Open session of the standing technical committee of the EUFMD- 2012 by EuFMD

OPEN SESSIONS OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES OF THE EuFMD COMMISSION APPLIANCE OF SCIENCE IN THE PROGRESSIVE CONTROL OF FMD 29-31 October 2012 Jerez de la Frontera, Spain

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¡Bienvenidos a Jerez! The 2012 Sessions will bring together people and organizations involved in the areas of FMD control, laboratory services, research and development, predominantly from Europe and its neighbourhood regions, but with important participation from free and endemic countries from across the world. In addition to the two Open Sessions – of the EuFMD Standing Technical and Research Committees the Annual Network Meeting of the OIE/FAO FMD reference Laboratory Network, and the Meeting of the Global Foot-and-Mouth Disease Research Alliance (GFRA), will use this opportunity to meet. The Open Sessions, as the name suggests, are Open to all persons having FMD control as a part of their job description. The Standing Technical Committee has responsibility to provide strategic guidance and advice to the EuFMD on technical issues and bring attention to new developments which are important for policy making. In 2011, the Commission established a Special Committee on Research (SCR), with 12 elected members, replacing the former “Research group of the Standing Technical Committee”. The SCR has a role to review the technical developments reported at the Open Session which are important to FMD surveillance and control in the member states and may be commissioned, or propose, studies to the STC. In 2009-12, studies were commissioned on full genome sequencing, epidemiology in wild boar, telemetry and non-invasive sampling of wild boar, NSP and LPBE diagnostics for SAT viruses, serotype specific PCR assays, software for surveillance design, vaccine effectiveness in the field, and a global survey on FMD Research (through GFRA). It is a requirement of these supported studies to report to the Open Sessions. More epidemiology, more control: since 2002, attendance at Open Session has grown from around 100 to about 250, with a shift from predominantly lab based studies to field based - with over half the papers in 2012 coming from epidemiology and risk management studies. What is behind this? More capacity to partner between north and south institutions? The success of projects linking the field and advanced labs? Better communications and networking? The ideas coming out of Open Sessions and other events that bring people together? The new confidence in endemic regions that comes from applying ideas locally? Or because FMD is becoming recognized in endemic countries as important and preventable? The Open Sessions have had a history of developing new ideas that are taken up by the international organizations – for example the Erice Session (2008) provided the concept of virus pools needing specific regional programmes, and the “Progressive Control Pathway (PCP-FMD)” developed by EuFMD with FAO as a framework for developing sustainable national programmes. The PCP-FMD has, since 2011, been a joint tool with the OIE, and provides a framework for the Global Strategy for FMD Control launched by FAO and OIE in Bangkok in June 2012. The Strategy has the aim of all endemic countries advancing two PCP Stages in the next 15 years; in other words, at the end of this period all countries will have at the least a control programme protecting their vulnerable sectors. So in the 4 years since Erice, a lot has happened that has lead to are revised system for promoting progressive control. An increased amount of field work is needed as part of the PCP application at national level, and in parallel a lot more basic and applied research is needed in almost all disciplines. The 2012 Open Session recognizes networking is essential, to develop and spread ideas. It recognizes that science will help us progress FMD control in every part of the affected world, and that what you publish and report at this Conference is part of the process of progressive control – your work is transforming the possibilities. So bring on the Open Session at Jerez de la Frontera – at the frontiers of FMD science, our ideas should not observe boundaries! Keith Sumption Secretary EuFMD

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Acknowledgements

Acknowledgements The EuFMD Commission gratefully acknowledges the support of the European Commission and the EuFMD Member States, for funding the Committee meetings, Working Groups, and Research Studies. Professor David Paton and Dr Aldo Dekker are thanked for their work as Chairpersons of the Standing Technical and Research Committees, for their ideas and enthusiasm for the Open Sessions and behind the scenes in the work of the Committees. The Open Session 2012 is made possible through the support of Dr Ulrich Herzog, President of the EuFMD Commission, Dr Alf Füssel (DG-SANCO), and our host, Dr Lucio Carbajo Goñi, Marta Caínzos and their team. We would like to acknowledge on your behalf the EuFMD team, and in particular Enrique Anton, who managed and undertook most of the many, major tasks involved with the Jerez Session. The city of Jerez is thanked for its hospitality. Organization of the 2012 Open Session Chairman of the Standing Technical Committee: Professor David Paton Members of the STC: Dr Christianne Bruschke Dr Matthias Kramer Prof David Paton Dr Preben Willeberg Chairman of the Special Committee on Research: Dr Aldo Dekker Members of the SCR: Dr Bernd Haas (Germany) Dr Emiliana Brocchi (Italy) Dr Naci Bulut (Turkey) Dr Stefan Zientara (France) Dr Labib Bakkali (France) Dr Jeff Hammond (WRL, Pirbright, UK) Dr Georgi Georgiev (Bulgaria) Dr Marisa Arias (Spain) Dr Eoin Ryan (Ireland) Dr Graham Belsham (Denmark) Dr Kris de Clercq (Belgium) Dr Michel Bellaiche (Israel) The EuFMD Team in Rome: Dr Keith Sumption (Secretary) Dr Eoin Ryan (Animal health officer) Dr Vesna Milicevic (Animal health officer, STP program) Dr Dimitrios Dilaveris (Animal health officer, STP program) Ms Nadia Rumich (Communications officer) Ms Rossana Cecchi (Operations officer) Ms Manuela Zingales (Clerk) Mr Leonardo Leon Perez (Clerk) Mr Enrique Anton (Manager of the Jerez meeting) Ms Claudia Ciarlantini and her team (graphic designer)

Sponsors

The generous support of our sponsors has greatly assisted to reduce the costs of the event, enabling us to widen participation, and is greatly appreciated.

And special thanks to our Hosts and Local Organiser:

Plenary Session

Day 1 :: Monday 29 October PLENARY ROOM Open Session of the Standing Technical Committee (STC): Focus on issues affecting FMD control in FMD free regions SESSION I. OPENING 08:15 – 08:45 08:45 – 09:15 09:15 – 09:45 09:45 – 10:00 10:00 – 10:30

Welcoming/Opening remarks. Government of Spain (Marm), FAO, EuFMD Frenkel lecture: Frenkel’s legacy: What is keeping us? (P.W. de Leeuw) Global surveillance – European neighbourhood and global FMD situation (J. Hammond) Minister of Agricultura, Alimentación y Medio Ambiente (M. Arias Cañete) Coffee/Tea break PLENARY SESSION

SESSION II. FOCUS ON ISSUES AFFECTING FMD CONTROL IN FMD FREE REGIONS What should the waiting periods be for reinstatement of FMD-free status after vaccination-to-live? 10:30 – 10:55 10:55 – 11:20 11:20 – 11:30

Aligning waiting periods for vaccinate to-Live & vaccinate-to-die (D. Geale) A quantitative approach to determining waiting periods for fmd freedom. (A. Cameron) Evaluation of the benefits and feasibility of a vaccination-to-live strategy in fmd free countries (D. Hadorn)

European Wild boar and their role in disease transmission: Lessons learnt and policy implications 11:30 – 12:00 12:00 – 12:30 12:30 – 12:40 12:40 – 13:40

FMD in wild boar and policy implications (K. Depner) FMD in wild boar: can the virus be maintained in wildlife? Experiences and consequences from Thrace (S. Khomenko) Discussion Lunch break

SESSION III. FOCUS ON ISSUES AFFECTING FMD CONTROL IN FMD FREE REGIONS 13:40 – 14:10

Fmd lab bio-risk management: what have we learnt from application of the 2009 minumum standards? (B. Haas)

Approaches to the evaluation of FMD emergency management options and control measures in Europe 14:10 – 14:40 14:40 – 15:10 15:10 – 15:20 15:20 – 15:50

Economic evaluation of fmd management options: Implications for science and policy (R. Bergevoet) Simple decision tools informed by model predictions when considering fmd emergency vaccination strategies (P. Willeberg) Panel discussion: With speakers and other invited panelists, to discuss ideas of where the work should go Coffee/Tea break

(cont. on page viii)

Research Group (RG): Immunology and Vaccine Research

SESSION P1. IMMUNOLOGY AND VACCINE RESEARCH 1 11:15 – 11:30 11:30 – 11:45 11:45 – 12:00 12:00 – 12:15 12:15 – 12:30 12:30 – 12:40 12:40 – 13:40

Parallel Session

PARALLEL SESSION

Keynote: The cellular innate immune response during acute infection of cattle and swine with FMDV (W. Golde) Adaptive immune responses in the respiratory tract of fmd-vaccinated cattle after oronasal infection (M. Pérez Filgueira) Characterization of opsonizing antibodies against fmd Virus (A. Summerfield) Whole 140s FMDV particles are needed to elicit specific cellular immunity in vivo and to stimulate recall responses in vitro (A.V. Capozzo) Intraserotype Chimeric Foot-and-Mouth Disease Vaccine Antigen elicit protection in cattle (F. Maree) Discussion Lunch break

SESSION P2: IMMUNOLOGY AND VACCINE RESEARCH 2 13:40 – 13:55 13:55 – 14:10 14:10 – 14:25 14:25 – 14:40 14:40 – 14:55 14:55 – 15:10 15:10 – 15:20 15:20 – 15:50 15:50 – 16:05 16:05 – 16:20 16:20 – 16:35 16:35 – 17:00 17:20 – 18:20

Rational development of FMD Virus Vaccines (B.Charleston) Development of a bovine Enterovirus-based vector that expresses Multi-Epitopes of Foot-and-Mouth Disease Virus (J.H.Park) Development and evaluation of an adenovirus vector based intranasal FMDV capsid vaccine in mice for increasing immune responses (A. Babu) Alternative FMD vaccine potency tests based on serology and payload (T. Willems) Improving challenge-free FMD vaccine batch acceptance (R. Reeve) Relation between antibody response and protection in FMD vaccine depends on vaccine quality (A. Dekker) Discussion Coffee/Tea break Cross protection against current Asia 1 field isolates is provided by a high potency Asia 1 Shamir Vaccine (Y. Li) Testing the efficacy of 01 Manisa high potency vaccine against challenge with 0/Vietnam/2010 (0 MYA98 topotype) in pigs (W. Vosloo) Antibody titres in fmd type a strains: comparison of methodologies to predict crossprotection (T. Tesfaalem) Discussion Poster session

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Plenary Session viii

Day 1 :: Monday 29 October (cont.) Open Session of the EuFMD Research Group (RG): Focus on science contributing to the roll-out of progressive control of FMD

SESSION IV. PROGRESSIVE CONTROL PATHWAY FOR FMD (PCP): RESULTS, TECHNICAL DEVELOPMENTS AND ISSUES 15:50 – 16:20 16:20 – 16:50 16:50 – 17:20 17:20 – 18:20 18:30**

PCP Stage 1– 3: Results, technical developments and issues. (C. Bartels, N. de Haan, M. McLaws) Socioeconomics: Enhanced fmd control through the integration of socio-economic approaches (C. Bartels, N. de Haan, M. McLaws) The role of the OIE in fmd prevention and control. How to translate science in standards and guidelines, how to develop tools and ensure their convergence (J. Domenech) Poster session Dinner offered by the Spanish MARM (buses from the venue to the ‘Yeguada’ la Cartuja)

Plenary Session

Day 2 :: Tuesday 30 October PLENARY ROOM Open Session of the EuFMD Research Group (RG): Focus on science contributing to the roll-out of progressive control of FMD SESSION V. FMD EPIDEMIOLOGY: TRANSMISSION, VIRUS CIRCULATION, RISK FACTOR 08:30 – 09:00 09:00 – 09:15 09:15 – 09:30 09:30 – 09:45 09:45 – 10:00 10:00 – 10:15 10:15 – 10:45

Keynote: Local differences in circulation, what have we learnt from patterns of fmd persistence and spread? (N. Knowles) Limited transmission of foot-and-mouth disease virus from infected sheep to naïve calves (C. Bravo de Rueda) FMDV infection in vaccinated and non-vaccinated sheep: transmission to contact animals and diagnostic aspects (P. Eblé) Trasnmission of fmdv from infected buffalo (Bubalus bubalis) to vaccinated and naïve buffalo and cattle (M. Madhanmohan) Within herd transmission and evaluation of the performance of clinical and serological diagnosis of foot and mouth disease in vaccinated cattle (J.L. Gonzales) Discussion Coffee/Tea break

SESSION VI. EPIDEMIOLOGY 2 10:45 – 11:00 11:00 – 11:15 11:15 – 11:30 11:30 – 11:45 11:45 – 12:00 12:00 – 12:15 12:15 – 12:30 12:30 – 12:45 12:45 – 13:45

Foot and Mouth Disease Virus (FMDV) in the african buffalo (Syncerus caffer) in Kenya (S. Wekesa) Seroprevalence profile of foot-and-mouth disease in wildlife populations of West and Central African Regions with special reference to syncerus caffer subspecies (A. di Nardo) Epidemiological patterns and risk factors for Foot-and-Mouth Disease exposure in traditional livestock-keeping systems of Northern Tanzania (T. Lembo) Retrospective serosurvey of Foot and Mouth Disease (FMD) in free ranging domestic pigs and wild suids in Sub-Saharan African Countries (M. Arias) Risks associated with unofficial livestock movements in the greater Mekong region (A. Cameron) Risk factors for foot and mouth disease in beef cattle herds in Israel (E. Klement) Risk mapping of foot-and-mouth disease prevalence in Central Asian Countries (A. di Nardo) Discussion Lunch break

SESSION VII. EPIDEMIOLOGY 3 13:45 – 14:00 14:00 – 14:15 14:15 – 14:30 14:30 – 14:45 14:45 – 15:00

Prevalence and risk factors for FMD-NSP-antibodies in cow and buffalo calves, and small ruminants in Egypt (V. Maanen) Determining the level of vaccine-induced versus field-virus induced antibodies in livestock in West-Azarbaijan, Iran (C. Bartels) Spatio-temporal origin and transmission of the Foot-and-Mouth Disease Virus outbreaks in Burgas Region (Bulgaria) in 2011 (B. Valdazo) Molecular epidemiology of Foot-and-Mouth Disease Virus in the african buffaloes in Southern Africa (C. Kasanga) Discussion

(cont. on page xii)

08:45 – 09:00 09:00 – 09:15 09:15 – 09:30 09:30 – 09:45 09:45 – 10:00 10:00 – 10:15 10:15 – 10:45

Antigenic cartography for analysis of antigenic variations in FMD Virus (B. Pattnaik) Evidence of further neutralisation after removal of five neutralising antigenic sites in serotype o FMDV (A. Asfor) Discussion Determining the Epitope dominance on the capsid of a SAT2 Foot-and-Mouth Disease Virus by mutational analysis (P.A. Opperman) Study of antigenic site variation in fmd virus serotype 0 grown under vaccinal serum antibodies in vitro (B. Pattnaik) Discussion Coffee/tea break

Parallel Session

SESSION P3. IMMUNOLOGY AND VACCINE RESEARCH 3

SESSION P4. FMD MANAGEMENT 1 10:45 – 11:00 11:00 – 11:15 11:15 – 11:30 11:30 – 11:45 11:45 – 12:00 12:00 – 12:15 12:15 – 12:45 12:45 – 13:45

Modelling into policy: How can an ‘Intelligent Customer’ ensure appropriate use of evidence? (F. Gauntlett) Scaling up from 1-to-1 animal transmission experiments to epidemiological models of national outbreaks (D. Schley) Epidemiological models of FMD in two different austrian regions (J. Hiesel) Multi-criteria decision analysis for evaluating control options during FMD outbreaks (K. Mintiens) Meta-Analysis on the efficacy of Foot-and-Mouth Disease Emergency Vaccination (T. Halasa) Evaluating vaccination for Foot-and-Mouth Disease Control – an international study (M.G. Garner) Discussion Lunch break

SESSION P5. COMPLEMENTARY RESEARCH 1 13:45 – 14:00 14:00 – 14:15 14:15 – 14:30

A new approach to the oldest disease developing an antiviral drug strategy for the containment of Foot-and-Mouth Disease outbreaks (N.Goris) The Pyrazinecarboxamide Derivative T-1105 offers protection against O1 Manisa Virus infection in Guinea pigs (De Vleeschauwer) Discussion

SESSION P6. FMD MANAGEMENT 2 15:30 – 16:00 16:00 – 16:15 16:15 – 16:30

Coffee/Tea break Simulated effects of introducing emergency vaccination or depopulation during fmd outbreaks in Denmark (A. Boklund) Modelling the spread of fmd in endemic regions (M. Tildelsey) (cont. on page xiii)

Plenary Session xii

Day 2 :: Tuesday 30 October

(cont.)

SESSION VIII. VACCINATION PROGRAMMES 15:00 – 15:30 15:30 – 16:00 16:00 – 16:15 16:15 – 16:30 16:30 – 16:45 16:45 – 17:00 17:00 – 17:15 17:15 – 17:30 19:30**

Keynote: Effectiveness of vaccination programmes (P. Fine) Coffee/tea break FMD Asia-1 vaccine effectiveness in Turkey (T. Knight-Jones) An investigation of vaccination effectiveness in two Cambodian villages facing an outbreak of Foot-and-Mouth Disease (A. Cameron) The field effectiveness of inactivated vaccine for prevention of foot and mouth disease (E. Klement) Foot and Mouth Disease: Vaccine impact and progressive control in India (S.N. Singh) A high throughput liquid phase blocking elisa for quantitative estimation of antibody titers against structural proteins of Foot-and-Mouth Disease Virus (G.K. Sharma) Discussion Meeting at the Alcazar for the “Afta” session AFTA – Thoughts Night session (also known as the Fred Brown event)

16:30 – 16:45 16:45-17:00 17:00-17-15 17:15-17:30

Assesing and comparing control strategies for FMD in endemic countries: adaptation of the North American animal disease spread models (NAADSM) (M.D.Salman) Geographically-grounded, cost-benefit based control policies: Built as equal circles or considering local connecting networks? (A.L. Rivas) Costs and benefits of FMDS practises in commercial dairy farms in central Ethiopia (A.F. Beyi) Discussion

Parallel Session

(cont.)

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Plenary Session

Day 3 :: Wednesday 31 October PLENARY ROOM Open Session of the EuFMD Research Group (RG): Focus on sciencecontributing to the roll-out of progressive control of FMD SESSION IX. DIAGNOSTIC DEVELOPMENTS AND LABORATORY NETWORKS 08:30 – 09:00 09:00 – 09:15 09:15 – 09:30 09:30 – 09:45 09:45 – 10:00 10:00 – 10:15 10:15 – 10:45 10:45 – 11:00 11:00 – 11:15

Keynote: New Elisa´s for FMD (E. Brocchi) FMD and SVD combined proficiency test studies 2011 (B. Armson) New roles for “Auxiliary labs” in the diagnosis of FMD? (B.Haas) Laboratory capacity for diagnosis of Foot-and-Mouth Disease in Eastern Africa: Implication on progressive control pathway (A. Namatovu) Evaluation of FTA® cards as a laboratory and field sampling device for the detection and serotyping of Foot-and-Mouth Disease Virus (M. Madhanmohan) Development of RNA transfection method for rescue of FMD virus in susceptible cell (B. Pattnaik) Coffee/Tea break Open fmd a resource for automatic and curated nomenclatures and tools for the FMD (epiphylogeography?) community (P. Claes) Development and evaluation of a real-time reverse Transcription-Loop-Mediated isothermal amplification assay for rapid serotyping of Foot-and-Mouth Disease Virus (M. Madhanmohan)

SESSION X. DIAGNOSTIC DEVELOPMENTS AND APPLICATIONS 11:15 – 11:30

11:30 – 11:45 11:45 – 12:00 12:00-12:15 12:15-12:45 12:45-13:45

Diagnostic performance of an immunochromatographic lateral-flow strip test using generic rapid assay device for detection and serotyping of Foot-and-Mouth Disease Virus serotypes 0 or Asia 1 in clinical samples (Z. Zhan) Development and evaluation of a one-step duplex real time RT-PCR for diagnosis of Foot-and-Mouth Disease (K. Gorna) The development and evaluation of a SAT-adapted 3ABC Diva Test for Foot-and-Mouth Disease Virus in the Southern Africa context (M. Chitray) Detection, isolation, and typing of Foot-and-Mouth Disease Virus from oral swab samples collected from balochistan province of Pakistan. (M. Assad Ullah) Discussion Lunch break

SESSION XI. WRAP UP AND CLOSURE 13:45

15:30

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FINAL Session of papers – or The way ahead. Conclusions given by leaders (keynote speakers) Recommendations Appreciations and prizes Closure

SESSION P7. GFRA MEETING 08:30 – 09:00 09:00 – 09:15 09:15 – 09:30 09:30 – 09:45 09:45 – 10:00 10:00 – 10:15 10:15 – 10:45

GFRA: State of the alliance overview (F. Marée) Role of buffalo in the maintenance of FMDV (B. Charleston) FMD virus ecology: Collaborative studies (L. Rodríguez) Development of a safe antigenic marker FMD vaccine platform (E. Rieder) Top priorities for research (D. Paton) GFRA workshop in 2013 and concluding remarks (F. Marée) Coffee/tea break

Parallel Session

GFRA MEETING: An African Perspective

Open Session of the EuFMD Research Group (RG): Focus on science contributing to the roll-out of progressive control of FMD SESSION P8. FMD MONITORING AND SURVEILLANCE: EXPERIENCE, METHODS ANDAPPROACHES 11:15 – 11:30 11:30 – 11:45 11:45 – 12:00 12:00 – 12:15 12:15 – 12:30 12:30 – 12:45 12:45 – 13:45

Estimating the incidence of foot and mouth disease (M. McLaws) Risk factors for transmission of foot-andmouth disease during an outbreak in Southern England in 2007 (K. Sharpe) The use of oral fluids from pig herds for pre-clinical diagnosis and monitoring in a FMD emergency: Current research and future directions (Z. Zhang) Foot-and-Mouth Disease Virus transboundary movements between Subsaharan Africa, North Africa and the Middle East (N. Knowles) Maximising efficiency with a surveillance strategy for Foot-and-Mouth Disease during an outbreak in a previously fmd-Free country (K. Walker) Discussion Lunch break

List of Appendices Session I: Opening - Monday 29 October: Plenary hall  Frenkel's legacy: What is keeping us? P.DeLeeuw  Global surveillance: European neighbourhood and global FMD situation, J.Hammond Session II: Focus on issues affecting FMD control in FMD free regions  Aligning waiting periods for vaccinate-to-live and vaccinate-to-die, D.Geale  A quantitative approach to determining waiting periods for FMD freedom, A.Cameron  Evaluation of the benefits and feasibility of a vaccination-to-live strategy in fmd free countries, D.Hadorn  FMD in wild boar and policy implications, K.Depner [Not available]  FMD in wild boar: can the virus be maintained in wildlife? Experiences and consequences from Thrace, S.Khomenko Session III: Focus on issues affecting FMD control in FMD free regions  FMD lab bio-risk management: what have we learnt from application of the 2009 minimum standards, B.Haas  Economic evaluation of fmd management options: implications for science and policy, R.Bergevoet  Simple decision tools informed by model predictions when considering FMD emergency vaccination strategies, P.Willeberg Session IV: Progressive Control Pathway for FMD (PCP-FMD): Results, Technical developments and Issues  Progressive Control Pathway (PCP) Stage 1-3 Results, Technical developments and issues, C.Bartels, N.DeHaan, M.Mclaws  Socioeconomics: Enhanced FMD control through the integration of socio-economic approaches, C.Bartels, N.DeHaan, M.Mclaws  The role of the OIE in FMD prevention and control. How to translate science in standards and guidelines, how to develop tools and ensure their convergence, J.Domenech Session P1: - Monday 29 October- Parallel Session: Immunology and Vaccine Research  The cellular innate immune response during acute infection of cattle and swine with FMDV, W.Golde  Adaptive immune responses in the respiratory tract of FMD-vaccinated cattle after oronasal infection, M. Perez Filgueira  Characterization of opsonizing antibodies against FMD virus, A. Summerfield  Whole 140s FMDV particles are needed to elict specific cellular immunity in vivo and to stimulate recal responses in vitro, A.V.Capozzo [Not available]  Intra-serotype chimeric FMD Vaccine Antigen elicit protection in cattle, F.Maree Session P2: -Immunology and Vaccine Research  Production and evaluation of FMDV stabilised capsids as potent, rapidly deployable vaccines, B. Charleston  Development of a bovine enterovirus-based vector that expresses multi-epitopes of FMDV, J.H. Park  Development and evaluation of an adenovirus vector based intranasal FMDV capsid vaccine, A. Babu  Alternative FMD vaccine potency tests based on serology and antigen payload, T. Willems  Towards challenge-free FMD vaccine batch acceptance, R. Reeve

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Relation between antibody response and protection in FMD vaccine depends on vaccine quality, A. Dekker  Cross-protection against current Asia1 field isolates is provided by a high potency Asia1 Shamir Vaccine, Y.Li  Testing the efficacy of 01 Manisa high potency vaccine against challenge with 0/Vietnam/2010 (0MYA98 tototype) in pigs, W.Vosloo  Antibody titres in fmd type A strains:comparison of methodologies to predict crossprotection, T.Tesfaalem Session V: Tuesday 30 October- Plenary Session: FMD Epidemiology: Transmission, virus circulation, risk factor  Local differences in circulation, what have we learnt from patterns of FMD persistence and spread, N.Knowles  Limited transmission of fmdv from infected sheep to naive calves, C.Bravo de Rueda  Fmdv infection in vaccinated and non-vaccinated sheep: transmission to contact animals and diagnostic aspects, P.Eble'  Transmission of fmdv from infected buffalo (Bubalus bubalis) to vaccinated and naive buffalo and cattle, M.Madhanmohan  Within herd transmission and evaluation of performance of clinical and serological diagosis of fmd in vaccinated cattle, J.L. Gonzales [Not available] Session VI: FMD Epidemiology 2  FMDV in the African buffalo (Syncerus caffer) in Kenya, S.Wekesa  Seroprevalence profile of fmd in wildlife populations of West and Central African Regions with special reference to syncerus caffer subspecies, A.DiNardo  Epidemiological patterns and risk factors for FMD exposure in traditional livestock keeping systems of Northern Tanzania, T.Lembo  Retrospective serosurvey of FMD in free ranging domestic pigs and wild suids in Sub-Saharan African countries, M.Arias  Risks associated with unofficial livestock movements in the greater Mekong region, A.Cameron  Risk factors for fmd in beef cattle herds in Israel, E.Klement  Risk mapping of fmd disease prevalence in Central Asian countries, A.DiNardo Session VII: FMD Epidemiology 3  Prevalence and risk factors for FMD-NSP antibodies in cow and buffalo calves and small ruminants in Egypt, C.Van Maanen  Determining the level of vaccine-induced versus field-virus induced antibodies in livestock in West Azerbaijan, Iran, C.Bartels  Spatio-temporal origin and transmission of the fmdv outbreaks in Burgas region (Bulgaria) in 2011, B.Valdazo  Molecular epidemiology of fmdv in the african buffaloes in Southern Africa, C.Kasanga [Not available] Session VIII: Vaccination programmes  Effectiveness of vaccination programmes, P.Fine  Fmd Asia1 vaccine effectivness in Turkey, T.Knight-Jones  An investigation of vaccine effectivness in two Cambodian villages facing an outbreak of fmd, A.Cameron  The field effectiveness of inactivated vaccine for prevention of fmd, E.Klement  Fmd: vaccine impact and Progressive Control in India, S.N.Singh  A high throughput liquid phase blocking elisa for quantitative estimation (...), G.K.Sharma[Not available]

Session P3: - Tuesday 30 October- Parallel Session: Immunology and Vaccine Research  Antigenic cartography for analysis of antigenic variations in fmdv, B.Pattnaik [Not available]  Evidence of further neutralisation after removal of 5 neutralising antigenic sites in serotype 0, A.Asfor  Determining the epitope dominance on the capsid of a SAT2 fmdv by mutational analysis, P.A.Opperman  Development of a predictive model for VM for sertotype 0, M.Mahapatra

Session P4: FMD management  Modelling into policy: How can an "Intelligent Customer"approach ensure appropriate use of evidence? F.Gauntlett  Scaling up from 1-to-1 animal transmission experiments to epidemiological (...), D. Schley  Epidemiological models of fmd in two different Austrian regions, J.Hiesel  Multi-criteria decision analysis for evaluating control options during fmd outbreaks, K.Mintiens  Meta-analysis on the efficacy of fmd emergency vaccination, T.Halasa  Evaluating vaccination for fmd control - an international study, M.G.Garner Session P5: Complementary research  A new approach to the oldest disease developing an antiviral drug (...), N.Goris  The Pyrazinecarboxamide derivative t-1105 offers protection against 01Manisa(...), A.De Vleeschauwer Session P6: FMD management  Optimizing the control of FMD in Denmark by simulation. Comparison of different control strategies, A.Boklund  Modelling the spread of fmd in endemic regios, M. Tildelsey  Assessing and comparing control strategies for fmd in endemic countries (...), M.S.Salman [Not available]  Geographically-grounded, cost-benefit based control policies: built as equal circles (...), A.L.Rivas  Costs and benefits of fmds practises in commercial dairy farms in central Ethiopia, A.F.Beyi Session IX: Wednesday 31 October- Plenary Session: Diagnostic Developments and Laboratory Networks  New ELISA's for fmd, E.Brocchi  FMD and SVD combined proficency test studies 2011, B.Armson  New roles for 'Auxiliary labs' in the diagnosis of fmd, B.Haas  Open fmd a resource for automatic and curated nomenclatures and tools (...), P.Claes  Development and evaluation of a realtime reverse transcription -loop-mediated isothermal (...), M.Madhanmohan [Not available] Session X: Diagnostic Developments and Applications  Diagnostic performance of an immunochromatographic lateral-flow strip test using generic rapid assay (...), Z.Zhan  Development and evaluation of a one-step duplex real-time RT-PCR for diagnosis of fmd, K.Gorna  The development and evaluation of a SAT-adapted 3ABC Diva test for fmdv in the Southern African context, M.Chitray



Detection, isolation and typing of fmd from oral swab samples collected from Balochistan province of Pakistan, M.Assad Ullah

Session P7: GFRA meeting: an African perspective  GFRA: State of the alliance overview, F.Maree  Role of buffalo in the maintenance of fmdv, B.Charleston  Fmdv ecology: collaborative studies, L.Rodriguez  Development of a safe antigenic marker fmd vaccine platform, E.Rieder  Top priorities for research, D.Paton  GFRA workshop in 2013 and concluding remarks, F.Maree Session P8: Fmd Monitoring and Surveillance: experience, methods and approaches  Estimating the incidence of fmd, M.Mclaws  Risk factors for transmission of fmd during an outbreak in Southern England in 2007, K.Sharpe  The use of oral fluids from pig herds for pre-clinical diagnosis and monitoring in an fmd emergency (...), Z.Zhang  Fmdv transboundary movements between Subsaharan Africa, North Africa and the Middle East, N.Knowles  Maximising efficiency with a surveillance strategy for fmd during an outbreak in a previously fmd-free country, K.Walker [Not available]

Draft Agenda – Jerez 2012- Open Session of the EuFMD Standing Technical Committee

EuFMD Jerez de la Frontera 8/1/2012

::Open Session of the EuFMD Standing Technical Committee:: Focus on issues affecting FMD control in FMD free regions 1

Waiting periods for reinstatement of FMD-free status after vaccination-to-live:  Two technical papers examining the basis for waiting periods after use of vaccination-to –live in free countries and achievement of desired outcomes of confidence in disease freedom. European Wild boar and their role in disease transmission : Lessons learnt and policy implications:  Two papers considering the lessons learnt from the FMD surveillance in wild boar in Bulgaria and Turkey in 2011-12 and the issues of improving surveillance for FMD and other infections in wild boar. Economic evaluation of FMD emergency management options and control measures in Europe: implications for science and policy:  Two papers considering the implications of recent epidemiologic-economic modeling of FMD emergency management options in European countries.

::Open Session of the EuFMD Special Committee on Research:: (formerly the “Research Group”)* 1

Progressive Control Pathway for FMD (PCP), technical developments and issues  Three invited papers reviewing how the PCP has been applied in endemic countries by EuFMD , the methodological issues and developments in identifying FMD risks and disease impact, risk management options, monitoring and evaluation of progress. Includes progress on developing a socio-economics toolbox to identify issues and potential solutions with private and public stakeholders in FMD control. FMD epidemiology; transmission, virus circulation, risk factors (17)  Keynote on viral threat emergence, and submitted papers on FMD epidemiology , including surveillance for new threats, experimental, molecular epidemiology, field studies. FMD monitoring and surveillance – experience, methods and approaches (6)  Submitted papers on FMD monitoring (focus on monitoring changes threatening health status) and surveillance (focus on detection of new events requiring response, in free countries or entry of exotic serotypes). Latter section includes issues of a. Efficiency in FMD case detection, by veterinary surveillance visits and sampling, in domestic animals and wildlife; b. Engaging livestock sector and animal health workers in surveillance during an animal health crisis: new tools for effective communication; c. the potential use of bulk milk screening (pooled sampling for pre-clinical diagnosis...). FMD vaccines (6)  Submitted papers on FMD vaccines: including selection, production, stability, cross-protection, quality control, duration of immunity, alternatives to challenge. Effectiveness of vaccination programmes (5)  Keynote and submitted papers on the monitoring of vaccination programmes, including measurement of FMD vaccine effectiveness against clinical disease. Diagnostic developments and laboratory networks (10)  Keynote papers on development of new diagnostic assays and on the application of the EuFMD Minimum Standards for FMD laboratory biorisk management. Open papers on development, validation and evaluation of diagnostics, and from leaders in the FMD lab networks in the seven virus pools. FMD management and the evaluating the impact of control measures (6)  Submitted papers from free and endemic countries; includes the issue of development of models for FMD spread and management options for use in endemic countries. Complementary research: pathogenesis, next generation methods, insights and ideas from research (6)  Submitted papers from basic or cutting edge research that improve understanding of FMD biology, risk and have potential to of high relevance to understanding FMD and indicate trends and ideas from technical fields of relevance to future management.

The approximate number of open (submitted) papers is given in brackets. Overall around 60 submitted papers will be accepted for oral presentation; the time allocated to each item will depend on the number and importance of the offered papers.

Appendix 3 Food and Agriculture Organization of the United Nations

Food and Agriculture Organization of the United Nations

1. Dr. H. S. Frenkel - his life, his work and his legacy -

Frenkel’s legacy: what is keeping us?

2. Headlines of the joint FAO/OIE Global FMD Control Strategy 3. The role of research - learning from Frenkel’s example -

Peter de Leeuw, DVM, PhD Senior veterinary advisor FAO

Food and Agriculture Organization of the United Nations

. 1940-1945: Dr. H.S. Frenkel was forced to resign as director of the SVRI and arrested by Nazi occupation SVRI, led by deputy Dr. Van Waveren, produced Waldmann/Köbe vaccine based on virus harvested from infected cattle in slaughterhouses 1946: Dr. H.S. Frenkel returned to Amsterdam after surviving Theresienstadt concentration camp and recovery period in Switzerland. Research again focused on in vitro production of FMD virus 1947 and 1949: Publications in OIE bulletin and Nature about FMD virus culture in surviving cattle tongue epithelium explants

Herman Salomon Frenkel (1891–1968) 1930: Appointed Director of the State Veterinary Research Institute (SVRI) with sole task: “Find the cause and develop methods to control FMD” 1930-1941: Crisis period: Improvised facilities in Rotterdam. Research focus immediately on virus propagation methods in vitro: fetal skin of guinea pigs, cattle, pig and sheep in buffer with serum and under aeration. Problems with bacterial contamination! 1941: SVRI moved to new facility in Amsterdam: isolation provided by location on marine establishment and surrounding city. Problem: animal facilities not included – slaughterhouses used for animal experiments 3

Food and Agriculture Organization of the United Nations

Machinery in the 50-ties:

• In 1950 the majority of FMD antigen was produced in vitro: o Superficial layer of cattle tongue removed o Epithelial layer separated from underlying layer o Cultivation in medium with antibiotics for 24 hours at 37 degrees Centigrade under aeration with O2/CO2 mixture o Crushing, centrifugation, filtration o Adsorption onto aluminum hydroxide and inactivation with formaldehyde • Epithelial production was mechanized and virus production scaled up to 40 liter vessels • One tongue provided antigen for approximately 120 vaccine doses, each with 10 PD50 • In 1965 a maximum of 130.000 tongues was used

-Brushing the tongues and harvesting tongue epithelium -Water bath for virus culture -Autoclaves for formaldehyde inactivation after virus adsorption onto aluminum hydroxide -Storage of readyto-use vaccine 5

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 3 Food and Agriculture Organization of the United Nations

Visit of Charles Merieux and C. Mackowiak to SVRI in Amsterdam in June 1958

Food and Agriculture Organization of the United Nations

Number of FMD cases in Western Europe Arrows indicate start of large scale vaccination campaigns

Institut Merieux was the first to produce the “Frenkel vaccine” commercially Method was deliberately not protected by patents

Food and Agriculture Organization of the United Nations

Since Frenkel’s time:

What are the lessons from these early experiences?

• FMD virus growth in BHK-21 cell cultures (Mowat, Chapman and Capstick, 1962) and in suspension cultures (Telling and Elsworth, 1965), allowing large scale production of cells, virus and vaccine • Better virus and antigen quantification methods • Improved inactivation methods with first order inactivants (AEI - Brown and Crick, 1959; (BEI) – Bahnemann, 1975) • Improved adjuvants – saponin, oil and double oil emulsions, providing longer lasting immunity and making vaccination of pigs effective • Better and faster diagnostic methods

1. FMD can be controlled with classical vaccines, in combination with veterinary police measures... 2. If vaccine is used in a systematic manner... 3. By only vaccinating cattle... 4. Even in countries with a high density of cattle and other susceptible species... 5. But this can only be done in a concerted effort within the region... 6. And the FMD vaccine production facility should be biosecure... 9

Food and Agriculture Organization of the United Nations

FMD is present in many parts of the world FMD continues to present a threat for FMD-free countries FMD blocks trade in animals and animal products FMD hampers animal husbandry development In developing countries, lowers production efficiency and FMD adds to food insecurity and to poverty at the household level

•Better understanding of FMD immunology •Better understanding of FMD epidemiology & risk factors •Development of molecular-epidemiological methods (sequencing) •Better understanding of biosecurity and bio-secure labs

and indeed...

and we are not talking peanuts….

•FMD has been controlled or largely controlled in several regions of the world where it occurred endemically (Western Europe, South America, parts of Southern and North Africa and South East Asia)

FMD damage worldwide is estimated at 5 billion US dollars per year and outbreaks in FMD-free countries have devastating effects – and cost 1 billion on average per year over the last 20 years (Rushton et al, Bangkok 2012)

but still... 11

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 3 Food and Agriculture Organization of the United Nations

Food and Agriculture Organization of the United Nations

The joint FAO/OIE Global FMD Control Strategy

If all basic tools for FMD control are available: what is keeping us? At the country level: A perceived lack of incentives and thus lack of political will Inadequate veterinary infrastructure (organization, staffing, expertise, laboratory support, legislation) Lack of funds and no or limited external support At the individual level: Only specific categories may see the advantages (dairy farmers) At both levels: Vaccines are expensive, not readily available, not always up to standard, not always matching the prevailing field strains and need to be handled with care (cold chain)

Notions and principles Component 1: FMD Control Basis: FMD control is not an utopia: we can do much better with existing means and methods – oFMD-endemic countries should be better aware of the damage caused by FMD and the opportunities lost [clear need for more socio-economic studies] oOnly regional approaches will be successful as history has shown (Western Europe, South America, SE Asia) oRegional approaches should take into account regional differences (for instance wildlife issue in Southern Africa) 13

Food and Agriculture Organization of the United Nations

oFocus should be on FMD-endemic countries using a progressive, risk-based approach, mainly based on the FMD Progressive Control Pathway oFMD-free (usually industrialized) countries should support the Global FMD Control Strategy, not just based on solidarity, but also on well-understood own interest (control at source) Component 2: Progressive FMD control in developing countries will go hand in hand with improvement of Veterinary Services (VS) Component 3: Improvement of VS will result in better possibilities to control other major diseases of livestock

Food and Agriculture Organization of the United Nations

2) Strengthening the vital disease control support functions: Laboratories (with a proposed structure of national and regional labs, a coordinating global lab and linked by networks; with some additional staff and support)  Epidemiology (similar structure proposed with national focal points, collaborative centers, coordinating center and networks; some additional staff and support)  Vaccines (improvement of availability; vaccine test centers for quality assurance and vaccine matching; improved vaccination planning and post -vaccination surveillance)

Food and Agriculture Organization of the United Nations

The 4 pillars of the FMD Control Strategy 1) Combination and integration of the tools and instruments of FAO and OIE: The Progressive Control Pathway (PCP), embedded in a regional approach with roadmaps jointly produced by the countries Performance of Veterinary Services Pathway (PVS), with FMDrelated critical competencies worked out per PCP stage and by using the GF-TADs platform for governance (with a possibility of acceptance of a PCP stage claimed by countries) OIE Terrestrial Animal Health Code to provide incentives o endorsement of a national FMD control program o recognition of FMD-free status (with or without vaccination)

Food and Agriculture Organization of the United Nations

3) Strengthening the “advanced-stage support functions”: Public/private partnerships Biosecurity Identification of farms and animals Emergency responses 4) Continuation of research The FMD control Strategy advocates for continued research, in particular in the fields of diagnostics, strain characterization, vaccine development, vaccine quality control and epidemiology

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 3 Food and Agriculture Organization of the United Nations

Food and Agriculture Organization of the United Nations

Conceptually: moving up means capacity building of Veterinary Services and vice versa 5

Aims The FMD Control Strategy aims at synchronous worldwide progress and spells out global targets on a 15-year horizon, divided into 5-year periods for evaluation and management, both for Component 1 (FMD) and Component 2 (Veterinary Services)

4 3 2

Component 3 of the Strategy (improvement of other disease control efforts and finding sensible combinations) has to be worked out for each (sub) region, whereby an important role is foreseen for the regional technical and economical organizations and the Regional GF-TADs Steering Committees

Confirm FMD free

Maintain zero circulation and incursions

Institutionalization

Implement Control strategy to eliminate circulation

Organization

Implement risk-based control

Identify risk and control options

Food and Agriculture Organization of the United Nations

Maintain zero circulation; withdraw vaccination

Studies

Incidence

Food and Agriculture Organization of the United Nations

Financial implications

Action plan

in USD as calculated by the World Bank for the first 5 years

Action plan (typical activities) was worked out  At country level – for each of the PCP stages and for each of the Strategy components  At regional level  At global level

- Cost of national FMD programmes (to support 79 initial PCP 0-2 Stage countries) - Vaccination cost (to support 45 initial PCP 1-3 Stage countries) - Regional level (reference labs/epidemiology support and networks) - Global level (coordination, evaluation)

The Global FMD Control Strategy and supporting documents are available on the websites www.FMDconference2012 http://www.oie.int

68 M 694 M 47 M 11 M

Food and Agriculture Organization of the United Nations

Bangkok and post-Bangkok

Is that a lot? The FMD damage in terms of production losses, trade opportunities, control costs etc is estimated at (Rushton et al, Bangkok 2012): 5 billion US dollars per year Outbreaks in FMD-free countries cost 1 billion US dollars on average per year over the last 20 years Although difficult to measure in figures, FMD can have severe effects at the household level in terms of food security and income Outbreak control methods used in hitherto FMD-free countries are under severe criticism and may be difficult to apply in the future

Bangkok was not a pledging conference, but over 100 countries, regional organizations, development partners and stakeholders supported the launch of the FAO/OIE Global FMD Control Strategy Roll out of the Global Strategy after Bangkok: 1.Countries where FMD is still endemic will be stimulated to step up their control efforts using the Progressive Control Pathway 2.Countries where FMD has been controlled, as well as development partners, will be requested to increase their support for FMD control at source (usually in developing countries) 23

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 3 Food and Agriculture Organization of the United Nations

Food and Agriculture Organization of the United Nations

Where does science come in? 3. Specific attention will be paid to countries in the FMD virus pool regions 3, 4, 5 and 6, the need of which was indicated by the port folio review of the Global Strategy 4. Socio-economic studies will be stimulated to provide further evidence of the damage caused by FMD at the country, sector and household levels 5. In regions where incentives for FMD control appear to be lacking, studies and workshops will be initiated to develop multi-disease approaches tailor-made for the (sub)region 6. Specific support will be requested from development partners to fund the studies and regional and global activities foreseen under the Global Strategy

1. Current and future research should assist in the roll out of the Strategy by providing effective means, methods and expertise to support and correctly implement the Progressive Control Pathway and move up the ladder 2. Science should help in improving the availability and quality of FMD vaccines as well as in simplifying vaccine quality control procedures 3. Science should help in solving problems and answering questions that inevitably arise in large-scale disease control efforts 4. Science should help to lower the cost of FMD preventive and control actions 26

Food and Agriculture Organization of the United Nations

Dr. H. S. Frenkel’s example should inspire both the scientific and the regulatory community:

 An enormous boost for the Global FMD Control Strategy would be the availability of a new generation of FMD vaccines that have the desired characteristics: safe, effective, long term immunity, broad spectrum of field strains covered, less temperature-dependent and cheaper

 With focus and dedication and despite  10 years of frustration during the economic crisis and  5 terrifying years during World War II

 Such a breakthrough may even be needed to convince skeptics that progress with FMD control indeed is no utopia and to mobilize forces on a global scale (Plan B - if we fail to convey our message that we can do much better with existing means and methods)

 He managed to move in just 5 years from the first experiments with FMD virus cultured on cattle tongue epithelium explants to large scale FMD vaccine use in practice!

Food and Agriculture Organization of the United Nations

GF-TADs joint FAO/OIE FMD Working Group

Thank you for your attention

•J. Domenech (OIE) and P. de Leeuw (FAO) - co-chairs; N. Leboucq and B. Todeschini (OIE); G. Ferrari and S. Metwally (FAO)

• Consultants: A. Donaldson and J. Rushton • Financial expertise: F. Legall and E. Fukase, World Bank

With indispensable contributions from regional organizations (EuFMD, AU-IBAR, SEACFMD; Panaftosa); country and regional representatives; individual experts; peer review group; OIE - SCAD; GF-TADs Management Committee and GF-TADs Global Steering Committee

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 4 FMD : Global Surveillance and European Neighbourhood Situation

Previously The Institute for Animal Health

EuFMD October 2012 The World Reference Laboratory for FMD

AVRI

NOW Dr Jef M. Hammond WRLFMD®

Donald King, Nick Knowles, Valerie Mioulet and Yanmin Li The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UNITED KINGDOM

FMD Reference Laboratories at Pirbright

World Reference Laboratory for FMD- WRLFMD

• European Community Reference Laboratories for FMD

FAO, OIE, EU and National Responsibilities

– Support and diagnosis for EU Member States

• OIE Reference Laboratory for FMD

– support of safeguarding and promoting international trade

• World Reference Laboratory for FMD designated by FAO – WRLFMD – Global surveillance and threat recognition – Reference Laboratory Network of OIE/FAO FMD Labs

Global Surveillance

• • • • • • • • • •

24/7 Diagnostic Service Global surveillance Strain characterisation Vaccine matching (Bulgaria, South Korea, Egypt) Extensive library of isolates Test improvement & Development, validation, Quality assurance Reagent supply Training Advice & Reports

Visualization of Regional Virus Pools as an Aid to Global Control Divides the Globe into 7 pools each with • Multiple serotypes but topotypes mainly confined to

Endemic

that pool

Intermediate, sporadic Free with vaccination Countries with multiples zones: FMD-free, free with vaccination or not free

• Each pool may need tailored vaccines and strategies

Free. Virus present in game parks Free

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4

The conjectured status of FMD showing approximate distribution of regional virus pools.

Pool 3 Pool 3

O, A, O, A, Asia Asia 1 1

Pool 5 Pool 5

O, A, O, A, SAT SAT 1, 1, 2 2

Pool 1 Pool 1

O, A, Asia 1

Pool 2 Pool 2

O, A, O, A, Asia Asia 1 1

Pool 4 Pool 4

A, O, A, O, SAT SAT 1, 1, 2, 2, 3 3

Pool 6 Pool 6

SAT 1, SAT 1, 2, 2, 3 3

The conjectured status of FMD showing approximate distribution of regional virus pools.

Pool 7 Pool 7 O, A

Enhanced Surveillance: OIE/FAO Lab network • • • • • • • • • • • •

WRLFMD: RRLSEA: LVRI: FGI ARRIAH: PDFMD: RRLSSA: FMD-Laboratory: PANAFTOSA: LFADLCT: ARC-OVI: PIADC: CODA-CERVA-VAR:

Pirbright, UK Pakchong, Thailand Lanzhou, China Vladimir, Russia Mukteswar, India Gabarone, Botswana Embakasi, Kenya Rio de Janeiro, Brazil Argentina Onderstepoort, RSA Plum Island, USA Ukkel, Belgium

Approximately ~2400 samples tested during 2011 OIE/FAO FMD Reference Laboratory Network Annual Report 2011 Editor: Dr Jef Hammond, IAH, Pirbright, UK.

Serotyping results for 2011

SAT 2 1% SAT 3 2% SAT 1 1% 0%

WRLFMD Serotyping results for 2012- upto 3rd Quarter ®

Asia 1 16%

ASIA 1 12%

O 40%

SAT2 19%

A 24% O 60%

The network labs received >2,400 samples in 2011 from 34 countries 60% were serotype O Increased Asia 1 activity…………………………..Serotype C was not detected

SAT1 2%

A 23%

From > 650 samples in 2012 from 25 countries 40% were serotype O

Increased Asia 1 activity Increased SAT 2 activity

Still No Serotype C (not reported since 2004)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4

25 countries ~650 samples ~ 300 positive

FMD Outbreaks 2012 ~ 650 650 Samples Samples from from 25 25 Countries Countries Sudan O, A & SAT 2

Egypt O, A & SAT 2 Libya O & SAT 2

PAT SAT 2 KSA O

Turkey O, A & Asia 1

UAE O

Turkey 2012- Dıstrıbutıon >1000 Outbreaks

Bahrain O & SAT 2

The majority of these have been Asia-1.

Kuwait O Iran O, A & Asia 1 Afghanistan O, A & Asia 1

Israel O

Russia O China O

Kazakhstan O&A

Chinese Taipei O Pakistan A & Asia 1 Zambia SAT 2

Namibia SAT 1

Kenya O, SAT 1 & SAT 2 FMD -Free

Endemic

Free. Virus present in game parks

Intermediate, sporadic

Tanzania O, A, SAT 1 & SAT 2

Vietnam O



Thailand O&A

Ethiopia O Eritrea O

Hong Kong O

South Africa SAT 2 Free with vaccination

Malaysia O&A

Countries with multiples zones: FMD-free, free with vaccination or not free

Total number of notifications of FMD received from governorates for Feb-March 2012

FMD Outbreaks 2012 SAT 2 in North Africa and Middle East

Diseased/Dead Animals per day

August 2012

FMD Outbreaks 2012 Vaccination against SAT 2 in Egypt

Vaccination with monovalent SAT2 – 1st round with locally produced vaccine Total= 827954 in 21 governorates . cattle 431568 Buffaloes 211352 sheep 159398 goat 25636 2nd round vaccination with monovalent locally produced SAT2 vaccine = 148741 cattle 114439 buffaloes 31946 sheep 1289 goat 1067 Information provided by Dr Soheir Hassan Abd El Kader

Under Secretary of centeral adminstration of preventive medicine General Organization for Veterinary Services Ministry of Agriculture Cairo, Egypt

serial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

governorates cairo Alex Port said Suez Domitta Dakahlia Sharkia Quliobia Kafrelshik Gharbia Menofia Behira Ismailia Giza Benisuef Fayoum Menia Assuit Sohag Quina Asswan Luxor Red sea New valley Matroh North sinia South sinia total

suspicious animals 1059 2229 451 4045 1799 8058 388 1219 1215 23766 1456 3588 171 2228 1296 744 1792 1107 1120 1054 227 701 0 168 59 1 0 59941

total No of dead animals 23 299 170 299 636 1176 36 176 933 3151 629 815 20 107 361 278 421 171 206 119 52 82 0 21 11 0 0 10192

Egypt- Recent FMD Outbreaks

Egypt has at least 3 serotypes of FMDV and a number of topotypes 1. 2. 3. 4. 5. 6. 7.

O/ME-SA/Egy-72 (2006-2009) O/ME-SA/PanAsia 2 (Egy-09) (2009 & 2011 A/Africa/G-VII (Ken-05) (2006 & 2009) A/Asia/Iran-05 (Bar-08) (2010-2011) A/Africa/G-IV (ISM-12) (2012) SAT2/VII/Alx-12 (2012) SAT2/VII/Ghb-12 (2012) SAT2/VII/Ghb

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4

Recent Observations- SAT 2 in Sudan 2010

Recent Observations- A in Sudan 2011

Regional Analysis- 2012

Regional Analysis- 2012 Asia 1 and SAT 2 on the move

Pool 3

Pool 5

O, A, SAT 1, 2

Pool 2

O, A

Pool 5

O, A, SAT 1, 2

Pool 1

O, A, Asia 1

Pool 7

A, O, SAT 1, 2, 3

Pool 6

SAT 1, 2, 3

Pool 1

O, A, Asia 1

Pool 7

Pool 2

O, A

O, A, Asia 1

Pool 4

Pool 3

O, A, Asia 1 O,&A,SAT Asia 1 2

O, A, Asia 1

Pool 4

A, O, SAT 1, 2, 3

Pool 6

SAT 1, 2, 3

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4

Current FMD Threat Analysis: from reported incidence Serotype O- widespread circulation – FMDV type O – ME-SA topotype – PanAsia-2 lineage – FMDV type O – SEA topotype – Mya-98 lineage Serotype A- widespread circulation – FMDV type A – ASIA topotype – FMDV type A – ASIA topotype

Current FMD Threat Analysis: Vaccine matching 2012 • Vaccine matching carried out on representative isolates from each submission • 2dm VNT carried out with a variety of Merial, Intervet (MSD) and ARRIAH

– Iran-05 lineage – other

bovine vaccinal reference sera • Results presented as traffic light system

Serotype Asia 1 – limited circulation- BUT risk of further spread – Reports from 6 countries in 2011 and now more in 2012 – Pirbright Vaccine trial carried out for EU– High potency vaccine protected animals challenged with current isolate

WRLFMD® Pirbright vaccine matching by 2dm VNT Good match

Result

Serotypes SAT – restricted circulation – Have not established outside of Africa – But recent spread of SAT 2 into North Africa and Middle East being monitored

Some matches No match

Serotype C - No reports of serotype C since 2004

Current FMD Threat Analysis: Vaccine matching 2012

Serotype O vaccine matching Country of Origin

Serotype

Topotype

Lineage/ strain

Sub Lineage

Afghanistan Bahrain Congo Egypt Ethiopia Iran

O O O O O O O O O O O O O O O

ME-SA ME-SA ME-SA ME-SA EA-3 ME-SA ME-SA ME-SA SEA EA-2 ME-SA ME-SA EA-3 SEA ME-SA

PanAsia-2 PanAsia-2 PanAsia PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 Mya 98 PanAsia-2 PanAsia-2 Mya-98 PanAsia-2

ANT-10 ANT-10 ANT-10 FAR-09 ANT-10 ANT-10 ANT-10 ANT-10

O O O O O O

EA-3 SEA ME-SA ME-SA ME-SA ME-SA

Mya-98 PanAsia PanAsia-2 PanAsia-2 PanAsia

ANT-10 ANT-10 -

Israel Japan Kenya Kuwait Libya Malaysia Kingdom Saudi Arabia Sudan Thailand Turkey UAE Vietnam

O 3039 O 4625 O Manisa O PA2

Serotype O vaccine matching Country of Origin

Serotype

Topotype

Lineage/ strain

Sub Lineage

Malaysia Thailand

O O O O

SEA SEA ME-SA ME-SA

Mya-98 Mya-98 PanAsia PanAsia

Vietnam

Current FMD Threat Analysis: Vaccine matching 2012

Serotype O vaccine matching Country of Origin

Serotype

Topotype

Lineage/ strain

Sub Lineage

O O O O O Israel O Kuwait O Libya O O Kingdom Saudi Arabia O

ME-SA ME-SA ME-SA ME-SA ME-SA ME-SA ME-SA ME-SA EA-3 ME-SA

PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2

ANT-10 ANT-10 ANT-10 FAR-09 ANT-10 ANT-10 ANT-10 ANT-10

O O O

EA-3 ME-SA ME-SA

PanAsia-2 PanAsia-2

ANT-10 ANT-10

Afghanistan Bahrain Egypt Iran

Sudan Turkey UAE

O 3039 O 4625 O Manisa O PA2

Serotype O vaccine matching O 3039 O 4625 O Manisa O PA2

Country of Origin

Serotype

Topotype

Lineage/ strain

Sub Lineage

Congo Ethiopia Kenya

O O O

ME-SA EA-3 EA-2

PanAsia -

O 3039 O 4625 O Manisa O PA2

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4 Current FMD Threat Analysis: Vaccine matching 2012

Current FMD Threat Analysis: Vaccine matching 2012

Serotype A vaccine matching Country of Origin

Serotype

Afghanistan Iran

A A A A A A

Pakistan Turkey

Topotype

Asia Asia Asia Asia Asia Asia

Lineage/ strain

Sub Lineage

Iran-05 Iran-05 Iran-05 Iran-05 Iran-05 Iran-05

HER-10 SIS-10 HER-10 AFG-07 HER-10 SIS-10

Serotype A vaccine matching

A Eri 98

A A Saudi Iran 95 A22 05

A A Tur MAY 06 97

Country of Origin

Serotype

Congo Egypt

A A A A

Sudan

Topotype

Africa Africa Asia Africa

Good match Some matches No match

Lineage/ strain

Sub Lineage

G-I G-IV Iran-05 G-IV

BAR-08 -

A Eri 98

A A Saudi Iran 95 A22 05

A A Tur MAY 06 97

Good match Some matches No match

Current FMD Threat Analysis: Vaccine matching 2012

Serotype A vaccine matching Country of Origin

Malaysia Thailand

Serotype

Topotype

A A

Asia Asia

Lineage/ strain

Sea-97 Sea-97

Sub Lineage

A Eri 98

A A Saudi Iran 95 A22 05

A A Tur MAY 97 06

Serotype Asia 1 vaccine Matching Country of Origin

Serotype Topotype

Asia 1 India

Afghanistan Asia 1 Iran Asia 1

SAT Serotypes Vaccine Matching Topotype

Kenya

SAT 1

I (NWZ)

SAT 2 SAT 2 SAT 2 SAT 2 SAT 2 SAT 2 SAT 2

SAT 1 Rho

IV VII IV VII VII VII IV Good match Some matches No match

Sindh-08 Sindh-08

Pakistan

Asia 1

Asia

Sindh-08

Asia 1

Asia

Sindh-08

Vaccine Recommendations (National & European Antigen Banks) HIGH PRIORITY

SAT 2 Zim SAT 2 Eri Bahrain Egypt Kenya Libya PAT Sudan Tanzania

Asia 1 Asia 1 Shamir Shamir >6PD50

Good match Some matches No match

Current FMD Threat Analysis: Vaccine matching 2012

Serotype

Asia Asia

Turkey

Good match Some matches No match

Country of Origin

Lineage/strain

MEDIUM PRIORITY

LOW PRIORITY

O Manisa* O PanAsia -2* * or additional O BFS or Campos strain A-Iran-05 A24 Cruzeiro A22 Iraq Asia 1 Shamir* SAT 2 Saudi Arabia (or equivalent - SAT 2 Eritrea) A Argentina 01 A Iran 96 A Iran 99 A Eritrea A Iran 87 or A Saudi Arabia 23/86 (or equivalent) A Malaysia 97 (or Thai equivalent such as A/Sak/97)* O Taiwan 97 (pig-adapted strain or Philippine equivalent)* SAT 1 South Africa SAT 2 Zimbabwe A15 Bangkok related strain A Kenya A87 Argentina related strain SAT 1 Kenya Within category: not in order of importance SAT 2 Kenya SAT 3 Zimbabwe C Noville

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4

European Neighbourhood Is that only pool 3?

European Neighbourhood?

WRLFMD Summary

Know Your Neighbours • • • • • • • •

What is it going to be? O? quite likely A? Asia 1? SAT 2? This is why we need the ref labs Improve and enhance Why we need to remain constantly on our guard

www.wrlfmd.org

• A major combined effort both National and Global is needed for control

• Accurate & Timely disease information is vital • The FMD Reference Laboratory Network provides the Engine Room for the Global Control Initiative Pirbright designated global coordinating lab for OIE/FAO GCP

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 4 Acknowledgements Jemma Wadsworth, Bob Statham, Pip Hamblin, Ginette Wilsden, Geoff Hutchings, Nigel Ferris, Claudia Doel, Miki Madi, Begona Valdazo-Gonzalez, Caroline Wright, Bryony Armson, Nicola Camping, Anna Ludi, Liz Wilson, Gareth Shimmon, Trish Ryder, & Suzie Hammond

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 5 Presentation Outline 

Aligning Waiting Periods for Vaccinate-to-live & Vaccinate-to-die

 

  

PLENARY Session II: FOCUS ON ISSUES AFFECTING FMD CONTROL IN FMD FREE REGIONS

 

 Project Leader: Project Team:

Context of the QUAD FMD Project What did the QUAD FMD Code Project conclude? What is the rationale for this conclusion?

Dorothy Geale (Canada) Paul Barnett (IAH, Pirbright, United Kingdom) Grant Clarke (New Zealand) Jennifer Davis (Australia) Thomas Kasari (United States)



Historical basis for 3 and 6 months Vaccinology/Carrier/Subclinical DIVA Post Outbreak Surveillance Animal Products

Conclusion/Recommendations Next Steps 1/5/2013

Context

In April, 2011 QUAD CVOs tasked a scientific literature review to see if support for alignment of waiting periods for vaccinate-to-live and vaccinate-to-die strategies Core Project Team  Desirable Outcomes 



Vaccinate-to-die

International FMD Strategic Reserves NETWORK project;

Vaccinate-to-live

live strategies

Collaboration with IAH, Pirbright  Timely decision making regarding FMD vaccination 



Dorothy Geale*of (Canada), Tomimpediment Kasari (USA), Grant Clarke (New  Removal economic for vaccinate-toZealand), Jennifer Davis (Australia), Paul Barnett (WRL FMD)



Global reduction of mass culling of livestock through

Work vaccination streams History ofoutbreak 3/6 mos, Vaccinology, DIVA, in a FMD Post-outbreak Surveillance & Trade in animal product 1/5/2013

1/5/2013

Conclusion

Presentation Outline   

Context of the QUAD FMD Project What did the QUAD FMD Code Project conclude? What is the rationale for this conclusion?    

 



Historical basis for 3 and 6 months Vaccinology/Carrier/Subclinical/DIVA Post Outbreak Surveillance Animal Products



Conclusions/ Recommendations Next Steps 1/5/2013



Alignment for vaccinate-to-live and vaccinate-todie is NOT feasible for all commodities But is feasible for vaccinated animal products using higher potency FMD vaccines Incremental risk of vaccinated animal products can be deemed negligible with additional risk mitigation measures to meet ALOP. Note Code Article 8.5.9.1 b) and c), deals ONLY with animal products not animals which are restricted by Article 8.5.12 3) 1/5/2013

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 5

Rationale

Presentation Outline   

   

 

HISTORICAL:  No specific scientific rationale for OIE waiting periods Years: achieve-with vacc; without vacc/recover-with vacc; without vacc

Context of the QUAD FMD Project What did the QUAD FMD Code Project conclude? What is the rationale for this conclusion?

Prior to 1992: 2 yr; 3 yr /6 mos 1992-1998: 2 yr; 12 mos/12 mos; 6 mos Vaccinate-to-live  1998-2002: 2 yr; 12 mos/12 mos; 3 mos; 3mos  2002 to present: 2 yr; 12 mos/6 mos (DIVA); 3mos; 3mos; 6mos (DIVA).

Historical basis for 3 and 6 months Vaccinology/Carrier/Subclinical/DIVA Post Outbreak Surveillance Animal Products

 

Conclusions/Recommendations Next Steps 1/5/2013



Relative risk determined by SCAD in 6 mos blocks for free with vacc and 3 mos for free without vacc

Vaccinate-to-live

1/5/2013

Rationale

VACCINOLOGY:  Higher potency (≥ 6PD50) vaccines protect earlier; single dose; last longer.  FMDV replication even inhibited in some animals experimentally proven relationship with potency  Infection chain is broken in 1/2 the time; less FMDV in environment exponentially = less challenge dose  No unequivocal experimental evidence that conventional vaccine which protects against disease also reduces susceptibility to infection, virus excretion or duration of persistence 1/5/2013

Rationale

CARRIER:  Anecdotal only; No experimental studies show cattlecattle transmission; only SAT2 African buffalo-cattle  Undefined trigger? Strain, serotype & challenge dose differences?  Modeling with high potency parameters suggests prevalence of carrier herds is very low 0.2% with one carrier per herd  Does waiting 6 vs 3 mos make a difference? Perhaps live animals but for animal products? 1/5/2013

Rationale

DIVA OR NSP ASSAYS:  PANAFTOSA tests, recognized by OIE, are the foundation to FMD eradication in South America  High potency vaccines are more purified  DIVA kits available with Se (68-94%) & Sp (9798%) but Se improved using tests in series.  DIVA validated at the herd level (appropriate for products) but lacks Se for individual animal level (already restrict live vaccinates) 1/5/2013

SURVEILLANCE:  Demonstrate absence of infection impossible in a vaccinated population (demonstrate is used in Article 8.5.9.1 c); use “substantiate” for “demonstrate” as NSP assays lack Se.  Even census surveillance (EU) does not provide absolute certainty; S Korea used <1% prevalence.  South America 5% @ 95%(follow-up per Code 8.5.49)  to change OIEuse paradigm waiting time to  Need Sentinels of limited due to from low transmission statistical certainty or concept of threshold of surveillance (long term solution) 1/5/2013 12

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 5

Rationale

Animal Products: [Commodity based trade]  Risk of FMDV from vaccinated products can be negligible with risk mitigation measures  Risk of mechanical contamination from carriers is negligible if correctly processed  Neutralizing antibodies are best guarantee of the absence of FMDV; No Code for milk from vaccinates  Embryos are not a risk provided handled as per IETS Manual (2007) 1/5/2013

1/5/2013

Presentation Outline   

  

 

Conclusions (repeat)

Context of the QUAD FMD Project What did the QUAD FMD Code Project conclude? What is the rationale for this conclusion? 



Historical basis for 3 and 6 months Vaccinology/Carrier/Subclinical/DIVA Post Outbreak Surveillance Animal Products



Conclusions/Recommendations Next Steps 1/5/2013

Alignment of a 3 month waiting period for vaccinate-tolive and vaccinate-to-die is feasible provided the incremental risk of vaccinated animal products is deemed negligible with additional risk mitigation. Article 8.5.9.1 b) and c), deals ONLY with animal products as animals are restricted by Article 8.5.12 3) Additional risk mitigation measures determined bilaterally to meet ALOP but may include bovine only (DIVA herd validated); animal identification & traceabilitiy; protection zone vaccination only; serology; no wildlife reservoir etc

1/5/2013



 



Code needs definitions for “emergency” vaccination, FMDV “circulation” versus “infection” OIE convene ad hoc group to define statistical certainty or threshold ,of surveillance to demonstrate the absence of FMDV infection and FMDV circulation. DIVA for higher potency vaccines for all species. Promote novel vaccine such as marker VP1 gene segment with duplicate DIVA capability. Encourage concurrent revision of EU 2003/85/EC.

