrian camels to other animals occurred.

•

FMDV not present in the mouth swabs: can explain no transmission to other camels and highly susceptible sheep.

•

FMD in Bactrian camels by natural infection could cause transitional decrease of their productivity.

• Dromedary camels were not suceptible to FMDV infection showed no clinical signs of FMD did not develop vireamia and specific antibodies did not transmit the FMDV to other camels or susceptible species

• Dromedary camels can not be considered as reservoirs of FMDV

WHY DIFFERENT • Differences in receptor expression? – integrin αvβ6, αvβ3, αvβ5, αvβ1 and αvβ8 or GAGs (heparan sulfate) • Cytokines (interferons)?

• Presence or amount of HCAbs (IgG2&3)?

THE END

Thanks also to Renate Wernery, Peter Nagy, Jutka Juhasz, Anita Varga and Winni Schiele at CVRL, Dubai, and staff at DFVF-Lindholm, and to Gitte Alexandersen, Barup, Denmark Research was supported by DFVF as well as by HH General Sheikh Mohammed Bin Rashid Al Maktoum

101

Appendix 22

Attempt for detection of FMD in wildlife by Infra Red Camera

Distance (range) of infrared detection

• By calculation, this model of camera can detect accurate temperatures of 1”x1”area 500” (41.6 feet) away (15°lens) • If area of foot (coronary band) on ungulate is 2.5”x2.5”, an accurate temperature could be detected at 34.7 yards.

Jack Rhyan, Mike Dunbar, Rony King 1

& Hagai Yadin.

Supported by the EUFMD

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

2

Summary

• Pronghorn - Infrared thermography detected the increase in coronary band temperature as early as 22 hrs post-inoculation (20 hours before lesions were observed)

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

Summary – Mule Deer

• FMD raised the coronary band temperature of pronghorns, mule deer, and cattle

3

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

• Mean foot temperature rose during the study from 24.2°C (ca 24 hrs p.e.; 3 days before lesions) to 29.7°C (between 70 hrs to 96 hrs p.e) on day of the 1st foot lesion occurrence to 33.0°C two days after the first lesions were observed.

• Temperature rose significantly (P < 0.05) from two days before 1st foot lesion to two days after • Can detect a rise in temperature 1 to 2 days before lesions.

4

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

Field Application – Gazelles and Cattle in

FMD in Feedlot Ramat Magshimim

Israel • Recent FMD epizootic in cattle and mountain gazelles (Gazella gazella) in Israel (April 2007) • Evaluated using infrared thermography to screen animals, including free ranging ungulates, in the field • Using infrared indicated that signs of infection could be detected up to a distance of ~ 45 yards from the ground or from the air using a helicopter.

5

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

6

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

102

Field Application – Gazelles and Cattle in Israel

Field Application – Gazelles and Cattle in Israel

• Recent FMD epizootic in cattle and mountain gazelles (Gazella gazella) in Israel (April 2007)

• Recent FMD epizootic in cattle and mountain gazelles (Gazella gazella) in Israel (April 2007)

• Evaluated using infrared thermography to screen animals, including free ranging ungulates, in the field

• Evaluated using infrared thermography to screen animals, including free ranging ungulates, in the field

• Using infrared indicated that signs of infection could be detected up to a distance of ~ 45 yards from the ground or from the air using a helicopter.

• Using infrared indicated that signs of infection could be detected up to a distance of ~ 45 yards from the ground or from the air using a helicopter.

7

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

8

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

10

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

Field Application – from the ground

Thermal image of a cow with possible FMD infection

• Taking thermal images of cows 9

Foot temperature 38.4°C (101.1°F) Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

Infrared thermography has use as tool for detecting signs of FMD

Field Application – from the air •

Gazelles from the air – fast moving, difficult to gain useful infrared information

• • • •

Unable to capture focused images from the helicopter for later analysis (would be possible if motion stability technology used in camera).

• Cattle from the air – more stationary, able to Inf Red Camera for FMD 01/05/2007 detc./Jack Rhyan & M.Dunbarevaluate

11

12

Screening animals difficult to handle Selecting animals for further testing Determine distribution of the disease Other potential applications – Remote detection (45 yards plus) – Earlier detection?