Article 62 of this Directive permits derogation of the OIE waiting periods of 3 and 6 mos. provided, “…the clinical and serological survey provided for in Article 56

and the measures provided for in Article 57 have been completed and confirmed the absence of foot-and-mouth disease virus infection” (EU, 2003). 1/5/2013

Presentation Outline: FMD vaccinate-to- live

Recommendations 

  

Context of the QUAD FMD Project What did the QUAD FMD Code Project conclude? What is the rationale for this conclusion?    

 

Historical basis for 3 and 6 months Vaccinology/Carrier/Subclinical/DIVA Post Outbreak Surveillance Animal Products

Conclusions/Recommendations Next Steps 1/5/2013

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 5

Current Status 1.

Next Steps

Propose alignment of waiting periods for vaccinate-to-live and vaccinate-to-die to the OIE (8.5.9 1. b) & c) a)

c) d)

Concept presentation was made at July 3-5 ad hoc FMD Group under SCAD Formal letter to Dr Vallat from QUAD CVOs on August 1 with revised QUAD paper with scientific evidence to support Code change

1/5/2013

Tabled at SCAD at end of August 2012 Referred back to ad hoc FMD Working Group with written rationale To be reviewed in Code Commission meeting February 2013 1/5/2013

Next Steps

Acknowledgements 

EU Support ? 

QUAD CVOs  

A principle conclusion of the EU Tervuren workshops in 2007 was “Vaccination-to-live

 



policy with subsequent freedom from infection substantiated by a survey system including NSP testing is a realistic and achievable option in FMD control.”

   



John Clifford (United States)Brian Evans/Francine Lord ( Canada) Mark Schipp (Australia) Matthew Stone (New Zealand)

Technical Reviewers 

   

Paul Barnett (IAH, NETWORK)- Vaccinology Grant Clarke (New Zealand) - DIVA Jennifer Davis (Australia) – Animal Products Thomas Kasari (United States)- Surveillance

QUAD CVOs



1/5/2013

Soren Alexandersen Alex Donaldson Paul Kitching Victor Saraiva Keith Sumption Gavin Thomson



Questions?

QUAD EMWG Reviewers    

Jane Rooney/ Pam Hullinger/Randy Crom/Hernando Duque Tom Smylie/ Jim Clark/ Al Barton/ Randy Morley Andre van Halderen/ Katie Owen/ Brendan Pollard Jill Mortier/ Dick Rubira

1/5/2013

Open Session of the EuFMD: 2012 Jerez de la Frontera, Spain

Appendix 6

Conclusions

A Quantitative Approach to Determining Waiting Periods for FMD Freedom Angus Cameron



Quantitative calculation of appropriate waiting periods is possible 



 

   

Waiting periods Surveillance standards Calculations Implications



Purpose of waiting periods Allow disease to spread 



What is the purpose? How were they determined? Are they correct?



Intuitive principles

1. Old surveillance loses value 2. Confidence accumulates over time

Rapid spread, very short period required

Vaccinated population 

Periods of 3, 6, 12, 18, 24 months depending on situation

Time and confidence in freedom

Reach a detectable level (design prevalence) Non-vaccinated non-immune population 



OIE Code for FMD 





Sensitivity of ongoing surveillance activities Probability of introduction of infection Use of vaccination

Waiting periods



From surveillance sensitivity to probability of freedom

Appropriate waiting periods depend on 

Overview

Need to change standard used to assess surveillance

Slower spread, may require more time



How to describe these effects quantitatively?

Allow disease to be detected by surveillance 

Ongoing surveillance (e.g. passive farmer reporting) 

More time = more surveillance = more confidence

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 6

Quantifying effects of time 

Decrease of value of old surveillance



Example:  Serosurvey achieving 95% surveillance sensitivity











Same survey conducted 5 years ago Are we confident that the population is free now?

Ongoing surveillance activities





Passive farmer reporting Abattoir surveillance Active negative clinical reporting

Surveillance sensitivity  



Relatively low sensitivity



Longer waiting time 

Contributing factors



Sample size Individual test sensitivity Design prevalence Risk-based sampling



Does not capture effect of time

  



More observations, higher surveillance sensitivity

Surveillance sensitivity 

Surveillance standards: Traditional approach

Individual observations 

No loss in confidence

Completed one month ago Are we confident that the population is free now?

Accumulation in confidence



No positive animals detected

Scenario 2: 



Due to risk of introduction of disease Perfect biosecurity

Scenario 1: 



Decay in confidence with time

Pr(S+ | D+) Probability of detecting at least on positive animal given that the population is infected at the design prevalence Only standard used in OIE code Sometimes called ‘confidence’ Usually set at 95%

Surveillance standards: Alternative approach 

Probability of freedom  

 

Pr(D- | S-) Probability that the population is free from disease (at the design prevalence), given that surveillance found no positive animals Calculated using Bayes’ Theorem Intuitively easier for regulators to understand

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 6

Calculation

Probability of freedom 

Contributing factors 





𝑡𝑛+1

Sample size, design prevalence, test Se, risk-based sampling

Multiple surveillance activities 



𝑃𝑟 𝐹𝑟𝑒𝑒

Surveillance sensitivity

1−Pr 𝐼𝑛𝑡𝑟𝑜 ×Pr(𝐹𝑟𝑒𝑒)𝑡𝑛 1−𝑆𝑆𝑒+(Pr 𝐹𝑟𝑒𝑒 𝑡𝑛 ×𝑆𝑆𝑒)

𝑛

e.g. serosurveillance + passive reporting + abattoir

𝑆𝑆𝑒 = 1 −

Accumulation of historical evidence over time Risk of introduction of disease over time

1 − 𝐶𝑆𝑒𝑘 𝑘=1

𝐶𝑆𝑒 = 1 − 1 − 𝑃∗ × 𝑆𝑒

𝑛

Tools for calculation 1 0.9

Probability

0.8

SSSe

0.7 0.6 0.5



Free on-line tool to do calculations

P(free)



P(intro)



0.4



0.3



0.2 0.1



0 0

Time Period

Multiple surveillance components Multiple time periods Herd-level data Herd- and animal-level risk based sampling

http://epitools.ausvet.com.au  

Access currently limited while under development Will be made public on completion of project

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 6

Implications 

Time to target probability of freedom 

Shorter with 

Higher sensitivity surveillance    

 

Calculating appropriate waiting periods 1. 2.

3. 4. 5.

Set target probability of freedom Identify surveillance components contributing evidence of freedom Estimate sensitivity of each component Estimate probability of introduction Calculate time to achieve target

Larger sample size Good risk-based sampling Higher sensitivity test system Higher design prevalence

More surveillance components Lower probability of introduction

Freedom with vaccination 

Effects of vaccination  



Lower within-herd design prevalence Lower sensitivity of clinical surveillance

Result  

Lower surveillance sensitivity Longer waiting period required

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 6

Conclusions 

Quantitative calculation of appropriate waiting periods is possible 

  

Need to change standard used to assess surveillance 



Acknowledgements Tony Martin Evan Sergeant Tripartite workshop participants 

 

From surveillance sensitivity to probability of freedom



Appropriate waiting periods depend on   

Sensitivity of ongoing surveillance activities Probability of introduction of infection Use of vaccination

Turkey

  

Pencho KAMENOV Tsviatko ALEXANDROV

Greece  



Adil ADIGÜZEL Abdulnaci BULUT

Bulgaria

Achilles SACHPATZIDIS Maria TOPKARIDO

EuFMD

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 7

Evaluation of FMD emergency vaccination in Switzerland

Federal Department of Economic Affairs FDEA Federal veterinary office FVO

Evaluation of the benefit and feasibility of a vaccination-to-live strategy in FMD free countries

Basic questions to be answered to adjust contingency planning: a) Target species for emergency vaccination (cattle – small ruminants – pigs) b) Dimension of vaccination zone (3 km = V3 or 10 km = V10) c) Time frame for emergency vaccination (immediately after detection of first case or later) d) Feasibility of a „vaccination-to-live“ strategy (Council Directive 2003/85/EC)

D.C. Hadorn1, S. Dürr2, B. Thür3, L. Perler1, T. Jemmi1 1

Swiss Federal Veterinary Office, 2 Veterinary Public Health Institute, 3 Institute of Virology and Immunoprophylaxis

…in order to achieve a benefit compared to conventional disease control only

EuFMD Open Session 2012

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

Materials and Methods •

Results

Simulation of FMD spread (serotype O „middle east“) within and between herds (local area spread, direct and indirect contacts) with Davis Animal Disease Simulation model1 General input parameters

Value1

Initial diagnosis delay (diagnosis delay for index case)

10 days

Second diagnosis delay (diagnosis delay for secondary cases)

4 days

Inputs for emergency vaccination

Value1

Application delay for 3 km vaccination zone

3 days

Application delay for 10 km vaccination zone

6 days

Protection delay for vaccination

14 days

Vaccine efficacy on herd level

0.9

a) Target species for emergency vaccination Cattle Small ruminants Pigs b) Dimension of vaccination zone and c) Time frame for emergency vaccination •

No benefit for emergency vaccination under Swiss situation* if vaccination campaign is started right after detection of first case (neither 3 km nor 10 km vaccination radius) * Low animal density (<167 ruminants and pigs / km2)

•

If the epidemic becomes extensive, vaccination with radius of 10 km around IP may be beneficial in terms of reducing the number of herds infected and the epidemic duration

1Dürr

et al. Evaluation of the benefit of emergency vaccination in a foot-and-mouth disease free country with low livestock density. Submitted to PVM. Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

Restriction Measures in terms of animal movement zone

d) Feasibility of vaccination-to-live strategy

Minimum duration

At least least15 15days daysplus plustime timefor forkilling killing&& No animal movement between holdings within, in At and out of the protection zone disposal & preliminary cleansing and disinfection At least least30 30days days Surveillance No animal movement out of surveillance zone; At Zone animal movement between holdings permitted after clinical inspection Vaccination zone At least 6 months in total

Protection Zone

Goal of vaccination-to-live strategy:

Phase 1 Phase 2 Phase 3

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

No animal movement between holdings within, in and out of vaccination zone No animal movement between holdings within, in and out of vaccination zone No animal movement out of vaccination zone; animal movement between holdings is subject to authorization; unvaccinated animals with restrictions (testing)

plus 30 30days days Time for vaccination plus Time for clinical and serological survey plus classification of herds 6 months minus time for phase 1 and phase 2

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 7

Discussion

d) Feasibility of vaccination-to-live strategy

Under Swiss conditions and in terms of the actual legal basis, the feasibility of a vaccination-to-live strategy is not given.

• Animal movement restriction within vaccination zone (phase 1 and 2) is as severe as in protection zone but more than twice as long

Points to be discussed: • Adaptation of restriction measures if vaccination zone is established outside protection/surveillance zone (different risk)

Significant increase in welfare culling, mainly in pig production sector, expected

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

•

Necessary duration of „stand-still“ within vaccination zone (only during vaccine application phase?)

•

Adaptation of restriction measures in holdings with and without vaccinated animals (target species of vaccination program?)

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

Thank you

Evaluation of benefit and feasibility of vaccination-to-live strategy | EuFMD Open Session Oct 2012 D. Hadorn

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 9

Triggered by FMD incursion to the EU SURVEILLANCE FOR FMD IN WILD BOAR IN 2011-2012

Jan-April 2011 Serotype “O”

RESULTS FROM BULGARIA AND TURKEY Sergei Khomenko Tsviatko Alexandrov Naci Bulut Sinan Aktas Keith Sumption

Scope of the surveys

Follow-up activities: • Wildlife surveillance in Thrace (TR+BG) – govt authorities / EU-FMD/FAO-EMPRES

ANATOLIA (TR) Dec 2011 – Feb 2012 N=252

• Anatolian wild boar survey for FMD (TR) – EU-FMD/FAO-EMPRES • Wild boar ecology telemetry project (BG) EU-FMD/FAO-EMPRES • Development of non-invasive wildlife surveillance methodology (BG) - EUFMD/FAO-EMPRES • Wild boar population mapping (N Eurasia) – FAO-EMPRES

Sero-positivity to FMDV:

THRACE (BG+TR) Jan 2011 – Jan 2012 N=1004

NSP + in Wild Boar in Thrace

Thrace (epidemic O) versus Anatolia (endemic O, A, Asia)

AGE GROUP

THRACE

ANATOLIA

NSP+ (95 % CI), %

ADULT

628

9.1 (6.9 – 11.6)

185

JUVENILE

358

5.6 (3.4 – 8.5)

ALL

1004

7.8 (6.2-9.6)

252

NSP+ (95 % CI), %

24.9

(18.3 - 32.4)

7.5 (2.5 - 16.6) 20.2

(15.5 - 25.7)

P <0.05 ns <0.05

NO DIFFERENCE BETWEEN SEXES FOUND, ONLY BETWEEN AGES

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 9 Spatio-temporal variation in seroprevalence in Thrace Prevalence in time (quarter yearly 2011) To outbreaks TOTAL, n 0 – 5 km 168 6 – 10 km 179 11 – 20 km 223 21 – 30 km 113 31 – 50 km 186 > 50 km 134

Prevalence by zones around outbreaks

Sero-prevalence in 5 provinces of concern

20,2

17,4

20 15 10

5,9 lower 95 % upper 95 % Prevalence

5 0

% Ab+ (95 % CI), % 10.1 (6.0 - 15.7) 17.9 (12.6 - 24.3) 8.5 (5.2 – 13.0) 5.4 (2.6 - 9.7) -

Anatolia: NSP+ in livestock v WB • Distinctly different from LR (P=0.1), but not SR (P=0.001); • Except for Samsun prevalence in WB does not differ from SR (P=0.6-0.8); • Prevalence in WB correlates best with that in SR (r=0.9, R² = 0,8), but not LR (ns).

Most closely related FMD Asia-1 viruses were found to co-circulate in this region in cattle

Conclusions on FMD epidemic in Thrace • Spread was spatially and temporary limited; • Transmission between livestock and wildlife was both ways (facilitated by humans?); • Disease event in wildlife developed in winter and died away end of spring • However, serology fails to identify sequence of the different stages of this particular disease event … EFSA, 2012; Alexandrov et al (in prep.), Dhollander et al (in prep.)

Regional variation in sero-prevalence: UNITS n ERZURUM 17 SAMSUN 73 GÜMÜŞHANE 58 KASTAMONU 76 RİZE 21 TOTAL 252

60,000

infected with FMD all over Turkey !

% NSP+ (95 % CI) 52,9 (27,8 - 77,0) 28,8 (18,8 - 40,6) 17,2 (8,6 - 29,4) 13,2 (6,5 - 22,9) 4,8 (0,1 - 23,8) 20,2 (15,5 - 25,7)

% ASIA + 11,8 12,1*

3,6

%O+ 41,2 28,8 5,2 13,2 4,8 16,7

ERZURUM is distinctly different from all other provinces (P<0.05) SAMSUN has higher prevalence than KASTAMONU (P=0.05) All other regional differences are NS * One virus positive animal in GÜMÜŞHANE

Serotypes in livestock and wild boar mismatch !

FMD outbreaks in livestock (5 provinces) 350 300 250 200

Isolate from wild boar

Closely related isolates from cattle

NO SEROTYPE “A” FOUND !

150 100 50 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

% outbreaks by serotype

TURKEY

Asia-1

100 90 80 70 60 50 40 30 20 10 0

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 9

Does age matter? 3 adult (3-5 yrs) wild boar might have been exposed to two serotypes during their life

Dec 2010

ASIA + (%) 5 (13) 3 (43) 1 (20) 9 (18)

O + (%) Total + 39 7 5 51

34 (87) 4 (57) 4 (80) 42 (82)

??? Hypothesis outline: ??? • FMD easily spills over to WB from SR (Kurban in 2010-2011?) and develops into epidemics in Nov – March; • Some serotypes (e.g. O, Asia) are better transmitted by WB, than the others (e.g. A) ?.. • Livestock (summer) and WB (winter) epidemics are in a seasonal antiphase (“old serotypes” detected); • Hunters can play important role in disease transmission from WB to livestock; • Mostly adult animals are involved (rut?); • Many piglets born thereafter have maternal Ab protection (low prevalence in juveniles); • This + low population density in early spring + higher temperatures bring R0 below 1.

Feb 2012 O

BG: TR:

Serotype O

Serotypes A, Asia, O

160

KURBAN

140

KURBAN

120

100 80 Livestock

Wild Boar

AGE GROUP ADULT (>21 month) SUB-ADULT (<21 and > 12 months) JUVENILE (<12 months) ALL

ANATOLIA: virus Asia-1 +

THRACE: virus O +

20 0 1

9 10 11 12 1

2010

Rut

Farrowing

9 10 11 12 1

NEEDS:

Rut

Farrowing

Experimental infection

Ropes (validated on farms and in wild boar for ASF (Chichikin et al, 2012). CSF vaccine baits with swabs; Swabs incorporated into food baits (e.g. maize cobs).

9 10 11 12

Rut

Farrowing

Rut

• Clinical signs on the 4 DPI (domestic 2 DPI) – e.g. incubation 4 days; • Most severe and evident lesions – 7 DPI; • Viraemia: 1 DPI through at least 9 DPI; • NSP antibodies detected 7-8 DPI; • RNA in saliva normally found up to 14 DPI and up to DPI 24 DPI intermittently.

CREDITS: A. Breithaupt, K. Depner, B. Haas, M. Beer (FLI – Federal Research Institute for Animal Health Institute of Diagnostic Virology)

EASY TO INCORPORATE ANY SWABS INTO

Chichikin et al, 2012

SOLUTIONS:

• Aims at detecting • virus by PCR; • Targets epi-unit (all animals attending a feeding site); • • As frequent as needed; • • Cost effective; • Logistically simple.

2012

EXPERIMENT WITH A TAME WILD BOAR

Non-invasive surveillance

2011

SUITS FOR COMMERCIAL FEEDERS

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 9 A male’s home range - 23.09 – 10.10.2012

Trapped …

Telemetry project Strandzha (10)

STRANDZHA

• Movements and habitat use of wild boar; • 20 GPS/GSM Tellus collars (1 year – 24 fixes a day); • Adult females (group leaders) targeted; • All adults and piglets eartagged.

Tutrakan (10)

BULGARIA

~ 4 km … and collared

>500 positions by now

http://www.followit.se/wildliferesearch.html

FMD in wild boar:

Historical range of Sus scrofa

Progress and challenges with telemetry project 5

• 90 animals trapped, but most juveniles + trap failures, poor GSM coverage, heat, lack of Zoletil … • 4 collars put into operation (two consistently give signal: a male in Strandzha and female in Tutrakan); • Field labor input underestimated – need more time and personnel; • Local collaborators were found and involved; • Potential cooperation with ASFORCE (+ 20 transmitters) is considered

1902 1908 1911 1917 1919 1925

2. Kazakhstan

1927 1931 1941 2011 ?

3. Kyrgyzstan

1953

4. Israel

19871999 2007 2011 ?

5. Europe

1920s? 2011

1 4

1. Caucasus

Marek & Hutÿra, 1931; Sludskiy, 1956; Danilkin, 2002

1*1 km resolution density data product

504 spatial objects 48 countries (NUTs 1) Population and/or harvest data (2005-2010) 3,600,000 – post harvest 2-2,500,000 harvested

Khomenko, Robinson, Gilbert (in prep.)

THANKS TO ALL

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 10

MINIMUM STANDARDS FOR LABORATORIES WORKING WITH FOOT-AND-MOUTH DISEASE VIRUS in vitro AND in vivo What have we learned from the FVO Inspections? Bernd Haas, FLI Riems, Germany

MINIMUM STANDARDS FOR LABORATORIES WORKING WITH FOOT-AND-MOUTH DISEASE VIRUS in vitro AND in vivo Conclusions: The „Minimum Standards“ proved to be a valuable tool for the management of laboratory bio-risks and the FVO inspections. The 2009 revision improved the „Minimum Standards“ significantly. However, it contains some ambiguities and duplications. Several member states maintain national laboratories able to diagnose FMD in case of an outbreak which are important for the whole European Community. However, they lack the funding for a facility fully complying with the „Minimum Standards“. A standard for diagnosic labs which don´t amplify virus nor use live FMDV as reagents is needed.

MINIMUM STANDARDS FOR LABORATORIES WORKING WITH FOOT-AND-MOUTH DISEASE VIRUS in vitro AND in vivo

Loeffler‘s animal facility: ‚High containment‘ anno 1900

History

Recommendations: Update Council Directive 2003/85/EC in respect to “auxiliary labs” Update “Minimum Standards“in respect to “auxiliary labs” and to remove some duplications and ambiguities Create an expert committee on lab bio-risk management to support the Commission as well as national competent authorities Allocate sufficient funding for diagnostic and research laboratries ‚...Germany could be considered free of FMD if there weren‘t Prof. Loeffler‘s experiments...‘

History

FLI 1990

1950th – present: „High Security buildings“

History Diagnostic Tests

• Double HEPA filters (exhaust)

UK, Dk, Ge, Nl, It, CH…

risk

• Constant negative pressure

high

• Automatic flaps (inlet and exhaust)

Containment Standards MINIMUM STANDARDS 1985/93

Large animal inoculation Guinea pigs

• Thermal decontamination of waste water (within containment)

Cell culture

• Chemical decontamination of equipment • Compulsory Shower-Out • Animal Carcass Rendering • Emergency Power Supply

MINIMUM STANDARDS 2009

ELISA low

PCR 1910

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

2001

2010

timeline

Appendix 10

History

Specific Requirements

Management COMMISSION WORKING DOCUMENT for adoption at the 38th General Session of the European Commission for the Control of FMD (EuFMD) - April 2009

Training Laboratory Biosecurity Personnel (limit access, shower, quarantine…) Facility Design Handling of FMD virus Airhandling systems (negative pressure, HEPA…)

MINIMUM STANDARDS FOR LABORATORIES WORKING WITH FOOT-AND-MOUTH DISEASE VIRUS in vitro AND in vivo

Waste and effluent treatment Removal of equipment, Removal of biological material Decommissioning

What was new in 2009? Terminology used and the biorisk management principles incorporated adapted from the Draft CEN/CWA “Laboratory Biorisk Management Standard” Bio-risk policy Responsibilities and independence of BRO Continual improvement process Risk/threat assessment Accident and incident reporting system Emergency procedures …. Almost no changes in respect to “hardware” and buildings

What was new in 2009?

Biosafety Laboratory biosafety describes the containment principles, technologies and practices that are implemented to prevent the unintentional exposure to biological agents and toxins, or their accidental release. Biosecurity Laboratory biosecurity describes the protection, control and accountability for valuable biological materials within laboratories, in order to prevent their loss, theft, misuse, diversion of, unauthorised access, or intentional release.

What was new in 2009?

Biorisk combination of the likelihood of the occurrence of an adverse event involving exposure to biological agents and toxins and the consequence (in terms of accidental infection, toxicity or allergy or unauthorised access, loss, theft, misuse, diversion or release of biological agents or VBMs) of such an exposure. Biorisk officer (BRO) or biorisk advisor (Biosafety / Biosecurity Officer) a staff member of an institution who has expertise in the biohazards encountered in the organisation and is competent to advise top management and staff on biorisk management issues

What was new in 2009? Primary containment layer: contains the live FMDV at source within closed containers or a class I, II or III microbiological safety cabinet, or in the case of infected animals, contains the live FMDV by physical containment in specially constructed rooms with treatment of all waste and the HEPA filtration of air Secondary containment layer: contains FMDV of infected materials and staff working with such materials within a closed and highly controlled physical environment and subject solids, fluids and air to a treatment by validated procedures that will remove or inactivate FMDV Tertiary containment layer: prevents contact between the live FMDV and susceptible livestock outside containment by appropriate measures, such as restrictions placed on access of staff to such livestock.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 10

Authorization of laboratories in respect to FMD

What was new in 2009? Risk-Analysis The main sources of FMDV are: high

infected pigs, cattle, sheep, goats and other susceptible large animals laboratory based physical and chemical processing of large quantities of virus infected laboratory animals, e.g. baby mice and guinea pigs infected tissue cultures diagnostic specimens

low

1. infection of experimental and/or large animals with FMDV;

„Real FMDV lab“

2. activities which produce high amounts of infectious FMDV,e.g. large scale virus production at a capacity that involves more than 10 litres of cell culture 3. activities involving the handling, and in particular, the propagation of infectious FMDV, but are limited to 10 litres of cell culture, and during which the FMDV is enclosed in containers which can be effectively autoclaved or disinfected; 4. test diagnostic samples for antibody to FMDV, by methods that do not involve live FMDV manipulation;

„Auxiliary FMD lab“

5. test diagnostic samples for FMDV genome by methods that do not involve live FMDV manipulation (e.g. RT-PCR); usually: „normal lab“ 6. apply on the genome of FMDV methods of molecular biology that do not involve live FMDV manipulation

What was new in 2009?

FVO-INSPECTIONS OF FMD FACILITIES Annex: Standard for Auxiliary Diagnostic Labs for Emergencies MINIMUM STANDARDS OF BIORISK MANAGEMENT FOR LABORATORIES UNDERTAKING DIAGNOSTIC INVESTIGATIONS OF LOW-RISK SAMPLES DURING AN OUTBREAK OF FMD relates to the use of laboratory tests which do not contain or require live FMD virus -for the testing of blood samples from holdings without clinical signs

2009-2012

In accordance with Article 66 of Directive 2003/85 EC, the FVO has inspected all 15 laboratories and 3 vaccine plants handling infectious FMDV and listed in Annex XI of the Directive. Results on specific laboratories are stricly confidential!

- any samples from any holding that have been treated in a way that ensures the inactivation of FMDV infectivity

FVO Inspections

FVO Inspections - Legal Aspects

The inspections were carried out by the Food and Veterinary Office (FVO) on the legal basis of Council Directive 2003/85/EC during 2010 - 2012. The inspection teams were comprised of one FVO inspector and two experts from European FMD laboratories with considerable experience in FMD work and bio-risk management. The “Minimum Standards” were used as the technical basis for the inspection of laboratories and vaccine plants handling infectious FMDV. After completion of the first round of inspections, it´s time for a critical review of the FMD risk management practises in Europe and a revision of the Minimum Standards.

Article 65 of Council Directive 2003/85/EC requires the Member States (MS) to ensure that: (a) laboratories and establishments, in which live foot-and-mouth disease virus, its genome, antigens or vaccines produced from such antigens are handled for research, diagnosis or manufacture, are strictly controlled by the competent authorities; (b) the handling of live foot-and-mouth disease virus for research and diagnosis is carried out only in approved laboratories listed in Part A of Annex XI; (c) the handling of live foot-and-mouth disease, virus for the manufacturing of either inactivated antigens for the production of vaccines or vaccines and research, is carried out only in the approved establishment and laboratories listed in Part B of the Annex XI.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 10

FVO Inspections - Findings

FVO Inspections - Legal Aspects

(d) the laboratories and establishments referred to in points (b) and (c) are operated at least according to the biosecurity standards set out in Annex XII.

= „Minimum Standards“

Transposition of law Article 65 of Council Directive 2003/85/EC has not been fully transposed into the national legislation. Designation of the competent authority (CA) Often unclear, ministry or local or regional authority without specific knowledge on bio-risk management Approval of the laboratory to handle live FMD virus Often no specific approval for FMD work

FVO Inspections - Findings

Organization of official controls Often no structured approach In most facilities controls were not carried out or did not sufficiently cover FMD bio-risk management

Qualification of the CA inspectors Entirely inadequate to inspect complex bio- risk management systems, in particular the technical installations of the air ventilation systems and the effluent treatment plants

If there was good bio-risk management, it was due to an internal structure of the lab!

Inspectors usually come from local or regional authority without specific knowledge on bio-risk management

Enforcement powers Usually in place Notification procedures in case of emergencies Often on an ad hoc basis

FVO Inspections - Findings

In several facilities:

Labs sometimes more or less OK

but

Conditions of HEPA filters unknown, no measurements, no knowledge

Effluent treatment plants in poor conditions, not in containment Situation sometimes made worse by a shunt between lab sinks and municipal sewage system „in case the plant doesn´t work“(!), with a control lever found unlooked and at the outer wall

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 10

FVO Inspections - Findings

In several facilities:

Scientists with (some) bio-risk responsibilities

Nice records on lab floor vs Reality downstairs

vs Staff running effluent and air-handling plants and autoclaves Sometimes made worse by outsourcing!

A senior staff member (BRO) should have felt responsible for the bio-risk aspects of the whole FMD facility - actually gone downstairs into the cellar and into the attic, - checked the plants, filter, tubes… - checked the autoclavs, - discussed with the craftsmen

FVO Inspections - Findings

In several facilities:

Nobody had ever asked - What can go wrong?

Flimsy „Secondary Containment“

- What could result from such a failure? - How long would it take until you realise something has gone wrong? Bad: FVO Inspektors find live animals in your effluent treatment tank Worse: FMD outbreak reported on TV

- What can be done do to control the risk?

Primary containment

e.g. plant in secondary containment, several valves in series, with pressure testing

Problem: Unacceptable for large animal infections and vaccine production Relies heavily on procedures and on discipline of staff

FVO Inspections - Findings

Suggested improvements

Murphy´s Law and the importance of a sturdy Secondary Containment

Primary containment

Competent authorities (CA) Delegate/support technical part of inspections by CAs to an expert group at European level

Primary containment

Problem: Such a type of secondary containment is very expensive; few member states can afford it! Do they all need one for lab diagnosis?

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 10

Suggested improvements

Suggested improvements EuFMD / DG SANCO Modify “Minimum Standards” according to risk:

Laboratories/Facilities Send BRO to major FMD labs for training! Allocate sufficient funds for contaiment!

“Real” FMD labs doing research and diagnostic work on foreign samples which contain or may contain live FMDV “in peace times” vs “Auxiliary” labs investigating only suspect samples from own country without using live FMDV as a reagent

Suggested improvements EuFMD / DG SANCO Consult

EuFMD Committee on Research FMD laboratory Bio-Risk Officers (BRO) Head Engineers of major FMD Laboratories

Consider: “EU/EuFMD Committee on Lab Bio-risk Management”

Thank you for your attention!

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

ECONOMIC EVALUATION OF FMD MANAGEMENT OPTIONS IMPLICATIONS FOR SCIENCE AND POLICY Ron Bergevoet and Marcel van Asseldonk Ron.Bergevoet@wur.nl

”The equation”

Cav = (1-p )* C no outbreak + p * C outbreak Cav = average annual costs of FMD p = probability of an outbreak C no outbreak = C annual surveillance C outbreak = C direct + C control + C trade

Socio-economic effects of FMD and its control

are determined by:

1. the probability of occurrence of an outbreak in one or more MS’s,

2. and the economic effects of a. the outbreak (the size and duration of the outbreak) and

b. the control measures taken by Competent Authorities and

3. the reaction of stakeholders/public and trade partners.

The probability of occurrence of an outbreak in one or more MS’s FMD

Source: WTO, international trade statistics (2012) Current prices

The world

Worldmapper.org Source worldmapper.org

Meat exports

Worldmapper.org Source worldmapper.org

Dairy exports

Worldmapper.org Source worldmapper.org

Export to countries outside the EU Export of Cheese from the Netherlands

Export value pig meat (2006) Million€

total

1767

3333

2458

intra EU

1543

2115

2200

extra EU

224

1218

257

fraction extra EU

13%

37%

10%

De Winter et al, LEI 2010

The economic effects of the outbreak and the control measures taken by Competent Authorities

Control of FMD in the EU Prophylactic vaccination in EU has been banned in the EU since 1992 (Directive 90/423/EEC)

EU minimal measures: ● culling of infected herds, ● pre-emptive slaughter of contact herds, ● establishment of control and surveillance zones

Additional measures: ● Ring culling and/or ● Emergency vaccination ● Delayed culling ● Vaccination to live

2001 FMD outbreak in NL EU minimal measures: ● culling of infected herds, ● pre-emptive slaughter of contact herds, ● establishment of control and surveillance zones

Additional measures: ● Ring culling and/or ● Emergency vaccination ● Delayed culling ● Vaccination to live

2001 FMD outbreak in NL

26 outbreaks were detected.

All susceptible animals on approximately 1800 farms were vaccinated. All farms subsequently were depopulated.

In total, approximately

260,000 animals were killed.

(Bouma, et. al.,Prev Vet Med. 2003, 20; 57 (3) :155-66.)

Economic effects of the outbreak

Direct costs:

● Compensation for depopulated animals ● Depopulation (taxation, culling, transport & destruction, cleansing & disinfection) Tracing Screening Vaccination Additional surveillance in movement restriction zone

● ● ● ● Indirect costs Business interruption ● Losses related to established movement restriction zones ● Repopulation of the farm. ● Losses from emergency vaccination

Economic effects of an outbreak

Direct costs:

Costs born by government (or PPP) & 60% by EU

● Compensation for depopulated animals ● Depopulation (taxation, culling, transport & destruction, cleansing & disinfection) Tracing Screening Vaccination (Additional surveillance in movement restriction zone)

● ● ● ● Costs born by directly Indirect costs affected farmers ● Business interruption ● Losses related to established movement restriction zones ● Repopulation of the farm. ● Losses from emergency vaccination

Consequential losses

Export market losses Ripple effects.

● upstream and downstream along the livestock value chain

Spill-over effects.

● During outbreaks e.g. tourism and other services

Export market losses The costs of animals and products, that because of an outbreak cannot be exported.

● During the outbreak and after completion of screening until EU lifts export bans

● After this period, this concerned the third countries

market for live animals, meat, meat products, milk and milk products from infected countries/compartments for another 3 months without vaccination and for another 6 months with vaccination-to-live. (OIE terrestrial code article 8.5.8)

● (Are markets after this period still available as before the outbreak?)

Costs of the 2001 FMD outbreak in NL

Total for Dutch society:

€900 million or 0.3% GNP

● Direct costs

€

90 million

e.g. enforcement costs, compensation of culled animals, screening etc. (had to be borne by the government)

● Farmers (Indirect and export market losses): ● Other parts of the livestock chain: ● Tourism and recreation sector:

€ 320 million € 215 million € 275 million

Source (CPB 2001 cited by Huirne et al., 2002)

Payments by the EU Emergency Fund (19972009)

Total payments by Emergency fund in this period: ● 1,109 million €

The reaction of stakeholders/public and trade partners

ECONOMICS OF THE ERADICATION OF FOOT-AND-MOUTH DISEASE EPIDEMICS WITH A VACCINATION TO LIVE STRATEGY

What has changed in the NL? ● No more images of large scale culling of animals

● Society is closely monitoring what is happening

● No welfare slaughter with

destruction but welfare slaughter with animals and products made available for consumption

● Vaccination to live strategy

Approach

Policy makers

Economic evaluation

Epidemiological modelling

Methodology (1) Definition of investigated policy options / Control strategies: The following strategies were evaluated:

1. EU basic strategy: EU minimal measures 2. EU basic strategy + Culling in 1 km around infected farms

3. EU basic strategy + Vaccination with radius of 2 or 5 km around infected farms

(culling 1st week)

Farm densities 2006

farms/km2 4 3 2 1 0

cattle 37 000 farms 3.7 mln animals

sheep 18 000 farms 1.5 mln animals

pigs 9 000 farms 11 mln animals

SPLA

farms/km2

DPLA

4 3 2 1 0

cattle

# infectious farms (50%)

Methodology (2) Epidemiological modelling

DPLA

1 km culling 2 km vaccination 5 km vaccination EU basic

control strategy

time (days) infection pressure over time detection time

within-herd module

infection time vaccination time (preemptive) culling time

for each species: - transmission rate - infectious period - clinical symptoms - vaccination effect - detection limit literature on cattle, sheep, pigs: - transmission experiments - vaccination experiments data FMD outbreak 2001

between-herd module

locations farm types

I&R database (cattle, sheep) GD database (pigs) data hobby farm surveys

course of hypothetical epidemic

transmission kernel

data FMD outbreak 2001 literature on cattle, sheep, pigs: - infectiousness - susceptibility

Backer et al, 2008, EU FMD conference

Methodology (3) Economic assessment

When vaccination-to-live strategy is applied

Products of vaccinated animals produced during

the outbreak: no difference with other animals in control and surveillance zones

Products of vaccinated animals still present after end of the outbreak until declared officially free:

the

● Logistic processing and sub-optimal value ● Market acceptance: products restricted to Dutch market

Estimated Average value loss due to lower revenues and logistic processing of vaccinated animals (in € per vaccinated animal). Category

Value loss

Dairy cows

450 €/ animal

Young stock

5 €/ animal

Veal calves

550 €/ animal

Other cattle

26 €/ animal

Sows

260 €/ animal

Fattening pigs

50 €/ animal

Sheep

34 €/ animal

FMD SPLA

(< 2 farms/km2)

NUMBER OF CULLED FARMS

area Friesland:

LAST WEEK OF DETECTION

TOTAL COSTS INCL COSTS OF OPERATION (in M€)

50%

95%

50%

95%

50%

95%

102

cul1

295

109

vac2

117

108

vac5

113

122

FMD PDLA

(>4 farms/km2):

Gelderse vallei

NUMBER OF CULLED FARMS

LAST WEEK OF DETECTION

TOTAL COSTS INCL COSTS OF OPERATION (in M€)

50%

95%

50%

95%

50%

95%

cul1

971

206

3217

236

615

vac2

260

707

227

526

vac5

230

571

228

106

504

Distribution of costs (median DPLA)

Culling 1 km

Vaccination 2 km

Distribution of costs

Culling 1 km

Vaccination 2 km

Implications for policy and research

1. Reduce the probability of occurrence of an outbreak in one or more MS’s,

1. preventive measures 2. public Private Partnerships

Share responsibility and costs between public and private sector (the PPP)

All farmers pay a levy to the compensation scheme. Sharing responsibility between government and stakeholders has to be established before decisions on cost sharing can be defined.

● Provides incentives for farmers to stimulate behavioural changes. ● Should impose biosecurity standards/quality assurance. ● Determining an appropriate base for cost sharing is a highly complex matter (no “one size fits all” solution).

● Should adequately consider national and regional differences ● Should be based on a EU set of basic requirements (and preferably recognized by the EU).

● Example is Dutch Animal Health Fund

Animal health fund Covenant of the Ministry of LNV with the Commodity Boards Cattle, Pigs, Poultry, Sheep and Goats

Covenant for financing outbreaks of animal disease ● Covers payments of the costs of outbreaks of contagious animal diseases designated by the Dutch government.

● The expenses for legal control of contagious animal diseases.

● Maximal contribution of different livestock sectors in 5 year period

Implications for policy and research (2) Research indicates that vaccination-to-live is alternative for large scale culling

Support with epi- and eco-models to continuous update during an outbreak

Harmonisation of regulation vaccination-to-live with culling or vaccination as delayed culling

Challenge is to put experiences from the past into perspective of the 21st century

Conclusion

Economic evaluation of different FMD management options:

● should to be based on universal principles, ● need to be tailored to local circumstances in discussion with stakeholders,

● is likely to result in different solutions for different

countries e.g. due to difference in livestock population density, trade patterns or acceptance of product originating from vaccinated animals, and

● should be supported by epidemiological and economic models.

Acknowledgements

Jantien Backer, Thomas Hagenaars, Herman van Roermund, Aldo Deckers, Gonnie Nodelijk WUR-CVI

Coen van Wagenberg, Nico Bondt, WUR- LEI The financial support of the Dutch Ministry of Economic Affairs,

Agriculture and Innovation for enabling much of the underlying research is highly appreciated.

FAO for the invitation

Appendix 12

Simple decision tools informed by model predictions when considering FMD emergency vaccination strategies Preben Willeberg DVM, PhD, Dr.med.vet., Dr.med.vet.h.c. Senior Veterinary Global Health Specialist Center for Animal Disease Modelling and Surveillance School of Veterinary Medicine University of California, Davis Adjunct Professor

Adjunct Professor

Contents 1. An update on EuFMD activities in FMD modeling and decision support 2. Simple decision tools to help optimize the control strategy 2 weeks into a Danish FMD epidemic

Background EuFMD Standing Technical committee discussed: • What is the role of EuFMD to assist countries in using FMD models? (Identify specific follow-up actions for EuFMD to assist) • Main proposal was a series of workshops at 3 levels: 1. CVO 2. Vet services-Contingency planners 3. Vet Services – modelers (maybe outside partner or contractor) • CVO workshop held in June 2012 to determine level of interest and commitment

”Knowing is not enough – we must apply. Willing is not enough – we must do”

Goethe

We have learned a lot from modelling of FMD – now it is time to put that knowledge into practical use We need not only accept and be willing to use models – more countries should be able to use modelling activities to support their contingency planning and emergency preparedness

Background Recommendations at EuFMD 39th General Session, Rome, 2011:

• Member states should consider the use of modeling tools as decision making aids, while ensuring that the output of such models are clearly understood by decision makers with respect to uncertainty and sensitivity. • Member states using such models should engage in comparisons with other states to constructively examine the issues affecting confidence in their use, and that support be given to assist countries to review the suitability of tools for their needs

Proposals for workshops 1. Introduction to disease spread models Objective: To familiarize participants with the principles, function, use and limitations of disease spread models. • An overview of commonly used models • The majority of the workshop will be hands-on with a representative model. • By the end of the workshop, participants should be able to initiate use of a model in their home country (collate required data, parameterization etc) Participants: Involved in contingency planning/disease control in the Veterinary services, but with little or no prior experience with disease spread models.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 12 First workshop, Vienna 15 – 19 Oct. 2012 • Two participants from each of the EuFMD member countries: Austria, Croatia, Czech Republic, Hungary, Malta, Serbia, Slovenia, and Slovakia.

• Most were state veterinarians (including two Chief Veterinary Officers) working in contingency planning • Some were epidemiologists and some were local veterinary inspectors • Apparently successful achievements for the group

Proposals for workshops 2. Integrating modeling and decision support tools into contingency planning: Objective: To familiarize participants with the tools available other than disease spread models, including multi-criteria decision models and economic models. To discuss best practices in modeling including communication of results, cross-border collaborations etc. Participants: for countries who already have some modeling capacity (could be from completing the 1st workshop)

• The regional approach was working well

Comparative Immunology, Microbiology & Infectious Diseases 25 (2002) 345–364 • The choice of whether or not to apply emergency vaccination is probably the most difficult decision facing the authorities when disease breaks out in an erstwhile FMD free country. • Effective computational models should be actively financed for a range of outbreak scenarios to assist objective decision-making and minimise bureaucratic delays in vaccine application.

• Contingency planning should include provision for emergency vaccination and must address the complex decisions of not only when, where, and how to apply vaccine but also its economic consequences. • Computer modelling may be a useful aid to cost benefit and decision support systems in this context. Planning must be detailed and regularly reviewed. • Although the option of emergency vaccination is included in the EU contingency plans, the qualifying conditions for vaccination have not been finally determined.

Council Directive 2003/85/EC

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 12 Original publication:

Argentina 2001 FMD epidemic

Vaccination strategies varied throughout the epidemic Number of detected herds by 17 provinces

ABSTRACT: First-fortnight incidence (FFI) is a modelling parameter that can be used to predict both the prevalence and duration of a foot-and-mouth disease (FMD) epidemic at regional and national levels. With an indication of how long an epidemic might last by the end of week two, it becomes possible to estimate whether vaccination would be economically viable from the start of an epidemic,

DTU-DADS Model results from Denmark: The median, 5th and 95th percentiles of epidemic outcome parameters in the three alternative control strategies for the 4,458 out of 5,000 simulated epidemics that lasted > 14 days following detection of the first infected herd.

Control scenarios Epidemic outcome parameters Cumulative number of infected herds Epidemic duration (days) Epidemic size (km) Epidemic costs (× €106)

Tomorrow in P6:

Use of FFI with DTU-DADS model simulations of FMD in Denmark

Basic scenario

Pre-emptive depopulation in 500 m zones

Suppressive emergency vaccination in 1,000 m zones

57 (11-273)

48 (8-174)

47 (9-182)

56 (19-151)

44 (15-105)

44 (15-97)

386 (80-698)

363 (63-646)

363 (67-646)

547 (411-947)

511 (395-770)

513 (395-751)

Use of FFI with DTU-DADS model simulations of FMD in Denmark Costs

Herds detected - day 14

1,6E+10

700 1,4E+10 600 y = 2,1298x - 0,913 R² = 0,346

400

Total - day 14

300

y = 3E+07x + 4E+09 R² = 0,2649

1,2E+10

Danish kroner

No. of herds

500

1E+10 Costs

8E+09 6E+09

Lineal (Costs)

Lineal (Total - day 14)

200

4E+09

100

2E+09

0 0

100

150

Day 14 detected herds

200

250

0 0

100

150

200

250

Day 14 detected herds

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 12 Decision tool step 1

Decision tool step 2

At day 14 during an outbreak the actual number of detected herds is known, e.g. 15 herds

•

Among the simulated epidemics under the basic control scenario we select the ones with 15 detected herds at day 14, i.e. 139 epidemics out of 4,458 simulations

•

The distribution of the number of detected herds at the end minus the number at day 14:

Basic scenario Histogram Detected Number of column # herds epidemics < 10

10 - 19

20 - 29

30 - 39

40 - 49

50 - 59

60 - 69

70 - 79 80 - 89

90 - 99

110 - 119

160 - 169

210 - 219

8 9

27 270 - 279 Grand total

Basic scenario 60

Number of epidemics

40 30 20 10

• Repeat step 1 with the vaccination scenario for the same 139 epidemics • Compare with effect of vaccination at day 14:

Histogram column #

Detected herds

Number of epidemics

< 10

10 - 19

20 - 29

30 - 39

40 - 49

50 - 59

60 - 69

70 - 79

1 1 139

130 - 139

Grand total

50 40 30 20 10 0

4 5 Column #

Column #

Subsequent period

Scenario A =<20 herds >20 herds Total

<100 herds

=>100 herds

Total

1879

110

1989

1697

772

2469

3576

882

4458

Specificity: 0.53

Decision scenario D Predictive values (p.v.): Neg. p.v.: 0.94 Pos. p.v.: 0.31

Sensitivity: 0.88

CONCLUSIONS (1) •

139

Decision scenario A

Day 14

Suppressive vaccination scenario

Number of epidemics

•

Each country should use modelling continuously adapted to suit their national situation with regard to: o Input parameters such as: • Population sizes and densities • Location of farms • Movement patterns • Available resources o Output relevance and priorities: • Economic losses • Number of animals killed • Number of herds infected o Strategic priorities: • Importing or exporting • Duration of basic measures phase • Vaccination ”to live” or ”to kill” and/or zonal culling

Subsequent period <100 =>100 Total herds herds

Scenario D

Day 14

Predictive values (p.v.):

=<50 herds

3377

526

3903

Neg. p.v.: 0.87

>50 herds

199

356

555

Pos. p.v.: 0.64

Total

3576

882

4458

Specificity: Sensitivity: 0.40 0.94

CONCLUSIONS (2) • Models are just one tool for providing scientific advice, and their results should be evaluated in conjunction with experience from experimental studies, field studies and scientific wisdom. • International collaborations such as those supported by the World Organisation for Animal Health (OIE) and the European Commission for the Control of Foot-and-Mouth Disease (EUFMD) can help address validation issues and improve the utility of models for emergency disease management.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 12

“Blind belief in authority is the greatest enemy of truth” Albert Einstein Transparency and documentation to substantiate implementation of FMD control measures are prerequisites in achieving political and public acceptance Few of us have any practical experience with FMD control Modelling may provide part of the answer to those needs

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 14

Enhanced FMD control through the integration of socio-economic approaches: the why and a toolbox Cryptic words: Impact and value chains To develop a strategy you need: Understand the problem, support and commitment, and options

C. Bartels, N. de Haan, J. Hinrichs, M. McLaws and J. Rushton FAO and RVC

Strategy development

Three specific constituencies and four issues with FMD

• How big is the problem? Epidemiology and economics – but does size matter?

National

Risk

Impact

• Who is going to fund it? Government? Which part? How much?

• Whose problem? Support and commitment

Incentives

Governance Farm level

Value chain (or sector)

The role of impact • Justification for decision making on public funding and awareness raising

Impact on the different constituencies

– Is the most requested activity for us – Different levels – including international and (more recently) livelihoods – Headlines of severe impact – what do we want to say?

• To inform the strategy

– Direct and indirect impacts • the cost of interventions

– Who is at risk? – Support and commitment

Work in progress!

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 14 FMD virus

National

Risk of virus Household real income levels

– wage earnings

Containment – slaughter and compensation –movement controls

– meat expenditures

Tourism

Animal welfare

Environmental concerns

Macro-economy – other sectors (inputs, transport) multiplier effects

National level

–foreign exchange – growth –Consumer meat prices

Livestock trade – production losses –profit losses (idle capacity, timing of sales

Risk management – preventive control (surveillance, fencing, zonation, movement controls) –maintain DVS capacity

Market access To export markets

Farm level

To local markets

• Some tools in the toolbox – SAM – CGE – Alive

Livestock production

– wage earnings – production losses (mortality, weight, milk losses, lameness) –treatment and containment costs –- other profit losses (idle capacity, timing of sales, price effects)

Farm level risk managements

Farm household real income levels

– own measures incl. vaccination –compulsory control measures (movement control) –traceability

–earnings –outputs –household welfare

Livelihoods Other products providing and income – manure –draft power –fuel –transport

– loss of asset –social capital –Increased vulnerability

Adapted from Perry and Randolph (2003)

SAM Example: Swine FMD in Missouri

National level tools • Social Accounting Matrix (SAM) Analysis

– Static national economic impact assessment tool to capture detailed interdependencies between institutions and sectors/regions. – Account for “multiplier” effects to attain better understanding of longer-term, more inclusive stakeholder interests

• Computable General Equilibrium (CGE) Modeling

– Dynamic modeling of market interactions – Extend SAM framework to simulate market activity – Highlight the role of prices and scarcity in determining the incidence of economic impacts

Source: David Roland-Holst at workshop ‘Harmonize the approach to determine socio-economic impacts of FMD Bangkok, 5-6 September 2012’

Alive toolkit

Data for Zambia

Question: GDP of livestock is underestimated A series of data and expert opinion A database on: • A more comprehensive calculation of GDP • Better characterization of the systems • Competitiveness of the sector • Degree of dependence on different species in nutrition and income • The role of diseases

Commodity Meat

Unit

LSIPT

Other references

Beef

TEC

56 000

60 000 42 000 52 000

FAOSTAT, 2010 SOFA, 2010 (referring FAO 2007) FAOSTAT, 2007

Goat Pork Poultry

TEC TEC TEC

3 100 32 000

8 500 16 500

42 000

42 500

FAOSTAT, 2010 FAOSTAT, 2010 FAOSTAT, 2010

Total meat

TEC

133 000

127 500

FAOSTAT, 2010

Cow milk

Liter

306 000

215 000 88 500

METAP, 2008 (commercialized milk) FAOSTAT, 2010

Hides

Tons

Other product from cattle

Energy (traction) Organic matter Cattle Poultry Pigs Total OM

*000 days Tons Tons Tons Tons

900

170 7 000

Leather industry assoc, 2010 FAOSTAT, 2010

28 000 37 000 70 000 190 107 000

ALive (2011)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

ALive (2011)

Appendix 14

On animal diseases

Farm level From national to farm level • The majority of impact studies have been done at national level – to influence public spending • Important but does not give any nuances or any understanding of the people being impacted, the people at risk or the incentives • Backyard – are out of the “system” – Depner: without them you will not succeed • Some tools in toolbox at farm level: – Livelihoods analysis – Direct costs – Household economy approach

Final scoring is indicative and reflects the relative impact of disease according to various socio economic structures of the Livestock Sector ALive (2011)

Livelihoods analysis: Contribution of livestock to

Farm level: case study Cambodia

income and protein requirement of households

120

Livestock as source of income % species Income level 1st tercile (poorest) 45% P 2nd tercile 30% P/C 3rd tercile (richest) 35% C Main activity Livestock dominant 70% P/C Crop dominant 20% P/P Off farm dominant 16% V Diversified activties 30% V C=cattle, G=goat, S=pig, P=poultry

Main source of protein LG MR species species C/S C/G/S C

G P/G/S C/P

C C C C

C G/S S/C S/C ALive (2011)

Small business, salaried work and other income

100

Agricultural labour

Rental income

Transfer and remittances

Other income from land and CRPs Livestock income

Crop income 0

all

Sample all infected households higher tertile medium tertile lower tertile

mean

median

7.4 4.4 7.3 11.7

std dev

3.9 3.3 5.8 5.8

9.6 4.3 13.5 13.5

A serious shock? Health burdens of malaria:

medium

highest

Sources of household income by income tertile

Farm level: case study Cambodia Disease burden: FMD costs as percentage of household income

lowest

Shankar et al. (2011)

Farm level • • • •

Under-represented? Smallholders: Diversification a means of survival Attribution at farm level On average – the nuances are missing on most of these studies so also limited data and information on exactly who gets impacted and who is at risk • But can inform decision makers about their own constituencies

• Need more research on less data intensive approaches – but this needs to be better informed by the whole debate and question being posed

Sri Lanka: 9.9 % (Attanayake et al. 2000) Malawi: 7.2 % (Ettling et al. 1994) Nigeria: 11.04 % (Onwujekwe et al. 2000) Shankar et al. (2011)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 14

Research on value chains shows • • • •

Value chains as one of the constituencies

Value chains are not value free: money needs to be made People in the value chains are not benign Value chains produce a product Value chains provide livelihoods

• Research within value chains can help identify: – – – – – – –

People involved with the chains (amounts and types) Livelihoods made from value chains (degree and importance) Risks within the chain The money circulating within the chains Power relationships within the chains Geographical reach Pathogens and toxins circulating

Value chains and FMD strategy

Value chain as a tool

• FMD is very dynamic and happens within a context – a value chain • What people do within the chain influences whether FMD spreads or not, and whether it is controlled or not • It shows where there are failures in the systems and thereby control points • If FMD hits, they might reshape themselves • Research on value chains allows those developing strategies to identify : – – – –

• Mapping the value chain and data collection – Initial mapping • Expert consultation

– Filling the value chain • • • • •

who these people are how they interact why they behave how to ensure support and commitment

Dairy farm

Male calves

Beef cattle

Female calves

Milking cows

Butchery Supermarke t Abattoir

Restaurant

Fattened animal

Carcass

Culled cow Illegally imported animal

Imported frozen meat

Meat

Consumer

Restaurant keeper

Shop keeper Abattoir

Farmer

Local village

Dairy farm

Illegal slaugther house

Shop keeper

Trading village

Butcher

Trading village

Trader

Trading village

Cold store Butcher

Neighbourin g country

Commercial Beef

Grilled meat

IVO office

Kebab

Producer

16,000

Collector

+15 % (32%)

Traditional Dairy

Emer. Dairy

20,000

Traditional Pork ZKM/ Unit or %

ZMK/lt. or %

ZMK/kg or %

Dealer

Carvansary

Value addition along the chain Traditional Beef

Dealer

Carvansary

Dealer

Neighbourin g country

Beef fattening farm

Local village

PRODUCT

Neighbourin g country

Beef fattening farm

Farmer

PLACE AND PERSONS INVOLVED

Value chain in Iran

Focus groups Semi-structured interviews Secondary data – sector development Primary surveys Tagging vehicles

2,000

2,200

180,000

+25 % (100%)

+10% (3%)

+55 % (50%)

25 % (17%)

+127% (63%)

+43% (50%)

80% (83%)

+26% (34%)

Market Butcher Abattoir/ Dairy Supermarket/ Retailer/Rest. Consumer

+35% (68%)

25,000

45,000

2,500

6,300

400,000

ALive (2011)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 14 The tip of the iceberg: incentives and governance • Value chains help us understand one of the main incentives – the economics since it is a product passing through • It helps in understanding who would have incentives and disincentive to participate in a control program – it starts to give an idea on how to develop public private partnerships – and who pays for what • Governance – who sets the rules, who decides what gets produced, who has the power…….legislation and beyond • Still need to unpack within systems and how to make them win win – and if not win win, stop from doing damage

Research requests • Any good strategy needs to have GOOD data on the socioeconomic impact of the disease, through doable data light approaches • And good cooperation with the people involved! • This means more research is needed :

– on understanding impacts, value chains, incentives, governance and risk (perceptions and actual) – links between the understanding and the legislation – to develop simpler approaches and models (a new generation of approaches) – to advise on the right data and models – a closer link with the epidemiologists – to develop a toolbox – started with a value chain risk assessment

Impact potential and level Human health

Global economy

National economy

Agriculture sector

Environmental

Cattle sector

Economic losses

Community

THANK YOU

Herd or household

Poverty/livelihoods

Impact metrics

Household real income levels

National and Sectoral

Global externalities (7)

Cattle sector impacts (2)

-wage earnings -meat expenditures

Containment

-slaughter & compensation - movement controls

Animal welfare

Value chain impacts (5)

National externalities (6)

National economy impacts (4)

Livestock / ag. sector impacts (3)

FMD

Overt disease

Farm / household impacts (1)

Tourism

Disease risk Macro-economy

- Other sectors (inputs, trannsport), multiplier effects - foreign exchange - growth - consumer meat prices

Livestock trade

- production losses - profit losses (idled capacity, timing of sales)

Environmental concerns Market Access To export markets To local markets

Livestock production

Farmlevel

- production losses (mortality, weight, milk loss, lameness) - Treatment, containment costs - other profit losses (idled capacity, timing of sales, price effects)

Farm household real income levels Household welfare

Other income activities

- crop production (manure, draught) - fuel, transport

Risk management

- own control measures (vaccination) - compulsory control measures (movement controls) - traceability

Natural resources

Livelihoods

-loss of insurance, financial, social networking functions -increased vulnerability

Source: David Roland-Holst at workshop ‘Harmonize the approach to determine socio-economic impacts of FMD Bangkok, 5-6 September 2012’

Risk management - preventive control (surveillance, fencing, zonation, movement controls) - maintain DVS capacity

- land use - settlement & migration - ecosystem sustainability

Source: Perry and Randolph (2003)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 14

Framework Situation analysis and preliminary risk appraisal

Detailed risk & value chain analysis to identify risk reduction options

• Value chain mapping (3P’s) • Stakeholders consultation and validation • Governance, incentives • Can FMD enter, survive, leave from each

• Understanding FMD • Risk hotspots Risk pathways • Identify CCPs • Identify risk reduction measures

Option appraisal

• Assess impact on stakeholder • FMD control strategy

FMD impact + FMD epidemiology

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 15 Animal diseases: a major problem for animal productions and human health:

The role of the OIE in FMD prevention and control How to translate science in standards and guidelines, how to develop tools and ensure their convergence

Food security Rural development Small holders livelihoods Trade: domestic, regional, international - Human health and well being

OIE has always supported FMD prevention and control as one of its major strategic objectives

Joseph Domenech EuFMD Open Session 2012 Appliance of Science in the Progressive Control of FMD 29-31 October 2012, Jerez-de-la-Frontera, Spain

OIE has always supported FMD prevention and control as one of its major strategic objectives

Disease Emergence

Global Hot Spots of Infectious Diseases

Repeated Crises

Continuing to consolidate major objectives of the 4th Strategic Plan

The OIE, an intergovernmental organisation 1924

1945

Creation of the Office International des Epizooties (OIE)

2003

Creation of the United Nations

World Organisation for Animal Health

Global cargo ship network

Transparency of world animal disease situation (including zoonoses)

Collect and publish veterinary scientific information, notably animal disease prevention and control methods

Sanitary safety of international trade in animals and their products under the mandate given by the WTO

In 2012

Headquarters in Paris (France)

5 Regional

Representations

6 Sub-Regional

Representations

Reinforcing priority missions of the Fifth Strategic Plan 2011-2015 Food security: Is a key public health concern Healthy animals guarantee food security and food safety

Food safety:

• Need for a global supply of safe food • The Veterinary Services play a key role in protecting consumers

Veterinary education Relation animal / environment and “One Health” A worldwide strategy for managing risks at the animal-human interface ecosystems

Developing international standards on animal welfare

OIE activities are a global public good

Scientific progress is incorporated into practice to achieve progressive control of diseases and food safety

Through the publication of standards, guidelines and recommendations Which will be translated in tools, methods, strategies and policies, laws & regulations

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 15 OIE standard setting process Based on responsive, transparent and rapid procedures. Well recognized and independent experts invited to participate to small groups which reports to the Specialist Commissions. Specialist Commissions play a central role in the OIE standard setting procedures Major source of OIE experts: OIE Reference Centres, comprising Reference Laboratories and Collaborating Centres (277 in 2012)

OIE standard setting process - Specialized Commissions: Scientific Commission for Animal Diseases, Code Com., Biological Standards Com., Aquatic animals Com. - Ad Hoc Groups: FMD Vaccine Quality, FMS Status, PPR, PRRS, Brucellosis, CSF, RVF, Epidemiology, Trade in animal products, Antimicrobial resistance…

- Working Groups: Wildlife Food safety…

Proposed Standards sent to all OIE Delegates Comments from all OIE Delegates Consultation of major partners Second round of discussions with Commissions… General Session May Adoption: vote of all Delegates during the World Assembly

Also: - Aquatic Animal Health Code - Manual of Diagnostic Tests for Aquatic Animals

The OIE’s scientific network OIE Reference Centers 2012

Reference Laboratories

236 laboratories, 112 diseases /topics in 37 countries

Collaborating Centres 41 Collaborating Centres 38 topics in 22 countries

Disease information

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 15 Permanent institutional cooperation Global Early Warning System (GLEWS) DISEASE TRACKING DATA ANALYSIS DISEASE INTELLIGENCE

FAO - Food and Agriculture Organization WHO - World Health Organization

WTO - World Trade Organization IPPC - International Plant Protection Convention

Fundamental activities

World Bank

for targeted surveillance, prediction,

CABI - CAB International

awareness and reporting to OIE And then for the appropriate response: choice of tools, methods and strategies

ILRI - International Livestock Research Institute

And cooperation with Regional public organisations and private sector bodies (more than 50 agreements)

Publications

www.oie.int

International Cooperation FAO - OIE GF TADS Global Framework for the Progressive Control of Transboundary Animal Diseases

OIE Scientific and Technical Review

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Appendix 15 International Conferences OIE Global Conference on Wildlife, Paris (France) 23-25 February 2011

Disease Information Summaries,“Disease cards

Disease Control strategies Underlying principles - It is better to prevent than to respond to crisis - To control at source where the pathogens are - To be science based - Disease prevention and control programmes against TADs are public goods - Applicable to bioterrorism

- Multisectoral approaches: Animal and human health, Agrosystems, Wildlife… - Private Public Parternership - Other factors than sciences to be adressed: Consumer perception Economical and political pressure

FMD Conf. :see below

- Based on surveillance and rapid response: Transparent health information Early warning and detection Immediate response - Risk based approaches, Risk analysis: Understanding the epidemiology of diseases Integrative and participatory approaches Multidisciplinary (socioeco., ecol…)

Disease Control strategies Underlying principles - Good governance of animal health systems: Quality of Veterinary Services to implement and certify Appropriate legislation - Use of OIE standards by importing and exporting countries

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 15 Capacity building

Some examples

Capacity building programme for Delegates and focal points Meetings organised by the OIE and its regional and subregional offices

Veterinary education “One Health”

OIE Worldwide Conference of Deans of veterinary Education establishments

Recognition of veterinary qualifications. Promotion of professional excellence throughout the world

- Minimum curriculum - Evaluation - Twinnings

Foot and Mouth Disease -

Code articles 1.6.2, 1.6.7 and 8.5. AHGs: FMD Statuts, Vaccine quality Transversal WGs and AHGs: Wildlife, Epid. Scientific Commission for Animal Diseases 9 FMD Reference Laboratories (including 4 OIE and FAO) *3 more FAO FMD Ref. Centers

- Publication on Deboned Beef trade: Qualitative Risk Assessment of spread by international trade in deboned beef (D Paton & All.)

OIE Stand. FMD official statuts, Endorsement of Nat. Cont. Programmes The Progressive Control Pathway for FMD (PCP-FMD)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 15 -

Laboratory twinnings Vet Faculty twinnings Support to Vet. Serv. Reg Programs: PAMA…

Field Projets

SEACFMD Networks - LabNet - EpiNet

Convergence of tools - Laboratories and networks - Epidemio Centers and networks - GLEWS, CMC-AH - Regional Animal Health Centers

- PVS-PCP Transparent animal Health information

FMD Reference Laboratories/Centers OIE: -

Buenos Aires, ARGENTINA Gaborone, BOTSWANA PANAFTOSA, Rio de Janeiro, BRASIL Vladimir, RUSSIA Onderstepoort, SOUTH AFRICA

OIE and FAO:

Plum Island, USA Pirbright , UK Pakchong, THAILAND Lanzhou, PEOPLE'S REP. Of CHINA

FAO:

- ICAR, Mukteswar, INDIA - Brescia, ITALY - CODA-CERVA, Brussels, BELGIUM

Linking the PCP-FMD stages to the OIE PVS levels of Critical Competencies (CCs) was one the very representative examples of why and how to improve the tool convergence.

Main OIE updates - Guidance issued on the management of Reference Centre networks (May 2012) - Guidance issued on the use of OIE emblem and titles (June 2012) - First “audit” expert mission conducted on a reference laboratory (August 2012) - Annual reports format under review (web-based online submission)

OIE/FAO synergies - Letters to designated FAO reference centres to request application as OIE Ref Centre - FAO and OIE discussions on how to recognise networks of reference centres - Task force members to meet and call for a meeting with WHO

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Appendix 15

Linking the OIE PVS and FMD PCP Tools

Component 2 of the Global Strategy The OIE Performance of the Veterinary Services (PVS) Pathway will be the major tool of Component 2 “Strengthening Veterinary Services” to structure and plan the activities and assess progress. To endorse National FMD control plans or to apply for FMD-free status recognition, countries progressing along the PCP Pathway will have to develop in parallel their VS to be able to fulfill the criteria.

Relationship between FMD PCP Stages and OIE PVS Critical competency Levels

The basic principle is that a country embarking on the PCP-FMD should acquire the appropriate capacity and capability of the VS to conduct activities aimed at the control or elimination of FMD (and other TADs). This is referred to as the ‘enabling environment’ in the PCP. A correspondence table has been worked out between the PCP Stages and the compliance level required for each of the PVS Critical competencies (CCs) relevant to FMD control.

33 OIE PVS Critical Competencies (CCs) among 46 are of particular relevance to the prevention and control of FMD: Animal Health (4 / 5) Veterinary Public Health (1 / 4)  Laboratory (2 / 2)  Trade (4 / 8)  General Management (17 / 31)  Resources (5 / 6)  

All countries reaching PCP Stage 3 must at least have reached compliancy level 3 (i.e. general agreement with OIE standards) for the 33 FMD-relevant CCs 42

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 15 Linking the PCP-FMD stages to the OIE PVS levels of Critical Competencies

Table I. Selection of FMD related PVS CCs

Table of Correspondance between OIE PVS CCs levels and FMD-PCP stages 

FMD PCP Stage 1: PVS level 3 required for 7 CCs



FMD PCP Stage 2: PVS level 3 required for 17 CCs



FMD PCP Stage 3 (request for official OIE endorsed FMD national control programme): PVS Level 3 for all 33 CCs



FMD PCP Stage 4 (transition towards OIEfree status with or without vaccination): PVS level 3 for all 33 CCs 44

Thank you for your attention

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16 The cellular innate immune response of cattle and swine to infection with foot-and-mouth disease virus (FMDV)

Jared Patch, Pervaiz Dar, Ryan Waters, Mary Kenney, Raisa Glabman, Felix Toka and Bill Golde

Innate Responses to viral infection • Production of anti-viral cytokines by infected cells, especially interferon • Interferon production from uninfected cells of the immune system Dendritic cells • Activation of other inflammatory cells following stimulation via pathogen associated molecular pattern receptors (PAMPs) For example, Toll-like receptors (TLRs)

Plum Island Animal Disease Center Agricultural Research Service, USDA

Innate Responses to FMDV infection/ DCs in swine

• Activation of antiviral cellular killing Natural Killer (NK) cells and gamma/delta T cells

Innate Responses to FMDV infection/ DCs in swine • Skin dendritic cells (DCs) in the epidermis (Langerhan cells) constitutively express interferon alpha and rapidly express interferon beta following FMDV exposure in vitro.

Innate Responses to FMDV infection/ DCs in swine

• Skin dendritic cells (DCs) in the epidermis (Langerhan cells) constitutively express interferon alpha and rapidly express interferon beta following FMDV exposure in vitro.

• Following FMDV infection in vivo, Langerhan cells lose the ability to secrete intereron alpha, ex vivo

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16 Innate Responses to FMDV infection/ DCs in swine

Innate Responses to FMDV infection/ DCs in swine

• Skin dendritic cells (DCs) in the epidermis (Langerhan cells) constitutively express interferon alpha and rapidly express interferon beta following FMDV exposure in vitro.

• Following FMDV infection in vivo, Langerhan cells lose the ability to secrete intereron alpha, ex vivo

• Monocyte derived DC from peripheral blood express moderate level of interferon alpha and beta following exposure to TLR 3 stimuli

• Monocyte derived DC isolated from peripheral blood of pigs infected with FMDV lose the ability to secrete intereron alpha

• Monocyte derived DC isolated from peripheral blood of pigs infected with FMDV lose the ability to secrete intereron alpha • Plasmacytoid DCs from the blood produce intereron alpha in response to TLR 9 mediated stimuli

Innate Responses to FMDV infection/ DCs in swine • Plasmacytoid DCs opsonized with anti-FMDV antibodies produce high concentrations of interferon alpha in response to FMDV

• Following FMDV infection in vivo, Langerhan cells lose the ability to secrete intereron alpha, ex vivo • Monocyte derived DC from peripheral blood express moderate level of interferon alpha and beta following exposure to TLR 3 stimuli • Monocyte derived DC isolated from peripheral blood of pigs infected with FMDV lose the ability to secrete intereron alpha • Plasmacytoid DCs from the blood produce intereron alpha in response to TLR 9 mediated stimuli • Following FMDV infection, Plasmacytoid DCs of swine lose the ability to secrete intereron alpha in response to TLR 9 mediated stimuli

Innate Responses to FMDV infection/ DCs in swine Natural killer (NK) cell induction in swine • Plasmacytoid DCs opsonized with anti-FMDV antibodies produce high concentrations of interferon alpha in response to FMDV • “Innate” recall response in convalescent animals?

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16 Stimulatory effect of cytokines for porcine NK cells

100

Stimulatory effect of cytokines for porcine NK cells

CD2+/CD8+/CD3- cells vs K562-GFP cells

100

non-st*

NK cell cytotoxicity against FMDV-infected SK6 cells

CD2+/CD8+/CD3- cells vs K562-GFP cells

non-st* IL-12/15

IL-12/18

IL-15/18 IL-12/15/18

NK cell cytotoxicity against FMDV-infected SK6 cells SK6 Cells 60

IL-2 IL-12 IL-15 IL-18 IFNa

Non-stim NK/non-infect. SK6 cells Non-stim NK/Infect. SK6 cells

20 Non-stim NK/non-infect. SK6 cells Non-stim NK/Infect. SK6 cells

0 40

25:1

12:1

6:1

E:T ratio

20 60

IL-12/15 IL-12/18 IL-15/18 IL-12/15/18

0 25:1

12:1

6:1

25:1

E: T ratio

12:1 E: T ratio

6:1

0 25:1

12:1

6:1

E:T ratio

NK cell cytotoxicity against FMDV-infected SK6 cells SK6 Cells 60

LL-FMDV infected SK6 Cells

IL-2 IL-12 IL-15 IL-18 IFNa

What happens if NK cells are derived from pigs infected with FMDV?

Non-stim NK/non-infect. SK6 cells Non-stim NK/Infect. SK6 cells

0 25:1

12:1

6:1

0 25:1

E:T ratio

12:1

6:1

E:T ratio

20 60

IL-12/15 IL-12/18 IL-15/18 IL-12/15/18

0 25:1

12:1 E: T ratio

6:1

IL-12/18 IL-12/15 IL-15/18 IL-12/15/18

0 25:1

12:1 E:T ratio

6:1

25:1

12:1

6:1

E:T ratio

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16 NK cell cytotoxicity in FMDV infection GFP-K562 cells as target

GFP-K562 cells as target

25 20 15

25 p912 p913 p914 p915 p924 p925

In swine, infection blocks NK activity

5 0

p5612 p5812 p622 p6412

5 0

Day 0 Day1 Day2 Day3 Day4 Day5 Day6 Day7

Time post infection (01 Campos)

What about cattle?? Day 0 Day1 Day2 Day3 Day4 Day5 Day6 Day7

Time post infection (01 Campos)

FMDV infected SK-6 cells 25 p5612 p5812 p622 p6412

20 15 10 5 0 Day 0

Day1

Day3

Day5

Time post infection (01 Campos)

Natural killer (NK) cell induction in cattle

Target cell for NK assays in human, mouse and swine is commonly the human T cell leukemia line K-562

What about cattle NK???

Natural killer (NK) cell induction in cattle

Target cell for NK assays in human, mouse and swine is the human T cell leukemia line K-562

Bovine B cell tumor BL3.1 provides appropriate target cell for bovine NK assays

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16 Natural killer (NK) cell induction in cattle

Natural killer (NK) cell induction in cattle

Test Ad5 expressed bovine cytokines for induction of IFN gamma or perforin

Test Ad5 expressed bovine cytokines for induction of NK activity

IFN-γ

Perforin

20 15 10 5 0

Natural killer (NK) cell induction in cattle following vaccination with killed virus vaccine

NK activity following FMD virus infection

B38

B43 Bov51

Bov52

B50 10

Bov54

B52

B58

-2 -5

Bov53 20

Days post vaccination

Days following infection

Intracellular IFNγ in γδ Tcells

What about the γδ T cells ?

Experiment 1

Experiment 2

Figure 4

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16 CD335 (NKp46) expression on γδ Tcells

Intracellular perforin in γδ Tcells

NK-like cytotoxicity of γδ Tcells

10 b8040 b8041 b8042

†

b8040 b8041

b8042

Experiment 1

b8040 b8041 b8042

6 4

Experiment 1

2 0

0 Day0

Day1

Day2

Day3

Day4

Day0

Day5

Day1

Day2

Day3

Day4

Day5

Days post infection

0 Day0

Day1

Day2

Day3

Day4

Day5

10 10 Bo8117 Bo8118 Bo8119

Bo963 Bo964 Bo965

Experiment 2

††

bo963 bo964 bo965

Experiment 2

0 Day0

Day1

Day2

Day3

Day4

Day0

Day5

Day1

Day2

Day3

Day4

Day5

Days post infection

0 Day0

Day 1

Day2

Day3

Day4

Day5

Days post infection

NK Killing without γδ T cells ?

NK Killing without γδ T cells ? 80

80 70

PBMC

n=5

CD3-

0 3

n=5

CD3-

0 0

Days following infection

Days following infection Removing CD3+ cells reveals increased killing, i.e. most killing in CD3- population

Killing Assays with BL3.1 70

Whole PBMC

Cellular innate immune response to FMDV infection

60 50 40 30 20 10 0 0

Days following infection

CD3- NK killing

WC-1+ NK “like” killing 70

60 50

BL3 61

BL3 65

50 40 30 20

0 1

Days following infection

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 16

Cellular innate immune response to FMDV infection

• Swine NK respond to FMDV infected cells

• Swine NK respond to kill FMDV infected cells

• Swine NK are inhibited during acute infection

• Swine NK are inhibited during acute infection • Cattle γδ T cells are activated to “NK-like” killing following infection • Cattle NK are activated during acute infection with FMDV • Killing is concentrated in NK cells, biphasic response • γδ T cells also contribute to “NK-like” killing, early (day 3)

Cellular innate immune response to FMDV infection • Swine NK respond to FMDV infected cells • Swine NK are inhibited during acute infection • Cattle γδ T cells are activated to “NK-like” killing following infection

Jared Patch

Pervaiz Dar, Raisa Glabman, Ryan Waters

• Cattle NK are activated during acute infection with FMDV • Killing is concentrated in NK cells • γδ T cells also contribute to “NK-like” killing

Does this explain why pigs amplify an outbreak???

Elida Bautista

Charles Nfon

Mary Kenney Felix Toka

Questions??

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 17

ADAPTATIVE IMMUNE RESPONSES IN THE RESPIRATORY TRACT OF FMD-VACCINATED CATTLE AFTER ORONASAL INFECTION J. Pega, S. Di Giacomo, D. Bucafusco, J. Schammas, D. Malacari, G. Stafforini, A. Capozzo, LL. Rodríguez, MV. Borca, M. Pérez-Filgueira *

Here, we demonstrated that local antibody responses can also be found in vaccinated cattle and that further infection stimulated a completely different immune response pattern than in naïve-infected animals

* Instituto de Virología, CICVyA, INTA Buenos Aires, Argentina

Progression of viremia and systemic adaptative antibody responses in naïve FMDV-infected cattle (B)

(A) Neutralized TCID 50%

# FMDV RNA copies/ml

systemic response FMDV RNA in serum

10000

1000

100

20,000

Lymphoid organs involved in local production of FMDV antibodies in naïve-infected cattle 1000

ELISA titer

* **

IgM IgG1 IgG2

30,000

1000

In a previous experiment we showed that naïve cattle, following aerogenous administration of infective FMDV O1 Campos, develop a rapid and vigorous genuine local antibody response throughout the respiratory tract

100

ML MRL LRL PhT TBL S

10 10,000

1 0.1

1 10 7 0

Days post-infection

 Viral RNA detection peaks at 3 dpi y disappears at 5 dpi; neutralizing Ab responses start at 4 dpi  IgM titers at 5 dpi constitute the main fraction of the Ab response against FMDV )

Days post-infection

 Onset of the local adaptative responses was detected at 4 dpi (n=3), no anti-FMDV ASC responses were observed at 3 dpi (n=3) or earlier (n=2)  At 4 and 5 dpi, TBL were the most stimulated organs, over the LN from the upper respiratory system

 Only at 6 dpi LN from the upper tract were able to exceed TBL ASC counts

Immunoglobulin isotype profiles induced in local adaptative immunity in naïve FMDV-infected cattle 5 days post-infection

4 days post-infection 1000 100

IgA IgM IgG1 IgG2

6 days post-infection

1000

100

Neutralizing activity of FMDV-specific serum IgM antibodies isolated from naïve-infected cattle 10000

whole sera purified IgM IgM-depleted normal sera

1000 100

10 1

ML MRL LRL PhT TBL

10 ML MRL LRL PhT TBL

 IgM was the dominant isotype among the FMDV-ASC developed at 4 dpi, followed by IgG1 ASC (3- to 4-fold below). Overall number of ASC increased at 5 dpi as well as the difference between IgM and IgG1 secreting cells (~ 10 to 15 fold)

 Isotype pattern at 6 dpi was still dominated by IgM ASC for all animals and organs assayed, although a relative rise in the number of IgG1 and IgA FMDV-specific ASC was also observed. ML and MRL were the most activated LN, followed by TBL

Days post-infection  Serum IgM fractions from pooled sera at 5 and 6 dpi neutralized infective FMDV with similar levels as the whole serum.  Neutralizing capacity of the IgM-depleted pooled serum fractions resembled that of the pooled normal serum for all time points.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 17 Experimental design for vaccinated-infected cattle Oronasal Infection

Vaccination (IM)

Days postvaccination (dpv)

Clinical symptoms and serum antibody neutralizing activity in vaccinated-infected cattle challenge

Days postinfection (dpi) 10000

 Mean neutralizing activity due to serum circulating antibodies rises up to 29 dpv and decreased after infection (within one log10)

1000

vaccinated-infected

100 10 1 0.1

 Neutralizing activity in naïveinfected cattle increased 3 log between 4 to 6 dpi, being lower than from vaccinated-infected animals at 6 dpi

Days post-vaccination

 Cattle were IM vaccinated with a monovalent 01 Campos vaccine (22 μg/dose) and challenged at 30 dpv with 107 50% SMLD of the homologous virus applied through the oronasal route  Lymphoid tissues, whole blood and serum samples were taken at 7 and 29 dpv, and at 2 to 6 dpi (n=2 at each time point)

Lymphoid organs involved in local production of FMDV antibodies in vaccinated-infected cattle PE

MRL

LRL

TBL

PhT

 None of the vaccinatedinfected animals showed clinical symptoms of the disease up to 6 dpi

Immunoglobulin isotype profiles induced in local adaptative immunity in vaccinated-infected cattle

7 days post-vaccination

1000

IgA

1000

29 days post-vaccination

1000

100

IgM

2 days post-infection

IgG1

100

days post-vaccination

 This pattern was sustained at 2 and 3 dpi but changed starting at 4 dpi with an increasing activation of respiratory LN (MRL and TBL in particular) and spleen

Immunoglobulin isotype profiles induced in local adaptative immunity in vaccinated-infected cattle 3 days post-infection 2 days post-infection 4 days post-infection 1000

1 LRL PhT TBL

ML MRL LRL PhT TBL

Immunoglobulin isotype profiles induced in local adaptative immunity in vaccinated-infected cattle 4 days post-infection

5 days post-infection 00

6 days post-infection 1000

IgM

IgG1

ML MRL

 Isotype profiles and magnitudes are mainly sustained 2 days following oronasal infection

IgG2

ML MRL LRL PhT TBL

 A clear isotype switch to IgG1 and IgG2 was observed in PE at 29 dpv; responses found at respiratory tract LN’s the were mainly driven by IgM ASC

1000

IgM IgG1

1 PE

IgA

100

 IgM was most frequent antibody isotype among the anti-FMDV ASC detected at 7 dpv

days post-infection

 At 29 dpv, a moderate though consistent FMDV-specific ASC activity was observed in all the mucosal lymphoid organs

1000

ML MRL LRL PhT TBL

 At 7 dpv, PE LN’s draining the vaccination site, were the most stimulated organs, followed by LN’s from the upper respiratory tract and spleen

IgG2

100

1 PE

ML MRL

LRL PhT TBL

ML MRL LRL PhT TBL

 3 days post-infection, isotype switches to IgA and IgG1 were observed in MRL/PhT and TBL, respectively. Overall ASC numbers remained stable.  At 4 dpi, IgG1 ASC increased to match or surpass IgM ASC in MRL and TBL, IgG2 and IgA ASC also increased their numbers at this time point, specially in MRL. Total FMDV-specific ASC numbers clearly increased in spleen.  A primary stimulation profile was still detectable in ML, LRL and PhT.

IgG2

100

ML MRL LRL PhT TBL

 5 days post-infection, TBL and MRL showed patterns dominated by IgG1, followed by IgG2 and IgM, and finally IgA ASC. Isotype switching became also evident in ML and LRL. ASC counts in PE decreased regarding 4 dpi  TBL and MRL continued as the most active LN at 6 dpi with all isotypes detected. Both ML and LRL increased their activity, mainly driven by IgG1 ASC, as observed for S. PhT profile also changed to be dominated by IgG1.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 17 Conclusions  Active FMDV-specific ASC, mainly IgM ASC, were found in lymphoid tissues along the respiratory tract from vaccinated cattle (29 dpv). Incipient ASC responses were already observed at early times postvaccination (7 dpv).

Isotype profiles in local lymphoid tissues in vaccinated-infected vs. naïve-infected cattle VACCINATED-INFECTED

NAÏVE-INFECTED IgA

1000

IgM

 TBL and MRL in vaccinated-infected resulted the most stimulated LN at 6 dpi, similar to naïve-infected animals.  Different patterns of isotype activation were observed depending on the LN, although IgG1 ASC dominated most the responses from 4 dpi

IgM

IgG1 IgG2

100

 Early isotype switch in vaccinated-infected animals was observed in MRL, PhT and TBL at 3 dpi, probably due to the infectious virus entering through the respiratory system. Significant isotype switching in naïve-infected cattle was only detected at 6 dpi.

5 days post-infection

1 PE

MRL LRL

PhT

TBL

IgA IgM IgG1 IgG2

1000

100

1000

IgM

1000

1 ML

MRL LRL

1000

IgG1

IgG2

PhT

TBL

100

MRL LRL

PhT

TBL

1000

100

1 PE

MRL LRL

IgA IgM

IgG1

IgG1 100

PhT

TBL

IgG2

MRL LRL

PhT

TBL

MRL LRL

PhT TBL

IgA

1000

IgM IgG1

100

IgG2

1 ML

MRL LRL PhT TBL

MRL LRL

J. Pega D. Bucafusco D. Malacari A. Capozzo

S. Di Giacomo J. Schammas G. Stafforini M. Pérez-Filgueira

MV. Borca LL. Rodríguez

Acknowledgements: PE

MRL LRL

PhT

TBL

J. Arzt, J. Pacheco (PIADC, USA), C. Pérez-Beascoechea, E. Maradei (SENASA, Arg.), E. Smitsaart (Biogenesis-Bagó S.A.), M. Wilda (CEVAN-CONICET, Arg.), G. König, O. Zábal, G. Zábal, D. Romero, R. Escobar, J. Vallejos

Participants and Acknowledgements

6 days post-infection

IgA

IgG2

4 days post-infection

IgG1

100

Isotype profiles in local lymphoid tissues in vaccinated-infected vs. naïve-infected cattle

IgA

1000

1 PE

MRL LRL

PhT

TBL

This work was funded by the ARS Agreement No: 58-1940-8-111F “Understanding Local and Systemic Protective Responses against FMDV Infection in Cattle: A Genomics Approach” and Proyecto Específico “Fiebre Aftosa” AESA 201721-INTA , Argentina

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 18

Hypothesis

Characterization of opsonizing antibodies against foot-and-mouth disease virus Artur Summerfield Institute of Virology and ophylaxis, Switzerland

The immunological value of antibodies may not be sufficiently determined in a simple VNT.

Protection against FMDV could involve antibodies opsonizing the virus for transfer on to Fc receptors (FcR):  virus destruction by phagocytes (McCullough et al 1992 J. Virol. 66:1835.)  IFN-a responses by plasmacytoid DC (Guzylack-Piriou et al. 2006 Eur. J. Immunol. 36:1674)  efficient antigen presentation to DC for T-cell activation In vivo evidence: In a mouse model, depletion of macrophages abrogates the protective effect of antibody transfer, and nonneutralizing can mediate protection (McCullough et al. 1988. Immunology 65:187).

FMDV stimulates plasmacytoid DC only in the presence of anti-FMDV antibodies

AIMS 1. Determine the relationship between the neutralising capacity and the opsonising activity of antibodies 2. Cross-reactivity of opsonizing antibodies 3. Use of a murine macrophage cell line expressing bovine Fc g RII to quantify opsonizing antibodies in cattle sera after vaccination

Immune complexed FMDV

FMDV

anti-FMDV Ig

FcR

FcR ssRNA

DNA

TLR7

TLR9

„plasmacytoid DC“

No/weak response

ssRNA

DNA

TLR7

TLR9

„plasmacytoid DC“

IFN-a Guzylack et al., Eur. J. Immunol, 2006

Serotype-crossreactivity of porcine opsonizing antibodies using pDC cultures

Relationship between neutralization and opsonization titres of porcine sera. anti-O Manisa serum NT in log10 OT* in log10 Viral Strain 4 4.1 O UKG 2001 Type-O 3 3.1 O VietNam 7/97 4 <1.5 A Brazil 10/93 Type-A <1.5 2 A Turkey /99 <1.5 Type Asia-1 Asia-1 Turkey /99 2 <1.5 C1-Noville Type-C 0 * Opsonizing titres representing minimum serum dilution able to enhance IFN-α responses by pDC Serotype

Serum: O Manisa-vaccinated pigs

 Reacts also with A Brazil 10/93

Lannes et al., 2012. Vet Res. 43:64

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 18

RAW264.7 murine macrophage cell line to characterize opsonizing antibodies

Anti-O Mab D9: against linear epitope near RGD of VP1

RAW_bovCD32 cells: murine macrophage cell line engineered to express bovine CD32 RAW264.7

Characterisitics: 1. Only (with exceptions) in presence of reactive mAb infection by FMDV 2. This can be detected by intracellular staining of the mAb/FMDV complex or by measurement of CPE

1st Vaccination challenge experiment in cattle Merial Aftovaxpur: inactivated O3039, C Noville, Asia1 Shamir, A Iran96 Adjuvant: aluminium hydroxide and saponin After 21 days infection with FMDV O BULGARIA (10000 ID50)

=> Cannot neutralize O Turkey, O Brazil, A serotype but still opsonize these viruses

VOT/VNT @ 21 days p.v. and protection full dose

¼ dose

1/20 dose

100 / 13

10 / 10

10 / 11

100 / 27 100 / 32

10 / 7

100 / 10

10 / 8

10 / 5

100 / 11 100 / 16

-/-

10 / 13

Group 1: 4 animals full dose vaccine Group 2: 5 animals 1/4 dose Group 3: 5 animals 1/20 dose

Red: not protected! No obvious relationship to protection

2nd Vaccination challenge experiment in cattle

But relationship to vaccination dose?

VOT/VNT @ 21 days p.v. and protection O3039 O 3039 and Manisa

O Manisa

Merial vaccine Adjuvant: aluminium hydroxide and saponin After 21 days infection with FMDV O BULGARIA (10000 ID50)

10 / 19 10 / 27

100 / 108

-/7

10 / 45

10 / 10 100 / 64

100 / 23

Group 1: 4 animals O3039 Group 1: 5 animals O3039 and O Manisa Group 3: 5 animals O Manisa

10 / 13 100 / 38

100 / 32

10 / 19

100 / 54

100/45

Higher levels of OT and NT against O Bulgaria after vaccination with O Manisa

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 18

Conclusions and recommendations

1. Opsonizing antibodies against FMDV can be broadly cross-reactive even amongst serotype.They enhance virus infection of macrophages and dendritic cells resulting in virus destruction and IFNa responses. 2. Often mAbs which efficiently neutralize are also efficient at opsonization. Non-neutralizing mAbs (against homologous virus) can probably not opsonize FMDV. However, opsonization of non-neutralized virus isolates is often seen. 3. Measurement of opsonizing antibody may represent a supplement to other immunological readouts to determine correlates of protection. 4. O Manisa protects against O Bulgaria 2001

Acknowledgements Heidi Gerber Trix Zumkehr Monika Gsell Albert Nils Lannes Lukas Bruckner Raffael Fricker

Dr. Satya Parida Dr. Santina Grazioli and Dr. Emiliana Brocchi. IZSLER, Brescia, Italy

Funding

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 20 Introduction • Effective control of FMD:

Intra-serotype chimeric FMD vaccine antigen elicit protection in cattle

- Physical barriers, including fencing - Immune barrier using vaccination

Vaccination

F.F. Maree Transboundary Animal Diseases Physical barriers, like fences

Introduction A key statement from discussions with DAFF • The control of FMD by vaccination is complicated by:

regarding what is needed in the control of FMD:

- Multiple genetic lineages (and antigenic variants) in different geographical regions. - Adaptations of field strains are cumbersome.

“We need better vaccines and

- Stability of the virus and/or antigen.

better fences”

- Longevity of antibody responses elicited by vaccines. - Developing countries: lack of vaccines tailored to their conditions

SAT2 Antigenic variation 1.2

SAT2 antigenic variation Vaccine match based on r-values

ZIM/07/83/2 KNP/19/89/2

ERI 12/89/2 RWA/02/01/2

0.8

0.6

Ref sera:

r-value criteria

ZIM/7/83

0.4

KNP/19/89

0.2

RWA/2/01

0 0

100

120

ERI/12/89

r-Values of SAT-2 virus isolates from different topotypes compared to antiserum prepared to the reference strains KNP19/89 (I), ZIM7/83 (II), RWA/2/01 (VIII) and ERI/12/89 (X).

SAT2 vaccine strains

SAT2 reference viruses

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 20 A joint approach between conventional, inactivated vaccine technology and genetic engineering

Rational design of vaccines • Vaccine containing a inter-serotype chimeric antigen elicited immune responses similar to that of the parental antigen.

FMDV capsid

• Functional amino acid residues involved in cell culture adaptation of SAT vaccine strains have been identified and introduced in field viruses using reverse genetics. • Genetically engineered SAT2 and SAT3 viruses revealed diverse stabilities to mild acidic conditions - stability of the capsid can be engineered.

• Yield of chimeric vaccine antigen and growth of viruses can potentially be improved by modification and improvement of enzymatic functions. Chimera capsid

• Epitope switching may be used to modify antigenicity of an infectious copy virus.

Construction of infectious chimeric particles

SAT2/ZIM/07/83

vSAT2

Vaccination of cattle with a SAT2/SAT2 vaccine

r-values for SAT2 viruses

1.20

ZIM/07/83/2 1.00

KNP/19/89/2 ERI 12/89/2

D14

D21

D32

0.80

SAT2/ZIM/17/91

vZIM17/SAT

SAT2/ZIM/14/90

vZIM14/SAT

chimera

0.60 0.40 0.20 0.00

KNP/19/89/2

ZIM/07/83/2

ZIM/17/91/2

ZIM/14/90/2

vZIM17/SAT2

Vaccinate 2ml containing 8 μg 146S:

vZIM14/SAT2

Challenge: ZIM/14/90

- ZIM/14/90 - Chimera - Placebo

Neutralising antibody titers

Vaccination of cattle with a SAT2/SAT2 vaccine Chimeric vaccine

Parental vaccine

3.0

2.5

2.0

1.5

Parental vaccine Cattle vaccinated with parental

3.00

2.50

2.00

1.50

1.00

1.5

1.0

0.5

0.0

Chimeric vaccine Cattle vaccinated with chimera

3.0

Day 0

Day 7

Day 14

Day 21

0.0

0.50

Day 0

Day 7

Day 14

•Cattle vaccinated with 8 ug of antigen •Challenge with 104 ID50 FMDV at 21 dpv •Sera tested with a SAT2 solid-phase competition ELISA (SPCE)

Day 21

0.50

0.00 Day 0

Day 7

Day 14

Days post-vaccination

Day 21

0.00

Day0

Day7

Day14

Day21

Days post-vaccination

•Cattle vaccinated with 8 ug of antigen •Challenge with 104 ID50 FMDV at 21 dpv •Sera tested with a SAT2 solid-phase competition ELISA (SPCE)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 20 Neutralisation titers compared to protection Vaccine

Animal number

SAT2/ZIM/14/90

vZIM14/SAT

Control

Neutralisation titers compared to protection

Temperature (ºC)

Neutralisation titer

Lesions postchallenge

P-1 P-2

39.5 39.3

2.06 2.38

None None

P-3 P-4

40.1 39.6

2.02 2.47

Mild None

P-5 P-6 P-7

40.0 38.6 39.7

2.15 2.38 2.00

Mild None None

2,5

C-1 C-2

39.6 39.7

2.43 2.82

None None

1,5

C-3 C-4

40.3 39.1

2.04 2.26

None None

C-5 C-6 C-7

38.7 39.6 39.2

2.59 2.40 2.61

None None None

0,5

1 2

39.6 38.9

<1.3 <1.3

Severe Severe

VNT titers 3

Protected parental

Unprotected parental

Structurally modified master seed viruses to enhance conventional foot-and-mouth disease virus vaccine production

Antigenic relatedness to SAT2 viruses r1= heterologous titer homologous titer

1,20

Protected chimera

1,00 0,80

good vaccine match

0,60 0,40

poor vaccine match

0,20 0,00

SAR/3/04

KNP/19/89

antiserum vZIM14-SAT2 antiserum SAT2/ZIM/7/83

ZIM/14/90

viruses

ZIM/7/83

antiserum SAT2/ZIM/14/90 antiserum SAT2/KNP/19/89

antiserum SAT2/SAR/3/04

Wellcome Trust translational award

Interpentamer residues aimed at stabilising the capsid

Global FMD Research Alliance Consortium consisting of: Institute for Animal Health, Pirbright

Structural model of SAT2 pentamer

Transboundary Animal Diseases, ARC-OVI Plum Island Animal Disease Centre, USDA University of Oxford

Side view of the pentamer, showing the interpentamer interface

MSD Animal Health

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 20 In vitro measurement of capsid stability Thermofluor assay

A24

vSAT2

Mut B

Neutralising Ab response in guinea pigs VNT titers of vSAT2 (wild-type) and mutant vaccinated GPs

---

A24

(55 °C)

---

Mutant B

(53 °C)

---

Mutant H

(52 °C)

---

Mutant F

(51 °C)

2.5

---

Mutant A

(51 °C)

---

vSAT2 WT

(47 °C)

---

Mutant C

(45 °C)

3.5

2 1.5 1

Shift of 6 °C 11.3 % increase in dissociation temperature using Thermofluor assay of mutant B.

0.5 0

vSAT2, 1 month

Conclusion

•

Intra-serotype Chimeric Vaccine VZIM/14-SAT2 elicited a protective immune response in cattle comparable to that of the conventional vaccine; SAT2/ZIM/14/90.

•

Similar antigenic profiles observed for both the chimera and conventional vaccines.

•

Thermo-stable SAT2 capsids were designed based on structure and infectious viruses based on a capsid stabilised mutation indeed shows an increase >10% in dissociation temperature

vSAT2, 6 months

Mutant, 1 month

Mutant, 6 months

Acknowledgements

P. Nsamba; K. Scott; B. Blignaut; P. Mutowemba;

Makarere University, Uganda TADP, ARC-OVI TADP, ARC-OVI TADP, ARC-OVI

A. Kotecha, E. Fry, D. Stuart B. Charleston E. Rieder D. Goovaerts, P .Guntram

Oxford Structural biology group IAH, Pirbright PIADC, USDA MSD Animal Health

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Bryan Charleston Global Foot-and-mouth disease Research Alliance (GFRA): Production and evaluation of Foot-and-mouth disease virus stabilised capsids as potent, rapidly deployable vaccines “emergency vaccination should be seen as a major tool of first resort … for controlling FMD outbreaks” Royal Society

Recombinant FMDV capsids

Translation strategy – selectively reduce 3C translation 142C>T/S

mRNA levels unaltered

Baculovirus derived FMDV capsids

Structure-based stabilisation of FMDV capsids

Proof of principle that an engineered mutation (his to cys) is consistent with capsid assembly. Similar approaches can be used for infectious copies.

Structure-based stabilisation of FMDV capsids

Proof of principle that an engineered mutation (his to cys) is consistent with capsid assembly. Similar approaches can be used for infectious copies.

Capsid production2

Wild-type and stable capsid structures2

Empty capsids with enhanced stability

Cattle vaccinated with 12ug of Baculovirus derived FMDV capsids in commercial oil adjuvant Day 0 and Day 21

Stabilised empty capsids - Improved storage characteristics: vaccine ready for deployment, less reliant on cold chain - Safe production: no live virus required, enhanced production capacity - Vaccine can be produced to new virus variants: no need to isolate virus and adapt to tissue culture, sequence → gene synthesis → expression - Opportunities for further development: enhance early response, increased antigenic breadth - No non-structural proteins: companion DIVA diagnostic tests, greater certainty of discriminating between vaccinated and infected animals

- Reagents for diagnostic test: safe production, improved shelf life

Julian Seago Nick Juleff Terry Jackson Claudine Porta Dave Stuart Liz Fry Abhay Kotecha Francois Maree Belinda Blignaut Katherine Scott Pamela Opperman Elizabeth Rieder Sabena Uddowla Ian Jones

Appendix 22

Conclusions and Recommendations Development of a bovine enterovirus-based vector that expresses multi-epitope of FMDV

Jong-Hyeon Park parkjhvet@korea.kr FMDV lab, FMD Division Animal Plant and Fisheries Quarantine and Inspection Agency (QIA), 480 Anyang 6 dong, Anyang, Republic of Korea

• A recombinant BEV vectors carrying B and T cell epitopes of FMDV were constructed and recovered as live viruses • BEV-based viral vector is able to deliver approximately 720 bps of foreign genes • The rBEVs show a proper growth without affecting of replication kinetics and plaque sizes compared to the parent virus. • The rBEVs were detected and isolated in feces and serum samples after administration to cattle. • The calves inoculated with rBEVs showed no clinical signs • The rBEVs show a possibility as live vaccine vector for FMD - Experiment for the immune and protective effect against FMDV is still undertaken. - Expression of larger sized FMDV molecule with molecular adjuvant should be tried

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Why use BEV-1 (LC R4 strain) as a vaccine vector?

Objectives

• Safe to animals (isolated from healthy dairy cattle, Kunin, 1957)

• To verify the possibility of BEV as live and delivery vaccine vector

- BEV infections are ubiquitous and are normally without significant or severe clinical symptoms

• The same Picornaviridae family with FMDV • Thermostable - maintenance of the cold chain is not always guaranteed from manufacturing to delivery)

• To investigate the multiplication and safety as a live vaccine candidate for FMD in vivo

• pH-stable - can be administered orally

• Very wide tissue tropism for cell types in vitro • Replicable in cattle (or other animals) • Generating high titers in various cell lines.

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Strategy for rBEV construction

FMDV and BEV genome P1 Poly(C)

L VP4 VP2 VP3 VP1 2A 2B

Cloning of full-length cDNA of BEV and recovery

3A 3B 3C

FMDV

• BEV Type 1 strain LCR4 (ATCC® Number: VR-248TM) • Cloned into pBluescript II SK (+) vector

VPg VP4 VP2

BEV

VP3

VP1

2A 2B

2C 3A 3B 3C

VPg T7 (Kbps)

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 22

EGFP and synthetic FMDV epitopes

Construction of rBEV containing foreign genes 2A Cleavage site (TSY/G) BEV_EGFP

EGFP (720 bps)

• EGFP for marker gene: 720 bps VP4

• O/Andong/SKR/2010 (O-SEA topotype, Mya-98 lineage isolated from epidemic of Andong region, Korea in Nov. 2010) • O1 Manisa (ME-SA topotype, PanAsian lineage, vaccine strain) - FMDV VP1 (a.a 141-160, 200-213) - FMDV VP4 (a.a 20-34) - FMDV 3A (a.a 21-35)

• T-helper epitope - Pan-HLA-DR (PADRE): a universal DR-restricted T-helper epitope (Hung CF et al. 2007; Alexander J et al. 1994)

VP2

VP3

EGFP

(A)16

BEV_O-AD-multi-epi/ O-Manisa-multi-epi VP4

VP2

VP3

VP1

2A Cleavage site (TSY/G) (GGSGG) linker

3A 3B

(A)16

2A Cleavage site (TSY/G)

327 bps(109aa) linker

FMDV_VP1200-213 PADRE EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

VP1

linker

FMDV_VP1141-160 3A21-35

linker

VP420-34

Hisx6

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

rBEV-EGFP

rBEV-AD

rBEV-Manisa

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Indirect immunofluorescence assay

EGFP expression from rBEV-EGFP ( 6 hours after infection in MDBK cells)

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 22

Search for pre-existing antibody against BEV-1 type in the field

Growth of rBEVs in MDBK cells 10

BEV-full clone BEV-EGFP BEV-AnDong multiepi BEV-Manisa multiepi

8 6

Distribution of BEV-1 (LC-R4 strain) VN titers in Korea 60

BEV-1 Cattle(N=172) BEV-1 Pigs (N=160)

• Positive rate of cattle is 48.3% • Positive rate of pigs is 70.6%

10 0

Hour post-infection

2.5

3.5

4.5

5.5

6.5

>7.0

BEV antibody titers (Log2) EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Infection of rBEVs to calves Calf No.

Virus titer

Inoculation virus

1180

rBEV-EGFP

9362 0522 0564

107.29

rBEV-AnDongMulti-epi

No clinical sign during 14 days after inoculation

Inoculation route

(TCID50/ml)

Intramuscular 2ml, Intranasal 2ml, Oral 2ml

107.44

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

RT-PCR for detection of rBEVs from infected calves

Virus isolation of rBEV in feces of inoculated calves Bright-field

Fluorescence

BEV in feces samples (DPI)

CPE

Groups

Calf No.

rBEV-EGFP

1180

+ + + +

rBEV-AD multi

0522

0564

Groups

Calf No.

rBEV-AD multi

1180

nd nd nd nd

+ + + +

9362

rBEV-EGFP No CPE

BEV in serum samples (DPI)

rBEV-EGFP

9362 0522 0564

CPE

rBEV-AnDong -Multi-epi

CPE

nd : not done EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 22 Virus neutralizing antibody against BEV and FMDV Serum Neutralization Titers using BEV-1 LCR4 Calf No. 1180 9362 0522 0564

Inoculation virus BEV-EGFP BEV-ADmulti-epi

0dpi

2dpi

4dpi

6dpi

8dpi

10dpi

12dpi

13dpi

Serum Neutralization Titers using O/AnDong/SKR/2010 Calf No. 1180 9362 0522 0564

Inoculation virus BEV-EGFP BEV-ADmulti-epi

0dpi

2dpi

8dpi

10dpi

12dpi

13dpi

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Conclusions and Recommendations • A recombinant BEV vectors carrying B and T cell epitopes of FMDV were constructed and recovered as live viruses • BEV-based viral vector is able to deliver approximately 720 bps of foreign genes • The rBEVs show a proper growth without affecting of replication kinetics and plaque sizes compared to the parent virus. • The rBEVs were detected and isolated in feces and serum samples after administration to cattle. • The calves inoculated with rBEVs showed no clinical signs • The rBEVs show a possibility as live vaccine vector for FMD - Experiment for the immune and protective effect against FMDV is still undertaken. - Expression of larger sized FMDV molecule with molecular adjuvant should be tried EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Acknowledgements Members of FMDV Lab, FMD Division, QIA. Korea. Jia-Qi Chu, Jeong-Nam Park, Yeo-Joo Lee, Rae-Hyung Kim, Su-Mi Kim, Kwang-Nyeong Lee, Hyang-Sim Lee, Young-Joon Ko, Byounghan Kim, Seo-Yong Lee, Soo-Jeong Jung

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 23 Background of the study Conventional FMDV vaccines induce protective humoral immunity but does not prevent the carrier state

Development and evaluation of an adenovirus vector based intranasal FMDV capsid vaccine Aravindh Babu1, Geraldine Taylor1, Sarah Gilbert2 and Satya Parida 1 1 The

2 The

Pirbright Institute, UK

Jenner Institute, UK

Salivary IgA is the indicator of oro-pharyngeal replication of FMD virus (Parida et al., 2006) IgA along with systemic antibody is insufficient to clear the infection Can a pre-existing IgA prevent viral colonisation?? Mucosal immunity against FMDV?

Hypothesis, aims and objectives

- a viral vector based vaccine containing FMD empty capsid delivered by intranasal route whether stimulate local and systemic immune responses that could block FMDV infection

Strategy for the recovery of Adeno-FMD recombinant virus

Expression of A22 FMDV empty capsids in rAdVH5-FMDV infected cells

HEK293A cells infected with AdVFMD Green = A22 FMDV capsid Blue = nucleus Scale bar = 40 M

Large arrows = FMDV empty capsids (~30nm) Small arrows = pentamers (~10nm) Scale bar = 100 m

Immunolabelling of the FMDV capsids in bovine airway epithelial cells and MoDCs infected with rAdVH5-FMD

Bovine airway epithelial cells (primary) Green = cytokeratin Red = A22 FMDV capsid Blue = nucleus Scale bar = 40 M

Lymphocyte proliferative responses of PBMCs from A22 FMDV vaccinated cattle re-stimulated with rAdV-FMD virus

Bovine monocyte derived dendritic cells Green = A22 FMDV capsid Blue = nucleus Scale bar = 80 M

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 23 Lymphocyte proliferative responses of PBMCs of A22 FMDV vaccinated cattle upon re-stimulation with rAdVFMD infected bovine MoDCs

Design of mice experiment

IFN responses in re-stimulated whole blood of A22 FMDV vaccinated cattle

FMDV specific serum IgG subclass antibody responses in rAdV-FMDV vaccinated mice 6

AdVFMD i.n. (n=16)

28 days

56 days

n=8 Killed n=8 boosted (i.n)

n=8 killed

IgG1

IgG2a IgG2b

AdVFMD i.m. (n=16)

n=8 Killed n=8 boosted (i.m)

n=8 killed

rAdV i.n. control (n=8)

n=4 Killed n=4 boosted (i.n)

n=4 killed

0 rAdVFMD i/n 28dpv

rAdVFMD i/n 56dpv

rAdVFMD i/m 28dpv

rAdVFMD i/m 56dpv

rAdV control

AdVFMD dose 5 x 108 IU per animal

Anti-FMDV IgA responses in lung wash of rAdV-FMDV vaccinated mice (56 dpv)

Proliferative responses of re-stimulated spleenocytes of rAdV-FMDV vaccinated mice

2,5

1,5

0,5

0 AdVFMD i/n 28dpv

AdVFMD i/n 56dpv

AdVFMD i/m 28dpv

AdVFMD i/m 56dpv

rAdV control 28dpv

rAdV control 56dpv

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 23 A

Summary • FMDV empty capsids are expressed in 293A cells when infected with rAdVFMD virus and their size was confirmed by EM • rAdVFMD virus infects bovine airway epithelial cells and dendritic cells • rAdVFMD virus re-stimulates lymphocytes of FMD vaccinated cattle for IFN- production and proliferation • rAdVFMD virus induces both systemic and mucosal antibody responses against FMDV in mice when administered intranasally • The intranasal vaccination generated balanced IgG1 and IgG2 antibody responses • Cattle experiment is being planned for intranasal administration of rAdVFMD virus and challenge virus using a face mask and nebuliser

• Satya Parida • Geraldine Taylor • Pippa Hawes

• Paul Smith • Members of the Vaccine Differentiation Group

Sarah Gilbert

Funding

• BBSRC+DFID (CIDLID) • EU-FP7 DISCONVAC

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 24 Objectives o In vivo PD50 Trials: • Animal- & Biosafety (live virus challenge) • Test variability Alternative foot-and-mouth disease vaccine potency tests based on serology and antigen payload CODA-CERVA ANSES MERIAL FGI ARRIAH

o Reduction of animal challenge • Challenge free vaccine potency test (serology)

o Replacement of animals • Animal free vaccine potency test

Altandi III consortium

EUFMD, Spain

Tom Willems

October 2012 2

Design

Conclusions

o D.O.E monovalent vaccines containing different Ag payloads

o Ag payload based AND Ag payload & vaccine dose model • Accurate PD50 estimates for 100 % Ag payload • ? Higher Ag payload than 100 % ?

o Strictly serology based model vs more complex model • Comparable sensitive and specific • Comparable PD50 estimates

(25, 50, 100, 200 & 400%, where 100% is expected to produce 10 PD50)

o All from same antigen batch FMDV O1 Manisa

o 3 vaccine potency tests (PD50) per Ag payload o Serum samples collected at 21 DPV and tested in LPBE

Modelling

Results (1)

o Ag payload based model

o In vivo • High variability

• Correlation protection & Ag payload •

•

First only Ag payload -> Ag payload model Second Ag payload x vaccine dose -> Ag payload vac.dose model

Antigen payload 1 25% 8

• Extrapolation to PD50 -> 50% Probability of Protection

• Correlation protection & Ag payload & serology

50%

3,5

100%

200% 400%

PD 50 2 10,6

3 8

2,6

PD50 95% CI Overall 8,7 5,5 - 13,9 4,2

2,9 - 6,7

13,9 10,6

10,7

6,7 - 16,8

18,4 18,4 24,3

20,2

13,9 - 29,2

17,9

11,6 - 26,6

24,3

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 24 Results (2)

Results (3) o Ag payload model

o Ag Payload model

• 50% : 4.4 % Ag payload • 22.7 [ 9.7 - 53.0] PD

• Correlation protection & Ag payload • •

Somer’s D : 0.42 -> 0.46 (1:16 dose out) 50% : 8.7 % Ag payload 100 % Ag payload -> 11.5 PD

o Ag payload vac.dose model

log10

0.5

• 50% : 6.6 % Ag payload • 15.3 [ 6.5 - 35.9] PD

payload %

1.5

log10

2.5

payload %

0.5

1.5

2.5

• Correlation protection & Ag payload & vac.dose • •

Somer’s D : 0.70 -> 0.74 (1:16 dose out) 50% : 7.0 % Ag payload 100 % Ag payload -> 14.3 PD

50% = -b0/b1

Results (4)

0.5

1 1.5 2 log 10 Ab ti

2.5

Quarter dose

0.5

1.5

log10 Ab ti

100

200

400

payload (%)

2.5

0.5

1.5

log10 Ab ti

2.5

o Estimated PD50 for different models

• All estimates are within range of in vivo PD50 • Similar to serology based model of Goris et.al.

•

25% & 50%  2 PD50 [ 1.4 - 6.4]  10 PD50 [ 4.2 - 22.1] 200%  13 PD50 [ 7.3 - 22.1] 400%

Model In vivo

Ag payload 100

PD50

[CI 95% ]

Vacc [CI 95% ]

Vcon [CI 95% ]

0.73 [0.64 - 0.83 ] 0.74 [0.66 - 0.86 ] 0.63 [0.55 - 0.73 ] 0.68 [0.60 - 0.79 ]

0.70 [0.62 - 0.82 ] 0.71 [0.63 - 0.83 ] 0.59 [0.52 - 0.68 ] 0.65 [0.58 - 0.77 ]

10.6

[6.7 - 16.8 ]

Payload.vac.dose & Serology Serology

100

6.6

[4.2 - 9.6 ]

100

4.5

[2.6 - 8.0 ]

Serology Goris et.al.

100

8.7

[5.0 - 13.9 ]

Conclusions

Results (6)

•

200

• Ag payload & vac.dose & serology model • Only serology based model

Sixteenth dose

•

• Sensitivity & specificity: • Increase with model complexity (A to C) • Small difference between: (C & D)

Serology based model Serology based model (Goris et.al.) Full dose

o Ag payload // vaccine dose // serology

• curves overlap with: •

Results (5)

o Ag payload vac.dose & serology model

payload (%)

•

50% = -b0/b1

o Ag payload based AND Ag payload & vaccine dose model • Accurate PD50 estimates for 100 % Ag payload • ? Higher Ag payload than 100 % ?

o Strictly serology based model vs more complex model • Comparable sensitive and specific • Comparable PD50 estimates

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 24

THANKS CODA-CERVA (VAR)

ANSES

Dr K De Clercq Dr A De Vleeschauwer Dr D Lefebvre T Willems

Dr L Bakkali-Kassimi Dr S Zientara

Pirbright Inst. Dr P Barnett

Merial Dr P Hudelet Dr P Dubourget Dr C Martin

FGI ARRIAH Dr V Diev Dr S Kremenchugskaya

Dr S Goutebroze

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 25

Towards challenge-free FMD vaccine batch acceptance

or...

Richard Reeve Boyd Orr Centre for Population and Ecosystem Health Institute of Biodiversity, Animal Health and Comparative Medicine University of Glasgow

Conclusions and Recommendations

• Conclusions:

What our current models are really saying while we’re not listening!

- Challenge-free PD50-based batch acceptance requires not just a serological threshold, but a measure for acceptance of the model itself - While it is possible to rank assays in order of preference, they can predict protection better in combination than individually

• Recommendations:

- A more detailed model of the assays themselves must be built to understand when results can and cannot be relied on - Sera from challenged animals should continue to be actively promoted for testing of novel assays

Existing serological models

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 25 Existing serological models

Existing serological models In particular this bit!

Existing serological models

• It says a titre of 1 (log titre of 0)

• is 75% likely to be protective

Existing serological models

- i.e. lower than the lowest titre we actually measure in practice

• A titre of 1 is 75% likely to be protective... • When we consider that this is the result we get from the unvaccinated controls, we can see that this is unlikely to be true!

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 25 Does it matter in practice though?

Predicting protection from vaccinated animals

●

Unprotected

Adding in the controls...

Protected

Ultimately we don’t care about them though

●

Unprotected

Protected

Improving prediction of protection

●

Unprotected

Protected

Does it matter in practice?

From 1137 sera... • Ignore controls: - On average, controls predicted to be >25% protected

• Include controls:

●

- Controls predicted to be 6% protected

Unprotected

Protected

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 25 Predicted protection of controls from serology

Does it matter in practice?

Predicting protection of unvaccinated animals

From 1137 sera... • Ignore controls: - On average, controls predicted to be >25% protected

• Include controls: - Controls predicted to be 6% protected ●

• But situation is much worse for individual vaccines...

●

With controls

Without controls

Why not include dose in model?!

It is better than titres across all animals...

Predicting protection using VNT, LPBE and dose

Predicting protection using VNT and LPBE

●

● ●

Unprotected

Protected

But problems with over-reliance on dose... Predicting protection of vaccinates using VNT, LPBE and dose

Unprotected

Protected

... makes the problem worse for vaccinates Predicting protection of vaccinates using VNT and LPBE

●

Unprotected

Protected

Unprotected

Protected

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 4

Appendix 25 Does it matter in practice?

And now some good news...

However, this just tells us that while we may have assays that will work - and do work in practice in some regions in a suitable regulatory framework - we have to be careful about applying them without sufficient attention in new situations.

ELISA

VNT Predicting protection of vaccinates using ELISA

Predicting protection of vaccinates using VNT

●

Unprotected

Protected

ELISA + VNT

Unprotected

Protected

ELISA + VNT

Predicting protection of vaccinates using VNT and LPBE

●

• Combining assays allows us to improve the predictive power of our models • Not surprising when we consider that different assays may measure subtly (or grossly!) different aspects of the overall picture

●

Unprotected

Protected

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 5

Appendix 25 VNT

Ongoing modelling problems Predicting protection of vaccinates using VNT

●

• Not correctly modelling negative assay results? - If we want to incorporate the controls into the picture, we have to accurately reflect what is actually going on in the individual wells

●

But what’s going on here? Unprotected

Protected

Conclusions and Recommendations

• Conclusions:

• Recommendations:

Acknowledgements

• Pirbright Institute - P. Barnett, S. Cox, S. Parida

• Argentina - E. Smitsaart, C. Perez Beascoechea, E. Maradei

• University of Glasgow - D.T. Haydon

• FLI - B. Haas

• VAR - K. De Clercq

Funders:

DEFRA EU FP7 BBSRC/DfID/ Scottish Government

Acknowledgements

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 6

Appendix 26

Relation between antibody response and protection in FMD vaccine depends on vaccine quality

Outline  Correlation between

antibody response and protection

● Purpose

Aldo Dekker

● History of research

 Current

study

● 447 cattle sera ● Forward logistic regression analysis ● Relation between antibody response and protection

in FMD vaccine depends on antigen and vaccine dose

 Best vaccine response

is the vaccine that induces the highest antibody

Correlation between antibody response and protection

Historical analysis Ab response protection

 Use for vaccine release

 Loeffler and Frosch,

● Producers develop own criteria ● Standard interval vaccination and measuring

 Van Bekkum

monitoring

● Variation between producers ● Different intervals vaccination and sampling

et al. 1969

● 566 cattle ● 2 weeks post vaccination type C (n=424) ● 9-49 months post vaccination 3 serotypes (n=142)

antibody response

 Use for post vaccination

1897

● Passive antibodies can protect against infection

 Pay and Hingley, 1987

● 360 vaccinated and challenged cattle ● 3 weeks post vaccination ● 3 serotypes

 Eblé et al. 2009

● Intradermal vaccination better protection at lower Ab dose

Historical analysis Ab response protection  Loeffler and Frosch,

1897

● Passive antibodies can protect against infection

Results van Bekkum et al. 1969 Relation Ab response protection Type C FMD

et al. 1969 ● 566 cattle ● 2 weeks post vaccination type C (n=424) ● 9-49 months post vaccination 3 serotypes (n=142)

vaccination at a lower Ab titre compared to 9 - 49 months after vaccination

 Cattle

● 360 vaccinated and challenged cattle ● 3 weeks post vaccination ● 3 serotypes

● Intradermal vaccination better protection at lower Ab dose

Ab and

● Protection 2 weeks after

 Pay and Hingley, 1987

 Eblé et al. 2009

 Relation between protection

 Van Bekkum

sampled at 9 - 49 months after last vaccination had been vaccinated 2 - 10 times

1.2

1.5

1.8

2.1 VNT titre

2.4

2.7

3.0

2 weeks post vaccination 9 to 49 months post vaccination

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 26 Results van Bekkum et al. 1969

Intradermal versus intramuscular vaccination

Frequency distribution Ab titres

 Titres 2 weeks

higher then 9 to 49 months post-vaccination

2 weeks post vaccination 9 to 49 months post vaccination <1.35

1.35 - 1.50

1.65 - 1.80

1.95 - 2.10

2.25 - 2.40

2.55 - 2.70

 Pigs  Difference

in relation between Ab titre and protection against virus shedding (mouth swabs)

 Intradermal

vaccination (in red ) better protection at lower Ab dose

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

VNT antibody titre

>2.70

VNT titre

Shelf life: Indication for degradation of 146S

Little decrease in Ab titre Trial Protection %

 Goris et al. 2008 (Vaccine 26: 3432-3437)  Clear decrease in vaccine efficiency in 10 months  Experimental vaccine  No data available from commercial producers

LPB ELISA mean titre Expected protection

100

2.50

93.8

2.66

89.9 91

93.8

2.15

80.4

81.3

2.21

87.5

2.38

87.3

2.49

88.9

Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests. Robiolo, B., La Torre, J., Maradei, E., Perez Beascoechea, C., Perez, A., Seki, C., Smitsaart, E., Fondevila, N., Palma, E., Goris, N., De Clercq, K., Mattion, N.

Lelystad vaccine registration dossier

Forward logistic regression analysis

 447  240

 Protection as result variable  Various explanatory variables

cattle used in challenge experiments

cattle used in potency tests (3 times 5 cattle vaccinated with 1, ¼ and 1/16th dose, challenged 4 weeks after vaccination

 9 different

strains

● A Iran 87, A TUR/14/98, A10Holland, A22Iraq,

A24Cruziero, Asia-1 Shamir, O Algeria, O1BFS, O1Manisa

 VNT titre obtained using primary porcine kidney cells  Forward logistic regression analysis ● Titre, log(dose), µg Ag, µg Ag in full dose, strain

● Titre, log(dose), µg Ag, µg Ag in full dose, strain

 Selection based on AIC Univariate analysis

 Antibody titre best predictor of protection  Logarithm of the dose second best predictor  Higher dose induces a higher antibody response

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 26 Forward logistic regression analysis  Multivariate

O Manisa antibody titre response curve

model

Logit(protection) ~ Antibody titre + strain + µg per full dose + log(dose) + strain:titre

Red full dose

 For each

µg of extra antigen in the vaccine the antibody titre that protected 50% of the cattle was reduced 0.04 (10log)

Blue 1/4 dose

 Cattle

vaccinated with a 4 fold higher dose need a 0.08 (10log) less antibody titre for 50% protection

Green 1/16 dose

 When

analysing 240 results from potency tests interaction is absent and each batch has a different result 0.0

0.5

1.0

1.5 Titre

2.0

2.5

3.0

10log

Conclusion

 Complete possible

replacement of standard potency tests is not

● Each vaccine producers should establish their own criteria based on protection experiments and use serology for batch release

 Monitoring antibody response is a good method for post vaccination monitoring

● Higher antibody titre correlate with higher level of protection

 Better vaccine induces higher antibody titres and protects already at a lower antibody titre

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 27 EUFMD 2012 open session, Jerez, Spain, 29-31 Oct 2012

Conclusion and recommendation

Cross protection against current Asia 1 isolates provided by a high potency Asia 1 Shamir vaccine Yanmin Li, Bob Statham, Bryony Armson, Pip Hamblin, Ginette Wilsden, Nicola Camping, Jef Hammond The EURL/WRL FMD, The Pirbright Institute, Pirbright, UK

Vaccine Matching (type Asia 1) 2000-2011 WRLFMD

 A high potency Asia 1 Shamir vaccine provided protection from current Asia 1 field isolates infection in cattle  r1 values obtained using pooled vaccinal sera correlated with the in vivo protection results

Asia 1 TUR 49/2011 virus

Asia1 Shamir No of tested

No of match 15

Vaccine matching studies for type Asia 1 FMDV by VNT-WRL FMD

6 4

4 2

<=2000

2001

2002

2005

2006

2008

WRL SAMPLE REF

Asia1 IND 8/79

Asia1 Shamir

Asia1 WBN 117/85

TUR 49/2011

TUR 51/2011

2009-Nov

r1 values for Asia 1 serotype -WRL FMD 2011 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

r1 values from Asia 1 TUR 49/2011 against Asia 1 Shamir using standard BVS (mean of two tests): 0.20

WRL ref no

Asia 1 Shamir Potency Test Heterologous challenge with Asia 1 TUR 49/2011 in Pirbright on March 2012

1 dose

1/4 dose

1/16 dose

Results - PD50

Unvaccinated controls

Asia 1 Shamir vaccine >6 PD50 Vaccinates and controls were challenged with FMDV isolate Asia 1 TUR 49/2011 at 21 days post single vaccination. Serum samples were collected at weekly basis for analysing the immune response Clinical scores have been read twice a day post challenge until 9dpc.

With one inconclusive (culled at 7dpc due to anorexia) gives the vaccine 13.92 PD50

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 27 Results - VNT

Results - ELISA

VNT Titre from full dose group 1600

Animal 302328

Animal 402329

Animal 504868

Animal 604869

Animal 702311

LPBE titre at 20dpv from all groups

1400

3500 3000

1200

LPBE Asia 1 Shamir

LPBE Asia 1 Tur 49/11

2500

1000

800

2000

VNT Asia 1 Shamir

1500

VNT Asia 1 Tur 49/11 600

VNT mean log titre

400

1000 500

LPBE mean titre

200

0 0

20 Dpv

Asia 1 Shamir

VNT titre at 20dpv from all groups 600

VNT Asia 1 Shamir

2.26/ 182.5

Full dose

1/4 dose

1/16 dose

Animal No

Control

Asia 1 Shamir

1414.83

SPCE Asia 1 Shamir at 20dpv from all groups

VNT Asia 1 Tur 49/11

120,00

500

Asia 1 Tur 49/11

400 300

1.68/ 47.8

80,00 60,00

100

40,00

Asia 1 Tur 49/11 1032.75

100,00

200

20,00 0,00

Full dose

1/4 dose

1/16 dose

Control

Animal No

Results - r1 values

Results - NSP ELISA

VNT titre from full dose group BVS NSP at 20dpv

100,00

NSP at 27/29dpv NSP at 35dpv

90,00

Asia 1 Shamir

Asia1 Tur 49/11

3.00 2.50 2.00

80,00

1.50

70,00

1.00

60,00

0.50 0.00 01/12

50,00

02/12

03/12

04/12

05/12

Animal ref no.

40,00 30,00

1.00

20,00

0.80

10,00

0.60

0,00

0.40

Pooled 01-05/12

Mean

r1 values from full dose group BVS vs Asia 1 TUR 49/11

0.20 0.00

Aniaml No

01/12

02/12

03/12

04/12

05/12

Pooled 01-05/12

Mean

Animal ref no.

Results - vaccine matching studies using high potency BVS

Conclusion and recommendation

Vaccine matching for serotype Asia 1 FMDV by 2dmVNT WRL FMD 2012 WRL REF

Asia1 IND 8/79

Asia1 Shamir

Asia1 Shamir(≥6PD50)

PAK 5/2012

PAK 91/2011

IRN 10/2012

IRN 54/2011

TUR 2/2012

TUR 65/2011

IRN 14/2012

IRN 23/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 27 Acknowledgements SAU/WRL/EURL FMD Geoff Hutchings Nigel Ferris Animal Unit

Defra and EC

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 28 Conclusions and Recommendations • In many cases high potency vaccines protect pigs against heterologous virus when vaccination is effective

TESTING THE EFFICACY OF O1 MANISA HIGH POTENCY VACCINE AGAINST CHALLENGE WITH O/VIETNAM/2010 (O MYA98 TOPOTYPE) IN PIGS

• Quality product • Effective cold chain • Careful administration

• Ropes show promise as surveillance tool for FMD infection in pigs

Vosloo, W.1, Morris, J 1, Nguyen, T.T.H.2, Kim, V.P. 2, Quach, V.N. 2, Le, T.T.P. 2, Dang, H2., Tran, X.H. 2, Pham, P.V.3, Vo, V.H. 3, Le, T.Q.A. 3, Mai, T.M. 3, Le, T.V.Q., Singanallur, N.1 BIOSECURITY FLAGSHIP 1 Australian Animal Health Laboratory,

5 Portarlington Road, Geelong, Australia Company, District 1, Ho Chi Minh City, Vietnam for Veterinary Diagnostics, Regional Animal Health Office No.6, Tan Binh dist., HCMC, Vietnam

2 National Veterinary 3 Center

FMD and Australia • The last outbreak of FMD was in 1872 • Trade advantage - stringent pre- and post border mitigation steps

FMD Risk Management Project • Vaccine efficacy testing and virus pathogenesis • Field validation of POC devices • Molecular epidemiology of FMD in SEA

• Australia has an antigen bank • Data needed on vaccine efficacy with heterologous challenge

Vaccine efficacy testing

• All live virus work - offshore

Relationship between the O1 Manisa (vaccine strain) with O VIT 05/2010 (OMya98 strain; challenge strain)

• Vaccinate with strains in Australian bank and challenge with SEA viruses • Progress on pig exp in Vietnam • Adapt O VIT 2010 (Mya 98 strain) in pigs • Perform first vaccine efficacy trial using O1 Manisa vaccine

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 28 Adapting O VIT2010 virus to pigs (AEC1465) • Successfully adapted the virus in 3 passes to pigs to reproduce infection via the heel bulb route

Testing O1 Manisa vaccine against O VIT2010 (AEC1497) • Vaccinate with high potency vaccine • Three groups of pigs • V7 : 5 pigs vaccinated 7 days prior to challenge • UVC7 : 5 pigs in close proximity • V4 : 5 pigs vaccinated 4 days prior to challenge • UVC4 : 5 pigs in close proximity • UV : 5 unvaccinated pigs challenged • UVC : 5 pigs in close proximity

Experimental lay-out

• Observe for 14 days •(>6PD50) Air samples once per day • Rope samples once per day on each side • Nasal swab, saliva and faeces samples once per day • Blood at regular intervals • Samples tested using qRT-PCR (Reid et al 2001) • Sera tested using LPBE, NSP and SPCE

Results of control group • Infected group (UV): • 1 pig died between 24-36 hours (heart lesions) • 3 pigs had lesions after 36 hours • 1 pig had lesions after 96 hours

• Contact group (UVC): • Day 9 - pigs broke partition and had direct contact • No lesions until day 13 - 1 pig with lesions • Day 14: no lesions, but heart lesions in one pig

Results of vaccine groups • Vaccinated 7 days before challenge • 3 pigs developed lesions at the site of inoculation • 1 pig with generalized disease at 2 DPC (temp 2-4 DPC) • RNA in saliva and nasal secretions from 1DPC up to 14 days • 80% protection

• None of the contact pigs (UVC7) showed disease • No viral RNA in secretions

• Vaccinated 4 days before challenge • 1 pig with generalized disease at 3 DPC (temp at 4-5 DPC) • 1 more pig had small lesion on the tongue at 4 DPC • Viral RNA in secretions 2 - 14 DPC • 60% protection

• None of the contact pigs (UVC4) showed disease • Viral RNA 3-4 DPC (intermittent) up to 14 days

Group

0 dpc

5 dpc

10 dpc

14 dpc

Negative

Positive

Negative

UVC

Negative

Positive

Negative

Positive

Negative

DEAD

UVC7

• From 1 DPC in infected animals • Only at Day 14 in contact animals

Group

Pig ID

0 dpc

5 dpc

10 dpc

14 dpc

PC001

Negative

Positive

PC002

Negative

Positive

Structural

7 dpc

PC003

Negative

Positive

PC004

Negative

Positive

PC005

Negative

Dead

Negative

PC001

Negative

Positive

PC002

Negative

Positive

Negative

PC003

Negative

Negative*

Negative

No Sample

Negative

Negative Positive

UVC

NSP

Negative Positive

Positive

PC004

Negative

PC005

Negative

PC001

Negative

Positive

PC002

Negative

Positive

Negative

Positive

PC003

Negative

Positive Negative

Negative

Positive

PC004

Negative

Positive

PC005

Negative

PC001

Negative

PC002

Negative

PC003

Negative

PC004

Negative

Negative V7

DEAD Negative

• Viral RNA in nasal secretions

Negative

UVC4

DEAD Negative

• From 1-14 DPC in infected animals • From 4 DPC in the contact animals

Negative

Negative V4

7 dpc

Negative Negative

• Viral RNA in saliva samples

Negative

Positive

Negative

Positive

Positive Negative Negative

UVC4

PC005

Negative

PC001

Negative

Positive

PC002*

Negative

Positive

PC003

Negative

Positive

PC004

Negative

Positive

Negative

Positive

PC005

Negative

Positive

Negative

PC001

Negative

Negative Negative

Negative

UVC7

Negative

PC002

Negative

PC003

Negative

PC004

Negative

PC005

Negative

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 28 Viral RNA in rope samples using qRT-PCR

Poster Agenda Item 8

Summary

Conclusions and Recommendations

• The O1 Manisa vaccine partially protected pigs from challenge with O/VIT/2010 as early as 4 DPV • Vaccination prevented clinical disease in the indirect contact groups • Viral RNA was present in the UVC4 group, but no disease occurred • No virus RNA was found in the UVC7 group • Ropes can be used for disease surveillance, but are more sensitive when virus loads are high

• In many cases high potency vaccines protect pigs against heterologous virus when vaccination is effective • Quality product • Effective cold chain • Careful administration

• Ropes show promise as surveillance tool for FMD infection in pigs

• More work is needed to improve on the collection methods

Acknowledgements • Chris Morrissy for helping with set-up and introductions • RAHO6 for lab space and technical assistance • NAVETCO for assisting with the animal work and providing the facilities • Funding provided by Meat and Livestock Australia • AHA for managing the contract and industry funding

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 29 Antibody titres in FMD virus serotype A strains: Comparison of methodologies to predict cross-protection

Conclusion 

LPB ELISA gives the best discriminatory capacity



LPB ELISA test demonstrated less variations when compared with VNT and NIT



LPB ELISA is recommended as an indirect serological method for vaccine matching purposes

Tesfaalem T.Sebhatu, Rob JM Moormann, Klaas Weerdmeester, Froukje van HemertKluitenberg, Aldo Dekker 29 October Jerez, Spain

Research goals:

Outline  

VP1 sequence of 10 selected FMDV type A strains Serological tests   

Virus neutralisation test (VNT) Neutralisation index test (NIT) Liquid Phase Blocking ELISA (LPBE)

 Comparison of test methodologies

 VNT, NIT, and LPBE  Which serological methodology confidence?



Statistical analysis



Mean titres Scaling titres  r1 values LPBE is a better test method than VNT and NIT

 Low variability within a group  Good discriminatory capacity

 

A/MAU/1/2006

Strains genetically different  Additional 3 strains from Asia topotype 



IRN/96 lineage • A/TUR/14/98 • A/IRN/2/97



A/SUD/2/84 A/ERI/2/98 A/ETH/13/2005

of vaccinated animals

Source of the 10 FMDV Serotype A strains used in the study WRLFMD, Pirbright, U.K.   