– Remove infected animals before viral shedding?

– At least 24 hours before shedding?

– Screening wild populations without capture?

– Screening from aircraft? Drones?

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

Further Research

The future: Screening from Aircraft • •

01/05/2007

• Continue investigating if FMD affects temperature similarly in other species

Over large geographic areas quickly Use of the unmanned aerial vehicles?

– Drones?

• Investigate if other herd diseases of domestic animals, pronghorns, and mule deer (e.g.vesicular stomatitis, bluetongue) can be detected with Infrared Thermography • Range of accuracy (45 yards?)

13

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

14

Inf Red Camera for FMD detc./Jack Rhyan & M.Dunbar

01/05/2007

103

Appendix 23

Vaccination of sheep; revision of guidelines

Do sheep need to be vaccinated?

Vaccination of sheep is effective at protecting against clinical disease and reducing virus replication and excretion. need to vaccinate sheep is controversial.

According to Donaldson (EUFMD review, 2000),

essential if the objective is to create an immune belt, for example when ring vaccination is implemented around an outbreak or when a buffer zone but NOT as part of routine prevention

104

Some FMD types can dissapear without effective sheep vaccination coverage

Turkey:

only about 5% sheep vaccinated, once a year type A epidemic has almost dissapeared (2007) and Asia-1 dissapeared (2002) without effective sheep vaccination but type O remains endemic, with epizootics (2007) exposure of sheep (NSP+ve) in south-east Anatolia is quite high

Endemic regions - no need to routinely vaccinate sheep?

evidence usually quoted is from South America (Uruguay) and Kenya in Argentina and Uruguay, after 8 years of vaccinating all species, serological studies indicated that sheep did not play a significant role in epidemics in S Am, sheep vaccination after 1990 restricted to emergency campaigns are these studies relevant to the Middle-east?

where ratio of sheep to cattle can be 10:1? with less restricted animal movement and contact rates?

105

Donaldson Paper on FMD control in populations with high density of sheep (EUFMD Session, 2000, Borovets)

•Buffer zone: vaccinate all species •Endemic: vaccinate only cattle, except emergency control of exotic strain •Free countries: include all species (below) Country or zone which is free from FMD and does not vaccinate

Eradicate the virus following an outbreak.

Contain the area of infection and reduce the probability of spread beyond the at risk zone. Appease public opinion against the slaughter of animals.

Apply total stamping out and zoo-sanitary measures to the infected premises. Establish protection and surveillance zones and restrict movement. Undertake a serological survey in the surveillance zone* to determine whether virus is circulating. Maintain movement restrictions until the absence of circulating virus has been verified.

Apply emergency vaccination to all livestock (including sheep) in a zone beyond the at risk area. Identify and record all vaccinated animals. Maintain restrictions on the movement of all livestock* and animal products from the vaccinated zone until serological and clinical surveys have confirmed that virus is not circulating.

Role of sheep in cattle epidemics

UK epidemic of 2001, sheep seeded infection throughout the country when they were moved through a series of markets during the period of acute infection.

On the other hand, where sheep are kept extensively at low density and separated from other species, then spread of infection can be inefficient and die out spontaneously. depends on virus type?

type A outbreaks, Turkey: little evidence sheep involved not NSP positive even on farms where disease had been seen in cattle

106

Emergency vaccination -sheep

in free country with non-vaccination status UK in 2001;

clear role in spread before the national movement ban unclear if sheep significant as source for cattle after this some evidence of local persistence in sheep

review of evidence needed additional studies : review if oldest lesions were in sheep in cattle (ie for evidence that sheep were the entry point into a holding)

Process

review epidemiological information: for/against vaccination post-vaccination surveillance:

what risk that non-vaccination of sheep would test positive and result in slaughter of all sheep/cattle on mixed holding?

if epidemiology indicates positive benefit:

consider impact on resources –vaccine and human resources to be considered exit strategy – for/against marketing of vaccinated sheep/products

107

Initial thoughts Emergency vaccination -sheep Setting

Objective

Action

Note

FMD free country which does not vaccinate

Eradicate the virus following an outbreak through stamping out plus emergency vaccination

......Apply sufficient intensity of emergency vaccination** in the risk area (PZ and SZ) that circulation dies out.