A/KEN/12/2005

gives the highest

 

A/ERI/2/98 A/SUD/2/84 A/KEN/12/2005 A/ETH/13/2005 A/MAU/1/2006

CVI, Lelystad, NL     

A22/IRQ/24/64 A/TUR/20/2006 A/TUR/14/98 A/IRN/2/97 A10/Holland/42

IRN/05 lineage • A/TUR/20/2006

A10/HOL/42 A22/IRQ/24/64

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 29 Serological test methods

Animal experiment

   

   



Virus neutralisation 

Vaccination in Eritrea 50 cattle plus 10 controls 10 different FMDV type A antigens



Neutralisation index 

BHK-21 BEI inactivated PEG concentration Sucrose gradient quantification of 146S



Fixed serum dilution versus variable virus concentration

Liquid Phase Blocking ELISA 

Vaccination with 10 µg antigen in 2 ml Aluminium hydroxide saponin adjuvant (formulation on site) Sera 21 day post-vaccination

Varying serum dilution versus fixed virus concentration

Using subtype specific Guinea-pig and Rabbit semipurified Ig fractions (saturated Ammonium Sulphate)

Statistical analysis

RESULTS



1 - A10/Holland/42

2 - A22/IRQ/24/64

Mean titres 1

9 10

3 - A ERI/2/98

9 10

4 - A ETH/13/2005

of the test strains are more easily neutralized by the vaccine sera than others, therefore scaling was applied

VNT (blue) NIT (red) LPBE (green) 



9 10

5 - A IRN/2/97

Huge dataset difficult to interpret

Mostly homologous titres are the highest (VNT: 6/10, NIT: 7/10 and LPBE: 8/10

Mean titres: Vaccine and test strain  Similar quality vaccines used  Neutralisation tests cannot be standardized, some

9 10

6 - A KEN/12/2005

 Due to low homologous and high heterologous responses, resulting r1 values are sometimes higher than 1 Vaccine mean titre

9 10

7 - A MAU/1/2006

9 10

8 - A SUD/2/84

Test strain mean titre

2,50

3,00 2,50

2,00

2,00 1,50 1,50 1

9 10

9 - A TUR/20/2006

9 10

10 - A TUR/14/98

VNT

1,00

0,50

9 10

Test strain

NIT 0,50

0,00

VNT

1,00

NIT

0,00

9 10

Vaccine

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Virus test strain

Appendix 29 1 - A10/Holland/42

2 - A22/IRQ/24/64

Statistical analysis using ANOVA

Scaled titres VNT (blue)

9 10

3 - A ERI/2/98

NIT (red)

9 10

4 - A ETH/13/2005

LPBE (green) 1



 

A clear difference in the scale of the response for each test strain For each test strain (titre-mean titre)/SD In all tests all homologous scaled titres are the highest

9 10

5 - A IRN/2/97

9 10

7 - A MAU/1/2006

9 10

8 - A SUD/2/84

9 10

9 - A TUR/20/2006

6 - A KEN/12/2005

9 10

10 - A TUR/14/98

9 10

Test strain

Statistical analysis using ANOVA

Testing for differences between vaccines VNT: (P=0.7) NIT: (p = 0.02) LPBE: (p << 0.001) SD per vaccinated group (5 cattle): VNT: 0.61 NIT: 0.43 LPBE: 0.37



LPBE has the best discriminatory capacity and lowest variation

9 10

The effect of scaling on r1 value Before scaling titre

After scaling titre

r1 values between 0-1 VNT 78%

r1 values between 0-1 93%

Using scaled titers 

Test

Testing for differences between vaccines VNT: (p = 0.09) NIT: (p = 0.0002) LPBE: (p << 0.00001)

Scaling improves discriminatory effect

Summary of the results 

Neutralisation tests (VNT & NIT) have high variability



NIT

92%

94%

LPBE

96%

Conclusion 

LPB ELISA gives the best discriminatory capacity

Scaled titres can reduce variations between and within tests



r1 values from VNT are difficult to interpret and not suitable for indirect vaccine matching as many values >1.0 are observed

LPB ELISA test demonstrated less variations when compared with VNT and NIT



LPB ELISA is recommended as an indirect serological method for vaccine matching purposes



LPBE has the best discriminatory capacity



LPBE has the lowest variation within a group of vaccinated cattle

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 29 Acknowledgement 



This work was funded by the International Atomic Energy agency (IAEA), Vienna, Austria We thank WRLFMD, The Pirbright Institute, UK for providing FMDV type A strains We would like to thank T. Kfleyesus, G.G. Egziabhier and T. Yemane, Animal Health staff,(MoA), Asmara, Eritrea for assisting in sample collection during animal experiment FAO, Representative Office in Eritrea

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 35 Virus Pools Pool 3

O, A, Asia 1

Local Differences in Circulation: What Have We Learnt from Patterns of FMD Persistence and Spread?

Pool 5

O, A, SAT 1, 2

Pool 1

O, A, Asia 1

Pool 7

Pool 2

O, A

O, A, Asia 1

Nick J. Knowles

Endemic

Molecular Characterisation & Diagnostics Group

Pool 4

Intermediate, sporadic

A, O, SAT 1, 2, 3

Free with vaccination Countries with multiple zones: FMD-free, free with vaccination or not free

Pool 6

SAT 1, 2, 3

Free. Virus present in game parks

Pool positions are approximate and colours indicate that there are three principal pools, two of which can be subdivided into overlapping areas

Free

East Asia – Pool 1

500 km

Pool 1 – FMDV type A

O, Asia 1

300 mi

Topotypes/lineages present:

Asia 1

ASIA / Sea-97

O, A O, A, Asia 1

O/CATHAY O/ME-SA/PanAsia O/ME-SA/PanAsia-2 O/SEA/Mya-98

1997

O, Asia 1

O, A O, A, O, A, Asia 1 O, A Asia 1

O/CATHAY

A/ASIA/Sea-97 Asia1/ASIA/Ind-76 Asia1/ASIA/Sea

O, A, Asia 1

1000 km

600 mi

A/VIT/2/2009 (HQ116378) A/VIT/8/2009 (HQ116384) A/HuBWH/CHA/2009 (JF792355) A/VIT/4/2008 (HQ116372) A/VIT/5/2008 (HQ116373) A/TAI/18/2008 (HQ116346) A/TAI/5/2009 (HQ116348) A/TAI/17/2008 (HQ116345) A/TAI/10/2009 (HQ116353) A/VIT/8/2008 (HQ116376) A/VIT/6/2008 (HQ116374) A/VIT/3/2009 (HQ116379) A/MAY/9/2009 (HQ116306) A/VIT/1/2009 (HQ116377) A/VIT/2/2008 (HQ116370) A/MAY/2/2009 (HQ116305) A/LAO/4/2008 (HQ116295) A/TAI/16/2008 (HQ116344) A/TAI/19/2008 (HQ116347) A/TAI/15/2008 (HQ116343) A/TAI/6/2009 (HQ116349) A/SKR/2/2010 (JQ070331) 99 A/Pochun/KOR/2010* (GU441855) A/VIT/3/2008 (HQ116371) A/VIT/5/2009 (HQ116381) A/VIT/6/2009 (HQ116382) A/VIT/7/2008 (HQ116375) A/TAI/13/2008 (HQ116341) A/TAI/14/2009 (HQ116354) A/TAI/4/2008 (HQ116336) 99 A/TAI/8/2009 (HQ116351) A/TAI/7/2009 (HQ116350) 92 A/VIT/1/2010 (JQ070332) 85 A/TAI/8/2008 (HQ116337) A/TAI/2/2006 (HQ116325) A/MAY/1/2008 (HQ116303) A/MAY/3/2007 (HQ116302) 99 A/TAI/11/2007 (HQ116335) A/CAM/1/2006 (HQ116292) A/CAM/2/2008 (HQ116294) 100 A/LAO/7/2006 (EU667458) A/LAO/1/2006 (2003)(EU667456) A/TAI/1/2006 (HQ116324) A/MAY/1/2007 (HQ116301) A/TAI/3/2007 (HQ116334) A/TAI/118/87* (EF208777) A/TAI/2/97 (EF208778)

0.01

O/MOG/3/2010 (JQ070310) O/MOG/4/2010 (JQ070311) O/MOG/56/2010* (JQ070326) O/MOG/1/2010 (JQ070308) O/MOG/2/2010 (JQ070309) O/RUS/Aug 2010‡ (JQ070330) O/MOG/CO3/2010* (JQ070325) O/MOG/5/2010 (JQ070312) O/MOG/7/2010 (JQ070314) O/MOG/77/2010* (JQ070328) O/MOG/6/2010 (JQ070313) O/MOG/9/2010 (JQ070315) 70 O/MOG/66/2010* (JQ070327) 99 O/TAI/12/2009 (HQ116260) O/TAI/13/2009 (HQ116261) O/MAY/20/2009 (HQ116218) O/TAI/19/2009 (HQ116266) O/TAI/23/2009 (HQ116270) O/VIT/2/2010 (JQ070322) O/LAO/1/2009 (HQ116182) O/TAI/3/2009 (HQ116258) O/MAY/8/2009 (HQ116216) O/TAI/18/2009 (HQ116265) O/MYA/2/2008 (HQ116226) O/TAI/4/2009 (HQ116259) O/TAI/5/2008 (HQ116252) O/TAI/2/2008 (HQ116250) O/TAI/6/2008 (HQ116253) O/TAI/2/2009 (HQ116257) O/LAO/2/2008 (HQ116180) O/MAY/5/2009 (HQ116213) O/LAO/1/2007 (HQ116175) 99 O/MYA/3/2008 (HQ116227) O/MYA/12/2009 (JQ070318) 86 O/TAI/20/2009 (HQ116267) 99 O/VIT/5/2010 (JQ070323) O/MYA/2/2006 (HQ116225) 100 O/MYA/3/2009 (JQ070317) O/MYA/1/2006 (JQ070316) 98 O/HLJOC12/03 (China) (DQ119643) O/MOG/2004 (JQ070324) O/MYA/3/2010 (JQ070319) O/MYA/11/2009 (HQ116232) O/MYA/5/2009 (HQ116228) O/TAI/22/2009 (HQ116269) 99 O/HKN/1/2010 (JQ070301) O/HKN/6/2010 (JQ070302) 72 O/HKN/19/2010 (JQ070305) 98 O/NC/CHA/2010 (HQ652080) O/MY/CHA/2010 (HQ652079) O/GZ/CHA/2010 (JN998086) O/SKR/4/2010 (JQ070320) O/VN/YB08/2010 (HQ260718) 72 O/HKN/7/2010 (JQ070303) O/HKN/9/2010 (JQ070304) O/HKN/1/2011 (JQ070306) 100 O/HKN/2/2011 (JQ070307) O/DY/CHA/2010 (HQ652078) O/CHA/31/2010* (JF792356) O/BY/CHA/2010 (JN998085) 96 O/TZ/CHA/2010 (HQ652081) O/JPN/MZ1/2010 (AB618503) O/RUS/Jul 2010† (JQ070329) O/SKR/5/2010 (JQ070321) O/TAI/189/87*

Pool 1 FMDV type O SEA / Mya-98 ‡

† 6

135 152

292

2 3

81 86

Southern Asia – Pool 2 © Daniel Dalet / d-maps.com

1000 km

Topotypes/lineages present:

600 mi

O (ME-SA/PanAsia) O (ME-SA/PanAsia-2) O (ME-SA/Ind-2001d) A (ASIA/unnamed) Asia 1 (ASIA/unnamed) O O, A, Asia 1

O O

0.01

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 35 O/BAN/25/2009 (HQ630686) O/BAN/27/2009 (HQ630688) O/BAN/26/2009 (HQ630687)

Pool 2

A/IND/128/2008* (HQ127712)

O/NEP/3/2010 O/BHU/19/2009 O/NEP/1/2010 O/BHU/8/2009 O/BHU/9/2009 O/BHU/10/2009 O/BAN/11/2009 (HQ630682) O/BHU/6/2009

O/APR190902/IND/2009 (JX070612) O/BHU/5/2009 O/NEP/15/2010 O/NEP/16/2010 O/BHU/4/2009 O/NEP/6/2010 O/BHU/7/2009

Iran 2009 (x1)

A/IND/226/2010* (HQ666873) A/IND/413/2007* (HQ127707)

O/BAN/9/2009 (HQ630681)

A/IND/245/2007* (HQ127704) A/IND/360/07* (FJ617243)

A/IND/360/2007* (HQ127705) A/IND/245/2007* (HQ832590) A/IND/13/2009* (2008) (HQ127718) A/IND/9/2009* (2008) (HQ127716)

A/IND/17/2009* (2008) (HQ127719) A/IND/17/2009* (HQ832592) A/IND/447/2005* (HQ127685) A/IND/447/2005* (HQ832583)

A/IND/152/2006* (HQ127700) A/IND/97/2006* (HQ127694)

A/IND/163/08* (FJ617242)

95 99

A/IND/136/2010* (HQ666878)

India 2005-2010

Ind-2001

A/IND/PD/283/2010* (HQ127731) A/IND/PD/135/2010* (HQ127730) A/IND/63/2010* (HQ666887) A/IND/PD/132/2010* (HQ127729) A/IND/61/2010* (HQ666881) A/IND/1/2010* (HQ666886)

A/IND/898/2009* (HQ127725) A/IND/749/2009* (HQ127724) A/IND/PD/98/2010* (HQ127728) A/IND/46/2010* (HQ666879)

O/ORKo0810/IND/2010 (JX070621) O/NEP/9/2010 O/UPMe1009/IND/2009 (JX070610)

A/IND/PD/94/2010* (HQ127727) A/IND/45/2010* (HQ666880)

Iran

ME-SA

O/KEKm2709/IND/2009 (JX070614) O/KAM0410/IND/2010 (JX070618) O/KEKm0610/IND/2010 (JX070619)

A/IND/53/08* (FJ617241)

A/IND/109/2006* (2005) (HQ127699)

A/IND/109/2006* (HQ832589)

Bhutan

O/CHRn0509/IND/2009 (JX070609)

O/TNNa1211/IND/2011 (JX070623) O/GUK2609/IND/2009 (JX070613) O/GUK2909/IND/2009 (JX070615)

85 99

O/UAE/4/2008 O/KUW/3/97 (DQ164904)

c a

O/OMN/7/2001 (DQ164941)

TN 2007 PanAsia-2 SRL 2011 Sri Lanka ? Southeast Asia

PanAsia-2

92 97 72

97 99

A/IND/26/2006* (HQ127688) A/IND/27/2006* (HQ127689)

A/IND/26/2006* (HQ832585)

A/IND/28/2006* (HQ127690) A/IND/106/2006* (HQ127698) A/IND/43/2006* (HQ127691)

A/IND/43/2006* (HQ832586)

Myanmar 1978

A/IND/152/2005* (HQ127683) A/IND/23/2006* (2005) (HQ127687) A/IND/22/2006* (2005) (HQ127686)

A/IND/22/2006* (HQ832584) A/IND/100/2005* (HQ127682) A/IND/50/2006* (2005) (HQ127692)

A/IND/50/2006* (HQ832587) 80

A/IND/195/2007* (HQ127702) A/IND/196/2007* (HQ127703) A/IND/49/2007* (HQ127701) A/IND/202/2010* (HQ666874) 99

72 94

O/UKG/35/2001 (AJ539141)

EA-3 EA-1

87 99

EA-2

99 99 99

99 78 73

A/IND/332/2009* (HQ127720) A/IND/332/2009* (HQ666884) A/IND/333/2009* (HQ666885) A/IND/333/2009* (HQ127721)

A/IND/744/2009* (HQ127722)

A/IND/744/2009* (HQ666883) A/IND/747/2009* (HQ127723)

Pak-98

Sri Lanka

ME-SA

O1/Manisa/TUR/69 (AY593823) 91

A/IND/192/2010* (HQ666877) A/IND/196/2010* (HQ666876)

O/IRN/67/2001 (DQ164897) O/Tibet/CHA/2/99 O/YEM/6/98

A/IND/197/2010* (HQ666875)

PanAsia

O/SAU/36/98 O/SAU/38/98 (AJ318852)

A/APR/51/05* (EF120400) A/APS/50/05/1* (IND)(EU109791)

A/IND/59/2006* (HQ127693)

2000 Bangladesh

O/ORC2103/IND/2003 (JX070597) O/TUR/2/2001 (DQ164985) 99 O/TUR/586/06/01* (DQ296517)

O/PAK/16/2003 (DQ165068) O/IND/53/79 (AF292107) O/IND/R2/75* (AF204276)

SRL 1999-2010 unnamed lineage possibly from Southeast Asia

A/IND/101/2006* (HQ127697)

O/POP14402/IND/2002 (JX070594) O/RAB0503/IND/2003 (JX070595) O/WBN13102/IND/2002 (JX070591)

A/IND/100/2006* (HQ127696)

O/APMb2205/IND/2005 (JX070604) O/APG10205/IND/2005 (JX070606) O/MAP0711/IND/2011 (JX070622)

A/IND/99/2006* (HQ127695)

2010

O/IND/77/00* (PD-FMD) 99

PanAsia

India 2011

India

Iran-2001

O/IRN/61/2001 (DQ164896)

A/APS/68/05* (EF120403) A/IND/174/2005* (HQ127684)

A/APS/45/05* (IND)(EU109792)

O/NEP/2/2009 (HQ630694) O/NEP/3/2009

70 99

Bangladesh

A/APS/44/05* (EF120402)

1997 2003 Bhutan

O/BAN/1/2009 (HQ630676) O/KEPt0710/IND/2010 (JX070620) 99

A/APS/66/05* (IND)(EU109788) A/APS/59/05* (IND)(EU109789)

O/PUJ0112/IND/2012 (JX070624) O/NEP/5/2008 (HQ630693) O/NEP/7/2008

ASIA

A/APS/58/05* (IND)(EU109790) 99

O/BAN/30/2009 (HQ630691) O/BAN/31/2009 (HQ630692) O/NEP/7/2010

India 2005-2010

A/IND/53/2008* (HQ127709) A/IND/88/2006* (HQ832588)

O/NEP/12/2010 O/BAN/28/2009 (HQ630689) O/BAN/29/2009 (HQ630690)

India

A/IND/1/2010* (HQ127726)

99 75

O/BHU/20/2009 O/BHU/23/2009

A/IND/163/2008* (HQ127713)

A/IND/PD/286/2010* (HQ127732) 89

O/BHU/26/2009

A/IND/152/06* (FJ617247)

99 94

A/IND/16/2005* (HQ127681)

O/BHU/40/2009 O/NEP/11/2010 O/BHU/25/2009

Iran

A/IND/11/2009* (2008) (HQ127717)

O/BHU/41/2009 O/BHU/37/2009

A/IND/417/2007* (HQ127708)

O/KAM0210/IND/2010 (JX070617) O/IRN/72/2009 O/BAN/2/2009 (HQ630677)

A/IND/437/2008* (HQ832591)

A/IND/438/2008* (HQ127715) A/IND/407/2007* (HQ127706)

O/KAM0110/IND/2010 (JX070616)

A/IND/437/2008* (HQ127714) 97

Serotype: A Topotype: ASIA

O/APR190901/IND/2009 (JX070611) 99

A/IND/124/08* (FJ617244) A/IND/124/2008* (HQ127711) 98

O/BHU/18/2009 O/NEP/17/2010 O/BAN/23/2009 (HQ630684)

Ind-2001d

Serotype: O Topotype: ME-SA

A/IND/123/2008* (HQ127710)

Pool 2

O/NEP/5/2010 O/BHU/3/2009 O/BHU/16/2009

India 2009-2012 Bangladesh 2009 Bhutan 2009 Nepal 2008, 2010

A/IND/128/08* (FJ617246)

O/BHU/2/2009

SEA

A/IRN/22/99 (EF208772) 99

A22 Iran-99

Sea-97

Type A has never been recorded in Sri Lanka

WA EA-4

A/IND/747/2009* (HQ666882)

Iran-96 A22/IRQ/64 (AY593763)

Thai-87 Iran-87 99

Iran-05 A15/Bangkok/TAI/60 (AY593755) A23/Kitale/KEN/64 (AY593766) A11/GER/29 (AGB)(EU553852)

CATHAY ISA-1 ISA-2 EURO-SA

EURO-SA AFRICA

0.02

Asia1/IND/396/01* (PD-FMD) Asia1/IND/397/01* (PD-FMD) Asia1/IND/387/01* (PD-FMD) Asia1/IND/438/01* (PD-FMD)

Pool 2

Middle East – Pool 3

Asia1/IND/388/01* (PD-FMD) Asia1/IND/423/01* (PD-FMD) Asia1/IND/389/01* (PD-FMD) Asia1/IND/139/02* (DQ101242) Asia1/IND/342/01* (PD-FMD) Asia1/IND/335/01* (PD-FMD) Asia1/IND/175/2004* (DQ101237) Asia1/IND/354/01* (PD-FMD) Asia1/IND/373/01* (PD-FMD) Asia1/IND/60/02* (DQ101243) Asia1/IND/61/02* (PD-FMD) Asia1/IND/180/02* (2001) (PD-FMD)

Serotype: Asia 1 Topotype: ASIA

Asia1/IND/141/02* (PD-FMD) Asia1/IND/198/02* (DQ101244) Asia1/IND/328/2004* (PD-FMD)

Asia1/IND/388/2004* (DQ101235)

100

Asia1/IND/389/2004* (PD-FMD)

Group III

Asia1/IND/140/01* (PD-FMD) Asia1/IND/148/01* (PD-FMD)

1000 km

Topotypes/lineages present:

Asia1/IND/149/01* (PD-FMD) Asia1/IND/160/01* (PD-FMD)

600 mi

Asia1/IND/114/2004* (2003)(DQ101239) Asia1/IND/147/2004* (2003)(PD-FMD) Asia1/IND/168/2004* (PD-FMD)

Asia1/IND/153/2004* (2003)(PD-FMD)

Asia1/IND/150/2004* (2003)(PD-FMD) Asia1/IND/165/2004* (DQ101238) Asia1/IND/158/2004* (2003)(PD-FMD) Asia1/IND/156/2004* (2003)(PD-FMD)

O (ME-SA/PanAsia) O (ME-SA/PanAsia-2) O (ME-SA/Ind-2001)

Asia1/IND/322/2004* (PD-FMD) Asia1/IND/268/2004* (DQ101236) Asia1/IND/325/2004* (PD-FMD)

100

Asia1/IND/327/2004* (PD-FMD)

Asia1/IND/152/01* (PD-FMD) Asia1/IND/673/2003* (DQ101241) Asia1/IND/762/2003* (DQ101240)

Asia1/IND/763/2003* (PD-FMD)

Iran

Asia1/IND 95/08* (HQ224558) Asia1/IND 137/08* (HQ224561) 100

Asia1/IND 96/08* (HQ224559) Asia1/IND 93/08* (HQ224557)

Asia1/IND 97/08* (HQ224560) Asia1/IND 121/07* (HQ224554) Asia1/IND 227/07* (HQ224555) Asia1/IND 32/08* (HQ224556)

1997 2002 Bhutan

Asia1/IND 12/07* (HQ224553)

O, A, Asia 1

Asia1/IND/155/2010* (JN247565)

Asia1/IND/509/2010* (JN247566)

O, A

Asia1/IND/321/01* (AY687333)

India

Asia1/IND/14/95* (AF390678)

1996 Bangladesh

Asia1/IRN/10/2004 (DQ121119)

Asia1/IND/367/00* (PD-FMD)

100

Asia1/IND/387/00* (PD-FMD)

Asia1/IND/248/01* (PD-FMD) Asia1/IND/383/00* (PD-FMD) Asia1/IND/384/00* (PD-FMD)

100

Asia1/IND/379/00* (PD-FMD) 73

O, A

A (ASIA/Iran-05)

O, A, Asia 1 O, A, Asia 1 O, A O, A Asia 1 Asia 1 O, A, O, A, O, A Asia 1 Asia 1 O

Asia1/BAR/8/2009 Asia1/IND/18/2011* (JN247567) Asia1/IND/107/01* (PD-FMD)

Asia1/IND/378/00* (PD-FMD) Asia1/IND/374/00* (PD-FMD) Asia1/IND/377/00* (PD-FMD) Asia1/IND/51/01* (PD-FMD) Asia1/IND/52/01* (PD-FMD) Asia1/Shamir/ISR/89

Asia 1 (ASIA/Sindh-08)

Asia1/IND/8/79 (EU553910) Asia1/IND/4/2004* (2003)(PD-FMD)

Asia1/WBN/117/85 (EU553911)

100

Asia1/HKN/19/74 Asia1/AFG/1/2001 (DQ121109)

Group I

Asia1/PAK/8/2008

100

Asia1/PAK/26/2009

Asia1/IRN/1/2012

Asia1/TUR/1/2012

100

Asia1/TUR/49/2011 Asia1/AFG/39/2011

Sindh-08

Asia1/PAK/1/2012 Asia1/PAK/106/2010

Sri Lanka

Asia1/BAR/1/2011 Asia1/IRN/33/2011 Asia1/PAK/6/2011

Type Asia 1 has never been recorded in Sri Lanka

100

Asia1/IND/2/71 (1964) Asia1/IND/63/72* (AY304994) Asia1/YNBS/CHA/58 (AY390432) Asia1/PAK/1/54 (AY593795) Asia1/IND/16/76 Asia1/IND/18/80 (DQ121116)

100 98

Group V

Asia1/IND/15/81 (DQ121117)

0.02

Pool 3 – O, A & Asia 1

PanAsia-2

FAR-09 BAL-09 PUN-10 100 O/IRN/8/2005

Asia 1 1973-1974 1983-1984 1999-2000 2011-2012

O/PanAsia

Kazakhstan Turkey

Georgia Azerbaijan Armenia

Kyrgyzstan O-PanAsia-2

Lebanon Syria

Kuwait

O-PanAsia-2

Tajikistan

Israel Jordan Iraq

A-Iran-05HER-10

TER-08 (O/Erzincan/257/2008) YAZ-09 (O/IRN/7/2009) FAR-09 (O/Agri/71/2010) SAN-09 (O/Sanliurfa/264/2009) BAL-09 (O/IRN/18/2010) ANT-10 (O/Gaziantep/153/2010) PUN-10 (O/PAK/16/2010)

100

73 100

Iran

Bahrain Saudi Arabia Qatar UAE

Asia1-Sindh-08

100

Yemen

PanAsia-2

SAN-09

Sub-lineages of O PanAsia-2

A-Iran-05

ANT-10

YAZ-09 O/UKG/35/2001 (AJ539141) O/SRL/1/2009 O/BAN/3/2009 99 O/BAN/4/2009 O/BAN/5/2009 O/BAN/24/2009 O/NEP/10/2009 O/BAN/20/2009 O/NEP/6/2009 O/NEP/15/2009 O/NEP/14/2009 100 O/NEP/11/2009 O/NEP/13/2009 O/IRN/61/2001 (DQ164896)

PanAsia

ME-SA

Iran-2001

Ind-2001 O/PAK/16/2003 (DQ165068) O/IND/53/79 (AF292107) O/IND/R2/75* (AF204276) O1/Manisa/TUR/69 (AY593823)

Pak-98

EURO-SA

0.02

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 35 FMDV O PanAsia-2 sub-lineages - 2009 East 54 84 Azerbaijan

60 62 64 70 71

7980 85 61 86

Zanjan Kordestan Kermanshah

Tehran 83

Hamedan

Khuzestan

56 87 88 Esfahan

7 40 57 89

Kohgiluyeh and Boyer-Ahmad

91 96 Tehran 75

Markazi 78 79 88 Lorestan

Pakistan

Khuzestan 83

113

86 Qom133 101 140 143 99

120

19 20 18

102

Razavi 64 66 69 Khorasan

Semnan

8 10 54 4 35 44 51 3Esfahan

95 104 105

Chaharmahal 33 59 and Bakhtiari 130

37 38

157

Kohgiluyeh and Boyer-Ahmad

Sistan and Baluchistan

Hormozgan

Saudi Arabia

100 mi © Daniel Dalet / d-maps.com

Virus Pools – Africa

13 14 22

>15% animals affected (2010) Mortalities observed (2010) PanAsia-2YAZ-09 PanAsia-2FAR-09 PanAsia-2ANT-10 PanAsia-2BAL-09 200 km PanAsia-2SAN-09 100 mi © Daniel Dalet / d-maps.com PanAsia-2PUN-10

Pakistan

5 Kerman 47 26

97 138 144

Fars

131

Afghanistan

39 43 South 106 141 Khorasan

2 11 12 Yazd 156

Location unknown 160 161 162 170 172 173

North 71 89 Khorasan

132 139 147

200 km

48 49 50 15 150 Hormozgan

124 129 Sistan and Baluchistan 154 155

United Arab Emirates

East Africa – Pool 4 Pool 4 – East Africa (O, A, SAT 1, SAT 2, SAT 3)

SAT 2

In Africa there are currently three FMD virus pools loosely defined as covering East Africa (Pool 4), West Africa (Pool 5) and Southern Africa (Pool 6). There is some overlap between Pools 4 and 5. It has been suggested to extend pool 4 southwards to include Tanzania and to contract Pool 6 to exclude that country. Thus Pool 6 contains only the SAT serotypes.

•

100

75 76 72Kerman

Fars

•

65 Hamedan

23 24 17 27 29 30 31 52 55 Mazandaran

146 158 127 142 Qazvin 148 149

South Khorasan

Yazd

Chaharmahal and Bakhtiari

Numbers represent WRLFMD Ref. Nos. (e.g. 88 = O/IRN/88/2009)

Kordestan 137

Afghanistan

126 Zanjan

82 109

Kermanshah 67

31 41 43

PanAsia-2YAZ-09 PanAsia-2FAR-09 PanAsia-2ANT-10 PanAsia-2BAL-09 PanAsia-2SAN-09 Ind-2001

135 East Azerbaijan 136

114 115 128

Khorasan

28 34 40

94 98

81Razavi 35 38 51 52

Semnan 42

Qom

Markazi Lorestan

Turkey

North Khorasan

66 65 68

Mazandaran 49 34

Qazvin

FMDV O PanAsia-2 sub-lineages - 2010

N.J. Knowles, 25 May 2010 (updated 09 August 2010)

Turkey

O, SAT 2 O, A, SAT 2

SAT 2

Eritrea Sudan O, A O O, A, O, A, SAT 2 SAT 1, O Central South SAT 2 Ethiopia Afr. Rep. Sudan O, A, O, A, Uganda Equatorial SAT 1,SAT 1, Guinea Gabon Congo O, A SAT 2 SAT 2 Kenya O, A, DRC SAT 1, SAT 2 Tanzania Chad

In the countries of the Maghreb (except for Mauritania) FMD is normally absent, however, periodic incursions occur either from the Middle East (Pool 3), West Africa (Pool 5) or more recently from Pool 4.

Burundi, Democratic Republic of the Congo, Djibouti, Eritrea, Ethiopia, Kenya, Rwanda, Somalia, Sudan, South Sudan, Tanzania, Uganda (with periodic spill-over into Egypt, Libya, northern Malawi, northern Zambia and the Yemen Arab Republic). Topotypes/lineages present: O (EA-1, EA-2, EA-3, EA-4) A (AFRICA/G-I, G-IV, G-VII) SAT 1 (I, IV, VI, VII, VIII, IX) SAT 2 (IV, VII, VIII, IX, X, XII, XIII, XIV) SAT 3 (V) SAT 1 topotype I is thought to have spread from southern Africa into Tanzania and Kenya (the only countries in East Africa where this topotype has been recorded) during the early 1970’s.

There is little or no information on FMD in Chad, the Central African Republic, Equitorial Guinea and Gabon. 1000 km 600 mi © Daniel Dalet / d-maps.com

East Africa – Pool 4

West Africa – Pool 5 Pool 5 – West Africa (O, A, SAT 2)

EA-3

EA-3 EA-3

Type O

EA-4

G-IV

EA-3

G-VII

Type A

EA-4 EA-4

EA-1 EA-2 EA-2EA-2

G-I

EA-2

Uganda

G-I G-VII G-I

G-I

O, SAT 2

Type SAT 1 IV

I I

O, A

G-I

VII X IV

IX IV

Chad

Central Afr. Rep.

Benin, Burkina Faso, Cameroon, Cote d'Ivoire, The Gambia, Ghana, Guinea, Guinea-Bissau, Liberia, Mali, Mauritania, Niger, Nigeria, Senegal, Sierra Leone, Togo (with periodic spill-over into Sudan). Although FMDV type SAT 1 has occurred in West Africa, the serotype has not been isolated from the region for 30 years [what about serology?]. Topotypes/lineages present: O (WA) A (AFRICA/G-VI) SAT 1 (V, VI) SAT 2 (V, VI, VII)

XIII

VIII IV

O, A, SAT 2 O, A, SAT 2

Equatorial Guinea Gabon Congo

VII

Type SAT 2

O, SAT 2

A/G-IV: various

O, A

VII XIII

VI Type SAT 3

G-IV

1000 km 600 mi © Daniel Dalet / d-maps.com

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 35 West Africa – Pool 5

Southern Africa – Pool 6

Type O WA WA WA

Type A WA

WA WA

G-IV G-VI G-VI

G-VI G-VI

WA WA WA

Pool 6 – Southern Africa (SAT 1, SAT 2, SAT 3)

G-VI

EA-3

G-VI G-VI

Angola, Botswana, Lesotho, Malawi, Mozambique, Namibia, South Africa, Swaziland, Zambia, Zimbabwe

G-IV G-IV G-IV

Topotypes/Lineages present: SAT 1 (I, II, III) SAT 2 (I, II, III, XI) SAT 3 (I, II, III, IV)

VII

VII Type SAT 2

VII 1000 km

VII

600 mi © Daniel Dalet / d-maps.com

Pool 6

Angola

Angola Zambia

Zambia

Malawi Mozambique

Zimbabwe

Namibia

Malawi Mozambique

Namibia Botswana

Botswana

Swaziland

South Africa

Swaziland

Lesotho

South Africa

Lesotho

FMD hotspots

Pool 6

South America – Pool 7 IV

III III

III

I II II

III III III II III I I I

O, A

IV I

Topotypes/lineages present:

O, A O, A

O/EURO-SA A/EURO-SA C/EURO-SA (not since 2004)

O, A

O, A, C O, A

IV II II

III

O, A

I I I 1000 km 600 mi

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 35 Questions (1)

Questions (2)

•

What are the precise mechanisms which result in the generation and expansion of new FMDV variants in Western Asia?

•

Is FMDV SAT 3 widespread in buffalo herds outside of Southern Africa as serology suggests?

•

Are West African buffalo persistently infected with FMDV and if so with which serotypes?

•

Why do we see relatively few African-Asian virus transfers?

•

Does SAT 1 occur in West Africa (not recorded in that region since 1981)?

•

No Asia 1 in Africa and few SAT outbreaks in Asia – why?

•

Which FMD viruses occur in Chad, Equatorial Guinea, Gabon and the Central African Republic?

Regular transfer of type O viruses from the Horn of Africa to Yemen, but not other serotypes – why?

•

What role do African buffalo play in the maintenance of FMD in East African cattle?

Are there any reservoirs of FMDV type C (which has not caused an outbreak since 2004)?

Acknowledgements Jemma Wadsworth Begoña Valdazo-González Antonello Di Nardo

SE 2939

Valerie Mioulet Claudia Doel Miki Madi Don King Jef Hammond David Paton Nigel Ferris (retired) Geoff Hutchings (retired)

World Organisation for Animal Health

Keith Sumption Botswana Vaccine Institute Lanzhou Veterinary Research Institute Onderstepoort Veterinary Institute …and many others…

The Pirbright campus is being redeveloped

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 36 (Limited) transmission of Foot-andMouth Disease virus from infected sheep to calves Carla Bravo de Rueda, de Jong M.C.M., Eblé P. L., van Hemert-Kluitenberg F., Dekker A.

Outline

 Aim: ● To quantify FMDV transmission from infected sheep to cattle

 Why: ● The proximity of sheep and cattle in the field ● The high susceptibility of cattle

 How: ● Two infected sheep in contact with one calf ● Laboratory tests and quantification of transmission

 Conclusion: ● Transmission from infected sheep to cattle occurred (R0 >1)

Interspecies transmission of FMD

Full screen image with title

Factors of risk

Sheep may not manifest clear clinical signs Sheep secrete and excrete considerable amounts of FMDV Sheep secrete and excrete FMDV for a long period Sheep and cattle are brought into close proximity Cattle are high susceptible to FMD

Material and methods

Results Laboratory tests

Animals:

●20 sheep inoculated with FMDV Asia1 Tur/11/2000 ●10 contact exposed calves

Samples and laboratory

tests:

●OPF and blood were analysed by Virus titration ●Probang (29, 30 and 31 dpi) and OPF were analysed by RT-PCR ●Serum were analysed by NS ELISA and VNT

Quantification

of transmission

●The next-generation matrix

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 36 Sp.

VI OPF

VI Blood

PCR OPF

PCR Probang

NSELISA

VNT

C.S.

Infected

Cattle Sheep Sheep

+ +

+ -

+ +

NO YES YES

Cattle Sheep Sheep

+ +

+ -

+ +

+ + +

+ -

YES YES YES

Cattle Sheep Sheep

+ +

NO YES YES

Cattle Sheep Sheep

+ +

+ -

+ +

NO YES YES

Cattle Sheep Sheep

+ +

NO YES YES

Cattle Sheep Sheep

+ +

NO YES YES

Cattle Sheep Sheep

+ + +

YES YES YES

Cattle Sheep Sheep

+ +

NO YES YES

Cattle Sheep Sheep

+ +

+ -

+ + +

+ +

+ + +

+ -

YES YES YES

Cattle Sheep Sheep

+ +

+ + +

+ +

YES YES YES

Quantification of transmission

Conclusions Laboratory tests

 100%

of the sheep (N=20) shed FMDV and became serological positive

 80% showed

clinical signs and 45% became carriers

How infectious are sheep compared to cattle?

 Only 1 or 2 calves (N=10) shed FMDV and 4 calves became serological positive

 1 calf showed

clinical signs and 1 calf became carrier

How susceptible are sheep compared to cattle?

 In average

2 calves get infected by 1 infected sheep

Would subclinical infected calves transmit FMD?

Quantification of transmission

 R0 is the average number of new cases caused by one typical infected individual in a susceptible population

 Previously R0 has been quantified in homologous

populations (i.e. sheep to sheep, cattle to cattle): R0 = 1.14 R0 = 14

 In our experiment a partial R0 was quantified:  We need to quantify transmission in heterologous

R=2

populations

 We used the Next-generation

matrix

Quantification of transmission

Results

The Next-Generation Matrix (NGM)

Quantification of transmission

 R0 is the dominant eigenvalue

of the next-generation matrix (Diekmann et al, 1990) wherein the typical infected individual is in the eigenvector

0.57

3.99

R0 = dominant eigenvalue of this matrix = 7.57

 

In a 50% cattle and 50% sheep population. We used Final Size estimates from experiments with Asia1 Tur/11/2000

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 36 Final conclusions

 Transmission occurred between infected sheep and naive calves (partial R0 = 2; R0 = 7.57 in a 50%s-50%c population)

 Considering

that cattle are highly susceptible to FMD, we found an unexpected “limited” transmission

 We estimated that sheep cattle

are 7 times less infectious than

 And that sheep are 0.57 times less susceptible than cattle

 It is unclear if subclinical animals could transmit the disease

Thank you for your attention

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 37 FMDV infection in non-vaccinated and vaccinated sheep Transmission to contact sheep and diagnostic aspects

Phaedra Eblé, Karin Orsel, Aldo Dekker

Introduction

 FMD infections in sheep ● Subclinical/unnoticed infections, risk?

 Transmission ● Self-limiting?

 Vaccination ● Added value? Example (one of many):

 Diagnosis ● which samples

Material and Methods: Animal experiments

Material and Methods: Samples and Tests

 Transmission experiments

 Clinical signs (daily): fever, lameness, lesion score  Virus detection:

● Non-vaccinated groups (6 groups 2Ix2C) ● Vaccinated groups (6 groups 2Ix2C) ● Control group: 4 vaccinated control sheep

 Vaccination ● Two weeks (-14dpi) before challenge ● Asia-1 Shamir

● OPF-swabs (daily 0-22 dpi): virus isolation (VI) ● plasma samples (daily 0-9 dpi): VI ● probang samples (31-32-33 dpi): RT-PCR

 Serology: ● serum samples (twice/week): VNT and NS-ELISA

 Challenge: ● Asia-1 Turkey 11/2000

 Comparison of detection methods ● infected sheep ● acute infections

Transmission and viral load

Results: Clinical signs

 Transmission

 Non-vaccinated groups (n=24):

● Final Size Method  Reproduction ratio R ● Homogeneity of occurrence of transmission

 Viral load pens ● Non-vaccinated vs vaccinated ● Weeks p.i. ● Pens with vs without transmission

● Fever 10 ● Lameness 3 Coinciding foot-rot 24 infection ● Lesions

 Vaccinated groups (n=24): ● Fever 0 ● Lameness 0 Coinciding foot-rot 24 infection ● Lesions

Open Session of the EuFMD: 2012,Jerez de la Frontera, Spain

Appendix 37 Results: VI and PCR non-vaccinated groups

Results: VI and PCR non-vaccinated groups All non-vaccinated sheep

Inoculated sheep

Non-vaccinated groups

Non-vaccinated groups dpi

C C I I

3.1

C C I I

2.9 2.3

C C I I

0.4 2.4

2.7 2.0

2.9 3.8

2.1 3.4

3.2 3.2

4.1 2.2

4.0 2.3

2.0 2.7

3.5 2.1

3.6 2.5

VI OPF and VI plasma 10 11 12

2.8 1.8

2.1 1.9

1.6

2.4 -

1.9 -

1.2

2.3 1.9

3.8 2.3

2.0 2.2

1.9 2.6

3.5 2.2

2.2 2.1

2.0 3.0

2.0 2.2

2.3 1.7

2.3 2.5

1.2 2.2

1.0

1.9 1.4

1.6 2.4

1.5

1.6 -

1.8 1.0

0.9

0.9 0.4

1.9 1.9

1.9 2.2

0.9 2.1

1.0 -

2.1 2.1

1.7

1.0

2.1 2.1

2.5 2.1

1.5

Probang 31-33

dpi

C C

0.7

1.9 -

0.7 -

2.1 2.2

2.1 1.7

1.6 2.2

1.2 1.2

2.0 1.4

1.8 0.4

1.7 2.0

0.7

0.7 0.9

+ +

I I

3.1

2.9 3.8

c 4.0 2.3

2.8 1.8

2.1 1.9

1.6

2.4 -

1.9 -

1.2

1.6 -

0.9

1.0

2.1 2.2

2.1 1.7

1.6 2.2

1.2 1.2

2.0 1.4

1.8 0.4

1.7 2.0

0.7

0.7 0.9

C C I I

2.9 2.3

2.1 3.4

2.0 2.7

2.3 1.9

1.9 2.6

2.0 3.0

2.7 2.9 2.3 2.5

2.8 2.9 1.9 1.4

3.1 2.7 1.8 1.0

1.2 1.2 0.9 0.4

1.1 1.9 2.2

0.4 2.1 2.1

2.2 1.7

2.2 2.4 2.0

1.9 0.7 2.0 1.6

1.5 0.7 2.3 2.6

1.7 1.8 2.0 2.1

2.1 1.8 0.7 1.8

1.9 1.9 1.2 1.9

2.1 1.0 1.9 2.3

1.7 1.7 1.5

C C I I

0.4 2.4

3.2 3.2

3.5 2.1

3.8 2.3

3.5 2.2

2.0 2.2

1.2 2.2

1.6 2.4

1.9 1.9

0.9 2.1

2.1 2.1

2.5 2.1

2.7 2.7

2.8 1.9

2.2 1.7

3.2 2.3

2.4 2.1

2.1 1.9

2.2 1.7

0.4 -

1.2 1.1

C C I I

2.7 2.0

4.1 2.2

3.6 2.5

2.0 2.2

2.2 2.1

2.3 1.7

3.0 1.0

3.7 1.5

2.9 1.0 -

2.1 -

1.9 1.7

0.4 1.5

0.7 1.4

1.4 2.2

1.1 1.8

1.2 1.8

0.4 1.8

0.9 1.9

1.9 0.4

0.7 2.3

1.0 1.4

C C I I

4.0 2.4

5.0 -

2.8 -

2.0 -

1.8 1.9

1.8 1.7

2.2 -

1.9 -

3.0 -

2.2 -

2.0 -

2.6 -

2.2 -

1.7 -

2.2 -

2.1 -

1.7 -

C C I I

2.2 3.2

1.4 3.5

2.2 3.0

2.4

1.4 1.9

2.3

0.4

1.7

0.9

1.9

0.9

0.4

1.7

0.7

2.4

0.7

0.4

2.2 1.7

2.7 2.7

1.4

2.4 2.0

2.8 1.9

2.2

2.0 1.6

2.2 1.7

1.8

2.3 2.6

3.2 2.3

1.8

2.0 2.1

2.4 2.1

1.8

0.7 1.8

2.1 1.9

1.9

1.2 1.9

2.2 1.7

0.4

1.9 2.3

0.4 -

2.3

1.7 1.5

1.2 1.1

1.4

4.0 2.4

5.0 -

2.8 -

2.0 -

1.8 1.9

1.8 1.7

2.2 -

1.9 -

3.0 -

2.2 -

2.0 -

2.6 -

2.2 -

1.7 -

2.2 -

2.1 -

1.7 -

C C I I

2.2 3.2

1.4 3.5

2.2 3.0

2.4

1.4 1.9

2.3

0.4

1.7

0.9

1.9

0.9

0.4

1.7

0.7

2.4

0.7

0.4

+ +

+ + + -

Results: VI and PCR vaccinated groups

C C I I C C I I C C I I C C I I

2.1 2.2

3.1

2.7

1.2

2.3

1.6 1.9

2.1 -

0.9 2.0

+ +

Vaccinated groups

All vaccinated sheep

Vaccinated groups

2.5

Results: VI and PCR vaccinated groups

Inoculated sheep

+ +

Contacts: 5 contact infections, 3x viraemia All: 11/17 VI OPF positive at 21 dpi, 10/17 carriers

All 12 inoculated sheep infectious, 9x viraemia

C C I I

Probang 31-33

C C I I

dpi C C I I

VI OPF and VI plasma 10 11 12

1.9

0.4 -

1.2 2.1

2.3

2.9

2.2 1.8

2.1 3.8

1.8 -

2.4

3.0

2.2

2.1 4.5

2.0 2.9

1.8

1.4

1.9 0.4

1.2

2.4 1.7

1.4

1.8

1.9 2.1

0.7 -

1.9 1.9

1.2

1.8

2.1 -

1.1 2.2

2.8 2.1

1.5

0.9 -

0.7

2.2 1.5

VI OPF and VI plasma 10 11 12

0.7

1.4

2.7

1.4

1.4 -

2.2

1.7 1.8

1.7

2.3 1.2

1.0 2.2

2.2 1.9

1.9 0.9

2.2

1.4 -

2.6

2.1 2.1

1.9

1.5

1.3

1.2

0.9

0.4

0.9

0.7

2.0

1.9 -

0.7 1.4

2.2 1.6

2.2

1.8

2.1 -

1.9

1.2 1.6

1.5

1.4 -

2.1

1.2 0.4

1.4

1.9 -

2.1

1.5 -

1.3

1.4

2.1 1.0

1.0

1.7

1.6 -

2.0

1.0 0.7

1.0

1.2 -

1.4 1.2

0.4 -

1.2

0.9 -

0.9 1.3

Probang 31-33

+ +

VI OPF and VI plasma 10 11 12 0.7 -

dpi C C I I

0 -

1 2.1 2.2

2 3.1

3 2.7

4 1.2

5 -

6 -

7 -

8 -

9 -

C C I I

2.5

2.3

2.4

1.8

1.4

1.2

1.4

C C I I

2.3

1.9

2.9

3.0

1.4

1.8

1.5

2.7

1.4

C C I I

1.6 1.9

0.4 -

2.2 1.8

2.2

1.9 0.4

1.9 2.1

2.1 -

0.9 -

1.4 -

C C I I

2.1 -

2.1 3.8

2.1 4.5

1.2

0.7 -

1.1 2.2

0.7

C C I I

0.9 2.0

1.2 2.1

1.8 -

2.0 2.9

2.4 1.7

1.9 1.9

2.8 2.1

2.2 1.5

Probang 31-33

13 1.9

14 1.9

15 1.5

16 1.5

17 1.3

18 1.2

19 0.9

20 0.4

21 0.9

2.2

0.7

2.2

1.4

1.0

1.2

2.2

2.0

1.8

1.5

1.4

1.3

1.7

1.4 -

1.9 -

2.1 -

1.4 -

1.9 -

1.6 -

1.2 -

0.4 -

0.9 -

1.7 1.8

1.7

1.0 2.2

2.6

0.7 1.4

1.9

2.1

1.4

2.0

2.3 1.2

2.2 1.9

1.9 0.9

2.1 2.1

2.2 1.6

1.2 1.6

1.2 0.4

1.5 -

2.1 1.0

1.0 0.7

1.4 1.2

0.9 1.3

+ -

+ + +

Contacts: 0 contact infections All: 5/10 VI OPF positive at 21 dpi, 6/10 carriers

10/12 inoculated sheep infectious, no viraemia

Results: Serology

Results: Comparison detection methods

 VNT (post vaccination): ● VN-titre against Asia-1 Shamir: 1.9 10log ● VN-titre against Asia-1 Turkey 11/2000: 1.1

 NS-ELISA (post challenge) ● Non-vac: 16/17 infected sheep detected ● Vac: 10/10 infected sheep detected

10log

fever lameness VI-OPF VI plasma NS-ELISA RT-PCR probang

Number of positive findings by several detection methods per week p.i. Infected non-vaccinated sheep (n=17) Infected vaccinated sheep (n=10) total # week 1 week 2 week 3 > week 4 total # week 1 week 2 week 3 > week 4 n=10 10 3** n.t. n= 0 0 0 n.t. n=3 3 0 0 n=0 0 0 0 n=17 17* 14 12 n=10 10 8 7 n=12 12 3 0 n= 0 0 0 0 n=16 2 13 15 14 n=10 0 8 9 10 n=10 10 n= 6 6

Many subclinical infections Virus detection (‘acute’ infection, 1-2 weeks p.i.): Gold standard (VI/PCR): vesicle material: subclinical infections, small lesions, ...

 % inhibition in positive

plasma samples: limited # positive, positive from 1-3 dpi

population varies!

Alternative: VI OPF (positive from 1-21 dpi)?

Open Session of the EuFMD: 2012,Jerez de la Frontera, Spain

Appendix 37 Results: Transmission

 Non-vaccinated

 Non-vaccinated groups: ● I’s: all (6x2)

Results: Comparison of viral loads (VI OPF) per pen

C’s: 2x2, 1x1, 3x0

● R= 1.14 [0.29-3.4]  major outbreaks can occur

vs vaccinated

● Whole period: non-vaccinated > vaccinated (p=0.01)

 Within non-vaccinated

/ vaccinated

● 1st week > 2nd and 3rd week

 Comparison

 Vaccinated groups: ● I’s: 4x2, 2x1

C’s: none

non-vaccinated pens with/without transmission

● No statistical difference

● R= 0 [0-0.81], significantly <1 (p=0.013)



Occurrence of transmission NOT homogeneously distributed

● Transmission events all in first week p.i.

Discussion/conclusions

 Long viral excretion in OPF swabs ● Strain dependent? ● Risk of transmission to other species?

 Choice for diagnostic samples ● OPF swabs instead of vesicle material/blood samples?

 Vaccination  R0<1  Transmission not homogeneously distributed ● implication for input in mathematical models? ● do viral load and/or other mechanisms play a role?

‘Black’ sheep...

- Is long viral excretion in OPF usual and if so, poses this a risk? - Should we use OPF-swabs as alternative sample for diagnosis? - Vaccination  R0 < 1 - Transmission predominantly in first week p.i.?

THANK YOU FOR YOU ATTENTION!!

Open Session of the EuFMD: 2012,Jerez de la Frontera, Spain

Appendix 38 Open Session of the EuFMD Research Group: Focus on science contributing to the roll-out of progressive control of FMD

Conclusions

TRASNMISSION OF FMDV FROM INFECTED BUFFALO (Bubalus bubalis) TO VACCINATED AND NAÏVE BUFFALO AND CATTLE M. Madhanmohan*,1, S. Yuvaraj1, Ralla Kumar1, Kankipati Manikumar1, Jangam Anil Kumar1, V. A. Srinivasan1 , David James Paton2, Satya Parida2 1 Foot-and-Mouth disease Virus laboratory, Research and Development Centre, Indian Immunologicals Limited, Gachibowli, Hyderabad 500 032, INDIA 2 The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK "The research leading to these results have received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 226556"

• In the present study, FMDV could be transmitted from infected buffalo to naïve buffalo and cattle by direct contact. • All the vaccinated cattle (7µg antigen payload) were protected. However, one third of vaccinated buffaloes were clinically infected with FMDV. • Higher antigen payload may be necessary to protect Buffaloes from FMDV infection. • This signifies the role of buffalo in FMDV transmission that may have an impact on future control strategy. 30/10/2012

Preparation of buffalo challenge virus O/HAS/34/05

Introduction

Passage 1 buffalo

• 98 million buffaloes in India (56% total buffalo population in the world) • The role of Indian buffalo in FMD epidemiology, transmission and immune response is limited • The transmission of FMDV infection from infected Indian buffalo to naïve and vaccinated cattle and buffalo 30/10/2012

Unvaccinated buffalo Sirsa district

Homologous (“r” value=>1) to current Indian type O vaccine strain

Passage 3 Buffalo

Maroudam et al., 2008: Journal of Comparative Pathology, 139,8185

Virus titration in buffalo and cattle calves 4

30/10/2012

Buffalo transmission studies groups Groups

No of animals

Vaccine (ISA 206)

Dose

Vaccination

Challenge

6 buffalo calves

O/ IND/R2/75 (7 µg/dose)

2 ml I/M

0 dpv

28 dpv

6 cattle calves

O/IND/R2/75 (7 µg/dose)

2 ml I/M

0 dpv

28 dpv

6 buffalo calves

Nil

28 dpv

6 cattle calves

Nil

28 dpv

12 donor buffalo calves

Nil

30/10/2012

Passage 2 buffalo

Haryana state

Preparation of buffalo challenge virus O/HAS/34/05

Buffalo passage 3 tongue lesion (24hrs post challenge)

Buffalo passage 3 foot lesion (5 days post challenge)

dpv - days post vaccination

30/10/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 38 Animal experiment Group 1

Group 2

Group 3

Samples collection schedule

Group 4

Days post challenge

Samples Nasal Swab

Donor buffaloes

Saliva

Direct contact challenge for 5 days Room 2 GI(B2) G2(C2) G3(B2) G4(C2) 2 DB

Room 1 GI(B1) G2(C1) G3(B1) G4(C1) 2 DB

Room 3 GI(B3) G2(C3) G3(B3) G4(C3) 2 DB

Room 4 GI(B4) G2(C4) G3(B4) G4(C4) 2 DB

Room 5 GI(B5) G2(C5) G3(B5) G4(C5) 2 DB

Room 6 GI(B6) G2(C6) G3(B6) G4(C6) 2 DB

30/10/2012

Protection percentage

0.04 M buffer

Lysis buffer

0.04 M buffer

Lysis buffer

Salivary and Nasal IgA

Serum

Whole blood assay

PBMC

OP fluid

Temperature

Clinical signs

30/10/2012

FMD clinical signs- Donor buffalo

Groups

No of animals

Vaccine

Percentage Protection

6 buffalo calves

O/IND R2/75 (7 µg/dose)

67 (4 out of 6)

6 cattle calves

O/IND R2/75 (7 µg/dose)

100 (6 out of 6)

6 buffalo calves

Nil

0 (0 out of 6)

6 cattle calves

Nil

0 (0 out of 6)

12 donor buffalo calves

Nil

0 (0 out of 12)

30/10/2012

Tongue lesion 2 dpc

Foot lesion 4 dpc 9

Foot lesion In-contact buffalo 2 dpc

30/10/2012

Tongue lesion In-contact unvaccinated cattle 2 dpc

30/10/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 38 In-contact unvaccinated cattle nasal and feet lesion 7dpc

Serum neutralizing antibody response

30/10/2012

NSP antibody response

NSP antibody response Groups

Gr1

Gr2

Animal No

0 DPV

28DPV/0DP C

5 DPC

10 DPC

15DPC

21 DPC

28 DPC

35 DPC

B-45770

B-45781

B-45575

B-45815

B-45666

B-45531

Groups

Gr3

Animal No

0 DPV

28DPV /0DPC

5 DPC

10 DPC

15DPC

21 DPC

28 DPC

35 DPC

B-0037

B-0043

B-0035

B-0040

B45611

B45633

C-46124

C-46443

C-0007

C-46512

C-0004

C-46341

C-0015

C-46374

C-0028

C-46534

C-6799

C-0021

Gr4 30/10/2012

30/10/2012

Conclusions

Acknowledgements

• Animal Holding farm, Indian Immunologicals Limited, Hyderabad

30/10/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 40 FOOT AND MOUTH DISEASE VIRUS (FMDV) IN THE AFRICAN BUFFALO ( SYNCERUS CAFFER) IN KENYA

CONCLUSIONS  FMD viruses are

circulating among buffaloes in Kenyan National parks

 Multiple serotypes are

in circulation (SAT1, SAT2 and probably serotype O)

 Overall, SAT

Sabenzia N. Wekesa

Presented at

prevalent

serotypes are more

EU-FMD JEREZ SPAIN 29 th -31 st October 2012

BACKGROUND • • • • • •

FMD is endemic in Kenya. First diagnosed 1932 Currently; O, A, SAT1 & SAT2; (Type C last isolated in 2004) FMD well-studied in Cattle but not wildlife Kenya has very large wildlife reserves – interaction between livestock and wildlife Diverse livestock production systems – e.g. free range and pastoralist

OBJECTIVES OF THIS STUDY 1. To determine the presence of FMDV in selected buffalo populations in Kenya 2.

To investigate the serotypes of FMDV in these buffalo populations

SAMPLING SITES •Focus on NP: Meru/Lewa (N=5331), Mara(N=4649) Tsavo (N=7402) Sample types; Probang Serum

Probang cups

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 40

LABORATORY TESTS •

•

Prio-CHECK® FMDV NS ELISA Serotyping: LPBE, VNT

Probang samples: rRT-PCR •

positive samples (Ct<32): BHK cell cultures •

antigen detection ELISA

LPBE

Mara Meru Lewa (n=39) (n=23) (n=6)

Overall positive; 

NSP ELISA: 53/68 (78%) positive:

Maasai-Mara (39), Meru (23) and Lewa (6)

Serology: •

•

RESULTS

68 buffalos from 3 national parks (NP):

O (9/68)

VNT Overall positive; 

O (18/68)

Mara (n=39)

Meru (n=23)

Lewa (n=6)



A (2/68)



C (14/68)



SAT1 (23/68)



SAT1 (36/68)



SAT2 (47/68)

SAT1 18

SAT2 (44/68)



SAT1



SAT2 35

SAT2

No positive results for A and C

SAT3 assays on-going



rRT-PCR •

28/68 (41%) positive(Ct<32).

DISCUSSION 

 %PCR positive



LPBE: antibodies against SAT 1/2 most prevalent followed by O - consistent with previous studies in East Africa (Ayebazibwe et al., 2010, Anderson et al.1979) VNT confirmed presence of antibodies against O, SAT 1 and SAT 2 – but not A and C The RT-PCR positive probangs show presence of FMDV in buffalo - may pose a threat to other animals (Vosloo et al.2002). Further virological assays and molecular analysis are on-going.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 40

CONCLUSIONS

RECOMMENDATIONS

 FMD viruses are

circulating among buffaloes in Kenyan National parks

 Multiple serotypes are

in circulation (SAT1, SAT2 and probably serotype O)

 Overall, SAT

prevalent

serotypes are more

Vincent B. Muwanika1, Hans R. Siegismund2, Graham J. Belsham3, Kirsten Tjørnehøj,3 Sheila N. Balinda1, Francis Gakuya5, Dominic Mijele 5, Vincent Obanda5, Alice Namatovu1, 6, Moses Dhikusooka1,6, Abraham K. Sangula4

Uganda.

 2Department of    



Further studies to establish the role of buffalo in the epidemiology of Non-SAT serotypes Further virological tests to ascertain serotypes, strains and genetic relationships (on-going)



More sampling to enrich the data



Continuous and consistent research in wildlife

ACKNOWLEDGEMENTS

COLLABORATORS

 1Makerere University, P.O. Box



7062/7298, Kampala,

Biology, Ole Maaløes Vej 5, DK-2200, Copenhagen N, Denmark 3 National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark 4 FMD Laboratory, Embakasi, P.O. Box 18021, 00500 Nairobi, Kenya 5Kenya Wildlife Service, Veterinary Department, P.O Box 40241 00100, Nairobi, Kenya 6 Ministry of Agriculture, Animal Industry and Fisheries, P. O. Box 513, Entebbe, Uganda

• • • •

• • • • • •

DANIDA Govt of Uganda- MAAIF Makerere Univ. Uganda Govt of Kenya – DVS,KWS FMD Kenya lab Director and colleagues University of Copenhagen Danish Tech. University UWA EuFMD FAO

I REST MY CASE

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 41 Rationale • The epidemiology of FMD in Africa is mainly sustained by two processes:

• Cycle where wildlife plays a major role in maintenance and spread of the disease to other susceptible domestic/wild ungulates

• Domestic cycle independent of wildlife

Seroprevalence Profile of FMD in Wildlife Populations of West and Central Africa Regions with Special Reference to Syncerus Caffer ssp. The Pirbright Institute, Pirbright, Woking, Surrey, UK

Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Campus International de Baillarguet, Montpellier, FRANCE African Union – Interafrican Bureau for Animal Resources, Nairobi, KENYA International Foundation for Wildlife Management, Paris, FRANCE Royal Veterinary College, Hatfield, UK European Commission for the Control of Foot-and-Mouth Disease, Food and Agriculture Organisation of the United Nations, Rome, ITALY

5 6

host for the SAT serotypes

• Contact transmission • Occasional transmission

acutely infected with susceptible individuals carriers with susceptible individuals

• Buffalo/wildlife role in FMD epidemiology has been investigated in Eastern and Southern Africa regions

• The role which West and African wildlife populations might play in the transmission dynamics of FMD is not know nor data were previously reported

Di Nardo1 A., Libeau2 G., Chardonnet3 B., Chardonnet4 P., Kock5 R., Parekh1 K., Hamblin1 P., Li1 Y., Parida1 S., Sumption6 K.J.

• Cape buffalo (Syncerus caffer) has been shown to serve as long-term maintenance

Study Design

NSP Prevalence

• PARC

and PACE programme implemented between 1986 and 2007

• Sera (n = 719) selected from a large collection stored at CIRAD as representative of wildlife population living in West and Central Africa according to:

• Country • Park • Specie

(n = 7) (n = 14) (n = 35)

• Buffalo samples (n = 196) Region West Africa

Central Africa

Country Benin Burkina Faso

Nigeria Cameroun CAR

Chad

DRC

Park Pendjari NP Arly NP Nazinga GR Pama R Borgu GP Benoue NP Aouk Aoukale FR Bamingui-Bangoran NP St. Floris NP Zemongo FR Aouk NP Ouadi Rime Ouadi Achim FR Zakouma NP Garamba NP

• African forest buffalo • Nile buffalo • West african buffalo

(S. c. nanus) (S. c. aequinocitalis) (S. c. brachyceros)

Nile Buffalo West African Buffalo African Forest Buffalo Other Wildlife Cattle TOT

• Sample tested with

• PrioCHECK® FMDV NS ELISA kit • SPCE Pirbright in-house test A, O, C, SAT 1, SAT 2, SAT 3

+ve/TOT 115/168 12/24 0/4 90/504 13/19 230/719

Observed [True] PREV† 68.45% [70.18%] 50.00% [51.04%] 0% [-] 17.86% [17.70%] 68.42% [70.14%] 31.99% [32.35%] †Adj-Wald

95% CI 61.03% - 75.04% 30.96% - 69.04% 14.75% - 21.46% 45.10% - 85.10% 28.67% - 35.50%

Test, F = 70.2854 (d.f. 4, 717) [p = 0.000]

• Confirmatory VNT for SPCE-positive

NSP – Wildlife

NSP – Region & Buffalo

West Africa

Central Africa

Buffalo spp.

No Positive/TOT

Apparent [True] Seroprevalence†

95% CI

Benin Burkina Faso Nigeria TOT CAR Chad Gabon DRC

8/18 4/5 0/1 12/24 52/81 30/53 0/4 33/34

44.44% [45.27%] 80.00% [82.16%] 0% [-] 50.00% [51.04%] 64.20% [65.77%] 56.60% [57.88%] 0% [-] 97.06% [99.85%]

23.85% - 67.15% 30.48% - 97.33% 30.85% - 69.15% 53.14% - 73.93% 42.99% - 69.29% 81.60% - 99.59%

TOT

115/172

66.86% [68.53%]

59.43% - 73.54%

African Forest Buffalo Nile Buffalo West African Buffalo TOT

0/4 115/168 12/24 127/196

0% [-] 68.45% [70.18%] 50.00% [51.04%] 64.80% [66.39%]

60.98% - 75.08% 30.85% - 69.15% 57.80% - 71.21%

†Adjusted-Wald test for

region, F = 2.2672 (d.f. 1, 195) [p=0.139] Adjusted-Wald test for buffalo spp., F = 3.5247 (d.f. 2, 194) [p=0.031]

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 41 NSP – Age & Year

Age Group

Year

SPCE Prevalence

No Positive/TOT

Apparent [True] Seroprevalence†

95% CI

2/4 2/2 21/31 15/20 37/56 41/69 30/44 29/55 13/22 49/62 6/13

50.00% [51.04%] 100% [100%] 67.74% [69.44%] 75.00% [76.97%] 66.07% [67.71%] 59.42% [60.81%] 68.18% [69.90%] 52.73% [53.87%] 59.09% [60.47%] 79.03% [81.15%] 46.15% [47.04%]

12.15% - 87.85% 49.54% - 81.79% 51.92% - 89.29% 52.70% - 77.29% 47.41% - 70.40% 53.05% - 80.25% 39.54% - 65.55% 38.00% - 77.30% 67.04% - 87.48% 22.20% - 72.03%

6m-1y 1-2ys 2-4ys 4-5ys 5-10ys >10ys 1999 2000 2001 2002 2003

†Adjusted-Wald test for

West Africa Serotype A O C SAT 1 SAT 2 Central Africa SAT 3

C SAT 1 SAT 2 SAT 3

age group, F = 0.8596 (d.f. 5, 177) [p=0.509] Adjusted-Wald test for year, F = 3.0306 (d.f. 4, 192) [p=0.019]

SPCE Pairwise ρ

A O C SAT1 SAT2 SAT3

A 0.060 0.410 0.648 0.404 0.562

O 0.060 0.160 0.059 0.162 0.084

C 0.410 0.160 0.444 0.532 0.646

SAT1 0.648 0.059 0.444 0.426 0.681

SAT2 0.404 0.162 0.532 0.426 0.391

Constant Park (Km2 ) Longitude Latitude Year Age

Discussion • Prevalence of FMD in West and Central African wildlife population • Several wildlife species (buffalo + antelope) tested as positive • High prevalence in both West african buffalo and African forest buffalo • Different patterns of FMDV Serotype involved with type O, SAT 2 and SAT 1 being dominant

95% CI 24.34% - 75.66% 95% CI - 75.66% 24.34% - 52.94% 30.71% - 81.56% - 88.09% 37.52% - 86.94% - 73.93% 44.74% - 91.75% - 80.29% 8.25% - 55.26% - 87.73% 34.71% - 52.68% - 55.27% 77.93% - 91.07%

Negative ≤1:11; Inconclusive >1:11&1:35≤ Adjusted-Wald test, F = 16.7064 (d.f. 5, 754) [p=0.000] 77/115 66.96% [66.93%] 57.85% - 74.94% 85/115 73.91% [73.84%] 65.12% - 81.13% 95/115 82.61% [82.54%] 74.55% - 88.51% 56/115 48.70% [48.34%] 39.68% - 57.79% †

†Adjusted-Wald test,

F = 16.7064 (d.f. 5, 754) [p=0.000]

Buffalo FMD Prev P(x)

SAT3 0.562 0.084 0.646 0.681 0.391 -

What has been highlighted?

Serotype No Positive/TOT Apparent [True] Seroprevalence† A 6/12 50.00% [49.95%] † No Positive/TOT Serotype Apparent VNT Titre O 6/12 [True] Seroprevalence 50.00% [49.95%] 56/127 A 44.09% [44.03%] 1:22[58.29%] 35.61% C 7/12 58.33% O8/12 82.26% [82.24%] 1:90[66.59%] 74.40% SAT 1 102/124 66.67% 84/127 C9/12 66.14% [66.11%] 1:178[74.90%] 57.37% SAT 2 75.00% 93/127 SAT 13/12 73.23% [73.16%] 1:256[24.47%] 64.75% SAT 3 25.00% 2 1:1024[43.42%] 74.10% 81.89% [81.82%] A 104/127 SAT 50/115 43.48% 3 1:355[85.69%] 37.86% 59/127 SAT 46.46% [46.08%] O 96/112 85.71%

Coef. [95% CI] 4.113 [1.566 to 6.661] -0.383 [-0.669 to -0.973] 0.129 [0.029 to 0.228] -0.235 [-0.366 to -0.103] -0.325 [-0.481 to -0.170] 0.007 [0.004 to 0.011]

SE 1.299 0.146 0.051 0.067 0.079 0.002

Z 3.16 -2.63 2.53 -3.50 -4.10 4.31

P 0.002 0.009 0.011 0.000 0.000 0.000

OR [95% CI] 61.147 [4.786 to 781.268] 0.681 [0.512 to 0.907] 1.137 [1.029 to 1.256] 0.791 [0.693 to 0.902] 0.722 [0.618 to 0.844] 1.007 [1.003 to 1.010]

σ2(SE) = 0.416(0.264); log-likelihood = -479.5846; AIC = 973.1693

Acknowledgements • • • • •

Genevieve Libeau (CIRAD) CIRAD staff PARC & PACE Keith Sumption (FAO) Nadia Rumich (FAO)

What needs to be answered?

• Livestock-Wildlife interface • Risk of transmission and role of buffalo in the epidemiological process (FMDV serotype normally present in domestic species)

• Knowledge gap of FMDV lineages circulating in Central Africa

Thank you!

The Pirbright campus is being redeveloped

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 42

CIDLID FMD PROJECT IN TANZANIA Tanzania Wildlife Research Institute

University of Glasgow

Epidemiological patterns and risk factors for foot-and-mouth disease exposure in traditional livestockkeeping systems of northern Tanzania

Sokoine University of Agriculture

Tiziana Lembo PDRA, Institute of Biodiversity, Animal Health and Comparative Medicine University of Glasgow

Conclusions and recommendations  FMD is very prevalent in traditional livestock-keeping systems across northern Tanzania, but infection patterns vary widely across ecosystems  Non-wildlife (anthropogenic and livestock) related factors appear to be important • Additional predictors (livestock management/movement) need evaluating • In some areas close interactions, hence transmission, amongst livestock and buffalo populations may occur • Risk factors for seropositivity should be further explored through serotype-specific analyses  Need to characterise the diversity of viruses to understand transmission patterns amongst livestock and buffalo  Urgent need to investigate options for disease control that address the needs of livestock-keepers while protecting the ecological integrity of wildlife-protected areas

Endemic FMD in Africa Key areas of research

Central Veterinary Laboratory, Ministry of Livestock and Fisheries Development

The Pirbright Institute

University of Edinburgh

Great concern for livestock-keeping communities in traditional systems • Livestock-keepers in much of Africa consider FMD as one of the most important diseases of livestock (loss of milk, calf mortality, reduced income from sale, etc.) • In rural Tanzania, FMD ranked second amongst the most important livestock diseases with multiple outbreaks each year (80 - 90% in the past year, up to 63% in the past four months) In herd in past year

In herd in past 4 months

1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00

The role of wildlife How much cattle infection is associated with spillover from wildlife? How can we best control FMD adjacent to wildlife protected areas?

? Relative importance of livestock- and wildlife-related factors in maintenance and transmission?

Open Session of the EUFMD: 2012, Jerez de la Frontera, Spain

Sian Brown

Appendix 42 Serengeti & Ngorongoro - NSP ELISA seroprevalence Cross-sectional surveys in northern Tanzania

Kenya

Agro-pastoralist

 Four ecosystems

Kenya

Tanzania

 Livestock surveys: • A range of production systems • Ten villages per ecosystem, two households per village • Serological surveys involving 30 - 40 livestock (cattle, sheep, goats) in each households and questionnaire conducted

Pastoralist

Serengeti livestock: Overall - 67% (n = 725) Cattle - 77% (n = 408) Sheep - 46% (n = 90) Goats - 59% (n = 225)

 Buffalo surveys: 25 animals per ecosystem from a range of herds  NSP ELISA serology and data analysed by GLMM

Serengeti

Agro-pastoralist Rural smallholder system

Pastoralist

Serengeti

Monduli

Pastoralist

SNP

Simanjiro

Livestock by village:

Buffaloes by individual:

POS

NEG

Tarangire - NSP ELISA seroprevalence

Serengeti buffalo - 69% (n = 26)

Arusha - NSP ELISA seroprevalence Rural smallholder system

Monduli District Arusha National Park

Pastoralist

Seroprevalence: Overall - 66% (n = 449) Cattle - 76% (n = 225) Sheep - 45% (n = 73) Goats - 60% (n = 149)

Simanjiro District

Seroprevalence: Overall - 34% (n = 615) Cattle - 38% (n = 296) Sheep - 36% (n = 105) Goats - 26% (n = 213)

Buffaloes - 96% (n = 24)

Tarangire National Park

POS NEG

Predictors of seropositivity

p-value

Estimate

Std error

Odds ratio

Odds ratio (95% CIs)

Age in years

Control

0.30 (per year)

0.026

1.35 (per year)

1.28 - 1.42

Management system

p < 10-3 0.66

0.25

1.93

1.18 - 3.15

Pastoralist

0.28

0.20

1.32

0.89 - 1.95

Rural smallholder

-1.3

0.30

0.27

0.15 - 0.49

Bovine

0.54

0.30

1.71

0.95 - 3.08

Caprine

-0.46

0.31

0.63

0.34 - 1.16

Ovine

-0.82

0.32

0.44

0.23 - 0.82

p < 10-15

Distance to protected-area boundaries

Frequency of interaction with buffalo

Frequency of interaction with other susceptible wildlife

Buffaloes by individual:

POS

NEG

Conclusions and recommendations

Agropastoralist

Distance walked for grazing/watering (wet and dry season)

Buffaloes - 48% (n = 23)

Livestock by village:

Buffaloes by individual:

NEG

Ngorongoro buffalo - 90% (n = 140)

Tarangire

Meru NCA

Species

Ngorongoro

Arusha

Loliondo

Livestock by village: POS

Ngorongoro livestock: Overall - 64% (n = 709) Cattle - 76% (n = 359) Sheep - 51% (n = 126) Goats - 54% (n = 224)

No significant wildlife-related predictors

 FMD is very prevalent in traditional livestock-keeping systems across northern Tanzania, but infection patterns vary widely across ecosystems  Non-wildlife (anthropogenic and livestock) related factors appear to be important • Additional predictors (livestock management/movement) need evaluating • In some areas close interactions, hence transmission, amongst livestock and buffalo populations may occur • Risk factors for seropositivity should be further explored through serotype-specific analyses  Need to characterise the diversity of viruses to understand transmission patterns amongst livestock and buffalo  Urgent need to investigate options for disease control that address the needs of livestock-keepers while protecting the ecological integrity of wildlife-protected areas

Open Session of the EUFMD: 2012, Jerez de la Frontera, Spain

Appendix 42

Thanks! University of Glasgow, UK Directorate Veterinary Services, TZ Tanzania Wildlife Research Institute, TZ • S. Cleaveland • F. Kivaria • R. Fyumagwa • D. Haydon • E. Ranga • J. Keyyu • R. Reeve • R. Hoare • H. Auty Central Veterinary Laboratory, TZ • M. Casey • M. Yongolo Sokoine University of Agriculture, TZ • K. Ferguson • C. Ngeleja • R. Kazwala The Pirbright Institute, UK • R. Sallu • C. Kasanga • D. King • E. Kamani • D. Paton Veterinary Investigation Centre-Arusha, TZ • A. Lugelo • S. Parida • D. Mshanga • M. Shabani • T. Knight-Jones • T. Kibona • N. Knowles University of Edinburgh, UK • R. Fupi • K. Parekh • B. Perry • V. Mioulet Onderstepoort Veterinary Institute, SA Washington State University, US • F. Maree • J. Yoder • T. Marsh

Deo Mshanga

Field team Enos Kamani

THANKS FOR PERMISSION • COSTECH, MoLFD, TAWIRI, TANAPA, NCAA, and WD for permission • All livestock owners for participating in the study with great patience and enthusiasm!

Mahemba Shabani and Paulo Tango

Robert Fyumagwa

Machunde Bigambo Ahmed Lugelo Raphael Fupi and Tito Kibona

Open Session of the EUFMD: 2012, Jerez de la Frontera, Spain

Appendix 43 RETROSPECTIVE SEROSURVEY OF FOOT AND MOUTH DISEASE (FMD) IN FREE RANGING DOMESTIC PIGS AND WILD SUIDS IN SUB-SAHARAN AFRICAN COUNTRIES

Situation of FMD in the last five years 2007

2009

2010

2011

Centro de Investigación en Sanidad Animal, CISA-INIA, Madrid, SPAIN. International Livestock Research Institute (ILRI), Nairobi, KENYA. Animal Pathology Dpt, National Veterinary Laboratory (LANAVET), CAMEROON. Ministry of Agriculture, Animal Industry & Fisheries. Entebbe, UGANDA.

Dr. MARISA ARIAS The role of swine species plays in the spread and maintenance of the disease in endemic areas is barely known.

EuFMD initiative Joint synergies with other Reference Laboratories

Conclusion Pilot Study

EU Reference Laboratory for African Swine Fever (ASF)- (CISA-INIA)

through an active cooperation with ILRI (Kenya) and the Ministries of Agriculture of the respective African countries ( Kenya, Cameroon; Uganda, Tanzania) Retrospective serosurvey on FMD using in domestic pigs and wild suids field serum samples collected during ASF surveillance programs, between 2004 and 2011 from sub-Saharan regions.. in collaboration with INIA and MAGRAMA

The low FMDV seropositivity rate observed in domestic pigs in FMD-endemic areas of Africa suggests that this species does not play a relevant role in the epidemiology of FMD in the studied areas.

All Wild suids samples examined from Kenya (eastern and western provinces, n: 77, 2006-2008) were negative against FMDV antibodies.

Spanish Ministry of Agriculture to carry out this work

SEROLOGICAL TEST Study performed by using: PERFORMING THE TECHNIQUES ACCORDING TO THE STANDARIZED PROTOCOLS AT CISA-INIA

FMDV recombinant 3ABC

(E.Coli)

Sampling areas

WEST AFRICA

EAST AFRICA

CAMEROON KENYA

non-structural protein (NSP) indirect ELISA.

Blanco et al, 2002. Veterinary Microbiology. 85, 13-21 Serological evidence of FMD subclinical infection in sheep population during the 1999 epidemic in Morocco.

TANZANIA

Blanco et al, 2000. European Commission for the control of FMD. Research group report 27, 211-221. Improved indirect ELISA based on the 3ABC polyprotein for differential infection from vaccination in foot and mouth disease.

UGANDA

Employed in the surveillance programme of Agricultural Ministry 2001, (more than 45.000 pig serum samples were analysed)

FMDV recombinant 3ABC non-structural protein (NSP) immunoblotting (IB). Optimized and evaluated (in house method).

Comparative study between PrioCHECK FMDV NS and In house 3ABC ELISA/IB

- To be in mind: Samples collected for ASF purpose. Insufficient amount

to perform all FMD serological test (such as VNT).

- Characteristic FMD clinical signs were not present in pigs sampled

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 43 EAST AFRICA

DOMESTIC PIGS

KENYA

SERA SAMPLES ANALYZED Wes tern

DOMESTIC PIGS

C ountry

2005 2006 2008 2009

K enya

Nº S eru m s am p les c o llec ted

P R O V INC E C entral P rovinc e Nyanz a P rovinc e Nyanz a P rovinc e C entral P rovinc e Nyanz a P rovinc e C entral P rovinc e C oas t P rovinc e C entral P rovinc e Wes tern P rovinc e Nyanz a P rovinc e

2010

2011

C ountry

WILD PIGS

C ollec tion D ate

2011

86 27 36 88 13 35 2 1 281 49

T OT AL

618

P R O V INC E

Nº S eru m s am p les c o llec ted

2006

Nort E as te P rovinc e

2007

Nort E as te P rovinc e

2008

C ollec tion D ate

Nyanz a P rovinc e T OT AL

281 T OT AL

Nyanz a P rovinc e

C entral P rovinc e

C ollec tion D ate

North E as tern P rovinc e C ollec tion D ate

2006 2007 2008

2008

T OT AL



6 Positive 2011

2

Positive 2006

1

Positive 2011

C oas t P rovinc e C ollec tion Date

P R OVINC E

Nº S erum s amples c ollec ted

2011

210

2 T O T AL

RESULTS

KENYA

Nº sa mple s c olle c ted

F MD 3AB C No pos itive s

% of pos itive s

2006

Nyanz a Provinc e

2008 2009

Nyanz a Provinc e

Central Provinc e

Nyanz a Provinc e

Central Provinc e

Coas t Provinc e

Central Provinc e

Wes tern Provinc e

281

Nyanz a Provinc e

6 1

2010

2011

Country

K enya

Collection Date

PROVINCE

DOMESTIC PIGS

Nº samples collected

FMD 3ABC No positives

% of positives

2006

North E as tern P rovince

2007

North E as tern P rovince

Nyanza P rovince

2008

DOMESTIC PIGS

86 88 35 1

Central Provinc e

WILD PIGS

2 T OT A L

Nº S erum s amples c ollec ted

K enya

C ollec tion D ate

Nº S e rum sa m ple s c olle c te d

2005

28 20 27 T OT AL

Nº S e ru m sa m p le s c o lle c te d

125

2005 2009 2010 2011

EAST AFRICA C ountry

C o llec tio n D ate

Nº S e rum sa m ple s c olle c te d

27 36 13 49 T OT AL

WILD PIGS

N y an z a P ro v in c e

281

2006 2008 2010 2011

Nº S e rum sa m ple s c olle c te d

WILD PIGS

0 Positive samples

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 43 EAST AFRICA

DOMESTIC PIGS

2004

TANZANIA

SERA SAMPLES ANALYZED

Arus ha C ollec tion Date

Nº S erum s amples collected

2004

DOMESTIC PIGS

C ountry

Tanzanya

C ollec tion Date

2004

Nº S erum s amples c ollec ted

P R OVINC E

DODOMA

MANY AR A

EAST AFRICA

Country Collection Date

Tanzanya 2004

PROVINCE

2004

DODOMA

MANYARA

ARUSHA

FMD 3ABC No % of positives positives

2 3

 3 Positive 2004  2 Positive 2004

DOMESTIC PIGS WHOL E C OUNTR Y C ollection Date

DOMESTIC PIGS

UGANDA

C ollec tion Date 2006

T OT AL

SERA SAMPLES ANALYZED

C ountry

Nº S erum s amples c ollec ted

Nº samples collected

EAST AFRICA

P R OVINC E WHOL E C OUNT R Y

Nº S erum s amples c ollec ted

2006

Nº S erum s amples collected

Nakas ang ola

C ollec tion Date TOTAL

Nº S erum s amples

2007

7 T O T AL

MUK ONO

NAK AS ANG OL A

WAK IS O

Ug anda 2007

Dodoma

TANZANIA

RESULTS

DOMESTIC PIGS

2004 T O T AL

27 TOTAL

Nº S erum s amples c ollec ted

C ollec tion Date

AR US HA

Manyara

27 TOTAL

T OT AL

0 Positive samples Mukono Wakis o C ollec tion Date 2007

C ollec tion Date

Nº S erum s amples c ollec ted

10 T O T AL

2007

Nº S erum s amples c ollec ted

9 T O T AL

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 43 WEST AFRICA

WEST AFRICA

CAMEROON

SERA SAMPLES ANALYZED

DOMESTIC PIGS

C ountry

CAMEROON

C AME R OON

C ollec tion Date

2010

North P rovinc e Nº S erum s amples c ollec ted

2010 TOTAL

K E NY A

KENYA

2006, 2007, 2008 DOME S TIC P IG S

TANZ ANY A

C AME R OON

Comparative study between Priockeck and In house 3ABC/IB

MANY AR A

Antibodies FMD 3ABC In house PrioCHECK ELISA and IB 3 ABC FMDV NS Nº positives

Nº positives

2 3

2 1 Antibodies FMD 3ABC

Country

Collection Date 2004

K E NY A

2011

NEGATIVES SAMPLES

PROVINCE

In house ELISA and IB 3 ABC

PrioCHECK FMDV NS

2 6 1

2 0 0

Nº positives

Nyanz a Wes tern Nyanz a

Nº positives

TANZANIA

UGANDA

2004 DOME S TIC P IG S 2006, 2007 DOME S TIC P IG S 2004

9/618 0/77 5/96 0/94 0/61

Conclusion Pilot Study The low FMDV seropositivity rate observed in domestic pigs in FMD-endemic areas of Africa suggests that this species does not play a relevant role in the epidemiology of FMD in the studied areas.

All Wild suids samples examined from Kenya (eastern and western provinces, n: 77, 2006-2008) were negative against FMDV antibodies.

n=60

100% concordance:

DOME S TIC P IG S 2005, 2006, 2008, 2009, 2010, 2011 WIL D P IG S

UG ANDA

T anzanya 2004

Nº POS / TOTAL SAMPLES

C ollec tion Date

DODOMA

TOTAL

SUMMARY

0 Positive samples

Country

EAST AFRICA

CAMEROON

Collection PROVINCE Date

Nº S erum s amples c ollec ted

NOR TH P R OVINC E

WEST AFRICA

DOMESTIC PIGS

POSITIVES SAMPLES

P R OVINC E

In house 3ABC ELISA / PrioCHECK FMDV NS

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 43 ACKNOWLEDGEMENTS

Cameroon

Tanzania

CISA-INIA TEAMS : Emerging and Transboundary Animal Diseases and the EU Reference Laboratory for ASF

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 44

Background:

Risks associated with unofficial livestock movements in the greater Mekong region. Chris Hawkins Jim Kerr Socheat Sieng Ben Madin Angus Cameron



Growing demand for livestock  

 

Cattle and pigs moved in large numbers Main sources  



Cambodia and Laos Also transit from Thailand and Myanmar

Volatile movements 





China and Vietnam Particularly for live animals to meet domestic requirements

dependent on price and exchange rates recognized movement pathways change rapidly

Rapid movements 

trans country in few hours (15-24); often travelling at night for coolness

Factors 

Traders   



Local, national, regional Tight margins, short-term high-interest finance Movement delays cost lots of money

Formal movement requirements  

Slow and expensive Checkpoints, quarantine stations, livestock depots 

Promote spread of disease

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 44

Response: unofficial movements  

Well documented Unquantified

Requirements for effective control of FMD requires: 



Regionally acceptable livestock ID and tracing system; Official processes that facilitate trade rather than hinder it; Appropriate recognition of disease status across borders 



Making movements safer 

Understanding livestock disease status 



Processing slaughter animals as a priority 

 

protect either the animals in transit or the animals at the destination not keeping them in depots or retaining some for other purposes (e.g. breeding)

Trader education Strengthening animal health management and biosecurity practices

Political will to address real issues, rather than tinker around the edges.

Conclusions 

Access to appropriate preventative strategies (vaccines) 



alerting destination about disease at origins/in transit

Why impose quarantine when disease status is the same both sides of a border?

Managing risk of FMD by understanding livestock movement  



Patterns change rapidly Requires ongoing real-time movement monitoring

Key components 

Policy and regulation 

 

Currently promote disease spread and informal movement

Traders Risk-based movement management 

Identification, traceability, vaccination status, surveillance

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 45

Conclusions 

Risk Factors for Foot and Mouth Disease in Beef Cattle Herds in Israel Eyal Klement – Associate Professor

Koret School of Veterinary Medicine Robert H. Smith Faculty of Agricultural, Food and Environmental Sciences The Hebrew University, Jerusalem, Israel

The Middle East

Small ruminant distribution in Israel



FMD morbidity is abundant in adult beef cattle despite multiple vaccinations. However, It appears that the risk for disease increases when the time gap from the last vaccination is more than 6 months The presence of calves under six month old is a significant risk factor for morbidity in adults The spatial pattern of FMD spread between beef cattle herds is different from its spread to feedlot cattle. The first spread by contact bewteen grazing cattle. The second by transportation of sick animals

Cattle distribution in Israel

Geographical distribution of FMD outbreaks in Israel South

North

Center

Open Session of the EuFDM: 2012, Jerez de la Frontera, Spain

Appendix 45

Seasonality of FMD outbreaks in Israel



FMD outbreak - Israel 2011 • Outbreak onset was on March and it involved 26 localities that included 30 beef herds, 11 feedlots, 4 dairy herds and 8 sheep flocks • Isolation of serotype O, Pan-Asia-2 viruses

Study design

Research Objectives

Reported Disease cases

Investigating risk factors for FMD morbidity in beef cattle herds

Regional Veterinary officers

Israel’s FMD reference lab

Golan heights study

Outbreak description

Case control study

Analysis of outbreak spread

Characterization of the FMD spread among beef cattle and feedlot cattle herds

Analysis of Risk factors

Analysis of virus spread • In order to analyze the pattern of virus spread we compared the distance between each affected herd and the most adjacent herd affected prior to it.

Analysis of risk factors for FMD among beef herds • Study area: Golan heights

• Average distance was compared between feedlots and beef herds. A B

Open Session of the EuFDM: 2012, Jerez de la Frontera, Spain

Appendix 45 - Druze beef cattle

Case-Control study Golan Heights  



• Herdsmen were interviewed by preformed questionnaires: – Group data:

25 FMD cases 26 controls

• • • •

Outbreak Case= Beef cattle herd, consisting of at least one sick animal (positively diagnosed)

• Time of clinical signs onset and extent of morbidity

• For feedlots the average distance from the previously affected adjacent herd was 19.9 Km (range 2-43.7 Km) while for beef herds it averaged only 3.2 Km (range 0.2-8.9 km) (p=0.01)

Conclusions



location number of animals Breed Age

• Origin • Number and date of vaccine administration prior to the outbreak

Results – outbreak spread



Data collection

- Jewish beef cattle

Results – risk factor analysis • In multi-variate analysis presence of calves under 6 months of age and vaccine administration more than six months before the outbreak were found to be significant risk factors for FMD in adult beef cattle

Risk factor

Odds ratio

presence of calves under 6 month

10.581

>6 months elapsed from last FMD vaccination

CI 95%

P value

1.73 to 64.7 0.011 1.2-53.2

0.032

Acknowledgments • Dr. Lior Zamir Israeli Kimron Veterinary institute Dr. Boris Gelman Hebrew University Dr. Udi Elnekave Ily Shlamovitz Hchaklait Dr. Benny Sharir Israeli Veterinary Services Dr. Boris Even-Tov Dr. Fares Hamed Dr. Yuval Hadani

Open Session of the EuFDM: 2012, Jerez de la Frontera, Spain

Appendix 46 Rationale • Within the PCP-FMD framework, seroprevalence data are important to assess risk and monitor control progress

Risk Mapping of Foot-and-Mouth Disease in Central Asia Region

•

STAGE 0 – STAGE 1 Serological data enable a preliminary assessment of the FMD epidemiological status and the level of risk of FMD infection

• The establishment of Regional Roadmaps provides an opportunity for

harmonising sero-survey and thus enhancing the precision of estimates at regional scale

• GTFS/INT/907/ITA •

September 2010 – March 2012, country-wide survey in Central Asia

Di Nardo1 A., Murvatulloev2 S., Tatov2 A., Eranov2 M., Khan2 E., Aslami2 A., Ferrari2 G.

• • •

The Pirbright Institute, Pirbright, Woking, Surrey, UK 2 Food and Agriculture Organization of the United Nations, Rome, ITALY 1

Survey Design Two-Stage Cluster Sampling Design

• 1st Stage (PSU = Village), required No calculated by Intensity Sampling

r N ( PSU )  Km 2

r = 25km

calculated by Simple Random Sampling Formula for Binomial Data

Villages (PSU) 196 235 132 61 110 734

Afghanistan Pakistan Tajikistan Turkmenistan Uzbekistan TOT

1.962  p  q d2

p = event proportion (0.5) q = non-event proportion (0.5) d = SE (25%) at 95% CI

Samples (SSU) 3243 3761 2112 976 1760 11852

2011

2012

Pakistan

Tajikistan

Turkmenistan

Uzbekistan

Harmonised cross-sectional surveys based on a two-stage cluster sampling design Subsistence farming system (village level) – age-stratified (0-6m; 6-18m; >18m) Diagnostic test by NSP-ELISA (in-house IZSLER Brescia, Italy)

Statistical Analysis

• Prevalence analysis

• Sample Base Weight and Finite Population Correction BWij 

1  z j   kij    Z   K   j   ij 

FPC jl 

N jl  n jl N jl  1

• Risk Analysis – Odds Ratio

• 2nd Stage (SSU = Animal), required No

N ( SSU ) 

2010 Afghanistan

Samples/Village 16.54 16.00 16.00 16.00 16.00 16.15

• Generalized Linear Model (Logit link) – categorical covariates • • • •

Age Vaccination distance Susceptibility Species, Farm System, Origin

(0-6m, 6-12m, 12-18m, >18m) (VaccinationDATE – SamplingDATE) (6monthDATE – VaccinationDATE) (data not shown)

• Cubic Spline Logistic Regression (combining continuous+categorical)

• Risk Map

• Mixed-Effect Model (via GLLAMM program – Stata 12.1 SE)

• Probability p(x) of FMD prevalence mapped in ArcGIS 10.1 using Kernel Smoothed Intensity map

p ( x) 

eg ( x) 1  eg ( x)

• Space-Time Permutation Model (via SaTScan 9.1.1)

FMD Prevalence

+ve/No Sampled

ORs - Age

Observed [True] PREV†

95% CI

Within-District PREV

Within-Village PREV

DEFF [DEFT]

Afghanistan

1504/3243

46.81% [46.93%]

43.40% - 50.24%

6.25% - 63.85%

0% - 100%

1.309 [1.144]

Pakistan

1591/3761

39.73% [39.79%]

36.53% - 43.03%

0% - 94.20%

0% - 100%

0.436 [0.660]

Tajikistan

851/2112

47.83% [47.96%]

43.96% - 51.73%

0% - 93.75%

0% - 100%

3.237 [1.799] 0.392 [0.626]

Turkmenistan Uzbekistan TOT

41.93% [42.01%]

38.26% - 45.69%

0% - 100%

281/1760

14.35% [14.18%]

11.49% - 17.78%

0% - 81.25%

0% - 82.00%

16.9 [4.111]

4692/11852

465/976

24.48% [24.40%]

22.24% - 26.87%

0% - 100%

8.963 [2.994]

Adj-Wald Test, F = 65.475 (d.f. 4, 11848) [p = 0.000]

†

+ve/No Sampled

Observed [True] PREV†

95% CI

OR [95% CI]

0 – 6 months

1599/3702

33.41% [33.41%]

28.90% – 38.25%

1.690 [1.306 – 2.188]

6 – 12 months

Status

902/2745

22.75% [22.65%]

19.06% – 26.91%

0.839 [0.645 – 1.092]

12 – 18 months

1090/2734

21.88% [21.78%]

18.50% – 25.69%

0.801 [0.616 – 1.041]

>18 months TOT

3/10

8.03% [7.80%]

1.18% – 39.01%

0.269 [0.037 – 1.980]

3594/9191

24.09% [24.01%]

21.79% – 26.54%

0.773 [0.660 – 0.906]

†Adj-Wald

Test, F = 7.3821 (d.f. 3, 9188) [p = 0.000]

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 46 ORs - Vaccination

Status

+ve/No Sampled

ORs - Susceptibility

Observed [True] PREV†

95% CI

OR [95% CI]

2665/6526

19.19% [19.06%]

17.01% – 21.58%

0.572 [0.450 – 0.726]

0>mpv<6

839/2668

14.57% [14.40%]

12.20% – 17.31%

0.378 [0.292 – 0.491]

6>mpv<12

723/1590

44.02% [44.12%]

39.88% – 48.25%

2.571 [2.069 – 3.195]

184/513

39.48% [39.53%]

32.07% – 47.41%

2.052 [1.449 – 2.907]

2027/5326

29.35% [29.31%]

25.61% – 33.39%

1.749 [1.377 – 2.220]

Vaccinated

>12mpv Unvaccinated

Adj-Wald Test, F = 19.1818 (d.f. 1, 11851) [p = 0.000]

†

P(x) Risk Map

Status

Observed [True] PREV†

95% CI

OR [95% CI]

3960/9689

28.89% [28.85%]

25.94% – 32.04%

2.339 [1.756 – 3.117]

1290/3147

27.45% [27.40%]

24.36% – 30.77%

1.206 [0.968 – 1.501]

732/2163

14.80% [14.63%]

11.96% – 18.17%

0.427 [0.321 – 0.569]

0>apv<6

280/1028

18.99% [18.86%]

14.64% – 24.27%

0.680 [0.484 – 0.957]

>6apv

176/596

8.65% [8.42%]

5.76% – 12.79%

0.256 [0.162 – 0.405]

Susceptible 6month age PV Not Susceptible

+ve/No Sampled

Adj-Wald Test, F = 20.3639 (d.f. 3, 8088) [p = 0.000]

†

Discussion • Validity of survey assumption and power of harmonised process • Trend of FMD Prevalence Increasing with Distance from Vaccination • Age as a confounding factor • Susceptibility Variable (naïve population) explains low Vaccination Effect • High Risk probability of FMD Prevalence found in bordering area between: • Afghanistan – Pakistan (n = 2) • Afghanistan – Tajikistan • Afghanistan – Turkmenistan

Country (Province)

Turkmenistan (Mary) Tajikistan (Rep. Subordination) Afghanistan (Herat) Afghanistan (Sar-e-Pol) Afghanistan (Takhar) Pakistan (Balochistan) Pakistan (Punjab) Pakistan (Sindh)

Radius (Km)

Observed/Expected

261.2 207.93 75.7 166.42 54.19 254.2 245.3 314.2

8.95 5.2 5.76 3.91 10.4 3.88 10.69 6.69

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

• Identified FMD Risk Areas can be used as Target Points for improving: • FMD surveillance • Joint effort for FMD Control

Acknowledgements • Gratitude to all the people involved in the project

• Giancarlo Ferrari (IZSLT) • Ciniglio Umberto (FAO) • Mauro Massoni (MoFA, Italy)

Thank you!

The Pirbright campus is being redeveloped

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 47

Conclusions from this study • FMD infection is highly endemic - continuous circulation of FMD virus throughout the year

Prevalence and risk factors for FMDNSP-antibodies in cow and buffalo calves, and small ruminants in Egypt Kees van Maanen

EUFMD/FAO consultant

Conclusions continued

• it seems more likely that sheep and goats are important for spread within the village rather than spread between villages

• FMD clinical outbreaks are seriously underreported • Risk factors are related to - Animal movements • Direct introduction of calves • Indirect through market. Effect local clinic? - Manure handling • Region; western region is relatively less FMD infected or turned around: no differences in FMD rates in Nile delta, Central, Upper and Eastern Egypt

Objective • Establish database about FMD (sero) prevalence - Estimate the within-region and within-village prevalence in 6 - 18 months old large ruminants and in (non-vaccinated) small ruminants - Identify high- and low-risk areas - Identify risk-factors to improve the control strategy

Context • Previous TCP in 2008-2009 - Five pilot governorates - Endemic FMD antibodies In this project nationwide serosurveillance alongside: • Passive surveillance • Outbreak investigation • Improving laboratory capacity

Design for sampling large ruminants • Five regions • Proportional random selection of villages with minimum of 30 per region (this was possible except for Western region) • Within each village sample collection of 14 large ruminants age between 6 - 18 months

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

156 56 33 27 31

Appendix 47

Design for sampling small ruminants • Small ruminants 15 villages per region (possible except for Nile delta)

156 13

56 15

• Samples of 14 small ruminants of all ages

33 23

27 16

• Sampled between May and December 2011

31 15

Data • 5299 animals with all information (lab results and questionnaire) from 310 different villages - 298 villages for cattle and buffalo - 82 villages for sheep and goats - 70 villages with both large & small ruminants - 12 villages with only sheep and goats

Sheep & goats

228 7012 Cattle and buffalos

% of animals with FMD-NSP antibodies Cattle

Positive 14.5%

Buffalos

Positive 26.0%

Sheep

Positive 12.7%

Goats

Positive 10.1%

Data FMD-NSP test results Questionnaire • Non-structural proteins • On animal indicate infection characteristics • Young Merging by animal number and orn, sex infectio unique code for village practices recentl - Manure, buying and selling of animals, • Expressed as • On village situation percentage inhibition - <= 50% is ‘Negative’ - > 50% is ‘Positive’

- Clinical signs of FMD, vaccination against FMD, presence of market, local clinic

Statistical analysis with STATA Use of appropriate statistical tests

Clustering of animals requires ‘multi-level’ modelling (random-effects) Applying statistical significance when P-value < 0.05 applying statistical trend when P-value between 0.05 and 0.10

The effect of FMD vaccination in relation to NSP seropositivity • Testing a Multi-vaccinated cattle (n=80) on two farms considered free from FMD for many years. • All samples from different age groups were tested negative. • Therefore vaccination does not seem to induce considerable bias in interpretation of test results.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 47

Effect FMD multi vaccination in relation to NSP seropositivity 50

Region

40 30 20 10 0 -10

100

-20 -30

Vaccinated cattle sorted by age-class (n=80)

Comparison of both studies for the same 5 governorates Level

Percentage FMD-NSP positive villages and animals by region and species

Current project 5 governorates

Large ruminants Animal

Village

Animal

Delta

21.6

14.8

Central

18.2

21.0

Upper

16.0

11.0

East

22.7

9.4

West

7.7

3.1

Total

78%

19.0%

57%

11.4%

Differences by region - Western compared with other regions and by species

Relation between age of calf (in months) and testing positive for FMD-NSP antibodies

• No relation with age • Buffalo calves test FMD-NSP positive more often than cow calves

Previous project 5 governorates

% positive

95% CI

% positive

95% CI

Village

86%

78 - 93%

78%

69 - 86%

Animal

20.6%

18.4 - 22.9%

24.0%

21.7 - 26.4%

No differences in FMD endemicity between two studies

Relation between age of sheep and goats with testing positive for FMD-NSP antibodies

Small ruminants

Village



Why do buffalo calves test positive more often

Effect of location of birth

• Relation with age • Older than 35 months, are mainly from Western region (low prevalence)

• When not born in village, higher percentage test FMD-NSP positive  animal movement is risk for FMD

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 47

Discrepancy between ‘what happens in villages’ and ‘what is known with GOVS’ In 98 villages (33%) clinical signs of FMD were seen (in the previous 12 months) where as a total of just 6 FMD outbreaks were officially notified to GOVS in the same time period

Risk factors for testing FMD-NSP antibody positive cattle and buffalo calves B u f f a l o

Manure dealing

Clinical FMD signs in village

Transported into village

Testing FMD-NSP positive No relation with age

Western region

Risk factors for testing FMD-NSP antibody positive sheep and goats

Veterinary clinic Animal market

Testing FMFMD-NSP popositive

Cattle and buffalos in the same household

Increasing age

Western region

• it seems more likely that sheep and goats are important for spread within the village rather than spread between villages

Conclusions from this study • FMD infection is highly endemic - continuous circulation of FMD virus throughout the year • FMD clinical outbreaks are seriously underreported • Risk factors are related to - Animal movements • Direct introduction of calves • Indirect through market. Effect local clinic? - Manure handling • Region; western region is relatively less FMD infected or turned around: no differences in FMD rates in Nile delta, Central, Upper and Eastern Egypt

FMD control strategy

• The current FDM control strategy is not effective in the field. • Need for risk-based control strategy, that requires more information on: - Value chain analysis - what socio-economic drivers make animals being transported - Interest of stakeholders, private and public - Knowledge, attitude and practice (KAPanalysis) of farmers - Additional studies (serological, virological) to learn more about spread of FMD

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 47

TEAMWORK!

Thank you for your attention! Questions?

Soheir Abd El-Kader Ahmed Hani

Chris Bartels

Iman Faraq

Nashwa Salah Hassan Ibrahim Yasser Basyouni

Kees v. Maanen Keith Sumption

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Ahmed Habashi

Appendix 48

Objective and Results Determining the level of vaccine-induced versus field-induced antibodies in youngstock in West Azarbaijan EuFMD: Chris Bartels, Melissa McLaws Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Brescia, Italy Emiliana Brocchi, Edoardo Spagnoli Santina Grazioli Iranian Veterinary Organisation: Naser Rasouli, Javad Emami

• Cattle vaccinated 3x/year 3-valent FMD vaccine (Razi) • 8378 serosamples from cattle in 281 epi-units • 30 cattle per epi-unit • Cattle: 6 and 24 months

Animal level: 3 outcome categories

NSP-Ab titers • Prionics test

• In consultation with the EuFMD Research Group (Bruxelles, Oct 2011) • To better distinguish positives from negatives: Inconclusive: at 75% inhibition • Inconclusive 14.2% of results • Positive: 39.6%

• 2 epi-units with 0 seropositive cattle • 6 epi-units with 30 seropositive cattle • 53.7% of samples positive for NSP-Ab (at cut-off 50%)

Retested with ELISA - IZSLER • To support results from testing in WAZB Laboratory Random sample of 540 samples retested in Brescia IZSLER - cutoff 10% positivity Prionics

Negative

Positive

Total

Negative

234

525

Inconclusive

Positive

197

209

Total

295

245

540

 excluding a laboratory artefact

• Measuring FMD infection prevalence in youngstock in West Azarbaijan, Iran

Negative

Research question • To what extent are NSP positive test induced by the use of non-purified vaccine (trivalent) in West Azarbaijan? • 540 samples tested for SP-antibodies (Brescia) - Serotype A, Asia1 and O - 3-fold diluations for positive samples (>=10)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 48

Hypothesis

Zooming in on NSP positives

• Vaccine-induced antibodies

• Leaving out 234 samples (negative in both ELISAs)

- Relative low NSP titers - increased levels of SP for all three serotypes

IZSLER

• Infection-induced antibodies - Relative high NSP titers - increased level of SP for only one serotype

Prionics

Negative

234

Inconclusive

Positive 30

Positive

197

311 samples

• However, there are a number of issues: -

Cross-reaction between serotypes Decay of antibodies over time Biological variation between animals Variation between assays

1 serotype +

233 (74.9%)

2 serotypes +

3 serotypes +

142

Negative

123

137

Positive

188 (60.4%)

174 (56%)

Example Serotype A

Serotype Asia1

Serotype O

540

4.5

6.3

Titer Log scale Difference between serotypes

 This related to 2 or more diluation steps •

SP-O

Conversion SP titer to log scale

• A distinct difference between serotypes of same sample defined as a difference greater than 2.2 on log scale.

Percent

SP-Asia1

to define differences between results from one sample • SP titers were converted to Log-scale

Number Negative

SP-A

2.3 A versus Asia1

Defined difference

2.2 Yes

A versus O

6.3

Asia1 versus O

Yes 1.8

 it only applies to results that are SP positive (thus titer of 10 and higher)

Matrix of SP positive and SP difference

• How to interprete: No difference One serotype between serotypes different (2 steps)

Two serotypes different (2 steps)

3 serotypes neg

No infection, no vaccination

1 serotype pos

2 serotypes pos

Infection

3 serotypes pos

Vaccination

Combination and or vaccination

Combination and/or infection

Infection

No difference between serotypes

One serotype different (2 steps)

Two serotypes different (2 steps)

3 serotypes neg

1 serotype pos

2 serotypes pos

3 serotypes pos

113

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 48

NSP results for different SP-defined categories

Same interpretation for IZSLER results

• Samples attributed to relate to none infected or vaccinated animals have lowest Percentage inhibition (left histogram)

• However, ‘infected’ (1 or 2 pos SP + 3 differences), have overall lower NSP titers compared with samples attributed to ‘vaccinated’ (3 pos SP + no differences between)

NSP results for different SP-defined categories

Reflecting on results • Our hypothesis is not supported: - SP testing reveals that ‘vaccinated’ are testing higher NSP titers compared with ‘infected’. This is the different from hypothesized

Open sessions of the Standing Technical and Research Committees

Discussion • Infection with 1 serotype in a vaccinated population can give rise of SP antibodies (booster-effect) • Effect of cross-reaction • No perfect match of the circulating antibodies with the antigens in immuno-assays

29-31 October 2012 Jerez de la Frontera, Spain

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 49

Index case: VP1 characterization © Daniel Dalet / d-maps.com

500 km © Daniel Dalet / d-maps.com

VP3 2xVP2

Spatiotemporal origin and transmission of the foot-and-mouth disease virus outbreaks in Burgas region (Bulgaria) in 2011

VP1 5x

VP2 VP3 VP3

VP2 2x

VP2

VP2 VP3 VP3

Capsid 1A VP4

1B VP2

1C VP3

Carboxy-terminal self-cleaving 1D VP12A

Membrane-binding Genome-linked (VPg) NTP binding* 2C

* putative functions 3’UTR

Protease 3A

Polymerase 3D AAA

VP1

(n)

Poly(C)

VP3 2x VP2

VP1VP1 5x

VP1 coding region 640 nt

Kocaeli

Gümüşhane Kastamonu

Bursa

Kastamonu Eskişehir

Eskişehir

Turkey

urkey

Si Gümüşhane

Sivas

Antalya

5th January 2011

O/BUL/1/2010 (Burgas 30/12/2010) O/TUR/926/2010* (Bursa 26/07/2010) O/TUR/1086/2010* (Antalya 16/08/2010) O/TUR/840/2010* (Agri 15/07/2010) O/TUR/1094/2010* (Giresun 11/08/2010) O/TUR/868/2010* (Eskisehir 20/07/2010) O/TUR/1003/2010* (Sivas 10/08/2010)

Bulgariaulgaria

AntalyT

Protease

300 mi

VPG

Valdazo -González et al 2011

Begoña Valdazo-González1*, Lilyana Polihronova2, Tsviatko Alexandrov2, Preben Normann3, Nick J. Knowles 1, Jef M. Hammond1, Georgi K. Georgiev2, Fuat Özyörük4, Keith J. Sumption5, Graham J. Belsham3, Donald P. King1 2

VP1 VP1

300 mi

5’UTR

O/TUR/18/2010 (Gumushane 09/07/2010)

Ağrı

Iran

O/TUR/883/2010* (Kastamonu 23/07/2010)

O/TUR/35/2010 (Erzincan 13/08/2010) O/TUR/153/2010* (Gaziantep) O/TUR/154/2010* (Gaziantep) O/IRN/94/2010 (West Azerbaijan 15/04/2010) O/IRN/92/2010 (West Azerbaijan 14/04/2010) 100 O/AFG/59/2010 (Samangan 14/05/2010) 97 O/PAK/36/2010 (Gilgit-Baltistan 29/07/2010) O/TUR/36/2010 (Kocaeli 13/08/2010) O/UKG/12/2001 (AJ311724) O1/Manisa/TUR/69 (AJ251477)

Iran

O/ME-SA/PanAsia-2ANT-10

0.01

Objectives:

Following outbreaks in livestock

1. Amplification, sequencing and analysis of the Bulgarian viruses (virus from the wild board and representative viruses from all the virus-positive outbreaks) plus 11 closely-related contemporary viruses from Anatolia (Turkey) and Israel

Virus positives Antibod

Wild boar First wave Second wave

2. Reconstruction of the spatiotemporal origin and transmission of the virus

2011

A. 47 days 30 Km apart

Affected

Designation

Affected/total

holdings

Maximum age of the

animals

lesions (days)

Index

1/1W

~15

≥3

1/194C, 14/117S , 12/149G, 8/72P

1/1C, 13/38S, 10/110G

1b/143C

2-5

6/133C

5-8

4/49C, 0/11P

1/81C

2/209C

3-5

Whole village

21/21C, 121/121S &G, 12/12P

121/121S&G, 72/72B

Whole village

11/45C, 5/356S&G, 0/6P

/92C, 0

~10 (1a); -(1b); 2-8 (1c) /77S&G

4/5-8

5-8 3-5

Valdazo -González et al in press

FG amplification and sequencing strategy

Origin of the outbreaks Who/what? - Statistical parsimony (TCS)

Original clinical sample (cattle or wild boar) - Epithelium (suspension) - 18 Cell culture - 1 RNA extraction RT-PCR Cycle sequencing (ABI PRISM 3730 DNA Analyzer) Data analysis: - Statistical parsimony (TCS) - Bayesian Markov chain Monte Carlo (MCMC) methods (Beast)

Valdazo -González et al in press

Open Session of the EUFMD: 2012, Jerez de la Frontera, Spain

Appendix 49 Origin of the outbreaks When/where - MCMC methods (Beast)

Transmission within the outbreaks Statistical parsimony (TCS) IAH2

TUR/36/2010

AY593815

O/IS R/2/2011

TUR/926/2010

30/12/2010

12 / 3

BUL/32/2011 BUL/30/2011

BUL/26/2011

12LPN3

4/1

26/07/2010 TUR/926/2010 Bursa (Closest Turkish virus)

Wild boar First wave Second wave

16/01/2011 12LPN3 Rezovo

TUR/27/2011

01/01/2010

First phase of outbreaks in livestock

TUR/840/2010

Second phase of outbreaks in livestock

TUR/868/2010

01/04/2010

01/07/2010

01/10/2010

Overall, wild boar included

01/01/2011

01/04/2011

Filling the gaps within the branches: seropositive-only outbreaks versus wildlife

Nucleotide substitution Amino acid substitution

18/03/2011

9/2

BUL/11/2011 Kirovo

14/3

24/03/2011

Wild boar First wave Second wave

23/03/2011

BUL/30/2011 Fakia

4/1

Putative common ancestor of Bulgarian outbreaks TUR/8/2011

TUR/883/2010 01/10/2009

Cottam et al 2008

TUR/1003/2010

0.999

IP5

12LPN1 Kosti

TUR/18/2010

IP7

IP2c

14/01/2011

14/7 40 nt changes 5 NS changes

12LPN1

IP8

IP2b

UK 2007 - Linear spread

BUL/1/2010 Wild boar

BUL/20/2011

0.999

IP6b IP3b

IP4b

BUL/11/2011 1

IP3c

IP1b(1)

Sampled virus P utative ancestor virus Nt change Aa change His to Arg Asp to Gly

Valdazo -González et al in press

BUL/1/2011

IP1b(2) MAH

IAH1

Valdazo -González et al in press

TUR/1086/2010

BUL/26/2011 Granichar 24/03/2011 BUL/20/2011

3/1

Goliamo Bukovo

7/2 2/1

Bulgaria 2011 - Radial spread

BUL/32/2011 M omin Tsakva 24/03/2011

Conclusions • A single putative ancestor exists for all the outbreaks in Bulgaria, early 2011. • The closest sequenced virus to this single putative ancestor is the virus found in a Bulgarian wild boar, more distant to an isolate from Bursa (Anatolia, Turkey) collected on July 2010. • The genetic distance between the closest Turkish virus and the Bulgarian viruses is consistent with active replication within the host when compared to data from the 2001 and 2007 outbreaks in UK.

Valdazo -González et al in press

• The estimated time for the common putative ancestor is in a temporal interval which is plausible with the Kurban Bavram festival (16/11/2010). However, the virus might have arrived the continent since April 2010. • Each wave of outbreaks in Bulgaria has its own unique common ancestor.

•

Only genetic data provide evidence that the two waves are connected via a single common ancestor

•

Long branch length represent undisclosed infection Sero-positive herds and/or wildlife hosts? Antibody detection in wild boar (and roe deer) is not conclusive

• The long branches between these secondary putative ancestors and the field viruses indicate un-sampled intermediate infection (either domesticated livestock on reported seropositive outbreaks or wildlife [wild boar]). • FGS analysis can be used as an effective real-time tool to support and help direct epidemiological investigations of field outbreaks.

Acknowledgements • FAO/OIE/EU FMD reference laboratory staff for undertaking the supporting laboratory analyses for the viruses included in this study • Dr David Paton (IAH, Pirbright, UK) for useful comments and criticism • This project was supported by the Department for Environment, Food and Rural Affairs, United Kingdom (Research grant no. SE2938) • Lilyana Polihronova was supported by an International Atomic Energy Agency (IAEA) Fellowship, code No BUL/09011

Open Session of the EUFMD: 2012, Jerez de la Frontera, Spain

Appendix 51

Effectiveness of vaccination programmes

Insights from human vaccination programmes

Paul Fine London School of Hygiene and Tropical Medicine

EUFMD 2012 “Appliance of Science in The Progressive Control of FMD” Jerez de la Frontera, Spain 29 - 31 October 2012

Effectiveness of vaccination programmes

Insights from human vaccination programmes A HUGE literature !

A few definitions “Vaccine potency”

Lab measure of vaccine contents - eg PD50 type measures

“Vaccine efficacy”

5th edition 76 chapters 1725 pages 3.6 kilograms 1000s of refs

A few definitions

Reduction in risk (of disease) in vaccinated individuals compared to non-vaccinated (under trial conditions)

“Vaccine effectiveness”

Vaccine efficacy under field conditions

“Vaccine programme effectiveness = impact” Reduction in disease (morbidity, mortality) attributable to a vaccination programme

A few definitions

“Vaccine potency”

“Vaccine efficacy”

So, if the risk of disease in a trial is 10 % in non vaccinated, 3 % in (equally exposed) vaccinated, “Vac the “vaccine efficacy“ is 70 % Re ie (10 - 3) / 10 = 0.7

“Vaccine effectiveness”

Lab measure of vaccine contents - eg PD50 type measures

Reduction in risk (of disease) in vaccinated individuals compared to non-vaccinated (under trial conditions)

“Vac Va

Lab measure of vaccine contents - eg PD50 type measures

Reduction in risk (of disease) in vaccinated individuals compared to non-vaccinated (under trial conditions)

Vaccine efficacy under field conditions

“Vaccine programme effectiveness = impact” Reduction in disease (morbidity, mortality) attributable to a vaccination programme

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 51

Effectiveness of a vaccine Depends upon:

Depends upon:

“Quality” of the vaccine (inc. “match”) Cold chain Quality of administration Number of doses Age at vaccination Time since last vaccination Level of exposure Environmental factors ?

• • • • • • • •

Effectiveness of a vaccine • • • • • • • •

Effectiveness of a vaccine Depends upon:

“Quality” of the vaccine Cold chain Quality of administration Number of doses Age at vaccination Time since last vaccination Level of exposure Environmental factors

• • • • • • • •

Vaccine effectiveness-examples Vaccine

Effectiveness 0 - 80 %

Comments Effectiveness generally high versus meningitis but varies greatly versus pulmonary disease. Lower in tropics than in temperate countries For 15 years ?

Pertussis

10 - 40 % 1 doses 30 - 60 % 2 doses 50 - 80 % 3 doses

Haemophilus

70 - 95 % 2,3 doses At least two doses needed

Measles

90 - 95 %

High if given after 9 months Lasts many years (decades )

Rotavirus

20 - 60 % 2 doses

Highest after three doses in wealthy (good hygiene) countries - lower in poor countries

Effectiveness differs greatly between different vaccines and by outcome (highest against severe disease) Wanes with time

“Quality” of the vaccine Cold chain Quality of administration Number of doses Age at vaccination Time since last vaccination Level of exposure Environmental factors

Effectiveness of a vaccination programme

(% reduction in risk in vaccinees, from field evaluations)

BCG

“Quality” of the vaccine Cold chain Quality of administration Number of doses Age at vaccination Time since last vaccination Level of exposure Environmental factors ?

Depends upon: •

Effectiveness of the vaccine

•

“Coverage” • Proportion of host population vaccinated • by age, • by risk group • geographic distribution ...

•

Other sources of infection etc

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 51

Effectiveness of a vaccination programme Depends upon:

The basic global “vaccination” programme (for humans): (Started

•

Effectiveness of the vaccine

•

“Coverage” • Proportion of host population vaccinated • by age • by risk group, • geographic distribution ...

by WHO in 1974)

“EPI“ (“Expanded Programme On Immunization”)

Other sources if infection etc

•

Basic EPI schedule (from 1970s) Purposefully simple Birth (or “first contact”)

BCG

6 weeks

10 weeks

14 weeks

√

9 months

Measles

Basic EPI schedule (from 1970s) Purposefully simple

BCG DTP Polio (OPV)

10 weeks

14 weeks

9 months

√ √

√

Measles

10 weeks

14 weeks

√

Measles

√

Example of a current schedule (England and Wales, 2012) Vaccine

Birth (“high risk”)

2 months

3 months

4 months

BCG

√

DTaP

√

IPV

√

Hib

√

PCV

√

MMR

12-13 months

To avoid maternal antibody

12-13 years (girls)

13-18 years

√

HPV

BCG = bacillus Calmette Guerin DTaP = diphtheria, tetanus, acellular pertussis IPV = Inactivated (killed) polio (trivalent) Hib = Haemophilus influenza B

3-4 years

√

√ √

Td 4-week spacing, to optimise boosting

To avoid maternal antibody

4-week spacing, to optimise boosting

MenC √

9 months

√

To avoid maternal antibody

4-week spacing, to optimise boosting

6 weeks

DTP Polio (OPV) √

Birth (or “first contact”)

BCG

√

DTP Polio (OPV)

Basic EPI schedule (from 1970s) Purposefully simple

√

PCV = Pneumococcal conjugate MenC = Meningococcus type C MMR = measles, mumps, rubella HPV = Human papilloma virus Td= Tetanus and diphtheria toxoids

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 51 Vaccine coverage

Examples of coverage and impact monitoring

Impact: linking of vaccine coverage and disease surveillance data

Vaccine coverage

Impact: eg at national level Pertussis, UK

Measles, UK 800

100

180 140 120 100

400

0 1950

1970

1980

1990

20 0 1940

0 2000

1950

1960

1970

1980

1990

2000

Year

Y ear

Measles incidence (routine notifications)

Campaigns in all children under 5

1960

Polio in: Cuba

160

200

Impact: eg at national level

200

600

These data are provided to WHO by most countries of the world, and are freely available

Effect of UNICEF’s “UCI 90“ (90 % by 1990) target

When EPI started, in 1974, only 15 % of the world’s children were receiving routine vaccines

Vaccine coverage (% of each cohort with “MMR”)

Brasil

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 4

Appendix 51

Programme evaluation “Herd immunity” thresholds have NOT proved useful for programme evaluation They are, in general, “optimistic”

Something is happening, now, with pertussis (whooping cough), in several countries …

Examples of vaccine effectiveness monitoring .....and responses

California, USA September 13 2012

England and Wales (Red line) - proportion of children who received only acellular pertussis vaccine

lab-confirmed cases, by quarter, 2007 - 2012

(Blue line) incidence of pertussis peaking between 8 and 12 years of age… (This is unusual !)

inc 10 infant deaths in 2012

UK policy change … From The Guardian , Friday 28 September 2012

Polio Global eradication initiative, since 1988

( 350,000 cases in 1988 ) -to increase maternal immunity... and thus protect very young infants

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 5

Appendix 51 Wild Poliovirus1, 10 Oct 2011 - 09 Oct 2012

Wild Poliovirus*, 06 Feb 2007 - 05 Feb 2008

Last 12 months *10 Oct 2011 - 09 Oct 2012 Wild virus type 1 Wild virus type 3 Wild virus type 1/3 Endemic country Country with WPV case in previous 6 months Country with WPV case 6-12 months ago

From AFP (acute flaccid paralysis) surveillance - all virologically confirmed

1Excludes viruses detected from environmental surveillance and vaccine derived polioviruses.

Wild virus type 1 Wild virus type 3 Wild virus type 1 & 3 Endemic countries Case or outbreak following importation (0 - 6 months) Case or outbreak following importation (6 - 12 months) As of 01 January 2006, Egypt and Niger were reclassified as non-endemic countries. *Excludes viruses detected from environmental surveillance and vaccine derived polio viruses. Data in WHO HQ as of 05 Feb 2008

Wild Poliovirus*, 06 Feb 2007 - 05 Feb 2008

5 years ago The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2008. All rights reserved

Wild Poliovirus*, 06 Feb 2007 - 05 Feb 2008

Afghanistan + Pakistan

Northern Nigeria Wild virus type 1

Low vaccine coverage due Case or outbreak following importation (0 - 6 months) Case or oto distrust of6 - 12 months) vaccine

Wild virus type 1 Wild virus type 3 Wild virus type 1 & 3 Endemic countries

Wild virus t Wild virus type 1 & 3 Endemic c

As of 01 January2006, Egypt and Niger were reclassified as non-endemic countries. surveillance and vaccine *Excludes viruses detected from environmental derived polio viruses. Data in WHO HQ as of 05 Feb 2008

War and breakdown of society

Case or outbreak following importation (0 - 6 months) Case or outbreak following importation (6 - 12 months) The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2008. All rights reserved

As of 01 January 2006, Egypt and Niger were reclassified as non-endemic countries. *Excludes viruses detected from environmental surveillance and vaccine derived polio viruses. Data in WHO HQ as of 05 Feb 2008

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2008. All rights reserved

Wild Poliovirus*, 06 Feb 2007 - 05 Feb 2008

North India (UP + Bihar)

Very low vaccine effectiveness (only 10 % per dose)

The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization or of its authorities, or concerning the legal status concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. WHO 2008. All rights reserved

Very low vaccine effectiveness (only 10 % per dose)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 6

Appendix 51 Wild Poliovirus1, 10 Oct 2011 - 09 Oct 2012

“Conclusion” Optimising effectiveness of a vaccination programme requires rigorous surveillance and analysis of

*10 Oct 2011 - 09 Oct 2012 Wild virus type 1 Wild virus type 3 Wild virus type 1/3 Endemic country Country with WPV case in previous 6 months Country with WPV case 6-12 months ago

Last 12 months

• disease trends, • vaccine “coverage”, • vaccine performance,

(and costs)

and appropriate response

1Excludes

viruses detected from environmental surveillance and vaccine derived polioviruses.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 7

Appendix 52 Conclusions & recommendations

1. Protection from Asia-1 field strain (Sindh08) by low potency Asia-1 Shamir vaccine was not detected in this field study

FMD Asia-1 vaccine effectiveness in Turkey

2. Protection from Asia-1 field strain by Asia-1 TUR11 vaccine detected for clinical disease but not infection 1. Reduced clinical signs => less virus shedding 2. Clinical vaccine effectiveness appeared to vary (32-74%)

Theo Knight-Jones,

3. Assess vaccine performance in the field

Naci Bulut, Paul Fine, Simon Gubbins, Keith Sumption & David Paton

1. As well as other methods (r-value, serology, challenge study)

FMD in Turkey

FMD vaccination in Turkey 4

Until 2012 >20 million doses/year of multivalent FMD vaccine (>€20million/year)

Target: cattle vaccinated every 6 months - 3 PD50 vaccines (low potency) - serotypes A, O & Asia-1

-FMD Free -Otherwise FMD Present

Target: sheep vaccinated once a year

Sumption et al., 2008

- serotypes A & O

Yet major FMD epidemics occur

Reported FMD outbreaks in Turkey 5

Key questions for a vaccination programme: 6

1. Are vaccinated animals protected?

Vaccine effectiveness 2. Are the animals being vaccinated (adequately)?

Vaccine coverage

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 52 Vaccine effectiveness

Vaccine effectiveness 8

Reduction in risk in similarly exposed vaccinated compared to unvaccinated animals in the field

Vaccine efficacy -> under controlled trial Vaccine effectiveness - > observational study (field study - program conditions)

Protection against what? - clinical disease, infection, shedding

VE = 1 - Incidence in Vaccinated Incidence in Unvaccinated

Vaccine effectiveness - study 1 9

Sampling-Retrospective cohort 10

1. Within a village - at end of outbreak:

Asia-1 outbreak investigation One week field study - Afyon, Turkey Jan, 2012

1. Selected households evenly spaced in village

- investigated new Asia-1 TUR11 vaccine (Sindh 08)

2. Within a household: 1. Examined all cattle

3. For each selected animal (n=229): 1. Asked owner about vaccination and FMD history 2. Examined for clinical signs 3. Assess infection history by serology (<30 months)

Asia-1 exotic virus -no prior exposure

Results

Proportion with clinical FMD: • Unvaccinated 50/114 (44%) vs one dose 9/80 (11%)

Clinical incidence risk: • Unvaccinated 50/114 (44%) vs one dose 9/80 (11%)

Vaccine effectiveness (Asia-1 TUR11 vaccine):

- Against clinical disease:

• Crude Vaccine Effectiveness=74% [95% conf int 55-85%]

- Against clinical disease:

• Crude Vaccine Effectiveness=74% [95% conf int 55-85%]

NSP seropositive:

- No protection against infection: • Vaccine Effectiveness=10% [95% conf int -12% to 27%]

- Same effect after adjusting for age confounding and using Asia-1 SP serology with NSP serology

• Unvaccinated 72/92 (78%) vs one dose 50/71 (70%)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 52 Studies 2 & 3: June/July 2012

Further FMD Asia-1 TUR11 results 14

Vaccine effectiveness (Asia-1 TUR11 vaccine): Study 2- Denizli:

- Reduced protection against clinical disease: • Crude Vaccine Effectiveness= 44% [95% conf int 30-55%] • Unvaccinated 55/68 (81%) vs one dose 134/297 (45%)

- No protection against infection: • Vaccine Effectiveness= 8% [95% conf int -11% to 23%] • NSP seropositive: Unvaccinated 28/29 (97%) vs one dose 125/140 (89%)

Further Asia-1 TUR 11 vaccine effectiveness studies Denizli & Afyon provinces central Turkey

Further FMD Asia-1 TUR11 results 15

Vaccine effectiveness (Asia-1 TUR11 vaccine): Study 2- Denizli:

- Reduced protection against clinical disease: • Crude Vaccine Effectiveness= 44% [95% conf int 30-55%] • Unvaccinated 55/68 (81%) vs one dose 134/297 (45%)

- No protection against infection: • Vaccine Effectiveness= 8% [95% conf int -11% to 23%] • NSP seropositive: Unvaccinated 28/29 (97%) vs one dose 125/140 (89%)

Further FMD Asia-1 TUR11 results 16

Vaccine effectiveness (Asia-1 TUR11 vaccine): Study 2- Denizli:

- Reduced protection against clinical disease: • Crude Vaccine Effectiveness= 44% [95% conf int 30-55%] • Unvaccinated 55/68 (81%) vs one dose 134/297 (45%)

- No protection against infection: • Vaccine Effectiveness= 8% [95% conf int -11% to 23%] • NSP seropositive: Unvaccinated 28/29 (97%) vs one dose 125/140 (89%)

Study 3- Afyon-2: - Reduced protection against clinical disease: • Crude Vaccine Effectiveness= 32% [95% conf int 14-47%] • Unvaccinated 71/124 (57%) vs one dose 69/177 (39%)

- No protection against infection: • Vaccine Effectiveness= -11% [95% conf int -25% to 1%] • NSP seropositive and Asia-1 SP: Unvaccinated 76/91 (84%) vs one dose 90/97 (93%)

Further FMD Asia-1 TUR11 results 17

Vaccine effectiveness (Asia-1 TUR11 vaccine): Study 2- Denizli:

- Reduced protection against clinical disease: • Crude Vaccine Effectiveness= 44% [95% conf int 30-55%] • Unvaccinated 55/68 (81%) vs one dose 134/297 (45%)

- No protection against infection: • Vaccine Effectiveness= 8% [95% conf int -11% to 23%] • NSP seropositive: Unvaccinated 28/29 (97%) vs one dose 125/140 (89%)

Study 3- Afyon-2: - Reduced protection against clinical disease: • Crude Vaccine Effectiveness= 32% [95% conf int 14-47%] • Unvaccinated 71/124 (57%) vs one dose 69/177 (39%)

- No protection against infection: • Vaccine Effectiveness= -11% [95% conf int -25% to 1%] • NSP seropositive and Asia-1 SP: Unvaccinated 76/91 (84%) vs one dose 90/97 (93%)

Asia-1 TUR11 reducing effectiveness?: -After adjusting for confounding (age, herd size, sex, management) Protection against clinical FMD - logistic regression Study 1 (VE 74%) Odds ratio - 0.09 [95%CI=0.02-0.43] Study 2 (VE 44%) Odds ratio - 0.12 [0.03 - 0.56] Study 3 (VE 32%) Odds ratio - 0.33 [0.13 - 0.88] Residual variation due to:

Random variation, residual confounding, age of vaccine, vaccine application, vaccine batch, field strain variation, host and management factors…

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 52 Study 2...

Study 4: Ardahan, Oct 2011 19

-Investigated Asia-1 Shamir vaccine: same design as example 1

Protection against infection (based on serology)

42% 77% sero-positive

82%

Maternal protection?

11%

No vaccine protection?

Same pattern for clinical disease

Conclusions & recommendations

1. Protection from Asia-1 field strain by low potency Asia-1 Shamir vaccine was not detected in this study 2. Protection from Asia-1 field strain by Asia-1 TUR11 vaccine detected for clinical disease but not infection

2-5

Effect of multiple doses or cross-protection from repeated A & O infections?