Vaccination ceases once the vaccination target is achieved and no more outbreaks have occurred outside of the PZ/SZ.

Vaccinate cattle.

Sheep included ?

•if significant role in transmission after movement bans in place •if high risk that they introduce infection to cattle •if movement cannot be controlled •if sufficient resources to vaccinate sheep •exit strategy acceptable ( marketing of vaccinated sheep/products)

Buffer zone Buffer zone (non-free country)

Maintain an immune barrier between an FMD free country from area with endemic infection

Apply routine mass vaccination to all clovenhoofed livestock* in the defined zone. Identify the vaccinated animals within the zone and restrict their movement by physical barriers and check-points. Prevent the movement of animals and potentially infected animal products to the free country/zone.

Vaccinate cattle.

Sheep included ?

• if significant role in transmission • if high risk that they introduce infection to cattle • if movement cannot be controlled • if sufficient resources to vaccinate sheep

108

Non-free countries Seasonal risk:

Vaccinate 4-6 weeks (under study) before lambing to protect lamb crop

Year round risk:

Schedule as required to maintain immunity (twice yearly)

Protect high value cattle population

Risk based vaccination program

Vaccinate cattle.

Sheep included • transhumant populations, vaccinate pre-movement • radius vaccination around dairy farms • zonal or district mass vaccination where mixed cattle/small ruminant husbandry

Prevent virus circulation, qualify for potential free zone/country

Risk based vaccination program

Cattle included.

Sheep included if: • if significant role in transmission • if movement cannot be controlled • if sufficient resources to vaccinate sheep Circumstances: • frequent finding of high and widespread NSP levels in (unvaccinated?) sheep indicative of exposure • uncontrollable movement of sheep • event based control fails because of lack of reporting or active surveillance • mixed cattle/sheep husbandry systems

Protect sheep health, including lamb crop

Setting

Objective

Action

Note

FMD free country which does not vaccinate

Eradicate the virus following an outbreak through stamping out plus emergency vaccination

......Apply sufficient intensity of emergency vaccination** in the risk area (PZ and SZ) that circulation dies out. Vaccination ceases once the vaccination target is achieved and no more outbreaks have occurred outside of the PZ/SZ.

Vaccinate cattle.

Sheep included ?

• if significant role in transmission after movement bans in place • if high risk that they introduce infection to cattle • if movement cannot be controlled • if sufficient resources to vaccinate sheep • exit strategy acceptable ( marketing of vaccinated sheep/products)

Buffer zone (non-free country)

Maintain an immune barrier between an FMD free country from area with endemic infection

Apply routine mass vaccination to all cloven-hoofed livestock* in the defined zone. Identify the vaccinated animals within the zone and restrict their movement by physical barriers and checkpoints. Prevent the movement of animals and potentially infected animal products to the free country/zone.

Vaccinate cattle.

Sheep included ?

• if significant role in transmission • if high risk that they introduce infection to cattle • if movement cannot be controlled • if sufficient resources to vaccinate sheep

Non-free country

Protect sheep health, including lamb crop

Seasonal risk:

Vaccinate 4-6 weeks (under study) before lambing to protect lamb crop

Year round risk:

Schedule as required to maintain immunity (twice yearly)

Protect high value cattle population

Risk based vaccination program

Vaccinate cattle.

Sheep included • transhumant populations, vaccinate pre-movement • radius vaccination around dairy farms • zonal or district mass vaccination where mixed cattle/small ruminant husbandry

Prevent virus circulation, qualify for potential free zone/country

Risk based vaccination program

Cattle included.

Sheep included if: • if significant role in transmission • if movement cannot be controlled • if sufficient resources to vaccinate sheep Circumstances: • frequent finding of high and widespread NSP levels in (unvaccinated?) sheep indicative of exposure • uncontrollable movement of sheep • event based control fails because of lack of

109