Acknowledgements

Supported by EuFMD and BBSRC Thanks to the SAP instutite, Turkey

1. Reduced clinical signs = less virus shedding & transmission 2. Clinical vaccine effectiveness appeared to vary (32-74%)

3. Assess vaccine performance in the field 1. As well as other methods (r-value, serology, challenge study)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 53

Conclusions An investigation of vaccination effectiveness in two Cambodian villages facing an outbreak of Foot-and-Mouth Disease



Broadening vaccine donor focus 



Need for systematic approach to control 

Socheat Sieng and Jim Kerr



Allocation of funds for monitoring all stages of administration and effectiveness Livestock movement, biosecurity

Poor use of vaccine may be worse than no vaccine 

Convincing farmers that it has no value

Context 

   

Advancing outbreak of FMD in Cambodia in 2010 Vietnam donated type O vaccine Local authorities managed administration Subsequent outbreaks in vaccinated villages Coincidental research project activities 

Opportunity for observational study of vaccine effectiveness

Village vaccination 

Village 1: Chrey Vien  



147 animals vaccinated (35%) on 2nd August First cases seen on 25th August (23 days later)

Village 2: Tropeang Ampil  

140 animals vaccinated (47%) on 3rd August First cases seen on 27th August (27 days later)

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 53

Morbidity during outbreak 

Village 1: Chrey Vien 

Attack rate  





Unvaccinated: Vaccinated:

Relative risk:

71.5% 51.4%

1.39

Village 2: Tropeang Ampil 

Attack rate  



Unvaccinated: Vaccinated:

Relative risk:

61.5% 57.8%

1.06

Attributable fractions (exposed) 

Proportion of cases in unvaccinated animals that could have been prevented by vaccination  

Village 1: 28% Village 2: 6%

Reasons for failure 

Low vaccination rates 



No herd immunity

Overwhelming exposure   

Roadside tethering Communal grazing Lack of hygiene when handling infected animals

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 53

Possible reasons for failure 

Poor planning / surveillance   



Date of first exposure uncertain Villages too close to existing outbreak Lack of movement control

Poor vaccination  

Cold chain, vaccination technique Inadequate dose (fee per animal)

Implications 

Examples of real-world use of vaccine 







Non-controlled, not part of research project

Farmer perceptions FMD vaccination more expensive and less important than killer diseases (e.g. haemorrhagic septicaemia) With failures illustrated here, vaccine does not prevent disease

Major challenge for future participation

Conclusions 

Broadening vaccine donor focus 



Need for systematic approach to control 



Allocation of funds for monitoring all stages of administration and effectiveness Livestock movement, biosecurity

Poor use of vaccine may be worse than no vaccine 

Convincing farmers that it has no value

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 53

Acknowledgements 

Australian Centre for Agricultural Research (ACIAR) project: 



Understanding livestock movement and the risk of spread of transboundary animal diseases Participating partners 



Cambodian Department of Animal Health and Production Department of Agriculture and Forestry, Western Australia AusVet Animal Health Services

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 54

Major conclusion • A cutoff of r1=0.3 for matching between vaccine and field strain may not suffice for routine vaccination, even if the vaccine is of high potency

The Field Effectiveness of an Inactivated Vaccine for Prevention of FMD

• However, such vaccine is effective when used during emergency vaccination, despite low titer of neutralizing antibodies

Eyal Klement – Associate Professor

Koret School of Veterinary Medicine Robert H. Smith Faculty of Agricultural, Food and Environmental Sciences The Hebrew University, Jerusalem, Israel

OIE recommendations • ≥3 PD50  routine vaccination • ≥6 PD50  emergency vaccination in naïve population

• r1 ≥ 0.3 indicates that the use of a vaccine based on this strain is likely to confer protection against challenge with the field isolate OIE 2009

Vaccine used in Israel • An inactivated vaccine with a PD50 ≥6, manufactured by Merial®. • Strains subtypes included in the vaccine were O1-Manisa, O1-4625, O1-3039, A-Iran 05, A-4165 and Asia1-Shamir

FMD in Israel • Israel suffers frequent outbreaks caused by FMD viruses, mostly of serotype O • Routine vaccination of sheep, goats, cattle and pigs in Israel is mandatory • The first FMDV vaccination is routinely administered to cattle between 2-6 months of age together with Brucella abortus live attenuated vaccine (two separate injections) • This is followed by an FMDV booster vaccination 12 months later and a repeated annual vaccination for FMDV only.

FMD outbreak Israel 2011 • Outbreak onset was on March and it involved 26 localities that included 30 beef herds, 11 feedlots, 4 dairy herds and 8 sheep flocks • Isolation of serotype O, Pan-Asia-2 viruses

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 54

FMD outbreak Ramat Magshimim

Data collection • Herdsmen and the veterinarian:

Ramat Magshimim Dairy herd n=931

– Group data:

Feedlot n=730

• • • • • • •

location number of animals Breed Age Origin Date of arrival to the farm Number and date of vaccine administration prior to the outbreak • Time of clinical signs onset and extent of morbidity

– Vaccination data confirmed by herd management software

Case definition • Feedlot – an animal defined by the herdman as showing lameness with or without excessive salivation and tongue lesions • Dairy cattle - an animal defined by the herdman and veterinarian as showing typical tongue lesions with or without excessive salivation, lip smacking or lameness

SN survey

NSP survey • 1-3 months after the outbreak 113 and 156 cattle heads were sampled from the dairy farm and feedlot, respectively, for the presence of anti NSP antibodies

Outbreak description

• Pre-outbreak sera were not available from the cattle in the affected farms. • in order to correlate the clinical outcome in the outbreak with the pre-exposure levels of neutralizing antibodies we tested sera collected from cows, heifers and calves, which represented parallel vaccination status to the outbreak groups (n=95)

16/6/2011

• These sera were tested by SN against: – O-4625 - The vaccine strain which showed the highest resemblance to the field strain – O ISR 11/11 – The field strain isolated during this outbreak in Ramat Magshimim

20/6/2011

1/6/2011

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

10/6/2011

11/6/2011

Appendix 54

Detailed outbreak results

Outbreak - summary Dairy Farm

SN results O ISR 11/11d

P value (t test)

NSP pos. (%)

O – 4625

Incidence (%)

Outbreak groups with Time since the same Farm Number of last vaccinationsa a vaccination vaccination status

Number of vaccinations

at least 3

at least 1

Time elapsed since last vaccination

7 months

14 days

3-11 months

1-9 days

Clinical morbidity

12%

none

~100%

<1%

anti NSP antibodies

78%

96%

55%

Summary 1 – duration of immunity

SN – GMTb (CI95%)

Vaccination status

Feedlot

0-9 days

Feedlot

16.94 (14.44-19.87)

6.97 (5.9-8.23)

<0.0001

14 days

L,M,R

Dairy farm

21.42 (15.99-28.69)

8.07 (5.92-10.99)

<0.0001

3.7

≥2

14 days

I, N,O,P,Q

Dairy farm

504.86 192.28 (360.45-707.12) (97.29-380)

0.0212

8.7

≥3 months

1-18

Feedlot

127.46 (87.99-184.63)

27.96 (14.54-53.79)

0.0012

~100

≥3

7 months

Dairy farm

95.52 (87.01-104.87) (87.01 104.87 104.87)

29.03 (26.57-31.72) (26.57 31.72

0.0004

17.54

100

r1 = 0.37

Summary 2 – emergency vaccination • The same vaccine elicited almost complete protection from clinical infection and partial protection from infection when administered up to 2 weeks before natural challenge. • This occurred even after administration of only one dose of vaccine that elicited only low antibody titers

• A 6 PD50 inactivated vaccine which contained an O virus with an r1=0.37 matching with the field strain was used for routine and emergency vaccination in cattle in Israel • The vaccine elicited only partial protection from clinical infection when natural challenge occurred 3 months or more after vaccination • NA GMT at this stage was more than 100 against the vaccine strain but only 28 against the field strain • This occurred despite previous multiple vaccinations.

Major conclusion • A cutoff of r1=0.3 for matching between vaccine and field strain may not suffice for routine vaccination, even if the vaccine is of high potency • However, such vaccine is effective when used during emergency vaccination, despite low titer of neutralizing antibodies

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 54

Acknowledgments • Dr. Udi Elnekave

Israeli Kimron Veterinary institute Dr. Boris Gelman

Hebrew University Dr. Lior Zamir Ily Shlamovitz

Israeli Veterinary Services Dr. Boris Even-Tov Dr. Fares Hamed WRLFMD, The Pirbright Institute Dr. Jeff Hammond Dr. Yanmin Li Dr. Pip Hamblin

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 55

Foot and Mouth Disease Vaccine Indian Economic Impact

LIVESTOCK POPULATION FAO

(in Mio)

Species Cattle Buffalo Sheep Goat Pig Poultry

1995

World 1295 150 1081 627 897 12273

2000

India 193 79 45 118 12 435

2012 India 217 112 85.5 162 12.2 800

No. 1 in Milk Production 130 - 135 MT LIVESTOCK IS AN ECONOMIC INSTRUMENT FOR RURAL SOCIO ECONOMIC TRANSFORMATION @ GRASS ROOT LEVEL

PRODUCTION PROCESS

Eco Friendly FMD Production Plant @ Malur - Bangalore, India

100 LTS

500 LTS

1000 LTS

2500 LTS

CRYO VIAL

T 175

CB1

CB2

CB3

CB4/CB5

VB1/VB2 /VB5

IT-01 BEI

CULTURE

FILLING PROCCESS

FINAL PRODUCT

OIL WITH

B UF F ER

ADJUVENT

Bio-safety & Bio-containment facility BSL3+Ag

-------

A O ASIA-1 05 05 05

CONCENTRATION

IT-02 INACTIVATION

Different destinations…. DESPATCH WITH COLD CHAIN

PACKING PROCESS

ANTIGEN BANKING • SERO TYPE • MIO DOSES

W I T H ANTIGEN BANK (-80C)

ANTIGEN

FMD VACCINE PRODUCTION 50 45 40 35 30 25

FMD VACCINE (IN MILLION DOSES)

20 15 10 5 0 2008-09

2009-10

2010-11

2011-12

TARGET ACHIEVED

05 MILLION DOSES MAINTAINED - AS INVENTORY

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 55 SEROCONVERSION AND POTENCY LPBE 1.8 Log10 (AVERAGE)

FMD- CATTLE SALIVATION

POTENCY (PD50)

2.2

4.5 4

2.15

3.5 2.1 3 2.05

2.5

2 LPBE 1.8 Log10 (AVERAGE)

1.95

POTENCY (PD50)

1.5 1

1.9

0.5

1.85

1.8 FMD SERO TYPE

Cattle are highly susceptible and readily show clinical disease

FMD SERO TYPE

ANTIGEN PURIFICATION & CONCENTRATION

Biovet PD50 EXPERIMENT

Antigen production Virus freshening

Virus titration

Experimental design

BSL3+ CONTAINMENT

purification

concentration

no concentration

no purification

highly purified

partly purified

non purified

NSP-free

NSP-reduced

NSP in vaccine

OIL ADJUVANT VACCINE

ANTIGEN • TCID50/ML

5.5- 6.5

• 146s (Sucrose Gradient) : 5-12µg/vaccinating cattle dose. • Adjuvant - SEPPIC OIL.

no purification

Oily phase

W/O

+ O/W

Aqueous phase

W/O/W Oil

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Water

Appendix 55 Biovet Focus Areas

STRATEGY FOR PURIFIED VACCINE Conventional FMD vaccines

Production of Purified FMD vaccines

Vaccine Launching :

Haemorrhagic Septicaemia Vaccine



Black quarter Vaccine



HS + BQ Vaccine



Enterotoxaemia Vaccine



Fowl cholera Vaccine

Product Development under Public Private Partnership:-

Purified FMD vaccines as marker vaccines



Paratuberculosis (Jhone`s Disease Vaccine)

 Rapid Diagnostic Kits - FMD, DIVA, JD & BT.  Blue tongue Vaccine  Salmonella Vaccine (Avian)  Adjuvant Development

Vaccination of Indian Cattle against “Foot and Mouth Disease” -1943, O,A,C confirmation, Asia-1 - 1956-57.

RINDERPEST

NETWORKING, COOPERATION AND COORDINATION

2 2004

Free INDIA

PD-FMD

Dream or Reality?

OIE/FAO/WRL GLOBAL FMD VACCINE STRAIN MATCHING

SERO MONITORING IN FIELD MAHARASHTRA STATE 1200

1000

800 Type O pre-vac Type O post-vac

600

Type A Pre-vac Type A post-vac 400

Type Asia1 pre-vac Type Asia1 post-vac

200

0 I

III

VII

VIII

XII

PHASE UNDER FMD-CP

GLOBAL VACCINE BANK….

REF. LAB. NETWORKING

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 55 FMD OUT BREAKS/CASES IN INDIA DURING 2006-07 TO 2010-11

SERO MONITORING IN FIELD HARYANA STATE 1600 1400 1200 1000

Type O pre-vac Type O post-vac

800

YEAR

SOUTH

NORTH

CENTRAL

WEST

EAST

NORTH EAST

TOTAL

2006-07

224

431

778

2007-08

445

258

877

2008-09

240

2009-10

365

598

2010-11

176

TOTAL

843

109

140

118

1149

310

2669

Type A Pre-vac Type A post-vac

600

Type Asia1 pre-vac Type Asia1 post-vac

400 200 0 III

VII VIII PHASE UNDER FMD-CP

XII

FIELD REPORT

FAO-ICAR INTERNATIONAL CONFERENCE ON 13-15th FEBRUARY 2012 REDUCTION OF OUTBREAK/CASES 50-80% POST VACCINATION

Economic farming system models

FMD OUT BREAK NEAR BANGALORE

Type “O”

Monovalent vaccination option - matching vaccine strain / field strain

One World…

One Health…

One Medicine… Global Control Framework… Dream or Reality?

snsingh_2002@yahoo.com

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 55 FMD vaccination strategy….. ‘’to live’’ ‘’not to kill’’

THANK YOU…...

snsingh_2002@yahoo.com

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Evidence of residual neutralisation after removal of five neutralising antigenic sites in serotype O FMDV By Amin Asfor, S. Upadhyaya, D. Paton, D. King, N. Knowles and M. Mahapatra Transmission Biology Group Pirbright Laboratory United Kingdom

Conclusion • FMDV O1K 5-site mutant virus has been produced for the first time using reverse genetics • Mutation of the amino acid residues of the neutralising antigenic sites result in rescue of a viable virus not critical for viability of the virus • Residual neutralisation observed after mutations of all the 5 known antigenic sites indicates presence of yet unidentified epitopes • Two Novel Residues (VP2-74 and VP2-191) could be part of Site 2. proved to be significantly important in reduction of virus neutralization titre

Genome orgnization • + sense ss RNA genome ~8300nt 1300nt

RNA 5'

(VPg) NH2

AUG

OPEN READING FRAME

1A Lpro

protease Selfcleavage

NONSTRUCTURAL

STRUCTURAL

myristoylation

VP4

3'UTR

AAA (n)

Polyprotein 805-7800 1B

VP2

VP3

VP1

2B B

1 B

2 B

3 B

AAA (n)

protease

Capsid

RNA polymerase Heparin sulphate receptor binding

G-H Loop Receptor binding

VPg

Antigenic sites of type O

Red- VP1, Blue- VP2, Light green- VP3, Green-VP4. Site 5 is not shown, but located on the GH loop of VP1 within site 1.

Project Background • Dunn et al (1998) reported that a mutant virus (5 sites mutant) which is resistant to neutralising murine monoclonal antibodies (Mabs) also resists neutralisation by bovine polyclonal sera • Guinea pigs were protected in cross-challenge studies from virulent wild-type and mutant viruses using either wild-type or mutant 146S antigen as inactivated whole virus vaccine. • The relative importance of different epitopes has not been ascertained. • This project is aiming to : • Define viral determinants of antibody mediated protection • Identifying new surface exposed neutralizing or non neutralising epitope/s using reverse genetic technique • This will contribute to the development of sequence-based vaccine selection methods and novel broadly cross-reactive vaccines

6 AIM To identify the contribution of different epitopes present on the surface of foot-and-mouth disease virus (FMDV) towards antibody mediated protection

Infectious clone(IC) O1 Kaufbeuren

CHALLENGE VIRUS

Addition of more mutations Design of neutral virus

in vitro neutralization using O1K antiserum

in-vivo experiment to check the lack of protection against challenge virus

Final construct

selected mutants Reconstitution of individual epitops

in vitro neutralization using O1K antiserum

Assessment of protection antisera

Disease generalization Body temperature

Neutral virus design and preparation (1) • Order a synthetic gene containing all the required mutations (26 amino acid changes): sub-cloned into the full length FMDV plasmid • No viable virus was recovered- too many mutations to be tolerated

•

G67 and O1K-WT G67: quadruple mAb escape mutant of O1K that bears point mutations conferring resistance to neutralization by mAbs, specific for each of the four major antigenic sites (site 1-4) defined previously O1 Kaüfbeuren (O1K-wt) – parent of G67

Preparation of G67 mAb mutant neutral virus (2) pT7S3/O1K (11.2 Kbp) Primer 1

AflII 5`

3` Primer 2

SpeI restriction site

Inverse PCR

Inverse PCR (16 cycle) with primer1 and primer2

AflII

SpeI

G67 capsid (4-site mutant)+ VP1 149 AflII

cloned wt capsid genes (2.5kb) containing all the required mutations in an intermediate vector WT capsid from which G67 was derived

SpeI

Results

In-vitro transcription of pT7S3-WT pT7S3-G67+ VP1 149 (5-site mutant) pT7S3-O1 Italy (Control)

• RNA was electroporated in to BHK 21 IB-RS 5,2 5,1 5 4,9 4,8 4,7 4,6 4,5 4,4 4,3

• Passage to BHK3

• Passage to BTY3

Viable virus was recovered from all construct

• In vitro assessment of the mutant viruses using virus neutralisation test GP post-vaccinal sera against WT

Bovine post-vaccinal sera

100

0 5M

0 O1Italy

O1Italy

Percent (%) reduction in neutralising antibody titre by mutant viruses

How can we increase the percentage of reduction in neutralisation?

Additional mutations Antigeni c sites/mut ants Viral protein position s O1K-wt 5M M1 M2 M3 M4 M5 DM1 DM2 DM3

Site 1

Site 2

Site 3

Site 4

Site 5

Additional mutations

VP1 133

VP1 138

VP1 148

VP1 150

VP2 72

VP1 43

VP3 58

VP1 149

VP3 85

VP2 74

VP2 191

K E E E E E E E E E

R K K K K K K K K K

L R R R R R R R R R

V A A A A A A A A A

S N N N N N N N N N

T K K K K K K K K K

E V V V V V V V V V

Q H H H H H H H H H

H H Q H H H H Q Q H

S S S P A S S P A P

T T T T T N A T T A

VP274

VP385

VP2191

O1 K reduced structure showing critical residues of 5 neut. antigenic sites (blue) and additional mutations (green/pink/red)

Growth- kinetics of single and double mutant viruses 6 5

O1K WT M1

2 Virus titre in log10 TCID50/ml

Phenotypic characterisation

M2 M3

0 Hr

3 hr

6 hr

9 hr

12 hr

24 hr

4 O1K WT

5M DM1

DM2

DM3

0 0 Hr

3 hr

6 hr

9 hr

Hours post-infection

12 hr

24 hr

DM1

DM3

mAbs binding activity of WT versus Mutant viruses

Optical density

• Technique used: Indirect ELISA • Murine mAbs specific for the five known antigenic sites Mab B2 D9 C6 C9 C8 14EH9 OC3

Site 1 1 2 2 3 4 5

D9, C8, C6, 14EH9, OC3

All (O1K)

Critical residues VP1-144, 148, 154 VP1-144 VP2-73 VP2–71, 72, 75, 77 VP1-43,44 VP3-58 VP1-149 VP1-43, VP1-138, VP1-133-VP1148, VP1-149, VP1-150, VP2-72, VP3-58

Critical residues of type O mAbs (Crowther et al., 1993)

Viruses

• In vitro assessment of the mutant viruses using virus neutralisation test Bovine O1BFS post-vaccinal sera

GP post-vaccinal sera

100

* *

80 60 40 20 0 5M

DM1

Percent (%) reduction in mutant viruses neutralising antibody titre Neutral virus 5 M + VP2.191(N-T)= M4

5 M +VP2.191(N-A)=M5

5 M +M5+M2 +DM3

5 M + VP2.74(S-P) = M2

DM2

DM3

Threshold of reduction in neutralizing antibody titre of GP sera •

To determine the threshold of reduction in neutralizing antibody titre of GP sera using different FMDV topotypes and serotypes (A22 IRQ, Asia1)

•

To confirm the specificity of the antibody and its homologous epitopes Topotypes

Serotypes

100 80 60 40 20 0

ME-SA AFRICA

CATHAY

% reduction in virus neutralisation antibody titre of GP post-vaccinal sera

Conclusion • FMDV O1K 5-site mutant virus has been produced for the first time using reverse genetics

• The amino acids residues of the neutralising antigenic sites (mutated so far) - not critical for viability of the virus • Residual neutralisation indicates presence of yet unidentified epitopes • Residues VP2-74 and VP2-191 could be part of Site 2. They are in a close proximity to VP2-72 and VP2-188 and proved be significantly important in reduction of virus neutralization titre

VP2-74 VP2-188 188 VP2-191

Future work • Production of hyper immune sera against the neutral virus (M5) in GPs • Reconstitution of individual epitopes – recover and characterise mutant viruses • In vitro and in-vivo studies: to determine the relative importance of different epitopes

Thank You Dr. Mana Mahapatra Prof. David Paton Dr. Donald King Mr. Nick Knowles Dr. Paul Barnett

Sasmita Upadhyaya Daryl Borley Fufa Bari D. J. Kalita

CODA-CERVA-VAR (Belgium) D. Lefebvre A. De Vleeschauwer K. De Clercq

Funding:

DETERMINING THE EPITOPE DOMINANCE ON THE CAPSID OF A SAT2 FOOT-ANDMOUTH DISEASE VIRUS BY MUTATIONAL ANALYSIS Opperman, P.A., Theron, J. and F.F. Maree

Introduction  Strong link between the protection of cattle against FMDV and the levels of virus-neutralizing antibodies produced following vaccination  Most important factor imparting vaccine-induced protection against FMDV  Humoral immune response

 Monoclonal antibodies (MAbs) have been used extensively to identify several antigenic sites on the structural proteins of virions

 Serotypes A, O, C and Asia-1  Located on structural protrusions on the virus surface  Loops connecting β-barrel structures of the three outer capsid proteins  βG-βH loop of 1D has been identified as immunodominant

 SAT2 serotype is most prominent in southern Africa:  Three antigenic sites have been identified  βG-βH loop of 1D, downstream of the RGD motif, is analogous to site 1 of serotype O1BFS  Residue 210 at the C terminus of the 1D  Residue 154 of 1D in combination with residue 79 of 1B  Importance of each of these sites in SAT2 viruses is still undefined

 Knowledge of the residues that comprise the antigenic determinants  Structural design of vaccine seed strains  Improved protection against specific outbreak isolates  Lack of information concerning the neutralizing antigenic determinants

of the SAT viruses

Objective Determine the role of known and predicted epitopes of SAT2 viruses and to present evidence of epitope dominance within the SAT2 serotype of FMDV

 Epitope-swapping approach in an infectious cDNA clone of a SAT2 virus  Selected and mutated residues located in ten of the structurally exposed loops of 1B, 1C and 1D  Measured the effect of these mutations on antigenicity with virus

neutralization (VN) assays  Polyclonal antisera raised against SAT2/ZIM/7/83, used as the genetic background, and SAT2/KNP/19/89, the epitope-donor

Predicting of Antigenic Sites  Capsid-coding region of FMDV strains found in sub-Saharan Africa analyzed using one-way antigenic relationships (r1-values)  Variable regions on the capsid proteins combined with structural data and

serological relatedness to identify possible epitope  Modelled SAT2 capsid structure using O1BFS as template  Based on the optimal alignment of the SAT2 virus, ZIM/7/83, P1 sequence corresponding to O1BFS  Variable residues on surface-exposed loops were regarded as immune

relevant and mapped to the SAT2 pentamer structure  Outside the 1D βG-βH loop were concentrated around the 5-fold and 3fold axis of the virion and the C-terminal of 1D (Maree et al., 2011)  Previously identified neutralizing epitopes of type A and O

1B (4) βA-βB loop (31-45) βB-βC loop (64-82) βC-βD βC βD loop (93 (93-101) 101) βE-βF loop (130-134/141) 134/141)

1C (4) N-terminus (30-45) βB-βC loop (63-77) βE-βF loop (125-142) βG-βH loop (165-183/172)

1D 1B 1C Predicted epitopes

1D (7) N-terminus terminus (9-40) (9 40) βB-βC loop (43-62/71) 62/71) βE-βF loop (80-103) 103) βF-βG loop (110-122) βG-βH loop (136-167) βH-βI loop (176-187) C-terminus (192-212)

Amino Acids • Regions of hypervariability • High entropy • Structurally exposed loops • Antibody recognition sites

10 structurally exposed loops of SAT2/KNP/19/89 were introduced into the pSAT2 plasmid, a SAT2/ZIM/7/83 infectious clone 1D 1B

DR→EK

Obscured by adjacent structural elements: • 1C: DR  EK • 1D: KP  NS

1D HNN→NKG HAD→YAS EHE→DHR KP→NS AFA→TFN

TKHK→IKHT

TQQS→ETPV

1B KD→RN

SD→PE

Near to residue that contributes to a discontinuous epitope

Cloning Strategy SAT2 P1

SAT2/ZIM/7/83 Ssp I

Site 2a SD

Site 2b KD

Site 4 DR

1B PE

C21

IRES

TFN

Xma I YAS NKG IKHT NS DHR ETPV

2A Lab

1B, 1C, 1D Ssp I

No viable virus: • 1C: DR EK • 1D: KP NS

TQQS

C-term HAD

SAT2/KNP/19/89

Site 1 TKHK

HNN

EHE

1C RN

T7 promoter S-frag

Site 3 AFA

3'UTR P2

A15

Xma I

KNPpSAT2

• 1D: TKHK TKHK

IKHT IKHK

Electrostatic Potential Of Mutations  Increase in the net positive charge in the 1D protein of the derived recombinant virus  EHE→DHR (84-86) and HNN→NKG (109-111) mutations of 1D  Strong effect on the local surface potential of the capsid  Distinct patch of surface area that was predominantly positively charged AFA→TFN

HNN→NKG EHE→DHR

KP→NS KD→RN

TKHK→IKHT TQQS→ETPV

SAT2/ZIM/7/83

vKNPSAT2

Antigenic Profiles Of The Epitope-Replaced Viruses

3500

N-terminal of βG-βH 40% increase

3000 2500 2000

1500 Antisera 1000

KNP/19/89

500

ZIM/7/83

Epitope Exchanged Viruses

Conclusions  Similar neutralization profiles obtained as for vSAT2 and SAT2/ZIM/7/83  SD→PE and the KD→RN mutations in 1B  TKHK→IKHK mutation in the βG-βH loop of 1D  HAD→YAS mutation at the C-terminus of 1D  Slight increase in neutralizing titre against the SAT2/KNP/19/89 antiserum and not significantly reduced against SAT2/ZIM/7/83  HNN→NKG and the AFA→TFN mutations in the 1D protein  Larger relative amount of neutralizing antibody against that particular epitope in the polyclonal serum.  Higher neutralization titres with SAT2/ZIM/7/83 antisera  EHE→DHR; HNN→NKG, AFA→TFN, TQQS→ETPV  Mutated epitope alters the binding of high affinity antibodies to the capsid

in such a way that the binding sites of lower affinity antibodies become available, resulting in a different neutralization kinetics/profiles.

 HNN→NKG change resulted in a predominantly positively-charged local surface potential

 Linked to the binding of SAT viruses to alternative receptors for cell entry

 Viruses propagated in cell culture  Adversely affect vaccine seed stocks  Selection of viruses that are altered at multiple sites on the capsid

Acknowledgements  Funding  MSD Animal Health  South African Department of Science and Technology (DST)

 Staff at the Transboundary Animal Disease Programme (ARC-OVI)  Jan Esterhuysen for providing the cattle anti-KNP/19/89 and antiZIM/7/83 sera and the assistance with virus neutralization assays

 Agricultural Research Council

Development of a predictive model for vaccine matching for serotype O FMDV from serology and capsid sequence

D. Borley, S. Upadhyaya, D. Paton, R. Reeve and Mana Mahapatra Pirbright Laboratory United Kingdom

FMDV Genome Organization AUG Lab

Cleavage sites L 3C 2A

AUG

Unknown 3B 1-3

VPg

VP0

VP3

VP1 2A

P1-2A

VP0

VP3

VP1 2A

3B 1-3

AAA n

3B1-3 VP0

VP4

VP3

VP1

VP2

3A 3A

3C 3C

3D 3D

Non-Structural Proteins

Structural Proteins

Capsid

RNA Synthesis/Replication, Helicase /polymerase/protease

Antigenicity, receptor binding

activity, virulence

Main Objectives - Develop a predictive model for vaccine matching for serotype O FMDV - Genetic and/or structural determinants of antibodymediated protection

To develop/improve FMDV vaccine strain selection, and design novel vaccine 3x

VP1- blue VP2- red VP3- green

Selection of suitable vaccine strains Vaccination using killed viral capsid antigens is very important for disease control Seven serotypes and multiple subtypes of FMDV Antigenic mis-match is one important cause for vaccine failure

Vaccine strains must be selected from the available pool and the need for new vaccines must be identified– eg. muiltiple outbreak in S. Korea SAT3

Current vaccine selection - VNT or ELISA tests SAT2 to measure the cross-reaction of a bovine vaccinal sera with the field strain in question Few studies have been carried out to measure cross-protection directly

SAT1 C

O Asia1

Problems with serological methods ● Time needed to grow up field viruses and not all grow

equally well

● Need for panels of vaccine strains and antisera ● Antisera are inherently variable

● Difficult to standardise tests or to have full confidence in results without many repetition ● Vaccine selection based on serological methods always not give same results as in vivo

Alternative approaches I.

Matching ELISA using type specific monoclonal antibodies (mAbs) -Need panel of well defined mAbs able to recognise differences between vaccine and field viruses -Need to know which antigenic sites are the most important (Mahapatra et al., 2008)

II. Antigenic Cartography

III. Sequencing of viral capsid and correlation of amino acid changes to antigenic matching -Still need to know which sites are the most significant

Viruses used -serotype O EURO-SA

Cathay

Asia

Africa

Serotype O viruses (n = 80) Bovine serum (n = 5)

Heatmap of viruses/antisera

III- Correlating capsid sequence to serology 5 sera X~80 viruses - Capsid sequence determined - Surface accessibility of each capsid residue determined

* 48 discrete regions (1-40 aa) * Optimised serology result * LME prediction model developed VNT

ELISA

O BFS Multimer: VP1- Red, VP2 – Blue, VP3 - Green (Borley et al. - manuscript in prep.)

Prediction model using VNT and Capsid sequence

For the first time serotype O vaccine strain can be selected without recourse to serology

Predicted regions using model VNT

Blue: VP2 70-79 Green: VP3 84-85 Red: VP3 219-220

ELISA

Blue: VP1 194-204 Green: VP3 84-85 Red: VP1 132-162

Individual aa tested VNT

ELISA VP2 191 VP1 198 VP1 138 VP3 219

VP2 191 Site 2 VP1 138 VP1 174 Site 1 VP2 194 O1 BFS

Neu. Ab Titre

500

Ab. repertoire of bovine sera

Epitope prediction (structure)

400 300

O BFS

200 100

O1K

Red. O BFS Red. O1K

Site1

Site 2

Site3

mAb escape mutants

(Mahapatra et al., 2012)

Site 4

Site 5

SAT-1

ASN 190

SER 190 ASN 190 ASP 190

THR 191

ASN 191 THR 191 GLN 191

GLU 192

ALA 192 THR 192

ASP 69

SER 70

ASP 71

ARG 218

ALA 219

GLU 220

ASP 71

ASP 69

SER 69

ASP 70

GLY 70

THR 71

SER 71

GLU 221 GLN 219 ARG 220 GLN 220 GLN 221

(Borley et al., submitted)

VP2

VP3

Reverse genetics to test the residues

Serum antibody titre

Type O cDNA clone Mutations introduced – VP2 191 and VP3 219 Recombinant virus - recovered, characterised Antigenic properties of the virus - serology 100 80 60 40 20 0 Parent VP3 219 VP2 191

Conclusion • Serological methods- time-consuming, still useful

• mAb-based assay- vaccine-sp, not feasible (limited resources) • Sequence vs serology model: -promising, needs further work - better rep. sera/viruses - testing and validation - may change in future

• VNT and ELISA – different results (aa) • New epitope – VP2 191, VP3 219

What we would like to do next • Refine the prediction model (type O):

-Include additional sera against antigenically distant vaccine -Develop further model – to confirm the residues predicted in the current model

• Test and validate the type O model: - Introduce in to use by FMD Ref. labs - Cross-protection studies • Test the predicted aa residues using a cDNA clone • Extend the work to serotype A

Acknowledgements Pirbright Daryl Borley Sasmita Upadhyaya Amin Asfor Fufa Bari David Paton Jef Hammond

Univ. of Glasgow Richard Reeve Dan Haydon

Nico Visser Danny Gooverts

Oxford Elizabeth Fry Dave Stuart

IZSLER, Brescia Emi Brocchi Santina Grazioli

MODELLING INTO POLICY: HOW CAN AN ‘INTELLIGENT CUSTOMER’ ENSURE APPROPRIATE USE OF EVIDENCE?

Francesca Gauntlett. AHVLA Epidemiology, Risk and Surveillance Group. Modelling coordinator and ICF Lead

Conclusions and Recommendations •

AHVLA‟s quantitative modelling ICF has liaised effectively with both policy makers and expert mathematical modellers.

•

ICF involvement has ensured that the modelling commissioned has been relevant and has contributed to the evidence informing policy.

•

ICF has provided: – expert challenge during the modelling process; – prevents modelling outputs being ignored through lack of time and capacity to make use of them. – should also lead to significant cost savings by working with officials commissioning work to prevent unnecessary modelling work

•

This is a valuable approach ensuring that model outputs are used appropriately in policy development, without each official engaged in a modelling project being required to develop sufficient technical understanding to commission and/or interpret the outputs.

Challenges • Epidemiological modelling may be used to aid the development of animal health policy, to support disease surveillance activities, and to evaluate existing or new intervention strategies.

• In order to use model outputs effectively, the decision maker has to consider a range of issues. •

In some areas, there is an increasing reliance on models across Government but few people who understand outputs.

• Need help to make evidence based decisions in complex systems • Danger of policy makers wanting a definitive answer for Ministers – modelling provides a level of certainty ..... – which may be limited!

Policy makers use evidence that comes from a variety of sources

Economic

Industry & other groups

Risk assessment

Scientific

Legal Policy makers

Modelling

Political

Veterinary Public acceptability Epidemiological Anaylses

Risk appetite

Modelling to inform policy development

• Different arenas for modellers – long term strategic questions, with time for „data gathering‟ vs. shorter, specific questions using available data • Dangers of rapid modelling from existing data and importance of obtaining good data in as close to real time as possible • Models are not reality, they are an approximation of possible outcomes • Modelling in haste = more assumptions, less data = less accurate • A particular problem in disease outbreaks, where decisions have to be made in absence of data • Models do not remove uncertainty, but may give an illusion of knowledge that is unfounded

Intelligent Customer Function (ICF)

• • • • • •

How does it work... Does this add value? Do policy makers benefit? Do the modellers benefit? Dependent on resource available. Are we asking modellers to do too much?

Intelligent Customer Function (ICF) Wikipedia: Intelligent Customer Function is • an in-house capability within an organisation which assists the organisation in the procurement of outsourced services. The 'Intelligent Customer' retains sufficient technical knowledge of the services being provided by a third party to competently specify requirements and manage delivery of the services. Nuclear industry definition: An intelligent customer should • know what is required • fully understand the need for a contractor's services, • specify requirements, • supervise the work • technically review output before, during & after implementation.

ICF for modelling: Skills/knowledge • AHVLA‟s QM ICF group made up of a mathematical modeller, epidemiologists and an economist. • Overview & understanding of Defra business • Speak "language" of policy development; ICF has the ability to translate the requirements from customers/users and the technical outputs of the modellers. • Aware of policy developments and issues;

• Ability to interpret and apply modelling derived evidence to guide policy decision making • Ability to influence and ensure best use of modelling outputs; ensures modelling outputs are not ignored as a result of a lack of time and capacity to make use of them.

Working with modelling - Dialogue helps

Ministers

Managers

Policy Interface (via Intelligent Customer)

Modeller Interface

Policy

Colleagues

Stakeholders

Expert Modellers

Constant Dialogue

Policy / ICF

Modeller

Clear Specification This is the problem This is why it‟s important Defined approach This is how we will model it These are our assumptions Review of approach Here are some better assumptions Have you included this effect?

Review of emerging findings Why does the model do that? Can you look at this?

Review of final findings Can you explain this better? Can you expand that ?

Emerging findings The model says this We need more data on that Does this look sensible?

More findings The model now says this This is what we can infer Does this explanation make sense?

Good commissioning and delivery

• Policy: – Set a good exam question – Try not to change your mind half way through – Explain who the outputs will be seen by

• Modellers: – Answer the exam question – Be clear and honest about what you don‟t know – Focus the outputs for the audience(s)

Broad Engagement

Help to identify parameter ranges and review model behaviours

Keep you honest. Build wider credibility

Experts

Peer Review

Stakeholders Help to ensure buy-in, and to force explanations to be clear and simple

Modeller

Other Modellers Allow alternative approaches to be compared

Lessons/What Next ?

• Modelling has an important role to play in policy development. • Modelling is not a substitute for data • Flexibility and willingness to engage in understanding the wider policy issues by “modellers” really helpful. • Policy customer should be clear at the outset what they want and what they want it for. • Can we put a value on modelling? – what are the benefits of modelling?

Conclusions and Recommendations •

AHVLA‟s quantitative modelling ICF has liaised effectively with both policy makers and expert mathematical modellers.

•

ICF involvement has ensured that the modelling commissioned has been relevant and has contributed to the evidence informing policy.

•

Acknowledgements

• Colin Birch– Biomaths & Statistics workgroup lead, AHVLA • Kate Sharpe – Veterinary Epidemiologist & QM Intelligent Customer, AHVLA • Simon Scanlon – Defra‟s lead animal health economist

Scaling up from one-to-one animal transmission experiments to epidemiological models of national outbreaks D.Schley, M.J.Tildesely & S.Gubbins

Disease control requires a good understanding of spread and transmission Transmission between individual animals

best understood from controlled experiments

Biosecurity practice

best informed by understanding what happens within farms

Control Policy

best informed by understanding what happens between farms

Individual infection model Pirbright challenge experiments

Science 332 (2011) 726

Day 0 Day 1

Day 2

Day 3 Day 4

Day 5

Day 6

Day 7

Day 8

! ! ! !

transmission clinical signs occurred first observed

! Science 332 (2011) 726

Day 0 Day 1

Day 2

Day 3 Day 4 Day 5

Day 6

Day 7

Day 8

incubation

transmission clinical signs occurred first observed

! latent

latent

infectious infectious

Incubation and latent period and correlation Infectious period and rate of transmission Posterior distributions fitted by Bayesian Monte-Carlo Markov-Chain (MCMC) Science 332 (2011) 726

Infectiousness profile - individual

Latent vs. incubation period Challenge outcome

Time that infectiousness occurs ahead of clinical signs within a group of animals Blood virus isolation

Nasal fluid virus isolation

Risk of undetected transmission

Any animal infectious before any animal shows clinical signs

Advanced warning of at least 1 day

Hidden infectiousness for at least 1 day

Transmission of infection P(Infected) = P(contact with source) X P(source infectious) X P(transmission occurs)

Initial infection in herd

Epidemiology and Infection 137 (2009) 1494-1504

Within herd contact: direct

14.4 distinct contacts per hour

Different contacts per animal recorded in 15 minutes

Within herd contact: indirect Gate post 43±6%

Within herd contact - faecal Single wet cowpat after 12hrs: 53±6% Single dry cowpat after 12hrs: 80±4%

Two dry cowpats after 24hrs: 90±7%

Within flock contact Physical contact network

Livestock Science 145 (2012) 34-43; Preventive Veterinary Medicine 106 (2012) 174-184

Within-farm herd model homogeneous-mixing individual-based compartmental stochastic “SLCRD” model uninfected Susceptible Latently infected

clinically Diseased

Clinically infectious Removed/recovered

Within-farm herd model Herd-level sample joint distribution of period parameters

Animal-level sample bivariate log-normal distributions to generate individual animal latent, incubation and infectious periods

Incorporates observed variation between individual animals and herds Incorporates observed correlation of latency and incubation

Disease dynamics

mean UK herd size

Disease detection

Infectiousness profile - herd

Regional between-farm model Warwick-Keeling (WaKe) regional model Distance-kernel transmission model UK livestock premises data Size-based farm infectiousness and susceptibility

Hierarchical Approximate Bayesian Computation (ABC) Monte-Carlo Markov-Chain (MCMC ) Hierarchical regional parameter structure Stochastic between farm spread driven by experimentally derived individual animal disease dynamics Science 294 (2001) 813-817; Nature 440 (2006) 83-86

Model fit ABC MCMC model captures parameter variability.

Potentially allows improved accuracy of simulations.

Insights into previous outbreaks 2001 UK (Cumbria region)

On-going & future work Improving accuracy of outbreak simulations Application to Japan 2010 outbreak (and other diseases) Development of different cattle management models Incorporation of within-farm dynamics for sheep and pigs

Acknowledgements The Pirbright Institute Simon Gubbins Dina Kleinlutzum Giles Weaver University of Warwick Michael Tildesley

Biotechnology and Biological Sciences Research Council The Pirbright campus is being redeveloped

Epidemiological Model for Outbreaks of Footand-Mouth Disease in two different Austrian Regions Jörg Anton Hiesel, Ian Kopacka, Angelika Loitsch, Peter Wagner and Josef Köfer 29.10.2012 – 31.10.2012, EU FMD OPEN SESSION, Jerez de la Frontera, Spain Veterinärmedizinische Universität Wien

Overview  FMD real time exercises in Austria  Key points/objectives

 FMD history in Austria  The two different regions  Model development    

Software Data collection and analysis Parameters Scenarios

 Results  Conclusions

FMD exercises in Austria  Real-time exercises in accordance with the Council directive 2003/85/EC in Austria 1) 2004 PICORNA 04 - Styria - Lower Austria - Burgenland 2) 2009 PICORNA 09 - Tyrol - Vorarlberg

Key points/objectives  Key assumptions    

Scenarios based on Picorna 04, Picorna 09 Constant index case Constant High Risk Period of 20 days Milk needs to be collected in case of an outbreak

 Objectives  Find answers:  How many farms could possibly be infected?  What could be the influence of the different control measures?  How much does it cost?

FMD History in Austria  FMD outbreak in 1972/1973  1.500 farms involved (Dangl, 2003)  app. 70.000 animals were culled (Dangl, 2003)  Costs? not published

 Last FMD outbreak in 1981 (Dangl, 2003)  2 farms infected  Costs? not published

The two regions Region I Total area: 39.541 km² Holding density: 1,1 holdings per km²

Region II Total area: 15.249 km² Holding density: 1,04 holdings per km²

The two regions Region I: cattle holdings

Region II: cattle holdings

The two regions Region I: small ruminant holdings Goat holdings Sheep holdings Mixed holdings

Region II: small ruminant holdings Goat holdings Sheep holdings Mixed holdings

The two regions Region I: pig holdings

Region II: pig holdings

The two regions Region I: animal density

Region II: animal density

The two regions Region I: vet. resources

Region II: vet. resources

Region I Lower Austria Styria Burgenland

Official Vets. Vets. in private Other Vets. (local level) practice

Official Vets. (administrative level)

Official Vets. (district level)

Vets. in the labs

Region II Tyrol Vorarlberg

Official Vets. (local level)

Official Vets. (administrative level)

Vets. in private practice

Official Vets. (district level)

Other Vets.

Vets. in the labs

Model development Definition of the system and objectives for modelling Analysis of data and knowledge relevant to the model Model formulation Validation Sensitivity analysis Use of the model Slightly adapted from Taylor, 2003

Model development – software  Interspread Plus® version 2.1.14   

provides a framework for modelling the spread of infectious disease among animal (and human) populations (Stern, 2003). Spatial, stochastic, state transition model (SIR – Model) Geo referenced

 ArcGIS ®  R ® (statistical software)  VIS (national animal database)

Model development – data collection and analysis  Scientific literature  Reports (e.g. Governmental reports, reports from the federal states)

 National animal database  National weather database  Vaccination data based on experiences with the Bluetongue vaccination program  Dairy data regarding milk tanker routes  Capacity of rendering plants

Model development – parameters  Spread Parameters    

Local spread (Sanson, 2003) Movements (generated from the national animal database) Airborne spread (Yoon, 2006; National weather database) Infectivity/susceptibility (Martinez-Lopez, 2010; Yoon, 2006)

 Control Parameters based on    

Council directive 2003/85/EC National legislation (TSG, MKS-VO) and contingency plans Personal resources Infrastructure already established, e.g., contracts with rendering plants and/or dairies

Model development – sensitivity analysis Evaluated parameters relative to the baseline scenario (100 Iterations each)    

High Risk Period (75%, 100 %, 125 %) Transmission probability of milk tanker (50 %, 100%, 150 %) Local spread (75 %, 100 %, 125 %) Culling resources (50 %, 100 %, 150 %)

Model development – scenarios  1. Culling of animals in detected farms;  2. Controlled collection of milk in combination with a culling of animals in the detected businesses.  3. Culling of animals in detected farms in combination with immunization within a perimeter of 0.5 to 10 kilometers  4. Culling of animals in detected farms in combination with a culling of animals within a perimeter of 0.5 kilometers;  5. Culling of animals in detected farms in combination with immunization within a perimeter of 0.5 to 10 kilometers and a culling of animals within a perimeter of 0.5 kilometers;

Results  Sensitivity Analysis Kruskal-Wallis test (p < 0,05  significant influence, p < 0,001 highly significant influence) Parameter

Mean decreased

Mean baseline

Mean increased

P-value

HRP

13.76

19.66

36.10

< 0.001

Local spread

13.72

19.66

29.65

< 0.001

Milk tanker

13.65

19.66

23.86

< 0.001

Resources

20.12

19.66

19.57

0.999

Results Region I

Region II

Number of infected farms for regions I and II and scenarios 1 = baseline, 2 = restrictions for dairy routes, 3 = vaccination, 4 = ring culling, 5 = vaccination + ring culling

Results Region I

Region II

1800

1600

1400

1200

1000 800

1000 Duration Farms infected

600

Animals infected

400

Animals culled

200 0

800

Duration Farms infected

600

Animals infected

400

Animals culled

200 0

Median Values: scenario 1 = baseline, scenario 2 = restrictions for dairy routes, scenario 3 = vaccination, scenario 4 = ring culling, scenario 5 = vaccination + ring culling

Results Influence of the control measures Scenario

Region I: mean value Infected farms

Region II: mean value Infected farms

Baseline scenario

20.16

23.81

Restrictions for dairy routes

18.13

20.43

Vaccination

18.27

18.37

Ring culling

17.85

17.83

Vaccination + ring culling

17.90

16.84

Results

Baseline = B; V = Vaccination; RC = Ring Culling; DR = Restrictions on dairy routes Region I

Region II

Compared Scenarios

obs. dif.

Crit. dif.

Sign. dif.

obs. dif.

Crit. dif.

Sign. dif.

B VS. DR

203.24

181.21

TRUE

215.36

181.21

TRUE

B VS. V

194.52

181.21

TRUE

371.07

181.21

TRUE

B VS. RC

247.94

181.21

TRUE

404.62

181.21

TRUE

B VS. RC + V

247.06

181.21

TRUE

485.42

181.21

TRUE

DR VS. V

8.73

181.21

FALSE

155.70

181.21

FALSE

DR VS. RC

44.69

181.21

FALSE

189.26

181.21

TRUE

DR VS. RC + V

43.82

181.21

FALSE

270.06

181.21

TRUE

V VS. RC

53.42

181.21

FALSE

33.55

181.21

FALSE

V VS. RC + V

52.55

181.21

FALSE

114.36

181.21

FALSE

RC VS RC + V

0.88

181.21

FALSE

80.80

181.21

FALSE

Conclusions  HRP, local spread and the probability of disease transmission by milk tanker have a significant influence on the number of infected farms.  Influences of control measures depend on the region/agricultural structure.  Number of infected farms can be significantly reduced by enforcing restrictions on milk tanker routes, immunisation and/or ring culling.  If milk needs to be collected, restrictions on milk tanker routes should be applied.  Pre-outbreak contracts with dairies/rendering plants seem to be reasonable.

Thanks to

Univ.-Prof. Dr. Josef Köfer

HR. Dr. Peter Wagner Dr. Angelika Loitsch

Dr. Ian Kopacka

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DECISION CONTEXT Control of FMD outbreaks involves: 1.  Early detec(on of infec(on (minimising the high-‐risk period) 2.  Elimina(on of infected animals (herds) 3.  Limita(on of popula(ons at risk: –  Using depopula(on of suscep(ble animals –  Using vaccina(on: to-‐live or to-‐death

4.  Compensa(on of economical losses

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&2N'1'_#%L"6'(026% U26+#)%

a#,+.2("$%"4%/#0')'"$% 123#-)%

&#2$)% O;>#0(5#)%

CRITERIA AND ATTRIBUTES 1

Social Criteria

Economic Criteria

Farmers welfare

Lost farm heritage Stress

Eradica(on costs Tes(ng costs Produc(on losses

Animal welfare # of animals killed Stress Suﬀering

Direct losses

Indirect losses

Trade restric(ons

Poli(cal Criteria Reputa(on of decision makers Public acceptance Time to eradicate Time for free status

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+ %

VALUING CONSEQUENCES

INPUTS •  Numbers: popula(ons @ risk

–  Diﬀerent zones: 1km, 3km, 10km, 15km –  Diﬀerent species

•  CapaciKes:

–  Culling –  Vaccina(on –  Sampling, tes(ng

•  Costs: vaccina(on, appraisal, euthanasia, rendering, cleaning and disinfec(on, sampling, tes(ng, trade restric(ons •  Sample sizes •  Time

CONSEQUENCE VALUES Em. Slaughter

PIGS Vacc. to live

Vacc. to death

Social Values 1. Farmers welfare Loss of heritage Stress

intermediate intermediate

none least

most most

# animals killed

61,431

1,121,851

Eradica(on costs Tes(ng costs

€ 37.16 M € 0.21 M

€ 4.49 M € 7.02 M

€ 683.14 M € 0

€ 197.94 M € 235.31 M

€ 9,827.59 M € 9,839.10 M

€ 3,541.64 M € 4,224.78 M

105 d 115 d

3,740 d 3,839 d

1,384 d 1,384 d

2. Animal welfare Economical Values 1. Direct costs

2. Indirect costs Trade restric(ons costs TOTAL COSTS PoliKcal Values 1. Reputa(on of decision maker (me to liking measures in SZ (me to free status

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SCALED CONSEQUENCE VALUES Em. Slaughter

PIGS Vacc. to live

Vacc. to death

Loss of heritage Stress

50 67

0 0

100 100

# animals killed

100

Eradica(on costs Tes(ng costs

5 3

0 100

100 0

0 0

100 100

35 42

0 100

100 0

35 334

Social Values 1. Farmers welfare

2. Animal welfare Economical Values 1. Direct costs

2. Indirect costs Trade restric(ons costs TOTAL COSTS PoliKcal Values 1. Reputa(on of decision maker (me to liking measures in SZ (me to free status

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Y =a1 ∗w1 +a2 ∗w2 +...+an ∗wn ADDITIVE VALUE MODEL

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Economic values%

Political values%

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FMD CASE ADDITIVE VALUES ADDITIVE VALUE MODEL

AddiKve Value

Decision

Mul( AAribute Value of Em. slaughter CaAle =

1286

Choose this alterna(ve

Mul( AAribute Value of Vacc. To live CaAle =

5285

Mul( AAribute Value of Vacc. To death CaAle =

8990

Mul( AAribute Value of Em. slaughter Small Ruminants =

1527

Choose this alterna(ve

Mul( AAribute Value of Vacc. To live Small Ruminants =

4711

Mul( AAribute Value of Vacc. To death Small Ruminants =

8620

Mul( AAribute Value of Em. slaughter Pigs =

1024

Choose this alterna(ve

Mul( AAribute Value of Vacc. To live Pigs =

4934

Mul( AAribute Value of Vacc. To death Pigs =

8620

AlternaKves

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Meta-analysis on the efficacy of foot-andmouth disease emergency vaccination Tariq Halasa Anette Boklund Sarah Cox Claes Enøe

Conclusions • Emergency vaccination provided clinical and virological protection against FMD in cattle, swine and sheep.

• No significant publication bias was identified in the analyses. • The outcomes of the meta-analysis can be used for further epidemiologic and economic assessment of emergency vaccination.

Objective • Conduct a comprehensive assessment of the efficacy of emergency vaccination using meta-analysis of available literature. • The outcome parameters can be used to assess the epidemiologic and economic consequences of emergency vaccination.

Definition of efficacy • Determine the efficacy of FMD emergency vaccination in terms of:

– Clinical protection. – Virological protection.

Outcome parameters • Parameters to represent protection:

– Relative risk (RR) of clinical disease. – RR of virological infection.

RR of clinical disease • RR = the incidence of clinical disease in the vaccinated group divided by the incidence in the non-vaccinated group. • Incidence of clinical disease = number of clinically diseased animals divided by the total number of animals per group.

Virological infection • Several tests were used to diagnose infection. – When an animal was positive to any of the tests, the animal was considered infected. • The tests were: – Virus isolation from the blood, oral, nasal, or esophagealpharyngeal fluid. – Presence of antibodies to non-structural proteins. – RT-PCR of oral, nasal, or esophageal-pharyngeal fluid.

RR of virological infection • The RR of virological infection = the incidence of FMD infection in the vaccinated group divided by the incidence in the non-vaccinated group. • The incidence = the number of FMD infected animals divided by the total number of animals per group.

Meta-analysis procedure • A statistical technique that summarize the results of different studies that address a related hypothesis. • A pooled RR was calculated for the clinical and virological protection for – Cattle – Swine – Sheep • Checked for publication bias.

Results - descriptive • • • • • • •

28 published studies fitted the criteria. 27 were included in the analyses. 10 conducted using cattle. 9 conducted using swine. 5 conducted using sheep. 3 had more than 1 species. 4 unpublished studies conducted on swine.

Results – Clinical protection in cattle Virus serotype

Study

A Brehm, 2008 A Goris, 2008 A Graves, 1968 A Mattion, 2004 Pooled RR A Asia 1 Salt, 1995 Pooled RR Asia 1 O Aggarwal, 2002 O Cox, 2005 O Cox, 2006 O Cox, 2007 O Doel, 1994 O Donaldson, 1989 O Goris, 2007 O Orsel, 2005 Pooled RR O Overall Pooled RR

Lower limit

Upper limit

0.051 0.123 0.075 0.225 0.126 0.124 0.124 0.100 0.026 0.026 0.294 0.019 0.179 0.090 0.059 0.118 0.123

0.003 0.071 0.016 0.069 0.079 0.052 0.052 0.007 0.002 0.002 0.177 0.001 0.089 0.037 0.008 0.062 0.087

0.770 0.214 0.352 0.737 0.202 0.295 0.295 1.490 0.406 0.406 0.488 0.297 0.361 0.219 0.408 0.226 0.175

RR and 95% CI

0.01

0.1

Vaccine

100

No vaccine

Results – Clinical protection in cattle - publication bias 0.0

Standard Error

0.5

1.0

1.5

2.0 -4

-3

-2

-1

Log relative risk

Results – Clinical protection in cattle - publication bias Duval and Tweedie trim and fill method 0.0

Standard Error

0.5

1.0

1.5

2.0 -4

-3

-2

-1

Log relative risk

Results – Virological protection in cattle • Vaccinated cattle • 0.65 (0.53 – 0.81) lower chance of FMD infection • No significant publication bias was identified.

Results – Protection in pigs and sheep • In pigs: • Clinical protection: 0.47 (0.36-0.62). • Virological protection: 0.68 (0.53-0.87). • In sheep: • Clinical protection: 0.31 (0.18-0.53). • Virological protection: 0.57 (0.39-0.82). • No significant publication bias was identified in the analyses.

Conclusions • Emergency vaccination provided clinical and virological protection against FMD in cattle, swine and sheep.

• No significant publication bias was identified in the analyses. • The outcomes of the meta-analysis can be used for further epidemiologic and economic assessment of emergency vaccination.

Characteristics of an emergency vaccine? •

Contain no residues of a live virus and have minimal side effects to newborns and adults

•

After a single application with the recommended dose, achieve a potency dose 50 (PD50) of ≥ 6

•

Be compatible with serological tests that identify infection in vaccinated animals

•

Induce reasonably long lasting immunity and provide a broad spectrum of antigenic protection

•

Be stable under storage once formulated

•

Provide a rapid protection after vaccination

•

Reduce the reproduction ratio (R0) to below 1

What is meta-analysis? • Statistical technique. • Summarize the results of different studies that address a related hypothesis. • Controls for study characteristics • Results in an overall average are more powerful than outcomes of individual studies.

Inclusion criteria • Experimental challenge with FMD emergency vaccination using cattle, swine, and/or sheep.

• Research or symposium papers published in English language. • Report the number of protected animals and the total number of animals in – vaccinated group – non-vaccinated control group. • Both groups should be challenged with a homologous virus.

Evaluating vaccination for foot-and-mouth disease control — an international study Garner MG, Gauntlett FA, Sanson RL, Stevenson MA, Forde-Folle K, Roche SE, Birch C, Owen K, Dube C, Rooney J, Corso B, Cook C, Rawdon T and Backer JA

Conclusions and Recommendations  

This multi-country modelling study utilises technical expertise and sophisticated FMD modelling capabilities. There is a need to look at operational issues around vaccination in FMD free countries: 

 

Calibrating models has been time consuming and complicated. So far, 2 vaccination scenarios have been modelled: 



How to use vaccine (strategies); What species to vaccinate; Assist with setting priorities.

looking at effect on size of outbreak (by number of IPs), geographical spread of outbreak, and duration of outbreak.

Preliminary data suggests that for a 3km suppressive vaccination zone: random vaccination of herds in the zone vs. targeted vaccination from outside-in appears to be similarly effective, when compared to a baseline control of stamping out only.

Context 

Vaccination increasingly being recognised as an important option for containing and eradicating FMD in previously-free countries



Well-designed collaborative studies can inform policy development --- Need to move beyond the simple question of whether vaccination should be used or not



Understand when vaccination may (and may not) offer benefits in terms of effective and efficient management of a FMD outbreak



Look at operational issues: How to use vaccine (strategies); What species to vaccinate; Setting priorities

Methods 

Multi-country modelling study – utilising technical expertise and sophisticated FMD modelling capabilities



Build on networks and successful collaboration 

2005-2010 QUADS EpiTeam Members completed a large model comparison and relative validation study (Ireland)

Study objectives 

Identify conditions under which vaccination may offer significant benefits



Identify key features and issues that influence the effectiveness of vaccination



Describe any differences between participating country FMD models that affect vaccination

Models 

Participating countries Australia (AusSpread)  New Zealand (InterSpread plus)  United States/Canada (NAADSM)  Great Britain (EXODIS) 





Netherlands (CVI)

Stage 1: Use data from UK’s Exercise Silver Birch (Nov 2010) – plausible multi-focal outbreak with ready to use data  Respective models set up to represent Silver Birch scenario





agreed vaccination strategies to be compared to a no vaccination baseline.

Stage 2: 

Findings checked under country-specific conditions

EXERCISE

Vaccination strategies 1. Approach: Suppressive vaccination (3km and 5 km rings); Protective vaccination (5-10km) 2. Timing: early (14 days); late (28 days, 50 IPs) 3. Species: all susceptible species; cattle only; cattle & sheep; cattle & pigs 4. Prioritisation: area; farm type 5. Deployment: inside-out; outside-in 6. Vaccination capacity: sensitivity analysis

Outcomes will be compared in terms of outbreak duration, size (IPs and spatial extent), and number of farms and animals vaccinated

Study area 

Central England and Wales 

 

65,000 holdings

Image plot shows the density distribution of the farm population at risk (herds per sq km) Model calibration   



Incorporate UK population data Adapt and modify parameters as appropriate Agree on standard settings e.g. probabilities of detection, resources Scenario – ‘Exercise Silver Birch’, 18 day silent spread phase. Simulate forward from day of detection (movement ban) --Run for 21 days without controls

Number of infected premises (n=100 iterations) (uncontrolled outbreak) Model

Min

Median

Max

Australia

137

158

167

182

222

New Zealand

132

154

166

224

North America

127

140

154

198

United Kingdom

328

439

477

523

639

Kruskal-Wallis rank sum test Kruskal-Wallis chi-squared = 93.943, df = 3, p-value < 0.01

Uncontrolled outbreak

Australia

New Zealand

North America

United Kingdom

Point maps showing the location of all predicted infected places (n=100 iterations) Australia

Image plots: density of predicted infected places IPs per sq km (100 iterations).

New Zealand

North America

United Kingdom

Size of outbreak area (sq km) (uncontrolled outbreak) Model

Min

Median

Max

Australia

14130

20010

22620

25510

32800

New Zealand

12190

17590

21050

23040

28750

North America

10720

22100

24870

28300

36600

United Kingdom

13700

17640

19030

21190

31770

Kruskal-Wallis rank sum test Kruskal-Wallis chi-squared = 93.943, df = 3, p-value < 0.01

Discussion  



Exodis: highest IPs (>2X), but smallest area AusSpead and IS+: similar findings NAADSM: smallest number of IPs, largest area

Explainable?  Exodis uses a spatial kernel (30km) to spread disease + maintains high infectiousness to end of infectious period = increased spread of disease in an uncontrolled situation



The other models use discrete pathways for local spread (3 km) and indirect contact spread (80 km) + infectiousness follows within-herd prevalence = less spread and more distributed patterns Differences in parameters and approaches used e.g. for wind-borne spread, implementing local spread

Preliminary data for vaccination scenarios (IPs) Baseline scenario – stamping out only

Suppressive vaccination – outside in (3km VZ)

Suppressive vaccination – random (3km VZ)

Conclusions and Recommendations 

International studies can: strengthen collaboration in the areas of modelling and epidemiology to support emergency disease management and response  provide significant rigor, relative validation and improve decision-maker confidence in model predictions  generate valuable information to inform policies on disease control 

Next steps 



Progressively implement control strategies – understand differences Compare vaccination outcomes

Thank you The QUADS EpiTeam would like to acknowledge the support of the participating countries and the Australian Department of Agriculture, Fisheries and Forestry who made funding available for a project team workshop in Victoria, Canada in April 2012

• UK: Francesca Gauntlett, Colin Birch and Charlotte Cook, AHVLA • Australia: Graeme Garner and Sharon Roche, DAFF. • New Zealand: Robert Sanson, Mark Stevenson, Jaimie Frazer, Katie Owen, Zhidong Yu, Tom Rawdon. • Canada: Caroline Dube, Neil Harvey. • United States: Kimberly Forde Folle, Barbara Corso, Kelly Patyk, Jane Rooney, APHIS, USDA. • The Netherlands: Jantien Backer, Central Veterinary Institute of Wageningen UR.

Appendix 63

Conclusions & Recommendations A NEW APPROACH TO THE OLDEST DISEASE Developing an Antiviral Drug Strategy for the Containment of FMD Outbreaks N. Goris, E. Kiss, L. Murao, J. Swinnen, D. Kollanur, A. Billiet, D. Lefebvre, A. De Vleeschauwer, K. De Clercq, A. Volny-Luraghi, A. Marchand, J. Neyts

• Significant progress towards the development of an antiviral drug against FMDV has been made • Fine-tuning of the molecular structure is ongoing - Physico-chemical properties (e.g. solubility, bio-availability) - Anti-FMDV activity (e.g. low nM EC50 to minimise dosage quantities)

• Antiviral drugs help contain outbreaks and are now worthy alternatives to emergency vaccination • Public-private partnerships in support of this control policy are crucial to align expectations with development efforts

Antiviral Drugs in Human Medicine

FMDV - Why Antivirals? • Rapid response needed - Highly contagious - Affects all cloven-hoofed animals

• Prevent spread of infection - Preemptive culling → public opposition - Emergency vaccination → strategic vaccine / antigen banks • Should rapidly prevent spread of infection after single application • Should provide sufficiently broad-spectrum antigenic protection • Should be sufficiently stable once formulated • Should allow trade to resume rapidly (waiting periods)

- Antiviral drug → rapid response stockpile • Logistically easier to apply • Act directly on virus replication • Tailored to be stable & serotype-independent

In Vitro Activity of Reference Compounds Reference compound

EC50 (µM)

FMDV strain

Literature reference

Ribavirin

1867 123-205 350 27

C1 Noville C1 O1 Manisa O/SKR/2002

Goris et al., 2007 De la Torré et al., 1987 Goris et al., 2007 Kim et al., 2012

5-azacytidine

Sierra et al., 2000

5-fluorouracil

770-7700

Sierra et al., 2000

2’-CMC

8.7

O1 Manisa

Goris et al., 2007

Guanine-HCl

5.5

O/SKR/2002

Kim et al., 2012

6-azauridine

0.32

O/SKR/2002

Kim et al., 2012

T-1105

O/JPN/2000

Futura et al., 2009

FMDV Hit Screening - Unique Collaboration

61,000 compounds

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 63

Hit Identification - Exploration - Optimisation Compounds circa 61,000

Hit Identification - Exploration - Optimisation

Desired compound profile - Active against 7 FMDV serotypes - Active in low nM range - >3 log10 reduction in VYA - Lipinski’s rule of five compliance - Favourable ADME-tox profile

Initial screen 1 or 2 conc. 159 active Hit rate: 0.26% compounds

Confirmation >2 runs 37 confirmed hit compounds

Analogue A Solubility higher EC50 = 4.5 µM

• Stable compound - Chemical stability - Plasma stability - Microsomal stability

Chemistry and IP checks 20 families selected for analogue testing

Hit compound identified through screen EC50 = 4.5 µM  Activity  Solubility  Stability

• Soluble compound

Analogue selection

- Easy synthesis - Low cost of goods - Favourable IP position

3 families selected for optimisation

Hit exploration: consistency in SAR demonstrated Compound class = chemically tractable

Hit Identification - Exploration - Optimisation Initial exploration

Analogue B Stability higher EC50 = 4.5 µM

Analogue C Combination A & B EC50 = 4.5 µM  Activity  Solubility  Stability

From Hit to Lead Compound

Optimisation 1 6 analogues

EC50 > 50 µM → keep centrale core Optimisation 2 Hit compound EC50 = 4.5 µM

Analogue C EC 50

Hit compound 66 analogues

= 4.5 µM

only Eurasian 4.5 µM √ √ ± ± √ ±

highly advantageous EC50 = 0.9 µM Optim on 3 81 analogues

highly advantageous EC50 = 0.9 µM

Combination compounds (n = 15) EC 50 ≈ 0.045 µM

What a difference a factor 100(0) makes... optimisation 0.9 µM

Lead

Aim

45 nM

4.5 nM

Combination compound

Compound profile Active against 7 FMDV serotypes Active in low nM range >3 log10 reduction in VYA Lipinski’s rule of five compliance Stable compound Soluble compound Easy synthesis Favourable ADME-tox profile

√ 45 nM √ √ √ ± √ under investigation

Conclusions & Recommendations • Significant progress towards the development of an antiviral drug against FMDV has been made • Fine-tuning of the molecular structure is ongoing - Physico-chemical properties (e.g. solubility, bio-availability) - Anti-FMDV activity (e.g. low nM EC50 to minimise dosage quantities)

FMD antiviral containment approach will be key

2’-CMC → 10 vials T-1105 → 13 vials

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 63 Thank You for Your Kind Attention Questions?

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 64 Conclusions o T-1105 offers significant clinical and virological protecion against O1 Manisa virus infection in guinea pigs The Pyrazinecarboxamide derivative T-1105 offers protection against O1 Manisa virus infection in guinea pigs De Vleeschauwer Annebel, Lefebvre David, Barnett Paul, Murao Lyre, Billiet Aino, Goris Nesya, Neyts johan, De Clercq Kris

30-10-2012

o O1 Manisa infection in guinea pigs represents an appropriate model for the preliminary in vivo evaluation of antiviral drugs

30/10/2012

Summary

Introduction

o Introduction - Development of antiviral drugs against FMDV - Development of an FMDV infection model in guinea pigs - T-1105 o Aim o Materials and Methods o Results - PK - Clinical and virological follow-up

o Development of antiviral drugs against FMDV (See also: presentation Dr. N. Goris, Okapi Sciences) Several phases: o Screening of chemical compounds and identification of antiviral activity in vitro o Optimization of antiviral activity in vitro o Study of safety, pharmacokinetics and antiviral activity in a rodent model (Lefebvre et al. 2010) o Study of safety, pharmacokinetics and residue-analysis in cloven-hoofed animals o Study of antiviral activity in cloven-hoofed animals: clinical protection and effect on transmission

o Conclusions o Acknowledgements 30/10/2012

30/10/2012

Introduction

Introduction o Development of an FMDV infection model in guinea pigs

o Development of antiviral drugs against FMDV (See also: presentation Dr. N. Goris, Okapi Sciences)

(See also: poster presentation) o o o o o

Several phases: Screening of chemical compounds and identification of antiviral activity in vitro Optimization of antiviral activity in vitro Study of safety, pharmacokinetics and antiviral activity in a rodent model (Lefebvre et al. 2010) Study of safety, pharmacokinetics and residue-analysis in cloven-hoofed animals Study of antiviral activity in cloven-hoofed animals: clinical protection and effect on transmission

30/10/2012

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Appendix 64

Introduction

Aim

o Pyrazinecarboxamides o Broad-spectrum inhibitors of RNA virus infections o Derivative T-1105

- 3-oxo-3,4-dihydro-2-pyrazinecarboxamide - Furuta et al.2009 In vitro anti-FMDV activity against O/Jpn/2000 strain, EC50 value of 12µM - Ohashi et al. 2008 In vivo anti-FMDV activity against O/Taw/97 in pigs (200mg/kg bid, po) -reduction of lesions -reduction of viremia -reduction of nasal virus excretion 30/10/2012

Assesment of the antiviral activity of the pyrazinecarboxamide derivative T-1105 in an O1 Manisa infection model in guinea pigs

30/10/2012

Materials and methods

o Pharmacokinetic profile of T-1105 - Dunkin Hartley guinea pigs - 200mg/kg, po, twice with an 8 hour interval - Blood collection at 0, 1, 2, 3, 4, 6 and 24 hours after 1st administration

- T-1105 treatment - 200mg/kg, po, bid with an 8 hour interval - 5 days - Challenge: O1 Manisa Gpp 13, intraplantary - Euthanasia at 4 and 10 dpi

o Assessment in vivo efficacy - Dunkin Hartley guinea pigs, 350-400g - 3 groups • T-1105 treatment and O1 Manisa challenge (n=16) • O1 Manisa challenge control (n=8) • T-1105 treatment control (n=4)

30/10/2012

- Efficacy parameters

• Lesion development and generalization • Viremia and virus generalization (RT-PCR)

30/10/2012

Results: Clinical protection

Results: PK profile of T-1105 o In vitro EC50 against O1 Manisa = 25µM

% animals with foot- and mouth lesions 100 80 60

o Peak serum concentration of 140µM at 2 hours after administration

Right hind footpad Left hind footpad Right front footpad Left front footpad Tongue Untreated control

40 20 0 1

4 dpi

Mean lesion score 3 2,5 2 1,5 1 0,5 0

30/10/2012

day.1

day.2

day.3

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 64 Results: Viral RNA load in serum

Results: Viral RNA load in organs at 4 dpi

% animals with viremia

% of animals with viral RNA

100 80 60 40 20

10 dpi T-1105 treated Untreated control

Mean Cp values in sera

15 20 25 30 35 45 50

Left Right Left Tongue Oral Heart Lung hind front front swab

T-1105 treated Untreated control

15 20 25 30 35 45 50

10 dpi

30/10/2012

Serum Right hind

Mean Cp values

Serum Right Left hind hind 13

Right front

Conclusions

Oral

Heart Lung 14

Acknowledgements

o T-1105 offers significant clinical and virological protecion against O1 Manisa virus infection in guinea pigs

Dr. David Lefebvre

Dr. Barnett Paul

Dr. Aino Billiet Dr. Lyre Murao

o O1 Manisa infection in guinea pigs represents an appropriate model for the preliminary in vivo evaluation of antiviral drugs

Prof. Dr. Johan Neyts

Dr. Kris De Clercq Lic. Tom Willems Dr. Nesya Goris

30/10/2012

Left Tongue front swab

30/10/2012

The Belgian Federal Public Service for Health, Food Chain Safety and Environment (RF 6203) DG Research of the European Commission: EC 7th Framework Programme (grant agreement n°226556, www.fmddisconvac.net) 30/10/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 65

Optimizing the control of foot-andmouth disease in Denmark by simulation

Conclusions • Extra control meassures will most often reduce size, duration and costs of an FMD epidemic in Denmark

Comparison of different control strategies on FMD in Denmark

• Depopulation in zones is preferable

Anette Boklund Tariq Halasa Lasse Engbo Christiansen Preben Willeberg Claes Enøe

• Protective vaccination preferable from an epidemiological point of view - BUT in Denmark NOT from an economic point of view!

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Inputs in the models

Control measures

• All Danish herds in 2006/2007 - Cattle (milking/not-milking) - Pigs (SPF/conventional, sow/finisher/farrow-finisher/hobby/nucleus) - Sheep or goat (hobby/commercial)

• Surveillance

• Movement data - Incl. abattoir movements

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• Depopulation • Movement restrictions • Vaccination

• Disease data - Time to infectious, time to clinical signs, etc. • Danish behavior - Probability of detecting and reporting disease, effect of movement restrictions, probability of tracking movements, etc. • Time from 1. herds is infected to 1. detection - 21 days 3

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

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Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Control scenarios

Results

• Basic - Basic EU regulation + 3 days national movement standstill + depopulation of herds that recieved animals from infected herds

• Epidemiologic results - Infected herds - Detected herds - Duration of epidemic • from first to last depopulation - Depopulated herds - Vaccinated herds

• Depopulation in zones (Depop) - varying radius • Suppressiv vaccination in zones (Vac-to-Cull - varying radius

• Economic results

• Protective vaccination in zones (Vac-to-Live) - varying radius

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

- 37 outputs (costs and losses) - Only total costs and losses are shown here!

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Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

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Appendix 65

Epidemiologic results - CattleHigh Control strategy

Epidemic duration (days)

Epidemiologic results - CattleHigh

Infected herds

Control strategy

Results as median (5-95) Basic

80 (5-255)

137 (3-696)

Basic

500 m

65 (5-178)

101 (3-426)

Depop10H

1000 m

46 (5-123)

1500 m

Depop10H

VacToCull10herds

VacToLive10herds

80 (5-255)

137 (3-696)

65 (5-178)

101 (3-426)

76 (3-287)

1000 m

46 (5-123)

76 (3-287)

41 (5-102)

66 (3-269)

1500 m

41 (5-102)

66 (3-269)

1km

58 (5-136)

86 (3-346)

1km

58 (5-136)

86 (3-346)

2km

48 (5-110)

69 (3-245)

2km

48 (5-110)

69 (3-245)

3km

44 (5-97)

66 (3-227)

3km

44 (5-97)

66 (3-227)

5km

42 (5-89)

65 (3-205)

5km

42 (5-89)

65 (3-205)

1km

53 (5-133)

81 (3-303)

1km

53 (5-133)

81 (3-303)

2km

43 (5-101)

64 (3-227)

2km

43 (5-101)

64 (3-227)

3km

39 (5-86)

57 (3-195)

3km

39 (5-86)

57 (3-195)

5km

37 (5-78)

52 (3-174)

5km

37 (5-78)

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Epidemiologic results - CattleHigh Control strategy

Epidemic duration (days)

VacToCull10herds

VacToLive10herds

VacToCull10herds

VacToLive10herds

Control strategy

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Epidemic duration (days)

Infected herds

Results as median (5-95) -

80 (5-255)

137 (3-696)

Basic

500 m

65 (5-178)

101 (3-426)

Depop10H

80 (5-255)

137 (3-696)

500 m

65 (5-178)

1000 m

46 (5-123)

101 (3-426)

76 (3-287)

1000 m

46 (5-123)

1500 m

76 (3-287)

41 (5-102)

66 (3-269)

1500 m

41 (5-102)

66 (3-269)

1km

58 (5-136)

86 (3-346)

1km

58 (5-136)

86 (3-346)

2km

48 (5-110)

69 (3-245)

2km

48 (5-110)

69 (3-245)

3km

44 (5-97)

66 (3-227)

3km

44 (5-97)

66 (3-227)

5km

42 (5-89)

65 (3-205)

5km

42 (5-89)

65 (3-205)

1km

53 (5-133)

81 (3-303)

1km

53 (5-133)

81 (3-303)

2km

43 (5-101)

64 (3-227)

2km

43 (5-101)

64 (3-227)

3km

39 (5-86)

57 (3-195)

3km

39 (5-86)

57 (3-195)

5km

37 (5-78)

52 (3-174)

5km

37 (5-78)

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

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Economics

VacToCull10herds

VacToLive10herds

52 (3-174)

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

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Epidemiologic results - CattleHigh

• Direct costs - Surveillance - Depopulation - Cleaning & desinfection - Empty stables - Compensations - Welfare slaughter - National stand still

Control strategy

Basic Depop10H

VacToCull10herds

• Export losses - Lost export of live animals and animal products - For EU and non-EU

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Epidemic duration (days)

Results as median (5-95)

- Vaccination

52 (3-174)

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Epidemiologic results - CattleHigh

Infected herds

Results as median (5-95) Depop10H

Infected herds

500 m

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Basic

Epidemic duration (days)

Results as median (5-95)

VacToLive10herds

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Infected herds

Economy (€x106)

80 (5-255)

137 (3-696)

665 (399-1137)

500 m

65 (5-178)

101 (3-426)

605 (397-921)

1000 m

46 (5-123)

76 (3-287)

549 (396-783)

1500 m

41 (5-102)

66 (3-269)

537 (394-754)

1km

58 (5-136)

86 (3-346)

567 (400-808)

2km

48 (5-110)

69 (3-245)

544 (406-726)

3km

44 (5-97)

66 (3-227)

538 (407-706)

5km

42 (5-89)

65 (3-205)

558 (422-730)

1km

53 (5-133)

81 (3-303)

631 (475-885)

2km

43 (5-101)

64 (3-227)

601 (474-767)

3km

39 (5-86)

57 (3-195)

592 (472-740)

5km

37 (5-78)

52 (3-174)

588 (477-736)

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

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Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 65

Epidemiologic results - CattleHigh Control strategy

Epidemic duration (days)

Results as median (5-95) Basic Depop10H

VacToCull10herds

VacToLive10herds

Infected herds

Epidemiologic results - CattleHigh Economy

Control strategy

(€x106)

Results as median (5-95)

80 (5-255)

137 (3-696)

665 (399-1137)

500 m

65 (5-178)

101 (3-426)

605 (397-921)

1000 m

46 (5-123)

76 (3-287)

1500 m

41 (5-102)

1km

665 (399-1137)

500 m

65 (5-178)

101 (3-426)

605 (397-921)

549 (396-783)

1000 m

46 (5-123)

76 (3-287)

549 (396-783)

66 (3-269)

537 (394-754)

1500 m

41 (5-102)

66 (3-269)

537 (394-754)

58 (5-136)

86 (3-346)

567 (400-808)

1km

58 (5-136)

86 (3-346)

567 (400-808)

2km

48 (5-110)

69 (3-245)

544 (406-726)

2km

48 (5-110)

69 (3-245)

544 (406-726)

3km

44 (5-97)

66 (3-227)

538 (407-706)

3km

44 (5-97)

66 (3-227)

538 (407-706)

5km

42 (5-89)

65 (3-205)

558 (422-730)

5km

42 (5-89)

65 (3-205)

558 (422-730)

1km

53 (5-133)

81 (3-303)

631 (475-885)

1km

53 (5-133)

81 (3-303)

631 (475-885)

2km

43 (5-101)

64 (3-227)

601 (474-767)

2km

43 (5-101)

64 (3-227)

601 (474-767)

3km

39 (5-86)

57 (3-195)

592 (472-740)

3km

39 (5-86)

57 (3-195)

592 (472-740)

5km

37 (5-78)

52 (3-174)

588 (477-736)

5km

37 (5-78)

52 (3-174)

588 (477-736)

Epidemic duration (days)

Results as median (5-95)

VacToCull10herds

VacToLive10herds

(€x106) 137 (3-696)

Control strategy

Depop10H

Economy

80 (5-255)

Eu-FMD Open Session

30/10/2012

Epidemiologic results - CattleHigh

Basic

Infected herds

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Depop10H

VacToCull10herds

VacToLive10herds

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Eu-FMD Open Session

30/10/2012

Eu-FMD Open Session

30/10/2012

1500

Economy

Infected herds

(€x106)

80 (5-255)

137 (3-696)

665 (399-1137)

500 m

65 (5-178)

101 (3-426)

605 (397-921)

1000 m

46 (5-123)

76 (3-287)

549 (396-783)

1500 m

41 (5-102)

66 (3-269)

537 (394-754)

1km

58 (5-136)

86 (3-346)

567 (400-808)

2km

48 (5-110)

69 (3-245)

544 (406-726)

3km

44 (5-97)

66 (3-227)

538 (407-706)

5km

42 (5-89)

65 (3-205)

558 (422-730)

1km

53 (5-133)

81 (3-303)

631 (475-885)

2km

43 (5-101)

64 (3-227)

601 (474-767)

3km

39 (5-86)

57 (3-195)

592 (472-740)

5km

37 (5-78)

52 (3-174)

588 (477-736)

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Basic

Epidemic duration (days)

Eu-FMD Open Session

30/10/2012

1000

500

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

1e+07

Ethics - numbers of depopulated animals

Costs and losses, CattleHigh 800,0 Export

Direct costs

700,0

1e+06

600,0

500,0

1e+05

400,0

300,0

200,0

1e+04

100,0

0,0

1e+03

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Eu-FMD Open Session

30/10/2012

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Eu-FMD Open Session

30/10/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 65

Conclusions

Thank you for your attention

• Extra control meassures will most often reduce size, duration and costs of an FMD epidemic in Denmark

Anders Stockmarr, DTU, Denmark

• Depopulation in zones is preferable • Protective vaccination preferable from an epidemiological point of view - BUT in Denmark NOT from an economic point of view!

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Aknowledgements:

Eu-FMD Open Session

30/10/2012

Lis Alban, Danish agriculture and food council, Denmark

Anette Bøtner, DTU, Denmark Graham Belsham, DTU, Denmark Kirsten Tjørnehøj, DTU, Denmark

Jan Dahl, Danish agriculture and food council, Denmark

Sten Mortensen, Danish Veterinary and Food Administration, Denmark

Erik Rattenborg , Knowledge Centre for Agriculture, Denmark

Torben Grubbe, Danish Veterinary and Food Administration, Denmark

Jørgen Nielsen, Knowledge Centre for Agriculture, Denmark

Kimberly N. FordeFolle, USDA, Fort Collins, USA

Simulated effects of introducing emergency vaccination or depopulation during FMD outbreaks in Denmark

Eu-FMD Open Session

30/10/2012

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 4

Appendix 66

Modelling the Spread of FMD in Endemic Regions

Mike Tildesley Matt Keeling Ellen Brooks Pollock

The Keeling/Warwick Model

In this model, the risk of spread is based upon various parameters: Sc,s

- The susceptibility of livestock (cattle, sheep, pigs etc.).

N c,i - number of livestock on a given farm. Tc - transmissibility of livestock. K(dij) - the distance kernel, giving probability of infection based on distance between farm i and farm j.

Kernel

Distance from source

Comparison between Model and Data

The 2001 Foot-and-Mouth Disease Epidemic For the UK we have demographic information on: • the location and size of all livestock farms • the movement of all animals For the UK we have epidemic information on: • the location of all infected & culled farms •epidemiological tracing of possible infection routes

So how would an FMD-endemic UK look? Livestock movement ban!

Comparison between Model and Data Very good agreement between the observed cases (black) and the mean predicted epidemic (red line). The cloud of points indicates the stochastic uncertainty in predictions. Nationwide livestock movement ban when first case is reported. Around 10,000 farms infected or culled as part of the control policy.

Keeling et al. (2001) Science.

Key Model Results: CP-Culling Contiguous Premises culling was hugely controversial. But did it work? In the Eastern UK, where animal numbers are low, CP culling is unnecessary (blue). In Cumbria and Devon (which were worst hit in 2001), the optimal strategy is to cull all CPs (dark red). Tildesley et al Proc. B. 2009

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

Appendix 66 Key Model Results: Ring Vaccination

Vaccine Efficacy

Not used in 2001.

Vaccination Radius (km)

Optimal size of ring dependent upon resource available. Optimally targeted vaccination preferred to CP culling.

As efficacy increases, epidemic size decreases. As efficacy increases, optimal vaccination ring size decreases.

Tildesley et al. 2006 Nature

Key Model Results - Endemic Scenario So what would happen if FMD were endemic in the UK? Livestock movements would play a major role in transmission dynamics. We seed the model from the 2001 cases, but allow movements to continue and do not control the outbreak. Assume animals are infectious for a period of 16 days then recover and are immune. So how would FMD behave in the UK under these conditions?

Infection burns very quickly through the high density regions. Cyclic behaviour post-2002.

Births

Susceptible population is depleted rapidly initially. The disease is maintained in the population by births. Two outbreak peaks each year - correlates with peak livestock movements in spring and autumn.

Number of farms infected peaks at just under 10,000. Some variation across the years - effects of variation in farm sizes across years - general trend for fewer, larger farms.

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

Appendix 66 Geographic Spread

Geographic Spread

So how does the disease spread in space?

Seed in Cumbria.

With a livestock movement ban, spread to nearby counties in North of England and Scottish Borders.

In the absence of a movement ban…

Endemic Disease: Bovine TB in the UK

Simulation Results

Model shows good temporal fit to reporting data.

Spatial Model Comparison

Where do we go from here?

2011

Good fit to the data.

DATA

MODEL

PERC

Adapt the endemic UK FMD model to look at disease spread in endemic regions. Consider circulation of multiple strains.

Model captures high density breakdown regions in the South East. Adapting the model to investigate targeted culling, standstill restrictions and environmental control.

Impact of interventions. Have investigated impact of reactive vaccination, but what about use as control in endemic situations? Can we devise cost effective control measures to reduce disease burden in endemic regions? Work in very early stages of development… 100

200

300

400

HBDs per county

500

Percen

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

Appendix 66

Acknowledgements University of Warwick

EuFMD

Matt Keeling Ellen Brooks Pollock Sam Mason Marleen Werkman Peter Dawson

Keith Sumption Eoin Ryan Melissa Mclaws Chris Bartels

U. Penn Gary Smith

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

Geographicallygrounded, cost-benefit based control policies: built as equal circles or considering local connecting networks? Ariel L. Rivas, Almira L. Hoogesteyn, Jim B. Hittner, Douglas J. Perkins Center for Global Health, Health Sciences Center, University of New Mexico, Albuquerque, NM 87131, USA, email: alrivas@unm.edu

Any chain is as strong as the weakest link –validation matters CONCLUSIONS: We will show that even very small datasets can give us more information, which may lead to lower costs/higher benefits.

• Dr. Dorothy Geale demonstrated how important it is to assess the validity of TEMPORAL measures. • Now we will talk about the validity of GEOGRAPHICAL measures

Equal-radius control zones –a common policy • The 2010 S. Korean FMD epidemic. • Park et al., (TBED, 2012)

Equal-radius control zones –a common policy • The 2010 Japanese FMD epidemic

• Muroga et al. (J. Vet. Med. Sci. 74: 399–404, 2012).

Are equal-radius zones optimal control policies?

• Can we extract more information from the same data so, hopefully, we can improve the efficacy of control policies? • Does that depend on LARGE datasets?

The 2007 British FMD epidemic (140 sq km area) The press reported the association between connectivity and epidemic spread .

Source: http://news.bbc.co.uk/2/ hi/uk_news/6990913.st m

Source: BBC

[http://news.bbc.co.uk/2/hi/uk_news/6990913.stm]

How much can we learn from this data structure?

Case density & Road density

Case density/sq km: 0.063 (=9/142.5) Road density/sq km: 0.22 (=33.34/142.5)

Are all cases equal?

* 0.144 (4/27.75,smallest circle), * 0.102 (5/48.79, intermediate circle) and * 0.056 (6/106.37, largest circle). Not all cases were homogeneously distributed over space: those closer to roads (and road intersections) were clustered.

Case density varied even within such a small area

Case density in the smallest polygon (which only partially connected all infected premises through fragmented roads) was 0.132 cases/km2 (7/52.86) Case density/sq km: 0.063 (=9/142.5)

Even EXPANDING (some) of the original boundaries, we can get higher case density and smaller area of intervention Case density

in the totally connected polygon – which included nonfragmented roads) was 0.078 (nine cases/ 114.42 km2), an area equivalent to 80 % of the original control zone (114.42/142.5)

Inter-farm distance The median distance from farms to the nearest intersection was less than half for the four farms included in the smallest circle than for farms located outside such circle (2.297 versus 4.721 km, respectively).

farm # 1

farm #2

farm #3

farm #4

farm #5

farm #6

farm #7

farm #8

farm #9

farm # 1

5.1

7.2

5.8

4.5

4.1

6.8

10.1

11.1

farm # 2

5.1

2.5

2.3

3.9

5.6

9.1

12.9

13.8

farm # 3

7.2

2.5

4.9

6.5

10.1

13.7

14.5

farm # 4

5.8

2.3

2.5

2.4

4.2

7.7

11.3

12.0

farm # 5

4.5

3.9

4.9

2.4

1.8

5.5

8.9

9.6

farm # 6

4.1

5.6

6.5

4.2

1.8

3.5

7.4

8.1

farm # 7

6.8

9.1

10.1

7.7

5.5

3.5

3.8

4.5

farm # 8

10.1

12.9

13.7

11.3

8.9

7.4

3.8

0.9

farm # 9

11.1

13.8

14.5

12.0

9.6

8.1

4.5

0.9

Farm median inter-farm distance (km)

6.3

5.35

6.85

4.7

4.9

6.15

9.0

10.3

Distance (km) between farms at risk

Global median inter-farm distance : 6.15 km

And how about the ‘too small sample size’? Contribution of each ER circle to the Chi-Square goodness-of-fit test

Contributed Value

1.2

Pareto-like data distribution 0.8

0.4

0.0

ER protection circle ID

Are all cases equal? • Apparently not. • Instead of building control zones of identical radius (an assumption based on the hypothesis that all cases are equal), we could consider the actual CONNECTING NETWORK. • Because in the early phase of ANY epidemic the number of cases is very small (close to zero), we will never have a large ‘sample size.’ Instead of waiting for something that will never happen, we can look at data distribution patterns, e.g., the ‘20:80’ pattern.

ACKLOWLEDGMENTS: We thank DEFRA for allowing us to utilize their maps.

Costs and benefits of foot and mouth disease vaccination in commercial dairy farms in Central Ethiopia Ashenafi Feyisa Beyi and Hogeveen Henk Wageningen University and Research Centre

Conclusions & Recommendation

• In previous FMD outbreak in Bishoftu, vaccination was not helpful because it did not reduce financial losses in commercial dairy farms. • Preventive biannual vaccination with quadrivalent vaccine coupled with treatment during an outbreak is economically feasible. • Biannual vaccination of dairy cattle with quadrivalent vaccine (O, A, SAT 1 & SAT 2) and medication during an outbreak is recommended.

Background (1) • Ethiopia has large livestock population, majorly extensive production system • Dairying is a booming industry, mainly exotic breeds and their crosses are kept • However, highly susceptible to FMD infection

Background(2) High seroprevalence of FMD in exotic dairy cattle reported • Pastoral, 21%

(Rufael et al., 2008)

• Mixed farms, 38.4% 2012)

(Negussie et al.,

• Intensive dairy, 80% 2012)

(Negussie et al.,

Background (3) • Severe economic losses in Ethiopia • Trade ban of 2005/2006 > US$14 million (Leforban, 2005)

• Serotype O, A, C, SAT1 and SAT2 are isolated

• Prophylactive vaccination in some dairy farms • No work on economics FMD vaccination in dairy sector in Ethiopia

Objective • To estimate benefits and costs of different FMD vaccination practices in commercial dairy farms in Central Ethiopia

Materials & Method (1) • Stochastic Monte Carlo simulation model was used Three scenarios No vaccination Reactive vaccination Preventive vaccination Two sub-scenarios: with & without treatment

@Risk software on Excel spread sheet

M & M (3) Input data Questionnaire survey Expert opinions Literature

Some considerations:

• Imported quadrivalent vaccine • Preventive vaccination, biannual • Treatment with antibiotics, multivitamin/Vit. B complex and antiseptics

Result(1)-economic costs of outbreak Economic costs/farm (%) Milk loss 0,23

21,8

Mortality loss

3,62 0,71 5,86 39,1

28,68

Abortion loss Culling loss Treatment expenses Other expenses Vet. Visiting fee

Result(2) – ANOVA output

Economic costs among farms of different vaccination status is not significant.

Result (3) – Simulation output

Result(3) - Summary of simulation output costs and benefits of different scenarios

900

800 700 600

€

500

Costs

400 300 200 100

Net benefits

0 1,1

1,2

2,1

2,2

Scenarios

3,1

3,2

Conclusions & Recommendation

• In previous FMD outbreak in Bishoftu, vaccination was not helpful from economic point of view because it did not reduce financial losses in commercial dairy farms. • The third Scenario, preventive biannual vaccination with quadrivalent vaccine coupled with treatment during an outbreak is economically feasible. • Biannual vaccination of dairy cattle with polyvalent vaccine (O, A, SAT 1 & SAT 2) and medication during an outbreak is recommended.

Thank you for your attention!

Questions?

Appendix 75

Open Session Vienna 2010 - Lecture Wilna Vosloo The changing landscape of diagnostics

New ELISAs for FMD diagnosis Emiliana Brocchi Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna

“Progress/developments of ‘old’ ideas” “Standardisation, Quality, Validation, Harmonisation”

Brescia, ITALY with collaboration of WRL

IZSLER EuFMD Open Session 2012

Why do we need “new generation” ELISA ?  Robust & simple diagnostic kits are essential complements of the measures for disease prevention and control  Particularly crucial in endemic countries adopting PCP  Emergencies, Diagnostics Bank ?

Develop “new generation” ready-to-use ELISA kits

 MAb based - sandwich ELISA  Lateral flow devices (LFDs)

For antibody detection

Properties User-friendly Limited assay steps Stability Robustness No need of sophisticated equipment

EuFMD Open Session 2012

Objectives For FMDV antigen detection / serotyping

 Limited availability for FMD diagnosis restrictions in handling of FMD viruses not easy accessibility to samples suited for assay validation

    

IZSLER

 Ab to FMDV serotypes O, A, Asia 1 (SP antibody) MAb based - Solid Phase Competitive ELISAs

 Ab to FMDV NSP = already available commercially

IZSLER

FMDV Antigen detection & serotyping Test purposes

EuFMD Open Session 2012

IZSLER

READY-TO-USE ELISA KIT for FMDV ANTIGEN DETECTION AND SEROTYPING Simple sandwich ELISA based on Monoclonal Antibodies  Serotype-specific MAbs coated to microplate wells as catching antibodies

 Direct testing of lesion materials

 Captured antigens detected by peroxidase-conjugated, cross-reactive MAbs

− Vesicular epithelium or fluid

 Indirect testing of all samples after amplification in cell cultures

Detector MAb

Traditional ELISA format

Target Ag

 Serotype specific reagents for double-sandwich ELISA

− Rabbit polyclonal capture antibody - Guinea Pig polyclonal indicator antibody (+anti-GP IgG conjugate) - Control antigens (inactivated) supplied separately - Four incubation steps

EuFMD Open Session 2012

IZSLER

Catching MAb

1h RT

1h RT HRP

HRP

TMB Substrate

ready-to-use

Fit for purpose: Rapid, direct diagnosis in vesicular epithelium or fluid Identification of viruses isolated in cell cultures EuFMD Open Session 2012

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

IZSLER

Appendix 75

Selection of typing MAbs (catching Abs) criterion: widest intra-type reactivity

WESTEURASIA Serotyping O, A, Asia 1, C

Final selection of catching MAbs 4D12 & 5F6

N. isolates tested

Period covered

Type A

130

1943 2010

MAbs N. isolates reactive

Type O

108

1950 2010

MAbs N. isolates reactive

3B11

Type Asia 1

1954 2009

MAbs N. isolates reactive

3D8

Type C

'60s 2005

MAbs N. isolates reactive

3E9

Type SAT 1

1948 2009

MAbs N. isolates reactive

HD7 +

Type SAT 2

1948 2009

MAbs N. isolates reactive

2H6 45

FMDV Serotype

122

The test is fast - 2.5 hours - and simple:

112

Microplates pre-coated with type-specific MAbs Positive control antigens already incorporated into corresponding wells The operator handles a unique or at most two immunological reagents, corresponding to crossreactive detector conjugates

105 53

 Kit shelf-life estimated ≥ 1 years at 5°C  Up to six samples can be tested in each plate  Layout flexible

32 FC12

28 +

AFRICA Serotyping O, A, SAT 1 , SAT 2

1F5 45

IZSLER

EuFMD Open Session 2012

FIELD EVALUATION

FMDV Serotyping kit for WestEurasia - types O, A, Asia 1 & C Summary of diagnostic performances (Vienna 2010)

Sensitivity FMDV types O A Asia C TOTAL Epithelium suspensions

Type-specific MAbs-based ELISAs

Polyclonal ELISA

Pan-FMD ELISA based on MAb 1F10

POS Sens.

136 103 30 29

116 67 22 19

85% 65% 76% 66%

129 41 26 20

95% 40% 90% 69%

118 67 21 20

87% 65% 72% 69%

298

224

75%

216

72%

226

76%

IZSLER

EuFMD Open Session 2012

FMDV Serotyping kit for WestEurasia Types O, A, C, Asia 1

FMDV Serotyping kit for AFRICA Types O, A, SAT1, SAT2

Initial feedback from 4 African Countries

Specificity  Epithelium suspensions Positive for O, A, Asia 1 and C cross-checked in heterologous tests Cross-reaction of some FMDV isolates of type O with one type A catching Mab

The kit identified in vesicular epithelium samples

 External evaluation

N. 12 samples pos type O

 Demonstration/Trainings

N. 1 sample pos type A

 Supply (FAO & EuFMD programs)

 N. 24 SATs and SVDV + Negative epithelium suspensions ALL NEGATIVE

N. 7 samples pos type SAT1 N. 29 samples pos type SAT2 N. 2 samples pos types SAT2 & O

EuFMD Open Session 2012

IZSLER

FMDV Antigen detection - Penside tests Lateral Flow Devices

Results in 10-20 minutes In use or evaluated in field & laboratory  Pan-FMDV LFD

EuFMD Open Session 2012

IZSLER

FMDV antigen detection by Multiplexing testing system LUMINEX TECHNOLOGY (POSTER)  Proof of concept: 7 different microspheres coupled with 7 different MAbs mixed for simultaneous analyses of FMDV serotypes O, A, Asia1, C and SAT1 in a single reaction  Represents an additional, flexible, though sophisticated tool, for FMD diagnosis

 SAT2-specific LFD  Asia 1-specific LFD

Based on same catching and detector MAbs of ELISA kits

 Potential to increase the number of simultaneous analyses against many more MAbscoupled microspheres (up to 100) makes the test appropriate for accurate serotyping and antigenic profiling

Under development  Type specific LFDs for missing serotypes  Multiplex serotyping in one strip

EuFMD Open Session 2012

IZSLER

EuFMD Open Session 2012

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

IZSLER

Appendix 75

“Ready to use” ELISA kits for SP-Ab FMDV serotypes O, A, ASIA 1

ELISAs for FMDV serotype specific serology SP-Ab

Test purposes

 Three prototypes kits developed and evaluated  Single protocol for the three SP-ELISAs  Test is fast - 2.5 h - and simple

 Diagnosis of suspect cases  Estimating prevalence and substantiating freedom  Trade certification

− Plates provided pre-coated with FMDV inactivated antigens − Only two incubation steps (sera & conjugated MAb) at RT

 Estimating vaccine efficacy and population immunity

Current ELISA formats

 Serotype specific reagents for LPBE or SPCE

− Rabbit polyclonal capture antibody - Guinea pig polyclonal indicator antibody (+anti-GP IgG conjugate) - FMDV antigens (inactivated) - Five incubation steps IZSLER

EuFMD Open Session 2012

IZSLER

EuFMD Open Session 2012

NEW ready to use ELISA kits for SP-Ab Diagnostic SPECIFICITY

NEW ready to use ELISA kits for SP-Ab FMDV serotypes O, A, ASIA 1

SP-type O Ab ELISA kit

SP-type A Ab ELISA kit

SP-type Asia1 Ab ELISA kit

derived from in-house MAbs based SPC-ELISA preliminary work  Conditions for stabilization of FMDV antigens trapped by homologous MAbs >1 year the shelf-life of plates sensitized with FMDV captured by MAbs

Percentage inhibition

 Conditions for stabilization of conjugated MAbs (competition phase) shelf-life ranging from 1,5 to 3 years

 Drawback : FMDV antigens tend to degrade to 12S during the stabilization need of initial excess of reagents process

IZSLER

EuFMD Open Session 2012

NEW ready to use ELISA kits for SP-Ab Diagnostic SENSITIVITY full dose vaccinated cattle (vaccine potency tests) SP-ELISAs vs homologous VNT Type O VNT

Neg

Type A

Neg

Type Asia1 LPBE

Pos/Db Neg

Pos/Db

VNT Pos/Db

or LPBE

Kit ELISA

Pos Neg

Kit ELISA Pos Neg

Type O

Bovine Ovine Porcine Total n.pos/n.tested 155/165 56/71 25/25 236/261

SP-ELISA O 85% VNT 92%

Sensitivity

Concordance 0.872

Type A

SP-ELISA A 90%

Concordance 0.857 SP-ELISA Asia1 100%

20 0

Concordance 1

VNT 100%

94%

79%

163/196

Sensitivity 83%

Type Asia1 Bovine Ovine Porcine n.pos/n.tested

49/49

4/4

Sensitivity

100%

EuFMD Open Session 2012

90%

100%

 100% for SP-A Ab kit (1621 sera tested)

 99.8% for SP-Asia1 Ab kit (5 false-positive in 2481 sera)

EuFMD Open Session 2012

IZSLER

Ready-to-use ELISA kits for SP-Ab FMDV serotypes O, A, ASIA 1 CURRENT PROTOTYPES: adequate diagnostic performances, irrespective of the strains that elicited antibodies Possible improvement: use of recombinant capsid particles

Bovine Ovine Porcine Total

n.pos/n.tested

VNT 96%

Kit ELISA Pos Neg

0 0

EXTERNAL VALIDATION - WRL vaccinated and/or infected animals positive to homologous VNT

 99.8% for SP-O Ab kit (4 false-positive in 2000 sera)

163/196

83%

Total 53/53

100%

IZSLER

EuFMD Open Session 2012

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

IZSLER

Appendix 75

Trainings

Other perspectives for FMD serology  Multiplex immunoassays (LUMINEX technology)

- Could enable profiling of humoral immunity in a single reaction (Garnier et al., POSTER)

 Pen-side tests (LFD)

− For NSP Ab (Gonzales et al., Dho et al., POSTERs) − For SP Ab

EuFMD Open Session 2012

Conclusions FMDV ANTIGEN DETECTION & SEROTYPING  ELISA kit suited for EURASIA validated and available  ELISA kit tailored for AFRICA: prototype under evaluation  LFD for pan-FMD detection (O, A, C, Asia1 + some SAT2) in use  LFDs SAT2-specific and Asia1-specific launched TO FOLLOW  SAT3 typing still missing  Typing 6-7 serotypes in one ELISA kit (new plate layout) ?  Complete serotype-specific LFDs and develop multiplex LFDs

FMDV ANTIBODY DETECTION & SEROTYPING  ELISA kits for SP-Ab to FMDV serotypes O, A, Asia 1 validated

IZSLER - Brescia

Development & Production • Santina Grazioli • Franco de Simone • Giovanna Dh0 • Edoardo Spagnoli • Massimiliano Bugatti

WRL - Pirbright

Collaboration & Validation • Nigel Ferris • Yanmin Li • David Paton

EUFMD Consultant

TO FOLLOW  Improve scale production for larger availability  ELISA kits for SATs-specific Ab (under development)  Recombinant virus particles EuFMD Open Session 2012

Acknowledgements

Feedback from trainings and evaluation in AFRICA • Kees Van Maanen IZSLER

Discussion points  Antigen detection & serotyping ELISA New ready-to-use kit:

• Sensitivity similar to polyclonal ELISA • Better type-specificity • User-friendly

Pros: easier than RT-PCR, enables serotyping Limit: sensitivity (lower than RT-PCR)

 Serotype specific-Ab detection Limit:

• Imperfect type-specificity • A different assay per each desired serotype

 Kits expiring date

− Usually kits can be used longer after expiration (check controls?) EuFMD Open Session 2012

IZSLER

Open Session of the EuFMD: 2012; Jerez de la Frontera, Spain

Appendix 76

Conclusions FMD and SVD Combined Proficiency Test Studies 2011 Bryony Armson

Introduction • The European Reference Laboratories (EURLs) are directed to organise annual proficiency testing for the National Reference Laboratories (NRLS) of EU Member States. • The WRL at Pirbright also fulfils this function for other regions for the control of FMD. • This enables the harmonisation of testing between national and international reference laboratories.

Sample preparation

• Many of the results from the labs corresponded well. • There were some discrepancies - e.g. due to sensitivity or specificity issues. • Generally a very good response from labs that are keen to monitor the quality of their diagnostic testing.

Aim • To complete a proficiency testing study for diagnostic methods for FMD and SVD during 2011. • To enable a clear picture of the scale of activities, the state of QA accreditation and the tests being used by Member States during 2010/2011 to be compiled.

Sample Testing

Four panels were prepared for those labs requesting them:

• The priority serotypes were O and Asia 1.

• Panel 1 : infectious material for virus detection

• Particular tests were not specified, but labs were invited to select tests and interpret results.

• Panel 2 : non-infectious material for virus genome/antigen detection. • Panel 3 : non-infectious material for FMD serology • Panel 4 : non-infectious material for SVD serology • Ten panel sets were tested in order to set up the criteria to assess all the labs’ performances. • Each sample was randomly assigned a number and labelled accordingly.

• Participating labs were asked to answer which samples were FMD/SVD positive or negative in which tests. Also an overall interpretation for each sample. • Labs were asked to supply us with copies of the standard operating procedures (or methodologies) for the tests carried out.

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 76 Participation and Sponsorship

Participation

2010

Panels supplied and results reported 60

• Countries invited = 78 • Total participants = 52

2011

2 53

3 48

2011 sponsorship

2 35

• Countries invited = 80 • Total participants = 56

Not reported Results reported

EC 26 23 (19+4)

EuFMD (FAO) Other

0 Panel 1

Panel 1

Panel 3

Tests: • Virus Isolation • Antigen ELISA • Molecular methods - e.g. RT-PCR

False results

Cell culture system used

4.5

20 18 16 14 12 10 8 6 4 2 0

4 3.5 3 2.5 2

False Positives

1.5

% Correct Results

3 False Negatives

VI & Ag: n=20

100

Panel 4

Results - Panel 1

Panel 1 : infectious material from 2 cases of suspected vesicular disease for virus detection

0.5

Incorrect 2

Correct

VI & Ag ELISA

Molecular methods: n=19

60 40

RT-PCR

Overall Interpretation: n=22

20 0 VI & Ag ELISA

Molecular methods Overall interpretation

Panel 2

Panel 3

Panel 2 : non-infectious material from cattle or pigs for virus genome/antigen detection. Tests: • Antigen ELISA • Molecular method - e.g. RT-PCR

Ag ELISA : n=36 Molecular Methods : n=43 Overall Interpretation : n=35

% of Correct Results

Results

100

45 40 35 30 25 20 15 10 5 0

90 80 70 60 50 40 30 20 10 Molecular methods Overall Interpretation

Panel 3 : non-infectious material for FMD serology Tests: •Liquid Phase Blocking ELISA (LPBE) •Virus Neutralisation Test (VNT) •Solid Phase Competition ELISA (SPCE) •Non Specific Protein ELISA (NSP) •PrioCheck O ELISA % of Correct results

Ag ELISA RT-PCR

Correct

0 Ag ELISA

Panel 2

False Cross False Positives Negativesreactions

LPBE : n=25

100 90 80 70 60 50 40 30 20 10 0

VNT : n=20 SPCE : n=13 NSP : n=55 PrioCheck O : n=22 LPBE

VNT

SPCE

NSP

PrioCheck O

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 76 Results - Panel 3

Choice of test

Panel 4

Results 60

PrioCheck O

Panel 4 : non-infectious material for SVD serology

50 40

NSP

LPBE

30 SPCE

VNT

SPCE

NSP

0 LPBE

PrioCheck O

25 0

NSP results

% of Correct Results 120

NSP choice

50 45 40 35 30 25 20 15 10 5 0

Tests: •ELISA •Virus Neutralisation Test (VNT) •Overall Interpretation

Priocheck In house 3ABC

ELISA n=45

100

VNT n=23

Checkit-3ABC Incorrect Correct

ARRIAH 3ABC

3ABC recomb E.coli

Overall Interpretation n=36

IZSLER IDEXX BIONOTE UBI3B indirect

5B7 Mac ELISA Priocheck In-house

VNT

ID VET

Isotype ELISA LPBE ELISA

c70 cELISA

Cypress 0

ELISA

VNT

Overall Interpretation

• Some labs did not send the results back as requested.

Choice of ELISA 36

Conclusion

Results - Panel 4 Choice of test

ID Screen SVD

• Many of the results from the labs corresponded well, especially panel 4. • There were some discrepancies - e.g. due to sensitivity or specificity issues, leading to false results or mistyping. • In some labs there may be the need to establish certain tests, or certain serotypes/SVDV within a test. • A number of QA systems are used by the labs, including ISO 9001, 17025 and in-house systems. • Generally a very good response from labs that are keen to monitor the quality of their diagnostic testing.

Future Work PTS 2013 • Results template - features a drop down menu, leaving less chance for error, and a more unified input. Future PTS • Development of computerised system to assist the conduction of the studies and analysis of the results. Also to monitor the performance from each test and each laboratory.

A WRL/EURL at Pirbright Dr. Jef Hammond Dr. Yanmin Li Ginette Wilsden Pip Hamblin Dr. Valerie Mioulet Dr Nigel Ferris Miki Madi Geoff Hutchings Sheila Wilsden Dr. Don King All participating laboratories

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 77 MINIMUM STANDARDS FOR LABORATORIES WORKING WITH FOOT-AND-MOUTH DISEASE VIRUS in vitro AND

New Roles for “Auxiliary Labs” in the Diagnosis of FMD? Bernd Haas, FLI Riems, Germany

in vivo

Conclusions: Several member states maintain national laboratories able to diagnose FMD in case of an outbreak which are important for the whole European Community.

However, they lack the funding for a facility fully complying with the „Minimum Standards“. Technically, modern diagnostic methods (PCR, ELISA) allow to test samples from suspect cases in „Auxiliary Labs“ in a safe, fast and efficient way. However, current regulations do not fully reflect this situation.

MINIMUM STANDARDS FOR LABORATORIES WORKING WITH FOOT-AND-MOUTH DISEASE VIRUS in vitro AND

in vivo

History

Loeffler‘s animal facility: ‚High containment‘ anno 1900

Recommendations: Update “Minimum Standards“ and Council Directive 2003/85/EC in respect to auxiliary labs Allocate sufficient funding for diagnostic and research laboratries

‚...Germany could be considered free of FMD if there weren‘t Prof. Loeffler‘s experiments...‘

History

Loeffler, 1906:

History

FLI 1990

... that the situation of a future institute should really be such that it of itself could completely exclude any spread of the infective matter. Undoubtedly, an island would be best suited for this purpose.

Loeffler, 1910

1950th - present: „High Security buildings“ • Constant negative pressure • Double HEPA filters (exhaust) • Automatic flaps (inlet and exhaust) • Thermal decontamination of waste water (within containment) • Chemical decontamination of equipment • Compulsory Shower-Out

FMD-Islands

Riems, Plum Island, Lindholm

• Animal Carcass Rendering • Emergency Power Supply

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 77 History Diagnostic Tests

Containment Standards

high

2001 epidemic, UK:

UK, Dk, Ge, Nl, It, CH…

risk

MINIMUM STANDARDS 1985/93

Large animal inoculation

MINIMUM STANDARDS 2009

Five auxiliary laboratories set up For serological testing by solid-phase-competition ELISA (SPCE)

Guinea pigs

3 million samples tested 200 000 samples a week

Cell culture

Required sample througput by far exceeded maximum performance of FMD lab (despite IAH/WRL worked in 3 shifts !)

ELISA low

First EuFMD draft of „Minimum Standards for auxiliary labs“

PCR 2001

1910

timeline

Reagent bank

2010

Minimum Standards - Hazard identification

What is a suspect case of FMD? Threshold for sending samples to lab for FMD testing

The main sources of FMDV are: high

high infected pigs, cattle, sheep, goats and other susceptible large animals

text-book clinical signs of FMD in several animals

laboratory based physical and chemical processing of large quantities of virus

oral/foot lesions (vesicles!) in single animal

infected laboratory animals, e.g. baby mice and guinea pigs

oral/foot lesions (vesicles) in several animals

infected tissue cultures

oral/foot lesions (vesicles) in single animal unspecific clinical signs several animals

diagnostic specimens (if FMD) low

unspecific clinical signs in single animal

low diagnostic specimens inactivated in microbiological safety cabinet diagnostic specimens inactivated on the premise

What is a suspect case of FMD? Threshold for sending samples to lab for FMD testing high

send samples to foreign lab (foreign language, legal aspects, special courier, costs…) send samples to national lab (trouble, costs, standstill..) send samples to regional lab screening/routine program in labs, including FMD as a DD

Sending samples to a foreign lab Language Legal aspects Special courier needed Time

Restricted numbers of samples (in a crisis foreign lab may not be available due to other/national outbreaks)

Data handling Costs

low

…

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 77 Minimum Standards - Authorization of Labs 1. infection of experimental and/or large animals with FMDV;

Legal Aspects „Real FMDV lab“

2. activities which produce high amounts of infectious FMDV,e.g. large scale virus production at a capacity that involves more than 10 litres of cell culture 3. activities involving the handling, and in particular, the propagation of infectious FMDV, but are limited to 10 litres of cell culture, and during which the FMDV is enclosed in containers which can be effectively autoclaved or disinfected; 4. test diagnostic samples for antibody to FMDV, „Auxiliary FMD lab“ by methods that do not involve live FMDV manipulation; Lower standards for 5. test diagnostic samples for FMDV genome by methods Secondary Containment

MINIMUM CONTAINMENT STANDARDS FOR FMD LABORATORIES: in application of Article 65(d) and Annex XII of Council Directive 2003/85/EC

MINIMUM STANDARDS OF BIORISK MANAGEMENT FOR LABORATORIES UNDERTAKING DIAGNOSTIC INVESTIGATIONS OF LOW-RISK SAMPLES DURING AN OUTBREAK OF FMD: in application of point 13 of Annex XV to Council Directive 2003/85/EC on laboratories for serology and for testing inactivated samples employed in case of a FMD outbreak

that do not involve live FMDV manipulation (e.g. RT-PCR); usually: „normal lab“ 6. apply on the genome of FMDV methods of molecular biology that do not involve live FMDV manipulation

Legal Aspects

point 13 of Annex XV to Council Directive 2003/85/EC:

Article 65 of Council Directive 2003/85/EC requires the Member States (MS) to ensure that:

National Laboratories shall cooperate with other laboratories designated by the competent authorities for performing tests, for example serological tests, that do not involve handling of live foot-and-mouth disease virus. These laboratories shall not carry out virus detection in samples taken from suspect cases of vesicular diseases. Such laboratories need not comply with the bio-security standards referred to in Annex XII, point 1, but must have established procedures which ensure that the possible spread of foot- and-mouth disease virus is effectively prevented. Samples giving inconclusive results in tests must be transmitted to the National Reference Laboratory for carrying out confirmatory tests.

(a) laboratories and establishments, in which live foot-and-mouth disease virus, its genome, antigens or vaccines produced from such antigens are handled for research, diagnosis or manufacture, are strictly controlled by the competent authorities; (b) the handling of live foot-and-mouth disease virus for research and diagnosis is carried out only in approved laboratories listed in Part A of Annex XI; …. (d) the laboratories and establishments referred to in points (b) and (c) are operated at least according to the biosecurity standards set out in Annex XII.

Suggested improvements EuFMD / DG SANCO Modify “Minimum Standards” according to risk: “Real” FMD labs doing research and diagnostic work on foreign samples which contain or may contain live FMDV “in peace times” vs

“Auxiliary” labs investigating only suspect samples from own country (PCR, ELISA in MSC) without using live FMDV as a reagent

Thank you for your attention!

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 81

Conclusions and recommendations

From OPENFLU to OPENFMD a resource for automatic and curated nomenclature and tools for the FMD community

Filip Claes, Philippe Le Mercier, Dmitry Kuznetsov, Robin Liechti, Anne Gleizes, Ioannis Xenarios, Gwenaelle Dauphin

OpenFLU - an open database of influenza data • • • •

http://openflu.vital-it.ch/ Contains only public data All data are accessible for browsing All users are welcome - standard free web registration is needed to upload and manage own or laboratory data

OpenFLU: data import/export

• Complete collection of FMDV (genetic) data is necessary for: - Surveillance of disease evolutionary trends - Scientific analysis - Infer gaps in epidemiological data

• OPENFLU-EMPRESi experience may be useful for FMDV community • OPENFMD might become a central portal for the community to store and share FMDV data - and move towards harmonized nomenclature

OpenFLU: an adequate data model for Influenza virus • Sequences grouped per isolates • Isolates accompanied by a comprehensive record of data for epidemiology: - Minimal data required: year, host species, country - Support for much more data: vaccination, patient status, resistance to antibiotics, animal domestication status, etc…

• Influenza-specific genetic knowledge and algorithms - Translation to viral proteins - Key mutations

OpenFLU: easy to find your isolate

• Import: - GENBANK - USER SUBMISSIONS

• CURATION !

daily

GenBank

OpenFlu

• Export: GenBank, data as a structured comment Dynamic fields showing an estimation

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Appendix 81

OpenFLU: more search criteria

OpenFlu: analysis of the data found

Multiple sequence alignment, BLAST, phylogenetic tree, SSM

OpenFLU example: SSM for H7N3

South American cluster

2001-2002

Eurasian cluster 1979-2011

North American cluster 1997-2012

A/Me x ico/InDRE7218/ 2012 A/chick e n/Jalisco/C PA1/ 2012

• Each sequence is a point on the map • Similar sequences are close to each other

• Zoom into a geo region also selects and highlights corresponding sequences on SSM

Epidemiological information

EMPRES-i Genetic Module Linking epi and genetics

• On outbreaks: date, location, species,… • On surveillance • Generated by field teams the day of the event, compiled and reported by vet services (epi unit) within a short time • Stored in databases: local, national, regional, global

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

Appendix 81

Virus information

How does it work

• Generated by labs:

- Detection of influenza virus, subtyping, RNA sequencing - Can take a few days (more)

• Sequence information deposited in a database - Genbank: universal repository - Other genetic databases, eg influenza specialized

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Appendix 81

OpenFMD: the project • A public database that will

- Store FMDV sequences and epidemiological data from various sources • Import from public repositories (like GenBank) • Direct user submission - Verify data consistency (e.g. control of user-submitted serotype by sequence similarity vs. collections of reference strain sequences - Eventually manually curate records - Analyse data using tools like phylo-trees or SSM - Serve all the curated and organized data together with analysis results back to the FMDV community - Towards a harmonized nomenclature (with parallel current names)

OpenFMD: a live DB • To be daily synchronized with all data sources • GenBank (EFetch) allows data export in computer-friendly form (XML) suitable for daily incremental updates • Precision of epidemiological data varies, but GenBank team collects and merges author’s updates • Authors might also correct/update data directly • Already >4000 (full and partial) sequences available in GenBank

OpenFMD: SSM try-out Color-coded by the available from GenBank serotype data N/A A Asia 1 C 1,3,4,5 O

Serotypes A and C

Serotype Asia 1

SAT 1,2,3

Serotype SAT

1,2,3

HKN/200 2

OpenFMD: a sandbox trial • FMDV specific data model • Simplified partial GenBank import • 257 full genome sequences • All 7 serotypes • 2 un-typed sequences - one is “HKN/2002”

SEROTYPE SEROSUBTYPE CNT A Asia 1 C O SAT SAT SAT -N/A-

1,3,4,5 1 2 3

72 41 17 113 10 5 5 2

OpenFMD: a closer look on the map for the type O • Clusters (topotypes) • No more precise annotation in the GenBank records • No serotype for “HKN/2002” (pink) • The serotype can be inferred from its neighbors, i.e. “O”

Serotype O

http://ssm.vital-it.ch/

The phylogenetic tree from

(609-621)

Samuel and Knowles, J Gen Virol 2001, vol. 82 no. 3

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 4

Appendix 81

How can it work ? DBASE A Genetic info

DBASE (P)B Genetic info

OpenFMD: HNK/2002 serotype?

DBASE C Genetic info

• Indeed, no serotype info in the GenBank record

Transfer public data Expert inputs

OPENFMD Interface - Bioinformatic tools - Curation

Science Comm.

Transfer public data EMPRES-i Epi info

OpenFMD: HNK/2002 serotype?

Conclusions and recommendations • Complete collection of FMDV (genetic) data is necessary for: - Surveillance of disease evolutionary trends - Scientific analysis - Infer gaps in epidemiological data

• OPENFLU-EMPRESi experience may be useful for FMDV community • OPENFMD might become a central portal for the community to store and share FMDV data Screenshots from Biochem Biophys Res Commun. 2004 Oct 8;323(1):254-63

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 5

Global Foot and Mouth Disease Research Alliance (GFRA): current actions and future perspective Cyril G Gay DVM, PhD Agricultural Research Service, USA

Francois Maree Onderstepoort Veterinary Institute Agricultural Research Council Fighting foot-and-mouth disease together

Mission Establish and sustain global research partnerships to generate scientific knowledge and discover the tools to successfully prevent, control and eradicate FMD

http://www.ars.usda.gov/GFRA/

Fighting foot-and-mouth disease together

Vision of GFRA 

A coordinated global alliance of scientists producing evidence and innovation that enables the progressive control and eradication of FMD

http://www.ars.usda.gov/GFRA/ Fighting foot-and-mouth disease together

History 

2003 - GFRA is first established with five founder members: Institute for Animal Health, Pirbright Laboratory (UK); Plum Island Animal Disease Center (USA); Australian Animal Health Laboratory; National Center for Foreign Animal Disease (Canada); and International Livestock Research Institute (Kenya).



2004 - Inaugural GFRA meeting is held at U.S. Department of Agriculture, Washington, DC.



2006 - Meeting is held in Agra, India, to explore the needs of countries in FMD-endemic settings.



January 2007. Funding by the United Kingdom Government (DEFRA) to IAH under GFRA umbrella to carry out collaborative research with the Plum Island Animal Disease Center.



May 2008. Experts meet at the Plum Island Animal Disease Center to develop a strategic plan to expand the Alliance and make GFRA more inclusive and relevant to countries combating FMD worldwide.

History 

Oct 2008 - First open GFRA meeting is held at the EUFMD Conference, Erice, Sicily



May 2009 - The second open GFRA meeting is held at the EPIZONE Conference, Antalya, Turkey



Sep 2009 - Wellcome Trust funds GFRA partners IAH-UK, PIADC-USA, OVI-SA, Intervet-NL to improve FMD vaccine production



Jan 2010 - Third GFRA open meeting on Pathogenesis at the Institute of Animal Health, UK



June 2011 - Fourth GFRA open meeting, at the Disconvac – Meeting: FMD Cross protection, B. Aires, Argentina



As of February 2012 - The GFRA is constituted by 35 institutions, public and private, distributed in five continents.



Apr 2012 – Fifth open meeting on Surveillance, Epidemiology, Vaccination and Control of FMD in Hazyview, South Africa.

Canadian Food Inspection Agency

http://www.ars.usda.gov/GFRA/ Fighting foot-and-mouth disease together

Membership 

Any organization interested in enabling the GFRA vision and mission is encouraged to join the alliance. There are three membership options: 1) 2) 3)

GFRA member. FMD research organization that has signed the GFRA MoU GFRA collaborator. Organization that collaborates on a research project with a GFRA member GFRA associate. Organization that benefits, shares or supports the GFRA mission

http://www.ars.usda.gov/GFRA/ Fighting foot-and-mouth disease together

Strategic Goals of GFRA 



Goal 1. Facilitate research collaborations and serve as a communication gateway for the global FMD research community Goal 2. Conduct strategic research to better understand FMD

Goal 3. Development of the next generation of control measures and strategies for their application Goal 4. Determine social and economic impacts of the new generation of improved FMD control Goal 5. Provide evidence to inform development of policies for safe trade of animals and animal products in FMDendemic areas

Research Activities Worldwide  

Report: 2010 - Research Activities worldwide Report: October 2011 - Update of Research Activities

http://www.ars.usda.gov/GFRA/ Fighting foot-and-mouth disease together

Purpose of the report 

This 2011 update report reflects current (2011) activities performed towards the first two goals of the GFRA: –

–

facilitate research collaborations and serve as a communication gateway for the global FMD research community and conduct strategic research to better understand FMD

It is envisaged that the report will be used to identify the gaps in strategic collaborations and research that may potentially prevent the progressive control and eradication of FMD in the future. http://www.ars.usda.gov/GFRA/

GFRA Collaborations 



ARS-PIADC / IAH Pirbright-UK –

Interaction between Ad5-FMD vector and bovine dendritic cells

–

Stabilization of FMDV capsid

ARS-PIADC / INTA-Argentina –

Developing predictive tools for prevention of FMD

–

Understanding Local and Systemic Protective Responses Against FMDV Infection in Cattle: A Genomics Approach

ARS-PIADC / Department of Animal Heath – Viet Nam / AAHLCSIRO - Australia –



Molecular Epidemiology, Surveillance and Predictive Tools for FMD Control In Vietnam

ARS-PIADC / NARC, NVL Pakistan/ UC-Davis/ FAO –

Real Time Data Analysis and Research Capacity Building toward Foot and Mouth Disease (FMD) Control in Pakistan.

GFRA Collaborations… 

IAH Pirbright / ARS-PIADC / ARC-OVI / Oxford U / Intervet –



ARS-PIADC / ICAR – India –



Structurally modified master seed viruses to enhance conventional foot-and-mouth disease virus vaccine production

Effective Molecular Vaccines Against Foot-and-Mouth Disease

IAH-UK/ ARC-OVI / Glasgow U / India Immunologicals –

Vaccine Matching for East and Southern Africa

–

Improved vaccine adjuvants

Fifth GFRA Scientific Meeting 

Surveillance, Epidemiology, Vaccination and Control of FMD – –

April 17-19, 2012 Hosted by the ARC-OVI, Hazyview, Kruger National Park, South Africa • •

Goal: To share the latest knowledge on FMD virus ecology, to improve vaccine efficacy and vaccine matching within the GFRA.

• • • • •

Program content: 1. FMDV-wildlife interaction and pathogenesis 2. Epidemiology and control in different regions of the globe 3. Vaccine efficacy 4. New and current approaches to vaccine matching

•

Participants: GFRA and non-GFRA members involved or interested in the topics mentioned.

Fifth GFRA Scientific Meeting 



Ninety-five delegates attended: –

FMD experts from Botswana, Kenya, Namibia, SA, Tanzania, Uganda, Cameroon, Nigeria, Eritrea, Egypt, also Thailand, Vietnam, China & India presented FMD situation in their region.

–

The FAO & OIE were represented

FMDV in wildlife –

An important concept to remember is that the SAT viruses are buffalo-adapted viruses. The buffalo-livestock interfaces are the critical area for disease prevention/ control

–

Current FMD management approaches in southern Africa are failing. What are the reasons for this?

–

It is clear from discussions that FMD and its control present complex socio-economic and environmental impacts in the region

Fifth GFRA Scientific Meeting 



FMD in southern Africa, Tanzania & Uganda –

more systematic surveillance, capacity building at all levels (incl. regional, national, inter-sectorial); and cross-sectorial collaboration on FMD research.

–

Describing the complex epidemiology and endemicity of FMD throughout Africa.

–

Cross-protection and vaccine-matching of the field isolates to available vaccines is required.

FMD in central and West Africa –

Experience continuous outbreaks of FMD: one of the main burdens to animal health and production.

–

Currently no control program, no vaccination and little is known about the viral strains circulating.

Fifth GFRA Scientific Meeting 



FMD in southern Africa, Tanzania & Uganda –

more systematic surveillance, capacity building at all levels (incl. regional, national, inter-sectorial); and cross-sectorial collaboration on FMD research.

–

Describing the complex epidemiology and endemicity of FMD throughout Africa.

–

Cross-protection and vaccine-matching of the field isolates to available vaccines is required.

FMD in central and West Africa –

Experience continuous outbreaks of FMD: one of the main burdens to animal health and production.

–

Currently no control program, no vaccination and little is known about the viral strains circulating.

Fifth GFRA Scientific Meeting 



Vaccine efficacy –

Quality of vaccines

–

Minimum requirements, needs, etc

Vaccine matching approaches –

Serology: VNT, ELISA

–

New approaches, like Antigenic cartography

–

Linear mixed effect models

–

etc

http://www.ars.usda.gov/GFRA/

Fighting Foot and Mouth Disease Together!

Role of buffalo in the maintenance of foot-and-mouth disease virus

Bryan Charleston

3 KNP buffalo isolates

Study design 6 month old Nguni cattle (n = 4)

SAT1/KNP/196/91 SAT2/KNP/19/89 SAT3/KNP/1/08/3

PK cells

SAT-3

OVI cattle titration ≈ 1 × 104 TCID50 SAT-1

SAT-2

Skukuza buffalo challenge study ≈ 5 × 105 TCID50 Necropsy N = 4 buffalo

Necropsy Necropsy N = 4 buffalo N = 4 buffalo

35DPC

95DPC

156DPC 163DPC 185DPC

1.25IU/kg

N = 4♀ Institute for Animal Health

400DPC

1.25IU/kg

ACTH stimulation (Synacthen®) N = 4 buffalo

N = 16 FMD free buffalo (7♂9♀) 10 – 30 months old Imfolozi Game Reserve, KZN

299DPC

Necropsy N = 4 buffalo

Day 302 Progesterone IUD Estrumate

Acute FMD: naïve buffalo

1DPC 2DPC

Acute FMD: naïve buffalo Conclusion • SAT co-infection in cattle – severe clinical FMD • 10 × dose in naïve buffalo: • mild clinical FMD • integrin αvβ6 expression in tissues typically associated with FMD lesions • viraemic for at least 5 to 7 days • No leucopenia • High levels type 1 IFN • seroconverted to all three serotypes by day 14

3D primer and probe

SAT1 primer and probe

SAT2 primer and probe SAT3 primer and probe

Chronic FMD: buffalo

Casteleyn et al.,

Budras, Bovine Anatomy

Follicles with central crypt

M Hofmeyr

Institute for Animal Health

Chronic FMD: buffalo Virus isolation (IBRS cells) Number of buffalo

Right tonsil swab Probang

7 6 5 4 3 2 1 0 50 DPC *109 DPC 35 35

*One round VI only () = passage number on IBRS cells

Institute for Animal Health

126 DPC 136 DPC 155 DPC 162 DPC 168 DPC 172 DPC 185 DPC

Day post challenge

Chronic FMD: buffalo 3D primer and probe

SAT1 primer and probe

SAT2 primer and probe SAT3 primer and probe

Institute for Animal Health

95DPC

Chronic FMD: buffalo

10µm 100µm

100µm Anti-3B (D5)

IB11

Merge

10µm Institute for Animal Health

FMD: buffalo

Discussion points • Despite close contact, +ve virus isolation and ACTH stim – no transmission/ seroconversion in cattle! • Consistent with previous reports: • no transmission/ transmission only after months of contact • Rx buffalo dexamethasone (Gainaru et al., 1986) • Rx cattle dexamethasone for 3 weeks (Ilott et al., 1997) – inhibited shedding of FMDV • Co-infection cattle rinderpest/ bovine herpes 1 viruses (McVicar et al., 1977) – no increase virus recovery/ transmission • Pattern of transmission from captive buffalo during acute infection is also variable • same pen: buffalo to buffalo/ buffalo to cattle× (Gainaru et al., 1986)/ buffalo to cattle× (Dawe et •

al., 1994)

adjoining pen: buffalo to impala× (Gainaru et al., 1986)

Knowledge gaps and questions • Improved methods to isolate virus/ viral sequences from carrier buffalo • Indentify sites of virus localisation and replication in buffalo • Propose hypothesise for mechanisms of persistence • Test potential “triggers” for transmission: short term stress? Long term stress? “childhood epizootic”? • How infectious are buffalo during acute infection? Virus excretion compared cattle/sheep/pigs. Transmission despite no/ limited FMD lesions? Institute for Animal Health

Future work •

FMDV maintains high force of infection in buffalo herds

•

Crucial that we understand how FMDV persists in isolated buffalo herds

•

3 year longitudinal study of isolated breeding herd in KNP

 > 98% buffalo in KNP exposed to all three SAT serotypes by age 2 (Thomson, 1992)  High rate of infection suggests common mechanism for inter-annual perpetuation  Avoids auto-extinction small isolated herds

 Primary objective to identify triggers to affect transmission from carriers.  If we can’t even understand this - how understand in livestock, large populations, global, multiple serotypes?

– – – – –

Account for environmental factors absent from experimental studies Identify factors affecting FMDV status (age, sex, seasonal, co-infections etc.) Phylogenetic and antigenic phenotype analysis of FMDV isolates Establish infection parameters in buffalo calves (naïve population) Test triggers under experimental conditions

•

We hypothesise:

•

To affect transmission:

– Previous demonstration of FMDV particles in lymph tissue on germinal centre FDCs provides a means of delayed viral clearance (extracellular reservoir)

– Immune stimulation (enhance immune-complexed FMDV replication in Fc receptor expressing cells) – Or suppress antibody response – for example protein-calorie restricted diet (Watson, 1985)

Acknowledgments Nick Juleff Lin-Mari de Klerk Lorist Roy Bengis Louis van Schalkwyk At Dekker Scalk van Dyk Dave Cooper

Ivan Morrison Francois Maree Berry Mutowembwa Dan Haydon Brenda Botha Livio Heath Katherine Scott Pamela Opperman Belinda Blignaut

Ferran JORI, Cirad Mammal Research Institute University of Pretoria Markus Hofmeyr Peter Buss Jenny Joubert

FMD Ecology: Collaborative Studies

Anna Ludi, Zaheer Ahmed, Steve Pauszek, Jonathan Arzt and Luis L. Rodriguez Plum Island Animal Disease Center

Disease Ecology • Differs from a traditional medical approach in that it’s not concerned with describing the pathology of individuals • Differs from epidemiology in that the emphasis is on general processes of host population, environment and agent interactions – long term maintenance

Epidemiology vs Ecology? • Epidemiology: the study of the determinants of disease and injuries – What causes disease? Where and When? – Mechanistic

• Disease ecology: the study of the underlying principles that influence the spatio-temporal patterns of disease – Adds the Why to the equation, (e.g. why do patterns of disease occur as they do?) – Conceptual: what variables play roles on disease

Pathogenesis Route of entry Dissemination Genetic background

Genetic determinants of virulence Patterns of Evolution

Climate change Environmental disruptions

FMD Ecology • Epidemiological knowledge on FMD clinical occurrence is large • Geographic distribution (pools) of serotypes/ topotypes derived primarily from clinical occurrence

• Little known about FMD ecology: – Where is FMD between outbreaks? – Role of persistence (e.g Asian buffalo) in long term maintenance? – What determines emergence of epidemic viral strains?

Objectives • • • •

1. To better understand the ecology of FMDV circulating in selected endemic regions of Asia and Africa 1.1 Acute Clinical Samples: conduct systematic FMD monitoring program to determine the molecular epidemiology of FMDV in local livestock 1.2 Collect samples from subclinical and persistently infected animals (longitudinal field study) 1.3 Understand role of Asian buffalo on FMD transmission

•

2. To determine the phylogenetic relationships of FMDV strains in endemic regions

•

3. To determine the antigenic relationship (vaccine matching) of FMDV strains circulating in the selected regions with currently available vaccine strains 3.1 Establish panel of reference cattle sera for vaccine matching testing 3.2 Measure antigenic relationship of field FMDV strains with reference vaccine sera

• •

PAKISTAN CHARACTERIZATION OF LOCAL ISOLATES OF FMDV AND DEVELOPMENT OF VECTOR BASED VACCINES 58-1940-7-161F; 057 002S 9/1/2007 - 8/31/2012

REAL TIME DATA ANALYSIS AND RESEARCH CAPACITY BUILDING TOWARDS FMD CONTROL IN PAKISTAN 1940-32000-052-14S 09/27/20 12 - 09/27/2014

Dr. Khalid Naeem – NARC Dr. Umer Farooq – NARC Dr. Muhammadimam Afzal, FAO Dr. Manzoor Hussain, NVL

Objectives • In collaboration with the Center for Animal Disease Modeling Systems at UC Davis carry out genomic evaluation of field isolates using BioPortal • Vaccine matching using sera derived from: – Trivalent Merial and ARRIAH vaccine – Cattle and buffaloes – Days 0, 7, 14, 28, 60 and 90

• Persistence studies in cattle and buffaloes in a natural setting – Sample collection at farms in high risk areas

• Anatomical pathology of persistence of FMDV in cattle and buffaloes – FMDV positive cases will be slaughtered and samples of Naso-pharynx, top of pharynx, dorsal of palate, regional lymph nodes and tonsils will be taken

Accomplishments TASK 1.1 – Acute Clinical Samples • 42 P1 FMDV sequences have been obtained • 85 samples currently in the process of being shipped

TASK 1.2 – Longitudinal Field Study • 4th rounds of sampling currently taking place of NSP positive animals • Initially 300 samples were collected with 233 being positive for NSP • NSP-ELISA, RT-PCR and virus isolation is currently being undertaken in Pakistan TASK 3.1 – Establish panel of reference sera for vaccine matching testing • 144 sera samples collected: – – –

against both Merial and ARRIAH vaccine Cattle and buffaloes Sera collected days 7, 14, 28, 60 and 90

OTHER ACCOMPLISHMENTS • Training session in Pakistan to teach epidemiology, bio-portal, cell culture and virus isolation

# of samples

Year

Region

Species

Serotype

2008

ICT

Cattle

2009

Sindh

Cattle & Buffaloes

A&O

2010

Punjab

Cattle & Buffaloes

2011

Punjab

Cattle

O & Asia 1

2011

Sindh

Buffaloes

Asia 1, O or mixed infection

Significant amino acid and nucleotide variations were associated with spatial distance, but not with differences in host species, consistent with the frequent multispecies infection of this serotype O FMDV.

Future Planned Work • Continued collaboration with UC Davis to utilize BioPortal to map all outbreaks detected • Carry out vaccine matching with serotype O, A and Asia 1 to answer following questions: • Is there a difference between neutralization titers obtained amongst cattle and buffaloes? • What is the neutralization responds of animals having maternal antibodies? • How are neutralization values affected by using trivalent vaccines (comparison will be made in collaboration with IAH, WRL) • If funding allows in vivo testing of results (ie vaccine challenge studies) • Characterization of persistent virus and phylogenetic comparison with strains causing outbreaks in the same regions

VIETNAM

MOLECULAR EPIDEMIOLOGY, SURVEILLANCE AND PREDICTIVE TOOLS FOR FMD CONTROL IN VIETNAM 58-1940-0-070F, 057 14S 6/1/2010-9/30/2012

Dr. Thanh Long Ngo, DAH, HCMC Dr. Ho Huu Dung, DAH, Hanoi

Dr. Carla Huston, Mississippi State University

Objectives • Molecular epidemiology of FMDV in local livestock including cattle, buffaloes and pigs • To better understand the transmission mechanism of persistently infected to susceptible livestock in natural setting • Enhance strategies for identification of persistently infected animals using new technology • Vaccine matching to asses efficacy against currently circulating strains.

Accomplishments

TASK 1.1 – Acute Clinical Samples • Received 69 samples: – Samples collected during outbreak – Pigs, buffaloes and cattle – Northern and Southern Vietnam • Preliminary sequencing from 43 samples identifies serotypes A and O TASK 1.2 – Longitudinal Field Study • HCMC region – finished collecting samples for the third time; probang after second sampling indicated 11 positive animals on 9 farms • Hanoi-SonLa region – third round of sampling now taking place; 9 carrier cattle and 5 carrier buffalo have been identified TASK 3.1 – Vaccine Matching Testing • Possibility of obtaining monovalent serotype O sera Other Accomplishments • Trained Saigon and Hanoi veterinarians in theoretical and practical aspects of probang sampling

Future Planned Work

• Dr. Jonathan Arzt to visit before the end of the year 18 persistent buffalos identified for necropsy and detailed tissue distribution analysis

CAMEROON

TRANSMISSION AND EVOLUTION STUDIES OF FMDV IN LIVESTOCK IN THE LAKE CHAD BASIN 58-1940-0-047N, 05713 – Non-Funded 04/01/2010 to 04/30/2015

Dr. Rebecca Garabed, Ohio State University

Dr. Simon Dickmu, LANAVET

Objective • To understand the epidemiology of infectious diseases in the ecological context of networks of host movement • How different networks of livestock movement affect disease epidemiology

Accomplishments TASK 1.1 – Acute Clinical Samples • Sequencing has shown serotypes O and SAT 2 – Currently in the process of obtaining full P1 sequences on 12 samples – Another 76 rRT-PCR positive samples to be processed

TASK 1.2 – Longitudinal Field Study • 385 serum samples have been analyzed using virus neutralization -Serotypes O, A and SAT2 Other Accomplishments • In the process of writing manuscript

Accomplishments

SAT 2 incursion into Northern Africa?

100 90 80

70 60

Serotype O

Serotype A

Serotype SAT1

Serotype SAT2

20 10

Serotype SAT3

Cattle infected with numerous serotypes Percentage of Cattle

Percentage of Cattle positive on SNT

FMDV serotypes prevalent in Cameroon from 2010-2012

100,00% 80,00% 60,00%

Market

40,00%

Mobility

20,00%

Sedimentary

0,00%

Number of serotypes

Outcomes • What new project ideas have been created? – Multiple FMDV serotypes co-infecting same animals, what is the mechanism? How many viruses co-exist in same animal? – FMDV strains SAT2 serotype detected in Cameroon 2010-2011, is this the SAT2 that incursioned into north Africa?

Future Planned Work UNDERSTANDING THE ECOLOGY OF FOOT-AND-MOUTH DISEASE VIRUSES CIRCULATING IN CAMEROON Not yet established - 057 42S 06/01/12-05/31/14 (pending signatures)

Development of a safe antigenic marker Foot-and-Mouth Disease Vaccine Platform

Elizabeth Rieder PhD. Foreign Animal Disease Research Unit, USDA-ARS

Plum Island Animal Disease Center, New York, USA. EUFMD 2012, Jerez de la Frontera, Spain

Vaccine recommendations made by the FMD-WRL are based on needs for the 7 pools

7a, b

4 6

Pool 1 East Asia

Pool 2 South Asia

Pool 3 Eur Asia

Pool 4 East Africa

Pool 5 West Africa

Pool 6

A, O, Asia 1

A,O, Sat 1-3

A,O, Sat1-2

Sat 1,2,3

Pool 7 South South Africa America A, O

Concerns with FMD Vaccines • Require adaptation and growth of large volumes of wild type virus in cells • Escape of virus from manufacturing facilities • Require banking of multiple antigen concentrates • Some antigens lack stability (low potency/short shelf life) • Onset of protection 7-14 days • Short duration of immunity <6 months • Difficult to differentiate vaccinated from infected animals (DIVA) due to presence of NS proteins • Vaccinated and exposed animals become carriers

Characteristics of an “Ideal” FMD Vaccine  Effective, rapid and long-lasting protection with one      

inoculation Prevents viral transmission Allow differentiation of infected from vaccinated animals (DIVA) Produced without the need for virulent FMDV Prevent development of carrier state Protection against multiple serotypes Stable antigen – long shelf life

Negative marker cDNA-derived killed FMDV Vaccine Platform 5’NTR

STRUCTURAL

IRES

RE1

Lpro deletion

3’NTR

3B23

δL 1A

poly(C)

NON-STRUCTURAL 2B

RE2

Negative marker in NSP Deletion of one 3B

Production of Vaccine antigens by Swapping Capsid sequences for those of Outbreak Strains

3B & 3Dpol

Wildtype and Marker FMD Viruses STRUCTURAL

NON STRUCTURAL 3B 123

A24 WT

VP4 VP2 VP3 VP1

5’NTR A24 LL3B3D

Asia1-LL3B3D

∆L

3’NTR VP4 VP2 VP3 VP1

RE2

∆L

2A VP4 VP2 VP3 VP1

5’NTR RE1

ATurkey06LL3B3D

3B2,3

5’NTR RE1

∆L

A (n)

3B2,3 2B

RE2

VP4 VP2 VP3 VP1

A (n) 3’NTR

3B2,3

5’NTR RE1

RE2

C3 Resende, SAT1, O1Campos, O1Ecuador

A (n) 3’NTR

Genetic based distinction between A24LL 3B3D and parental viruses Lanes 1, 4, 7, 10: A24 Cru Lanes 2, 5, 8, 11: A24LL3B3D Lanes 3, 6, 9, 12: Asia1 LL3B3D

10 11 12

1. full genome sequence

Leader Asia1 capsid

2. RT-PCR screening

3B3 (WT) 3B2 (LL) A24 capsid

+ Absence of leader

+ + Presence of A24 capsid

+ Presence of Asia1 capsid

# of 3B copies

Antigenic based distinction of marker A24LL 3B3D from parental virus F19

F44

Western Blot Vaccine Source

anti3B MAb F8

Anti 3Dpol- MAbs F19

F44

Mock A24Cru (WT) A24Cru 3D

A24 Cru (WT)

A24LL 3B3D

A24Cru 3B3D

Asia1-LL 3B3D

A24LL

AturkeyLL-3B3D

A24LL 3D A24LL 3B3D

Two deleted epitopes and corresponding MAbs present opportunities for development of two DIVA diagnostic companion tests

Competitive 3B- and 3D-based ELISA (DIVA) Indirect ELISA

Virus

Anti-3B antibodies

Anti-3D antibodies

A24 WT

A24 WT 3B3D

Competitive DIVA 3B and 3Dpol ELISA 3B cELISA

Competing 3B and 3D MAbs recognize specific epitopes that are absent in the marker virus

3D cELISA

A24 WT A24 WT 3B3D(9143) A24 WT 3B3D (9144)

3B and 3D cELISA can differentiate immune response to WT from marker vaccine-exposed animals

Safety Data Pigs single marker A24FMD-LL3D Pig #40 Viremia b Virus in oral swab b Virus in nasal swab b Neutralization titer d Clinical score

0 dpi a Negative c Negative Negative < 0.9 Negative

1 dpi Negative Negative Negative < 0.9 Negative

2 dpi Negative Negative Negative < 0.9 Negative

3 dpi Negative Negative Negative < 0.9 Negative

4 dpi Negative Negative Negative < 0.9 Negative

5 dpi Negative Negative Negative < 0.9 Negative

6 dpi Negative Negative Negative < 0.9 Negative

Pig #41 Viremia Virus in oral swab Virus in nasal swab Neutralization titer Clinical score

0 dpi Negative Negative Negative < 0.9 Negative

1 dpi 5.44 Negative Negative < 0.9 Negative

2 dpi Negative 5.45 Negative < 0.9 Negative

3 dpi Negative Negative Negative < 0.9 Negative

4 dpi Negative Negative Negative 0.9 Negative

5 dpi Negative Negative Negative 1.2 Negative

6 dpi Negative Negative Negative 1.2 Negative

Pig #42 Viremia Virus in oral swab Virus in nasal swab Neutralization titer Clinical score

0 dpc e Negative Negative Negative < 0.9 Negative

1 dpc Negative Negative Negative < 0.9 Negative

2 dpc Negative Negative Negative < 0.9 Negative

3 dpc Negative Negative Negative < 0.9 Negative

4 dpc Negative Negative Negative < 0.9 Negative

5 dpc Negative Negative Negative < 0.9 Negative

6 dpc Negative Negative Negative < 0.9 Negative

Pig #43 Viremia Virus in oral swab Virus in nasal swab Neutralization titer Clinical score

0 dpc Negative Negative Negative < 0.9 Negative

1 dpc Negative Negative Negative < 0.9 Negative

2 dpc Negative Negative Negative < 0.9 Negative

3 dpc Negative Negative Negative < 0.9 Negative

4 dpc Negative Negative Negative < 0.9 Negative

5 dpc Negative Negative Negative < 0.9 Negative

6 dpc Negative Negative Negative < 0.9 Negative

All room air samples negative

FMD-LL3D is attenuated and non-transmissible in pigs!

Direct Inoculation

Direct Contact

Safety Data in Pigs

Safety Data Cattle Viremia, Maximum Titer. b (DPI) c

Virus in Saliva, Maximum Titer b (DPI) c

Fever d (DPI) e

Maximum Clinical Score/ Maximum achievable (DPI) g

Neutralization Titer maximun (Starting DPI)

Shedding in air. Maximun Titer i (DPI) c

Bovine #a

Virus

7109

A24WT

7.60 (3)

8.90 (3)

Yes (2,3)

5/5 (7)

2.4 (5)

5.57 (5)

7110

A24WT

7.31 (4)

10.18 (3)

Yes (2-5)

5/5 (5)

2.4 (6)

9143

A24WT3B3D

7.40 (4)

9.03 (5)

Yes (3)

1/5 (5)

3.6

6.29 (6)

9144

A24WT3B3D

7.92 (4)

8.85 (5)

Yes (4)

4/5 (7)

3.0

5.45 (7)

9145

A24LL3B3D

Negative

0/5

1.5

ND j

9146

A24LL3B3D

Negative

0/5

2.4

Negative

Efficacy Data Serological Response Cattle Vaccine consisted of 15 μg/dose A24LL3D or A24LL3B3D chemically inactivated and oil adjuvanted

Challenge

No clinical disease (0/4)*

Single Marker FMD vaccine Double Marker FMD vaccine

100 % clinical disease (4/4) Cattle were challenged by intradermolingual inoculation of 10,000 bovine infectious doses of FMDV A24Cru

Marker FMD-LL3B3D Vaccine Candidate  Safe production: attenuated      

in cattle and pigs Easy production: uses same production system as current FMD vaccines Simplified downstream processing: no need for NSP removal Non transmissible from cattle and swine Negative markers: 2 independent DIVA compatible markers Immunogenic: same as current inactivated vaccine Cassette construct allows to rapidly insert capsid-coding region from emerging strains

IRES δL 1A 1B

3’NTR

3B 1C

1D 2B UCS2

lacking epitopes: DIVA marker

Acknowledgements Rieder Laboratory • Sabena Uddowla • Paul Lawrence • Devendra Rai • Elizabeth Schafer • Joe Conderino Former members • Jason Hollister • Ariel Vagnozzi • Chris Larson APHIS: Ian Olsen

• •

Luis Rodriguez Juan Pacheco

GLOBAL PARTNERSHIPS • • • • • •

South Africa Argentina UK Russia Netherland Uganda

• Israel

FMD Research Priorities David Paton

Research Priorities What is needed – what would make the most impact? What is possible – available technologies and opportunities? Applied research • Goal oriented on practical translation and uptake of research findings

Fundamental research

• Understanding of principles/mechanisms applicable to different problems

Quantitative approaches

• Problem weighting, expected value of information, payback

Research is unpredictable – should not set priorities? Why set research priorities? • Maximise benefit from limited resources • Focus, coordinate, influence effort

Global roadmap for improving the tools for FMD control in endemic settings (GFRA 2006) Two principal priorities both requiring a combination of fundamental and applied research Better vaccines – the ideal and what would be enough to make a difference – likely success, timescale and expense

Better understanding of animal production systems and FMD dynamics within them – epidemiological studies to identify critical control points and alternatives to mass vaccination – cost-benefit of disease control

Scientific Developments & Technical Challenges in the Progressive Control of FMD in South Asia Vaccines with longer duration of immunity, greater stability and broader crossprotection Understand viral ecology, genetic evolution, and infection and transmission dynamics Tools for local diagnosis and management and better vaccine monitoring

New Delhi Conference 13-15 Feb 2012

DG Research Call 2008 KBBE-2008-1-3-02: FMD: improve and/or develop vaccines, vaccination strategies and diagnostics assays for free and endemic settings

• Substitution of vaccine potency tests • Assessment / improvement of heterologous vaccinal protection • Development of vaccines / anti-virals with rapid onset / long duration • Improvement in 'DIVA' tests • Improve knowledge on FMDV transmission in recently vaccinated animals • Development or adaptation of computerised FMD-spread models to optimise vaccination schemes.

GFRA Strategic Goals PIADC – 2008 Goal 1- Facilitate research collaborations Goal 2 - Conduct strategic research to better understand FMD Goal 3 - Development of the next generation of control measures and strategies for their application Goal 4 - Determine social and economic impacts of new generation of improved FMD control Goal 5 - Provide evidence to inform development of policies for safe trade of animals and animal products in FMD endemic areas

GFRA Strategic Goals Goal 2 - Conduct strategic research to better understand FMD Objective 1 Host-pathogen interaction Objective 2 Understand the mechanisms of viral persistence Objective 3 Understand the ecology of FMD

GFRA Strategic Goals Goal 3 – development of next generation control measures and strategies for their application Objective 1 Discover vaccines specifically designed for the control and eradication of FMDV Objective 2 Improved diagnostics for surveillance, outbreaks, and recovery Objective 3 Discover biotherapeutics that will provide rapid protection Objective 4 Determine scientific information to implement effective biosecurity measures

Fundamental Issues Cellular replication Pathogenesis The host response to infection Transmission between hosts Viral evolution

Policy Needs Recovering free status • Early recognition • Dealing with wildlife and carriers • Vaccinate-to-live • Decision support tools

Progressive control • Incentives and cost-benefit • Husbandry, trading, pastoralism • Dealing with multiple hosts and wildlife • Surveillance strategies • Movement control strategies – including zones and compartments and commodities • Vaccination programmes • Monitor progress

EuFMD Supported Technical Studies

Theo Knight-Jones

Wild boar surveillance methods and prevalence studies Estimating vaccine effectiveness NSP serology validation in water buffalo and African buffalo Application of full genome sequencing to Asian FMDV outbreaks Quantification of surveillance components

Tsviatko Alexandrov

New Opportunities Studies in countries introducing disease control measures GPS and satellite tracking Modelling approaches to help resolve complex situations Miniaturisation and automation High throughput screening Next generation sequencing – host and virus Insights on immune mechanisms and susceptibility traits Bioimaging and microdissection Genetic manipulation – host and virus Limitations of the species worked upon?

Epidemiology Networks of interaction and trade Differences between serotypes and strains Host-specific epidemiological characteristics, including wildlife Understanding/predicting epidemic cycles Required levels/distribution of population immunity Importance of different transmission routes and mechanisms Spread models that can be adapted to different circumstances Need for multidisciplinary approaches combining expertise available in free and endemic countries

Vaccines and anti-virals Safe, stable, DIVA – making progress Stronger and longer duration – antigen persistence, stronger innate signals, T cell support, live vaccines, new delivery routes and systems Cross-protection – progress on strain selection Conserved B cell epitopes or other broadly X-protective mechanisms? Vaccination programme monitoring Need for fundamental studies of host and viral determinants of pathogenesis and immune responses. Where are we with rapid onset protection?

Diagnosis Early and preclinical detection High throughput & non-invasive / distance methodologies Unscrambling complex epidemiological situations Simplified and penside diagnostic developments Combining surveillance measures of freedom Molecular tracing tools Mix of technology transfers and validations of applications in relevant field settings

Other Product safety • FMDV survival in commodities and products • Residual bone marrow and lymph nodes in deboned beef

Socio-economic and cost-benefit studies

Conclusion Important to have a clear message of what is needed and what is possible

Presentations at this meeting Immunology

• Antibody responses – epitopes, mechanisms, correlations

Vaccine research

• Viral vectors, capsid expression, capsid chimeras, mucosal delivery

Epidemiology • • • •

Risk factors – wildlife, husbandry systems, prevalence studies, movements Transmission experiments – with/without vaccines Models – simulations, decision support, economics Molecular epidemiology

Vaccine implementation

• Predicting protection – strain selection • Vaccine effectiveness and impact

Diagnostics

• Simplified formats and submission methods

Anti-virals

Vision of GFRA 

A coordinated global alliance of scientists producing evidence and innovation that enables the progressive control and eradication of FMD

http://www.ars.usda.gov/GFRA/ Fighting foot-and-mouth disease together

Fifth GFRA Scientific Meeting 

Ecology & immunology – –

September, 2013 Arusha, Tanzania

• • •

Goal: To share the latest knowledge on FMDV Ecology, pathogenesis, immunology.

• • • • •

Program content: 1. Ecology 2. Transmission 3. Pathogenesis 4. Immunology

• • •

Participants: GFRA and non-GFRA members involved or interested in the topics mentioned.

Measuring and Monitoring FMD Occurrence Melissa McLaws EuFMD

Acknowledgements •Chris Bartels: •Naci Bulut:

EuFMD

FMD Institute, Ankara, Turkey

•Theo Knight Jones: Institute for Animal Health, Pirbright, UK •Shams Amin Abo Gabal, Rehab Abdel-Kader El Bassal, Animal Health Research Institute, Cairo, Egypt Soheir Hassan Abdel-Kader, Iman Ali Farag, Amaal Ibrahim Attya Mansour, General Organization for Veterinary Services, Cairo, Egypt •General Directorate for Food and Control, Ankara, Turkey •Iranian Veterinary Organization, Tehran, Iran

Key points • Data from Turkey and Iran show: – Yearly incidence rate 10-30 X higher when measured using NSP serosurveys compared to reports of suspected cases – Provincial (or district) level FMD incidence measured by 1) reports and 2) serological data were poorly correlated

• FMD reporting should be encouraged and reports analysed continuously (monthly) to detect ‘events’ • Carefully designed serosurveys should be conducted regularly (annually if possible) – Unbiased measure of FMD infection, best information about risk factors

Monitoring FMD is key principle of PCP Measuring FMD occurrence to: 1. Assess FMD risk o Species, sector, husbandry system, area o To target control and inform FMD impact assessment 2. Monitor changes over time – Detect ‘events’ (epidemics) – Efficacy of control strategy

But what is the best way to do it?

Monitoring FMD Occurrence: Passive Surveillance All FMD reports

2009-2010 300

Turkey:2009-2010

number of reports 100 200

Turkey Case Reports

month

no. villages reporting in 2010 / ttl villages in province

Incidence (%) 6.00 - 12.00 4.00 - 6.00 2.00 - 4.00 1.00 - 2.00 0.50 - 1.00 0.00 - 0.50 No data

20 10 ct O

01 0 Ju l2

Ap r2 01 0

20 10 Ja n

20 09 O

00 9 Ju l2

Ap r2 00 9

2010 Village Incidence FMD reports

Ja n

20 09

2010 Village level incidence

Month 0

number of outbreaks 200 150 100

number of outbreaks 100 150 200

250

Month 20 01 Ja n 20 02 Ja n 20 03 Ja n 20 04 Ja n 20 05 Ja n 20 06 Ja n 20 07 Ja n 20 08 Ja n 20 09 Ja n 20 10 Ja n 20 11 Ja n 20 12

number of outbreaks 100 150 200

Serotype O

Ja n

20 01 Ja n 20 02 Ja n 20 03 Ja n 20 04 Ja n 20 05 Ja n 20 06 Ja n 20 07 Ja n 20 08 Ja n 20 09 Ja n 20 10 Ja n 20 11 Ja n 20 12

Ja n

number of outbreaks 100 200 300 400

Turkey: 2001-2012

Ja n

20 01 Ja n 20 02 Ja n 20 03 Ja n 20 04 Ja n 20 05 Ja n 20 06 Ja n 20 07 Ja n 20 08 Ja n 20 09 Ja n 20 10 Ja n 20 11 Ja n 20 12

Ja n

Monitoring FMD Occurrence: Case Reports All Outbreaks

• Most are laboratory confirmed & serotyped

Month

Serotype A

Asia-1 Serotype Asia1

Month

Monitoring FMD Occurrence: Active surveillance • Clinical cases • NSP serology – Sampling young stock gives picture of FMD infection in recent months 2010 Village Seroprevalence

Incidence 80.00 - 100.00 60.00 - 80.00 40.00 - 60.00 20.00 - 40.00 0.00 - 20.00 No data

Turkey: 2010

How to relate reporting and serological data? • Compare FMD incidence measured using survey data and reports in Turkey and Iran • Well developed passive reporting systems • Extensive serosurveys Turkey (spring 2009)

Turkey (spring 2010) W. Azerbaijan, June 2011

-32,670 samples

-64,765 samples

-8349 samples

78 provinces, 334 districts, 554 villages

74 provinces, 460 districts, 946 villages

1 province, 14 districts 281 epi-units

-60 samples /village

- 30 samples/epi unit

-cattle only -mostly 4-18 months

50% cattle, 50% SR -mostly 4-18 months

-cattle only -6-24 months

How to relate reporting and serological data? 2009

Serology

2010 2010 Village Seroprevalence

2009 Village Seroprevalence

Incidence 80.00 - 100.00 60.00 - 80.00 40.00 - 60.00 20.00 - 40.00 0.00 - 20.00 No data

Reports

2010 Village Incidence FMD reports

no. villages reporting in 2010 / ttl villages in province

2009 Village Incidence FMD reports

Incidence (%) 6.00 - 12.00 4.00 - 6.00 2.00 - 4.00 1.00 - 2.00 0.50 - 1.00 0.00 - 0.50 No data

no. villages reporting in 2010/ttl villages in province

Incidence 6.00 - 12.00 4.00 - 6.00 2.00 - 4.00 1.00 - 2.00 0.50 - 1.00 0.00 - 0.50 No data

All FMD reports

month

01 0

20 10 ct

Ju l2

20 10

Ap r2 01 0

Ja n

00 9

20 09 ct

Ju l2

20 09 Ja n

Ap r2 00 9

number of reports 100 200

300

Turkey:2009-2010

Relative comparison (serological ÷ report incidence) Serological/Report Village Incidence 2009

Relative Incidence 200.00 - 500.00 100.00 - 200.00 80.00 - 100.00 60.00 - 80.00 40.00 - 60.00 20.00 - 40.00 0.00 - 20.00 No data

2009: Median 30X

Serological/Report Village Incidence 2010

Relative Incidence 200.00 - 500.00 100.00 - 200.00 80.00 - 100.00 60.00 - 80.00 40.00 - 60.00 20.00 - 40.00 0.00 - 20.00 No data

2010: Median 11X

Absolute comparison (Serology minus Report incidence)

Serological and Report Village Incidence 2009: Absolute difference

Incidence difference (%) 80 - 100 60 - 80 40 - 60 20 - 40 0 - 20 -10 - 0 No data

Median difference: 20%

Serological and Report Village Incidence 2010: Absolute difference

Incidence difference (%) 80 - 100 60 - 80 40 - 60 20 - 40 0 - 20 -10 - 0 No data

Median difference: 29%

W. Azerbaijan serosurvey • 80.2% of epi-units had at least 5 calves with a high titre (>70% inhibition) • 18% observed clinical signs in their stock in the previous 12 months (questionnaire) District-level FMD Incidence: Serosurvey

Clinical signs: Survey

2011 Incidence FMD serosurvey

Incidence FMD reports (sample)

Clinical signs: Offical reports Incidence FMD reports 16 mo prior to survey

Incidence 85.71 - 100.00 80.71 - 85.71 71.43 - 80.71 66.67 - 71.43 50.00 - 66.67

Incidence 28.57 - 40.00 27.27 - 28.57 19.81 - 27.27 16.67 - 19.81 8.57 - 16.67 0.00 - 8.57

Incidence 13.77 - 32.76 9.97 - 13.77 9.18 - 9.97 7.81 - 9.18 7.37 - 7.81 1.52 - 7.37

How to relate reporting and serological data? Epi-unit level

Turkey 2009

Median serological incidence

Turkey 2010

20%

W. AZB

33%

81% GISVet survey

Median report incidence

0.22%

2.8%

20%

Median relative incidence

30.8

11.8

8.2

3.7

Median incidence difference

20%

29%

69%

65%

100 60

serological incidence 40 60 80

20 0

No significant correlation (Spearman’s)

W. Azerbaijan serosurvey

serological incidence 70 80 90

100

Turkey 2010

report incidence

0.00

10.00

20.00 report incidence

30.00

40.00

Differences: Serological and Report data May be due to: 1. Under-reporting 2. Subclinical infection • FMD signs observed by farmers on: – 17% NSP+ epi-units in W. Azerbaijan – 41% NSP+ calves (0-12 months) in Asia-1 outbreak investigation in Turkey – 86% NSP+ calves (0-12 months) in Asia-1 outbreak investigation in Turkey

• Will vary by FMD strain, species infected, vaccination status

Differences: Serological and Report data May be due to: 3. NSP antibody induced by vaccine rather than infection • •

Especially if use unpurified vaccine Less likely if youngstock targeted in survey (?)

4. Reflection of previous year’s cases? • Compared Turkish serosurvey results to previous year’s reports and correlation not improved

Both approaches contribute to assessing and monitoring FMD risk • Reports of suspect clinical cases – Essential for early detection & response (PCP Stage 3 and higher) – Real-time, linked to control – Laboratory confirmation including serotype – Cost-effective – Effectiveness dependent on favourable ‘attitude’ to reporting – Subject to bias, under-reporting

Egypt

• Nationwide serosurvey in 2011 – 5299 ruminant samples tested in 310 villages

• NSP-Ab positive: – 17.6% samples (95%CI: 16.6 – 18.6%) – 78% villages (95% CI: 73-82%)

• 2011: 15 reports of suspect disease

350

300

250

200

150

30 20

100

Nile delta

Central

Upper

% villages with >=2 seropositive

East % cattle

West % buffalo

Total

Nr villages tested

number villages sampled

% positive

Egypt 2011: Large ruminant results in serosurvey

Both approaches contribute to assessing and monitoring FMD risk • NSP serology – Detects clinical and subclinical FMD (ie measures FMD infection) – Less biased (with careful design!) • Unit of analysis: what is a ‘case’? • Target young stock – Combine with questionnaire about risk factors – Resource intensive • Survey can be combined with post-vaccination monitoring (SP serosurvey)

Thank you! •EuFMD •FMD Institute, Ankara, Turkey •Institute for Animal Health, Pirbright, UK •Animal Health Research Institute, Cairo, Egypt •General Organization for Veterinary Services, Cairo, Egypt •General Directorate for Food and Control, Ankara, Turkey •Iranian Veterinary Organization, Tehran, Iran

Appendix 88 Background – FMD outbreak: the basic facts  1st case 3rd August 2007 (IP1)  Confirmed strain O1BFS 1860  Origin Pirbright site (lab and vaccine plant)  Further case in 1st cluster (IP2)  10 km control/protection zone – no further cases  2nd cluster – first case confirmed 12th September 2007 (IP3)  Subsequent cases – IP3, 4, 5, 6, 7 & 8

UK Foot & Mouth Disease 2007 – A case-control study during an outbreak Kate Sharpe Head of Field Epidemiology AHVLA

M4 WINDSOR

HEATHROW

6b 7

EGHAM

4b 5

X 3a

3d X 3eX

Pirbright site 2b 1b

ALDERSHOT

WOKING X3g X

M25

GUILDFORD

GODALMING 1a X

Defra Nobel House 15th July 2008

Objectives

Conclusions  Successfully carried out a case control study during exotic disease outbreak (limiting recall bias)  IP1 – flooding may have contributed to entry of virus; potential source for all farms in phase 1  IP5 – evidence consistent with it being a pIP – genome sequencing suggests it was a sIP – how did FMD get there?  pIP had relatively more young cattle present– curiosity? Teething?  sIP – risk increased by:

 To determine whether any features of the infected farms (cases) were different to a sample of uninfected farms within the protection zone in the risk period (controls)  To identify any factors that were associated with: □ the risk of entry of FMD virus to infected premises □ susceptibility of livestock to infection, given exposure  To investigate factors that might have been associated with the risk of transmission between farms

Calvings Proximity to pIP Public roads Greater environmental score Greater biosecurity score

 No evidence that horses, other livestock, wildlife, footpaths or other enterprises impacted FMD risk

Questionnaire design

Farm selection

 Aim – to complete in about 1 hour  Questions defined after “brainstorm”– ideas gathered into themes: □ Livestock:

Cases  7/8 IPs

1. numbers, location, species, purpose, husbandry

Controls (22 in total)

□ Geography:

□ Phase 1 – 8 farms randomly selected from 29 potential

1. location of fields, streams, roads etc plus weather

controls □ Phase 2 – all farms (18) in PZ that met control definition

□ Intercurrent disease or health issues:

1. Damage to skin or mucosal surfaces or immunosuppressive

□ Entry to fields or parts of the farm with

 Participation was voluntary

livestock

1. Eg vehicles, farm staff, feed, visitors

□ Features that increased human or vehicle 5

traffic

□ Eg farm shop, horse livery, footpaths

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 88

Questionnaire design cont.

Other data  Maps used for visual validation of questions □ Fields with woodland, streams, footpaths, roads  Data from HSE on high-risk lorry routes to IP1 & IP2 provided (but same detail not available for all farms)  Straight-line distance from closest margin of a field with susceptible livestock to nearest road & nearest high-risk road  Farms classified as “primary” (infection from Pirbright) or “secondary” (from another IP)  Distance & direction from nearest IP  Distance & direction from Pirbright  Any connection between land parcel via footpaths or watercourses  Elevation

□ Presence of wildlife 1. Badgers, foxes, deer etc – resident/ transitory □ Biosecurity 1. State of fencing & buildings, access to stock, builders etc □ Land parcels 1. Inc shared grazing, rented land, field work (eg repair fences)

□ Unusual events – people, wildlife, material □ Eg trespassing, camping, large flocks of geese, fly-tipping

Analysis

Key observations

 Descriptive analysis

Weather  Flooding (affected IP1, one sIP and 4 controls)

□ Each variable from each farm considered independently

 Univariable analysis

□ Each variable considered for evidence of association with outcome (pIP vs control, sIP vs control)

 Livestock age Secondary IPs

Primary IPs

Multivariable analysis

2% 5%

13%

□ Definition of composite variable ‘entry risk’, biosecurity risk’

Control farms

13%

39%

44%

‘environmental risk’

Primary v secondary IPs

41%

45%

85%

□ IP1 primary □ IPs 2,3, 4 and 6-8 secondary □ IP5 at time of study field evidence suggested primary.

Cows

Calves

Youngstock

Other

pIPs had proportionally more cattle <=18 months old (85% v 44%)

Sequencing later indicated secondary. Analysed according to each assumption 9

Relation of sIPs to pIP’s On sIPs, calving was associated with an increased risk of FMD (p=0.002)

Number of calvings

Mean

Range

sIPs

12/ farm

0-41

Controls

0.77/ farm

0-12

 sIPs North of pIPs  Controls mainly South or East of pIPs

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 88

Entry risk

Biosecurity risk

 Farm vehicle risk

 Sum of risks derived from the following variables: □ Open daytime or no gate at entrance: risk = 1 □ No visitor/staff changing area: risk=1 □ No visitor car park away from animal area: risk=1 □ No physical barrier to animal area: risk=1 □ No signs at main entrance: risk=1 □ No boot dip provided: risk=1 □ Free running dogs with access to livestock areas: risk =1 □ Unlocked gates in boundary: risk =1 □ Co-grazing susceptible animals with other herds: risk=1 □ Presence of any other enterprises: risk= 1 □ Experiences/allowing unusual events on premise: risk=1 □ Providing regulars with no farm specific clothing: risk=0.5 □ Providing regulars with no farm specific footwear: risk = 0.5  ASSOCIATED with FMD risk (p=0.030)

□ (# regular vehicles entered per risk-day) + (# feed

deliveries per risk-day) + (# machines entered per riskday) – (daily vehicle washing/10)

 Farm people risk

□ (# of people regularly entering per risk-day) + (# of visitors with animal contact per risk-day)

 Farm animal risk

□ (# of animals moved onto premise per risk-day)

 NO evidence of association with FMD risk 13

Environmental risk

Multivariable model

 Sum of risks derived from the following variables: □ No woodland or copse on field: risk=1 □ Public road adjacent to livestock field: risk =1 □ High risk road adjacent to livestock field: risk=1 □ Parking areas next to livestock fields: risk=1 □ Flooding during risk period: risk=1 □ Surface water running through field: risk =1 □ Public foot paths through fields: risk=1 □ Animals grazing adjacent to susceptible livestock fields: risk = 1 □ Distance to the Pirbright Laboratory: risk = 1/distance (km) □ Distance to likeliest primary IP: risk = 1/distance (km) □ Distance to IP1: risk = 1/distance (km)  ASSOCIATED with FMD risk (p=0.063 all IPs & 0.022 re sIPs)

Composite Variable

Odds ratio (95% ci) p-value

Biosecurity risk

6.2 (1.2 – 32.0) 0.038

Environmental risk

3.2 (0.9 – 11.0) 0.073

Entry Risk

0.3 (0.1 – 1.4) 0.135

sIPs tended to be closer to (nearest) pIPs than controls were (3 km v 5 km; p = 0.084) Clean parking had particular impact on biosecurity – cf salmonella

Conclusions  Collection of systematic & standard data valuable  Maps invaluable  Study contributed positively to knowledge from outbreak & complemented other actions  If outbreak had continued, initial findings could have informed farmer advice (biosecurity risk) and prioritisation of surveillance visits (calving, young stock)

Calvings Proximity to pIP Public roads Greater environmental score Greater biosecurity score

 No evidence that horses, other livestock, wildlife, footpaths or other enterprises impacted FMD risk

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 88

Acknowledgements  AHVLA  Alex Cook, Joey Ellis-Iversen and Richard Smith,  Alan Hurst, Alan Shuttleworth and Maria Dominguez, Jane Gibbens, Giles Paiba, Andy Paterson, John Wilesmith, Helen Roberts and Charlie Byrne,  GIS team Jon Hicks & Justine Robilliard  All farmers who took part 19

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 89 the Eufmd Open Session 2012

Conclusion

The use of oral fluids from pigs for pre-clinical diagnosis and monitoring in an FMD emergency: current research and future directions

Z. Zhang*, and S. Alexandersen National Centres for Animal Disease, Canadian Food Inspection Agency, 1015 Arlington Street, Winnipeg, Manitoba, M3E 3M4, Canada

Why pre-clinical diagnosis of FMD •

FMD is highly contagious

•

Infected animals are infectious before appearance of clinical signs

The stages of FMDV infection

Reproduced from S Alexandersen,,M. Quan, C. Murphy, J. Knight, Z. Zhang 2003 Journal of Comparative Pathology, 129: 268– 282

The stages of FMDV infection

Pre-clinical diagnosis of FMD •

Preclinical RT-PCR screening was used during 2007 FMD outbreak in UK by Pirbright Institute

•

Based on sera Collection of blood samples is associated with intensive labour and time consuming • Invasively collect • Difficult to collect • Stress for people and animals

UK 2001

•

Impossible on large-scale

•

Not penside

Japan 2010

South Korea 2011

Reproduced from S Alexandersen,,M. Quan, C. Murphy, J. Knight, Z. Zhang 2003 Journal of Comparative Pathology, 129: 268– 282

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 89 Oral fluid as a diagnostic fluid

•

Advantages over samples of serum/tissues

•

Virus detection using oral fluids • •

Non-invasively collect Easy to collect, by individuals with limited training • Simple to collect: no special equipment needed • Reduced stress for people and animals • Scalability

•

In human

Increasing usage of oral fluid in human medicine and recent in veterinary medicine

• • • • •

•

In pigs (reviewed in Prickett et al 2012) • • • • • •

Known methods used to detect animal viruses in oral fluids Assays

More than twenty three viruses in oral fluids samples Herpes viruses (reviewed in Malamud et al 2011). HIV Human Papillomavirus (HPV) Influenza virus Poliovirus Hepatitis B virus (Portilho et al 2012) rubella virus (Domonova et al 2012) measles (Hutse et al 2010) Vesicular stomatitis virus (Stallknecht 1999) FMDV (Alexandersen et al 2003; Zhang et al 2004) Porcine respiratory reproductive syndrome virus (Prickett et al 2008) Circovirus (Prickett et al 2008) Swine Influenza A Virus (Romagosa et al 2011; Richt et al 2012) ASF

Comparison of FMDV loads in different samples from pigs

Viruses

Virus isolation

FMDV, VSV

Ag ELISA

FMDV

Real-time RT-PCR

PPRV, FMDV, SIV, PCV2,SVD

IV for Vesicular stomatitis virus (Stallknecht 1999) RT-PCR for FMDV (Alexandersen et al 2003; Zhang et al 2004) RT-PCR for PPRSV (Prickett et al 2008) • RT-PCR for PV2 (Prickett et al 2008) • RT-PCR for SIV (Romagosa et al 2011; Richt et al 2012) • • •

S Alexandersen, M. Quan, C. Murphy, J. Knight, Z. Zhang 2003 Journal of Comparative Pathology, 129: 268–282

Comparison of FMDV loads in different samples from pigs • A longer detection

window in oral fluids compared to blood • Oral fluid reflect levels of

FMD virus detected in serum/blood

• Similar to blood, viral RNA

detected at preclinical stage (1dpi)

• Oral fluids can be used for

early diagnosis

Challenges • Assay optimization

• RT-PCR • Extraction • PCR protocols • Unknown inhibitor in the oral fluid matrix • Ag ELISA and penside test • Inadequate sensitivity

S Alexandersen, M. Quan, C. Murphy, J. Knight, Z. Zhang 2003 Journal of Comparative Pathology, 129: 268–282

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 89 Challenges

•

Conclusion

Collection of oral fluids

Inconsistent oral fluid Elution buffer volume • Individual vs pen-based oral fluid •

http://www.offlu.net

• • •

Individual based: Salivette, cotton buds Pen-based • Cotton rope with FMDV in pigs (Vosloo et al

2012, EUFMD )

• Cotton rope has been successfully used to

detect PPRSV, SIV and PCV2 • Bait with swabs: wild boars (Khomento et al 2012, EUFMD) • SIV pen-based oral fluids for SIV to 80% with a possibility of detection ranging from 99% when the pen prevalence was higher than 18% (Romagosa et al 2011)

http://www.farminguk.com/news

Pre-clinical viral presence and a longer detection window support that oral fluid-based testing could facilitate rapid detection of FMD • However, there are still some technical issues to be resolved before its full potential diagnostic value could be reached, including inconsistent oral fluid and elution buffer volume, variable virus recoveries and as yet inadequate assay sensitivity for oral fluid testing •

Acknowledgements •

Eion Ryan, FAO

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 90

Conclusions and Recommendations

Foot-and-Mouth Disease Virus Transboundary Movements Between Sub-Saharan Africa, North Africa and the Middle East Nick J. Knowles, Begoña Valdazo-González, Jemma Wadsworth, Antonello Di Nardo, Valerie Mioulet, Jef M. Hammond and Donald P. King

• Increased trade and political/social upheaval have probably increased the transboundary transmission of FMD at the interfaces between virus pools. • Recommend continued (and expanded) monitoring of viruses (sampling & sequencing) both in ‘at-risk’ countries and endemic countries from which viruses originate.

Introduction

Materials & Methods

• Foot-and-mouth disease (FMD) viruses are frequently geographically restricted, both at the serotype and topotype level, and thus transboundary spread of virus can be traced using VP1 sequence information.

•

FMD viruses, isolated in cell cultures from clinical samples received by the FAO World Reference Laboratory for FMD, were subjected to RT-PCR of the VP1-coding region using previously described methods.

•

The resultant amplicons were sequenced using an ABI 3730 Automated Sequencer.

•

VP1 sequences were assembled using SeqMan Pro 10 (DNAStar Inc.) and phylogenetic trees were constructed using MEGA 5.05 software.

• We have examined the recent and historical spread of FMD viruses between sub-Saharan Africa, North Africa and the Middle East to assess current and future threats.

Europe (SA) to North Africa

Middle East to North Africa

1977 1979 1981 1983

1972 - type O – Libya 1972 – type O – Egypt 1987 – type O – Egypt 1988 (-1989) – type O – Libya 1989 (-1992) – type O – Tunisia / Algeria / Morocco 1993 – type O – Egypt 1994 – type O – Tunisia / Libya 2006 (-2011) – type O PanAsia-2 - Egypt 2009 – type A Iran-05BAR-08 – Libya 2010 (-2011) – type A Iran-05BAR-08 – Egypt

– type A - South America (via Europe?) to Morocco / Algeria – type A5 - Europe to Libya / Tunisia (-1982) – type O1 – Europe to Libya – type A5 – Europe (Spain?) to Morocco

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 90 Sub-Saharan Africa to North Africa 1972 1974 1999 2003 2006 2012 2012 2012 2012 2012 2012

Sub-Saharan Africa to Middle East • 1962 (-1965) – type SAT 1 – Bahrain (Israel, Lebanon, Jordan, Syria, Turkey, Iran, Iraq, Greece) • 1970 – type SAT 1 – Saudi Arabia & Kuwait • 1984 – type SAT 1 VI - Yemen • 1985 – type A G-II - Yemen • 1990 – type SAT 2 IV – Yemen • 2012 – type SAT 2 VII – Palestinian AT • 2012 – type SAT 2 IV - Bahrain

– type A – Egypt – type O EA-1 – Egypt – type O WA - Algeria, Morocco & Tunisia – type SAT 2 VII Lib-03 - Libya (-2009) – type A G-VII - Egypt – type SAT 2 VII Lib-12 - Libya – type SAT 2 VII Gbh-12 & Alx-12 Egypt – type SAT 2 VII Lib-12 - Libya – type A G-IV – Egypt – type O EA-3 - Libya – type O EA-3 - Egypt

Africa to Yemen AR FMDV O East Africa 3 topotype 1974 1977 1982-1983 1985-1987 1989-1990 1992 1995 2003-2004 2006 2008-2009

A/LIB/93/2009

O/YEM/5/2009 O/YEM/21/2009 O/YEM/16/2009 O/YEM/18/2009 O/YEM/19/2009 O/YEM/20/2009 O/YEM/17/2009 O/YEM/22/2009 O/YEM/35/2009 O/YEM/30/2009 O/YEM/43/2009 O/YEM/44/2009 O/YEM/45/2009 O/YEM/46/2009 O/YEM/8/2009 O/YEM/6/2009 O/YEM/7/2009 O/YEM/11/2009 O/YEM/57/2009 O/YEM/58/2009 O/YEM/59/2009 O/YEM/41/2009 O/YEM/53/2009 O/YEM/42/2009 O/YEM/56/2009 O/YEM/63/2009 O/YEM/64/2009 O/YEM/29/2009 O/YEM/9/2009 O/YEM/12/2009 O/ETH/26/2009 O/ETH/24/2009 O/ETH/25/2009 O/ETH/27/2009 O/ETH/28/2009 O/ETH/9/2009 O/ETH/10/2009 O/ETH/6/2009 O/ETH/7/2009 O/ETH/1/2009 O/ETH/3/2009 O/ETH/4/2009 O/ETH/8/2009 O/ETH/11/2009 O/ETH/5/2009 O/YEM/21/2004 O/YEM/66/2004 O/YEM/28/2004 O/YEM/35/2004 O/ETH/54/2006 O/YEM/57/2004 O/ETH/38/2005 (2003/4) O/ETH/3/2004 O/YEM/70/2004 O/ETH/48/2005 O/ETH/61/2005 O/ETH/49/2005 O/ETH/53/2005 O/ETH/62/2005 O/ETH/51/2005 O/ETH/52/2005 O/NYE/10/87 O/NYE/9/90 O/ETH/57/2005 O/ETH/67/2005 O/ETH/54/2005 O/ETH/66/2005 O/ETH/55/2005 O/ETH/56/2005 O/ETH/65/2005 O/ETH/63/2005 O/ETH/64/2005 O/ETH/1/2005 O/ETH/29/2008 O/ETH/1/2007 O/ETH/27/2007 O/ETH/28/2007 O/ETH/26/2007 O/ETH/24/2008 O/ETH/25/2008 O/ETH/20/2008 O/ETH/21/2008 O/ETH/13/2008 O/ETH/15/2008 O/ETH/19/2008 O/YEM/6/83 O/ETH/48/2006 O/ETH/46/2006 O/ETH/62/2006 O/YEM/4/95 O/SOM/1/2007 O/SOM/2/2007 O/SOM/4/2007 O/YEM/22/2003 O/YEM/28/2003 O/YEM/1/2004 O/YEM/48/2004 O/YEM/3/2006 O/YEM/4/2006 O/YEM/29/2006 O/YEM/36/2009 O/YEM/37/2009 O/YEM/4/2008 O/YEM/7/2008 O/YEM/5/2008 O/YEM/8/2008 O/YEM/6/2008 O/ETH/31/2008 O/ETH/32/2008 O/YEM/23/2009 O/YEM/24/2009 O/YEM/10/2008 O/YEM/34/2009 O/K101/80* O/K8/84* O/K22/82* O/K201/83* O/ETH/11/2005 (1996) O/ETH/1/79 (1977)(AY283376)

▲ Yemen ▲ Ethiopia ▲ Somalia

A/LIB/94/2009

A/CAR/14/2000

FMDV A

100

A/LIB/92/2009

A/CAR/15/2000 (EF208755)

A/LIB/91/2009 A/LIB/57/2009

A/CAR/13/2000

A/LIB/56/2009 A/LIB/54/2009

A/CAR/12/2000

A/LIB/51/2009 A/LIB/50/2009

A/CAR/P19/2000 (mfa/057/01)

A/LIB/48/2009 A/LIB/47/2009

A/TOG/9/2005 A/MAI/4/2004

A/LIB/44/2009 A/LIB/43/2009 A/LIB/42/2009 A/LIB/21/2009

A/MAI/12/2006

100

A/LIB/18/2009 A/LIB/17/2009

A/NIG/36/2009

A/LIB/16/2009

A/NIG/38/2009

100

A/LIB/15/2009 A/LIB/14/2009

A/NIG/39/2009

A/LIB/13/2009 A/LIB/117/2009

A/NIG/3/2009

A/LIB/11/2009 A/LIB/10/2009

A/ERI/2/98

100

A/LIB/9/2009 A/LIB/5/2009

A/ERI/3/98

100

A/LIB/4/2009 A/LIB/3/2009

A/ERI/3/97

A/LIB/2/2009

A/CAR/P22/2000 (fke/066/08)

from SubSaharan Africa

A/CAR/36/2005 A/CAR/115/2005

87 100

A/LIB/1/2009 A/ISR/1/2009

A/KUW/5/2009 A/ISR/5/2009

A/IRQ/9/2009 A/IRQ/10/2009 A/IRQ/12/2009 A/BAR/6/2008 A/ISR/3/2009

A/ERI/1/2006 77

A/SUD/1/2006 (GU566069)

A/IRQ/19/2009

A/ERI/5/2006

A/IRQ/17/2009

A/IRQ/15/2009 A/IRQ/21/2009 A/ISR/7/2009

A/ERI/16/2009 A/ERI/4/2007

A/IRN/28/2009 A/KUW/6/2009

A/ERI/1/2008 A/SUD/3/77 (GU566064) A/SUD/2/84 (GU566067)

A/SUD/1/85 (GU566068)

G-II

G-I G-III G-VI G-V

81 89

A/EGY/9/2011 A/EGY-12-2011* A/EGY-14-2011* A/EGY/5/2011 97 A/EGY-8-2011* A/EGY/1/2010 A/EGY/2/2010

A/EGY/3/2010 A/EGY-2-2011* A/EGY-3-2011*

A/AFG/6/2007 A/AFG/10/2010 A/IRN/78/2009 A/IRN/125/2010 A/TUR/1/2008 99 87

A/IRN/1/2005 (EF208769) A/TUR/33/2008 A/IRN/9/2011 A/IRN/9/2010 A/TAI/118/87* (EF208777)

A23/Kitale/KEN/64 (AY593766)

99 98

A/TAI/7/2003 (HQ116312) A/TAI/2/97 (EF208778) A/IRN/22/99 (EF208772)

ASIA

EA-2

A/EGY/2/2011 A/EGY-5-2011*

EURO-SA

O/EGY/1/74 O/UGA/3/72 O/TAN/2/2004 O/MAL/1/98 (DQ165074) O/KEN/5/2002 (DQ165073) O/UGA/3/2002 (DQ165077)

A/EGY/8/2011 A/EGY-11-2011*

A15/Bangkok/TAI/60 (AY593755)

Sudan 1986-1989

A/KUW/4/2009

87 94

G-VII

A/ISR/16/2009

from the Middle East

A/SUD/1/81 (GU566065)

A/IRN/2/87 (EF208770) A22/IRQ/64 (AY593763) A/IRN/1/96 (EF208771)

O/YEM/8/85 O/SYE/1/86

A15/Bangkok/TAI/60 (AY593755)

O/YEM/8/95 O/YEM/15/98 O/YEM/3/98 O/YEM/6/98

EURO-SA AFRICA

0.02 O1/BFS 1860/UK/67 (J02185)

ASIA

A/IRQ/11/2009

A/ERI/5/2008

Iran-05

A/IRN/22/2009 A/IRN/29/2009

A/ERI/40/2009

98 99

BAR-08

A/ISR/2/2009 A/IRN/2/2009 A/IRN/21/2009

AFRICA

A/SUD/3/2006 (GU566070)

100

A/LIB/8/2009 A/LIB/19/2009

A/CAR/116/2005 A/EGY/1/2012 100 A/EGY-B-2012*

EA-3

A/PAT/6/2009

G-IV

AFG-07 HER-10 FAR-09 SIS-10 ARD-07 EZM-07 QAZ-11 ESF-10

Thai-87 Sea-97 Iran-99 Iran-87 A22 Iran-96 A15

EURO-SA

0.02

O/ISR/3/2007 (FJ561313) O/TUR/4/2007 O/JOR/6/2006 (FJ561317) O/TUR/28/2007

FMDV O

O/TUR/1/2007 O/TUR/3/2007

ME-SA

SAT 2

O/JOR/7/2006 (FJ561318)

O/SAU/1/2007 O/SAU/2/2007 94

SEA EA-2 EA-1

O/SAU/4/2007 O/SAU/5/2007 O/JOR/5/2006

O/TUR/16/2007 O/TUR/15/2007

EA-4

O/TUR/30/2007 O/IRN/20/2007 O/IRN/20/2006

O/ETH/3/2004 (FJ798109)

O/IRN/6/2007

O/ETH/1/2007 (FJ798137)

O/IRN/23/2006 O/IRN/1/2007

O/ETH/2/2006 (FJ798127)

O/TUR/731/2007* (FMDI) O/IRN/17/2006

O/ERI/21/2004 O/ERI/2/2004

O/IRN/8/2006

O/ERI/1/2004

O/IRN/9/2006

O/ERI/3/2004 85 75

O/IRN/10/2006 O/IRN/35/2006

O/SUD/3/89 (1987)(GU566038)

O/SUD/2/89 (GU566037) O/ETH/3/95

O/ETH/26/2011 O/ETH/59/2011

70 99

O/IRN/88/2009

99 96

O/SUD/25/2004 (GU566053)

O/SUD/3/2004 (GU566046)

Irn-2001 Ind-2001 Pak-98

O/SUD/16/2004 (GU566052)

O/IND/R2/75* (AF204276)

O/SUD/14/2004 (GU566050)

O1/Manisa/TUR/69 (AY593823) 84

O/SUD/1/2004 (GU566045)

81 81

TER-08 SAN-09 FAR-09 BAL-09

from the Middle East

74 93 100

100

O/SUD/26/2004 (GU566054)

CATHAY ISA-1 ISA-2 EURO-SA

100 100 78 74

SEA EA-2 EA-3 EA-1 WA EA-4 CATHAY ISA-1 100 ISA-2 EURO-SA

PUN-10

III

SAT2/KEN/1/84 (K7/84) (AY344505) SAT2/ETH/1/90 (1989)(AY343935)

PanAsia 100

SAT2/ETH/2/90 (1989)(AY343936)

100

O/IND/53/79 (AF292107)

O/SUD/4/2004 (GU566047)

O/IRN/31/2009 O/IRN/18/2010 O/UKG/35/2001 (AJ539141)

O/SUD/9/2004 (GU566048)

O/SUD/15/2004 (GU566051)

O/EGY/7/2011

O/TUR/264/2009* (FMDI)

O/PAK/16/2010

O/SUD/30/2004 (GU566055)

O/EGY/10/2011 O/EGY/6/2011

100

O/TUR/257/2008* (FMDI) 98

O/SUD/12/2004 (GU566049)

ANT-10

O/EQ57/EGY/2009 (AHRI) 96

O/SUD/3/2005 (GU566058)

O/IRN/53/2006

O/IRN/34/2006 (RJS-1) O/IRN/34/2006 (RJS-2)

O/SUD/1/2005 (GU566056) O/SUD/2/2005 (GU566057) O/NIG/1/2007

99 98

O/IRN/29/2006 O/IRN/43/2006

O/NIG/15/2009

O/SUD/8/2008 (GU566063) 98

O/IRN/10/2007

O/SUD/1/99 (DQ165076)

SAT2/UGA/MBF-4/2002 (buffalo)(FJ461346)

O/PAK/5/2007 O/IRN/18/2007 O/IRN/45/2006

O/SUD/4/99 (GU566044)

Ghb-12

SAT2/EGY/5/2012

XIV XIII VIII IX XII X XI V

O/PAK/53/2007 (FJ798179)

O/IRN/50/2006

O/SUD/3/99 (GU566043) 99

VII

SAT2/EGY/4/2012

O/IRN/11/2006

O/SUD/6/2008 (GU566062)

Alx-12

SAT2/PAT/1/2012

O/PAK/3/2007 75

O/SUD/4/2008 (GU566060)

Lib-12

SAT2/EGY/15/2012

O/PAK/10/2006 (EF494503) O/PAK/Lahore vaccine (EU244455)

EA-3

O/LIB/54/2012 O/SUD/3/2008 (GU566059)

O/SUD/5/2008 (GU566061)

SAT2/EGY/10/2012

O/PAK/6/2006 (EF494501)

O/ETH/28/2011 99

SAT2/EGY/14/2012 SAT2/EGY/6/2012

O/PAK/8/2006 (EF494502)

O/ETH/3/96 (EU919240)

100

ME-SA

O/PAK/4/2006 (EF494500)

O/ETH/3/96 (HM211079)

SAT2/LIB/41/2012

SAT2/EGY/3/2012 SAT2/EGY/13/2012

O/PAK/16/2006

O/ETH/30/94 (HM211078)

SAT2/LIB/40/2012

SAT2/EGY/11/2012

O/PAK/8/2006 O/PAK/10/2006

O/SUD/2/86 (DQ165075)

100

SAT2/EGY/2/2012 SAT2/EGY/9/2012

PanAsia-2

O/IRN/8/2005 O/PAK/4/2006 O/PAK/6/2006

O/SUD/7/89 (GU566042)

O/PAK/9/2006

O/SUD/6/89 (1988)(GU566041)

100

SAT2/SUD/1/2007 (GU566071) SAT2/LIB/39/2012

100

O/IRN/56/2006

O/SUD/4/89 (1987)(GU566039) O/SUD/5/89 (1987)(GU566040)

87 86

Lib-03

SAT2/CAR/8/2005 SAT2/NIG/2/2007

PAT

O/IRN/12/2006 O/IRN/19/2006

O/ERI/22/2004

SAT2/LIB/7/2003

100

Egypt

O/IRN/24/2007

O/YEM/6/83

SAT2/ERI/12/98 (AF367126)

Libya

O/TUR/18/2007

O/NYE/9/90 O/YEM/4/95

SAT2/ERI/4/98 (AY343934) SAT2/CAR/P12/2000 (VDI 44/1)(HM211082) SAT2/LIB/1/2003 SAT2/CAR/1/2005

O/IRN/51/2006

O/YEM/3/74 O/YEM/1/82

SAT2/ERI/1/98 (AY343933)

SAT2/SAU/6/2000 (AF367135) 100

O/IRN/39/2006 O/TUR/14/2007

O/ETH/1/79 (1977)(HM211077)

100 99

O/TUR/23/2007

O/ETH/11/2005 (1996)(FJ798107)

from SubSaharan Africa

O/SAU/3/2007

SAT2/KEN/11/2009

Bahrain

SAT2/KEN/13/2009

SAT2/KEN/122/2009 SAT2/BAR/12/2012

100

SAT2/BAR/16/2012 100

Ken-09

SAT2/BAR/10/2012 SAT2/BAR/13/2012 SAT2/BAR/28/2012

0.02

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain

Appendix 90 SAT2/NIG/3/2008

SAT2/ERI/1/98 (AY343933)

SAT2/NIG/2/2008 SAT2/NIG/1/2008

SAT2/ERI/12/98 (AF367126)

SAT2/NIG/4/2008 SAT2/NIG/2/2007 (JX570636)

SAT2/SAU/6/2000 (AF367135) SAT2/LIB/1/2003 SAT2/LIB/7/2003

100

SAT2/NIG/5/2008 SAT2/NIG/6/2008

Lib-03

100

SAT2/LIB/39/2012 (JX570633) SAT2/LIB/40/2012 (JX570634)

100

SAT2/LIB/40/2012 SAT2/LIB/41/2012

SAT2/EGY/2/2012 SAT2/EGY/9/2012

Lib-12 Alx-12

SAT2/CAR/104/2005 SAT2/CAR/105/2005 SAT2/CAR/8/2005 (JX570616) SAT2/CAR/58/2005

VII

SAT2/CAR/95/2005 SAT2/CAR/38/2005 SAT2/CAR/39/2005 SAT2/CAR/1/2005 (JX570615)

SAT2/EGY/11/2012

SAT2/CAR/119/2005

SAT2/EGY/3/2012 SAT2/EGY/13/2012

100 100

SAT2/EGY/14/2012 SAT2/EGY/6/2012 SAT2/EGY/10/2012

SAT2/LIB/1/2003 (JX570631) SAT2/LIB/7/2003 (JX570632)

Ghb-12

SAT2/ERI/12/98 (AF367126) SAT2/ERI/1/98 (AY343933)

SAT2/EGY/4/2012

SAT2/ERI/4/98 (AY343934)

SAT2/EGY/5/2012

SAT2/EGY/2/2012 (JX570617) SAT2/SUD/4/2010

SAT2/UGA/MBF-4/2002 (buffalo)(FJ461346)

VIII IX XII X XI V

74 93 100

SAT2/EGY/3/2012 (JX570618) SAT2/EGY/26/2012* (JX013979) SAT2/PAT/1/2012 (JX570637) SAT2/EGY/7/2012* (JX013978) SAT2/EGY/15/2012 (JX570627)

SAT2/EGY/23/2012* (JX013980)

SAT2/EGY/10/2012 (JX570623) SAT2/EGY/4/2012 (JX570619) SAT2/EGY/5/2012 (JX570620)

SAT2/KEN/13/2009

XIV XIII

SAT2/KEN/122/2009 SAT2/BAR/12/2012

100

SAT2/BAR/16/2012 100

Ghb-12

SAT2/EGY/6/2012 (JX570621) SAT2/EGY/14/2012 (JX570626)

SAT2/KEN/11/2009

Middle East to Libya O PanAsia-2ANT-10 A ASIA Iran-05BAR-08

SAT2/EGY/13/2012 (JX570625)

SAT2/ETH/2/90 (1989)(AY343936)

100

Alx-12

SAT2/EGY/11/2012 (JX570624)

SAT2/KEN/1/84 (K7/84) (AY344505) SAT2/ETH/1/90 (1989)(AY343935)

100

Sub-Saharan Africa to Egypt O EA-3 A SAT 2 VII Ghb-12 & Alx-12

SAT2/EGY/9/2012 (JX570622)

III

SAT2/CAR/P12/2000 (VDI 44/1)(HM211082)

XIV XIII

VII

SAT2/SEN/27/2009 SAT2/SAU/6/2000 (AF367135)

SAT2/PAT/1/2012

Lib-03

SAT2/NGR/15/2005

SAT2/EGY/15/2012

Lib-12

SAT2/LIB/41/2012 (JX570635)

SAT2/SUD/1/2007 (GU566071) SAT2/LIB/39/2012

Sub-Saharan Africa to Libya O EA-3 SAT 2 VII Lib-12

SAT2/NIG/7/2008 SAT2/SUD/1/2007 (GU566071)

SAT2/CAR/1/2005 SAT2/CAR/8/2005 SAT2/NIG/2/2007

100

Recent Virus Introductions

SAT2/NIG/8/2008

SAT2/ERI/4/98 (AY343934) SAT2/CAR/P12/2000 (VDI 44/1)(HM211082)

O, SAT2

100 99

O, A, SAT2

SAT 2

VIII IX XII X XI V

Ken-09

SAT2/BAR/10/2012 SAT2/BAR/13/2012 SAT2/BAR/28/2012

Middle East to Egypt O PanAsia-2ANT-10 A ASIA Iran-05BAR-08

SAT2/UGA/MBF-4/2002 (buffalo)(FJ461346)

1000 km

III IV

Conclusions

Conclusions and Recommendations

• Until recently the spread of FMD viruses into North Africa from sub-Saharan countries has been a relatively rare event. However, in 2012, examples of trans-Saharan FMD virus movements have been identified involving three serotypes, O, A and SAT 2 (three lineages).

• Increased trade and political/social upheaval have probably increased the transboundary transmission of FMD at the interfaces between virus pools.

• In the same period movement of FMD virus type SAT 2 from East Africa to Bahrain was also detected.

• Recommend continued (and expanded) monitoring of viruses (sampling & sequencing’) both in ‘at-risk’ countries and endemic countries from which viruses originate.

Acknowledgements Claudia Doel Miki Madi Nigel Ferris (retired) Geoff Hutchings (retired) Keith Sumption Kees van Maanan Egypt

World Organisation for Animal Health

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Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain