Open session of the standing technical committee of the EUFMD- 2014 by EuFMD

Where science and policy meet: FMD RISK MANAGEMENT

in a world of changing disease landscapes Open sessio oft 1e S anding Technical and Research Com 11ittees ofthe EuF �D Cavta (Croatia) 29-31 october 2014

Food nd Agriculture Organi tion of the United ations

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OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Contents DAY 1 – PLENARY: The changing disease landscape and its implications ………………………………… 9

Session I: Opening Research advances over the last ten years (W.Vosloo) State of FMD research review (T. Knight Jones) The pressures affecting the current and emerging disease landscape (H.J. Ormel)

Session II: FMD trends global landscape: global challenge Changing pathways: Lessons from recent pathogen migrations for FMD risk assessment (S. Alexandersen) Update on current global situation for FMD: New outbreaks and threats (D. King) Changing landscape for livestock production in Europe; directions and expected change in the next 20-30 years (includes wildlife issues) (A. Mottet) Changing landscape for livestock production and health in China and neighbouring countries; directions and implications for FMD management (J. Edwards)

Session III: The changing landscape for FMD management Contribution of the Food and Veterinary Office of the European Commission to reinforce FMD import risk management measures, and animal disease emergency preparedness and early warnings systems in the EU (F. J. Pérez Pérez) Prospects for FMD control (K. Sumption) Engaging livestock keepers as actors in animal health (A. Cameron) Control of FMD and other major infectious transboundary diseases towards better integration of control programmes (J. Domenech) Available upon request

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Session IV: The management landscape: looking ahead, human creativity and new business Business and Livelihoods in African livestock: A twin-track approach to livestock development (S. Nuala) Foot and Mouth Disease Continuity of Business Planning for the U.S. Dairy Industry (P.J. Hullinger)

DAY 1 – PARALLEL: The changing disease landscape and its implications …………………………… 86 Session P1: Virology and vaccinology Quantifying and predicting antigenic relationships: A comparison of two alternative approaches investigated using data from FMDV and influenza A (W. Harvey) The introduction of positively-charged residues at the five-fold axis of the FMD virus SAT-type capsid enhances infection of cultured cells (M. Chitray) A Thiazepino[4,5-a]Benzimidazole derivative inhibits the in vitro replication of Eurasian serotypes of FMD virus (D.J. Lefebvre) Identification of FMDV strain in multivalent vaccines by using Loop Mediated Isothermal amplification (M. Fiorucci) Identification of novel antibody binding determinants of serotype O FMDV (M.Mahapatra ) Establishment of a persistent FMD virus infection in MDBK cells (L.Kopliku, S.Blaise-Boisseau) Comparative utility of the fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for virus isolation from clinical samples collected from animals experimentally infected with a foot-and-mouth disease virus (K.Fukai et al)

Session P2: Diagnostic methods (1)- antigen/antibody Results of the 2013 Proficiency Testing Scheme (Anna B. Ludi) Development and validation of multiple non-structural protein antibody tests to confirm FMD infection in vaccinated animals (K.Parekh) Ready-to-use ELISA kits for antibody to FMDV SAT1 and SAT (E. Brochi) Validation of VNTs for all FMDV serotypes to determine sero-prevalence in cattle in Eritrea (A. Dekker) Development of Lateral Flow Assay for antigen detection and serotyping of FMDV (K. Morioka et al.)

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Development and Comparisons of Various ELISA for the Diagnosis of Foot and Mouth Disease Virus (FMDV) ( S. Bose, S. Aman, M. Ali) Available upon request Diagnostic observations with IZSLER Antigen ELISA kits for detection and serotyping of FMDV serotype O, A, SAT1 and SAT2 in several African countries (K. van Maanen) Available upon request New competitive ELISAs for detection of non-structural or structural FMDV antibodies (L. Comtet) Available upon request

DAY 2 – PLENARY: The FMD science and policy development landscape …………………………… 125

Session V: FMD risk management in free countries (Pillar I) – risk assessment and epidemiology Risk analysis framework to compare the importance of the different source regions for entry of FMDV into Europe (M. McLaws) The enhanced passive surveillance system: a solution supporting data collection, integration and analysis for disease surveillance (L.Holstrom) Quantitative risk assesment evaluating the transmission of FMD via fresh deboned beef produced froman endemic region (G.T. Fosgate) Available upon request Modelling FMD transmission in a feral pig-domestic cattle ecosystem (M. Ward) Transmission and survival of FMDV on environmental fomites (E.Brown) Sero-epidemiological Study of Foot-and-mouth disease in livestock in Tripoli, Lybia (A. Dayhum) Available upon request FMD in Lybia and the Control Strategy (I. Eldaghayes) Available upon request

Session VI: FMD risk management in free countries (Pillar I) Resource estimations in contingency planning for FMD (S. Mortensen) Maximising efficiency with a surveillance strategy for FMD during an outbreak in a previously FMD-free country (K. Walker) Evaluating vaccination strategies to control FMD: A model comparison study (C. Cook) Impact of stakeholders influence, geographic level and risk perception on strategic decisions in simulated FMD epizootics in France (M. Marsot) An adaptive management approach to foot-and-mouth disease control (M.J. Tildesley) 3

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Session VII: GFRA led session (1): vaccine stability Thermofluor analysis of the FMDV capsid and the effects of different solutions on stability (J. Seago) Available upon request A method based on the use of specific llama antibodies for quality control testing of FMD vaccines (E. Pérez Martín) Evaluation of the immune responses of Nguni cattle vaccinated with foot- and-mouth disease virus stabilised SAT2 antigens (K. Scott) Demonstration of a high potency SAT2 vaccine in cattle and confirmation of efficacy in pigs against virulent challenge (L. Mouton)

Session VIII: GFRA led session (2): FMD ecology Looking Forward from FMD Epidemiology to FMD Ecology (R. Garabed) Mechanisms of persistence of FMDV in African buffalo populations: A briefing on current work at Kruger National Park (A. Jolles) Available upon request Foot-and Mouth Disease Ecological Studies In Endemic Settings: Ongoing Studies in Vietnam and Pakistan (J. Arzt, L. Rodríguez) An Emergent Strain Of Foot-And-Mouth Disease Virus, Serotype Sat 3, Isolated From A Long-Horned Ankole Calf In The Queen Elizabeth National Park In Uganda (G.J. Belsham) Available upon request Recovery of Viral RNA and Infectious Foot-And-Mouth Disease Virus from Positive Lateral-Flow Devices (V.L. Fowler) Available upon request

DAY 2 – PARALLEL: The FMD science and policy development landscape …………………………… 196

Session P3: Progressive control of FMD: technical developments and issues for non-free countries (Pillar II) Outbreaks of FMDV in Libya and Saudi Arabia during 2013 due to an exotic O/ME-SA/IND-2001 Lineage (K. Bachanek- Bankowska) Mass vaccination, immunity and coverage: Modelling population protection against FMD in Turkish cattle (T.J.D. Knight-Jones) Modelling endemic FMD in Turkey (P.M. Dawson) FMD health situation in Tunisia (H. A. Heni)

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Serological survey in Libya to asses FMD viruses circulation and vaccine immune response (G. Ferrari)

Session P4: Progressive control of FMD: technical developments and issues for non-free countries (Pillar II) Promoting a risk-based strategy plan for FMD control in endemically infected countries: Development and examples from 4 countries (C.J.M. Bartels) Serosurveillance and the PCP-FMD: Why are serosurveys useful, how are they being used and what are the gaps? (M.McLaws) ELITE: An electronic laboratory information tracking environment for supporting the Progressive Control of FMD in Pakistan (K. Biggers) Knowledge and perceptions of communal farmers concerning FMD at the wildlife/livestock interface of the Kruger National Park (D. D. Lazarus)

Section P5: PCP-FMD Asia SEAC FMD Roadmap: A risk-based approach to FMD control in SE Asia and China (R.C. Abila) Epidemiological investigation of Foot and Mouth Disease incidences in southern peninsular India during 2013 (G. K. Sharma) Using risk assesment to inform FMD policy in Mongolia (M.E. Schuppers) Pig, cattle and buffalo value and social network analysis in Xayabury province of LAO PDR (J. Hinrichs) Isolation and identification of FMDV types and its sequence analysis on the basis of P1 (Capsid protein gene) in Pakistan (U. Waheed)

DAY 3 – PLENARY: FMD future: responding to change in the global landscape …………..……… 274

Section IX: Impact and Africa Global landscape of FMD control (S. Metwally)

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

What do we know about the economic impact of FMD in smallholder production? - Summary of the evidence (J. Rushton) Impact of FMD on milk yield, mastitis and culling on a large-scale dairy farm in Kenya (N.A. Lyons) Household level impacts of FMD on traditional livestock-keeping systems of Northern Tanzania (M. Casey) Emerging massive FMDV outbreaks in Uganda and possible impact on PCP (A. Chrisostom) Sero-survey to understand transmission pathways for foot-and-mouth disease spread between and within different regions of Kenya (B. Kibore)

Section X: Immunisation and vaccines Vaccine evaluation on large-scale dairy farms using routine prophylactic schedules for FMD (N.A. Lyons) Influence of the colostrum in the immune responses of calves to current FMD vaccines (A. V. Capozzo) Serotype O vaccine efficacy and challenge with different viruses from South East Asia in various species (V. Vosloo) Simultaneous immunization of cattle with FMD and live anthrax vaccines (A. Capozzo) Early protection in sheep against heterologous challenge with Serotype O Foot-and-mouth disease virus using high potency vaccine ( J.Horsington et al) Overview on the performance of FMD vaccines in South America: a manufacturer perspective (O. Mozzer)

Section XI: The future of vaccination Developing technologies to track the adaptive immune response following FMD infection or vaccination of livestock (T. Golde ) Available upon request Novel vaccine strategies for the control of FMD in Africa (F.F. Maree) Available upon request Recombinant adenovirus expressing empty capsid of serotype A22 FMDV provides sterile immunity in cattle following homologous prime-boost vaccination (S. Parida) Available upon request Defining requirements and execution of international field trials for next generation FMD vaccines and diagnostics (M. Colby) 6

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Follicular dendritic cells: a key player in the pathogenesis of Foot-and-mouth disease virus (M.H.A Doudo) Development of a Novel, Recombinant, Potent, and Safe DIVA Vaccine for Foot-and-Mouth Disease Virus (FMDV) (M.Ali, S.Aman, D.Raghavan) Vaccine development, challenges and strategies for FMD control in Indian subcontinent (S.N. Singh) Available upon request Session XII: Final keynote, conclusions and closing Lessons from delivery and impact assessment of human vaccination programmes: the GAVI experience (S. Malvolti) Available upon request

DAY 3 – PARALLEL: FMD future: responding to change in the global landscape …………..……… 335

Session P6: Diagnostics (2): Molecular diagnostic developments Development of probe-based real time RT-PCR assays for detection and serotyping of FMDVs circulating in West EurAsia (S.M.Jamal) Development and evaluation of a multiplex conventional RT-PCR for simultaneous detection and typing of FMDV in West Africa (K. Górna (Blaise- Boisseau)) Development of tailored specific real-time RT-PCR assays for detection of FMDV serotypes A, O, SAT 1 and SAT 2 circulating in East Africa (K. Bachanek- Bankowska) Real-time RT-PCR for the rapid detection of FMDV in milk (B. Armson) Development and evaluation of multiplex reverse transcription loop mediated isothermal amplification assays combined with lateral-flow visualisation for the discrimination of FMD from other vesicular diseases (V. L. Fowler) Realising the potential of simple isothermal molecular tools for field diagnosis of FMD (E. Howson)

Session P7: Transmission and FMD diagnosis in non-African wildlife From sequence to prevalence: Phylodynamics of FMDV (A. Di Nardo)

OPEN SESSION OF THE STANDING TECHNICAL AND RESEARCH COMMITTEES (EUFMD)

CAVTAT, 29-31 OCTOBER 2014

Beyond the consensus: Investigating intra-herd variability of FMDV using the Illumina Miseq (D.J. King) Probability of infection of cattle, sheep and pigs exposed to FMD virus aerosols (J.L. Gonzales) A novel protocol to generate consensus level genome sequences for FMD virus and its application to sequencing a large outbreak (G. Freimanis) Non-invasive sampling systems for the detection of FMDV in wild boar (S. Mouchantat)

Session P8: Understanding FMDV diversity in Africa Evolution of FMDV during persistence in African buffalo (M. Cortey) Genetic characterization of circulating FMD viruses from African buffalo (Syncerus caffer) and cattle in Kenya: Evidence for independent virus populations (S Wekesa) Multiple FMDV serotypes identified in Uganda during 2010-2013 (K. Tjørnehøj) Identification of novel genotypes of FMDV recovered from African buffalo in Marromeu, Mozambique (C.J. Kasanga) Emergence of antigenic variants of SAT2 FMD viruses at the wildlife/livestock interface in South Africa (B. Blignaut)

Please note the Book of Abstracts is available online and as a separate document on the EuFMD website.

LE AR he changing disease ̀ landscape and its iplications ̀

Historical events during the last 116 years

Research advances over the last 10 years Wilna Vosloo

Principal Research Scientist, CSIRO-Australian Animal Health Laboratory, Geelong, Australia

Introduction

Frequently listed as the most economically important disease of livestock in developed and developing countries Little data on socio-economic impact in endemic countries Global impact due to production losses and vaccination in endemic countries US$6.5 21 billion per year*

FMDV was the first animal virus to be described by Loeffler and Frosch in 1898 116 years later

Impact on free countries enormous

Only 2 viruses eradicated: small pox and rinderpest Many gaps in our understanding and control of FMDV

Outbreaks in free countries >US$1.5 billion per year* Australia AUD50 billion over ten years**

Vaccines Pathogenesis Immunology Carrier state, etc

*Knight-Jones and Rushton, 2013 **Buetre, 2013

Assigned incidence and prevalence index for cattle (Sumption et al., 2008) 4

Major developments over the last 10 years

More rational approach to control by dividing the world into virus pools PCP and OIE endorsement of control plans

Different categories of freedom Countries/zones free without vaccination Countries/zones free with vaccination Endemic countries/zones Compartmentalisation Commodity based trade (?)

Emphasis on regional collaboration Improved advice on vaccines and reagents for diagnostics

Vaccination now accepted as general component of response to incursions in free countries General acceptance of NSP tests to prove freedom from infection, but with limitations 5

Understanding the virus

Epidemiology and control

Pathogenesis Initial replication occurs in the nasopharynx, followed by expansion in the lung and systemic viraemia Local Type I and III interferon response at the primary sites of infection Virulence determinants at the point of infection

Improved data to feed into models both from field epidemiology and laboratory studies Relationship between onset of clinical signs and the FMD transmission window in cattle The infectious period is ~ 1.7 days The mean latent period 4.6 days The mean incubation period 4.1 days

Virus-intrinsic factors, host defence mechanisms and route of exposure Arzt et al., 2011

Charleston et al 2011 7

Epidemiology and control

Diagnostics Automation of testing

Identified predictors of clinical signs (virus present in the OPF, blood or nasal fluid - above a measured threshold) Transmission is strongly associated with detectable levels of virus in the air - air-borne spread not a major route of transmission

Real-time RT-PCR made testing of clinical samples in large numbers possible Sample type Epithelial suspension Tissue suspension Vesicular fluid Fluid Serum EDTA -blood Probang Swab Faecal suspension Total samples tested % of samples tested

Chase-Topping et al 2013

Number rRT-PCR submitted 50 50 1 2 3 3115 32 36 6 1 3246

1 1 3 3086 32 36 6 1 3216 99.1

AgELISA 49

1 1 1 52 1.6

1 1 585 32 36 3 1 709 21.8

Diagnostics

Diagnostics Full genome sequencing

Retrospective analysis of UK 2001 outbreak - no virus circulation on 23% of farms deemed infected and culled

Improvements in sequencing technologies and protocols Development of computational phylogenetics Ability to sequence the full genome routinely in real time Improvement in resolution between closely related viruses Reconstruct the likely origins and transmission pathways Substantiate the temporal dynamics of an outbreak, point out possible missed premises

2007 outbreak in the UK One holding associated with a case, no clinical signs evident 19/58 animals rRT-PCR positive blood samples, confirmed by VI Indicated near simultaneous infection on multiple animals First use of real-time preclinical diagnosis for FMD in the field Intensively Patrolled Areas (IPA), reduced un-necessary slaughter

Valdazo-Gonzalez et al 2012 12

Novel Ad5-FMD Vaccine

Diagnostics Next generation sequencing

A novel FMD vaccine was developed by ARS-USDA at the PIADC Utilizes a defective human adenovirus vector to deliver genes coding for FMDV structural proteins First US licensed recombinant FMD vaccine Efficacy shown against FMD A(s), O(s), Asia 1, SAT 1, SAT 2 and SAT 3

Detect minority sequence variants Explore within-host virus evolution and selection High-frequency polymorphisms at specific sites Intermediate stages in evolution Gradual change in major variant and overtaking of another under selective pressure

Substantially advancing knowledge of FMDV population dynamics within the animal

Human Defective Adenovirus 5 vector - Lacks necessary proteins for growth - Delivers and expresses transgenes in target cells

M. Grubman Wright et al 2011 13

Implications of Ad5-FMD vaccines

addressed by Ad5-FMD What does this vaccines add for preparedness?

Possibility of domestic production in free countries Rapid response to new strains Opened regulatory path to other recombinant vaccines

Effective, rapid (5-7 days) and long-lasting (at least 6 months) protection with one dose Prevents viral transmission Allow easy differentiation of infected from vaccinated animals Safe: produced without live FMDV No need for adaptation of field strains to cell culture Prevent development of carrier state Broad antigenic coverage Stable antigen long shelf life (stable for >2 yrs at 4oC)

Implications to global control and eradication

Potential application in final stages of eradication?

Work in progress

Lower dose, cost

Vaccines

Conclusions

Current developments

New technologies have facilitated significant advances

In vitro synthesis of empty capsids (VLPs) Decrease in 3C toxicity Stabilising capsids to exposure to heat and low pH Mutations to enhance cell culture adaptation (lysine at VP1100)

Increased understanding of virus-host interactions Improved diagnostics More accurate modelling Rational design of improved vaccines New registered FMD vaccine

New attitudes/approaches have been beneficial

New policy acceptance based on improved knowledge Global, concerted effort to control FMD

Despite large investment, many gaps remain Porta et al 2013 17

Acknowledgements Lucy Robinson and Theo Knight-Jones (GFRA global report 2014) Don King, Pirbright Institute

State of FMD research

GFRA ExCo

Theo Knight-Jones

Luis Rodriguez Bryan Charleston Cyril Gay Francois Maree

Epidemiologist, ILRI

Lucy Robinson

Senior Partner, Insight Editing London

FMD Research historical breakthroughs

Our ability to control a disease is constrained by the tools available

Our ability to control a disease is restrained by the tools available Progress accelerates and stagnates as breakthroughs are made Control FMD where control not previously possible Disease control

Same level of control but more efficient (time, cost)Diagnostics

-DIVA methods -Phylogenetics & typing Increased certainty in disease/infection status (trade, rapid detection) -Immune correlates

Immunisation

Increased understanding Veterinary services

Epidemiology -Passive -Strategies -Vaccines -Mass produced vaccines -Modelling -Improved & stable vaccines

-Coordinated control

Global control with current technologies

Countries with outbreaks in free zones Jan 2005-Aug 2014

FMD research review - approach

FMD research needs

Literature review FMD publications 2011-14

Free countries

Institute updates on current research

Preparedness, early detection and control

Free and endemic with vaccination

Contacted >50 FMD research institutes (GFRA/EuFMD)

Vaccine immunity - duration, match

Response from 33 Europe 10 (30%) North America 5 (15%) South America 2 (6%) Asia 6 (3) (18%) Africa 8 (24%) Australia/New Zealand 2 (6%)

Vaccine stability, safety, strategy & evaluation Proving freedom

Limited control activities

Review by GFRA experts

Impact estimation Feasibility of FMD control effectiveness, cost, convenience/compliance 6

Peer-reviewed publications June 2011-14 Research Category Epidemiology

Papers (n) 116 (23%)

Report: Key findings Vaccines The ideal FMD vaccine

Half modelling studies

Vaccines

94 (19%)

Pathogenesis

74 (15%)

Molecular biology

57 (11%)

Prevents viral transmission & carrier state

Diagnostics

55 (11%)

Immunology

50 (10%)

Allow differentiation of infected from vaccinated animals (DIVA)

Effective, rapid and long-lasting protection with one inoculation

Policy, preparedness and trade

38 (8%)

Produced without the need for virulent FMDV

Wildlife

19 (4%)

Protection against multiple serotypes

Vaccine evaluation (quality, efficacy, effectiveness)

16 (3%)

Other

15 (3%)

Economics

14 (3%)

Biotherapeutics

13 (3%)

Total

505 (100%)

Stable antigen long shelf life Reasonable cost to enable eradication programs

Economic studies needed by those with least resources

Gay et al. 2013 8

Vaccines

Diagnostics

Progress

Empty capsid vaccines

Faster, cheaper, simpler diagnostics

Adenovirus vector, Baculovirus expression system

RT-LAMP, high-speed, portable PCR, sequencing Simple pen-side tests

Inactivated molecular chimeric vaccine Early stages but:

More powerful molecular techniques Next generation sequencing Tools for molecular epidemiology

Produced without live FMDV Improved DIVA Improved stability

Better understanding of within-host virus evolution, between animal transmission, population level virus ecology

Gaps

Gaps Cost Duration and breadth of immunity important for endemic settings African vaccine strains- SAT 2 vaccines Lack of standardised approach to novel vaccine evaluation

Continued progress in rapid/simple diagnostics & molecular techniques Vaccine matching beyond small scale, imprecise antibody assays Wildlife diagnostics & sampling (under investigation)

Disease management

EuFMD 3 Pillars: Research needs

Progress

1. Improve readiness for FMD crisis management by Members

Modelling approaches developed & widely used Importance of field evaluation of control programmes Alternatives to stamping out [vaccinate-to-live] Incentivisation through commodity-based trade

Vaccine matching tests & measures of cross-protective immunity Further development of modelling approaches for policy guidance Improved, cross-protective, DIVA vaccines

2. Reduce risk to Members of an FMD incursion from the neighbourhood Cheaper vaccines with long lasting, broad spectrum immunity Capacity building technical skills, regional collaboration, vaccine quality assurance Improved knowledge and monitoring of wildlife and illegal trade

Gaps Evaluation & validation of models Practicalities of vaccinate-to-live Safety & acceptance of commodity-based trade Strategies & guidance for endemic countries

3. Promote the global FMD control strategy Cheaper vaccines with long lasting, broad spectrum immunity [SAT 2, African strains] Can FMD be controlled with current vaccines without effective biosecurity? Economic impact of FMD & when are control efforts cost-effective Evaluation of commodity based trade as a safe way of exporting from wildlife FMD endemic areas

Knowledge sharing South America

Progress in genetic and molecular technologies will benefit all 13

EuFMD 3 Pillars: Research needs

1. Improve readiness for FMD crisis management by Members

Vaccine matching tests & measures of cross-protective immunity Further development of modelling approaches for policy guidance Improved, cross-protective, DIVA vaccines

2. Reduce risk to Members of an FMD incursion from the neighbourhood

Cheaper vaccines with long lasting, broad spectrum immunity Capacity building technical skills, regional collaboration, vaccine quality assurance Improved knowledge and monitoring of wildlife and illegal trade

3. Promote the global FMD control strategy

Cheaper vaccines with long lasting, broad spectrum immunity [SAT 2, African strains] Can FMD be controlled with current vaccines without effective biosecurity? Economic impact of FMD & when are control efforts cost-effective Evaluation of commodity based trade as a safe way of exporting from wildlife FMD endemic areas

Cheaper vaccines with long lasting, broad spectrum immunity Can FMD be controlled with current vaccines without effective biosecurity? Economic impact of FMD & when are control efforts cost-effective Evaluation of commodity based trade as a safe way of exporting from wildlife FMD endemic areas

Progress in genetic and molecular technologies will benefit all

Progress in genetic and molecular technologies will benefit all 14

EuFMD 3 Pillars: Research needs

Conclusion

1. Improve readiness for FMD crisis management by Members Vaccine matching tests & measures of cross-protective immunity Further development of modelling approaches for policy guidance Improved, cross-protective, DIVA vaccines

3. Promote the global FMD control strategy Cheaper vaccines with long lasting, broad spectrum immunity Can FMD be controlled with current vaccines without effective biosecurity? Economic impact of FMD & when are control efforts cost-effective Evaluation of commodity based trade as a safe way of exporting from wildlife FMD endemic areas

Progress in genetic and molecular technologies is crucial & will benefit all 16

Acknowledgements GFRA Exec. Committee: Bryan Charleston, Wilna Vosloo, Luis Rodriguez, Cyril Gay. All institutions & scientists that provided input. EuFMD [funding and support]

A changing world and changing disease landscapes J. Lubroth and H.J. Ormel

Questions and Focus Why and how pathogens of animal origin have become a major global public health threat and what might be done about it? Disease dynamics at the human-animalecosystems interface and how to curb these.

a changing world

DISEASE

PRESSURE

STATE - RESPONSE

ANALYSIS FRAMEWORK

Pressure

MITIGATION State

ADAPTATION

Response

Pressure1: rise of the world population 9 billion people in 2050

Consumption of livestock products is growing rapidly...

Pressure 2: change in geo-politics

Eggs

Meat

Milk Cereals Roots and tubers 1960

1970

1980

1990

Roots and tubers Meat Eggs Per caput consumption of major food items in developing countries

Source: FAO-SOFA 2009

2000

2010

Cereals Milk

kg per caput per year (index numbers 1961=100)

Pressure 3: Climate change

Pressure 4: Urbanization

Pressure 5: economic growth remains uneven

Pressure 6: an ever more interconnected

world POULTRY

CDL - Drivers

DISEASE

PRESSURE

STATE - RESPONSE

ANALYSIS FRAMEWORK

Four distinct driver-disease complexes: 1. the poverty-related endemic disease burdens in humans and livestock.

Pressure

MITIGATION State

Response

ADAPTATION

POVERTY

CDL - Drivers

ENDEMIC DISEASE

Adopting a health-in-development perspective, with emphasis on selfhelp safety practices at the grassroot level Addressing health, food and income security in conjunction with the other Sustainable Development Goals

2. the biosafety challenges posed by globalization and climate change.

Pressure Deficient or absent Sanitation infrastructure Poverty

State

Endemic disease burden in humans and Livestock Constraining health and well being of the poor (and livestock)

Restoring malfunctioning health systems: clinics,vaccinations, parasiticides , etc.

Response

GLOBALIZATION AND CLIMATE CHANGE

CDL - Drivers

ADDRESSING THE DRIVERS OF DISEASE EMERGENCE AND SPREAD

Develop policies and strategies aimed at mitigating health and biosecurity risks associated with travel, trade and traffic Mitigate GHG emissions through healthier and more productive livestock

Pressure

State

International collaboration to establish a virtual radar screen on microbial dynamics and hazards at the human-animal-ecosystem interface

3. the food and agriculture-related public health threats.

Globalization and Climate Change

Global redistribution of pathogens, vectors and hosts; evolution of new forms of disease

Response

CDL - Drivers

STATE

disease

Number of infections

4. Risks in species jumps of disease agents from wildlife to livestock and humans.

time

RESPONSE

to the

STATE

RESPONSE

to the

STATE

time

IF WE DO NOT REACT:

time

DISEASE IMPACTS INCREASE SPILLOVER TO OTHER SPECIES HIGHER LOSSES DECREASED CONSUMER CONFIDENCE TRADE RESTRICTIONS MORE HUNGER AND MORE POVERTY

disease

PANDEMIC

Number of infections

EARLY WARNING EARLY REACTION

disease

Number of infections

disease

Number of infections

time

SHIFT TO THE LEFT

J Newcomb 2005

Agriculture and Consumer Protection Department December 2013

Changing Pathways: Lessons from Recent Pathogen Migrations for FMD Risk Assessment Executive Director, Prof. Soren Alexandersen National Centres for Animal Disease (NCAD) Winnipeg and Lethbridge Laboratories Canadian Food Inspection Agency 30 October 2014

NCFAD established in 1998 in Winnipeg in the Canadian Science Centre for Human and Animal Health as a partner with the Public Health Agency of Canada (PHAC) One Facility: Two Labs together: One Health

UNIQUE FACILITY with both human and animal CL 4 labs

NCAD = L+W

Only CL 4 labs in Canada National Centres for Animal Disease NCAD: Lethbridge Laboratory + National Centre for Foreign Animal Disease (NCFAD)-Winnipeg

A unique approach that encourages collaboration and provides better science and policy 2

National Centres for Animal Disease (NCAD) NCFAD-Winnipeg and Lethbridge Laboratory of the Canadian Food Inspection Agency (CFIA)

Containment Level 2 - 25% Containment Level 4 - 5% Can handle all FAD, zoonotic and emerging infections

INDIRECT CONTACT Traditionally: looked mainly at potential international/transboundary transmission as happening by specific (biological) products and a few things like vectors or boots or clothing

CL 3+/ag - 70 % Staff of ~ 65 + facility Expertise: virology, mol biol, pathology, immunology/serology and reagents

LETHBRIDGE LABORATORY ~35 staff + facility. Expertise: BSE, TSE, Anthrax, endemic viruses, CL3 large animals etc

Now 2010

ANYTHING by ANY route Amount and complexity are increasing and contact to susceptible species by all possible routes very complex

Transport and travel are increasing Farming changing/intensifying Improbable are changing to possible! 5

Canadian Examples FMD Saskatchewan 1952 Foreign worker/on purpose??

25 February 2001

Rabbit Hemorrhagic Disease A single pet rabbit in apartment Winnipeg 2011

pH1N1 2009 into swine in Alberta Likely from a returning foreign worker H5N2 AIV into poultry in Manitoba 2010 close match to circulating wild bird sequences

Also anaplasmosis in Manitoba in 2010 and in Ontario and Manitoba in 2013

How???

BTV 11 BC 2013 Likely culicoides (wind)

FMD UK 2007 escape from site faulty effluent lines? 6

EHDV 2 Alberta Deer crossing 7

Danish Example

PORCINE EPIDEMIC DIARRHEA VIRUS (PEDV) IN THE USA AND THEN IN CANADA An Alphacoronavirus, first seen in the UK in 1971, then other parts of Europe and later Asia. Affects only swine; not zoonotic - maybe originated from bats! From late April 2013, Iowa State University (ISU) got samples from farms with diarrhea/vomiting in all ages and 90 95% mortality in suckling piglets. Severe atrophy of villi in the small intestines, rota- and TGE virus negative; Coronavirus-like particles by EM, pan-coronavirus RT-PCR positive. Sequencing: 99.6 100% identity among RT-PCR products from first 4 farms, highest identity (>99%) to PEDV strains from China in 2012. NVSL: confirmed using 3 nested PCRs; close to 2012 China strains.

International Examples Schmallenberg virus MERS-CoV

Whole genome sequences of virus from 2 farms in 2 states had 99.5% identity with 2011 2012 PEDV China strains.

Culicoides?

The nearly simultaneous outbreaks and high degree of homology (99.6 100%) between strains from unrelated farms, suggested a common source of virus.

Dromedary camels???

BSE

MBM 8

Source into USA still not identified

but source into Canada is! 9

NCFAD preparedness:

Heads-up from NVSL-Ames on 17 May 2013 Work closely with NVSL and our CAHSN labs Set up conventional RT-PCRs (NVSL but modified primers) and partial sequencing AHL-Guelph develop real time RT-PCR which we all test for suitability Get positive RNA from ISU and an European virus isolate (BR1/87) and positive serum from helpful Danish colleagues grow virus in Vero cells and ship positive RNA to several labs Set up serology by IFA and an ELISA using mabs from The Netherlands.

From: Status Update on Swine Coronaviruses Recently Identified in US Swine 2-27-2014 (Iowa State University)

Canada (Ontario) case 1 Late on 22 Jan 2014 heads up from AHL-Guelph receive samples at NCFAD on 23 Jan late afternoon

Huang, Dickerman, Piñeyro et al. mBio 4(5):doi:10.1128/mBio.00737-13. Origin, Evolution, and Genotyping of Porcine Epidemic Diarrhea Virus Strains in the United States 2013.

Initial European strain from 70ties

Report preliminary positive rRT-PCR before midnight (within 8 hours) Report positive conventional RT-PCR results (3 targets) on 24 Jan at noon (within 20 hours)

from late 2013/ early 2014

Plus swine deltacoronavirus from early 2014

Report confirmatory sequences on 25 Jan early morning - consistent with initial USA strain (within 40 hours)

Reported to the OIE as emerging infection by USA, Japan and Canada on 21, 24 and 26 of April 2014, respectively

Subsequent full S gene sequenced (4.2 kb) and virus isolated!

Further testing/confirmation done at NCFAD

Direct testing on Ontario plasma and feed (PEDV real time and small S gene RT-PCR)

Ontario cases 2-11 (partial S sequence 14 Feb) Manitoba case 1 (13, 14 and 15 Feb) PEI case 1 (14, 14 and 18 Feb) Quebec case 1 (26, 26 and 27 Feb)

5 plasma samples all weak positives (Ct 36.2-36.97) Also positive in S gene RT-PCR and sequence as initial USA strain 3 plasma samples for bioassay. Only 1/5 feed samples had a dubious reaction in the rRT-PCR (Ct 42.88). This sample had a dubious reaction (wrong size) in conventional S RT-PCR while a third sample was clearly positive. The clearly positive sample and the real time RT-PCR reactive sample selected for bioassay The one feed sample S amplicon sequence is PEDV but not sufficient for good/quality sequence

Follow up epidemiological investigation on the first 11 Ontario cases and the PEI case pointed to feed and one lot of spray dried porcine plasma from the USA as a common risk factor Ontario feed & dried plasma RT-PCR POSITIVE BIOASSAY Other feed and feed ingredients mainly negative, a very few considered non-negative/dubious 12

BIOASSAY 1 Clinical observations

BIOASSAY 1 and BIOASSAY 2

No clinical signs observed in negative controls. Positive control group: depression, off feed and diarrhea/soft faeces from 1 day after inoculation; temperatures within normal range. Plasma group: a few piglets with diarrhea/soft faeces from day 2; temperatures within normal range. Feed group: mildly depressed and one or two piglets with diarrhea/soft faeces from day 1 and decreased feed intake day 3-7; temperatures within normal range. Such mild signs may be caused by many things can only be related to a specific cause by testing and should not be used for any conclusions!

BIOASSAY 1 (started 14 Feb 2014) 40 piglets, 10 positive control, 10 negative control, 3 x 4 piglets for plasma and 2 x 4 piglets for feed 3-4 piglets as contacts from day 7 Given a 10% suspension 25 ml by gastric tube and 25 ml orally RESULTS TO BE DISCUSSED BIOASSAY 2 (started 21 Feb) 60 piglets, 12 negative controls 3 x 8 piglets given feed as above and 1 x 8 piglets also fed this feed for two more days 4 contact piglets introduced from day 2 BIOASSAY 2 ALL NEGATIVE FOR 7 DAYS 14

S gene RT-PCR and amplicon sequence on BIOASSAY 1 rectal swabs at day 3

BIOASSAY 1 rRT-PCR on rectal swabs

All 12 plasma inoculated piglets strongly positive sequence similar to the initial USA strain One feed inoculated piglet (35) had very weak band of right size - also PEDV sequence but poor quality. Cloned and sequenced: 1/ 8 clones PEDV sequence like plasma piglets. A few others had weak bands of a different size unspecific amplification unrelated to PEDV

Piglet number, Ct (Y axis) and day (X axis). Contact piglets from day 7 as black. Negative as Ct 45. All piglets at beginning of trial, all controls and all contact piglets at day 7 tested negative.

All feed inoculated piglets NEGATIVE (BUT!) - also negative in Bioassay 2. 16

Positive control (5 days post contact)

Manitoba case

Plasma feed group (7 days post inoculation)

Negative control

Plasma direct

Ontario Case 1 Ontario case

Plasma direct Quebec case

Colorado Iowa 1 Plasma piglets Ontario case

PEI Plasma piglets

Ontario cases

Plasma piglet Minnesota Indiana

SK environmental Second USA strain

Carissa Embury-Hyatt

Conclusions!

Additional evidence Electron microscopy

Plasma piglet d7

This imported lot of spray-dried porcine plasma contains infectious PEDV:

Pos cont contact d5

Virus excretion for many days by rRT-PCR and S gene sequence typical for the USA/Canada PEDV strain Lynn Burton

Serology

Spread to contact piglets on day 7 & seroconversion

clear seroconversion in plasma inoculated and positive control piglets!

Positive Control Group

Tested feed is non-conclusive/not possible to determine as infectious by laboratory trial (Bioassay):

Spray Dried Plasma Group

Weak positive by RT-PCR, but variable results and PEDV sequence obtained not of good quality A single piglet weak positive band day 3 sequence weak but typical PEDV of the same sequence Repeat (Bioassay 2) using more piglets and contacts: all negative but feed sample Done using a monoclonal antibodies-based ELISA to detect antibodies to the

PEDV spike (S) protein and supported by immunofluorescence assay (IFA) 20

However, strong indication that PEDV was introduced into Canada by contaminated porcine plasma in feed! 21

Swine/Porcine deltacoronavirus (SDCV/PDCV) detected in the USA

+MB 19 & 24 Sept week 35/36

SDCV/PDCV initially described by Hong Kong scientists in 2012 based on sequence findings (not disease) related to Asian Leopard Cat and Sparrow deltacoronavirus *30 *9 April May

*13 June

Detected in the USA by Ohio State University in early 2014 and by AHL-Guelph in March 2014 in samples from 6 premises in Ontario.

*21 & 30 July 2nd strain

We have confirmed SDCV in 2 of their first submissions using rRT-PCR and a pan-CoV RT-PCR followed by sequencing.

*Jan 2014

Speculation that this virus may also have been in feed: We tested feed and plasma samples received including those positive for PEDV: All clearly negative for SDCV in our test. All 40 piglets from Bioassay 1 also negative for SDCV. 22

CSHIN 12, 24 June, 21, 30 July 2014

SUMMARY

Acknowledgements

Constant risk of introduction of new or emerging infections Importance and impacts may be both health and economic Economic impacts can be from production losses, treatment and control costs, loss of international market access or loss of domestic consumer confidence Effects are highly inter-connected and inter-dependant and global events can quickly come close to home Consider: Movement from A to ..Z of ANYTHING by ANY route (spray dried milk powder next???)

I thank all staff at NCAD for their contributions and dedication and other staff at CFIA and at NML for productive collaboration. Also Thank: NVSL&ISU-Ames, LindholmDenmark, Lelystad-Netherlands, CAHSN labs (in particular AHL-Guelph), Biovet and many others

It is important for everyone to have optimal: Awareness, bio-security and a good veterinarianclient relationship and excellent laboratory services.

Thanks for listening! 24

Update on current global situation for FMD: New outbreaks and threats Donald King (donald.king@pirbright.ac.uk) WRLFMD Team: Valerie Mioulet, Nick Knowles, Anna Ludi, Ginette Wilsden, Bryony Armson, Pip Hamblin, Kasia Bachanek-Bankowska, Kelly Adams, Jemma Wadsworth, Begoña Valdazo-González, Britta Wood, Barsha Thapa, Bob Statham, Abid Bin-Tarif, Ashley Gray, Emma Fishbourne, Beth Johns, Debbie Gibson, Trish Ryder, Sarah Belgrave. 26

Recent submissions to WRLFMD Pirbright

Recent FMD outbreaks (Jan 2013-Sept 2014):

October 2012 October 2014

28 countries have submitted samples during 2014 SAT 2 and O North Africa and West Eurasia

No reported clinical outbreaks during 2013/14 Last report 2012 in Paraguay

East-West Transboundary movements

Israel

Multiple lineages East Asia

Outbreaks reported to the OIE (change of epidemiological status): http://www.oie.int/wahid-prod/public.php?page=home

PAT

Clinical samples

South Korea Bhutan

Viral sequences

Nepal Trinidad

Chinese Taipei Hong Kong SAR

Sri Lanka

Samples being processed: Vietnam Rwanda Mozambique Zimbabwe Botswana

Reports for these samples can be found at www.wrlfmd.org

North Africa: Changing patterns:

Serotypes recovered

serotype SAT 2 in Egypt during 2012

100

During 2014: 544 samples generating 335 Isolates Additional 37 samples positive by rRT-PCR

Camer oo n 20 05

S AT2/LIB/40/201 2 100

First FMD cases in Egypt due to serotype SAT 2 since 1950 Sequence data supported at least two introductions of SAT 2 FMDV into Egypt

O A Asia 1 SAT 1 SAT 2

S AT2/LIB/41/201 2 S AT2/LIB/39/201 2

S AT2/SUD/1 /2007 (GU5 66071) SAT2/NIG/2/200 7 97

SAT2/NIG/5/200 8 SAT2/NIG/6/200 8 89

100

SAT2/NIG/7/200 8 S AT2/NIG/4/200 8 SAT2/NIG/1/200 8

SAT2/NIG/2/200 8 SAT2/NIG/3/200 8 SAT2/NIG/8/200 8

100

S AT2/LIB/1/2003 S AT2/LIB/7/2003 S AT2/NG R/15/2005

SAT2/SEN/27/2009 SAT2/EG Y/2 /2012 SAT2/SAU/6/2000 (AF36 7135)

S AT2/CAR/P12 /2000 (VDI 44 /1)(HM2 11082)

Within SAT 2 topotype VII

S AT2/ERI/1 2/98 (AF3 67126) S AT2/ERI/1 /98 (AY3 43933)

100 100

S AT2/ERI/4 /98 (AY3 43934)

S AT2/EG Y/9 /2012 100

S AT2/EG Y/1 1/2012 S AT2/EG Y/1 3/2012

Distinct from other contemporary SAT 2 lineages in the Middle East and North Africa

S AT2/EG Y/6 /2012 S AT2/EG Y/1 0/2012 S AT2/EG Y/1 4/2012 SAT2/EG Y/1 5/2012 S AT2/EG Y/3 /2012 S AT2/EG Y/4 /2012 S AT2/EG Y/5 /2012

0.01

topotype VII from Libya topotype IV from Bahrain

No serotype C since 2004 Serotype-specific rRT-PCR assays have been used recently for VI negative samples from Tanzania

SAT 2 (Alx-12) detected in Egypt during 2014 In partnership with the Animal Health Research Institute, Egypt

North Africa: Changing patterns:

Continued spread of O/ME-SA/Ind-2001

2013/14: rapid spread of O/ME-SA/Ind-2001

Tunisia and Algeria

O/BHU/1/2013 O/SAU/1/2013 O/SAU/4/2013 O/SAU/3/2013 95 O/SAU/6/2013 O/SAU/7/2013 85 O/SAU/8/2013

Initial reports Libya in Sept/Oct 2013

Saudi Arabia in Aug/Nov 2013 UAE received during Jan 2014

O/ME-SA/Ind-2001d lineage most closely related to viruses from Indian sub-continent India dominant serotype O strain for 2012/3 Bhutan in 2013 Nepal in 2014 Sri Lanka in 2014

Tunisia: reported 29/04/14 (134 outbreaks) Algeria: reported 27/07/14 (418 outbreaks) Closely related to earlier viruses from Libya Increased threats to Morocco and Europe?

O/LIB/16/2013 O/LIB/1/2013 O/LIB/4/2013 O/LIB/5/2013 O/LIB/7/2013 O/LIB/11/2013 O/LIB/12/2013 O/LIB/22/2013 O/LIB/13/2013 O/LIB/17/2013 O/LIB/2/2013 O/LIB/3/2013 O/LIB/6/2013

Dr Ibrahim Eldaghayes

See presentations by: Bankowska, Heni, Ferrari, Dayhum, Eldaghayes Tomorrow - 09.00 hrs

Source: OIE (12/10/2014)

East Asia: continued outbreaks due to exotic FMDVs

Vaccine matching for O/ME-SA/Ind-2001

FMD outbreaks since 2008

22 field isolates tested by VNT

Vaccine strain Field Isolate LIB 1/2013 LIB 7/2013 LIB 17/2013 LIB 22/2013 NEP 13/2012 NEP 6/2012 NEP 21/2012 NEP 6/2013 NEP 18/2013 NEP 1/2014 NEP 6/2014 SAU 1/2013 SAU 4/2013 SAU 6/2013 SAU 7/2013 SAU 1/2014 SRL 1/2013 SRL 1/2014 UAE 1/2014 UAE 2/2014 ALG 1/2014 TUN 1/2014

O-3039

O Manisa

O BFS

O/TAW/98

Since 2009-2014 Spread of three FMD virus lineages from Southeast Asia: O/ME-SA/PanAsia China, Russia, Mongolia, Kazakhstan O/SEA/Mya-98 China, Japan, South Korea, North Korea, Russia, Mongolia, Taiwan A/ASIA/Sea-97 South Korea, China, Mongolia, Russia, Kazakhstan

O/TUR/5/09

nd nd

borderline borderline

borderline borderline borderline borderline

nd nd nd nd nd nd

Matched Not matched

borderline

nd nd nd nd nd nd nd nd borderline nd

borderline borderline

(Chin a, Japan , RoK

OIE: WAHID

borderline borderline

East Asia: O/SEA/Mya-98

East Asia: O/ME-SA/PanAsia

Recent detection in South Korea

Recent detection in the Russia Federation and Mongolia

O/SKR/1/2011 O/SKR/3/2011

New FMDV outbreaks in South Korea

Reported to OIE on 24/07/2014

85 80

Cases in pigs

FMD virus closely related to recent outbreaks in the Eastern part of the Russian Federation

75 82

O/HKN/15/2010

O/SKR/4/2010 (JQ070320) O/KOR/1/2010* (HM143846) O/GZ/CHA/2010 (JN998086)

86 98

O/VIT/NCVD-20/2010 (NVRQS) O/GZ-MT/CHA/2013 (KJ646655) O/Primorskiy/RUS/2014 (ARRIAH) O/SKR/6/2014 99 100 O/SKR/01/2014* (APQA)

100 92

O/VIT/12/2013 O/VIT/32/2013 O/VIT/33/2013 O/VIT/13/2013 O/VIT/15/2013 O/VIT/38/2013 O/VIT/46/2013

O/VIT/47/2013 O/VIT/51/2013 O/VIT/40/2013 O/MOG/4/2014 O/Zabaikalsky/RUS/2014* (ARRIAH) O/MOG/5/2014 O/MOG/2/2014 O/MOG/1/2014

Appear to be separate from sequences sent from China in 2011

O/NC/CHA/2010 (HQ652080) O/GSLX/CHA/2010 (JQ900581) O/VN/YB10/2010 (HQ260720) O/VIT/NCVD-8/2010 (NVRQS) O/VN/YB08/2010 (HQ260718) O/VIT/NCVD-9/2010 (NVRQS) O/VN/YB09/2010 (HQ260719) O/TAI/22/2009 (HQ116269) O/VIT/NCVD-19/2010 (NVRQS)

100

O/VIT/16/2013 O/VIT/39/2013 O/VIT/36/2013 86 O/VIT/11/2013

76 91

O/ME-SA/PanAsia not O/SEA/Mya-98

Mya-98

PanAsia

O/CAM/3/2010 O/CAM/6/2010 O/VIT/4/2010 95 O/VIT/14/2013

Outbreaks during 2014 in Eastern Russia (Zabaikalsky)

O/JPN/MZ1/2010 (AB618503) O/HKN/14/2010 O/HKN/7/2010 (JQ070303) O/HKN/8/2010 O/HKN/13/2010

O/VN/LC169/2009 (HM055510) O/MY/CHA/2010 (HQ652079) 85

Appears to be a separate introduction into South Korea from cases in 2010-11

O/SKR/12/2010 (KC438373)

O/33-P/CHA/2010 (JQ973889)

Recent samples from Mongolia Sequences from ARRIAH

O/Yeoncheon/SKR/2010 (NVRQS) O/Yangju/SKR/2010 (NVRQS) O/SKR/8/2011 O/CHA/31/2010* (JF792356)

O/HKN/9/2010 (JQ070304) O/HKN/10/2010 O/HKN/11/2010 O/HKN/12/2010

O/VIT/1/2011 O/CAM/2/2010 O/VIT/13/2010 O/CAM/1/2010

O/Paju/SKR/2010 (NVRQS) O/SKR/13/2010 O/SKR/7/2010 O/SKR/10/2010

O/MOG/3/2014 O/UKG/35/2001 (AJ539141)

PanAsia-2 Irn-2001 Ind-2001 Pak-98

O/IND/53/79 (AF292107) O/IND/R2/75* (AF204276) O1/Manisa/TUR/69 (AY593823)

87 90

100

SEA EA-2 EA-3 EA-4 WA EA-1

ME-SA

West Eurasia

East Asia: A/ASIA/Sea-97

Antigenic patterns (A/ASIA/Irn-05)

Recent detection in the Russia Federation

In the past 3 years, at least 9 sub-lineages are co-circulating in the region

A/MOG/2/2013 A/MOG/4/2013 A/MOG/1/2013

Sequences provided from ARRIAH

A/MOG/2013 (ARRIAH) A/MOG/5/2013 A/MOG/3/2013

Outbreaks during 2014 in Eastern Russia (Zabaikalsky)

A/KAZ/2/2013* (ARRIAH) A/Zabaikalsky/RUS/2013 (ARRIAH) A/GDMM-CHA-2013-S (LVRI) A/KAZ/1/2013* (ARRIAH) A/Amur/RUS/2013* (ARRIAH)

Distinct from outbreaks in Black Sea region A/ASIA/Iran-05

Vaccine matching are quite variable and require close monitoring

A/Amur/3/RUS/2013 (ARRIAH)

A/Zabaikalsky/5/RUS/2013 (ARRIAH) A/Zabaikalsky/2/RUS/2014* (ARRIAH)

A/Amur/2/RUS/2013 (ARRIAH) A/Amur/4/RUS/2013 (ARRIAH) A/Zabaikalsky/6/RUS/2013 (ARRIAH)

Sept 2014: new outbreaks due to Sea-97 in Tibet, PR China.

A/Zabaikalsky/1/RUS/2014* (ARRIAH)

Approaches are being developed for routine capsid sequencing

A/MOG/11/2013 93

99 73

A/MOG/12/2013 A/MOG/13/2013 A/QHXN-CHA-2013-B (LVRI) A/VIT/25/2012

Vaccine Strain Sublineage Egypt 2013

BAR-08

Afghanistan 2013

FAR-11

Iran 2014 Iran 2013/14

FAR-11 SIS-10

Pakistan 2012 Pakistan 2012 Pakistan 2013/14 Pakistan 2014

HER-10 SIS-12 FAR-11 FAR-09

A/Iran A22Irq A/TUR/ 2005 24/64 2006

Palestinian AT 2013 BAR-08 Turkey 2013/14*

SIS-10

No Nt

A/VIT/26/2012

Mixed data for different isolates *Includes viruses with a 4 amino acid deletion in VP1

A/TAI/15/2012

Vaccine bank recommendations

(Increasing) threats to Europe (?)

Vaccine strain (for each category are not listed in order of Importance)

Mainly from: 1. The Middle East 2. East Asia 3. North Africa

New FMD lineages in North Africa Outbreaks in FMD-free countries

Outbreaks in UK in 2001 Increased FMD circulation in East Asia

High Priority

O Manisa O PanAsia-2 (or equivalent) O BFS or Campos A24 Cruzeiro Asia 1 Shamir A Iran-05 (or A TUR 06) A22 Iraq SAT 2 Saudi Arabia (or equivalent i.e. SAT 2 Eritrea)

Medium Priority

A Eritrea SAT 2 Zimbabwe SAT 1 South Africa A Malaysia 97 (or Thai equivalent such as A/NPT/TAI/86) A Argentina 2001 O Taiwan 97 (pig-adapted strain or Philippine equivalent)

Low priority

2010-2011 Outbreaks in Bulgaria O/ME-SA/PanAsia-2 A/ASIA/Irn-05 Asia-1

A Iran 87 or A Saudi Arabia 23/86 (or equivalent) A15 Bangkok related strain A87 Argentina related strain C Noville SAT 2 Kenya SAT 1 Kenya SAT 3 Zimbabwe A Kenya

Considerations for FMD-free countries

Revised vaccine bank recommendations?

Suggestions

Vaccine strain (for each category are not listed in order of Importance)

Threats to FMD-free countries (based on historical evidence of trans-boundary movements of viruses) and frequency of FMD outbreaks in endemic pools West EurAsia (pool 3) O/ME-SA/PanAsia-2 A/ASIA/Iran-05 Asia-1

East Asia (via southeast Asia: Pool 1) O/SEA/Mya-98 O/ME-SA/PanAsia A/ASIA/Sea-97

Medium Priority

A Eritrea SAT 2 Zimbabwe SAT 1 South Africa A Malaysia 97 (or Thai equivalent such as A/NPT/TAI/86) A Argentina 2001 O Taiwan 97 (pig-adapted strain or Philippine equivalent)

A Iran 87 or A Saudi Arabia 23/86 (or equivalent) A15 Bangkok related strain A87 Argentina related strain C Noville SAT 2 Kenya SAT 1 Kenya SAT 3 Zimbabwe A Kenya

O/ME-SA/Ind2001 SAT 2 O/EA-3

Remove/reduce redundancy in vaccine coverage

Enhanced global surveillance & improved tools

Acknowledgements

Via the OIE/FAO FMD Lab Network

Improved tools for vaccine matching New tools for sequence exchange and analysis

O Manisa O PanAsia-2 (or equivalent) O BFS or Campos A24 Cruzeiro Asia 1 Shamir A Iran-05 (or A TUR 06) A22 Iraq SAT 2 Saudi Arabia (or equivalent i.e. SAT 2 Eritrea)

Low priority

North Africa (via sub-Saharan Africa: Pool 4)

1. To understand global virus distribution and patterns 2. To improve the quality of laboratory testing carried out by international and national reference laboratories.

High Priority

Support for the WRLFMD and research projects David Paton Collaborating FMD Reference Laboratories and field teams Partners within the OIE/FAO FMD Lab Network

1034 Samples tested during 2013

Outline EU projections for the main sub-sectors: meat and milk production should continue to grow over the next decade and exports may increase But more economic volatility is expected (e.g. dairy sector) The CAP reform could result in some geographical shifts Adaptation to climate change will be a constraint, but

Changing landscape for livestock production in Europe Directions and expected change in the next 20-30 years

Anne Mottet Livestock Policy Officer, FAO

EU per capita meat consumption is expected to recover from economic crisis levels

Beef: decrease

Herd reduction Low export growth in South America Demand declines

Source: DG Agri, EC 3

Source: DG Agri, EC 4

Pork: rebound

Poultry: growth

Slightly slower than recent growth due to low beef and pork supplies High demand, both domestic and for export

Welfare regulation had slowed down the growth High demand for exports

Source: DG Agri, EC

Global milk production is growing

Europe remains a global milk supplier

Removing quotas could lead to a limited growth of production, especially if competition from NZ, Argentina and Brazil remains high

Source: OCDE-FAO 7

Source: IDF 8

Further concentration of milk production

Milk production will grow in already biggest member states

Modeling production 2023/2012 (%) under certain price and market assumptions (JRC)

Higher animal density?

Source: DG Agri, EC 9

Exports of dairy products will continue to grow

Dynamic cheese market development

Billion

2.5

High demand for exports with emerging

2.0

1.5

EU market not yet saturated (esp. new member states)

1.0

0.5

0.0

DE 2002

DK 2003

FR 2004

2005

UK 2006

GR 2007

IE 2008

IT 2009

2010

NL 2011

PL 2012

2013

Source: Eurostat 11

Source: DG Agri, EC 12

Impact of the 2014 Russian ban

Less cows, more milk

Private Storage Aid for butter, Skimmed Milk Powder and certain cheeses ? Reserve for Crises in the agricultural sector (CAP 2014) 424m in 2014 by cutting

Source: Eurostat-Comext/Treatment INRA SMART LERECO

Source: DG Agri, EC 13

Milk price volatility higher after 2015?

Income volatility slows down investments

Source: Institut de 16

Dairy farm consolidation - more animals per farm

More animals per farmers 90

180

160

Livestock units per annual work unit

Specialist dairy

80 70 60 50

Nb dairy cows/farm

140

30 20

120

10 0 Denmark

100 80

Ireland 1993

Livestock units per

1995

Spain 1997

France 2000

Netherlands 2003

2005

Poland 2007

United Kingdom

Specialist monogastrics

300 annual work unit

250

200

20 0

Germany 1990

150 100

Denmark

Germany

1990

Ireland

2000

Spain

2010

France

Netherlands

2020*

Poland

2035*

United Kingdom

50 0 Denmark

Source: Eurostat

Germany 1990

Italy 1993

Spain 1995

1997

France 2000

Netherlands 2003

2005

Poland 2007

United Kingdom

Efficiency gains

Feed use efficiency Cereals and oil crops used as feed per protein output (index 100 in 1980)

Production of milk protein per cow, index 1000 in 1961 200

200

Southern Asia

190 180

180

Northern America

170

South America

160 150 140

Africa

World

140

Africa

120

Europe

130 Europe

120

Southern Asia

160

South America

100

110 100

Northern America

Eastern Asia

90 80 70

1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010

50 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 Source: FAOSTAT 19

Improve feed use efficiency (precision feeding, increased use of by-products, biotech and protein extraction from grass?) Source: FAOSTAT 20

2014 CAP reform : impact on production spatial distribution?

What about greening the CAP?

External convergence by 2020 (1/3 of the gap to 90% of EU average)

Crop diversification min. 3 crops on arable land; each crop between 5 70% of area Permanent grassland maintain at least 95% of hectares in 2014 Ecological focus areas no production on 7% of all eligible ha except perma. grassland

Climate change winners and losers?

Climate change and animal health

Spread of bluetongue virus (sheep) across Europe artly attributed to CC through increased seasonal activity of the Culicoides vector. Gastro-intestinal parasites amplified with global warming and South species spreading to North. Ticks (primary arthropod vectors of zoonotic diseases in Europe, e.g. Lyme disease and tick-borne encephalitis) distribution towards higher altitudes and latitudes (IPCC, 2014). Impact on wildlife?

Wild animals population implications for animal health

Conclusions

Densities of wild boar in Europe and wild boars numbers in the Russian Federation

European livestock farmers are exposed to more and more Further concentration of livestock production in Northern Europe is expected, but what about animal densities? The trend of a CAP less market/income oriented and greener will continue To maintain a comparative advantage, European livestock needs to continue gaining in efficiency

Khomenko et al, 2013, Empres Watch vol 28 25

Scope of Presentation The changing landscape and livestock and ecological systems in China and neighbouring countries Changing movement pathways Epidemiology of FMD in China and neighbouring countries Example from China-Mongolia-Russia

Changing landscape for livestock production and health in China and neighbouring countries; directions and implications for FMD management Professor John Edwards Team Leader, FAO ECTAD China

The changing landscape, livestock and ecological systems

China and Neighbouring Countries

14 direct land borders and several other close neighbours Southeast Asia, South Asia, Northeast Asia, Central Asian Republics

Diversity of geographical features Climate variation and change Production systems FAO

The changing landscape and livestock and ecological systems - Trends

Livestock (FMD susceptible species) demographics

Varying livestock systems by location and country - Epizones Urbanisation Cultural diversity Capacity of animal health systems Varying levels of collaboration among countries Social/political/legal issues Transport and risk pathways Value chains drivers and continuing changes

Pigs Rapid growth Growing intensification Mainly eastern parts of country

Ruminants Cattle and buffalo Sheep, goats, camels, yaks Wildlife

Varying livestock systems by location and country - Ecosystems and epizones 47

Changing livestock movements 2000-5

2005-10

2010-15

China looking at new approaches to managing these risks by creating buffer zone for value adding, slaughter and movement of lower risk products 8

Large scale movements of small ruminants in 2014

Epidemiology of FMD in China and neighbouring countries Current status for FMD in the region China Mongolia Russia Collaboration as an example

Eg. PPR in 2014 FMD in 2005

Current status of FMD

Epizones for FMD

Pool 3 O, A, Asia 1

Most recent types of FMD Type O - Pan-Asia topotype closest strain Vietnam 2011 - Myanmar 98 was dominant but now in decline - Hong Kong reports Cathay (pig adapted strain) Type A closest related strains in Southeast Asia - Two separate variations detected - Variation in matching with vaccines (needed to change vaccine strains) - Initially limited coverage of type A vaccines No recent reports of type Asia 1

Pool 2 O, A, Asia 1

Pool 1 O, A, Asia 1

Pool 7 O&A

Pool 4 O, A, SAT1,2,3

Pool 5 O, A,SAT1,2 Pool 6 O, A, SAT1,2,3

Current status of FMD (Cont)

Status of FMD

Pathways to Russia and Mongolia through east Asia (including China). Also through central Asia (eg. Type Asia 1) Various pathways live animals (legal and illegal), livestock products and human movements long and short distances Currently vaccines used are trivalent O, A and Asia 1. Type A more prevalent recently China high level of vaccination and particularly in border areas. Suppression of disease. 49

Ruminants, mainly cattle, sheep and goats. Camels, yaks also Variation in grazing systems (Nomadic in Mongolia and more intensive and controlled grazing in neighbouring parts of China and Russia) Pigs small numbers (some cases FMD in Russia, first report CSF in Mongolia) Gazelles evidence of disease but expert opinion is that in Mongolia do not play a significant role in persistence of the virus FMD in Mongolia 10 new cases in last 12 years. Always a different strain of FMD and previous strains do not persist. Neighbouring areas similar?

Next steps for China-Russia-Mongolia Collaboration Agreed on two joint projects to study the ecosystems, value chains and risk pathways for TADs including FMD Develop processes for: Harmonisation on standards for conduct of surveillance for FMD in the three countries sharing of information in real time Final design, oversight and implementation of the two projects

Get mongolia slide

Hostile and extreme environment Livestock in standstill for long periods of time Control measures Map Ex Dr Purevhkuu Tsedenkhuu, Mongolia

Conclusions Need to study and understand the landscape, climate, ecosystems risk pathways and value chains so that the risks can be managed Understand that the epidemiology of FMD will vary in different landscapes and production systems Monitoring and acting on this knowledge is required Address the risks of livestock movements by creating safe pathways, safe products and managing value chains Countries sharing information and working together is necessary for the control of TADs

Questions/comments

Thank you

The Food and Veterinary Office in brief We are a service of the European Commission responsible for verifying compliance and/or equivalence with EU standards concerning food & feed safety, animal health, animal welfare, and plant health Mandate recently extended. The FVO is based in Grange, Ireland, since 2002 as such since 1997. 170 staff 90 auditors (7 animal health) Main activity 250 audits per year covering EU MS and non-EU countries (some 25 on animal health). In addition, regular contribution to development of policy and official control systems.

CONTRIBUTION OF THE FOOD AND VETERINARY OFFICE OF THE EUROPEAN COMMISSION TO REINFORCE FMD IMPORT RISK MANAGEMENT MEASURES, AND ANIMAL DISEASE EMERGENCY PREPAREDNESS AND EARLY WARNING SYSTEMS IN THE EU Francisco Javier Pérez Pérez

Unit F6 Animal health & welfare Food and Veterinary Office (FVO) Directorate General for Health and Consumers (DG SANCO) European Commission

What do we do and how do we do it?

What do we do in relation to animal health? A broad variety of areas to check:

We conduct audits and other related activities in order to provide :

Main activities in MS of the EU:

assurances necessary to increase the confidence of consumers, facilitate trade and contribute to economic growth. the Commission with critical oversight of how control systems (EU and non-EU) operate the Commission can proactively take the necessary steps to minimise risk.

EU co-funded disease control and eradication programmes. Compliance with animal health rules for Intra-EU trade. Contingency planning Emergency preparedness and early warning systems. Compliance with rules post-outbreak of an epizootic disease. Aquatic animal health.

How do we do that: We gather audit evidence (questionnaires, review of documentation, interviews, observations, data analysis, verification on-the-spot) aimed at answering four basic questions: IS THERE a system? SANCO CAN it work? FVO DOES it work? WHY? If not, WHY not? PLANNING EU and international (e.g. OIE) standards Compliance / Equivalence / Effectiveness Basis for: FVO recommendations in reports Identification of best practice Transposition (MS) Enforcement (MS) Equivalence (TC)

AUDIT INSPECTIONS

Main activity in non-EU countries - Equivalence of animal health control

New or changed legislation

FINDINGS RECOMMENDA TIONS

Priorities

POLICY & RISK MANAGEMENT

Safeguard measures (MS) Infringements (MS) Delisting (TC)

FOLLOW UP

systems before authorisation is granted to export live animals and commodities of animal origin to the EU: Audits adapted to animal species, commodity and hazard identification pre-defined certification conditions e.g. FMD (milk, beef), AI (poultry and ratites meat, hatching eggs), AHS and VEE (live horses), CSF and ASF (pork, semen), ISA, IHN and VHN (live salmonids and products thereof). Analysis of risks to inform import risk management process operation of measures at origin to mitigate risk of disease introduction in the EU. 4

Historical perspective in MS of the EU

Main activities in relation to FMD

Country

Series of inspections on contingency planning have been carried out regularly since the 90s in addition, checks related to FMD outbreaks. We are carrying out a multi-annual series of audits in MS of the EU that since 2012 aims to reinforce the existing exotic animal disease emergency preparedness and early warning systems FMD contingency planning is one of the main priorities. Frequent audits in non-EU countries to evaluate FMD risks associated with import of animal commodities. A multi-annual series of audits in MS of the EU on verification of compliance with bio-security standards applied in laboratories and establishments handling live FMD virus. Verification of the effective implementation of FMD eradication measures in Bulgaria in response to the outbreak of FMD in 2011 and of FMD controls in general, and vaccination in particular, in Turkey. 5

Finland

Netherlands

Austria

Germany

Slovakia

Switzerland

Latvia

France

FMD crises

2009-8210 2009-8253 2003-9087 2003-9101 2013 (pre-access.) 2013-6781 2003-9185 2003-9084 2003-9100 2003-9151 2015 (planned) 2003-9193 2003-9078 2009-8265 2003-9190 2009-8259

2013-6778

2000-1280 2012-6401 2014-7044

2001-3323 2001-3333

2015 (planned)

2013-6777

2003-9196 2008-7789 2003-9102

2013-6775

2003-9197 2009-8267 2014-7046

2013-6780

2004-7267

2013-6776

2001-3324

2012-6402

2001(3318, 3328, 3331), 2007-7416

What have we been looking at to evaluate the effectiveness of early warning and emergency preparedness systems to prevent worst-case situations from happening?

2014

Portugal

2002-8545

3rd visit

Emergency preparedness and early warning systems (2)

Why? A legal mandate to ensure that the EU has prepared effective strategies to control disease outbreaks causing the least possible economic damage for agricultural and non-agricultural sectors of the economy. The MS believe that FVO involvement in evaluating the state of readiness of their emergency preparedness systems is relevant and effective, and that we should carry on doing our audits in this area. It has been a priority to evaluate the main 'risk areas' for large animal disease outbreaks; e.g. impact of the MS administrative organization, high density of animals and animal trade, periphery of the EU, and also to learn about best practice. 2013

Peace time 2nd visit 2014-7043

Emergency preparedness and early warning systems (1)

2012

AT BE BU CY CZ DE DK HR EE EL ES FI FR HU IE IT LT LU LV MT NL PL PT RO SE SI SK

1st visit 2000-1094 1999-1019 2007-7527 2005-7618 2005-7574 2000-1097 1999-1215 2010 (pre-access.) 2005-7616 2002-8551 2001-3163 2000-1096 2001-3381 2005-7619 2002-8511 1999-1143 2005-7621 2002-8655 2005-7617 2005-7620 2002-8535 2005-7612 2000-1245 2007-7526 2000-1108 2005-7615 2005-7609

Estonia Sweden United Kingdom 7

Animal health surveillance systems: to ensure early detection of unusual disease events (e.g. awareness, exclusion diagnosis, risk-based and syndromic surveillance). Legal powers and compensation and cost-sharing schemes. Cooperation and coordination at all levels a clear chain of command and adequate instructions and guidance for staff involved in managing a disease outbreak training and operations manuals. Availability of technical and epidemiological expertise definition of surveillance, targeting of outbreak investigation and response, economic analysis to inform prioritisation of actions. Laboratory capacity - ready to perform in highly demanding emergency situations. Availability of data analysis and information management tools. Adaptable and tailored communication arrangements in place. Availability of equipment and material resources to cope with a major disease outbreak. Arrangements for application of emergency vaccination. Respect of EU requirements on depopulation of animals in the event of an outbreak. Rendering and incineration capacity. Self-evaluation real-time and simulation exercises, alarm drills lessons learnt. 8

Emergency preparedness and early warning systems (3)

Emergency preparedness and early warning systems (4)

What have we found so far and what implications may that have in the event of a large FMD outbreak? An ample variety of approaches, options and solutions, some gaps and a lot of good practice. Integration of animal health crisis management in regional and national crisis management centres availability of expertise and all sorts of resources.

Diversity of approaches in relation to compensation and cost-sharing biosecurity a bonus? Availability of adequate technical and epidemiological expertise and laboratory capacity. Adequate arrangements in relation to animal depopulation, and rendering and incineration. Room for improvement in relation to emergency vaccination and organisation of simulation exercises decision-making protocols and development of practical arrangements to implement vaccination or alternative options.

Well-developed chains of command irrespective of the type and complexity of the veterinary administration no major problems in relation to coordination and cooperation. Excellent examples of web-based interactive platforms substituting paperbased CPs and integrated information management tools that ease up and enhance responsiveness to emergency situations.

What are we doing with all of this? Apart from the individual reports addressed to the MS; i.e. recommendations for improving the system, an overview report will be produced in 2015 highlighting generalised or systematic gaps and best practice.

Satisfactory animal health surveillance systems that should contribute to the early detection of any disease outbreak and prevent its escalation out of control. With some exceptions, largely effective animal traceability systems animal density and trade networking analysis anticipating possible spread routes for epizootic diseases.

Annual workshop organised in the FVO to discuss the various areas evaluated and share good practice two workshops so far (2013 and 2014). 9

Activities in non-EU countries

Most of the audits in non-EU countries; e.g. in South America and in Southern African countries, focus on the evaluation of:

Audits in non-EU countries to evaluate FMD risks associated with import of animal commodities: Since 2011, the FMD situation has been evaluated in countries in South America (five countries), Southern Africa (three countries) and the EU-neighbouring region (e.g. Turkey, Belarus, Serbia). The outcome of those audits has informed the decisions of the EU FMD import risk management system coordinated by the European Commission. Country BR BY AR UY PY CL BW NA ZW SZ ZA TR RS

Audit 2009-8212 2010-8418 (milk and milk products) 2009-8211 2007-7397 2009-8240 2013-6802 2009-8325 2009-8326 2001-3178 2005-7396 2005-7605 2011-6112 (EU funded vaccination) 2011-6225 (live animals)

The effectiveness of the vaccination programmes to prevent outbreaks of FMD in cattle, buffalos and sheep: Selection and quality control of vaccines. Planning, implementation and verification of the vaccination campaigns cold chain! Monitoring of the immunity levels achieved in the different age cohorts.

The ability of the competent authorities to demonstrate the absence of circulating FMD virus in cloven hoofed animals: Awareness, notification and investigation of suspect cases technically valid diagnostic results. Epidemiological surveys design, implementation, reliability of the diagnostic system (NCP), coverage of wildlife?

2011-6099 2012-6399 2013-6327 2014-7249

Their capacity to prevent the entry of the FMD virus in the country and the ability to promptly detect the presence of the virus and take measures to prevent its spreading: Import risk assessment and implementation of import controls neighbouring countries (risks?).

2013-6792 2013-6782

official controls in areas bordering

Other risk mitigation measures de-boning, maturation. The reliability of the certification system in place for animal health requirements: Respect of EU and OIE standards for international veterinary certification knowledge of the certifying officers. Animal and product traceability all along the certification chain.

2014-7089

independence and

Use of those activities in EU import risk management

Compliance with bio-risk management standards in laboratories and other establishments handling live FMD virus Series of audits between 2009 and 2012 - 19 FMD laboratories in 15 MS (including three vaccine manufacturers) at least one FVO auditor accompanied by two experts from MS (one on FMD and the other on bio-risk management in laboratories) EuFMD Minimum Standards. The outcome was that: Bio-risk management systems in the FMD laboratories are in general sufficient to prevent escape or release of FMD virus from the primary containment area. Some deficiencies were identified in air handling and in the management of fluid and solid waste (risk of FMD virus escape!), but prompt corrective action was taken in each one of these cases to rectify the shortcomings. The evaluation of the procedures showed that most MS had difficulties in providing the required level of official controls lack of highly specific technical competences needed for assessing the bio-risk management systems for airflow and waste. The audits have also identified and disseminated best practices related to official controls, qualification of inspectors and laboratory bio-security. A new version of the Minimum Standards has been agreed on the basis of this series of audits. Overview report to be released shortly workshop to discuss it in January 2015. 13

THANK YOU FOR YOUR ATTENTION AND ENJOY THE MEETING!!

Prospects for FMD control

http://ec.europa.eu/food/fvo/how_en.htm Keith Sumption1, Emerson Tuttle and Ugo PicaCiamarra2 Executive Secretary, European Commission for the Control of FMD Food and Agriculture Organization (FAO) of the United Nations, 2Livestock Sector Analysis Branch, AGA FAO

United Kingdom Special Inquiry on FMD , 1925 (after a massive epidemic in which stamping out had temporarily been abandoned)

2012: Bangkok Conference and Jerez Open Session

Extra-ordinary infectivity results in a very high global burden

Preface

30 (-100+?) million LIVESTOCK UNITS per year ( 2% of world's cattle population)

The real trouble with foot-and-mouth disease is not its deadliness but its extra- ordinary infectivity, and we do not have to consider its effects upon the animals actually attacked but upon the flocks and herds of the United Kingdom as a whole. Movement, both human and animal, is now much more extended than it used to be, and infection would constantly be carried from one part of the kingdom to another.

Knight-Jones and Rushton, 2013

Erice, 2008 and the Ecosystems approach: 7 virus pools, Regional Roadmaps for each pool

The development of FMD Control: five ages 1.

The age of Isolation and quarantine (+prayer: 1514 to 1890)

The age of stamping out and keepit-out policies 1890-(1955) : successful application in control in the United Kingdom from before the virus was discovered.

- pools where FMDV lineages and epidemics occur independently.

Given the scale of infectivity and global burden what are the prospects of extending control beyond the current free regions? Legend: Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 Pool 7

Serotype O Serotype A Serotype Asia 1 Serotype SAT 1 Serotype SAT 2 Serotype SAT 3

Until mid-1950s, use of isolation/quarantine was the response in most of continental Europe, but epidemics involved thousands of infected farms.

Fracastoro: principles for control by isolation from and spread by fomites

The Progressive Control Pathway for Foot and Mouth Disease (PCP-FMD) :

The development of FMD Control: five ages 3.

Regional control through generalised vaccination: develop vaccines (192565) and control through generalised vaccination, 1955-1980 (Europe, later S America, elsewhere) Development of free regions : Europe (later S America) pushes for FMD free regions without vaccination: 1980(Advanced countries/OECD) The Progressive Control age (PCP); the move to widen access to the benefits of FMD control for the many, progress in attainable steps, reducing global risk (Global Strategy 2012-2027)

Objective: Assist countries where FMD is endemic to progressively reduce the impact of FMD Progressively increase the level of FMD control over 5 stages In use since 2008 Joint FAO-EuFMD-OIE Tool

Foot and Mouth Disease control

West and South Eurasia Roadmap

E. Africa Roadmap

PCP based projects supporting the Hemispheric Plan for Eradication PHEFA

S. Africa Roadma p

Past ages have shown what works in advanced countries Controlling Disease ..and Controlling FMD virus circulation..are different Outcomes Free countries usually want to control circulation rapidly to eradicate virus. Endemic countries may want to control the disease to reduce impact. Sectors in each category may have different desired outcomes The Progressive Control Pathway (PCP-FMD) is a policy development tool for sustainable strategies Market based solutions might provide a new way to widen access to FMD vaccines in the lower PCP stages

Countries in the SEACFMD 2020 Roadmap

SAARC Roadma p

No magic formula fits all settings

Prospects for extending FMD Control beyond currently free regions : overview

PCP Stage 2 and upwards: countries must have RiskBased Strategic Plans 1. Situation analysis 2. Benefits of FMD Control 3. Goal, objectives, tactics and activities 4. Monitoring and evaluation 5. Operational plan 6. Technical assistance

Successful control models Regional successes >120 years experience Deep knowledge base Vaccination options Processes/pathways to freedom (PCP-FMD) Global Strategy (FAO/OIE)

Global demand for livestock (productivity, trade) Private producer demand for FMD control at the base of the pyramid Benefit/cost of control POSITIVE Awakened Government interest (Export Africa and Eurasia) Economic development (a new Africa..) Appropriate policy setting tools (PCP-FMD) Stage of vaccine development Communication revolution

Extreme infectivity Instability (borders, gains rapidly lost) poor adoption of state control models (~100 countries) Barriers to access quality vaccines Quality of national policy and strategy Duration of immunity (vaccine) Expertise and knowledge transfer

Strengths

Weaknesses

Opport unities

Threats

Strengths Successful control models Regional successes >120 years experience Deep knowledge base Vaccination options Processes/pathways to freedom (PCPFMD) Global Strategy (FAO/OIE)

Protectionism (vaccines, trade) Reduction in R&D low hanging fruit ROT Weakening UN /international technical support to countries Wars and instability

Control options from Herd to Global Level Herd level: Quality vaccines now far cheaper National level Processes for gaining freedom (OIE) OIE Code: standards for safe trade Pathways out of endemicity (PCP-FMD) Tested emergency management systems (options include vaccination, to regain freedom) Eco-system and Regional level(Roadmaps in each POOL) Global level Strategy for FMD (Strengthened animal health systems) from 2012 International framework (GF-TADS FAO/OIE)

Strengths

Successful control models Regional successes >120 years experience Deep knowledge base Vaccination options Processes/pathways to freedom (PCP-FMD) Global Strategy (FAO/OIE)

Strengths

Successful National and Regional control models Western Europe, Americas One Serotype (C) disappeared (globally gone?)

Successful control models Regional successes >120 years experience Deep knowledge base Vaccination options Processes/pathways to freedom (PCPFMD) Global Strategy (FAO/OIE)

Deep knowledge base

Controlled in the UK before the virus was identified British experience- focus on isolation, tracing and slaughter; understand every outbreak and improve control of the next >100 years of research on transmission

Vaccination options

Strengths Successful control models Regional successes >120 years experience Deep knowledge base Vaccination options Processes/pathways to freedom (PCP-FMD) Global Strategy (FAO/OIE)

Vaccines used in emergency (rings) since 1938 and preventively since WW2 (Frenkel, NL) Emergency vaccination (free countries) (EU) with or without slaughter (DIVA) of vaccinates EU vaccine/antigen bank Preventive vaccination programmes, non-free countries : Strategic options Disease reduction (PCP2) vs eliminating virus circulation (PCP3) Outcome level : Herd, zone, risk targetted, national Duration of immunity: limited - little improvement since 1938? Cost: fell greatly 1950-1980, then little change (quality producers)

Strengths Processes/pathways to freedom (PCP-FMD) Global Strategy (FAO/OIE)

Global Progress As part of Regional Long Term Roadmaps Supporting sustainable National Strategies

FMD Global Strategy - Financial implications

in USD as calculated by the World Bank for the first 5 years The GLOBAL FMD CONTROL STRATEGY

- Cost of national FMD programmes 68 M (to support 79 initial PCP 0-2 Stage countries) - Vaccination cost 694 M (to support 45 initial PCP 1-3 Stage countries) - Regional level 47 M (reference labs/epidemiology support and networks) - Global level 11 M (coordination, evaluation)

Adopted in Bangkok, 2012 Action plan

THREE Phases of 5 years Aim: every country to progress by 2 PCP Stages By 2027 all countries to be in minimum PCP Stage 2 (= implementing control programmes) Action plan (typical activities) were worked out At country level for each of the PCP stages and for each of the Strategy components At regional level At global level The Global FMD Control Strategy and supporting documents are available on the websites www.FMDconference2012 http://www.oie.int

SEVERAL RESULTS: EC VIA EUFMD: INCREASED FUNDING TO PIRBRIGHT TO COORDINATE THE GLOBAL FMD LAB NETWORK MORE FREQUENT REGIONAL ROADMAP MEETINGS EURASIA, MID-EAST, EAST AFRICA

Weaknesses

Extreme infectivity Instability (borders, gains rapidly lost) Poor adoption of state control models (~100 countries) Quality of national policy and strategy Barriers to access quality vaccines Duration of immunity (vaccine) Expertise and knowledge transfer

Add PCP map

FMD a consequence of barriers to accessing FMD prevention options

Barriers to accessing quality FMD vaccines

Weaknesses Extreme infectivity Instability (borders, gains rapidly lost) Poor adoption of state control models (~100 countries) Barriers to access quality vaccines Quality of national policy and strategy Duration of immunity (vaccine) Expertise and knowledge transfer

-Decreased resilience -Negative livelihoods impact -Decreased market access -Limited ability to trade products internationally

Demand-side (buyer) 1) Lack of trust: in suppliers and products 2) Limited resources (Government, private) 3) Lack of delivery to point of need 4) Lack of perceived benefits 5) FMD as one of many concerns

Disease outbreak

Supply-side (Vaccine producer to local deliverer) 1) Lack of harmonised registration systems (Africa, Asia) 2) Impediments to pharmaceutical importation (cost of registration, bureuacracy, corruption, local monopolies) 3) Exclusive government capture of FMD control (private sector excluded) 4) Risks relating to spatial distribution of consumers and Temporal distribution of demand 5) Lack access to appropriate epidemiological info

Increased circulation of virus Minimal/no preventive rural control

Poor access of rural livestock holders to quality FMD prevention options

Demand-side barriers

Supply-side barriers

Underlying socioeconomic issues create the environment for disease to persist

Farmers can

cope with FMD. Government has other things to do. FMD is too complex.

Weaknesses Limited duration of vaccinal immunity

Expertise and knowledge transfer

Three times per year in some risk settings Protection further reduced where vaccine not well matched to challenge Guidance on vaccination intervals comes mainly from vaccine producer Few laboratories qualified to undertake antibody kinetic tests to predict optimised regimes

FMD is good for antibiotic sales. But there is no vaccine.

How close is FMD to me? What can I do? Internet or local vet?

Opportunities Global demand for livestock (productivity, trade) Private producer demand for FMD control at the base of the pyramid Benefit/cost of control POSITIVE Awakened Government interest (Export - Africa and Eurasia) Economic development (a new Africa..) Appropriate policy setting tools (PCP-FMD) Stage of vaccine development Communication revolution

Global ranking of food and agriculture commodities in value terms (2010) Rank 1 2 3 4 5 6 7 8 9 10

Source: FAOSTAT

Commodity Rice, paddy Cow milk, whole, fresh Indigenous Cattle Meat Indigenous Pig meat Indigenous Chicken Meat Wheat Soybeans Tomatoes Sugar cane Maize

Production value ($ billion) 180 180 172 168 122 81 66 55 54 54

Knight-Jones and Rushton, 2013:

Opportunities Global demand for livestock (productivity, trade) Benefit/cost of control POSITIVE Private producer demand for FMD control at the base of the pyramid Awakened Government interest (Export - Africa and Eurasia) Economic development (a new Africa..) Appropriate policy setting tools (PCP-FMD) Stage of vaccine development Communication revolution

Average cost per vaccination: 1 USD Average loss per case (cattle) : 100 USD Benefit/cost usually >>1 in free countries; in endemic countries, few studies but positive in affected systems (cattle) Endemic setting, pastoralists, Sth Sudan : 11.5

Global loss: US$ 11 billion (6.5 to 21 billion, 90% range) Global estimate burden: 32 million livestock units (LSU; range 28 -79 million) (based on reported cases; true cases (serology) much higher (e.g. 8 fold- Iran)

Estimated 2% of world's cattle population has FMD in a year (90% uncertainty range: 2 5%)

Opportunities Global demand for livestock (productivity, trade) Benefit/cost of control POSITIVE Market based solutions meet demand for FMD control at the base of the pyramid Awakened Government interest (Export - Africa and Eurasia) Economic development (a new Africa..) Appropriate policy setting tools (PCP-FMD) Stage of vaccine development Communication revolution

The new market place is the BoP

Opportunity: Market-based solutions (MBS) Most of the worlds population are consumers with relatively little purchasing power

(Base of the Pyramid)

Globally: 4 billion people living on <$2.50/day Ethiopia: $84 billion of wealth in the BoP3, 22 million rural poor Kenya: 15 million rural poor4

Target this consumer class through high volume/low value business models

http://www.generationim.com/sustainability/challenges/realneeds-base.html

Background

A new business model for FMD: Viewing the poor as a conscious consumers as opposed to victims

WB FAO ILRI AU-IBAR Livestock Data Innovation in Africa Project

Rapid and continued growth projected for Sub-Saharan Africa trend of privatization the poor as a conscious consumers as opposed to victims Challenge the idea that the poorest are not willing to pay for goods and services Design interventions targeting those at the BoP

Objective institutional linkages to improve the (quantity and) quality of livestock data and promote pro-poor investments in

Many consumers low purchasing power

Impact investments also include a social dimension

http://moonofthesouth.com/kenyan-media-accused-stereotyping-2/

% rural households keeping livestock

Rural livestock keepers

Characterising livestock keeping

Sub saharan Africa: The majority of rural households keep livestock

1. Business-oriented livestock farming cash from selling meat, milk and other products to the market

2. Livelihood-oriented livestock farming food, manure, insurance, etc. as a step in a ladder out of poverty

Business-oriented livestock keepers

Livelihood-oriented and business-oriented livestock keepers The majority of rural households are livelihoodoriented few are business.oriented (from 5% to max 25%)

1. Business-oriented livestock farming cash from selling meat, milk and other products to the market) relatively large herds / flocks (e.g. > 3 cattle equivalent) livestock key for income of cash income from livestock) major incentives to tap into the growing market for animal foods

FMD control: What is the potential market? One example

Different value chains

LIVESTOCK PRODUCER

DEMAND FOR INPUTS

SUPPLY OF OUTPUTS

Livelihoodsoriented

Many farmers with low purchasing power/ low-cost technologies / scattered / demand is irregular

Business-oriented

Few farmers with purchasing power / small-scale affordable tech / scattered / demand is regular

Focus on cattle in the East African region, specifically Ethiopia and Kenya:

Implications FMD

Public (State) subsidised

Low volume / heterogeneous quality

Willingness to pay depends on risk : Market potential, PPP

Ethiopia 54 million head6 Kenya 17 million head7 14.5 million dairy cattle combined

Business of $142 million per year for control strictly through vaccination Robinson and Siembieda, 2011 39

Policy setting: Opportunities Global demand for livestock (productivity, trade) Benefit/cost of control POSITIVE Market based solutions meet demand for FMD control at the base of the pyramid Awakened Government interest (Export - Africa and Eurasia) Economic development (a new Africa..) Appropriate policy setting tools (PCP-FMD) Stage of vaccine development Communication revolution

Opportunities

BLUE- areas producing surplus bovine (potential export) Robinson, 2010

Global demand for livestock (productivity, trade) Benefit/cost of control POSITIVE Market based solutions meet demand for FMD control at the base of the pyramid Awakened Government interest (Export - Africa and Eurasia) Economic development (a new Africa..) Appropriate policy setting tools (PCP-FMD) Stage of vaccine development Communication revolution

Who will benefit? Who is willing to pay? Reduce disease (PCP2) or reduce virus circulation(PCP3)? Government as facilitator - of access to vaccines and information or service provider and enforcer? Heavy Investment cost to move up

Livestock ownership and trade is almost all in private hands - our biggest opportunity is to enhance their participation in FMD risk management

Add

Opportunities Stage of vaccine development (fruits of recent research) Communication revolution

Private benefits and incentives are crucial to participation

5.9 billion of 7 billion people use a mobile phone (2013)

In 2014, we can reach almost all livestock producers with biosecurity information , by cell phone. What an opportunity!

FMD is common and damaging disease but who benefits and who should pay for control?

Shipping Forecast

OH ..AND Reduction in R&D - low hanging fruit ROT

Threats Protectionism (vaccines,

And Perish the Thought Failure to maintain global expertise and centres of excellence

trade) Weakening UN /international technical support to countries

Wars and instability (Agro-terrorism)

And Woolly thinking

Veteriological Forecast at 0900

Conclusions

GMT 29th October 2014

The challenge of progressive control is to extend access to those who need it in the rest of the world

Global demand for livestock products will result in an increasing demand for FMD control

Market based solutions (MBS) hold promise for delivery to the business oriented livestock producers

Removing barriers affecting availability of quality vaccines needs international attention

The complexity and cost of eradication programmes is formidable barrier the drivers to

Adopters of the PCP approach are found in every continent - but support is needed to share experience and develop management capacity

Optimising control programmes to control virus circulation is complex

The communications revolution is a huge opportunity to develop the expertise needed in every country and engage owners in risk management

Threats to global progress are real and include the downturn in funding on FMD research.

achieve this may weaken as countries become net meat importers.

-and needs more epi and economic modelling and experts

Engaging livestock keepers as actors in animal health

Context Areas of interest Animal health surveillance Animal health information systems

Principles applicable to other areas of animal health

Angus Cameron AusVet Animal Health Services 66

Starting assumptions Main purpose of surveillance

Early detection

report to OIE and FAO

Difficult and expensive so focus resources on: priority diseases in priority areas use reliable (high Se and Sp) tests

Focus on priority diseases Collecting data on routine diseases is a waste of time and money

Passive surveillance (farmer reporting) Hard to make farmers report disease

Designing surveillance

Farmers are stupid

Senior veterinary officials or epidemiologists at the central level

Theory Reasons for surveillance Demonstration of freedom Describe level of disease Case finding Early detection

Requirements for early detection and case finding:

Sampling possible

Complete coverage of the population Continuous surveillance

Conclusion Farmer reporting is the only practical and affordable approach Not just another component Must be the core of almost all surveillance systems 67

Early detection / Case finding

Why?

Objective

Farmers fail to report Why?

Every case (suspect of being a priority disease) Reported immediately, and Investigated to provide definitive diagnosis (or at least rule out priority disease)

Farmers are not stupid Rational decision based on:

intuitive cost-benefit analysis

Reality Almost always fail

Example

Conclusion

Notifiable disease

Farmers must get direct, personal benefit from reporting

(authorities not very interested in routine diseases) Cost:

Must work with all diseases of interest to the farmer

Definite disruption Probable quarantine Possible culling of all animals

Must provide animal health service (give) not do surveillance (take)

Benefit for the farmer Almost none All benefits at higher level 68

Staff Incentives

Philosophical framework (part 1) 1. Farmer must get direct personal benefit from participating in disease reporting

100 90 80 70

2. Passive reporting systems must work with all diseases, not just priority diseases

60 50 40 30 20

Philosophical framework (part 2)

Consequences

3. All players must get direct personal benefit from participating in disease reporting

Aim: Collect data on all diseases instantly

Result: Too much paper, too much data

4. Information system must be designed by field and local staff to meet their needs, not central staff

Traditional solution Hierarchical compilation and filtering Slow Data of very little value 69

Jan-10

Sep-09

May-09

Jan-09

Sep-08

May-08

Jan-08

Sep-07

May-07

Jan-07

Sep-06

May-06

Jan-06

Sep-05

May-05

Jan-05

New approach: Big Surveillance

Theory Practice

Components

Philosophy implemented in Indonesia

Powerful central database Field staff capture data in electronic format SMS, mobile Apps, etc.

Direct, immediate submission to central database Automated checking, analysis and reporting Immediate feedback for case management Atomic data handling

National integrated animal health information system

Result 30 fold increase in reporting Sustainable No incentives

More information YouTube Search for iSIKHNAS - user testimonies iSIKHNAS - how the system works

Web http://wiki.isikhnas.com Full system documentation Indonesian and English

Thank you

Acknowledgements Indonesian funding DGLAHS

Team members M Syibli Sigit Nurtanto S Yulianti CK Yohana Priyono RN Muhammad Soegiarto a

Australian funding AusAID Department of Agriculture

Team members Jonathan Happold Albertus Muljono Catriona Mackenzie a

Outline

African Union

Interafrican Bureau for Animal Resources

Africa: Economic context Demand for livestock products

A twin-track approach to livestock development

African Livestock: Opportunities Typology of African livestock keepers

www.au-ibar.org

Business and Livelihoods in African Livestock

African livestock markets by size

Policy Implications Key Messages 71

Demand for livestock Products

Africa: economic context

Per capita consumption of livestock products will increase

slow

Meat: from 14 kg in 2005/07 to 26 kg in 2050

down by the current Ebola outbreaks in West Africa)

Milk: From 30 liters of milk in 2005/2007 to 64 liters in 2050.

5.4% per year over the period 2004-2012

Total meat consumption, 2005/07 to 2050

4.8% in 2013

Meat: from 11 to 35 million tons (+218%)

5.1% in 2014

Milk from 32 to 83 million tons (+159%)

Projected to grow at 5.8% in 2015.

Total market value, 2005/07to 2050

Africa population is growing fast -

Meat products from US$ 33 to US$ 108 billion (+227%),

from 0.9 Billion in 2005/2007 to 2.2 Billion in 2050

Milk from US$ 17 to US$ 44 (+158%).

African livestock markets by size

Milk is and will be the largest market, followed by beef and poultry

Mio tons

African livestock markets by size

2nd

3rd

1st

African milk markets: regional variatons

Western

Eastern

Central

Milk 2005/07 = 32.4 mio tons

Northern

African beef markets: regional variations

Southern

Western

Eastern

Beef 2005/07 = 4.7 mio tons

Milk 2050 = 82.6 mio tons Africa and Northern Africa are Eastern

Central

Northern

Southern

No,

Buffaloes

4,000,025

Cattle

Poultry 2050 = 11.8 mio tons Poultry market is dominated by Southern and Nothern Africa Over 56 % of the increased demand in 2005/07- 2050 from Southern and Northern Africa; 20 % from West Africa -6% in all regions

Beef 2050 = 13.6 mio tons

LIVESTOCK

Camels

Poultry 2005/07 = 2.9 mio tons

Southern

African Livestock: Opportunities

African poultry markets: regional variations Eastern

Northern

Beef market is relatively uniform in terms of its importance across regions Over 70 % of the increased demand for beef in 2005/07- 2050 from Western, Southern and Eastern Africa -3% in all regions

the largest markets Over 60 % of the increased demand from milk in 2005/07- 2050 from Eastern and Northern Africa -3% in all regions

Western

Central

282,925,261

Chickens (1000)

1,646,024

Horses

5,337,831

Pigs Sheep and Goats

20,735,087

29,698,920 609,922,313

African Livestock: Opportunities

Typology of Rural livestock keepers

Markets present business opportunities for livestock keepers: % rural households keeping livestock

Economic growth (livestock account for > 1/3 of agricultural GDP) Poverty reducution (60 percent of rural households keep animals) Food security (214 mio people are undernourished)

For these opportunities to realize the livestock sector should grow by about 5-6%/yr compared to actual 2% What does this mean eg: Kenyan milk yield average: 600 kg/lactation In 20 years at 6%/yr growth rate: 1815 kg/lactation We need to triple milk yields

QUESTION Who can ultimately benefit from the growing market for animal-source food and contribute to increase livestock productivity?

Typology of rural livestock keepers

The majority of rural households keep few animals (mean TLU / median TLU)*

1. Business-oriented livestock farming

*TLU = 250 kg live weight; about one cattle

(1.41 / 0.60)

(1.25 / 0.45)

(2.20 / 0.88)

(3.17 / 0.33)

(0.47 / 0.13)

(1.93 / 0.50)

cash from selling meat, milk and other products to the market (0.80 / 0.50)

% rural households keeping livestock

Typology of rural livestock keepers

The majority of rural households keep livestock (60%)

2. Livelihood-oriented livestock farming food, manure, insurance, etc. as a step in a ladder out of poverty

-oriented and business-oriented livestock keepers

-oriented livestock keepers

The majority of rural households are livelihood-oriented few are business.oriented (from 5% to max 25%)

Livelihood-oriented livestock farming Food, manure, insurance, etc. as a step in a ladder out of poverty relatively small herds / flocks (e.g. < 3 cattle equivalent) livestock not key for income (< 25% of cash income from livestock) little incentives to tap into the growing market for animal foods

-oriented livestock

Policy Implications

keepers

Twin-track approach for livestock development in Africa

Business-oriented livestock farming

1. Support to Livelihood-oriented livestock farming to make full use of their livestock assets Rethinking the role of government or the notion of public goods infrastructures system of animal health disease and control

cash from selling meat, milk and other products to the market) relatively large herds / flocks (e.g. > 3 cattle equivalent) livestock key for income of cash income from livestock)

bolster the purchasing power of rural livestock keepers

major incentives to tap into the growing market for animal foods

Policy Implications

Key Messages

Twin-track approach for livestock development in Africa

African is growing fast Development of African livestock anticipated to influence the shape of the agricultural economy in the continent, and pattern of poverty reduction Inclusive growth of livestock requires a twintrack approach: business-oriented and livestockoriented livestock keepers Animal health service delivery shaped around the two typologies of livestock keepers

2. Support to Business-oriented livestock farming heightening regulation of veterinary drug quality providing incentives for private business model experimentation through grant funding or other means, developing an institutional and regulatory framework that allows private actors to freely enter and exit the animal disease control market Invest in animal health research

livelihoodentrepreneurs / international community can facilitate business oriented: traditional businesses

Authors

Acknowledgement WB FAO ILRI AU-IBAR Livestock Data Innovation in Africa Project

Simplice Nouala, AU-IBAR Ugo Pica-Ciamarra, FAO

Objective institutional linkages to improve the (quantity and) quality of livestock data and promote pro-poor investments in

Cheikh Ly, FAO Nancy Morgan, FAO Derek Baker, UNE (former ILRI)

AU-IBAR: Providing leadership in the development of animal resources for Africa

Thank You

Foot and Mouth Disease Continuity of Business Planning for the U.S. Dairy Industry Pam Hullinger DVM, MPVM, DACVPM Veterinary Epidemiologist, Lecturer Department of Veterinary Medicine and Epidemiology University of California, Davis phullinger@ucdavis.edu

AU-IBAR: Providing leadership in the development of animal resources for Africa 1

U.S. Foreign Animal Disease Response Planning is Moving in a New Direction

Why Secure Food Supply (SFS) Plans? Size, structure, efficiency, extensive movement inherent in North American livestock industries will present unprecedented challenges in an FAD outbreak

U.S. Secure Food Supply (SFS) Plans are underway !

Goal of SFS Plans: Business Continuity for U.S. Agriculture

Common Components of Secure Food Supply Plans

Minimize unintended negative effects of disease and disease response, while achieving response goals

Voluntary pre-outbreak preparedness components Biosecurity, surveillance, epidemiology questionnaires, movement permits Proactive risk assessments (completed and in-process) Plans must be based on current capabilities and will evolve with science, risk assessments and new capabilities Guidelines only: Final decisions made by responsible officials during outbreak Outreach and training pre- and postoutbreak

Control or eradicate disease without

Secure Food Supply Plans under development

U.S. FMD response is based on USDA VS guidance

Secure Milk Supply

Foot and Mouth Disease (FMD) Movement of milk

Animal/product movements from concentrated dairy, beef and swine sectors present a huge challenge Mass depopulation unlikely - inadequate resources/political will May take months/years to gain freedom from the disease

Secure Pork Supply

FMD, Classical Swine Fever, African Swine Fever, and Swine Vesicular Disease Movement of animals

Secure Beef Supply

FMD Movement of animals

Secure Egg Supply

High Path Avian Influenza (HPAI) Eggs and egg products

Recent policy enhancements

Secure Turkey Supply HPAI Movement of birds

Continuity of business planning Early consideration of vaccination

Secure Broiler Supply

HPAI Movement of birds, hatching chicks and eggs

There are many tools for the control of FMD

Modern U.S. FMD response plans must be scalable to different size outbreaks FMD response and management strategies: Depend upon the magnitude, location, other characteristics of the outbreak

Biosecurity Quarantine and Managed Movement Stamping Out Slaughter of all clinically affected and in-contact susceptible animals (within 24 hours or as soon as possible) Trace back/Trace forward 2 incubation periods prior to outbreak (OIE incubation period for FMD is 14 days) Rapid Diagnostics Vaccination

Depend on the stage

Vaccinate to kill/slaughter/live 8

If we get FMD, we likely will be 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0

The U.S. National Secure Milk Supply Plan

Days

U.S. Disease Freedom Recognition - Effective Date

OIE Disease Freedom Recognition Length of Outbreak

Japan 2000

United Kingdom 2000

France 2001

United Kingdom 2007

Japan (VAX) 2010

Country, Year of Outbreak

Source: USDA-APHIS FMD Response, Ready Reference Guide Overview of FMD Freedom and Vaccination, March 2013

National SMS Initial Focus: Raw Milk Movement from Farm to Processing

What is the Secure Milk Supply Plan ? U.S. Dairy Industry Continuity of Business Planning for FMD Initial Goal To maintain milk movement in a Foot-and-Mouth Disease (FMD) outbreak and to provide a continuous supply of wholesome milk and milk products for consumers

Public-Private Partnership Industry, State, Federal, Academia

Voluntary

The SMS must accommodate the diversity of the US dairy industry State

Total Milk (million lbs)

Milk Cows (1,000)

# Farms

California

41,256

1,780

1,515

Wisconsin

27,572

1,271

10,860 13.7

New York

13,469

610

5,030

6.69

Idaho

13,431

573

550

6.67

Pennsylvania

10,565

533

7,200

5.25

Top 5 U.S. dairy states, 2013

National and Regional SMS Partners National Partners

% US Milk Production

Industry

20.5

Working groups, topic experts

Academia Iowa State University University of California, Davis University of Minnesota

USDA-APHIS-VS National Preparedness and Incident Coordination (NPIC)

Regional Partners

California Colorado New England States Animal Agricultural Security Alliance (NESAASA) CT, MA, ME, NH, RI, VT

Mid-Atlantic States

VA, MD, TN, NC, SC, DE, WV, NJ, PA

Michigan Pacific Northwest WA, OR

Wisconsin

SMS Plan Components Biosecurity performance standards Dairy premises, milk haulers, processing plants

Pre-event risk assessments Identify needed mitigation steps to minimize FMD virus spread

Milk movement decision support tools Guidance documents Herd monitoring/surveillance tools Handling of milk from FMD infected farms Surge capacity for FMD vaccination

Line of Separation (LOS)

Management Issues in a Large FMD Outbreak

(Milk tanker does not cross in this example)

On-farm calf rearing and management of replacement heifers Enabling other necessary activities (cropping, manure handling, feed, etc.) Milk handling from FMD infected or vaccinated dairies

Farm dedicated hose Licensed weigher/sampler on farm Hauler does not cross LOS

Dairy export loss mitigation opportunities for industry

Wears gloves

Export loss mitigation opportunities for the dairy industry

Bulk tank milk testing strategy for FMD

U.S. Dairy Export Council funded initial review Review conducted by CFSPH, ISU Encouraging industry to pursue evaluation of exported dairy commodities (whey powder, lactose, NDMP and cheeses)

There is a bulk tank milk PCR test for FMDv It is currently undergoing final review at the national level for deployment to selected US NAHLN laboratories The robust industry bulk tank milk sampling program could be leveraged during an outbreak to find FMD cases more quickly Testing strategies for various scenarios are being developed

www.securemilksupply.org

Messaging will be critical to maintaining U.S. consumer confidence

U.S. FMD vaccination contingency

FMD is NOT a public health concern NOT hand-foot-mouth disease that affects children NOT

Industry has invested heavily on crisis communications preparednesswww.FootAndMou th DiseaseInfo.org Industry representatives suggest using term messaging

Hoof

Development of producer training materials

How do we increase availability of emergency vaccine?

A SMS- HMD Emergency Response Plan

It is estimated that $150M over 5 years is needed to enhance the U.S. FMD antigen bank to properly protect the $100B U.S. animal industries

Perimeter Bio-security

Cleaning & Disinfection

Herd Monitoring for Disease

There are technical issues and

Creating a HMD Emergency Response

It is a voluntary program Economics and competing challenges in animal agriculture and government can make sustained FAD planning efforts very challenging Information technology to support management and scalable permitting 83

For More SFS Information

www.securemilksupply.org

http://www.cfsph.iastate.edu/Secure-Food-Supply/index.php

Secure Food Supply Plans USDA Foot-and-Mouth Disease Response Plan "The Red Phases & Types of an FMD Outbreak NAHEMS Guidelines: Continuity of Business NAHEMS guidelines: Vaccination for contagious diseases; Appendix A: Vaccination for Foot-and-Mouth Disease FMD Vaccine Surge Capacity for Emergency Use in the United States Inactivation of Foot-and-Mouth Disease Virus in Milk Products Foot and Mouth Disease in Pigs - Progression of Lesions

FMD Info Dairy Industry Manual Phases and Types Inactivation of FMDV in dairy products Vaccination info FMD Response Plan OIE resources

Acknowledgements

Thank you!

USDA, APHIS, VS funding Industry State and federal government partners Academic partners Iowa State University University of Minnesota 84

ARALLEL he changing disease ̀ landscape and its iplications ̀

The introduction of positively-charged residues at the five-fold axis of foot-and-mouth virus SAT-type capsids enhances infection of cultured cells Melanie Chitray Agricultural Research Council Onderstepoort Veterinary Institute South Africa

CHO-677

Introduction

(13)

A biological characteristic of SAT viruses that complicates protective efficacy of FMD vaccines is the intra- and inter-serotype variability.

CHO

3.1 x 106 5.5 x 104 3.2 x 103

One of the challenges in the control of FMD is to timely and cost-effectively produce vaccines tailored for specific geographical regions that will afford adequate protection against circulating antigenic subtypes of FMDV field strains.

1.24 x 103 9.4 x 103 3.5 x 10 5 2.7 x 10 5

In addition, the production of new vaccines is critically dependant upon adaptation of viruses from the field for growth in BHK-21 cell culture.

1.7 x 105 7.5 x 10 5 1.37 x 105

However, not all field strains can be adapted to suspension BHK-21 cell cultures and do not necessarily produce desirable amounts of stable antigen.

1.05 x 105

The production of custom-engineered FMD vaccines can be facilitated using infectious cDNA technology, which not only makes it possible to engineer chimeric viruses containing the antigenic region of a field strain, but also allows for the introduction of cell-culture receptor binding sites and antigen-stabilising mutations.

1.58 x 105

This will facilitate fast and effective cell-culture adaptation of engineered viruses: Speedy amplification within a few passages in BHK-21 cells to create master virus seed stocks. Circumvents need to isolate on primary cell lines for further adaptation.

Int+, HS+

Int-, HS+ CS+ SA+

HS-

1.02 x 10

Adaptation of field virus (SAT2/Sau/6/00)

Adaptation of SAT viruses to cultured cells

1. Plaque morphology: medium-milky to large clear plaques = indicative of adaptation

SAT1

2. Genetic characterisation: BHK-adapted SAT2/SAU/6/00 vs parental strain BHK-21 P1 region: 5 amino acid substitutions accumulated in encoded proteins

CT1:

VP3 135 175

SAT2

F. F. Maree

VP1 D55N

Capsid protein

Amino acid change

Observed structural effect (predicted 58x BHK-21 model)

VP3

D193N

VP2

T99A

Residues overshadowed by surface loops; Side chains face inwards; not CT1 BHK58 exposed. Minor role in adaptation.

VP1

V50L

Surface exposed; Conservative change. CT = Calf thyroid cells

VP1

D55N

= Babyinhamster Surface exposed;BHK Change local kidney cells surface charge.

VP1

T158K

Surface exposed; Change in local surface charge to strong (+); Cterminal base of GH loop; within known epitope.

V50L

T158K

VP3

VP2

T99A

D193N

Modeled structure of SAT2/SAU/6/00 crystallographic protomer

Courtesy of Abhay Kotecha University of Oxford

Results

AIM

Introduction of inter-serotype vSAT2

VP4

vSAT2

mutations

VP2

VP3

VP1

E135K E135K,E175K 1110

Plaque assays of the pSAT2/Nam/307/98 Mutants VP3 9

2074

LOG TITRE

Effect of the residue changes on cell entry? BHK-21, CHO-K1, CHO-677, CHO-745 cells, CHO-lec2

BHK-21: Integrin+, HS+

6.9 6.1

CHO-K1: HS+, CS+, SA+

CHO-677: HS-, CS+, SA+

CHO-745: HS-, CS-, SA+

2 1

0 BHK-21 cells

VP2

8.4

1112

VP1

To investigate the effect of amino acids identified to enhance cell culture adaptation on intra-serotype Sau) and inter-serotype (vSAT2 (vSAT2 ) FMDV chimeric viruses in cell entry

CHO-K1 cells NAM307/98WT

CHO-677 cells CELL LINES vSAT2NAM1C135

CHO-745 cells vSAT2NAM1C135,175

CHO-lec2 cells

CHO-lec2: HS+, CS+, SA-

Results

Introduction of intra-serotype (vSAT2

VP4

vSAT2

VP2

Sau)

mutations

VP3

VP4

vSAT2

VP1

VP2

Sau)

mutations

VP3

VP1

T158K

Plaque assays of vSAT2/Sau1D158

V50LD55N

Plaque assays of vSAT2/Sau1D50,55

7 6.8 6

7 6

BHK-21:

LOG TITRES

5.2

4 3 2 1 0

0 BHK-21 cells

CHO-K1 cells

CHO-677 cells CELL LINES

vSAT2Sau_WT

CHO-745 cells

CHO-lec2 cells

VP4

HS+

BHK-21: Integrin+, HS+

5.2 4.5

4.1

CHO-677: HS-, CS+, SA+

CHO-745: HS-, CS-, SA+

CHO-lec2: HS+, CS+, SA-

CHO-745:

HS-,

CS-,

CHO-lec2:

HS+,

SA+

CS+,

SA-

CHO-K1 cells

vSAT2Sau1D50,55

CHO-677 cells CELL LINES

vSAT2Sau_WT

Sau)

VP2

VP3

CHO-K1: HS+, CS+, SA+

BHK-21 cells

Introduction of intra-serotype (vSAT2

vSAT2

Integrin+,

LOG TITRE

6.4

mutations

CHO-745 cells

CHO-lec2 cells

vSAT2Sau1D158

Sau)

Introduction of intra-serotype (vSAT2

VP1

VP4

vSAT2

VP2

VP3

mutations

VP1 KGG110-112KRR

E83K

Plaque assays of vSAT2/Sau1D110-112

Plaque assays of vSAT2/Sau1D83 10 7

LOG TITRE

6.6

6.4

BHK-21: Integrin+, HS+

5.2

2 1 0

0 BHK-21 cells

CHO-K1 cells

CHO-677 cells CELL LINES

CHO-745 cells

0 CHO-lec2 cells

BHK-21: Integrin+, HS+

CHO-677: HS-, CS+, SA+ CHO-745: HS-, CS-, SA+

CHO-K1: HS+, CS+, SA+

vSAT2Sau1D83

5.7

5.2

CHO-745: HS-, CS-, SA+

CHO-lec2: HS+, CS+, SA-

CHO-K1: HS+, CS+, SA+ CHO-677: HS-, CS+, SA+

BHK-21 cells vSAT2Sau_WT

8.7

8.6

6.2

LOG TITRE

CHO-K1 cells

CHO-677 cells CELL LINES

vSAT2Sau_WT

CHO-745 cells

vSAT2Sau1D110-112

0 CHO-lec2 cells

CHO-lec2: HS+, CS+, SA-

Summary

Energetically favourable binding site - GRID

The virus strains adapted via frequent cell culture passage resulted in some strains possibly utilising chondroitin sulphate for cell entry and two of these viruses could be using sialic acid as a receptor. The 5 1 and v 5 receptor usage is also unknown. Further investigation is required. The introduction of the positively charged mutations for each of the chimeras, increased the affinity of the mutants to utilise the integrin receptors for cell entry. The change in plaque morphology of the WT vs the chimera viruses observed is indicative of adaptation. Amino acid changes in VP1 positions: T158K, E83K, KGG110-112KRR allows for heparan sulphate usage without the need for consistent cell culture passaging.

This study has shown to be beneficial for SAT type vaccine production where viruses that were previously impossible to adapt to cell culture can be designed with improved growth properties.

VP1 110-112 Heparan Sulphate molecule

Courtesy of Abhay Kotecha University of Oxford

Acknowledgements

Dr Francois Maree

Prof Jacques Theron

A THIAZEPINO[4,5-a]BENZIMIDAZOLE DERIVATIVE INHIBITS THE IN VITRO REPLICATION OF EURASIAN SEROTYPES OF FOOT-AND-MOUTH DISEASE VIRUS

Dr Sonja Maree

David Lefebvre Scientist, CODA-CERVA, Belgium Dr Peninnah Nsamba

Emergency vaccination against FMD

Alternative strategies Interferons (Dias et al, J Interferon Cytokine Res, 2011; Perez-Martin et al, J Virol, 2012)

Council Directive 2003/85/EC

CpG oligonucleotides (Kamstrup et al, Antiviral Res, 2006)

Emergency FMD vaccines:

Poly IC

Induce protective, neutralizing antibodies 4 to 7 days post

(Dias et al, J Interferon Cytokine Res, 2012)

RNA or DNA(-like) interference

Serotype- and subtype-specific (7 FMDV serotypes, multiple subtypes)

(Chen et al, J Virol, 2006; Vagnozzi et al, J Virol, 2007; Kim et al, Antiviral Res, 2008)

Small chemical molecules (Furuta et al, Antiviral Res, 2009; Lefebvre et al, Transbound Emerg Dis, 2013; Rai et al, Antiviral Res, 2013; De Vleeschauwer et al, Transbound Emerg Dis, 2014)

Combined therapy (Kim et al, Antiviral Res, 2012) 2

Reduction of CPE in SK-6 cells

Virus strain

EC50 (

)

EC90 (

)

FMDV O1/MAN/TUR/69

15.6 8.6

44.2 14.3

FMDV A22/IRQ/24/64

18.4 8.2

41.1 4.3

FMDV A/IRN/11/96

8.0 1.5

28.5 16.7

FMDV C1/Noville/SWI/65

39.7 27.8

58.8 32.9

FMDV Asia1/Shamir/ISR/89

51.4 27.1

83.8 39.6

FMDV Asia1/CAM/9/80

26.6 5.8

51.2 11.6

FMDV SAT1/ZIM/25/89

>100

FMDV SAT2/ZIM/3/97

>100

FMDV SAT3/ZIM/4/99

>100

SVDV UKG/27/72

>100

Dose-activity curve for FMDV RNA reduction

Effect of delayed addition on FMDV RNA levels Viral RNA (% of virus control at 6 hpi)

% Inhibition of viral RNA (relative to the virus control)

120 100 80 60 40 20 0 50

2.5

1.25

120 100 80 60 40 20 0 0

Concentration of lead compound ( M)

Time point of addition (hpi)

Conclusions

Acknowledgements Annebel De Vleeschauwer Nesya Goris Steven Van Borm Kris De Clercq

The present study further illustrates the potential of small molecule inhibitors to interfere with the replication cycle of FMDV

Johan Neyts

Further research and development are ongoing

Alba Chimirri Anna Maria Monforte

Begona Valdazo-Gonzalez Don King

Agenda LAMP presentation LAMP characteristics and mechanism of action Application for Identification of strains in MERIAL active ingredient

Loop Mediated Isothermal amplification (LAMP): application for MERIAL vaccine identification

Application for identification of strains in MERIAL vaccine Conclusion

Marc FIORUCCI, PhD MERIAL, France

Loop Mediated Isothermal Amplification

Loop Mediated Isothermal Amplification: Primers 4 Primers recognizing 6 different region (minimun)

Tsugunori Notomi et al. Eiken Chemicals Japan in 2000 (NAR 2000)

F1c

°C) (No thermocycler required)

FIP

F3 Primers

F2 LF

Primers: 4 (or 6) different primers with high specificity and particularities

F3c

F2c

F1c

Target DNA

B1c

B2c

B3c B3

Specific enzyme BST:

DNA polymerase DNA dependent High strain displacement capacities No exonuclease -5

B3 Primers B2

FIP/BIP: Inner Primers: Ends should not be AT-rich

B1c BIP

-65°C

Applications:

Primers purity could be crucial-HPLC-purified FIP and BIP recommended

LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases It helps in the identification of genus and species-specific parasites

[FIP or BIP]>[F3-B3]

Loop Mediated Isothermal Amplification: Read out

Loop Mediated Isothermal Amplification:

Generation of amplicon with different sizes: inverted repeats of the target and cauliflower-like structures with multiple loops

Agarose Gel 2% dsDNA Dye SYBR Green Typical LAMP amplicon product

J. Trop. Med. Hyg. (2011)

Implementation of LAMP on Active Ingredient (AI)

MERIAL FMDV Vaccine FMDV- Eu MA: double oil emulsion vaccine: Mono, bi or tri valent

Method: Extraction RNA

LAMP

Asia 1 Shamir O1 BFS, O1 Manisa, O Tawain A Turkey, A 24 Cruzeiro, A 22 Irak

Is LAMP usable for identity test? Primers design in variable region: VP1 gene

Specificity of Asia 1 Shamir is demonstrated 8

Implementation of LAMP on Active Ingredient firstly

Implementation of RT-LAMP on Active Ingredient AMV Reverse Transcriptase: 60 minutes at 42°C

Method

46°C 48°C 50°C 53°C 57°C 61°C 63°C 64°C

Extraction RNA from AI

RT Thermoscript: Avian reverse transcriptase with high thermal stability (up to 70°C)

RT Mix O1BFS

Mix O1 Manisa

Mix O Taïwan

LAMP LAMP Asia 1 Shamir Specificity for all strain demonstrated

RT-LAMP one step Is feasible

RT-LAMP Asia1 Shamir Mix A22 Irak

Mix A 24 Cruzeiro

Mix A Turkey

RT-LAMP on vaccine with new Read Out Negative

Positive

RT-LAMP on vaccine without genome extraction

Negative Positive

MERIAL Vaccine Monovalent

RT LAMP

MERIAL Vaccine Trivalent Visually Naked eye

Neg ctl

(orange)

Asia 1

+ (yellow)

Fluorescence

RT-LAMP Asia 1 Shamir RNA extraction

7 RT-LAMP

identification

O1 BFS

A24 Cruzeiro

A22 Irak

O1 Manissa

O Tawain

A Turkey

95°C 5min

RT-LAMP

identification

Extraction, RT, LAMP, agarose gel read out (4 hours )

Boiling, RT-LAMP, SYBR read out (1 hour)

100

Conclusion

Acknowledgement

High specificity demonstrated between MERIAL strains: O1 Manisa, O1 BFS, O Taïwan/A 22Irak, A24 Cruzeiro, A Turkey/Asia 1 shamir

Marc

lab

LAMP and RT LAMP can be run together by using RT Thermoscript

Marc Cyril

RT-LAMP is completed in 1 hours on MERIAL double oil emulsion vaccine Without any genomic extraction

Guillaume

Stéphanie Sandra

Céline

Delphine

Powerful tool for vaccine identification and easily transferable to QC department

Viral capsid 60 copies of each of the structural proteins

Identification of novel antibody binding determinants of serotype O Foot-andmouth disease virus

Five protomers make a pentamer Twelve pentamers make the capsid b)

Mana Mahapatra, S. Upadhyaya, A. P. Babu, A. S. Asfor, D. J. Paton and Satya Parida 3x

101

VP1- blue VP2- red VP3- green

Reactivity of 5M virus with mAbs Dunn et al (1998)

A mutant virus (5 sites mutant- 5M) resistant to neutralising murine monoclonal antibodies (Mabs) also resists neutralisation by bovine polyclonal sera

mAb

Cross-challenge studies (GP) - virulent wild-type and mutant antigen as inactivated whole virus vaccine: all animals protected

Main aim: 1 8 1 8 9 1

7 74 2

1 74 1 1 88 4 5 9 0

1 3 8

Critical residue

O Lausanne murine mAbs B2 (Site-1) VP1 144, 148, 150 D9 (Site-1) VP1 148 C6 (Site-2) VP2 73 C8 (Site-3) VP1 43, 44 C9 (Site-2) VP2 73 14 EH9 (Site-4) VP3 58 OC3 (Site-5) VP1 149 O Manisa murine mAbs

4 3

Unidentified epitopes: neutralising/non-neutralising

Optical density

5M- O1K cDNA clone

rO1K-wt

SA85 (Site-2 and 5) VP2 72, VP1 149 SA107 (Site-2) VP2 73 SA113 (Site-2) VP2 72 SA127 (Site-2) VP2 73 O Lausanne bovine mAbs C2 (Site-2) VP2 188

8 5 5 8

Define viral determinants of antibody mediated protection

C96 (Site-2 and 4) MH5 (Site-3)

Development of novel broadly cross-reactive vaccines and sequence-based vaccine selection methods

100

Optical density (%)

Background

VP2 78, VP3 58 VP1 46

80 60 40 20 0 SA85

SA 107

SA113

SA127

C96

MH5

Neutralising antigenic site 2: immunodominant (Mahapatra et al., 2012)

VP2 sequence- Site 2 O Kau/66/X00871 O/BFS/AY593815 O/Lau/65 O/Manisa/69/AJ251477 O/Caseros/U82271 O/Campos/AJ320488.1 O/TAW/97/ AF154271 O/HKN/06/83 O/TUR/03/87 O/ITL/01/93 O/BUL/01/93 O/PER/06/94 O/TUR/03/94 O/PHI/02/95 O/PHI/09/95 O/ALG/02/99 O/China/01/99/AF506822.2 O/JPN/2000/AB079061.1 O/SAR/19/2000/AJ539140.1 O/SKR/2000/AY312587.1 O/UKG/35/2001/AJ539141 O/HKN/2002/AY317098.1 O/SKR/2002/AY312589.1 O/UGA/03/2002 O/UGA/07/2003/EU919243 O/HKN/03/2004 O/ETH/03/2004 O/VN/SL22/2006/GU125647 O/YEM/04/2006 O/UAE/04/2009 O/KUW/02/2006 O/UAE/03/2008 O/VN/QB88/2009/GU582115 O/SKR/04/2010

66 75 85 ....|....|....|....| LFDWVTSDSFGRCHLLELPT .................... ............Y....... ........P........... ........P........... .................... ........P........... ........P........... ........R........... ........P........... ........R........... ........P........... ........R........... ........P........... ........P........... ........P........... ......G.P....Y...... ........P........... ........P...-Y...... ........P....Y...... ........P....Y...... ....G.N......Y...... ........P....Y...... ........P........... .................... ....G.N............. ........P........... .................... .................... ........P........... ........P........... ........P........... ........P........... ........P...Y.......

181 190 200 ....|....|....|....|....| VMVVAPLTVNTEGAPQIKVYANIAP ......................... ......................... .........KS.............. ......................... ......................... ..........N.............. ..........N.............. ......................... ......................... ..........N.............. ..............A.......... ..........N.............. ..........N.............. ..........N.............. ..........N.............. ......................... ......................... ......................... ......................... ......................... ..........N.............. ......................... ......................... ..........N.............. ..........N.............. ......................... ......................... ..........N.............. ..........S.............. ..........S.............. ..........S.............. ......................... .........................

Site 1

Antigenic sites/mut ants

Site 2

Site 3

Site 4

Site 5

Additional mutations

Viral protein positions

VP1 138

VP1 148

VP1 150

VP2 72

VP1 43

VP3 58

VP1 149

VP3 85

VP2 74

VP2 191

O1K-wt 5M 5M1 5M2 5M3 5M4 5M5 5M1/2 DM1/3 DM2/5

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

VP2: 74 and 191 VP3: 85

10 different recombinant viruses

VP274

102

VP385

VP2191 O1 K reduced structure showing position of critical residues of 5 neut. Antigenic sites (shown in blue) and additional residues mutated in this study (shown in green/pink/red).

Reactivity with bovine mAbs

* Optical density (%)

% reduction in VN titre

Neutralising Ab

Bovine antisera

Functional relationship between VP2 74 and 191

VP2 191 could be an epitope

Context FMDV is able to cause persistent infection in 15-50% of ruminants. can occur in both initially naïve and vaccinated animals. can be maintained for many months (up to 2 years in cattle) -level production of virus in pharyngeal tissues Potential risk for transmission of virus to susceptible animals.

Establishment of a persistent FMD virus infection in MDBK cells

Molecular Mechanisms of viral persistence and determining factors remain unknown.

Aim

Lela Kopliku & Sandra Blaise-Boisseau

To identify the viral and cellular factors involved in the establishment and maintenance of FMDV persistence

ANSES, Animal Health Laboratory Virology Unit (ANSES-INRA-ENVA joint research unit)

103

Development of a relevant in vitro bovine cellular model of FMDV persistence 2

MDBK cells persistently infected with FMDV O/FRA/1/2001

Choice of the host cell line: MDBK cells

MDBK cells were maintained during 42 passages post initial infection (representing 5 months of culture) Viral RNA and viral proteins are still detected in highly passaged cells Evidence of infectious particles produced by MDBKp cells

MDBK cells are permissive to FMDV O/FRA/1/2001 infection Regrowth of infected MDBK monolayers (48-72 h p.i.) Viral antigen detection in reconstituted monolayers 72 h p.i.

MDBK

Mock

BHK-21

24 h p.i.

48 h p.i.

72 h p.i.

Conclusion & Perspectives

Aknowledgments « BIOPIC » Team

Establishment of a FMDV persistent infection in a bovine cell line. Viral Such cells could constitute a useful tool to study virus-host interplay and understand mechanisms of the carrier state.

Anthony Relmy

Kamila Gorna

Characterization of persistent viruses will be continued

Aurore Romey

Lela Kopliku

To investigate mutations linked to persistence

Labib Bakkali Kassimi

104

Stephan Zientara & All members of the Virology Unit 6

Objectives Two established cell lines, ZZ-R 127 and LFBKgroups to have highly sensitivity to FMDVs.

v 6,

were recently reported by different research

The groups individually evaluated the susceptibility of both the cell lines using cell culture supernatants and epithelial suspensions. However, it is generally difficult to collect fresh epithelial and vesicular materials in the field. Therefore, it is important to evaluate any primary cells and cell lines for virus isolation using clinical samples, which can be collected more easily than epithelial materials. However, the susceptibility of the LFBK- v 6 cells to virus isolation has never been evaluated using the clinical samples other than epithelial materials. Therefore, to evaluate the susceptibility of LFBK- v 6 cells to virus isolation was the first objective of this study.

Comparative utility of the fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK- v 6 for virus isolation from clinical samples collected from animals experimentally infected with a foot-and-mouth disease virus

The susceptibility levels of both the cell lines have never been compared under the same conditions. Therefore, to compare the susceptibility levels of both the cell lines to FMDV isolation was the second objective of this study.

K. Fukai, K. Morioka, M. Yamada, K. Yoshida, T. Nishi, R. Kitano, R. Yamazoe, T. Kanno

D.V.M., Ph.D., Exotic Disease Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization

Results

Discussion

A total of 481 samples were collected from experimentally infected cattle. Viruses were isolated from 50 (10.4%) and 59 (12.3%) of the samples by the ZZ-R 127 and LFBK- v 6 cells, respectively. The virus isolation rates for both the cell lines were not different statistically.

Although slight difference in virus isolation rates was observed between both the cell lines across the animal species from which the samples were collected, viruses were isolated sufficiently from the samples by both the cell lines. The availability of the LFBK- v 6 cells for FMDV isolation from clinical samples other than epithelial suspensions was confirmed in this study.

In total, 292 and 372 samples were collected from experimentally infected goats and pigs, respectively. As with the experimental infections using cattle, viruses were isolated from approximately the same number of samples by both the cell lines. The virus isolation rates were not different statistically.

The virus isolation rates by both the cell lines were not different statistically. However, there were several samples from which viruses were isolated only by one or the other of the cell lines. Therefore, diagnostic work for FMD will be improved sufficiently by using a combination of both the cell lines.

105

Purpose of PTS

Ensure countries are able to correctly identify FMD in their country Achieved by feedback on PTS, training and consultation

Observations from the 2013 Proficiency Testing Study (PTS)

Harmonisation amongst reference laboratories Presenting results on how reference laboratories compare, distribution of reagents

Anna Ludi*,

Yanmin Li, Ginette Wilsden, Valerie Mioulet, Bryony Armson, Kelly Adams, Trish Ryder, Sarah Belgrave, Jeffrey Hammond, Donald P. King * Manager of the Serum Assay Unit within the VDRL, The Pirbright Institute

Differentiation between SVDV and FMDV 1

Participants

56 countries worldwide (26 National Reference Laboratory for EU)

106

Panel 1 - Virus Isolation n = 108 tests

Graphs Explained

Agreement

No Agreement

6 samples 18 laboratories

Reference Testing

Agreement

positive

10X testing negative

positive

negative

1 + 1 inconclusive

negative

10X testing positive

Participants

positive negative

Results from participating labs

Reference testing

*4 SVD samples not tested

Panel 1 Antigen ELISA n = 108 tests

6 samples 17 laboratories

positive

*4 SVD samples not tested

Reference testing

107

positive

negative

positive

negative

Participants

positive

negative

Participants

Reference testing

negative

6 samples 18 laboratories

Panel 1 rRT-PCR n = 102 tests

*4 SVD samples not tested

Panel 2 rRT-PCR n = 352 tests

Panel 2

8 samples 44 laboratories

80 60 40 20

rRT-PCR

Antigen ELISA

negative

positive

100

positive

290

negative

Reference testing

Participants

% of laboratories (n=56)

Non-infectious material for virus genome/antigen detection

9 SVD sample *3 SVD samples not tested

Panel 2 Antigen ELISA n = 328 tests 8 samples 41 laboratories

Panel 3 Reference testing

248 Inconclusive 7 Mistype 7

Non-infectious material for serology testing for nonstructural and structural proteins.

negative

100

% of laboratories

positive negative

Participants

positive

60 40 20

16 SVD sample

NSP

LPBE

SPBE

Priocheck O

VNT

0 *3 SVD samples not tested

108

Panel 3 NSP ELISA n = 424 tests

Panel 3 LPBE ELISA n = 192 tests 8 samples 24 laboratories

Inconclusive 5

253

positive

Participants

positive

146

negative

Participants

positive

Reference testing

positive

Reference testing

negative

8 samples 53 laboratories

101 Inconclusive 10 Mistype 9

negative

0 46

*1 laboratory all inconclusive

Panel 3 SPBE ELISA n = 120 tests

8 samples 20 laboratories

61 Inconclusive 9 Mistype 4

negative

Reference testing

Participants

positive negative

Participants

positive

28 109

positive

negative

positive

Reference testing

Inconclusive 7

Mistype 1

negative

8 samples 15 laboratories

Panel 3 PrioCheck O ELISA n = 160 tests

*6 Asia 1 samples correctly identified

Panel 3 VNT n = 160 tests

Conclusions for Panel 1-3:

8 samples 20 laboratories

Reference testing

positive

96 Inconclusive 8 Mistype 4

negative

Participants

positive

Panel 1 - The 5

negative

rRT-PCR was unable to detect a SAT 2 sample.

Panel 2 - Certain laboratories do not have the antigen ELISA available for the SAT serotypes.

Panel 3 - Cross-reactivity and mis-typing was seen with different serological tests. It is suggested that multiple tests are used to derive the final conclusion. - Not all labs could determine if an animal was vaccinated.

Laboratories had an increase chance of correctly identifying a sample by using multiple tests per sample

Acknowledgement: Central Service Unit (CSU) ***Sheila Wilsden

Development & validation of confirmatory NSP antibody ELISAs to detect infection in vaccinated animals (DIVA)

European Unit, FAO, OIE and DEFRA

YOU!!! *** 2014 Proficiency Testing Scheme under way anna.ludi@pirbright.ac.uk

A.Tewari, H. Ambrose, A. Di Nardo, K.G. Parekh, T. Inoue, J. Guitian, D.J. Paton, S.Parida

110

Sera used to estimate diagnostic specificity

Sera used to estimate diagnostic sensitivity

991 naïve cattle sera from Italy

A.) To detect seroconversion or infection Group

Number of animals

Number of samples

1.) Unvaccinated infected recovered

a.) Contact challenged

b.) Needle challenged

2.) Vaccinated infected recovered

185

261

a.) Contact challenged

130

b.) Needle challenged

131

Group

Number of animals

Number of samples

1.) Unvaccinated infected carriers

a.) Contact challenged

b.) Needle challenged

2.) Vaccinated infected carriers

154

a.) Contact challenged

b.) Needle challenged

130 vaccinated cattle sera

Six in-house tests developed and validated

B.) To detect carriers

2B and 3B peptide tests 3ABC, 3D, 3CD, 2C proteins

36 Bovine NSP panel sera, 159 clinically infected field sera

Detection of infection and/or carrier status in unvaccinated infected cattle Test N AUC [95% CI 1) Unvaccinated infected recovered Prionics 1038 1.00[0.99-1.00] 2B 1038 0.99[0.99-1.00] 3B 1038 0.99[0.99-0.99] 3ABC 1038 0.99[0.99-1.00] 3D 1038 0.99[0.99-0.99] 3CD 1038 0.99[0.99-0.99] 2C 1038 0.85[0.79-0.89] a.) Contact challenge Prionics 1022 1.00[0.99-1.00] 2B 1022 0.99[0.99-1.00] 3B 1022 0.99[0.99-0.99] 3ABC 1022 0.99[0.99-1.00] 3D 1022 0.99[0.99-0.99] 3CD 1022 0.99[0.99-0.99] 2C 1022 0.85[0.79-0.91] b.)Needle challenge Prionics 1007 0.99[0.99-1.00] 2B 1007 0.99[0.99-1.00] 3B 1007 0.99[0.99-1.00] 3ABC 1007 0.99[0.99-1.00] 3D 1007 0.99[0.99-0.99] 3CD 1007 0.99[0.99-1.00] 2C 1007 0.85[0.74-0.96] 2.) Unvaccinated infected carrier Prionics 1013 1.00[0.99-1.00] 2B 1013 0.99[0.99-1.00] 3B 1013 0.99[0.99-1.00] 3ABC 1013 0.99[0.99-0.99] 3D 1013 0.99[0.98-0.99] 3CD 1013 0.99[0.99-1.00] 2C 1013 0.77[0.68-0.87] a.) Contact challenge Prionics 1007 1.00[1.00-1.00] 2B 1007 0.99[0.99-1.00] 3B 1007 0.99[0.99-1.00] 3ABC 1007 0.99[0.99-1.00] 3D 1007 0.99[0.98-0.99] 3CD 1007 0.99[0.99-1.00] 2C 1007 0.79[0.70-0.87] b.) Needle challenge Prionics 997 0.99[0.99-1.00] 2B 997 0.99[0.99-1.00] 3B 997 0.99[0.99-1.00] 3ABC 997 0.99[0.99-1.00] 3D 997 0.99[0.99-1.00] 3CD 997 0.99[0.99-1.00] 2C 997 0.73[0.46-1.00]

Sp )

LR+

LR-

Ref 0.59 0.13 0.33 0.21 0.21 0.00

Se ) 100.00 100.00 100.00 100.00 97.87 97.87 82.98

99.39 99.10 98.39 99.09 97.17 98.59 76.69

165.16 110.11 61.93 110.11 34.63 64.66 3.55

0.00 0.00 0.00 0.00 0.02 0.02 0.22

Ref 0.87 0.82 0.87 0.81 0.81 0.17

Ref 98.75 98.17 98.75 96.82 98.07 76.69

Ref 0.69 0.13 0.41 0.32 0.23 0.00

100.00 100.00 100.00 100.00 96.77 96.77 80.65

99.39 99.10 98.39 99.09 97.17 98.59 76.69

165.16 110.11 61.93 110.11 34.25 63.93 3.45

0.00 0.00 0.00 0.00 0.03 0.03 0.25

Ref 0.82 0.85 0.82 0.82 0.83 0.11

Ref 98.73 98.14 98.73 96.77 98.04 76.52

Ref 0.80 1.00 0.36 0.13 1.00 0.00

100.00 100.00 100.00 100.00 100.00 100.00 87.50

99.39 99.10 98.39 99.09 97.17 98.59 76.69

165.16 110.11 61.93 110.11 35.39 66.06 3.75

0 0 0 0 0 0 0.16

Ref 0.90 0.81 0.90 0.88 0.81 0.07

Ref 98.71 98.11 98.71 96.82 98.11 76.56

Ref 0.46 0.32 0.27 0.21 0.40 0.000

100.00 100.00 100.00 100.00 95.45 95.45 68.18

99.39 99.10 98.39 99.09 97.17 98.59 76.69

165.16 110.11 61.93 110.11 33.78 63.06 2.92

0.00 0.00 0.00 0.00 0.04 0.04 0.41

Ref 0.86 0.88 0.86 0.84 0.86 0.06

Ref 98.72 98.12 98.72 96.74 98.03 76.21

Ref 0.60 0.43 0.36 0.10 0.62 0

100.00 100.00 100.00 100.00 93.75 93.75 68.75

99.39 99.10 98.39 99.09 97.17 98.59 76.69

165.16 110.11 61.93 110.11 33.18 61.93 2.94

0 0 0 0 0.06 0.06 0.40

Ref 0.82 0.81 0.83 0.86 0.89 0.041

Ref 98.71 98.11 98.71 96.72 98.01 76.27

Ref 0.81 1.00 0.59 1.00 1.00 0.35

100.00 100.00 100.00 100.00 100.00 100.00 66.67

99.39 99.10 98.39 99.09 97.17 98.59 76.69

165.16 110.11 61.93 110.11 35.39 66.06 2.86

0 0 0 0 0 0 0.43

Ref 0.87 0.87 0.87 0.86 0.87 0.012

Ref 98.70 98.09 98.70 96.79 98.09 76.33

Detection of infection and/or carrier status in vaccinated infected cattle

Agreement Test

111

AUC [95% CI

1.) Vaccinated infected recovered Prionics 1252 0.92[0.90-0.95] 2B 1252 0.83[0.80-0.87] 3B 1252 0.89[0.86-0.91] 3ABC 1252 0.79[0.75-0.83] 0.92[0.90-0.94] 3D 1252 0.92[0.90-0.94] 3CD 1252 0.59[0.54-0.63] 2C 1252 a.) Contact challenge Prionics 1121 0.85[0.80-0.90] 2B 1121 0.70[0.64-0.77] 3B 1121 0.78[0.72-0.84] 3ABC 1121 0.68[0.61-0.75] 0.87[0.84-0.91] 3D 1121 0.87[0.83-0.91] 3CD 1121 0.48[0.42-0.54] 2C 1121 b.)Needle challenge 1122 0.99[0.98-1.00] Prionics 0.96[0.95-0.98] 2B 1122 3B 1122 0.98[0.96-0.99] 3ABC 1122 0.92[0.88-0.96] 3D 1122 0.97[0.96-0.98] 3CD 1122 0.97[0.96-0.98] 2C 1122 0.70[0.65-0.75] 2.) Vaccinated infected carrier 0.96[0.94-0.98] Prionics 1145 0.90[0.87-0.93] 2B 1145 0.90[0.87-0.94] 3B 1145 3ABC 1145 0.89[0.85-0.93] 3D 1145 0.92[0.90-0.94] 3CD 1145 0.93[0.90-0.95] 2C 1145 0.68[0.63-0.72] a.) Contact challenge 0.93[0.89-0.98] Prionics 1065 0.82[0.76-0.88] 2B 1065 0.83[0.77-0.89] 3B 1065 3ABC 1065 0.82[0.76-0.89] 3D 1065 0.86[0.82-0.90] 3CD 1065 0.88[0.84-0.92] 2C 1065 0.59[0.52-0.65] b.) Needle challenge 0.99[0.97-1.00] Prionics 1071 0.97[0.95-0.99] 2B 1071 0.97[0.95-0.99] 3B 1071 0.95[0.91-0.99] 3ABC 1071 3D 1071 0.97[0.96-0.98] 3CD 1071 0.97[0.95-0.99] 2C 1071 0.76[0.71-0.81]

Agreement %

Se (%)

Sp (%)

LR+

LR-

Ref 0.00 0.00 0.00 1.00 1.00 0.00

82.49 59.14 72.37 59.92 66.15 57.59 43.19

99.39 99.10 98.39 99.09 97.17 98.59 76.69

118.55 66.10 42.82 60.28 19.57 30.49 1.872

0.17 0.41 0.28 0.40 0.35 0.43 0.73

Ref 0.74 0.85 0.77 0.67 0.66 0.18

Ref 93.51 95.96 94.14 91.05 91.29 71.65

Ref 0.00 0.01 0.00 1.00 1.00 0.00

69.83 37.93 51.72 39.66 43.97 35.34 29.31

99.39 99.10 98.39 99.09 97.17 98.59 76.69

116.96 42.35 32.48 44.28 12.99 18.69 1.25

0.30 0.62 0.49 0.60 0.58 0.65 0.92

Ref 0.61 0.71 0.63 0.38 0.40 0.04

Ref 95.45 96.25 95.63 91.44 92.77 73.42

Ref 0.10 0.20 0.06 0.06 0.09 0.00

93.89 82.44 90.84 80.92 87.02 78.63 57.25

99.39 99.10 98.39 99.09 97.17 98.59 76.69

155.08 102.12 56.26 89.09 30.79 55.65 2.47

0.06 0.17 0.09 0.19 0.13 0.21 0.55

Ref 0.83 0.88 0.86 0.81 0.79 0.20

Ref 96.88 97.59 97.33 95.99 96.08 74.42

Ref 0.00 0.00 0.00 0.01 0.06 0.00

90.26 70.13 75.97 70.78 61.04 56.49 49.35

99.39 99.10 98.39 99.09 97.17 98.59 76.69

149.07 86.87 47.05 77.93 21.60 39.98 2.13

0.09 0.30 0.24 0.29 0.40 0.44 0.65

Ref 0.77 0.81 0.81 0.64 0.64 0.17

Ref 95.55 96.07 96.24 92.75 93.19 73.28

Ref 0.00 0.00 0.00 0.06 0.18 0.00

87.84 50.00 58.11 51.35 35.14 31.08 35.14

99.39 99.10 98.39 99.09 97.17 98.59 76.69

145.07 55.05 35.99 56.54 12.89 20.53 1.50

0.12 0.50 0.42 0.49 0.66 0.69 0.84

Ref 0.62 0.65 0.63 0.37 0.38 0.03

Ref 96.15 96.15 96.24 93.15 94.08 73.62

Ref 1.00 0.49 0.42 0.25 0.70 0.00

92.50 88.75 92.50 88.75 85.00 80.00 62.50

99.39 99.10 98.39 99.09 97.17 98.59 76.69

152.78 109.93 57.29 97.72 30.08 56.62 2.70

0.07 0.11 0.07 0.11 0.15 0.20 0.48

Ref 0.84 0.85 0.89 0.73 0.76 0.18

Ref 97.85 97.85 98.51 96.08 96.83 75.84

Detection of infection in vaccinated infected cattle using Bovine NSP panel Test Prionics 2B 3B 3ABC 3D 3CD 2C

N 1027 1027 1027 1027 1027 1027 1027

AUC [95% CI 0.99[0.99-1.00] 0.99[0.99-0.99] 0.96[0.91-1.00] 0.96[0.92-1.00] 0.91[0.84-0.98] 0.94[0.89-0.98] 0.78[0.70-0.85]

p Se (%) Ref 91.67 1.00 91.67 0.80 88.89 0.67 75.00 0.09 72.20 0.06 72.20 0.00 63.80

Sp (%) 99.39 99.10 98.39 99.09 97.17 98.59 76.69

LR+ 151.40 113.55 55.05 92.90 61.17 66.85 2.97

LR0.08 0.08 0.11 0.25 0.45 0.53 0.40

Ref 0.77 0.70 0.68 0.50 0.60 0.10

Detection of infection in cattle in field

Agreement(%) Ref 98.34 97.66 97.86 95.81 97.08 76.24

Non-parametric ROC curve comparison of in-house tests & Prionics 3ABC test

Test Prionics 2B 3B 3ABC 3D 3CD 2C

Non-parametric ROC curve comparison of in-house tests & Prionics 3ABC test

AUC [95% CI 0.99[0.98-0.00] 0.98[0.97-1.00] 0.98[0.97-0.99] 0.98[0.97-0.99] 0.87[0.83-0.92] 0.84[0.79-0.89] 0.76[0.69-0.83]

p Ref 1.00 1.00 0.73 0.00 0.00 0.00

Se (%) 96.86 96.23 97.48 96.23 75.47 71.07 81.76

Sp (%) 99.39 99.10 98.39 99.09 97.17 98.59 76.69

LR+ 159.97 105.95 60.37 105.95 26.71 46.95 3.50

LR0.03 0.03 0.02 0.03 0.25 0.29 0.23

Ref 0.93 0.92 0.94 0.74 0.75 0.37

Agreement(%) Ref 98.43 98.26 98.61 94.09 94.61 77.39

Performance of joint-simultaneous testing with the PrioCHECK® FMDV NS test for vaccinated infected animals

Performance of joint-simultaneous testing with the PrioCHECK® FMDV NS test for Unvaccinated infected animals Serial Testing Test Se Sp 1.) Unvaccinated infected recovered Prionics Ref Ref 2B 0.93[0.82-0.99] 0.99[0.99-1.00] 3B 0.93[0.82-0.99] 0.99[0.99-1.00] 3ABC 0.93[0.82-0.99] 0.99[0.99-1.00] 3D 0.92[0.82-0.98] 0.99[0.99-1.00] 3CD 0.92[0.82-0.98] 0.99[0.99-1.00] 2C 0.80[0.72-0.84] 0.99[0.99-0.99] a.) Contact challenge Ref Ref Prionics 0.93[0.81-0.99] 0.99[0.99-1.00] 2B 0.93[0.81-0.99] 0.99[0.99-1.00] 3B 0.93[0.81-0.99] 0.99[0.99-1.00] 3ABC 0.92[0.81-0.97] 0.99[0.99-0.99] 3D 0.92[0.81-0.97] 0.99[0.99-1.00] 3CD 0.77[0.69-0.84] 0.99[0.99-0.99] 2C b.) Needle challenge Ref Ref Prionics 0.93[0.81-0.99] 0.99[0.99-1.00] 2B 0.93[0.81-0.99] 0.99[0.99-0.99] 3B 0.93[0.81-0.99] 0.99[0.99-1.00] 3ABC 0.93[0.81-0.99] 0.99[0.99-0.99] 3D 0.93[0.81-0.99] 0.99[0.99-1.00] 3CD 0.83[0.73-0.91] 0.99[0.99-0.99] 2C 2.) Unvaccinated infected carriers Prionics Ref Ref 2B 0.93[0.81-0.99] 0.99[0.99-1.00] 3B 0.93[0.81-0.99] 0.99[0.99-1.00] 3ABC 0.93[0.81-0.99] 0.99[0.99-1.00] 3D 0.91[0.81-0.96] 0.99[0.99-0.99] 3CD 0.91[0.81-0.96] 0.99[0.99-1.00] 2C 0.65[0.54-0.73] 0.99[0.99-0.99] a.) Contact challenge Ref Ref Prionics 0.93[0.81-0.99] 0.99[0.99-1.00] 2B 0.93[0.81-0.99] 0.99[0.99-1.00] 3B 0.93[0.81-0.99] 0.99[0.99-1.00] 3ABC 0.90[0.81-0.95] 0.99[0.99-0.99] 3D 0.90[0.81-0.95] 0.99[0.99-1.00] 3CD 0.65[0.54-0.74] 0.99[0.99-0.97] 2C b.) Needle challenge Ref Ref Prionics 0.94[0.81-0.99] 0.999[0.99-1.00] 2B 0.94[0.81-0.99] 0.99[0.99-0.99] 3B 0.94[0.81-0.99] 0.99[0.99-1.00] 3ABC 0.93[0.81-0.99] 0.99[0.99-0.99] 3D 0.93[0.81-0.99] 0.99[0.99-1.00] 3CD 0.64[0.55-0.71] 0.99[0.99-0.97] 2C

N 159 159 159 159 159 159 159

Parallel Testing

Ref 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.97-0.99]

Ref 0.97[0.95-0.99] 0.97[0.99-1.00] 0.99[0.99-1.00] 0.95[0.93-0.97] 0.97[0.94-0.98] 0.75[0.73-0.78]

Ref 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.97-0.98]

Ref 0.97[0.96-0.99] 0.97[0.95-0.98] 0.97[0.96-0.99] 0.96[0.94-0.97] 0.97[0.95-0.98] 0.76[0.73-0.78]

Ref 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.97-0.99]

Ref 0.98[0.96-0.98] 0.97[0.95-0.98] 0.98[0.96-0.98] 0.96[0.94-0.97] 0.97[0.95-0.98] 0.76[0.73-0.78]

Ref 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.98-1.00] 0.99[0.98-1.00] 0.99[0.95-0.99]

Ref 0.97[0.95-0.98] 0.97[0.95-0.98] 0.97[0.95-0.98] 0.95[0.93--.97] 0.97[0.95-0.98] 0.76[0.73-0.78]

Ref 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.98-0.99] 0.99[0.98-0.99] 0.99[0.95-0.99]

Ref 0.98[0.96-0.99] 0.97[0.95-0.98] 0.98[0.96-0.99] 0.96[0.94-0.97] 0.97[0.95-0.98] 0.76[0.73-0.78]

Ref 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.99-1.00] 0.99[0.95-0.99]

Ref 0.98[0.96-0.98] 0.97[0.95-0.98] 0.98[0.96-0.98] 0.96[0.94-0.97] 0.97[0.96-0.98] 0.76[0.73-0.78]

Serial Testing Test Se Sp 1.) vaccinated infected recovered Prionics Ref Ref 2B 0.50[0.45-0.56] 0.99[0.99-1.00] 3B 0.61[0.58-0.64] 0.99[0.99-1.00] 3ABC 0.51[0.46-0.57] 0.99[0.99-1.00] 3D 0.55[0.50-0.60] 0.99[0.99-1.00] 3CD 0.48[0.43-0.54] 0.99[0.99-1.00] 2C 0.36[0.33-0.39] 0.99[0.99-0.99] a.) Contact challenge Prionics Ref Ref 0.28[0.21-0.36] 0.99[0.99-1.00] 2B 0.38[0.31-0.45] 0.99[0.99-1.00] 3B 0.30[0.22-0.38] 0.99[0.99-1.00] 3ABC 0.30[0.24-0.35] 0.99[0.99-1.00] 3D 0.24[0.18-0.30] 0.99[0.99-1.00] 3CD 0.20[0.13-0.29] 0.99[0.99-0.98] 2C b.) Needle challenge Prionics Ref Ref 0.77[0.73-0.80] 0.99[0.99-1.00] 2B 3B 0.84[0.78-0.89] 0.99[0.99-1.00] 0.76[0.70-0.81] 0.99[0.99-1.00] 3ABC 0.81[0.74-0.86] 0.99[0.99-1.00] 3D 0.73[0.66-0.80] 0.99[0.99-1.00] 3CD 0.53[0.46-0.60] 0.99[0.99-0.98] 2C 2.) Vaccinated infected carriers Prionics Ref Ref 2B 0.63[0.57-0.69] 0.99[0.99-1.00] 3B 0.69[0.62-0.74] 0.99[0.99-1.00] 3ABC 0.64[0.58-0.70] 0.99[0.99-1.00] 3D 0.55[0.49-0.61] 0.99[0.99-1.00] 3CD 0.51[0.44-0.57] 0.99[0.99-1.00] 2C 0.44[0.37-0.52] 0.99[0.99-0.99] a.) Contact challenge Ref Ref Prionics 0.44[0.37-0.50] 0.99[0.99-1.00] 2B 0.51[0.42-0.59] 0.99[0.99-1.00] 3B 0.45[0.38-0.52] 0.99[0.99-1.00] 3ABC 0.30[0.24--.36] 0.99[0.99-1.00] 3D 3CD 0.26[0.20-0.33] 0.99[0.99-1.00] 0.29[0.24-0.35] 0.99[0.99-0.98] 2C b.) Needle challenge Ref Ref Prionics 0.80[0.73-0.87] 0.99[0.99-1.00] 2B 0.85[0.81-0.88] 0.99[0.99-1.00] 3B 3AB C 0.81[0.75-0.87] 0.99[0.99-1.00] 3D 0.78[0.72-0.82] 0.99[0.99-1.00] 0.73[0.68-0.78] 0.99[0.99-1.00] 3CD 0.57[0.54-0.60] 0.99[0.99-0.99] 2C

112

Parallel Testing Se

Ref 0.91[0.89-0.93] 0.93[0.92-0.94] 0.91[0.89-0.93] 0.93[0.90-0.95] 0.91[0.89-0.94] 0.89[0.87-0.91]

Ref 0.97[0.94-0.99] 0.97[0.93-0.99] 0.97[0.94-0.99] 0.95[0.91-0.97] 0.96[0.93-0.98] 0.75[0.72-0.79]

Ref 0.79[0.71-0.86] 0.82[0.75-0.88] 0.79[0.71-0.87] 0.83[0.76-0.89] 0.80[0.72-0.87] 0.79[0.69-0.87]

Ref 0.97[0.95-0.99] 0.97[0.94-0.99] 0.97[0.94-0.99] 0.95[0.92-0.97] 0.96[0.93-0.98] 0.76[0.72-0.79]

Ref 0.98[0.97-0.99] 0.99[0.98-0.99] 0.98[0.97-0.99] 0.98[0.97-0.99] 0.98[0.96-0.99] 0.97[0.95-0.98]

Ref 0.97[0.94-0.99] 0.97[0.93-0.98] 0.98[0.94-0.99] 0.95[0.92-0.97] 0.97[0.93-0.98] 0.75[0.72-0.78]

Ref 0.96[0.94-0.98] 0.97[0.94-0.98] 0.96[0.93-0.98] 0.95[0.92-0.97] 0.95[0.92-0.97] 0.94[0.91-0.97]

Ref 0.97[0.94-0.99] 0.97[0.93-0.99] 0.97[0.94-0.99] 0.95[0.92-0.98] 0.97[0.93-0.99] 0.75[0.72-0.78]

Ref 0.93[0.88-0.96] 0.94[0.89-0.97] 0.93[0.88-0.96] 0.92[0.86-0.95] 0.91[0.85-0.95] 0.92[0.86-0.95]

Ref 0.98[0.95-0.99] 0.97[0.94-0.98] 0.98[0.95-0.99] 0.96[0.93-0.97] 0.97[0.95-0.99] 0.75[0.73-0.78]

Ref 0.98[0.97-0.99] 0.99[0.98-0.99] 0.98[0.97--.99] 0.98[0.97-0.99] 0.98[0.97-0.98] 0.97[0.95-0.98]

Ref 0.97[0.94-0.99] 0.97[0.94-0.98] 0.98[0.94-0.99] 0.95[0.92-0.97] 0.97[0.94-0.98] 0.76[0.72-0.78]

Performance of joint-simultaneous testing with the PrioCHECK® FMDV NS test for panel and field samples

Serial Testing Test Se Sp Panel Prionics Ref Ref 2B 0.84[0.81-0.86] 0.99[0.99-1.00] 3B 0.79[0.70-0.86] 0.99[0.99-1.00] 3ABC 0.70[0.62-0.73] 0.99[0.99-1.00] 3D 0.66[0.63-0.68] 0.99[0.99-1.00] 3CD 0.66[0.63-0.68] 0.99[0.99-1.00] 2C 0.58[0.54-0.62] 0.99[0.99-0.99] Field outbreak sera Prionics Ref Ref 2B 0.90[0.83-0.95] 0.99[0.99-1.00] 3B 0.91[0.83-0.96] 0.99[0.99-1.00] 3ABC 0.90[0.83-0.95] 0.99[0.99-1.00] 3D 0.71[0.64-0.76] 0.99[0.99-1.00] 3CD 0.66[0.60-0.72] 0.99[0.99-1.00] 2C 0.77[0.69-0.83] 0.99[0.99-0.99]

Summary Six in-house NSP antibody ELISAs were developed and validated. Bovine sera extracted from naïve animals, experimental vaccine challenged animals,a well-established bovine serum panel and sera field outbreak were used In-house tests were further evaluated to detect infection and carrier status in vaccinated and/or unvaccinated FMD infected and subsequently recovered and carrier animals, respectively In-house tests were used as confirmatory tests to the PrioCHECK® FMDV NS screening test and the increased sensitivity and specificity have been calculated using a Bayesian approach

Parallel Testing Se

Ref 0.99[0.98-0.99] 0.98[0.97-0.99] 0.97[0.96-0.98] 0.97[0.96-0.98] 0.97[0.96-0.98] 0.96[0.95-0.97]

Ref 0.97[0.95-0.98] 0.97[0.95-0.98] 0.98[0.96-0.99] 0.96[0.94-0.97] 0.97[0.95-0.98] 0.76[0.73-0.78]

Ref 0.99[0.98-0.99] 0.99[0.99-0.99] 0.99[0.98-0.99] 0.98[0.96-0.99] 0.98[0.96-0.99] 0.99[0.97-0.99]

Ref 0.98[0.95-0.99] 0.97[0.94-0.98] 0.98[0.95-0.99] 0.96[0.93-0.97] 0.97[0.94-0.98] 0.75[0.72-0.77]

Good news

Acknowledgements

Preliminarily evaluated ID Vet c-ELISA NSP kits (Over night incubation and 2 hour incubation)

Funding

36 Bovine serum panel and 91 naïve serum samples were used and all were found negative

ICAR IF, New DelhDefra, UK

Similar results as Prionics 33/36 (91.6%) were positive Only one common sample was found negative by both the tests

Defra, UK

Joining both the commercial tests 35/36 (97.2%) samples were seen positive Test 1) Panel ID Vet short 2B 3B 3ABC 3D 3CD 2C 2.) Panel ID Vet o/n 2B 3B 3ABC 3D 3CD 2C

AUC

Se (%)

Sp (%)

LR+

LR-

128 128 128 128 128 128 128

0.99 1 0.96 0.94 0.89 0.91 0.84

Ref 1 0.67 0.26 0.08 0.08 0.00

91.67 91.67 88.89 75.00 72.20 72.20 63.80

99.39 99.10 98.39 99.09 97.17 98.59 76.69

151.40 113.55 55.05 92.90 61.17 66.85 2.97

0.08 0.08 0.11 0.25 0.45 0.53 0.40

Ref 0.89 0.81 0.65 0.65 0.68 0.60

Agreement % Ref 96.09 92.90 87.50 87.50 89.06 85.06

128 128 128 128 128 128 128

1.00 1 0.96 0.94 0.89 0.91 0.84

Ref 1 0.65 0.25 0.08 0.08 0.00

91.67 91.67 88.89 75.00 72.20 72.20 63.80

99.39 99.10 98.39 99.09 97.17 98.59 76.69

151.40 113.55 55.05 92.90 61.17 66.85 2.97

0.08 0.08 0.11 0.25 0.45 0.53 0.40

Ref 0.91 0.83 0.67 0.67 0.71 0.63

Ref 96.88 93.75 88.28 88.28 8980 85.90

EU DISCONVAC

113

Objective: Complete the portfolio of ELISA kits for FMDV antibody detection AVAILABLE: Kits for Ab to FMDV serotypes O, A, Asia 1

(Open Session 2012)

NEW: Kits for Ab to FMDV serotypes SAT1 and SAT2

READY-TO-USE KITS FOR THE DETECTION OF ANTIBODY TO FMDV SEROTYPES SAT1 and SAT2

Product: ready-to-use ELISA kits

User-friendly Limited assay steps Stability Robustness No need of sophisticated equipment

G. Dho1, S. Grazioli1, M. Bugnetti1, G. Pezzoni1, F.F Maree2, J. Esterhuysen2, M. Chitray2, K. Scott2 and E. Brocchi1 1

Onderstepoort Veterinary Institute, Transboundary Animal Diseases Programme, Private Bag X05, Onderstepoort 0110, South Africa 2

Test Principle: MAb-based Solid Phase Competitive ELISAs 1

Development and Validation process for the SAT1 and SAT2 Ab kits

FMDV anti-SP Ab

Feasibility study

Single protocol for the five SP-ELISAs Serotypes O, A, Asia1, SAT1 and SAT2

Selection of most suited MAbs, both for resistance to the stabilization process and for test performance Stabilization process causes degradation of capsids in 12S-like particles

Test is fast - 2.5 h - and simple Plates provided pre-coated with FMDV inactivated antigens trapped by capture mAbs Only two incubation steps (sera & conjugated MAb) at RT

Evaluation of diagnostic performances ROC analyses Threshold Diagnostic Specificity Diagnostic Sensitivity

114

ROC curve: Kit for Ab to FMDV SAT1

2301 negative sera (naïve)

AUC = 0,986 P <0,0001

Sample size : Positive group : Negative group :

AUC = 0,986 P <0,0001

(cattle, buffalo and small ruminant)

sensitivity

Threshold > 57%

93 sera from exp. infected cattle (sequential samples from two animals)

100-specificity

4 cattle - 21 dpv and 28 dpv 2 cattle - 28 dpv 162 positive field cattle

Sensitivity Specificity

2567 266 2301

Se 98.1 97.4 96.6 92.1

Sp 94.4 97.7 97.8 99.1

60% 96.6 (95% CI 93.7-98.4) 97.8 (95% CI 97.1-98.3)

100-specificity

(Zimbabwe, herds C, D, E - positive to VNT)

Note: results of the ELISA kit were compared with those of VNT or LPBE obtained against homologous strains 5

ROC curve: Kit for Ab to FMDV SAT2

ROC curve: Kit for Ab to FMDV SAT2 2790 negative sera

(cattle, buffalo and small ruminant)

AUC= 0,954 P<0,0001

Sample size : Positive group : Negative group :

AUC= 0,954 P<0,0001

sensitivity

Threshold 30 exp. Vaccinated cattle (SA)

(21, 42 dpv, 28 days post-second vacc.)

10 buffalo (SA)

(outbreak in 2008)

30 cattle - 36 dpv (Libya) 29 reference sera (SA)

91.4

89.0

> 66 %

88.1

96.4

85.9

98.0

70% 85.9 (95% CI 82.9-88.5) 98 (95% CI 97.4-98.5)

(pi and pv cattle)

272 field positive cattle (SA) 100-specificity

3458 636 2822

(positive to LPBE)

100-specificity

237 field positive cattle (Zimbabwe) (herds A,B,C - positive to VNT)

115

Comparative results of SAT2-Ab kit and LPBE

Comparative results of SAT2-Ab kit and LPBE post-outbreak sera

KIT

+ -

VNT

SAT 2 KIT

+ -

+ 232 0

4 1

416 field samples (South Africa) Seroprevalence rate: SAT-2 Ab kit 59% LPBE 65% Concordance 0.728

N. cattle

SAT 2

LPBE + 202 43 70 101

sequential samples from 30 experim. vaccinated cattle

237 field samples (Zimbabwe) Seroprevalence rate: SAT-2 Ab kit 98% VNT 99.5% Concordance 0.983

0 dpv

2 dpv

4 dpv

7 dpv 9 dpv 11 dpv 14 dpv 21 dpv 28 dpv 42 dpv 56 dpv 70 dpv

Booster vaccination

Conclusion

The availability of new ready-to-use ELISAs kits for detection of Ab to FMDV type SAT 1 and SAT 2 fills a gap in the spectrum of FMD diagnostic tools

Analysis of serotype-specificity Investigations on how the antigenic divergence between antigens in the kits and FMD viruses eliciting Ab in the field may affect sensitivity

The ready-to-use kits contribute to standardization and harmonization of results obtained in different laboratories

Identification of a protective threshold

The validation process proved evidence of adequate or at least acceptable diagnostic performances.

Investigation whether different thresholds may be needed for different vaccine strains

The SAT-2 Ab kit was applied and evaluated in Ondesterpoort Lab proving satisfactory robustness

116

Validation of VNTs for all FMDV serotypes to determine sero-prevalence in cattle in Eritrea Aldo Dekker

Central Veterinary Institute, part of Wageningen UR

Conclusion

Testing 451 456 negative Dutch cattle A Eritrea

Different cut-offs for different VNTs are necessary Current study used a cut-off based on specificity, further research is needed on sensitivity Type O and A mainly found in Eritrea ± 15% infection in first year in cattle

O Manisa

96 % < 0.6

0.0

1.0

2.0

VNT titre (log

93 % < 0.6

3.0

0.0

117

1.0

2.0

3.0

0.0

10)

0.0

1.0

2.0

VNT titre (log

2.0

83 % < 0.6

3.0

0.0

10)

SAT 3 KNP 1000 TCID50

0.0

1.0

2.0

VNT titre (log

2.0

3.0 10)

Asia 1 Shamir

98 % < 0.6

3.0 10)

1.0

VNT titre (log

89 % < 0.6

3.0 10)

1.0

VNT titre (log

39 % < 0.6

3.0 10)

2.0

SAT 1 Zimbabwe

99 % < 0.6

SAT 3 Kruger Nat Park

85 % < 0.6

VNT titre (log

1.0

VNT titre (log

10)

SAT 2 Saudi Arabia

0.0

C Detmold

0.0

1.0

2.0

VNT titre (log

3.0 10)

Selection based on agreement VNT and NSP test Cut-off 99% percentile + 0 99% percentile + 0.3 99% percentile + 0.45 99% percentile + 0.6 99% percentile + 0.75 99% percentile + 0.9

Kappa 0.325 0.400 0.443 0.438 0.381 0.404

DEVELOPMENT OF LATERAL FLOW ASSAY FOR ANTIGEN DETECTION AND SEROTYPING OF FMDV K. Morioka1, K. Yoshida1, K. Fukai1, R. Yamazoe1, R. Kitano1, K. Sakamoto1, T. Kanno1 Exotic Disease Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization, 6-20-1 Josui-honcho, Kodaira, Tokyo 187-0022, Japan. 1

Comparison of the results of antigen detection

The Serotyping strip for FMD

Serotyping strip Virus & Sample

Absorbent Pad

Control FMDV (multi serotype) Type C Type Asia1 Type A Type O Sample Pad

multi serotype

No result

Control FMDV Type C Type Asia1 Type A Type O

O/JPN/2000 vesicular fluid

experimental infection

A15 TAI 1/60 vesicular fluid 1 experimental infection

Freeze dried colloidal gold labelled MAb (G-MAb) (multi serotype reactive)

O/JPN/2010 vesicular epithelials 2 field samples

Sensitivity *The antigen detection limits of the Serotyping strip are almost equal to from 103 to 104 TCID50 . 2

118

1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8

+ + ++ ++ ++ W+ + ++ + ++ ++ ++ + ++ ++ ++ ++ ++ + ++ ++ + ++ -

se rotyping O or A

mABS

result

82.8 73.0 385.8 136.8 196.2 13.3 87.1 294.8 21.7 236.8 36.7 309.4 66.9 427.6 403.7 275.7 165.4 240.1 34.1 350.0 114.6 53.2 388.5 n.d.

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

mABS

122.7 159.8 731.1 351.5 496.3 32.3 212.5 572.8 68.6 328.0 125.4 427.6 69.2 681.1 574.6 508.6 335.1 349.6 228.6 490.7 160.5 112.2 574.0 n.d.

MSD-ELISA

type O 3

RT-PCR

1.63 1.29 2.21 2.58 2.49 0.33 1.65 3.23 1.65 2.75 2.64 3.63 1.72 3.06 3.13 3.08 2.43 2.42 2.31 2.65 2.78 2.66 2.67

+ + + + + + + + + + + + + + + + + + + + + + + +

0.03

of vesicular fluid was added of running buffer. 2

% vesicular epithelial emulsion of running buffer.

3 MSD-ELISA is MAb based antigen detection ELISA (Morioka et.al., 2009, 2014). 4 n.d.= not detected 5 evaluation criteria ++ strong positive + positive W+ weak positive - negative (invisible)

The Serotyping strip was able to detect all 16 samples of vesicular fluid, and seven-eighths samples of 10% vesicular epithelial emulsions. The correlation coefficient between mABS and MSD-ELISA OD was significantly high (R=0.67). 3

Conclusion This Serotyping strip enables rapid antigen detection and serotyping of FMDV without the extra-devices. Early antigen detection and serotyping is important to implement the contingency plan for disease control (e.g. preparing for vaccine and immunological diagnosis kit ). The Serotyping strip will be useful in not only FMD-free countries but also FMD infected countries where it could not be conducted laboratory diagnosis. in vast countries. in countries lacking transportation facilities.

Development and Comparisons of Various ELISA for the Diagnosis of Foot and Mouth Disease Virus (FMDV) Sumita Bose, Shela Aman, and Masarrat Ali*

Further study is ongoing

Alpha Diagnostic Intl. Inc., San Antonio, Texas, USA

SAT1, SAT2 and SAT3 specific MAbs have been produced. We are already applying for seven serotypes, and ongoing sensitizing and manufacturing with private companies.

untyped

www.4adi.com

Arsh Bio 4

FMD Vaccine (inactivated, O, A, A1) BioFMD, India generate antiVP1 to O, A, A1, SAT1-2 and NSPs in rabbits

FMDV Vaccines (Whole Virus/Inactivated) Vaccine status with anti-VP1 ELISA ELISA not quantitative Vaccines contaminated with NSPs DIVA tests (3ABC) ELISA not reliable Assess various NSPs ELISA 2ABC, 3AB3, 3ABC, 3D ELISA

Antibodies to O, A, A1, and also SAT1, SAT2 VP1s

119

Antibodies to 2ABC and 3ABC NSPs making DIVA Test difficult

Natural Infection with FMD Virus (O, A) produced cross-reactive VP1 antibodies in bovine

FMDV Antibodies in random Bovine Samples Sample Analyses 1 = Negative 2 = Natural Infection (DIVA) 3 = Vaccinated/Infected 4 = Vaccinated

Bovine Infected with FMD O (28 dpi)

Bovine Infected with FMD A (28 dpi)

Arsh Biotech. India

2nd Generation Quantitative ELISA kits Developed Quantitative Antibody ELISA Kits FMDVP1 IgG: NSPs:

Serotypes O, A, A1, SAT1-3 2ABC, 3AB3, 3ABC, and 3D

Quantitative Antigen ELISA Kits VP1 Proteins: NSPs:

Serotypes O, A, A1, SAT1-3 2ABC, 3AB3, 3ABC, and 3D

ELISA Kits: 105 mins at RT (60+30+15), all reagents (Antigen or antibody controls), buffers, sample diluent supplied in ready to use stable liquid format. Sensitivity ~<1 ng/ml of antigen or antibody Stability ~ 1 year

120

Natural Infection with FMD Virus (A1, SAT1) produced cross-reactive VP1 antibodies in bovine

Bovine Infected with FMD A1 (28 dpi)

Natural Infection with FMD Virus (SAT2-3) produced cross-reactive VP1 antibodies in bovine

Bovine Infected with FMD SAT1 (42 dpi)

Bovine Infected with FMD SAT2 (28 dpi)

Bovine Infected with FMD SAT3 (28 dpi)

ELISA kits, ready to use reagents, precoated plates

Diagnostic observations with IZSLER Antigen ELISA kits for detection and serotyping of FMDV serotype O, A, SAT1 and SAT2 in several African countries Kees van Maanen EuFMD consultant

121

Plate lay-

African

serotyping ELISA kit

Conclusions Robust Userfriendly Mainly unequivocal typing results Good agreement with results WRL Implemented in many different countries Diagnostic performance depends on quality and type of samples 30 samples per kit, no big investments needed to buy antigen ELISA kits Much less risk for expiration of kits 3

Aims Offer Solid Phase Competitive ELISA kits for FMDV Ab detection anti-NSP 3ABC Ab serotype specific Ab ELISAs (FMD O and FMD A)

New competitive ELISAs for detection of non-structural or structural FMDV antibodies

Objective :

Mickaël ROCHE, Fabien DONNET, Muriel MALZAC, Loïc COMTET, Philippe POURQUIER

easy stable diagnostic performance 1

122

ID Screen® FMDV NSP

ID Screen® FMDV NSP Specificity (n=960)

cELISA detects Ab to the 3ABC NSP protein

Specificity for all species was high

coloured and ready-to-use reagents

Sensitivity The IDvet kit detects a high number of experimentallyinfected animals

overnight / short sample incubation protocol / results in 3 hours only

Species

Specificity (%) (number of samples)

CI95%

Bovine

99.5 (364)

98.0 99.9

Swine

99.7 (295)

99.7-99.9

Caprine

100.0 (111)

96.9-99.9

Ovine

99.4 (400)

96.5-99.9

Results obtained at ANSES, FRANCE

Results obtained at Pirbright, UK

ID Screen® FMD NSP

cutoff 50% night incub detected /total

35/36

cutoff 50% short incub

34/36

cutoff 50% night incub

cutoff 50% short incub

33/36*

32/36**

* ** sera closed to the cut-off

Parida, S. et al. 2007. J Vet Diagn Invest 19:539 544

Results kindly provided by ANSES & Pirbright

ID Screen® FMDV type O and type A FMD type O

Specificity (n=1120 & 640) Excellent specificity

Exclusivity

Species

Specificity (%) (number of samples)

CI95%

Specificity (%) (number of samples)

CI95%

Bovine

100 (248)

98.5 100

100 (208)

98.2-100

Swine

100 (400)

99.1-100

100 (216)

98.3-100

Caprine

99.6 (263)

97.9-99.9

100 (88)

95.9-100

Ovine

99.1 (210)

96.6-99.7

100 (128)

95.9-100

3 new cELISA for FMD diagnosis anti-NSP 3ABC anti-serotype specific ELISA (FMD O and FMD A)

IAEA reference sera

type O ELISA

100

Conclusions

FMD type A

Objective :

type A ELISA

100

easy

Asia 1

SAT 1

serum

SAT 2

SAT 3

Asia 1

SAT 1

serum

SAT 2

SAT 3

All sera correctly identified. Cross-reactions with other serotypes may occur 5

123

ready-to-use (no freeze-dried reagents) shorter incubation time use of a single protocol for serotype ELISAs stable 18 months shelf-life diagnostic performance validated / ongoing 6

Thank you for your attention ! Acknowledgements: S. Zientara, L. Bakkali Kassimi, S. Blaise-Boisseau, A. Relmy - ANSES, FR S. Parida & collegues - Pirbright Institute, UK E. Brocchi - IZS, IT K. de Clercq - CODA-CERVA, BE P. Hudelet, P. Dubourget

MERIAL, FR

124

LE AR he ̀ FMD science and polic ̀ deelopent landscape ̀

125

Context The selection of vaccine strains to be represented in the EU FMD vaccine bank should be based on risk assessment informed by up-to-date knowledge of the global distribution of FMDV serotypes and strains and of the likelihood of their spreading to the EU SANCO/7070/2010

Risk analysis framework to compare the importance of source regions for FMDV entry into Europe Lucie Collineau2 , Melissa McLaws1, Caroline Dubé3, Katharina Stärk2, Keith Sumption1 1

EuFMD, FAO Rome, Italy SAFOSO, Liebefeld, Switzerland 3 Canadian Food Inspection Agency, Ottawa, Canada 2

Objective

Method Initial model: McLaws et al., FMD Week 2010, Vienna, Austria

To develop a risk analysis framework to compare the importance of source regions for FMDV entry into Europe

FMD-infected country (FMD prevalence)

Source regions = FMDV pools FMDV entry = crossing the EU border (not necessarily leading to animal exposure) Europe = EU as a whole (no distinction between EU countries)

FMDV-contaminated animal products: FMD infected animal entering EU via: Road, Ferry Foot PROXIMITY

1. Carried by individuals via: Road, Ferry, Foot PROXIMITY

Air: AIR PASSENGERS

2. Illegal commercial trade (cargo) Concealed contents LEGAL TRADE Falsified origin: regional pool level?)

FMDVcontaminated fomite (negligiblelow risk)

Cross border wildlife Share land border with EU PROXIMITY

EU 4

126

Method

Initial model:

What conditions are required for FMDV to enter the EU?

McLaws et al., FMD Week 2010, Vienna, Austria

1. FMDV must be present in the country of origin 2. FMDV must be transported via one of these routes: Illegal import of infected animals or contaminated products and no detection at the border Entry of infected wildlife (wild boars, deers) Returning trucks Assumption: FMDV has a negligible risk to enter via legal trade of animals and animal products

Update: FMD prevalence level, proxies Refinement: considering additional transmission pathways , weighting proxies Application of a more systematic approach (scenario tree)

3. FMDV must survive in animals and contaminated products

Presence of infected animals or products at origin

Pathway 2: illegal import of animal products for personal consumption

Pathway 3: illegal import of animal products for commercial purposes (by road)

Possible pathways

I2 no

Presence of Infected animals or products

yes

Attempt to smuggle meat products (by road)

Pathway 6: returning contaminated trucks

yes

Illegal import passed the BIP (road)

Pathway 1: illegal import of live animals

yes

Pathway 2: illegal import of animal products for personal consumption

FMD virus survives

yes

P6 no

Number of passengers arrivals, number of residents

Infected animals or animal products enter EU

FMD virus survives

Illegal import passed the BIP (airport)

yes

Attempt to bring back meat products (by air)

yes

FMD virus survives

yes P3

P7 no

Illegal import passed the BIP (port)

yes I4

yes no

yes

FMD virus survives

P7 no

Excess of meat, meat price, no

yes

Infected deer or wildboar crossing the EU border

FMD virus survives

I5 no

Trucks transporting live animals visiting infected country

127

yes I6 no

Amount of live animals exported/imported from/to EU by road

yes

Trucks not properly cleaned and disinfected

yes P5 no

FMD virus survives

yes P9 no

Pathway 4: illegal import of animal products for commercial purposes (by boats)

Pathway 5: infected wildlife crossing the EU border

P8 no

Density, proximity

No FMD introduction into the EU

Pathway 3: illegal import of animal products for commercial purposes (by road)

Excess of meat, meat price, proximity

FMD incidence score Attempt to smuggle meat products (by boat)

Pathway 5: infected wildlife crossing the EU border

yes

Illegal import passed the BIP (road check points)

Proximity, meat price

(FMD incidence score)

Pathway 4: illegal import of animal products for commercial purposes (by boat)

yes

Attempt to cross the border with live animals (by road)

Infected animals or meat products entering into the EU

Pathway 1: illegal import of live animals (road)

Pathway 6: returning contaminated trucks

Model parameterization:Transmission pathways

Model parameterization : FMD incidence score

Proxy

Score 1-4, based on: K. Sumption et al. 2008 OIE WAHID database until 2014 National seroprevalence studies Progressive control pathway stage (when available) Performance of the veterinary services (PVS OIE reports when available)

Pathway

Data sources

Score

Uncertainty

Proximity of country X to the EU

1,3,5

Google maps

{0; 1}

Meat price differential between EU and country X

3,4

FAOSTAT 2012, FAO GIEWS 2013, OECD 2013

{1; 2; 3; Medium 4}

Number of passenger arrivals in EU from country X

Eurostat 2013

{1; 2; 3; Medium 4}

Number of EU residents with country X nationality

Eurostat 2013 + national databases

{1; 2; 3; 4}

Low

Excess of meat in country X

3,4

FAO STAT 2009

{1; 2; 3; 4}

Medium

Density of wildboars and deer in country X

FAO EMPRES 2010, Burbaite et al. 2010

{1; 2; 3; Low 4}

Amount of live animals traded between country X and EU by road

Eurostat 2013

{1; 2; 3; Medium 4}

Null

Model parameterization:Transmission pathways

Results: FMD Score

Expert elicitation Relative importance of each proxy on the I1 to I6 Probability of illegal imports of animals/animal products to be detected at the border inspection points

Literature FMDV survival in live animals / animal products / manure

Model outcome: 1 score per transmission pathway per country Qualitative description of the country contribution to FMD entry into EU 12

128

1. low sporadic incidence, effective reporting 2. sporadic incidence, ineffective reporting 3. yearly outbreaks (seasonal &/or restricted) 4. outbreaks throughout the year 13

Estimated relative importance of transmission pathways

Discussion

(preliminary results: pie size reflects overall importance of country, map colours for virus pools)

Model construction Additional transmission pathways to be considered? Legal trade of animals (eg risk if very recent FMD incursion) But not informative to vaccine bank

Waste from international planes and ships Semen importation Most probably at neglibible risk

Selection of the proxies How representative are they of risk pathways?

Relative importance of pathways 14

Discussion

Next steps

Model parameterization

Finalization of the model parameterization

Main data gaps

Sensitivity analysis

Routine seizures data at border inpection points (only publicly available for UK at the moment) Origin of ships / flights / passengers arriving at the EU border Comparable national meat prices (same calculation method)

Combine risk assessment with data on FMDV serotypes and strains circulations (WRL Pirbright)

From which countries should transmission by road be considered? How often do we need to update the model?

Recommendations for EU vaccine banks

Risk assessment expanded to include exposure of EU susceptible animals Better conducted at EU country level?

Your input is very welcome! 16

129

Thank you for your attention

The Enhanced Passive Surveillance System:

A Solution Supporting Data Collection, Integration and Analysis for Disease Surveillance Lindsey Holmstrom, DVM

Epidemiologist Institute for Infectious Animal Diseases A Department of Homeland Security Science and Technology Center of Excellence Texas A&M University, College Station, Texas, USA 18

Disease Surveillance Systems

Integrated Disease Surveillance System Active Surveillance

Future disease surveillance is focused on broader surveillance methods

Implement novel ways to collect & evaluate data

Develop new surveillance streams & strategies

Encourages new partnerships

Rewards & incentivizes participation

Participatory, community-based Opportunistic Disease Agnostic Vets/Producers

Analytical Risk-based, targeted Slaughter Programmatic, OIE list diseases

Moving away from one disease/one surveillance plan Application of innovative information science and technology systems Integrated animal disease surveillance systems

Leverage new tools & technologies

Enhanced Passive Surveillance

Internationally recognized assays ELISA, PCR Sequencing

Ensure information is available when needed

Optimize Surveillance Competencies: Priorities

Diagnostic Laboratory

LIMS/submission form data Test orders Test results

Animal Disease Surveillance 2

130

Project Background

EPS System: Concept

Enhances situational awareness and demonstrates the usefulness of multiple-data stream syndromic surveillance by: Applying the power of real-time reporting and data aggregation to animal and wildlife surveillance

Automatic or near real-time data collection and analysis

Leveraging first responders (veterinarians) for animal and public health surveillance

Standardized, disease agnostic reporting Multiple data streams

Estimating baseline prevalence rates of observed animal health anomalies for each stream

Data collection in the field using mobile applications Diagnostic laboratory data

Evaluating the significance of resulting signals as disease detection events

Use data, methods to allow for identification of subtle trends not visible to an individual Provide indicators to anomaly detection, investigation, quantification, localization, communication, and outbreak management

Providing early warning information obtained from validated signaling activity to stakeholders to assist in making decisions

Integration of Data Streams

Enhanced Passive Surveillance System BFES Mobile Application

EPS Analyst Workstation

Surveillance Data Industry

Vet

Wildlife Biologist

Syndromic Reports Production-level Indicator Data

Laboratory Data BFES is a mobile application for real-time collection and reporting of enhanced surveillance data Veterinarians, producers, and production managers can enter healthy and syndromic animal health data from livestock/poultry premises and livestock markets A web-based analyst workstation allows for the integration of surveillance data with other data streams (e.g., diagnostic laboratories, wildlife, and environmental data sources) into a common display for analysis

Test Order Counts

Epidemiologists and other analysts can evaluate and analyze the data in real-time through the use of different visual, geospatial, and temporal analysis tools

Environmental Data

EPS Analyst Workstation

Test Results

Embedded surveillance algorithms are included to help determine baseline conditions in order to detect any anomalies that may signal the onset of an animal disease outbreak

Season Rainfall Temperature Vegetation Soil

Common Integrated Picture

131

EPS Analyst Workstation

Current Reporting by EPS Users

State Government

Federal Government

Cattle/Small Ruminant:

Over 22,215 reports submitted since July 2012 Represents the health status of over 1.1 million animals in 3 states Reporting activity 70-80%

Equine:

Aggregation of all data streams into one common operating picture Web-based, user permissioned access for industry, States, and USDA Immediate access to data as surveillance reports are submitted from the field in real-time Automated visual, geospatial, and temporal analysis tools Surveillance algorithms to calculate baselines and detect animal health anomalies Data can be downloaded and imported into other statistical and analytical programs User generated reports to end users

Over 4,229 reports submitted since August 2013 Represents the health status of over 45,131 horses in Colorado Reporting activity is 80-100%

Poultry:

40 syndromic reports submitted since August 2013 Represents the health status of over 317,050 birds in Texas

EPS Analyst Workstation: Deployed in Federal Government (USDA) and 3 State Governments 11 government analysts trained on its use 8

Program Management

Expansion of the EPS System (Year 1)

Program Management Structure EPS Integrated Project Team (IPT)

Swine

Fed-only Working Group

IIAD Working Group

Data Access, Protection and Use

(Federal Partners Only)

Joint Working Groups

Data Access, Protection and Use (Feds, States, and Industry) Data Standardization and Ontology Lab Data Standardization/ Test Categorization Industry*

Mixed Animal

(Industry and State Partners Only)

EPS Analyst Workstation Design EPS Data Analysis and Baseline Determination Methodologies

10 Swine Companies/Practices Medium/large integrated operations, genetics operations , swine practices 30+ swine veterinarians in 13 states Coverage: ~5.2 million pigs monitored

EPS System Assessment and Detection Window Exercises Expansion, Sustainability, and Transition

3 Veterinary Diagnostic Labs 3+ State Government EPS AWS Deployed

*There will be separate WGs for each industry 10

132

Beef cattle, dairy cattle, equine, poultry, small ruminants, swine, and wildlife 100+ Veterinary Practitioners in 6 states 58 BFES users currently

4 Veterinary Diagnostic Labs 6 State Government EPS AWS Deployed

Enhanced Passive Surveillance System

Expansion to Other Industries

Facilitates data sharing and communication among stakeholders Users engaged, customization of technology to meet requirements Creates new and strengthens current partnerships User permissioned access, data protection, security, and standardization

Year 2 Focus Industry Expansion Feedlot: targeting 3 large feedlot regions/states and labs Wildlife: working with USDA APHIS Wildlife Services and USGS

Contributes evidence for: Claims of disease freedom and quantification of how much we are looking for disease Emerging disease detection or changes in endemic disease status Identifying areas for more intensive surveillance Identifying data trends, change in prevalence and geographic/temporal

Big Data/Data Analytics partners Year 3 Focus Industry Expansion Poultry: targeting 3 production systems (commercial egg layers, broilers/breeders, turkeys) and 2 labs Dairy: targeting 3 large dairy regions/states and labs

Provides a template supporting national implementation

Perform an exercise to evaluate the efficacy/utility of the system Transition a low-cost, low-maintenance system

Outcome:

A sophisticated system that serves as a central point for multiple groups to collaborate and leverage their collective resources, facilitating access to critical, cutting-edge technologies to better assess animal health and inform decision-making 12

The EPS System: International Applications Incorporate community-based and participatory methods to enhance conventional surveillance Tracking of human and animal health activities Education/outreach of the health environment and risk factors Engage populations from being passive recipients of information to active participants in a collaborative community

Challenges Country-specific reporting requirements Reporting timeliness Data source authentication Data validation Data needs Continuous reporting and sustainability

Questions?

Solutions Integration of existing database systems Adaptable system responsive to changing human/animal health Automated, customizable tools Auto-population of data Data standardization Value back to participants 14

133

Modelling FMD Transmission in a

Outline

- the problem - the study area - study methods - study results - implications

MICHAEL WARD | Faculty of Veterinary Science

Outline

Do wildlife play a role in FMD outbreaks?

- the problem - the study area - study methods - study results - implications 134

Do wildlife play a role in FMD outbreaks?

The outbreak among these wild animals was due to spread from infected cattle which had been slaughtered in this same region.

Texas, September 1924: - herd of zebu cattle south of Houston - FMDV probably entered via a Gulf port - outbreak controlled within 30 days

" a population of feral pig reservoirs Total number of deer exterminated: 20,819

complicated eradication "

Do wildlife play a role in FMD outbreaks?

boar in Southeastern Bulgaria scores of feral and domestic swine were comparable. However, feral swine exhibited a higher tolerance for the disease, and their thicker, darker skin made vesicular lesions difficult to detect

135

Feral pigs in Australia Livestock

Feral Pigs

27 million cattle, 72 million sheep, 3 million pigs

Garner, M.G. (2002) Rev.Sci.Tech.Off.Int.Epiz.

~13.5million (3.5 23.5m) (Hone 1991)

West, P. (2008) Assessing Feral Animals in Australia 9

What role does wildlife play in emergency disease?

Outline

The case of the feral pig

- the problem

1. quantify endemic disease transmission in feral pigs and cattle

- the study area

2. forecast the potential role that feral

- study methods

pigs might play in exotic animal disease (e.g. FMD) incursions

- study results - implications

3. define appropriate surveillance and mitigation strategies

136

The Study Area

18.2 S, 125.6 E The Kimberley

Fitzroy Crossing study site ~10 000 km2 Kimberley region >400 000 km2 13

137

138

Outline

Study Methods

- the problem - the study area - study methods - study results - implications

139

140

141

Study Methods: Disease Simulation Model

Outline

Results: Sampling

- the problem - the study area - study methods - study results - implications 142

Cattle Distribution

)

Feral Pig Distribution

Simulated cattle herds

Simulated feral pig herds

3,500 feral pigs in 275 pig

Decision support system

Decision support system incursion scenario pig-only cattle-only both (start in pigs) both (start in cattle) transmission parameters shared watering points, proximity, indirect, cattle movements incursion response stop movement surveillance

143

feral pig culling 40

Feral pig model

Outline

- the problem - the study area - study methods - study results - implications

Informed policy

Informed policy pig only simulations: FMD inevitably died out without intervention - short infectious period of FMD in small pig groups - discontinuous nature of the population - generally small daily movement distances

cattle only simulations: FMD much more likely to persist - wider cattle distribution - greater movement distances - mixing of herds: within paddocks, at watering points, management practices

simulation of interspecies transmission: persistence with larger outbreaks - infection in pigs maintained via exposure to infected cattle

144

Informed policy

The Joys of Field Work

control strategies targeting feral pigs only not predicted to be successful control based on cattle only successfully eradicated disease targeting both pigs and cattle resulted in smaller outbreaks

The Joys of Field Work

145

Further details Cowled, B.D., Garner, M.G., Negus K., Ward, M.P. Controlling disease outbreaks in wildlife using limited culling: modelling classical swine fever incursions in wild pigs in Australia. Veterinary Research, 2012; 43:3. Cowled, B.D., Ward, M.P., Negus, K., Galea, F., Garner, M.G., Laffan, S., Marsh, I., Sarre, S., Quasim, S., MacDonald, A., Woolnough, A., 2012. Endemic Salmonella in wild pigs: prevalence, risk factors, transmission and implications for control of wildlife disease. PLoS One 7, e46310. Leslie, E., Cowled, B., Garner, M.G., Ward, M.P. Effective surveillance strategies following a potential Classical Swine Fever incursion in a remote feral pig population in northwestern Australia. Transboundary and Emerging Diseases, doi:10.1111/tbed.12044 Ward, M.P., Cowled, B.D., Galea, F., Garner, M.G., Laffan, S.W., Marsh, I., Negus, K., Sarre, S.D., Woolnough, A.P., 2013. Salmonella infection in a remote, isolated wild pig population. Veterinary Microbiology 162, 921 929.

146

Acknowledgements Brendan Cowled, Edwina Leslie, Katherine Negus University of Sydney

Shawn Laffan University of New South Wales

Graeme Garner

Transmission and survival of FMDV on environmental fomites

Dept. of Agriculture, Fisheries & Forestry

Andrew Woolnough, Mick Everett Dept. of Agriculture & Food WA

Ian Marsh, Fran Galea NSW Dept. of Primary Industries

Steve Sarre

Emma Brown

University of Canberra

Research assistant, The Pirbright Institute 1

Environmental results- Aerosol, wall & floor data Relative (PCR) Log 10 TCID50/m3 of air

Experimental design

3.5

Calf

Room- Calf present

Room- Calf absent

3.0 2.5 2.0 1.5 1.0 0.5 0.0 Room 2

Room 3

Room 4

Room 5

Relative (PCR) Log10 TCID50/m2

Room number where infected calves were housed

C1-Four inoculated cattle (housed separately) C2- Four contact cattle (housed with inoculated cattle- 1:1) C3- Four fomite challenged cattle (housed separately in the dirty rooms)

3.5

wall

3.0 2.5 2.0 1.5 1.0 0.5 0.0 Room 2

147

floor

Room 3

Room 4

Room 5

Room where infected calves were housed 3

Applications of study RESOURCE ESTIMATIONS IN CONTINGENCY PLANNING FOR FOOT-ANDMOUTH DISEASE (FMD)

The study presents preliminary findings on environmental contamination and the risk of transmission, a area of research which is poorly studied. The findings of this study are being used to design direct new studies on indirect transmission that will aim to estimate transmission rates by aerosol and fomite exposure.

Anette Boklund1 Sten Mortensen2 Maren Holm Johansen3 Tariq Halasa1

Will hopefully give a better understanding of the magnitude of fomite transmission and will help in prioritizing and targeting preventive measures in the field.

1 2 3

Technical University of Denmark, National Veterinary Institute, Denmark The Danish Veterinary and Food Administration, Head Office, Denmark Veterinary Control Office North, Denmark

Background

Foot and mouth disease (FMD)

Contingency plans should include

highly contagious disease ruminants and pigs (and other cloven-hoofed) can lead to a large economic damage.

Provision for adequate resources Personnel Equipment Laboratory capacity

EU legislation must provide contingency plans be prepared for an outbreak

Take into account resources needed to control a large number of outbreaks occurring within a short time Ensure methods for mass disposal of animal carcasses and animal waste without endangering human health or environment

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

148

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods

Questions

simulation model Movement data - movements of animals Semen - movements to abattoir - milk tanker routes - Semen distances

What are the needs in an outbreak of FMD?

Herd data ID-numbers Numbers of animals Coordinates Herd types

Abattoir

Transport vehicle

Data related to FMD - time to symptoms - within-herd spread Infected herd Neighbouring herd

Movements of pigs

Danish contingency - are symptoms recognised - are rules followed Person contacts - are things reported (symptoms, contacts mv.) 4

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods

simulation model

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods - Sampling 100/1000 epidemics

All epidemics started in a cattle herd in a cattle dense area Abattoir

Semen

1000 epidemics simulated Semen

1000 randomly chosen herds Transport vehicle

Infected herd Neighbouring herd

Movements of pigs

Person contacts

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

149

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods - Sampling 100/1000 epidemics

Zone culling/vaccination

100 epidemics around the median are extracted

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods - Data from simulation model

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods

Data from simulation model

Average of herds in 100 epidemics Detected (118)

Depopulated (143)

Daily averages used

Suspicions (5*detected) In zones

Protection zones (1768) Surveillance zones (8080) Duration (87 days)

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

150

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Methods

Data from experts

Ressource estimations

Examples:

Task Which type of people should each team consist of?

How much time will they spend in one herd?

VETdays

Assistantdays

1 suspicion with symptoms

1 traced contacts

1 zone visit

What equipment do they need?

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Results

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

151

staff needed (Local crisis center)

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Results

staff needed (VETs - local)

Danish Emergency Managentment Agency is responsible for the hygiene barrier, i.e. getting people, equiment and trrucks in and out of outbreak farms 16

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Results

rendering

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Results (worst case) Virus isolation in cell culture: Antigen ELISA (virus detection): RT-PCR analysis: Serological analyses:

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

152

lab capacity 40 samples per week 200 samples per week 2000 samples per week 25000 samples per week

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Perspective

Are we prepared?

Compare with the current staff Identify staff for different functions Identify training need by function Develop training programme by function

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Perspective

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Perspective

Compare with the current staff numbers Identify staff for different functions Identify training needs by function Develop training programme by function

Changing herd structure?

Changing herd structure? Other control measures Depopulation Emergency vaccination

Identify other ressource needs and secure by contracts Killing equipment Wellies, protective chlothing Cars, hotels Cleaning and disinfection 22

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

153

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Acknowledgements Thanks to our co-authors: Tariq Halasa and Maren Holm Johansen

Thanks to the expert group for their great effort in this work: The Danish Veterinary and Food Administration: Majbritt Birkmose, Deputy head of Veterinary Control Office, North Jesper Valbak, Official Veterinarian Annelise Pallesen, Official Veterinarian Peter Lybecker Larsen, Official Veterinarian Tina Mørk, Veterinary Officer (Head Office) Stig Mellergaard, Deputy Head of Division for Animal Health Kim Vandrup Sigsgaard, Head of Danish Alert Unit for Food Erik Jepsen, Head of information The Danish Emergency Management Agency: Hans Kaj Henrik Bruhn, Major (CP) 24

MAXIMISING EFFICIENCY WITH A SURVEILLANCE STRATEGY FOR FOOT-AND-MOUTH DISEASE DURING AN OUTBREAK IN A PREVIOUSLY FMD-FREE COUNTRY.

Kylee Walker Incursion Investigator and Veterinary Epidemiologist, Ministry for Primary Industries, New Zealand

Resource estimations in contingency planning for foot-and-mouth disease (FMD)

Reasons for a new surveillance strategy for FMD

New Zealand has never had an outbreak of foot-and-mouth disease, but preparedness planning for responding to an incursion is a Primary Industries because of the devastating effect the disease would have on our country.

Our current plan for surveillance, once index case testing established the disease is present, would be based around regular surveillance visits to at-risk properties, to identify clinical signs at their onset. But this is not ideal: Waiting for the advent of clinical signs provides greater periods of virus shedding, and greater spread of infection We have large numbers of sheep as well as deer and goats which may not have overt clinical signs It is resource-heavy to send out vets to all at-risk or surveillance zone properties every 2nd day for 14 days, whereas with testing most farms would have 1 or 2 visits to confirm or rule-out infection

As part of FMD preparedness in New Zealand, a sampling and diagnostic surveillance strategy was developed to guide rapid and efficient detection of infected farms, through to proof-of-disease-freedom surveillance.

MPI Investigation and Diagnostic Centre Wallaceville

And now we have better tools available:

Advancements in laboratory testing for agent identification (RT-PCRs) means we can identify pre-clinical viraemic animals early detection NAIT animal movement tracking system in NZ allows more informed tracing better idea of risk of spread

Other advantages:

Knowing if antibodies or virus are present on farms could aid epidemiological investigation Will use serology in Proof-of-Freedom phase, so should use during outbreak phase too

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Timeline of response to FMD virus infection in an individual animal, including detectable components for testing

Aim of surveillance strategy The strategy: establishes the appropriate diagnostic testing algorithm, specimen types and sample size numbers to use for different farm situations, allowing rapid deployment of an effective, pre-validated surveillance programme to diagnose infected properties, including pre-clinical ones, with the greatest efficiency, accuracy and speed.

Incubation period - Within farm 2-14 days - Between farm 4-14 days - Generally 2-6 days

Exposure to infective dose

The diagnostic surveillance strategy is designed to improve efficiency in an outbreak by streamlining and pre-planning decision-making at the farm and the laboratory.

Development of detectable viraemia

Vesicles rupture

Clearance of virus Cattle by 2 weeks Pigs by 3-4 weeks post-infection

Circulating Virus-neutralising antibodies appear antibodies appear

Detection of clinical signs

1wk

2wk

3wk

4wk

Detectable seroconversion: to SPs to NSPs

It will reduce reliance on clinical surveillance and enhance pre-clinical diagnosis, hopefully assisting in a faster delimitation of an outbreak and ongoing management.

Detectable component: Cattle

Pigs

Carriers -->

Virus in OP fluid Virus in milk Virus in blood Virus in vesicular tissue

Other advantages would be: assisting good decision making for confirmation or rule-out of infection and avoid unnecessary large-scale culls, to allow the collecting of appropriate samples to describe the epidemiology of the outbreak, which may be used both real-time and retrospectively, and providing guidelines for the farm-level sampling and testing for a proof-of-freedom claim.

Clinical signs -->

Antibodies

Key: Less likely to observe at this time

Information adapted from: Laboratory Testing and Sampling in a Foot and Mouth Disease Response by Farm Type, Kylee Walker, November 2011, MPI Internal Document 4

Table 1: Minimum screening sample sizes per farm required by purpose of testing and herd size, when clinical signs are absent.

Table 2: Minimum confirmatory sample sizes per farm required by purpose of testing and herd size, when clinical signs are present. Suspect Infected Places.

Herd Size (single management group and species) Purpose of visit Property status Property in Protection Zone *3

Species

Serum

Dairy cattle

At-Risk Place*3 (<3 days post-exposure) At-Risk Place (4-7 days post-exposure)

At-Risk Place (>7 days post-exposure) At-Risk Places (every visit) End of surveillance period (Protection Zone or At-Risk Place)

Sample type *1

OP fluid or Throat, nasal or oral swabs Bulk tank milk and Residue milk Serum

Deer Sheep Goats Camelids Buffalo

Tests employed, in order of use *2

PCR SP Ab-ELISA NSP Ab-ELISA PCR

R (at every surveillance visit)

PCR

OP fluid

PCR

Serum

PCR SP Ab-ELISA NSP Ab-ELISA PCR

OP fluid or Throat, nasal or oral swabs Serum

Dairy cattle

Required or Optional

Bulk tank milk and Residue milk Serum

R (if no sentinel *4 species present) O (cattle, pigs, or listed species with sentinels present)

SP Ab-ELISA NSP Ab-ELISA PCR PCR

SP Ab-ELISA NSP Ab-ELISA PCR *5

1 - 40

41 - 60

61 - 80

81 - 125

126 200

201 2000

All

100ml

All

100ml

All

Herd Size (single management group and species) Purpose of visit Confirmatory testing for herds with clinical signs (Suspect Infected Places)

Animals to sample Animals with vesicles

Animals with clinical signs as sampling priority Animals with clinical signs (increase numbers if need to sample non-clinicals) Animals with clinical signs

Other management groups/species on a Suspect Infected Place, without clinical signs

Sample type *1 Vesicular fluid, epithelial tags Serum

Required or Optional R (if present)

Tests employed *2 PCR Ag ELISA

SP Ab ELISA NSP Ab ELISA PCR

OP fluid

PCR

Epithelium (not directly related to a vesicle) Serum

O (if animals culled at the visit)

PCR

PrioCheck NSP Ab-ELISA In series with SP Ab-ELISA PCR

1 - 40

41 - 60

61 - 80

81 - 125

126 - 200

201 2000

All

*1 Take 2 serum tubes per animal sampled. OP = oropharyngeal. 2 * PCR on serum calculated with test parameters of 100% Sp and 99% Se @ 5% prevalence. Screening serum sample numbers also fit with the requirements for SP Ab-ELISA if a minimum 10% prevalence of antibodies is assumed in a clinically-recovered herd. All sample sizes calculated for 95% confidence of detecting 1 infected animal. 3 * Protection Zone: within approximately 3km of any Infected Place. At-Risk Place: Determined by Intelligence and Planning Team to be at-risk because of tracing, spatial proximity or air-borne spread. 4 * Sentinel species are regarded as cattle and pigs, where clinical signs are typically more overtly displayed. 5 * Tests are not validated for these species.

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* Take 2 serum tubes per animal sampled. OP = oropharyngeal. *2 All sample sizes calculated for 95% confidence of detecting 1 infected animal. Serum sample size based on SP ELISA with test parameters of Se 96.4% and Sp 99.4% @10% prevalence of antibody-positive animals assumed in a clinically-affected herd, or serial interpretation of PrioCheck NSP Ab-ELISA with SP Ab-ELISA @ 5% prevalence. Serum sample size also accurate for PCR on serum calculated with test parameters of Sp 100% and Se 99% @ 5% prevalence. Vesicular fluid sample size based on PCR test parameters of Sp 100% and Se 99% @ 50% prevalence of FMD-infected animals within those with vesicular lesions. OP fluid and epithelium calculated for an assumed prevalence of 30% FMD-infected animals within clinically-affected animals. For rationale on these assumptions, please refer to the main body of text.

Table 3: Sample sizes per farm required in Proof of Freedom stage.

So how would this work practically? One submission form to rule them all... The purpose of the visit to the property (risk status) is prescribed already by the Planning and Intelligence Team on the laboratory submission form, which also serves as the sampling guidelines for the veterinarian.

Herd Size (single management group and species) Purpose of visit

Sample type *1

Freedom from disease surveillance property

Serum

Freedom from disease surveillance property

Serum

Follow-up visit to property with positive Ab-ELISA results

OP fluid or Throat, nasal or oral swabs

Required or Optional R

Tests employed *2 PrioCheck NSP Ab-ELISA In series with SP Ab-ELISA Idexx NSP Ab-ELISA In series with SP Ab-ELISA

1 - 40

41 - 60

61 - 80

81 - 125

126 - 200

201 - 2000

All

N/A *3

The veterinarian then assesses whether clinical signs are present or absent on the farm. The sampling guidelines from the tables are presented as a flowchart (separated into clinical signs present or absent) on the pre-specified laboratory submission form, enabling the field veterinarian to easily determine the appropriate samples to take for a particular farm.

PCR 15 *4

The same submission form identifies for the Animal Health Laboratory which of the preestablished testing algorithms the samples should follow (by identifying testing stream 14), and their priority.

*1 Only 1 serum tube required per animal sampled. OP = oropharyngeal. 2 * Serum for 5% prevalence for proof of freedom with 1) PrioCheck NSPp + SP ELISA serial sensitivity of 96.4% and serial specificity of 100%, 2) Idexx NSP + SP ELISA serial sensitivity of 89.3% and serial specificity of 100%. OP fluid or swabs to detect virus carriers at minimum prevalence of 15% (carrier rates 15-50%) with PCRs in parallel or series (combined se 95%, 99.94%; sp 100%, 99.8% respectively). 3 * Use Idexx and PrioCheck NSP ELISAs in parallel before SP ELISA in series, or use PrioCheck NSP in series with SP ELISA, to provide necessary sensitivity this is not attainable in small herds from the screening use of Idexx NSP ELISA. Take samples from all animals in herd of up to 40 animals. 4 * use PCRs in parallel for necessary sensitivity or take 20 samples.

The results are reported back, along with the pre-specified farm details through a Diagnostic Interpretation role in the Planning and Intelligence Team, who assess the diagnostic results in context of the farm's epidemiology (including risk status).

Testing streams 1-4 for Testing Algorithms Purpose of visit Property status

Tests employed, in order of use

Testing algorithm order (negative farms proceed past first algorithm)

Testing stream

Property in Protection Zone *1

PCR SP Ab-ELISA NSP Ab-ELISA

Agent detection priority 1 Serology priority 2

At-Risk Place*2 (<3 days post-exposure)

PCR

Agent detection only

At-Risk Place (4-7 days post-exposure)

PCR SP Ab-ELISA NSP Ab-ELISA

Agent detection priority 1 Serology priority 2

At-Risk Place (>7 days post-exposure)

SP Ab-ELISA NSP Ab-ELISA PCR

Serology priority 1 Agent detection priority 2

End of surveillance period (Protection Zone or At-Risk Place)

SP Ab-ELISA NSP Ab-ELISA PCR *3

Serology priority 1 Agent detection priority 2

Confirmatory testing for herds with clinical signs (Suspect Infected Places)

SP Ab ELISA NSP Ab ELISA PCR

Serology priority 1 Agent detection priority 2

Other management groups/species on a Suspect Infected Place, without clinical signs

PrioCheck NSP Ab-ELISA In series with SP Ab-ELISA PCR

Serology priority 1 (but alternate testing algorithm) Agent detection priority 2

All sample types other than serum

PCR

Agent detection only

* Protection Zone: within approximately 3km of any Infected Place. *2 - At-Risk Place: Determined by Intelligence and Planning Team to be at-risk of infection because of tracing, spatial proximity or air-borne spread. *3 Testing other species of animal (Deer, Sheep, Goats, Camelids, Buffalo) in herds without sentinel species present (which are regarded as cattle and pigs, where clinical signs are typically more overtly displayed). Tests are not validated for these other species.

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Further work which has used these testing protocols as a base Report on Expected Laboratory Testing Workload in a Foot and Mouth Disease Outbreak in New Zealand, March 5 2014 Projected sample numbers to inform the business case for the sizing of New Zealand's new National Biocontainment Laboratory.

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Cumulative Outbreak Graphs for Enhanced Risk Model in South Auckland

Further work to do Establish prioritisation system for which farms' samples to test based on what resources we have. This requires knowledge of the capacity of the laboratory (current and future), which has now been done.

Acknowledgements

Establishing how samples will come into specimen reception and proceed into the appropriate testing algorithm. This is underway now with templates for the laboratory information management system being written, and logistics being worked out.

In particular, Dr Richard Clough at MPI AHL IDC Wallaceville for the details of the available diagnostics, and the validation work done on them, and helping formulate and adapt the testing algorithms.

A vaccination policy is under discussion now in New Zealand, if this is implemented, will need to adapt the protocols for DIVA testing. Also as additional tests become available, creating an active system for incorporating them and adapting the protocols.

The scientists at MPI Animal Health Laboratory IDC Wallaceville, in particular the other members of the AHL FMD Preparedness Planning Group, for example Reinhold Kittelberger who is here today, for accommodating this strategy and helping make it achievable! My manager and team of Surveillance and Incursion Investigation (Animals and Marine) at MPI IDC Wallaceville for support and advice.

Problems Establishing a practical system for this process to work. The current software we use doesn't support it and a paper-based system would be difficult to maintain. In the event of an outbreak in New Zealand, it is likely that one of the two main islands would be affected predominantly. This means that establishing compartment freedom for one island to resume trade would be a priority along with controlling the epidemic in the other island. In this instance, the sample collection and testing resources required for compartment proof-of-freedom and epidemic control may be demanded simultaneously. 17

Introduction Evaluating vaccination strategies to control foot-and-mouth disease: a model comparison study

Vaccination is being recognised as an option for containing and eradicating FMD in previously diseasefree countries For major exporting countries implications of vaccination, including management of vaccinated animals, will require careful consideration Simulation models ideal for situations where data is scarce but country specific context is vital Well designed collaborative studies can inform policy development

Roche SE, Garner MG, Sanson RL, Cook C, Birch C, Backer JA, Dube C, Patyk KA, Stevenson MA, Yu ZD, Rawdon T, Gauntlett F Epidemiology and Infection 2014, July 31:1-20

158

Study Objectives

Model Comparison Approach

Compare vaccination strategies for FMD outbreak control What factors affect impact of vaccination

Multiple countries and models participating Shared problem approach All countries have same starting data Multi-focal hypothetical outbreak

Time of deployment Deployment strategy (outside-in, insideSize of vaccination zone Approach to vaccination (Suppressive or Preventative)

Transmission parameters harmonised across models Some compromises needed where models incompatible

Comparison metrics for simulation results

Simulate scenarios in different models and compare results Understand and explain the differences occurring between the scenarios

Total number of infected premises (IPs) Duration of outbreak (days) Geographical distribution of IPs

Vaccination Strategies Investigated Timing of Vaccine delay (days)

Species vaccinated

Results

Vaccination Approach Suppressive versus Preventative

Vaccination zone size (km)

Stamping Out

Alla

Random

All

Outside in

All

Large farm size first

V4 V5 V6 V7 V8

SV SV SV SV SV

3 3 1 5 3

7 28 14 14 14

All All All All Cattle only

P P P P P

Outside in Outside in Outside in Outside in Outside in

3-7

All

Inside out

V10

All

Outside in

V11

5-10

All

Inside out

Retrospective (R) or Prospective (P)

Order of vaccine deployment

Epidemic Size (Number of IPs)

700

Cattle Only

1 Km

AusSpread

Median (5-95) number of IPs

600

Netherlands Exodis

500

InterSpread Plus 400 NAADSM 300

200

100

0 SO

159

V10

V11

Results

Epidemic Duration (Days)

Median (5-95) epidemic duration (days)

Cattle Only

1 Km

500

Discussion All results are in the context of this specific outbreak

450

All vaccination scenarios result in significantly smaller and shorter outbreaks than stamping out alone Certain vaccination scenarios are robust to substantial differences in models design

400 350 300 250

Vaccination use has implications

200

Time to disease freedom declaration

150

Implications of vaccinated animals in population

100

Tracking & identification of vaccinated animals Animal products and by-products of vaccinated animals Further economic impact assessment

50 0 SO

V5 V6 Control strategy

V10

V11

Acknowledgments and Thanks We thank Kim Forde-Folle (US Department of Agriculture), Neil Harvey (University of Guelph), Naya Brangenberg (NZ Ministry for Primary Industries), Tom Smylie (Canadian Food Inspection Agency), and Katie Owen (NZ Ministry for Primary Industries) for providing policy advice and input throughout the project The QUADS Epi-team would also like to acknowledge the government departments involved for continued support of this and other projects

Impact of stakeholders influence, geographic level and risk-perception on strategic decision in simulated footand-mouth disease epizootics in France Maud Marsot Epidemiologist (PhD) ANSES, France

160

Ralph Steadman

"Animal farm " illustration

G. Orwell

Decision-making in case of epizootics

Objectives

Simulation models Test influence of parameters on FMD spread

Geographic level

Compare control strategies

Riskperception

VS Preemptive slaughter

Agro-food industries Regional

Government National

Protective vaccination

Optimal control strategy ?

Public opinion

Keeling 2001 Science, Ferguson 2001 Nature, Mahul 2000 Prev.Vet.Med.

Neutral

Averse

Fixed

Adaptive

Decision-making

Indirect costs

Type of strategy

Stakeholders

Scientific rationality Direct costs Social impact 2

Simulation model of FMD epizootics

FMD control strategies SO

Transmission between herds

stamping-out

Herd x Susceptible

Latent

Subclinically Infectious

Clinically infectious

Immune

SPS

Within-batch transmission

SPV

selective PS

Herd y

selective PV

suppressive vaccination Between-batch transmission

preemptive slaughter

protective vaccination

SPSV

selective PS and PV Rautureau 2012 Trans. Emer. Dis.

161

Methodology 1st step

National fixed control strategy Public costs (millions euros)

Simulations of FMD epizootics 50 introduction points / region

Number of slaughtered herds

Export losses (millions euros)

21 regions X

7 fixed control strategies

= 7350 simulations

Risk-neutral

Impact of simulated epizootics Government Public opinion

public costs number of slaughtered herds

Agro-food industries

National level

Risk-neutral

mean

Regional level

Risk-averse

variance

SPV

export losses Risk-averse

Regional fixed control strategy Public costs

Number of slaughtered herds

Methodology 2nd step Export losses

D42 D35

3 control strategies SO, PS, PV

D28

D21 PV

PV PV

PV PS

PV PV

PS PV

PV PV

PS PV

PV PV

162

PS PV

Initial conditions

PV PS

Risk-averse

PV PV

D14

PS PV

PV PV

= 35 decision paths D7

PS PV

7 time steps - duration : 45 days - interval : 7 days Risk-neutral

PV PV PV PV PV

Decision tree

Methodology 2nd step Simulations of FMD epizootics

Public costs - D14

1000 introduction points / region X 21 regions X 35 decision paths

Prediction yes

1? no

Incidence < 5 ?

Protective vaccination

yes

= 735 000 simulations

Preemptive slaughter

Comparison of scenarios - national fixed - national adaptive - regional fixed - regional adaptive

Control panel indicators Incidence, cattle density CART

Protective vaccination

0.85 0.61

decision trees from D0 to D42

0.51

Government public costs Agro-food industries export losses Public opinion number of slaughtered herds PS

Comparison of scenarios Scenario

National fixed

National adaptive

Regional fixed

Public costs (millions euros)

17,44

17,18

16,94

[16,74 ; 18,07]

[16,5 ; 17,8]

[16,28 ; 17,57]

Synthesis Number of slaughtered herds

325

314

296

[311 ; 339]

[306 ; 321]

[284 ; 307]

- National vaccination-based strategies

Export losses (millions euros)

45,81

45,78

45,81

- Regional strong heterogeneity ~ stakeholders + regional densities of animals

[43,99 ; 47,84]

regional adaptation = improvement / national level Marsot M., Rautureau S., Durand B., Impact of Stakeholders Influence, Geographic Level and Risk Perception on Strategic Decisions in Simulated Foot and Mouth Disease Epizootics in France, Plos One, 2014, 9(1): e86323.

[43,66 ; 47,69]

- design of a steering tool for the control of FMD epizootics : combination control panel + decision procedure based on control panel indicators

[43,69 ; 47,66]

- regional decision-making seems to be the best in general Regional adaptive

17,43

[16,77 ; 18,1]

315

[307 ; 323]

45,37

[43,31 ; 47,22]

steering tool is still in development

163

Thank you for your attention Acknowledgements Expert committee

Partners

Co-authors - Benoit Durand - Séverine Rautureau

- Labib Bakkali - Barbara Dufour - Benoit Durand - Yves Leforban - François Moutou - Séverine Rautureau - Xavier Rosières - Bernard Toma - Gina Zanella - Stephan Zientara

An adaptive management approach to foot and mouth disease control Mike Tildesley

Adaptive Management

Critical decisions are necessary in the face of uncertainty

Critical decisions are necessary in the face of uncertainty If we have a new outbreak, we cannot necessarily assume that the epidemic will spread in the same way and control will have the same effect.

164

Adaptive Management

Critical decisions are necessary in the face of uncertainty

If we have a new outbreak, we cannot necessarily assume that the epidemic will spread in the same way and control will have the same effect. Adaptive Management

Formalized method to learn about impact of control in the event of model uncertainty

Formalized method to learn about impact of control in the event of model uncertainty Assessing the cost of making the right decision depends on the projected outcome of the intervention conditional on each model, AND likelihood of each model being correct

Questions

How limiting is model uncertainty to the development of policy?

How limiting is model uncertainty to the development of policy? Though there may be things we want to learn to advance biological understanding, if the same control strategy is still optimal even with this uncertainty, it doesn't represent a limitation to policy.

165

Questions

Simulation Exercise

How limiting is model uncertainty to the development of policy?

Simulation of a theoretical FMD outbreak in Cumbria, UK

Though there may be things we want to learn to advance biological understanding, if the same control strategy is still optimal even with this uncertainty, it doesn't represent a limitation to policy. Is it better to use multiple models to predict the spread of disease?

Simulation Exercise

Simulation of a theoretical FMD outbreak in Cumbria, UK Run using 3 current models

Warwick, NAADSM, AusSpread

100 simulations using each of 5 control options from start to end Impact of competing control strategies investigated for each model.

166

Control Options

Culling of Infected Premises only (IP)

Culling of Infected Premises only (IP) Dangerous Contacts + Infected Premises (DC)

Control Options

Culling of Infected Premises only (IP) Dangerous Contacts + Infected Premises (DC) IP+DC+3km Ring Culling (RC)

Culling of Infected Premises only (IP) Dangerous Contacts + Infected Premises (DC) IP+DC+3km Ring Culling (RC) IP+DC+3km Ring Vaccination (V3)

167

Control Options

Objective metrics The optimal control strategy may be highly dependent upon the objective of control:

Culling of Infected Premises only (IP) Dangerous Contacts + Infected Premises (DC) IP+DC+3km Ring Culling (RC) IP+DC+3km Ring Vaccination (V3) IP+DC+10km Ring Vaccination (V10)

For instance we may seek to minimize: Cost of response: i.e. cost of livestock + cost of vaccination Outbreak duration (days) Number of livestock culled

Number of livestock culled

Epidemic Duration AusSpread

NAADSM

AusSpread

Warwick

NAADSM

Warwick

100000

400

75000

300

200

50000

100

25000

0 v10

pi_dc

v10

Control

pi_dc

v10

pi_dc

v10

Model predictions of epidemic duration are different across the five control strategies. The three models predict different impact of control on epidemic duration.

pi_dc

v10

Control

pi_dc

v10

Warwick and AusSpread predict similar impact of control on livestock culled.

168

pi_dc

Example

Objectives clearly matter!

How would we try to control a new outbreak of FMD in the UK? 3 Alternative Models 1. UK 2001 Model (UK)

So how do we incorporate uncertainty in model belief?

2. Alternate Model 1 (M1) 3. Alternate Model 2 (M2)

Example

How would we try to control a new outbreak of FMD in the UK? 3 Alternative Models

Dangerous Contacts (DC)

3. Alternate Model 2 (M2)

Contiguous Premises (CP)

1. UK 2001 Model (UK)

Candidate Interventions Infected Premises (IP)

2. Alternate Model 1 (M1)

Dangerous Contacts (DC)

3. Alternate Model 2 (M2)

Contiguous Premises (CP)

Objective Minimize total cost epidemic Total cost = 1000 * cattle culled + 100 * sheep culled -- based on compensation costs from 2001 outbreak

169

severity

2. Alternate Model 1 (M1)

3 Alternative Models

severity

1. UK 2001 Model (UK)

Candidate Interventions Infected Premises (IP)

How would we try to control a new outbreak of FMD in the UK?

Kernel weight

Cost Matrix

Interventions

Best

Kernel weight

Best

.25

8.4

5.5

8.2

5.5

.25

8.4

5.5

8.2

5.5

.25

28.4

22.1

37.8

22.1

.25

28.4

22.1

37.8

22.1

512.9

190.1

116.2

512.9

190.1

116.2

265.7

103.0

69.6

65.0

265.7

103.0

69.6

65.0

Cost in units of ~ £5 million

Cost in units of ~ £5 million Best conditional intervention is to cull contiguous premises

Cost Matrix

Weight of Belief in Models The best strategy is dependent upon the initial model weights

Interventions Kernel weight

Best

.25

8.4

5.5

8.2

5.5

.25

28.4

22.1

37.8

22.1

512.9

190.1

116.2

265.7

103.0

69.6

65.0

Cost in units of ~ £5 million Best conditional intervention is to cull contiguous premises

P(M2)=0

Future outbreaks in the UK might be presumed to follow prior pattern 170

Weight of Belief in Models

The best strategy is dependent upon the initial model weights

Severity of expected outcomes under UK kernel model dominates until weights are very low.

Severity of expected outcomes under UK kernel model dominates until weights are very low. So unless we strongly believe that the UK model

P(M2)=0

recommend severe culling from the onset.

Adaptive Control Now we consider the effect of control if we can update strategies based upon observations.

Phase 1

Adaptive Control Phase 2

Now we consider the effect of control if we can update strategies based upon observations. What is the best 1st phase intervention, when there is an opportunity to update?

171

Phase 1

Phase 2

Expected Value of Adaptive Strategy

DC culling is optimal 1st stage strategy for a broader range of initial weights

DC culling is optimal 1st stage strategy for a broader range of initial weights If an adaptive strategy is adopted, costs can be reduced by adopting a less intensive DC culling strategy at the onset and switching if necessary later in the epidemic.

P(M2)=0

Adaptive Management Conclusions

There is clear value to planning to manage adaptively

There is clear value to planning to manage adaptively Costs can be saved by adopting the optimal strategy at the start of an outbreak, given that there is uncertainty in how the outbreak will spread.

172

Adaptive Management Conclusions

There is clear value to planning to manage adaptively

Costs can be saved by adopting the optimal strategy at the start of an outbreak, given that there is uncertainty in how the outbreak will spread.

Strategies can then be modified as uncertainty is resolved.

Strategies can then be modified as uncertainty is resolved. Multiple models can be used in an ensemble framework to provide a single prediction regarding how the disease will spread and strategies for disease control.

FMDV particles: quantification and monitorization 146S

75S

12S

> 56°C pH < 6.5

Neutralizing antibodies

Quantification methods:

USE OF SPECIFIC LLAMA ANTIBODIES FOR QUALITY CONTROL TESTING OF FMD VACCINES

Affinity chromatography, S-E chromatography, UV spectrophotometry, thermo fluor assay, sucrose density gradient: o 146S (inactivated vaccines) o Final production of a vaccine

Eva Perez-Martin Cavtat, October 2014

173

DAS ELISA based on VHH antibodies: Quantification of the content of antigen and integrity: o 146S (inactivated vaccines) o 75S (subunit vaccines) o 12S o in different steps of production (optimization of expression, storage conditions) o detection of antigen expressed at small scale: quick identification of new vaccine candidates

VHH llama Ab (from CVI, Lelystad)

Fab 50 KDa

13 KDa

Fab 50 KDa

150 KDa

13 KDa

75 KDa

O1 Manisa

Asia 1

M170: 146S/75S M3:12S

VHH llama Ab (from CVI, Lelystad)

Standard: O1Manisa BEI (0.5ng/ml) %146S M3 (12s)

Fab 50 KDa

ng/ml of antigen (lines)

700

13 KDa

M377: 146S/75S M311: 146/75S + 12S

O1 Manisa binders: M170 (146S) and M3 (12S) 800

75 KDa

SAT2

M332: 146S/75S M3:12S M98: 146/75S + 12S

(Harmsen MM et al., 2011)

ScFv 25 KDa

150 KDa

ScFv 25 KDa

150 KDa

Homolog sandwich elisas: M170 (146S) antigenic site I (GH-loop), trypsin-sensitive, neutralizing ab (Mateu and Verdaguer,2004) M3 (12S) antigenic site II of VP2? (Mateu and Verdaguer,2004)

M170 (146S) Total ag

600

500

400

300

200

100

%146S (columns)

75 KDa

ScFv 25 KDa

-Signal in M170 (146S) elisa disappears when antigen is dissociated -Signal in M3 (12S) elisa increases when antigen is dissociated

0 40°C

45°C

50°C

55°C

60°C

65°C

70°C

75°C

Temperature (15 minutes) 2500

M170: 146S/75S M3:12S

(Harmsen MM et al., 2011)

Asia 1 M332: 146S/75S M3:12S M98: 146/75S + 12S

SAT2

2000

M377: 146S/75S

antigen ng/ml

O1 Manisa

M311: 146/75S + 12S

Homologous or heterologous double antibody sandwich elisa -Standard: Sucrose purified antigen with a known quantification (UV260nm) 146S elisa: intact antigen 12S elisa: heat treated at 56°C for complete dissociation to 12S -Screening of antigen: quantification by interpolation

Vaccine antigens at 1ug/ml (UV) M3 (12S) M170 (146S)

1500

Quantification of antigen by 146S elisa coincides with quantification by UV absorbance 50% of antigen is 12S but still, it is a good vaccine antigen (146S)

1000

500

Batch discarded

174

O1Manisa BEI OOUKG UKG BEI BEI O1M BEI

Commercial O1Manisa O1Manisa Commercial O1M vaccine vaccine O1M vaccine (RT) (RT) vaccine

Detection of O1Manisa VLPs by VHH:

1000

900

M170 (146S/75S)

800

M3 (12s)

700

600

500

400

300

200

100

ng/ml

%146/75S)

ng/ml

Thermal stability 100

0 40°C 45°C 50°C 55°C 60°C 65°C 70°C 75°C O1Manisa BEI

40°C 50°C 55°C 60°C 65°C 70°C 75°C

100

900

M170 (146S/75S)

800

M3 (12s)

O1M mutant VLPs cell lysate

40 °C 45 °C 50 °C 55 °C 60 °C 65 °C 70 °C 75°C

O1Manisa mutant VLPs

O1M BEI

%146S

%146/75S)

Thermal stability %146S

1000

0 40°C 45°C 50°C 55°C 60°C 65°C 70°C 75°C

O1Manisa mutant VLPs (fraction 3)

40°C 50°C 55°C 60°C 65°C 70°C 75°C

O1Manisa BEI

O1M mutant VLPs sucrose purifed

40 °C 45 °C 50 °C 55 °C 60 °C 65 °C 70 °C 75°C

O1Manisa mutant VLPs

O1M BEI

O1Manisa mutant VLPs (fraction 3)

O1M mutant VLPs cell lysate

O1M mutant VLPs sucrose purifed

pH stability 900

100

%75S M170 (146S/75S) M3 (12S)

800 700

90 80 60

ng/ml

500

400

300

200

100

0 ph 4.47

5.3

5.6

5.87

6.14

6.3

7.5

ph 4.47

5.3

O1Man-BEI

1/2

1.6

1/4

0.9

0.7

1/4

1.4

1/8

0.8

0.2

Asia Shamir Asia Bahrain O1Manisa BEI SAT2 ZIM BEI sup sup

intact

40°C

47°C

M3 (12S)

0 Asia1 Shamir wt Asia1 Shamir T. ni mut P2 T.ni

Asia1 Shamir mut P2 sf9

175

SAT2 WT

52°C

50°C

48°C

54°C

intact-4°C

47°C

56°C

%12S %146S

52°C

75S Asia1 Shamir Mut VLPs > wt VLPs T.ni> sf9 insect cells

40°C

Thermal stability: SAT2 wt and mutants

12S 75S

intact

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 50°C

VLPs: cell lysates

M332 VHHs are subtype specific: 146S Asia1 Shamir No cross-reaction with other serotypes

48°C

56°C

SAT2 ZIM 8/96 BEI

SAT2- mut1

SAT2- mut2

SAT2-mut3

54°C

AsiaShamir BEI

52°C

AsiaShamir Asia Shamir Asia Bahrain O1Manisa SAT2 ZIM BEI BEI sup sup BEI

0.1

intact 56°C intact 56°C intact 56°C intact 56°C intact 56°C

50°C

intact 56°C intact 56°C intact 56°C intact 56°C intact 56°C

48°C

1/3200

0.3

0.2

0.1

1/1600

0.4

54°C

0.4

1/800

0.5

intact-4°C

0.2

1/400

52°C

0.6

1/200

0.6

0.8

0.3

7.5

Polyclonal antiSAT2

50°C

0.4

M377 (146S)

48°C

1/16

6.3

0.7

1/16

0.5

1.2

1/8

ug/ml

1/2

0.8 0.6

6.14

M3 12S

1.8

intact-4°C

5.87

O1Manisa mutant VLPs

54°C

M332 146S

5.6

SAT2 VHHs binders (M377)

Asia1 VHHs binders (M332): 146S and 75S VLPs 0.9

%146/75S

600

Other applications of VHHs

SAT2 VHHs binders (M377)

Control (no primary)

Cell supernatant: Eritrea and ZIM isolates 3500

146S (M377F)

3000

Polyclonal ab

M3 VHH (12S)

M170 VHH (146S)

Detection of 146S SAT2 ZIM> Eritrea SAT2 SAT2 Egypt?

2500 ng/ml

IB11 Mab

2000 1500 1000 500 0

% of 75S relative to antigen at 4°C

sat2 Eritrea SUP

120

%75S

Immunofluorescence on epithelial tissue: Cattle infected with O1Manisa FMDV intradermalingually at 3 dpi

sat2 ZIM 8/96 p6 SUP

(Green: O1 Manisa) (Blue: DAPI)

Rabbit antiO1Manisa

SAT2 VLPs cell lysate

M3 VHH (12S)

(Nick Juleff)

M23 VHH (12S+146S)

100 80 60 40 20 0

4°C 40°C 47°C 56°C

SAT2-wt MVA

SAT2-mut1 MVA

IBRS2 cells

Goat ZZR cells

IPMA on IB-R2 and goat epithelial ZZ-R cells infected with O1Manisa FMDV at MOI 0.5 for 3.5h

Other applications of VHHs

Summary

Immunofluorescence on monolayer

Red: O1Manisa Blue: DAPI Green: His

Anti-His Mab

M23 VHH (12S+146/75S)

Sf9 cells infected with O1Manisa-His baculovirus at MOI 0.1

176

146S/ 75S VLPs

Serotype ab binders

Isolates

O1 Manisa O Turkey O UKG O BFS

M170 VHH

M3 VHH

Asia1

Asia1 Shamir

M332 VHH

M3 VHH

SAT2

SAT2 Zim Eritrea SAT2 Egypt?

M377 VHH

12S

Content and integrity of FMDV antigen Inactivated vaccines Subunit vaccines

Antigen detected during the whole process of production Antigen detected even when produced at smallscale: Optimization of expression (cells, buffers, time of infection) Test the viability of modifications/mutations of antigen Storage conditions

Acknowledgements

Viral Immunology group Bryan Charleston Julian Seago Nick Juleff Miriam Windsor Mohammed Doudo Ben Jackson Liz Reid Marti Cortey Fuquan Zhang

Michiel Harmsen Aldo Dekker

OXFORD University Division of Structural Biology Claudine Porta Dave Stuart Abhey Kotecha

Evaluation of the immune responses of Nguni cattle vaccinated with FMD stabilized SAT2 antigens.

Ian Jones Silvia Oliveiros

Katherine Scott, N.M Rathogwa, A.V. Capozzo, F.F. Maree

Pip Hamling Claudia Doel

Agricultural Research Council (ARC-OVI), South Africa

FMD in South Africa

Quarantine was first introduced in 1377 in Dubrovnik on Croatia's Dalmatian Coast

177

Introduction

FMD virus inherently unstable: infectivity relying on disassembly caused by endosomal vesicle acidification. FMD vaccine antigens are unstable in mild acidic pH & elevated temperatures. Instability of capsid (146S) causes virus to dissociate into smaller 12S subunits. Most unstable: O & SAT2 serotypes, linked to a reduction in vaccine efficacy (Doel & Baccarini, 1981). Unstable antigen believed to be less immunogenic due to degradation before & after inoculation. Therefore to be effective, FMD vaccines require frequent booster vaccinations.

Stability of the FMD vaccine is of crucial importance in global FMD control vaccines with improved stability & less reliant on a cold chain are needed.

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

Aims

Stability of the FMD vaccine is of crucial importance in global FMD control

1. To evaluate of the immune responses of Nguni cattle vaccinated with FMD stabilized SAT2 antigens compared to wild-type antigen ZIM/7/83

vaccines with improved stability and less reliant on a cold chain are needed.

FMD virus is unstable due to: infectivity relying on disassembly caused by acidification by endosomal vesicles. As a result, FMD vaccine antigens are unstable in mild acidic pH and elevated temperatures. The instability of the capsid (146S) which causes the virus to dissociate into smaller 12S subunits FMD vaccines but especially for the serotypes O and SAT2 (Doel and Baccarini, 1981). Project: 2010-2012 Unstable antigen are believed to be less immunogenic due to degradation Output: before and after inoculation. Mutations in SAT2 capsid that Relative instability of the antigen of SAT2 has been linked to a reduction in lead to increased temperature vaccine efficacy (Doel and Baccarini, 1981). stability. Therefore, to be effective, FMD vaccines require frequent booster vaccinations. Chimeric viruses showed increased stability both in vitro & in vivo (guinea pigs)

2. To compare the efficiency of 2 adjuvants, ISA206 and Quil A saponin.

178

Liquid Phase Blocking ELISA

EXPERIMENTAL GROUPS GROUP 2 7 cattle vaccinated with Mutant A stabilised SAT2 vaccine + ISA 206 adjuvant

GROUP 1 7 cattle vaccinated with Wild-type SAT2 ZIM 7/83 vaccine + ISA 206 adjuvant

GROUP 3 7 cattle vaccinated with Mutant B stabilised SAT2 vaccine + ISA 206 adjuvant

Mutant B ISA 206 Mutant B Saponin Mutant A WT Control Group

Compare 3 Antigens 8ug per dose GROUP 5 2 cattle No vaccination Control Group

Compare 2 Adjuvants

M4 M5

GROUP 4 7 cattle vaccinated with Mutant B stabilised SAT2 vaccine + Saponin adjuvant

WT, Mut B ISA-206, Mut B Saponin peaked at 9 dpv. Mut A peaked at 14 dpv after first vaccine dose. After 14 dpv, all groups increased their titers, except Mut B Saponin. The second vaccination elicited similar titers in all vaccinated groups. Mut B Saponin showed decreasing curve, starting one month after the 2nd vaccine.

Virus Neutralisation Test

IgG1 & IgG2 isotyping

IgG1 3

Mutant B ISA 206 Mutant B Saponin Mutant A WT Control Group

2nd Vaccination 42-133dpv: 56dpv: Mut B Isa > Mut B Sap > Wt > Mut A

VNT- 42 dpv 4

IgG1: 1st vaccination 0-42dpv: 21dpv: Mut B Sap - sharp decline 28dpv: Mut B Isa > Mut A & Wt

b a

3 2

DPV

100

125

BSap A

BIsa

dpv. VNT titers higher for WT & Mut B ISA 206 (b) compared to others (a). From 21-42 DPV: WT > Mut B ISA 206 & Mut A > Mut B Sap & control. From second dose 42 DPV no statistical differences in VNT titers between vaccinated groups . VNT titers were more comparable between animals of the same group than LPBE titers.

IgG2

179

IgG2: 1st vaccination 0-42dpv: 21dpv: Mut B Isa 42dpv: Wt 2nd Vaccination 42-133dpv: 56dpv: Mut B Isa & Mut B Sap > Wt & Mut A

Challenge - LPBE

Temperature

Clinical score

Challenge - Clinical data

Con Days post challenge

BSap

BIsa

Con

BSap

BIsa

Days post challenge

9 DPI: stabilized vaccines achieved higher LPBE titers than WT Animals with clinical symptoms: WT = 0/7 full protection Mut A = 0/7 full protection Mut B ISA206 = 0/7 full protection Mut B Sap = 1/7 partial protection Control = 2/2 (no vaccination)

Challenge

Virus Isolations & real-time PCR

Days post-challenge Probangs Antigen Mut A

Animal 8 14 22 37 38 61 68

D0 Neg Neg Neg Neg Neg Neg Neg

D2 Neg Neg Neg 18 Neg Neg Neg

D4 D7 36 37 41 31 26 36 27 34 34 36 34 Neg 33 40

5 21 25 26 33 56 67

Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg 18 Neg Neg Neg

36 30 33 27 33 25 30

Control

34 53

Neg Neg

18 18

Mut B ISA206

17 35 39 47 62 71 73

Neg Neg Neg Neg Neg Neg Neg

Mut B Saponin

6 9 20 32 43 59 69

Neg Neg Neg Neg Neg Neg Neg

Days post-challenge Retropharyngeal tonsil swabs

Concluding remarks All vaccines were protective. Mut A, Mut B ISA206 & WT were 100 %. Mut B Sap partially. Kinetics of specific Abs seems to be more stable over time when oil adjuvant (ISA206) used by maintaining Ab levels longer than the aqueous adjuvant (Sap) (breadth of the immune response). Mut B ISA206 gave higher responses (final titers & breath) than Mut A, and similar to the WT. IgG1 > IgG2 indicative of correlation to protection Further work on interferon gamma response, measurements of Avidity and CMI will help to identify differences in the immune responses elicited by the different stabilised vaccines. SAT2 vaccine: results confirm the recommended strategy to perform 1 st vaccine with 2nd vaccine 4-6 weeks later. Possible that this design could not differentiate true stability of stable vs WT antigens. Future aims to test storage, heat dissociation and potency might show better comparisons

Clinical Scores Protected

D9 D11 33 35 29 29 33 34 33 ND 34 Neg 35 34 34 32

D0 Neg Neg Neg Neg Neg Neg Neg

D2 Neg Neg Neg Neg Neg Neg Neg

D4 32 33 29 29 33 34 32

D7 33 33 34 34 33 Neg 36

D9 32 32 32 34 32 31 32

D11 30 33 36 ND 32 32 35

Y Y Y Y Y Y Y

D0 0 0 0 0 0 0 0

D2 0 0 0 0 0 0 0

D4 0 0 0 0 0 0 0

D7 0 0 0 0 0 0 0

32 33 38 29 44 32 35

29 35 34 32 33 Neg 29 33 33 34 33 35 33 33

Neg Neg Neg Neg Neg Neg Neg

26 29 33 30 33 31 32

32 34 34 38 35 37 Neg

33 32 33 32 32 30 32

31 33 34 33 31 32 32

Y Y Y Y Y Y Y

0 0 0 0 0 0 0

29 32

25 31

28 30

33 34

Neg Neg

34 33

29 42

24 32

30 30

N N

0 0

Neg 18 18 18 Neg 14 Neg

36 23 23 25 27 26 32

36 32 31 33 36 34 35

33 32 34 33 33 33 33

32 34 34 32 33 34 34

Neg Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg 16 Neg

32 24 25 31 21 27 33

33 Neg 34 34 Neg 36 35

32 34 32 33 31 31 30

32 31 30 31 32 34 33

Y Y Y Y Y Y Y

0 0 0 0 0 0 0

Neg Neg 18 Neg 18 Neg 18

35 34 23 34 23 33 25

32 33 32 37 33 35 33

33 38 34 32 31 32 34 Neg 28 34 34 34 33 33

Neg Neg Neg Neg Neg Neg Neg

34 32 25 35 28 37 27

33 33 31 Neg 33 Neg 35

33 32 31 32 32 31 32

32 31 34 41 30 33 32

Y Y N Y Y Y Y

0 0 0 0 0 0 0

0 0

8 6

D9 D11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

8 6

4 4 0 0 0 0 0 0 0

2 0 0 0 0

2 2 0 0 0 0 0 0 0

0 0 2

0 0 0 0

0 0 0 0 0 0 0

0 0 1 0 0 0 0

180

Elizabeth Rieder

Bryan Charleston Julian Seago

ARC-OVI TADP team: Francois Maree Maclaughlin Rathogwa Kedibone Mawela Raksha Bhoora Liz Botha P. Mutowemba B. Botha J. Esterhuysen D.Semenya

Demonstration of a high potency SAT 2 Saudi Arabia vaccine in cattle and confirmation of efficacy in pigs

Dave Stuart Liz Fry Abhay Kotecha Alejandra Capozzo

L. Mouton1 , A. Dekker2, M.J. Bleijenberg2, M. Blanchet1, C. Hamers1, J. Coco-Martin3, P. Hudelet1, S. Goutebroze1

Danny Goovaerts Nico Visser

1: Merial S.A.S., Lyon, France; 2: Central Veterinary Institute (CVI), Lelystad, The Netherlands; 3: Merial B.V., Lelystad, The Netherlands 1

Background

FMDV South African Territories (SAT) types 1-3 are endemic to sub-Saharan Africa Recent incursions of SAT 2 topotype VII virus into Middle East countries have been recorded Genotyped SAT 2 strain isolates reported from 2003 to 2013 in Africa (Tekleghiorghis et al., Onpress)

SAT 2 Saudi Arabia (SAU) is listed as high priority by WRL Pirbright

Relationship between SAT 2 topotype VI isolates (Ahmed et al., 2012)

FMDV infection with SAT strains have been well studied in cattle but not in pigs, as their role in the epidemiology is likely to be of minor importance

For type SAT-2 the topotypes and genotypes were: I

III

VII

XIII 2

181

PD50 study in cattle

Materials and methods

Serology monitoring

Vaccination SAT 2 SAU FMD antigen on D0 (injected dose)

10.000 cattle ID50 SAT 2 SAU FMD challenge on D28

Aftovaxpur DOE (full dose : 2 ml)

Yes

Aftovaxpur DOE (1/4 dose: 0.5 ml)

Yes

Aftovaxpur DOE (1/16 dose: 0.125 ml)

Yes

Aftovaxpur DOE (1/64 dose: 0.031 ml)

Yes

Non-vaccinated controls

Yes

Vaccination G1-G4 (decreasing dose, SC route)

Challenge G1-G5 (10.000 cattle ID50, ID into tongue)

D28

D0 BS

4.00 3.50 3.00

Titres (Log10 VN titre)

Group

Number of cattle

2.50 2.00 G1 (vaccinated 2 ml): 5 cattle

1.50

G2 (vaccinated 0,5 ml): 5 cattle

1.00

G3 (vaccinated 0,125 ml): 5 cattle*

0.50

G5 (controls): 2 cattle*

G4 (vaccinated 0,031 ml): 5 cattle*

0.00

Necropsy Sedation G1-G5 G1-G5 (FMD signs) (FMD signs)

*On D32, 1 cattle in G3, 1 cattle in G4 and both cattle in G5 presented FMD lesions at inspection. They were humanly euthanised on D32.

D32 BS* Rectal temperature

D36 BS

All vaccinated cattle (groups G1 to G4) clearly seroconverted after vaccination (by D28), with an effect of the vaccine dose

General clinical signs BS: blood sample (VNT)

* If euthanised on D32

PD50 study in cattle

Rectal temperature after challenge

Protective dose

42.0

Mean rectal temperature (°C)

Days

G1 (vaccinated 2ml): 5 cattle G2 (vaccinated 0.5ml): 5 cattle G3 (vaccinated 0.125ml): 5 cattle* G4 (vaccinated 0.031ml): 5 cattle* G5 (controls): 2 cattle*

41.5 41.0 40.5 40.0 39.5

Group

SAT 2 SAU FMD challenge (D28)

Protected animals (D32 or D36)

G1 (vaccinated 2 ml) G2 (vaccinated 0.5 ml) G3 (vaccinated 0.125 ml) G4 (vaccinated 0.031 ml) G5 (controls)

Yes Yes Yes Yes Yes

5/5 5/5 3/5 3/5 0/2

39.0 38.5 38.0 D27

D28

D29

D30

D31

D32

D33

D34

D35

Vaccine

D36

Days *On D32, 1 cattle in G3, 1 cattle in G4 and both cattle in G5 presented FMD lesions at inspection. They were humanly euthanised on D32.

Aftovaxpur DOE

Transient increase of temperature observed in all groups after challenge 6

182

Experiment results (global logistic regression model)

1 PD50 (ml)

PD50 per dose

0.03

59.1

The vaccine tested provides a very high level of clinical protection against virulent SAT 2 challenge in cattle 7

Efficacy in pigs

Materials and methods

Serology monitoring

Number of pigs

Vaccination SAT 2 SAU FMD antigen on D0 (injected dose)

100.000 TCID50 SAT 2 SAU P2 FMD challenge on D28

Aftovaxpur DOE (dose = 2 ml)

Yes

Non-vaccinated controls

Yes

Challenge G1-G2 (100.000 TCID50, 0,4 ml; ID bulb heel)

Vaccination G1 (2 ml, IM)

3.50

FMD VNT (Log10 VN titres)

Group

Sedation Necropsy G1-G2 G1-G2 (FMD signs) (FMD signs)

3.00 2.50 2.00 1.50 1.00

G1 Vaccinated: 5 pigs

0.50

G2 Controls: 5 pigs*

0.00 0

Days of study

* One pig of group G2 was euthanised on D31

D0 BS

Left hind foot challenge (Pacheco and Mason, 2010)

D31 BS*

D28

D36 BS

All vaccinated pigs (G1) clearly seroconverted after vaccination An increase of mean antibody titres was observed in both groups after challenge

Rectal temperature

Injection into the bulb of the claw (4 * 0,1 ml)

General clinical signs Virus isolation (mouth swabs) BS: blood sample (VNT) * If euthanised on D31 8

Efficacy in pigs

Rectal temperature after challenge

Virus isolation and FMD specific lesions

40.5 G1 Vaccinated: 5 pigs

Group

G2 Control: 5 pigs*

Vaccinated

39.5 39.0 38.5

Non-vaccinated

Temperature ( °C)

40.0

38.0 D27

D28

D29

D30

D31 D32 Days of study

D33

D34

D35

D36

* One pig of group G2 (# 8026) was euthanised on D31 (hyperthermia > 40°C since 2 days and lameness)

Pig #

Titre of mouth swabs (log10 pfu/ml)

Lesions* inspection

Lesions* post-mortem D36

D28

D29

D30

D31

D34

D35

D31

8019

8020

8021

8022

RF (±)

8023

8024

2.0

3.1

8025

3.7

4.1

LF (±), RF (±), RH, T

8026 8027

0.3 1.4

3.7 3.2

3.8 3.2

RF, T -

8028

2.0

3.4

4.2

*Lesions on left hind foot were not reported (site of challenge inoculation) RH: right hind foot; LF: left front foot; RF: right front foot; T: tongue (±) Doubtful lesion Not applicable (euthanised)

None of the pigs in G1 (vaccinated) showed hyperthermia after challenge (D28) 10

183

Efficacy in pigs

Conclusion

Protection Group

SAT 2 SAU FMD challenge

Virus positive animals

Protected animals

G1 (vaccinated 2ml)

Yes

0/5

4/5*

G2 (controls)

Yes

5/5

2/5

The vaccine tested provided a very high level of clinical protection against SAT 2 SAU challenge in cattle Its efficacy against virulent challenge was confirmed in pigs, although live virus challenges in pigs or even wild porcine have been rarely reported

* Considering # 8022 (doubtful lesion) unprotected

Pathogenicity of SAT 2 SAU was demonstrated in pigs A complete prevention of viral excretion was evidenced Partial clinical protection in pigs was shown, although not significant under conditions of the test

Internal and outsourced collaborations

Looking forward from FMD epidemiology to FMD ecology Rebecca Garabed Associate Professor, The Ohio State University

184

Two Paradigms

Similarities

Epidemiology and Ecology

Both provide information about a disease. Both can provide information about control. Both can be descriptive or analytic. Both summarize results as they apply to the population(s).

Epidemiology describes risks of disease in a population. Ecology describes interactions among populations including, but not limited to disease. Human Decisions Cattle Virus Small Ruminant s

Swine Wildlife

Epidemiologic Studies Descriptive

Differences Ecology provides context and for epidemiology. Ecology has more inherent complexity and non-linearity. Ecology is mechanistic while epidemiology is statistic.

without History of Vaccination in the Far North Region of Cameroon." Transboundary and Emerging Diseases (2014).

185

Ecological Studies Virus and Host

Current Questions in FMDV Transmission What is the role of movement in disease transmission? What are carriers and how do they contribute to disease transmission? Where and how do wildlife spread disease? If we control clinical disease does subclinical disease still spread significantly? Do co-circulating strains and serotypes enhance or

Garabed, Rebecca B., Wes O. Johnson, and Mark C. Thurmond. "Analytical Disease Virus." Transboundary and Emerging Diseases 56.4 (2009): 142-156.

How do human dimensions (economics, compliance, motivation for control, regulatory decision-making) influence transmission and how can we influence them?

What can ecology tell us?

Land Use Water Management and Human Health

Pictures from Paul Scholte and Mark Moritz

186

Pictures from Jessica Healy Profitos

Case Study 1 The Role of Movement in Cameroon Risk of disease due to transhumance

What about FMD(V)?

Bronsvoort et al. 2004 odds ratio 2.6 Garabed et al. (2010 data) relative risk 1.32

Modeling dynamic transmission

Movement accounts for an average of 10.8% of the incidence in endemic models. The difference in incidence is not statistically significant when accounting for variation among simulations.

(not yet published)

Differences in reproductive parameters can explain some differences in risk.

Work with Patrick Schnell, Yibo Shao, and Joseph Tien Work with Patrick Schnell, Yibo Shao, and Joseph Tien

187

Case Study 2 Human Dimensions of FMD in the UK

Effects of humans on FMD Kao, Rowland R. "The impact of local heterogeneity on alternative control strategies for foot-and-mouth disease." Proceedings of the Royal Society of London. Series B: Biological Sciences 270.1533 (2003): 2557-2564. Uses actual mechanisms of human behavior, animal movement, and species differences (all interacting) in key inferences and conclusions.

Pictures from bbc.co.uk and telegraph.co.uk

Effects of FMD on humans

Human Dimensions of FMD The interactions of humans and FMD Adaptive decision-making Modeling the complete social-ecological system

188

Methods We Need

Teams We Need Quantitative and qualitative Virology, genetics, and immunology Movement from the geographic perspective and movement from the human perspective Policy, economics, and psychology Wildlife and livestock Anthropology and other social sciences International Communicators (translators within teams)

Looking across scales Hierarchical models Differential equation models Agent-based models

Looking at feedback, adaptation, and endogeneity Emergent simplicity Emergent complexity Perturbations to the system

Data We Need

Summary Epidemiology is about risk and ecology is about interactions. Both are needed in FMDV research. Questions like the role of movement and human dimensions of FMD need to be examined under the ecological paradigm. New multi-scale models and interdisciplinary teams are needed to answer these questions. We also need small-scale, high-resolution data.

Some questions are theoretical questions that do not require data. Wide-spread animal tracking and large scale surveillance have a place. Data from controlled laboratory studies. Small scale detailed data that looks at all of the complexity is needed. 189

What does this mean for me?

Acknowledgements

Make a new friend here today. Make the effort to understand other disciplines and settings. If you are doing a study consider including additional questions, tests and samples. Support data collection at all scales.

Co-authors Laura Pomeroy, Mark Moritz, Shweta Bansal, Ningchuan Xiao, Michael Tildesley, Joseph Tien, Luis Rodriguez Collaborators Disease Ecology and Computer Modeling Laboratory, National Veterinary Laboratory (Cameroon), and Center for the Support of Research and Pastoralism (Cameroon), PIADC-ARS Funding PHPID and IPR at the Ohio State University, National Science Foundation - Ecology and Evolution of Infectious Diseases Program, USDA FADDL, and USDA CEAH

Think Ecologically!

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

Foot-and Mouth Disease Ecological Studies In Endemic Settings: Ongoing Studies in Vietnam and 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 Dr. Zaheer Ahmed Dr. Anna Ludi

Luis Rodriguez (Jonathan Arzt)

Research leader, USDA-ARS Plum Island 1

190

Study Design Acute Clinical Samples Clinical case reports, geographic location, demographics, etc Clinical samples, viruses, sequence Longitudinal Field Study Population: buffalo premises all located near Islamabad Serological survey of 40 farms for NSP positive animals, probang, history Selection of 29 farms (300 buffalo) for sampling (serum and probang) 4X year for 1 year NSP-ELISA, RT-PCR and virus isolation Establish panel of reference sera for in country vaccine evaluation Vaccination of 10 cattle and 10 buffalo with commercial vaccine to be used in field Serum collection at 0, 21 dpv, boost vaccination Serum collection at 42 dpv Carry out vaccine matching studies

Significant amino acid and nucleotide variation, wide spatial distribution of genetic lineages, no with differences in host species, all consistent with frequent multispecies infection of this serotype O FMDV in a highly mobile population

Obtained multiple viral strains from persistent buffalo opportunity to look mechanism of strain emergence 8 /22 Animals 2 viruses of same serotype isolated at different sampling time points from same animal [8x2=16 viruses]

ASIA 1 = 14 O

TOTAL = 16

3/22 Animals

22/51 Animals VI positive 2 or more times, yielding 47 virus isolates

3 viruses of same serotype isolated from each Id at different sampling time points [3x3=9 viruses isolated]

9/22 Animals 2 viruses of 2 different serotypes isolated from each animal at different sampling time points [9x2=18 viruses isolated]

ASIA 1

TOTAL = 9

ASIA 1/A

8X2=16

ASIA 1/O

1X2=2

TOTAL

=18

1/22 Animal (3 viruses of 2 different serotypes isolated from each Id at different sampling time points) [1x3=3 viruses isolated]

191

ASIA 1/A =3

Phylogeny of Carrier vs Clinical Serotype A Viruses

Vaccine Evaluation Serotype O FMDV Pak-O 2010, 2012, 2012 SGD/PAK/19/2011 JX170755

Green: Carrier viruses Red: Clinical local Blue: Clinical non-local

KHI/PAK/42/2011 JX170757 ISR/7/2007 AJ294910

PanAsia 2

OKAB/AFG/L2826/2009 HQ439234 MAY/2/2004 HQ116194 MAY/2/2006 HQ116205 PAK/39/2008 GU384685 97

Found close relationship between persistent and acute lineages suggesting transmission

PAK/63/2007 FJ798183

99 76

BAG/AFG/L1494/2009 HQ439234 MAY/11/2009 HQ116217

PAK/1/2008 FJ798190 PAK/29/2008 GU384684 CAM/1/2008 HQ116174

Directionality of transmission not yet determined

99 90

VIT/124/2010 VIT/169/2010

PanAsia

VIT/17/2005 HQ116283 VIT/7/2002 HQ116273

UKG/3802/2001 DQ164982*** SKR/1/2002 DQ164972 VIT/3/2005 HQ116277

TUR/2/2001 DQ164982 80

Phylogeny Reconstruction Statistical Method -------------- Maximum Likelihood Test of Phylogeny --------------- Bootstrap method (500 replicates) Substitutions Type -------------- Nucleotide Model/Method ------------------ General Time Reversible model Rates among Sites --------------- Gamma distributed with Invariant sites (G+I) No of Discrete Gamma Categories - 5 ML Heuristic Method ------------- Nearest-Neighbor-Interchange (NNI) Initial Tree for ML ------------------Make initial tree automatically No. of Seqs : 33 No. of Sites : 623 *** - reference strain

Ankara/TUR/377/10/02 DQ296523 TUR/4/2005 FJ561321 TUR/2/2000 DQ164982 PAK/18/2002 DQ164982

IRN/20/2004 DQ164982

ME-SA

99 97

Mersin/TUR/13/01/04 DQ164982

IND/53/73_AF292107 O1 Manisa iso87 AY593823 Vaccine Virus representative India/R2/75 AF204276

SEA CAM-94

CAM/3/98 AJ294910

0.02

Vaccine Evaluation Serotype A FMDV

Serotype O Vaccine Matching against O1 Manisa

100 87

PAK/CHK/11/2012* PAK/KCH/15/2012*

PUN/PAK/L1354/2009_HQ439251 PAK/76/2009_GU384686

O/KHI/41/11

O/JGH/6/12

O/ISB/255/12

O/FSD/266/12

SIN/PAK/L694/2009_HQ439247 SAR/AFG/L1435/2009_HQ439274 IRN/5/2008_FJ755068 TUR/1/2008_FJ755133 IRN/1/2005_EF208769*** vaccine virus representative PAK/1/2006_FJ755082 PAK/ICT/2/2008* PAK/ICT/1/2008

neut.

r1-value

neut.

r1-value

neut.

r1-value

neut.

r1-value

2.33

1.8

0.71

2.18

1.65

0.5

2.48

1.95

2.25

1.95

1.8

1.5

0.5

2.4

1.95

0.5

1.5

0.5

2.03

1.35

0.35

1.5

1.8

PAK/ICT/3/2008 100

PAK/KCH/5/2009* PAK/KCH/6/2009 PAK/KCH/7/2009

AFG/131/2004_EF457981

Phylogeny Reconstruction Statistical Method -------------- Maximum Likelihood Test of Phylogeny --------------- Bootstrap method (500 rep) Substitutions Type -------------- Nucleotide Model/Method ------------------ General Time Reversible model Rates among Sites --------------- Gamma distributed with Invariant sites (G+I) No of Discrete Gamma Categories - 5 ML Heuristic Method ---------Nearest-Neighbor-Interchange (NNI) Initial Tree for ML ------------- Make initial tree automatically No. of Seqs : 24 No. of Sites : 623 *** Ref Strains * used for vaccine matching

IRN/41/2003_FJ655020

* All values given in log10

PUN/PAK/L1364/2009_HQ439253

Conclusion: r1-values are above 0.3 suggesting the vaccine is protective against the serotype O viruses tested.

IRN/22/99_EF208772*** IRN/2/87_EF208770*** A22 Iraq64 iso86_AJ251474*** vaccine virus representative IRN/1/96_EF208771*** 100

192

IRN/5/2003_FJ775018

0.05

VIETNAM

neut.

r1 A22

1.65

1.65 1.41F

2.03

1.65

0.7

MOLECULAR EPIDEMIOLOGY, SURVEILLANCE AND PREDICTIVE TOOLS FOR FMD CONTROL IN VIETNAM 58-1940-0-070F, 057 14S 6/1/2010-9/30/2012 A/KCH/15/12

A/KCH/5/2009

A/ICT/2/2008

A/CHK/11/2012

Serotype A Vaccine Matching against A-IRN05 and A22 IRQ

R1 IRN

neut.

r1 A22

r1 IRN

neut

1.73

1.95

1.41

1.8

<1.2

1.95

<1.2

0.25

2.25

1.65

1.41

0.5

2.03

<1.2

2.48

<1.2

1.58

<1.2

Collaborators: Dr. Jonathan Arzt Dr. Helena Ferreira Dr. Thanh Long Ngo, DAH, HCMC Dr. Ho Huu Dung, DAH, Hanoi Dr. Carla Huston, Mississippi State University

Conclusion: * All values given in log10 VN titers above 1.6 observed for 2008-2009 isolates VN titers below 1.2 for 2012 isolates, suggesting that the vaccine is not a good match to these strains.

Design

Objectives 1- Molecular epidemiology of FMDV in local livestock including cattle, buffaloes and pigs

Objective 1 Acute Clinical Samples Samples collected during outbreak Pigs, buffalo and cattle Northern and Southern Vietnam

2- To better understand the transmission mechanism of persistently infected to susceptible livestock in natural setting

Objective 2 Longitudinal Field Study HCMC region tranmsission cells (9 farms) Hanoi-SonLa region carrier cattle and buffalo (field necropsy)

3- Enhance strategies for identification of persistently infected animals using new technology

Objective 3 Persistent buffalo studies Field necropsies Molecular characterization (tissue level, cytokine mRNA, protein expression)

193

Objective 2. Ecology of FMDV carriers

Objective 1. Phylogenetics of novel strains of FMDV

Two Provinces identified with history of FMD outbreaks in cattle, buffalo SonLa (north)

Multiple lineages in multiple species no species specific lineages Novel strains (bold) found in this study

Jan 2011

LongAn (south) Feb. 2011

Established study sites: Targeted surveillance study Buffalo necropsy study / cattle transmission study

Objective 3. Transmission Study From Carrier Cattle to Sentinel Cattle

Targeted surveillance study

Study site in southern Vietnam no clinical activity >1yr Individual small premises - pastures, surrounding premises vaccinated bi-annually 9 farms 2 donor carrier buffalo or cattle (NSP +, Probang +) housed in direct contact with 2 -, Probang -) Direct contact - one year study serum and probang every 2-3 months Probang rRT-PCR, VI -

Risk of being FMDV infected and/or FMDV carrier: Buffalo - highest risk of being FMDV-infected, intermediate carrier risk Dairy cattle lower risk of being FMDV-infected (OR=0.2), lowest carrier risk (OR=0.2) Beef cattle - lowest risk of being FMDV-infected (OR=0.4), highest carrier risk (OR=4.9)

Carrier infected : 3ABC+ and Probang +; Ever inf.: 3ABC+; Never inf. : 3ABC neg

194

Preliminary Results

Thank you!

All donors shed virus intermittently throughout the exposure time probang negative until the end

of the study Multiple viral sequences were obtained from persistent animals

SUPPORT:

195

ARALLEL he ̀ FMD science and polic ̀ deelopent landscape ̀

196

FMDV-O 0.02

Outbreaks of FMD in North Africa and the Middle East during 2013 and 2014 due to an exotic O/ME-SA/Ind-2001 lineage Kasia Bankowska, PhD The Pirbright Institute

Vesicular Disease Reference Laboratory Group

EA-3 1

O-Ind-2001

O-Ind-2001 VP1

Limited outbreaks: O/SAU/11/2001 (DQ164970) O/UAE/6/2001 (DQ164997) O/OMN/4/2001 (DQ164940) 100 O/BAR/1/2001 (DQ164863) O/ISR-Ramala 1-02* (HM561396) 99 O/PAT/2/2002 (DQ164944) O/KUW/3/97 (DQ164904) O/BAR/2/97 (AJ318824) 99 96 O/UAE/7/97 (DQ164992) O/UAE/1/2008 100 O/UAE/1/2009 O/SAU/2/97 (AJ318851) O/ISR-Gush Halav-96* (HM561410) 98 O/JOR/2/95 (DQ164903) O/IRN/72/2009 84

1995- Jordan

1996- Israel 1997- Bahrain, Kuwait, Saudi Arabia and the United Arab Emirates (UAE)

2001- Bahrain, Oman, Saudi Arabia and UAE 2002- Palestinian Autonomous Territories and Israel

2008-2009- UAE 2009- Iran

Ind-2001b

Ind-2001c Ind-2001a Ind-2001d

0.01

The O-Ind-2001 lineage has been divided into four sub-lineages: a, b, c and d.

197

O-Ind-2001 during 1995-2009

O-Ind-2001d during 2013-2014 - based on reported outbreaks

a b1 b2 c d

O-Ind-2001d

O-Ind-2001d during 2013-2014 0.005

Libya India Saudi Arabia Nepal UAE Bhutan Tunisia Algeria Sri Lanka Sequences were determined at The Pirbright Institute (WRLFMD), PD-FMD, Mukteswar, India and IZSLER, Brescia, Italy 7

198

Real-time RT-PCR (VP1)

rRT-PCR

Samples 0

0 3D Ind2001 13.69 13.58 17.41 18.92 18.17 19.48 16.76 17.62 16.89 18.21 16.9 17.96 15.15 15.29 16.02 17.39 16.52 17.08 15.08 15.3 18.04 18.82 10.85 11.35 11.83 11.36 11.87 11.47 13.61 14.58 15.34 15.34 32.97 32.56 15.4 15.94 13.98 13.82 15.67 16.21 14.6 15.15 16.73 14.99 15.28 14.05 13.34 14.27 15.33 15.63 14.28 14.16 13.33 13.45 30.24 30.8 13.88 13.77 14.96 13.8 16.55 16.56 15.95 15.26 17.57 15.92 15.32 15.92 14.95 15.27

10 0.005

Ct value

Topotype Lineage ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001 ME-SA Ind-2001

3D 30

VP1

40 50

No60 Ct

Samples 0

0 10

Ct value

Isolate BHU/1/2013 LIB/1/2013 LIB/2/2013 LIB/3/2013 LIB/4/2013 LIB/5/2013 LIB/6/2013 LIB/7/2013 SAU/1/2013 SAU/3/2013 SAU/4/2013 SAU/6/2013 SAU/7/2013 SAU/8/2013 SAU/1/2014 NEP/3/2013 NEP/4/2013 NEP/6/2013 NEP/11/2013 NEP/12/2013 NEP/15/2013 NEP/16/2013 NEP/17/2013 NEP/18/2013 NEP/1/2014 NEP/2/2014 NEP/4/2014 NEP/15/2013 NEP/5/2014 NEP/6/2014 NEP/7/2014 NEP/8/2014 NEP/9/2014 UAE/1/2014 UAE/2/2014

20 30 40 50

No60 Ct

Knowles et al. In press 9

Isolate LIB/1/2012 LIB/2/2012 LIB/3/2012

Topotype ME-SA ME-SA ME-SA

Lineage PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 unnamed PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2 PanAsia-2

3D 18.57 20.61 15.48 22.77 25.19 22.23 24.54 17 14.24 13.15 12.37 13.2 13.04

Ind2001 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct

LIB/5/2012 LIB/7/2012 LIB/48/2012 LIB/74/2012 3D LIB/54/2012 PAK/5/12 VP1 PAK/53/12 PAK/55/12 PAK/3/13 PAK/4/13

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

PAK/8/13 PAK/12/13 PAK/14/13 PAK/15/13 PAK/16/13 PAK/20/13 PAK/22/13

ME-SA ME-SA ME-SA

PanAsia-2 PanAsia-2 PanAsia-2

12.91 15.52 12.7

No Ct No Ct No Ct

ME-SA ME-SA ME-SA ME-SA

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

11.45 11.78 15.49 12.77

No Ct No Ct No Ct No Ct

Conclusions

Acknowledgments The Pirbright Institute:

VP1 sequence data supports independent introductions of O-Ind2001 sub-lineage d into Gulf States area (probably in 2013) as well as to Libya, Tunisia and Algeria.

Nick Knowles Don King Jemma Wadsworth Begoña Valdazo-González Valerie Mioulet and the WRLFMD team Libya:

Ibrahim Mohamed Eldaghayes, Abdulwahab Kammon & Monier Sharif

We have determined complete genome sequences of representative viruses form Bhutan, Saudi Arabia and Libya (Valdazo-González et al. 2014).

Italy (IZSLER, Brescia):

Santina Grazioli & Emi Brocchi India (PD-FMD, Mukteswar):

Saravanan Subramaniam & Bramhadev Pattnaik

A recent separate introduction of the virus to Sri Lanka has occurred.

Saudi Arabia:

Scott Waight & Arafa Shamia Bhutan:

The spread of this lineage needs to be monitored and the lineage specific real time RT-PCR can be applied for this purpose.

Sangay Tenzin UAE:

Ulrich Wernery 11

199

Mass vaccination FMD vaccine protection: - Requires several 50) - Declines with time since vaccination

Mass vaccination, immunity and coverage:

Mass vaccination

Modelling population protection against FMD in Turkish cattle

[No. of doses, time since last dose]

Theo Knight-Jones Epidemiologist, ILRI

Mass vaccination twice a year: Population age-sex-vaccination distribution

Beef suckler cattle: Beef fattener cattle: Different production system = different age structure = different population immunity Males

Females Females Age 4-5 years 9-10 doses 7-8 doses

3-4 years

5-6 doses

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

Percentage of population 3

200

Percentage of population 4

Population vaccine history & immunity varies by region

Structure changes with births & deaths over the annual production cycle

New births = New unvaccinated animals Population immunity is constantly changing with population turnover & declining antibodies

If multiple doses needed, variation in immunity resulting from variation in coverage becomes exaggerated

Post-vaccination immunity also declines with time depending vaccine history Vaccinated Spring+Autumn 2012

Vaccinated Autumn 2012

Vaccinated Autumn 2011+ Spring+Autumn 2012

Log10 (SP titre) 2.4 2.3

once

twice

3 times

District with 100% coverage:

Protection threshold

After 3 rounds: 100% of cattle vaccinated 3 times

District with 50% coverage:

After 3 rounds: 50% x 50% x 50% = 12.5% vaccinated 3 times

Autumn mass vaccination 7

201

Percentage never vaccinated 6 months after mass vaccination if eligible cattle always vaccinated

Population coverage model

median values reported

Describe population immunity over the production cycle with 2012 Turkish mass vaccination policy Simulated the Turkish cattle population for each province Age-structure by day and month of birth Using data from national random surveys for each province and census data

Dynamic population model representing the changing age structure for each province over the annual production cycle

Unvaccinated = Cattle too young at prior vaccination + New births since prior vaccination 9

But not all eligible cattle will be vaccinated

if eligible cattle always vaccinated

Field studies and routine data found 40 99.9% vaccinated Betapert distribution (minimum=40%, maximum=100%, most likely=80%)

Results: Six months after the last round of vaccination almost remain unvaccinated

Vaccinated

Only 50% of all cattle would have been vaccinated more than once with the last dose received

Adult cattle

202

From coverage to immunity

Population immunity predictions

Predict immunity for simulated population

Log10 (SP titre)

Two-dose primary course: Increases proportion of 6-12 month cattle above threshold by 25-40%

: 30% [24%-38%]

LPBE SP titre = Time since vaccination + No. of times vaccinated Log10 (SP titre) : 27% [20%-35%] Threshold titre is useful but Antigenic similarity of: 1) Vaccine 2) Test 3) Field virus

Using regression models fitted to data from extensive post-vaccination sero-monitoring study [n=647]

Log10

-40%]

Log10(SP titre)

Autumn mass vaccination

District coverage and population immunity

Primary course 14

Sustained antibodies after single dose Immunity reflects coverage Fewer problems

Modelled proportion vaccinated in a district at autumn vaccination against the percentage 2 in mid February of cattle with 15

203

From: Selman P, Chénard G, Dekker A (2006) Cedivac-FMD; Duration of Immunity in cattle, sheep and pigs. Open session of the EuFMD, Paphos, Cyprus, 17-19 October 2006 16

Conclusions: Mass vaccination in Turkey 2012 Major immunity gaps despite biannual mass vaccination Improved vaccine required Two-dose primary course used in certain areas

Immunity gaps will still exist Each round of vaccination may exclude a quarter of all cattle Often unavoidable

Improved biosecurity measures required Avoid over reliance on vaccine protection 17

Outline Disease modelling network

Modelling foot-and-mouth disease in Turkey

Data available Movements Births/deaths Cleaning

Peter Dawson Cavtat, 30th October 2014

Full network model Case study Future work 204

P.M.Dawson@Warwick.ac.uk

Disease modelling

Standard method is to use a compartmental model

Individual based model Model transmission as a Poisson process with daily time-steps

Move through compartments at rates determined from data Can write down a system of ODEs and solve

With infectious individuals recovering independently at a rate

P.M.Dawson@Warwick.ac.uk

Disease Modelling So far we have assumed homogenous mixing within the population Can make heterogeneous by incorporating an age or spatial structure For our case we will consider a network structure

P.M.Dawson@Warwick.ac.uk

205

P.M.Dawson@Warwick.ac.uk

Movements look good after 2006

Data Movement data Birth and death data Outbreaks Serosurvey Vaccination data

P.M.Dawson@Warwick.ac.uk

206

P.M.Dawson@Warwick.ac.uk

Movements to district from other districts

Births and Deaths

P.M.Dawson@Warwick.ac.uk

Births and Deaths

All is not lost

P.M.Dawson@Warwick.ac.uk

207

P.M.Dawson@Warwick.ac.uk

Cleaned data

Full network model

P.M.Dawson@Warwick.ac.uk

Specific farm model One farm with many outbreaks of different serotypes Look at different models using different control strategies Give a better idea of the within herd dynamics

P.M.Dawson@Warwick.ac.uk

208

P.M.Dawson@Warwick.ac.uk

SEIRS w regular vaccination

P.M.Dawson@Warwick.ac.uk

SEIRS w reactive vaccination

SEIRS w both vaccination strategies

P.M.Dawson@Warwick.ac.uk

209

P.M.Dawson@Warwick.ac.uk

Future work Will begin to scale the model up to a district level before moving to full nationwide model Add local spread A Bayesian parameter inference scheme such as MCMC or ABC will be run Test out various control strategies

P.M.Dawson@Warwick.ac.uk

Thank you

FMD in Tunisia: Heath situation, crises management, and future action plan

HAJ AMMAR Heni, ZRELLI Malek, KHORCHANI Roukaya General Direction of Veterinary Services EuFMD Cavtat Croatia le 28th October 2014

P.M.Dawson@Warwick.ac.uk

210

Plan

Plan Introduction Emergency measures Health situation Action plan Conclusion

Introduction Emergency measures Health situation Action plan Conclusion

Strategy fighting against FMD

Animal density (heads/Km²)

Official Control Plan

Medical prophylaxis

Surveillance

BV: trivalent vaccine

Laboratory

SR: bivalent vaccine

Training and sensibilization Risk analysis and economic studies Contingency plan FMD Simulation exercise Bovine density / delegation

Sheep density /delegation

Goats density / delegation

vaccination)

211

Maitrise et contrôle de la maladie

Plan

General results of FMD control plan Sub-comosante

visit numbers

negative

positive

Total

Pourcentage

Slaughter house

147

1059

1103

3,99

Markets

108

1038

1068

2,81

Sentinels

478

492

2,85

Transhumance

414

415

0,24

Fattening units (small ruminants )

304

1141

1152

0,95

Wildlife

105

0,00

Total

646

4235

100

4335

2,30

Introduction Emergency measures Health situation Action plan Conclusion

Evénement sanitaire en 2014 Nabeul : Friday, 25th of April 2014

Saturday 26th of April 2014 (samedi) :

Heard : 17 Bovine including 2 affected animals

Sampling and sending to laboratory (IRVT)

Diagnosis (IRVT) : rt-PCR Clinical signs : T , salivation, mouth lesions

Animal trader

Group health monitoring is compound Information of the Agriculture Ministry Immediate Meeting with regional VS of Nabeul:

Implementation of immediate measures (killing, confinement, vaccination peri-focal)

Objectives of implemented measures Sunday 27 avril 2014

Thursday : 29th of April 2014

Strengthening the immunization coverage of sensitive animals Ovoid the spread and dissemination of the disease

Memorandum note: immediate measure to implement in the hole country (last update 09th of august 2014)

Information of others departments

notification to OIE

Determine the origin of the infection Stock vaccine: good distribution of available vaccine doses

of 29th of April 2014

Management and control of animal movement

of 26th August 2014

Communication plan : activate of the plan (established in 2013)

212

Emergency Vaccination

Specific measures in the affected governorates

the measures are dependent on several factors. immunization, depended on the available vaccine in stock. Regarding the movement of animals, veterinary services have collaborated with the other departments In case of confirmation of the disease in Small Ruminants: vaccination of cattle and small ruminants in the outbreak(s) and peri-focal vaccination of all susceptible species within 5 km and vaccination of all cattle in the governorate affected In case of confirmation of the disease in cattle only: vaccination BV and PR in the outbreak(s) and vaccination of all cattle in the governorate affected Vaccination of fattening units (2653 farmers in PR 83581)

The search for the origin of the infection

Implementation of vaccination posts in livestock markets Ban output and input measures of susceptible animals to and from the affected governorates Information and awareness of Private Veterinarians and official health control and notification of suspected cases cleaning and disinfecting the (soda to 8 p1000)

Vaccination campagn Vaccine used: Cattle: O manisa; O maghreb99, A22, SAT 2 Small ruminants: O manisa; O maghreb99, SAT 2

213

Vaccination campagn in 2013 Vaccination coverge % in 2013 200

200.0

TCV PR

Objectif

79258

Cattle

407177

543444

3713008

818729 83581 (FU)

Emergency vaccination 622702 Number of vaccinated cattle during 2014

150

140.0 120.0

100

100.0

80000

80.0

60000

40000 20000

Reliquat de 2013

Communication

Vaccination of Small ruminants Started 1st week of september Ongoing 2900000 heads 60p100

Cumulative pourcentage of vaccination campaign of small ruminants

communication Plan establishing in 2013, Management of the health nformation, Communications in the media (Radio, Télé )

70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00

Weekly %

cumulative %

214

Medenine

Kebilli

Tozeur

Tataouine

Gafsa

Gabes

Sfax

Campagne d'urgence

Sidi Bouzid

Sousse

mahdia

Nabeul

Monastir

Kairouan

Le kef

kasserine

Jendouba

Beja

Tunis

Zaghouan

Tozeur

Tatauine

Silina

Sousse

Sfax

SidiBouzid

Nabeul

Monastir

Manouba

Medenine

Kef

Mahdia

Kebilli

Kairouan

Kasserine

Gafsa

Jendouba

Gabes

Bizerte

Beja

B arous

0.0

Ariana

20.0

Siliana

40.0

Bizerte

60.0

Zaghouan

(2011, 2012 et 2013).

TCV BV

160.0

Complementary

Ariana

Strengthen of the vaccination in the south of the country following the appearance of the disease in Libya

180.0

Emergency

La Manouba

67,8 p100 (3713008) in the small ruminants

2014

Annual

Ben Arous

In 2013: TCV total 78,9 p100 (407177 ) in the cattle

2013

Training and sensibilization

International coordination Tow missions :

Training for regional veterinarians

FAO (Eufmd , CMC) UE , Italie, OIE

Sensibilisation four sessions for livestocks techniciens (september 2014) sensibilization of the farmers

Juin 2014 Recommandations Donation UE:1000000 cattle doses (1 C = 2 Sheep) Coordination with UMA countries

Scientific and technic FMD commission

International coordination

Decision of the Minister of Agriculture Mains partners:

Reception of the vaccine doses of CE donation : 21 August 2014 First outbreaks: 26th of April 2014 Donation serotyping : O BFS, A Iran05, SAT 2 Eri (Cattle & Small ruminant )

ENMV CNVZ CONMVT Syndicat

Three meeting :

Vaccine used and matching vaccine test Experimentation of the vaccine (OBFS)

215

Tunisia strain = Algeria Strain 100 % homology

Origin : Ind-2001 99% homology

Plan

Matching strain

Introduction Emergency measures Health situation Action plan Conclusion 216

Clinical signs bovine

Clinical signs SR

Vidéo Aymen HENI

Geographic repartition

ARIANA BEJA BEN AROUS BIZERTE GAFSA JENDOUBA KAIROUAN KASSERINE LE KEF MAHDIA MANNOUBA MEDENINE MONASTIR NABEUL SFAX SIDI BOUZID SILIANA TATAOUINE TUNIS ZAGHOUAN Total général

Repartition of outbreaks by present species in the farms

Nombre de foyers 9 19 5 5 6 6 5 5 4 4 10 1 3 14 8 23 1 5 4 3 140

Affected species

Outbreaks

104

BV/OV

TOTAL

Répartition desfoyers enfonction des espèces animales atteintes dans les foyers

BV/OV, 10, 7% CP, 3, 2% OV, 18, 13%

Other, 26, 19%

BV, 104, 74%

140 PR, 5, 4% BV

217

BV/OV

Outbreaks

140

Morbidity rate 14,8

Sensitive-Bv Case_Bv

2500 435

Bovine

6548 7,6 546

Sheep

17/10/2014 - 22/10/2014

10/10/2014 - 16/10/2014

26/09/2014 - 02/10/2014

05/09/2014 - 11/09/2014

29/08/2014 - 04/09/2014

22/08/2014 - 28/08/2014

15/08/2014 - 21/08/2014

08/08/2014 - 14/08/2014

01/08/2014 - 07/08/2014

618

Epidemiologic indicators

Goats

Sensitives_O Case_Ov Sensitive_Cp Case_Cp v Small farms

7,3

218 Outbreaks sensitives cases % Morbidity

73 389 221 56,8

Medium farms ]10-50[ 34 706 156 22,1

Large farms 7 1391 60 4,3

<=10

> 50

12/09/2014 - 15/09/2014

05/09/2014 - 11/09/2014

29/08/2014 - 04/09/2014

22/08/2014 - 28/08/2014

15/08/2014 - 21/08/2014

08/08/2014 - 14/08/2014

18/07/2014 - 24/07/2014

11/07/2014 - 17/07/2014

04/07/2014 - 10/07/2014

27/06/2014 - 03/07/2014

20/06/2014 - 26/06/2014

13/06/2014 - 19/06/2014

06/06/2014 - 12/06/2014

30/05/2014 - 05/06/2014

23/05/2014 - 29/05/2014

16/05/2014 - 22/05/2014

y = 0.1251x2 - 4.8077x + 47.87 R² = 0.6971

09/05/2014 - 15/05/2014

02/05/2014 - 08/05/2014

Weekly evolution of FMD suspicions

25/04/2014 - 01/05/2014

25/07/2014 - 31/07/2014

18/07/2014 - 24/07/2014

11/07/2014 - 17/07/2014

04/07/2014 - 10/07/2014

27/06/2014 - 03/07/2014

20/06/2014 - 26/06/2014

13/06/2014 - 19/06/2014

06/06/2014 - 12/06/2014

30/05/2014 - 05/06/2014

23/05/2014 - 29/05/2014

16/05/2014 - 22/05/2014

09/05/2014 - 15/05/2014

02/05/2014 - 08/05/2014

25/04/2014 - 01/05/2014

Weekly evolution of the number of suspicions (until October 2014)

Weekly evolution of the outbreaks (until October 2014) 20

Evolution hebdomadaire de nombre de foyers

y = 0.0385x2 - 1.4889x + 17.255 R² = 0.6783

Total

Poly. (Total)

Epidemiologic indicators

Origin of infection Neighberhood

Immunity of affected farms (vaccination )

13.9

Selling

27.8

Entry of products and transport engines

The majority of affected herd: not vaccinated Affected animal are both young and adults (NSD) Affected animals are not vaccinated oven if the heard is immunized

72.2

Entry of persons

66.7

Entry of animals

69.4

Outgoing of products and transport engines

58.3

Outgoing of persons

77.8

Outgoing of animals (new, return)

55.6 0.0

10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

Plan

Action plan Objective: mastery of the health situation (September 2014) Immunization of small ruminants (PR):

Introduction Emergency measures Health situation Action plan Conclusion

total workforce estimated at 6000000,

Vaccination campaign exceptional reminder: small ruminants and cattle. Effective Cattle: 650000, Effective Small ruminants: 6000000.

Study of the immune status (January 2015) Evaluation of the vaccination (January 2015) Review official control programme for FMD Apply for endorsement of official control program for FMD

219

Plan

Comments

Introduction Emergency measures Health situation Action plan Conclusion

Comments Egypte 2012/13: A-Iran-2005 & A-Afr G-IV, Sat-2, O-Panasia & O-EA3

Zone endémique ???

Current situation: WHY ? Patterns predictable ? Underestimate threats ?

Border endemic area

Palestine (2013/14) O-PanAsia A-Iran-2005 Irak (2013) A-Iran-05 Asia-1 Iran (2014) A-Iran-05 O PanAsia-2 Asia-1

Algérie (July 2014) O-India -2001

TUNISIE (April 2014) O-India-2001

Libya (2013) O-India-2001

Libya 2003 FMDV-SAT2 2009 FMDV A 2010 FMDV-O 2012 FMDV SAT2

Emirates AU: 2013: O PanAsia-2 2014: O-India-2001 Arabie Saoudite (2013/14) O-India-2001

Zone endémique ??? Source: http://www.wrlfmd.org/

220

Conclusion

Thank you

Challenge: (Urgent Actions) FMD epizootic controlled Vaccine appropriate or1 value oHigh potency ( > 6 PD50) oVaccination twice per year

Enhanced coordination with Libyan Vet Authorities oSpecific program for southern Tunisia oControl animal movements oReview and evaluation of the control plan

Review official control program for FMD

oBeing in compliance with provisions chapter on FMD oApply for endorsement of official control program for FMD

COOPERATION with LIBYA supported by Italian MINISTRY OF HEALTH Understanding epidemiology (PCP stage 1) surveillance design IZS Brescia-IZS Rome, Libyan authorities, EuFMD

Serological survey in Libya to assess FMD viruses circulation and vaccine immune response

IZSLER support to:

Emiliana Brocchi, Santina Grazioli, Giovanna Dho, Ibrahim Eldaghayes, Abdunaser Dayhum, Abdulwahab Kammon, Monier Sharif and Giancarlo Ferrari

Serological testing ( Virological diagnosis Lab Training Supply of diagnostic kits

Name of the presenter: Giancarlo Ferrari

221

Laboratory diagnostic capacity in Libya

Component 2

COOPERATION with LIBYA supported by Italian MINISTRY OF HEALTH

Immune response of vaccinated animals.

Component 1: investigations in FMD suspected

Criteria: The outcome to be measured was the level of

outbreaks to detect circulating strains.

NSP and SP antibodies before the vaccine is injected and the level of antibodies at 30 dpv in animals between 6 and 12 months of age (providing animals would have remained NSP negative).

Component 2: immune response of vaccinated animals. Component 3: serology as a mean to investigate on the level of FMD virus circulation (anti-NSP antibodies) and on the serotypes present (anti-SP serotype-specific antibodies)

Component 3

Component 3 Serology as a mean to investigate the level of FMD

SMALL RUMINANTS: the target for the small ruminants population were sheep with a random selection of 30 owners for each of the three main regions of Lybia.

virus circulation (anti-NSP antibodies) and on the serotypes present (anti-SP serotype-specific antibodies)

Criteria for being enrolled in the study was to have more than 100 individual heads in their flock. For each flock a total of 48 blood samples were collected divided into the following agecategories: (i) 16 samples from the 6-12 months age group; (ii) 16 from animals between 1 and 2 years of age and (iii) 16 from animals with more than 2 years of age.

Criteria: LARGE RUMINANTS: For cattle (and buffaloes)

a random selection of 300 owners for each of the Western and Eastern region and from each of the randomly selected owner all animals belonging to agecategory 6-18 months was supposed to be sampled. 222

Results - component 1

Results - component 2 Immune response in SR (NSP negative)

Of the three serotypes O, A and SAT2 reported in Libya, type O has been the only one to be detected from tissue samples in FMD clinical outbreaks in cattle with the new emergent of the Indian strain O/ME-SA/Ind2001 for the first time in North Africa in 2013.

SR FMDV type O

180 160

before vacc.

140

30 days PV Number of sera

Number of sera

140

SR FMDV type A

160

120 100 80 60 40

before vacc.

120

30 days PV

100 80 60 40 20

20 0

0 <10

10-30

30-90

90-180

180->810

<10

10-30

Range of Titres

30-90

90-180

180->810

Range of Titres

Results - component 3

Results - component 2 Immune response in LR (NSP negative)

Total number of cattle sampled and tested: 1273 Total number of small ruminants sampled and tested: 2542 Period of sampling: January-May 2013 Vaccination period: November-December 2012 (cattle with trivalent O, A, SAT2 and small ruminants against O, A)

223

Results - component 3

Overall crude prevalence (large ruminants):

Overall crude prevalence (small ruminants):

P (NSP+) = 235/1273 (18.46%)

P (NSP+) = 371/2542 (14.59%)

P (Type O+) = 766/1273 (60.17%)

P (Type O+) = 1324/2542 (52.08%)

P (Type A+) = 825/1273 (64.81%)

P (Type A+) = 1337/2542 (52.6%)

P (Type SAT2+) = 548/1273 (43.05%)

P (Type SAT2+) = 146/2542 (5.74%)

Serological findings (component 3) Prevalence of NSP+ in small ruminants by age-group: Three main questions to be answered:

p (0_12 months) = 26/645 (4.03%) avg age 8.8 months

Data support the hypothesis of endemicity?

Lower limit 1.07% - Upper limit 6.99% [DEFT 1.95]

p (12_24 months) = 103/739 (13.93%) avg age 19.8 months

Is there any indirect evidence of type A or SAT2 virus circulation?

Lower limit 7.43% - Upper limit 20.44% [DEFT 2.60]

p (> 24 months) = 184/785 (23.44%) avg age 47.9 months

Can the level of immunity induced by vaccination be

Lower limit 14.89% - Upper limit 31.99% [DEFT 2.88]

considered adequate?

A similar pattern was observed in each region of Libya

224

Evidence of SAT2 virus circulation (in SR)

Evidence of SAT2 virus circulation

In small ruminants antibodies against SAT2 were found in 5.74% (146/2542) of the animals tested. Study of the association between being positive for serotype SAT2 and serotype A and O respectively (through estimation of stratified Odds Ratio)

Identification of serotypes present by serology REGION Alzawia

FARM

Green Mountain

Tripoli

West Mountain

MEAN TITRES MEAN TITRES O A

MEAN TITRES SAT2

SPECIES

Alzawia City

205

Subrata

211

488

Ajdabiya

309 316

Al Abyar

Stratified Odds Ratio A vs SAT2

Among A+ = 4.93

Among O+ = 1.58

Among A- = 4.61

Among O- = 1.48

No modification effect

Odds Ratio (Mantel-Haenszel) = 4.80

Odds Ratio (Mantel-Haenszel) = 1.56

95% CI for ORmh = 8.54 2.70

95% CI for ORmh = 2.58 0.95

Conclusions

EVIDENCE for serotype

ZONE

Benghazi

N. NSP +

Stratified Odds Ratio O vs SAT2

480

Benghazi

Suluq

1660 207 99

Al Bayda

209

Almarig

100

Shahhat

Tobruk

900

Al Qubbah

Al Sahel

Masallatah

657

177

Misratah

Tarhunah

106

157 623

Tripoli

Tripoli City

172

Az Zintan

3328

Gharyan

405

278

Mizdah

859

Yafran

532

Color code

1. The serconversion rate and the mean antibody titres at 30 DPV was considered to be adequate. 2. Test results on NSP antibodies level suggest a level of endemicity at low rate. In the small ruminant population there is a gradient in the proportion of positives among the three age-categories considered. The hypothesis (to be worked out) is then that the virus is maintained into the small ruminant population (the ratio between small ruminants and large ruminants is around 30. Approximately 6,000,000 of small ruminants vs 200,000 large ruminants).

Blue POS A Titre vs type A 10X higher

Pink POS O Titre vs type O 10X higher

225

Conclusions 3. The data available from the survey suggest that the positive results against SAT2 obtained in small ruminants (small ruminants were not vaccinated against SAT2) may be the result of a significant association with being positive also for O 4. Concurrent circulation of serotype A cannot be ruled out

Objective and Study Design Objective

to understand the epidemiology of the disease by identification the risk factor associated with FMD To estimate the sero-prevalence of FMD among cattle, sheep and goats in Tripoli region Libya.

Study Design

Sero-epidemiolgical Study of Foot and Mouth Disease in Livestock in Tripoli, LIBYA

A cross sectional studyand questionnaire survey Ten percent of sheep and goat herds (with 100 animals or more) and then select 48 animals from the selected herds (16 from each age group ; < year, 1 - 2 years, and > 2 years). Ten percent of cattle herds (all animals in the herd if less than 5 animals are sampled, if the herds more than 5, only 5 animals are selected. The preferred age is 6 18 months.

Abdunaser Dayhum, DVM, PhD

Director, National Center of Animal Health-Libya

226

Result

The Sero-prevalence The herd prevalence of FMD was 100% and 72% for SR and cattle respectively. Mean within-herd prevalence 15% (95% CI: 8.8% - 21.3%) with FMD prevalence in an infected herd ranged from 3.8% to 45.8% for SR and 42% (95% CI: 32.9% 51.1%) with FMD prevalence in an infected herd ranged from 0% to 100% for cattle.

Seroprevalence 42.3

42.3

29.0 23.5 15.0

12.5

Cattle

Goat

Sheep

Total

*Statistical Significance with P-value less than 0.05

Only Management system, sex and type of small ruminant were identified to be risk factor for SR. For Cattle; sex and age were the only significant risk factor in this study by applying the Logistic Regression Model 3

Conclusion

Thank You For Your Attention

The present study is part of the project to control FMD. To control FMD , understanding of the livestock production, data collection on the management system and risk factors analysis is needed. Vaccination alone is not enough to control the disease. Other control measures should be included like animal movement control, animal management practice, good surveillance plan, vaccine matching, vaccine quality control, the circulating of the virus, regional cooperation especially on border area, early detection and early responses. It is clear that even though all cattle arise in closed system; it still has high sero-prevalence of FMD. Lack of biosecurity measures at the farm level and insufficient immunity at herd level may be one of the important reasons. Farmer education about the importance of biosecurity , targeted the cattle at commercial dairy by vaccination twice per year should be applied. 5

227

Since 1959 3 Sero-types (O, A and SAT2) Sero-type O (ME-SA Pan Asia 2ANT-10) ME-SA Ind-2001 lineage Sep. 2013

FMD in Libya and the Control Strategy

Mass vaccination Dr Ibrahim Eldaghayes National Center of Animal Health Faculty of Veterinary Medicine University of Tripoli Libya

Vaccine: O Manisa O 3039 ; A Iran 05 Collaboration with IZSLER, Brescia, Italy 1

Vaccination: Cattle: twice/year, sheep and goats: once/year

Components 1, 2 and 3

Same 3 components have been carried out this year 2014.

FMD lectures

Samples have been collected and will be sent to IZSLER lab.

RRT - Pen-Side tests

Collaboration with IZSLER in Italy will be continued.

Lab training

Surveillance along borders will be planned.

Workshops

RBSP for FMD in Libya will be drafted.

FMD Scientific Day

228

Thank you

229

230

231

232

233

234

235

Meeting of the Modeling Network: 15:30 on Thursday, parallel session Network objective: promote a better understanding of existing decision support tools for contingency planning, Improve dialog and awareness between developers and users

Serosurveillance and the PCP-FMD

Open to: Model developers and users with an interest in the objective

Melissa McLaws, Theo Knight Jones, Chris Bartels EuFMD

Current users, and those with a strong interest

Speak to Melissa to get copy of discussion paper before the meeting 1

236

Serosurveillance for FMD Infection

Serosurveillance and the PCP

Useful:

To define/monitor level of infection Gives picture over time (cumulative)

To differentiate risk in different regions, populations & measure economic impact

Complements outbreak surveillance (clinical FMD) Advantages: Captures subclinical infection, unreported disease Limitations: Resource intensive

2 1

This study: Review use of serosurveillance globally Survey objectives, methodology, results

Maintain zero circulation and incursions

Measuring impact of control

Level of infection

Implement Control strategy to eliminate circulation

Implement risk-based control

Proving absence of virus circulation

Identify risk and control options

Towards elimination of circulation

Objective Assessment of Progress of PCP for FMD

Methods Literature review:

Results 48 surveys identified:

1. Google Scholar search: (2005-2015) 1. 2. 3.

Maintain zero circulation; withdraw vaccination

9 reported species-specific results separately report 22 different countries represented, virus pool 1-6

serosurveillance

2. Look at references in papers 3. Limit to domestic species, non-free countries

Number of studies by virus pool: 2005-2014

Studies from colleagues (EuFMD, FAO, WRL) Develop database :

Number of samples per virus pool 2005-2014 1244

1: E. Asia 2: S. Asia

64717

1093 73248

3: W. Eurasia 4: NE Africa

Study date, objective , species, number of samples, number epi-units, number regions, lab test used, Adjust for Se/Sp, vaccination, age Results: animal-level, epi-unit level, regional-level

5: NW Africa 6: S. Africa

237

173354 212034

Results

Results Survey objective:

Number of studies per year 10 9

At wildlife interface (3) Inform plans for zoning (3) Post outbreak (1) Economic impact on exports to Arabic countries (Ethiopia) Surveillance for eradication (Taiwan)

8 7 6 5 4

Literature

internal report

report

47% (27/57) were national surveys, rest focused on a particular region within the country 4 studies used sera from rinderpest eradication campaign

1 0

year data collection completed

Results

Sample size varied from 46 to > 53,000 Species

Number Surveys

Sample size (mean, range)

Large ruminants

11,671 (228-52,224)

Small ruminants

6,000 (46-32,000)

Pigs

27,262 (766-53,759)

Mixed

3414 (448-9,241)

Not reported

1,716 (923-2,510)

Test used: NSP ELISA: 56% (32) liquid phase blocking ELISA: 12 % (7) LPB and NSP ELISAs : 28% (16) (NSP results reported) virus neutralisation test: 4% (2)

4 studies reported adjustment for Se/Sp of test

238

Results: How the surveys dealt with....

Results: animal level

1. Vaccination:

Mean survey seroprevalence, by virus pool (Bubble size proportional to number of samples)

60% (34) studies did not report if animals vaccinated or not 19% (11) : animals not vaccinated 16% (9) mix of vaccinated and unvaccinated 5% (3) animals were vaccinated

% population seropositive

2. Age: 49% (27) did not report 31% (17) included in risk factor analysis 27% (15) found higher seroprevalence in adults; 4% (2) found no difference

Large ruminants Small Ruminants

16% (9) young animals only 2% (1) only adult animals

Virus Pool

Results: animal level Large Ruminants: % seropositive

80 60 40 20 0

1000000 100000 10000 1000 100 10 1

Results: Regional level, Epi-unit level 1. Regional level (= animal level prevalence in different regions)

no. samples

% seropositive

100

49% (27) studies measured prevalence in different regions Regional difference reported varied enormously: 3-100% absolute difference (18% on average)

2. Epi(definition of positive varies from 1-5 infected animals)

30 25 20 15 10 5 0

100000 10000 1000 100 10 1

mean seropositive

no. samples

% seropositive

Small Ruminants: % seropositive

1. Assessed in 20% (11) studies 2. % positive epi-units ranged from 20-87%

number of samples 13

239

Discussion Number of studies increasing over time (?)

Discussion Study design will influence results enormously

Pools 5, 6, 7 (W. & S. Africa, S. America seem under-represented)... BUT many studies may be unpublished

Age of animals, vaccination status, study area, diagnostic test Not consistently reported or analysed

Reporting of results: animal vs epi-unit level Epi-unit level analysis appropriate because FMD is so infectious

Impacts interpretation and comparability of results

Need for guidelines?? Objectives: when to do serosurvey, why? Study design, including how to minimise bias/confounding Data analysis: animal level, epi unit level Interpretation of results

Discussion: Serosurveys and the PCP

Discussion: Value of serosurveys Address specific policy or research questions: Strategy development (eg zoning), role of different species incl. wildlife Demonstrate subclincial disease, under reporting, freedom from disease

PCP Stage

Some countries invest large amount annually detailed tracking and analysis at subnational level

Stage 1 FOCUS Getting an understanding about FMD virus transmission and impact

Use of Define risks Serosurvey

Stage 2 FOCUS Implement riskbased control to reduce impact of clinical FMD

Monitor risk and FMD as RBSP is implemented

Stage 3 FOCUS Implement control targeted at eliminating FMDV circulation Demonstrate reduced virus circulation

Stage 4 FOCUS Zero-tolerance of FMD outbreaks, with vaccination

Stage 5 FOCUS Keeping zerotolerance of FMD outbreaks, without vaccination

Demonstrate FMD freedom

With appropriate design, analysis and interpretation!

240

Thank you for your attention!

ELITE: An Electronic Laboratory Information Tracking Environment for Supporting the Progressive Control of FMD in Pakistan Keith Biggers, Ph.D.

Institute for Infectious Animal Diseases A Department of Homeland Security Science and Technology Center of Excellence Texas A&M University, College Station, Texas, USA

Project Background

Project Overview project will design, implement, and integrate a laboratory information management system (LIMS) with a robust information dashboard capable of depicting an accurate and timely common operating picture as a means of building a national biosurveillance capability for situational awareness. Statement of Work Design, develop, and deploy a state-of-the art LIMS to support the full workflow of a laboratory Design, develop, and deploy a biosurveillance system that has a suite of visual and analytical tools capable of harvesting and examining data on FMD Coordinate with in country personnel to ensure that the effort complements the ongoing FAS-FAO project for progressive control of FMD Yield an integrated technology solution that:

241

o o o o

Streamlines the flow of information between laboratories working to manage animal diseases Allows for sharing of standard operating procedures Supports biosurveillance analysis and monitoring Provides a robust and reliable solution 3

Data Sharing Across Multiple Levels

National Lab LIMS

National Lab

FMD Database

Updated Form and Report Templates

Three ways the system can be used: Lab Operations

LIMS Administration

Receive samples, perform requested tests, record and report results to the Lab 1

other IT functions.

Lab 2

Lab 3

Biosurveillance Monitor key results from laboratory operations, analyze data, produce reports, provide aggregate level data to individual laboratories on program specific diseases.

LIMS Administration Create/edit accessioning forms, define new test specifications, disseminate updates to laboratories (i.e., new/updated test specifications and report templates).

Local Lab Standalone LIMS

Provincial Labs District Labs

Program Specific Results

Areas of Focus

Local Lab Networked LIMS

Submitted to Provincial

ELITE Organization

ELITE Suite of Tools

Lab Information

Form Designer

ELITE

Report Designer

Allows design of forms: Visual form building Exporting of form data

Facilitates lab operations and biosurveillance: Data entry through test process Management of users/groups Biosurveillance visualizations Exporting of lab data Biosurveillance and LIMS reporting Import test result forms Create test specifications Import report designs for use by provincial and national labs

Allows design of reports: Visual report building Exporting of report data

Available to LIMS Administrator only.

Permissions based access to features. Available to all users.

Available to LIMS Administrator only.

Data Exports Form Designer

Report Designer

Reports

Biosurveillance Support 6

242

Design, Development, and Fielding Process

Lab Operations: Accessioning

Worked closely with FAO, USDA, and a working group of lab representatives throughout the design, development, and piloting process Supported by the National University of Sciences and Technology Piloted with a subset of labs before full deployment Final system has been deployed to the National Veterinary Lab and the network of provincial labs

Case Details

Source and Sample Details

Program Specific Details

Lab Operations: Test Assignment

Lab Operations: Performing Tests

Assignments

Section and Individual Assignment

Test Results SOPs

Review Case Details

Accessioning Information

243

Audit History

Biosurveillance Form-based Querying

Research Results Data Export/Download

Developed ELITE and used it as the basis of PKLIMS provincial laboratories Over 1,300 biosurveillance reports for FMD have been submitted Trained IT personnel and the final system is being transitioned to Pakistan Actively meeting with international groups to discuss other potential applications for the technology

Interactive Graphical and Geospatial Visualizations

Future Work

Questions?

Expanded set of program diseases Refine day-to-day laboratory capabilities More sophisticated biosurveillance tools Additional national level IT training

244

Characterization of the virus on basis of the VP1 Protease VPG

Capsid

1A VP4

1B VP2

1C VP3

Carboxy-terminal self-cleaving 1D VP1 2A

Membrane-binding Genome-linked (VPg) Protease

* putative functions

NTP binding* 2C

Polymerase

3D AAA (n)

Poly(C)

VP1 region

Molecular variability within the FMD virus O/ME-SA/PanAsia-2 lineage

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) Kocaeli O/TUR/1094/2010* (Giresun 11/08/2010) Kastamonu O/TUR/868/2010* (Eskisehir 20/07/2010) Kastamonu O/TUR/1003/2010* (Sivas 10/08/2010) Bursa Gümü hane O/TUR/18/2010 (Gumushane 09/07/2010) Sivas Bursa Turkey Eski ehir O/TUR/883/2010* (Kastamonu 23/07/2010) A r Sivas Antalya Iran Turkey O/TUR/35/2010 (Erzincan 13/08/2010) Iran Antalya 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) O/PAK/36/2010 (Gilgit-Baltistan 29/07/2010) 97 O/TUR/36/2010 (Kocaeli 13/08/2010) O/UKG/12/2001 (AJ311724) O1/Manisa/TUR/69 (AJ251477)

Bulgaria Bulgaria

Begoña Valdazo-González

PhD, MRCVS, The Pirbright Institute, Surrey, United Kingdom

O/ME-SA/PanAsia-2ANT-10

640 nt

Valdazo González et al 2011

0.01

Since 2002, new emerging lineage that spread over much of southern Asia reaching not only Europe (Bulgaria) but also North Africa (Egypt and Libya) 1

Variability, origin and evolution rate of O/ME-SA/PanAsia-2

Preliminary results: Genetic variability of O/ME-SA/PanAsia-2 A.

1,129 VP1 sequences of O/ME-SA/PanAsia-2

Classification:

- FAO World Reference Laboratory for FMD (WRLFMD) - 2002-2014 - 24 countries - Cattle, small ruminants, pigs and wild life - Water buffalo, wild board, oryx and gazelle

Sub-lineage TER-08 BAL-09 FAR-09 SAN-09 PUN-10 ANT-10 Unnamed

Analysis - Molecular Evolutionary Genetic Analysis (MEGA) - Bayesian Evolutionary Analysis Sampling Trees (BEAST)

245

Frequency 0.4% 0.6% 8.1% 0.2% 0.6% 50.6% 39.5%

Origin of the name Unknown Balkh, Afghanistan Fars, Iran

Punjab, Pakistan Gaziantep, Turkey -

Per country

Per year

Per host species

Preliminary analysis: Molecular clock of O/ME-SA/PanAsia-2

Further analysis Standardization of the classification of these viruses Comparison classification using different analysis Comparison with classification by other authors

HKN model of base substitution Relaxed molecular clock Bayesian skyline plot

Jamal et al 2011. Infect Genet Evol. Yuvaraj et al 2013. Infect Genet Evol.

Understanding of the epidemiological dynamics of these viruses

Nt substitution - 9.06×10-3/site/year

Origin and evolution rates of different sub-lineages Phylogeography

(95% HPD: 8.28×10-3 - 9.90×10-3)

Ancestor ~1999

Development of approaches to predict the emergence of new viral lineages and sub-lineages in endemic countries

(95% HPD= 1995 - 2011)

Preliminary MCC tree (1000 sampling trees) 5

Acknowledgements The Pirbright Institute, United Kingdom - Nick J. Knowles - Donald P. King - Jemma Wadsworth - Katarzyna Bachanek-Bankowska

SEACFMD Roadmap: A risk-based approach to FMD control in SE Asia and China

Department for Environment, Food&Rural Affairs (Research grant no. SE2940), United Kingdom

Ronello Abila Sub-Regional Representative OIE Sub-Regional Representation for South-East Asia

Thank you for your attention! 7

246

The South East Asia and China Foot and Mouth Diseases (SEACFMD) Campaign

1997

Coordination and Governance

2010 Approved by the OIE General Assembly in May 25, 2010, PARIS

SEACFMD LabNet

Role of EpiNet

Rapid diagnosis of FMD viruses

A regional network of epidemiological expertise to support the SEACFMD Campaign FMD reporting, analysis FMD data, design and implementation of surveillance activities Training on outbreak investigation, disease, information systems, and other epidemiological tools

Early detection and confirmation of FMD virus serotypes Use of appropriate vaccine strain

Facilitate sending of field isolates to Ref. Labs. Strengthen surveillance Molecular epidemiology Mapping on the evolution of FMDV serotypes

247

Communication Network

SEACFMD 2020 Roadmap

SEACFMD Regional Communication Plan Align with the OIE Animal Health Communication Strategy for SE Asia

Development of a Field Manual for Communication Launching of the SRR SEA Website, including SEACFMD Newsletter and E-news

SEACFMD Roadmap 2020 Strategies

Roadmap principles

First Strategy

Rapid identification of the foci of infection

Reduce FMD prevalence by targeting hotspots and critical points

surveillance

Prevention of infection of susceptible hosts restrict infected to contact healthy herd

critical points along the animal movement pathways

Elimination of the source of FMDV disinfection

Hotspots = foci, endemic source Critical points = amplification point

Increasing herd and animal immunity vaccination 248

SaTScan

Probable Hotspots

All the outbreaks over the last 10 years Type O clusters detected using SaTScan Also note the O outbreaks which are not clusters

Source: Ben Madin, 2011

Animal movement pattern vis-à-vis population and price

Risk Pathway: Cattle/Buffalo movement

249

The likelihood of an FMD infected shipment of cattle entering Viet Nam from Cambodia

Source: C. Hawkins, 2011

Source; Socheat, 2010

Network of traders involved in the movement of livestock from the source in central Myanmar, through to the destination in the MTM Zones of Malaysia and Thailand.

Lao PDR

Cambodia

Source :Polly Smith, 2012

250

Malaysia

Important contributors to the risk of FMD incursions into the MTM Zone : Prevalence of FMD in Myanmar, Prevalence of FMD in Thailand, the probability that an infected animal does not show clinical signs probability that an animal enters the MTM Zone unofficially

1997

Roadmap Strategies

SEAFMD 1997

Second Strategy

OIE FMD Free Zone

FMD Infected Zone

Pursue zoning in the most advanced area of FMD control Set up control/eradication zones in priority areas once disease incidence decreased to low levels and the likelihood of recurrence is reduced.

2004

SEAFMD 2004

2011

SEAFMD 2008

OIE FMD Free Zone FMD Infected Areas

Free zone Infected zone Buffer zone Control zone Eradication zone

251

Roadmap Strategies

FMD Zones in China

Third Strategy Maintain and expand FMD free zones Zones which are currently free will be protected by increasing the focus on quarantine and movement management at zone or countries borders.

PCP stages and SEACFMD Strategy

Maintenance and Expansion of FMD Free zones

3rd Strategy: Maintenance and expansion of FMD free zones 2nd Strategy: Progressive zoning

1st Strategy: Reduction of FMD prevalence by targetting hotspots and critical points

252

Pilot Comprehensive FMD Control

Indicative PCP stages SEACFMD Members

Northern Lao PDR Country

2012

2013

2014

2015

2016

2017

2018

Controlling FMD in critical points along the risk pathway

2020

Cambodia China

Central Myanmar Controlling FMD in Hotspots

Thailand Malaysia Myanmar Lao PDR Vietnam

Pilot Comprehensive FMD Control Mass vaccination in the high risk villages and districts Use of high potent (6PD 50) double oil emulsion vaccine Surveillance Animal movement Stakeholder enagement

253

Intervention at the source and critical point along the Risk Pathway:

Intervention high risk production system Dairy farms in Thailand Farms near livestock markets in Thailand and Vietnam

Population Growth

Future Challenges

254

Projected Population Movement

255

Source: OECD-FAO Agricultural Outlook, 2013-2022. http://www.oecd.org/site/oecd-faoagriculturaloutlook/highlights-2013-EN.pdf

ASEAN Economic Community( AEC ) * a single market and production base * a highly competitive economic region * a region of equitable economic development * a region fully integrated into the global economy

Future production systems

Laos

Thailand

Will adjust to markets and environmental factors Demand though will be strong in China Movements increasing Abattoir developments??

Laos Laos

Malaysia

Free movement of goods Services

Cambodia

USA

Skilled laboured Singapore

2015

Japan

Australia

(Economic scale)

Flow of capital

EU Philippines

Myanmar

S. Korea 256

India

Vietnam

AEC

Investment

Source: Peter Black, 2014

China

Brunei

Indonesia

Asean+3

New Z. Asean+6

3rd Edition of SEACFMD Roadmap

Amidst these challenges

Re-affirm and strengthen risk-based approach to FMD control; Provide a description of the strategic framework and directions to achieve strategic objectives by 2020; Identify linkages with the FMD Global strategy structure; Define FMD control strategies at regional and national levels; Emphasize the importance of strengthening veterinary services to control FMD and other TADs.

Need to update the SEACFMD Roadmap ; Adjust to consider changes in the epidemiology of FMD and economic development in the region; Application of new technologies in vaccines and vaccination, diagnostics and surveillance; Strengthen political engagement to get more support to then programme.

Thank you for your attention! Using risk assessment to inform FMD policy in Mongolia M. Schuppers, B. Batsukh, S. Enktuvshin, P. Bolortuya, B. Wieland

Animal Health Matters. For Safe Food Solutions.

257

Objectives of the risk assessment

Background Mongolia has a susceptible animal population of approximately 50 mio heads (livestock plus wildlife) Livestock density is low, traditional nomadic herding system Mongolia faces regular FMD outbreaks, but there is no evidence supporting virus circulation between outbreaks There are regional differences in terms of risk: East vs West Routine, semi-annual vaccination with seromonitoring in the East Annual surveys to document freedom in the West National FMD strategy called for risk assessment

To estimate the likelihood of entry, exposure and spread of FMD in Mongolia To provide recommendations for strengthening FMD prevention and control measures To build national capacity to conduct risk assessments

Methodology April-May

First workshop: risk pathways & data needs

First workshop Separate for each region Focus: risk pathways, identification of data needs

June-August

Data collection by veterinarians Herder survey by Master students Data entry and analysis

September

Second workshop Joint for both regions Focus: risk estimation, recommendations

Stage

Eastern region

Western region

Entry

Exposure

Infection in first herd

Spread before detection

Spread after detection

FMD at origin

Vehicle in contact with virus

Vehicle brought to Mongolia

Virus survives on vehicle

National FMD policy meeting May

258

Poor disinfection at border

Entry via vehicle

Data collection

Second workshop: risk estimation

Workshop participants compiled existing data using standardized data collection sheets Master students conducted herder survey among 180 herders in Eastern and Western region

Qualitative estimation with 6 levels: negligible-very high individual risk estimates Delphi approach: individual estimates plenary discussion individual estimates Individual estimates recorded by electronic audience-response system Uncertainty measured by level of disagreement between participants

- FMD knowledge, understanding of FMD risks, presence of risk factors

Master students distributed visitor and movement logs among 90 herders - Contact structure of herder families, mixing of herds

Data entry by Master students 6

Second workshop: recommendations

National FMD policy meeting

Opinion-forming game Group discussions Formulation of recommendations for each step of the risk assessment Presentation of recommendations on pinboard Prioritizing of recommendations by stickers

Approximately 80 participants from across the country Results and recommendations of the risk assessment were presented Discussion about national FMD strategy Decisions about changes to FMD control measures

259

Discussion Thank you for your attention

Workshop participants involved in entire process: - Essential to create understanding of risk assessment and develop ability to provide sound recommendations

manon.schuppers@safoso.ch

Very high level of commitment, lively and constructive discussions Data collection phase essential to improve quality of risk estimation Huge amount of information to process for risk estimation

Acknowledgements:

- Logistical and human resource challenge

All workshop participants from soums and aimags and Master students of the School of Veterinary Medicine and Biotechnology in Mongolia for participation and data collection

Findings of risk assessment are reflected in revised FMD control strategy

Swiss Agency for Development and Cooperation for funding of the workshops and data collection

Animal Health Matters. For Safe Food Solutions.

Role of Livestock in the Economics of Pakistan Share of Agriculture in GDP: 21.0%

Livestock Crops

Share of Livestock in agriculture : 56 % Share of Livestock in GDP: 11.8%

Identification of FMDV types from Pakistan and its sequence analysis on the basis of P1 (Capsid protein gene)

44% 56%

Main source of labour: 43.7% Earnings through Livestock sector: Rs. 776.5 billion (2013-14)

Usman Waheed, Lizhe Xu, Kate R. Schumann, Fred Grau, Qaiser

Livestock progress: 2.9% annually

Mahmood Khan, Michael T. McIntosh Assistant Professor in Microbiology University of Veterinary & Animal Sciences, Lahore, Pakistan

260

Source: Economic Survey of Pakistan 2013-14 2

FMD IN PAKISTAN: IMPACT ON ECONOMY

LIVESTOCK POPULATION IN PAKISTAN-2014 Species

Population (Million Nos.)

Milk Production (000 tones)

Cattle/Cow

39.7

18,027

Buffalo

34.6

31,252

Sheep

29.1

Goat

66.6

822

Total

170.0

50,990

FMDV strains in Pakistan: O, A and Asia 1 Losses due to FMD in Pakistan Rs ~20.5 Billion The economic losses are due to: Direct Losses (Deaths, Loss of production draught capacity) Indirect Losses (Restrictions on trade, import and export of live animals and dairy products) Treatment and vaccination costs

Source: Ministry of National Food Security & Research, Govt. of Pakistan 3

METHODOLOGY

SAMPLE COLLECTION AREAS IN PAKISTAN

Collection of samples

RNA extraction

Real Time PCR

Universal FMDV LPro-P1 RTPCR

Gel Electrophoresis

Sequencing of amplified products

Phylogenetic Analysis (MEGA 6.0)

261

http://d-maps.com/pays.php?lib=pakistan_maps&num_pay=102&lang=en. 114x119mm (300 x 300 DPI) 6

CYCLING CONDITIONS AND PRIMER SET FOR AMPLIFICATION OF P1 OF FMDV O AND A

RNA EXTRACTION AND Lpro-P1 RT-PCR RNA extraction by Trizole ® Reagent L-P1 RT-PCR reactions: Primers: 10 µM of each primer 2X reaction buffer: 25 µl Platinum® Taq Hi Fi enzyme mix: 1.0 µl RNA template: 5 µl Final reaction volume: 50 µl

Action

Temperature Duration

Reverse transcription

55 C

30 min

Activation of Platinum® Taq Hi Fi

94 C

2 min

Denaturation

94 C

15 s

Annealing

55 C

30 s

Extention

68 C

5 min

No. of cycles 39

Primer Name

Sequence (5/ ---- 3/)

Area of Genome

Orientation

Univ F

TGG TGA CAG GCT AAG GAT G

L-P1

Forward

Univ R

GCC CRG GGT TGG ACT C

L-P1

Reverse Xu et al., 2013 8

RESULTS

REAL TIME PCR PROFILE AND INTERPRETATION Temperature 60 oC 95 oC (Denaturation) 60 oC (Annealing/ Extension)

Time Length No. of Cycles Optics 10 Min Hold Off 30 Sec x 45 cycles On 60 Sec

Samples which gave amplification from 0 -40 cycles were considered positive, from 40 45 as inconclusive & repeated and above 45 as negative.

262

BLAST COMPARISON FOR PRESUMPTIVE GENOTYPE/ SEROTYPE

LINEAR VIEW OF REAL TIME PCR

Linear view of the rt PCR amplification plots of samples from field outbreaks of Pakistan occurred in different areas

Molecular Phylogenetic analysis of FMDV O isolates of present study

Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete genome Foot-and-mouth disease virus O isolate o1skr iso85, complete genome Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genome Foot-and-mouth disease virus O, strain SKR/2000, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete genome Foot-and-mouth disease virus O isolate o1skr iso85, complete genome Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genome Foot-and-mouth disease virus O, strain SKR/2000, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete gen Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 BOV, complete genome Foot-and-mouth disease virus O isolate o1skr iso85, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete genome Foot-and-mouth disease virus O isolate o1skr iso85, complete genome Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genome Foot-and-mouth disease virus O, strain SKR/2000, complete genome Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete genome Foot-and-mouth disease virus O isolate o1skr iso85, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 BOV, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 TC, complete genome Foot-and-mouth disease virus O, strain Tibet/CHA/99, complete genome Foot-and-mouth disease virus O, strain TAW/2/99 BOV, complete genome Foot-and-mouth disease virus O isolate o1skr iso85, complete genome

Molecular Phylogenetic analysis of FMDV A isolates of present study

EU244451 EU244450 EU244446 EF494469 EF494470 EU244452 EF494473 EF494472 EF494471 EF494476

NB: The isolates of present study are highlighted with blue dots.

EU244449 12

EF494477 EF494467 EU244453 EF494478 EF494468 EU244440 EF494465

EU244444 EF494474 EF494475 EU244447 EF494464 EU244448

FJ755083

64 58

FJ755082 EF494466 EF494479 EU244445 EU244443

EF494484 EF494483

57 95 49

EF494485 EF494482

FJ755084 GU384686 present study

EU244442 EU244441 EU244439 FJ798194 present study EF494481 100

MEGA 6.06 (Tamura et al., 2013)

263

0.1

EF494480

MEGA 6.06 (Tamura et al., 2013)

Evolutionary relationships of 121 FMD type O viruses

Accession Numbers of the Distinct P1 sequences

Sr. No.

Ref. No.

Accession No.

FMDV Strain

Location

PAK/29/2008

GU384684

PAK/39/2008

GU384685

PAK/76/2009

GU384686

100

Waheed et al., 2011

O/PAK/12/2006 (EF494505) O/PAK/14/2006 (EF494506) O/PAK/11/2006 (EF494504) O/PAK/2/2006 (EF494499) O/PAK 52 9 (EF494498) O/PAK/Lahore vaccine (EU244455) O/PAK 232 8 (EF494491) O/PAK 241 8 (EF494493) O/PAK/6/2006 (EF494501) 87 O/PAK/8/2006 (EF494502) O/PAK/4/2006 (EF494500) O/PAK/10/2006 (EF494503) O/PAK/53/2007 (FJ798 179) O/PAK 231 8 (EF494490) 89 O/PAK/6/2008 (FJ7981 93) 91 O/PAK/61/2006 (FJ798 169) 85 87 O/PAK/2/2008 (FJ7981 91) O/PAK/3/2008 (FJ7981 92) O/PAK/71/2007 (FJ798 189) O/BHU/41/2003 (DQ165041) O/NEP/4/2003 (DQ165059) O/AFG/120/2004* (EF457984) 94 O/AFG/201/2004* (EF457985) O/BHU/47/2003 (DQ165042) O/BHU/49/2003 (DQ164867) O/NEP/5/2003 (DQ165060) O/BHU/15/2003 (DQ164865) O/NEP/6/2003 (DQ165061) O/BHU/33/2004 (DQ165046) 98 O/BHU/39/2004 (DQ164870) O/BHU/40/2004 (DQ165047) PanAsia-2 89 O/BHU/31/2004 (DQ164869) O/BHU/30/2004 (DQ165045) O/BHU/28/2004 (DQ165044) O/BHU/26/2004 (DQ165043) O/BHU/27/2004 (DQ164868) 99 O/PAK/38/2005 (FJ798 167) O/PAK/1/2008 (FJ7981 90) 99 O/PAK/70/2007 (FJ798 188) 100 O/PAK/66/2007 (B)(FJ79818 5) 82 O/PAK/68/2007 (FJ798 186) O/PAK/69/2007 (FJ798 187) O/PAK/66/2007 (A)(FJ79818 4) O/PAK/33/2005 (FJ798 164) O/PAK/71/2006 (FJ798 175) 91 O/PAK/35/2005 (FJ798 166) 100 O/PAK/56/2007 (FJ798 180) O/PAK/63/2006 (FJ798 170) O/PAK/60/2006 (FJ798 168) O/PAK/73/2006 (FJ798 177) O/PAK/63/2007 (FJ798 183) O/PAK/32/2005 (FJ798 163) O/PAK/74/2006 (FJ798 178) 89 O/PAK/67/2006 (FJ798 172) O/PAK/61/2007 (FJ798 182) O/PAK/66/2006 (FJ798 171) O/PAK/70/2006 (FJ798 174) O/PAK/34/2005 (FJ798 165) O/PAK/31/2005 (FJ798 162) O/PAK/72/2006 (FJ798 176) O/PAK/60/2007 (FJ798 181) 100 O/IRN/1/2000 (DQ164892) O/IRN/16/2000 (AJ318840) 80 O/IRN/41/2001 (DQ164894) 79 O/IRN/58/2001 (DQ164895) 99 O/IRN/16/2003 (DQ165052) O/IRN/2/2003 (DQ165048) O/PAK/68/2006 (FJ798 173) 84 O/AFG/210/2004* (EF457986) O/IRN/67/2001 (DQ164897) 87 O/IRN/9/99 (AJ318838) O/IRN/24/99 (AJ318839) O/IRN/16/2001 (DQ164893) O/AFG/50/2003 (DQ165036) 100 O/AFG/16/2003 (DQ165035) O/PAK/45/2003 (DQ164942) 100 O/BHU/2/2002 (DQ165037) O/BHU/38/2002 (DQ165038) O/IRN/6/2004 (DQ165053) O/IRN/8/2004 (DQ165054) O/BHU/22/2003 (DQ165039) 100 O/BHU/24/2003 (DQ165040) 78 O/BHU/25/2003 (DQ164866) 100 O/NEP/12/2000 (DQ164938) O/NEP/13/2000 (DQ164939) O/BHU/7/2002 (DQ164864) 99 O/PAK/1/2003 (DQ165065) 100 O/PAK/53/2003 (DQ164943) 88 O/PAK/15/2002 (DQ165062) 96 100 O/PAK/16/2002 (DQ165063) O/PAK/18/2002 (DQ165064) O/IRN/61/2001 (DQ164896) Iran-200 1 O/IRN/15/2004 (DQ165055) 71 O/IRN/4/2003 (DQ165049) O/IRN/8/2003 (DQ165051) O/IRN/15/2003 (DQ164898) O/IRN/6/2003 (DQ165050) O/IRN/20/2004 (DQ165056) O/IND/53/79 (AF292107) 72 O5/IND/1/62 (DQ164890) O/IND/R2/75* (AF204276) O1/Manis a/TUR/69 (AJ251477) 88 O/PAK/16/2003 (DQ165068) O/PAK/73/2003 (DQ165070) 100 O/PAK/17/2003 (DQ165069) Pak -9 8 OA/PAK/12/2003 (DQ165066) 81 O/PAK/14/2003 (DQ165067) O/IND/APR/74/03/1* (EU109785) 100 O/IND/APR/74/03/4* (EU109786) 100 O/IND/APNz/66/04* (EU109783) O/IND/APNg/85/05* (EU109782) O/IND/APMb/79/04* (EU109784) O/IND/APV/48/04* (EU109776) O/IND/APMb/67/04/2* (EU109780) 100 O/IND/APP/96/04/2* (EU109787) O/IND/APP/98/04/1* (EU109781) O/IND/APMb/83/04/1* (EU109778) O/IND/APKu/91/04* (EU109779) 99 O/IND/APWg/52/04* (EU109777) 97 O/IND/APV/42/04* (EU109774) 88 100 O/IND/APV/84/04* (EU109775)

PanAsia

0.05

Evolutionary relationships of 121 FMD type O viruses

CONCLUSION

Iran-05

Upon phylogenetic analysis it is confirmed A/IRN/2/2002 (EU414527) A/IRN/7/2003 (EU414528) A/IRN/32/2001 (FJ755017) A/IRN/34/2001 (EU414526)

that;

A/PAK/73/2007 (FJ798194)

Iran-87

A/IRN/10/2003 (EU414529) A/PAK/28/2002 (EU414534) A/PAK/9/2003 (EU414535)

The isolated sequences are in close relation with the previous and circulating FMDV in the areas of study Same viruses were the cause of outbreak of FMD in the area of study with minor changes in nucleotide sequences

Iran-99

A/IND/61/88* (AF390657) A/IND/160/90* (AF390608) A/WBN 17/82* (A/IND/490/97*) (AF390652) A/IND/17/82* (IndImm) A/IND/17/82 (1981) A/IND/7/82 (1980)

India 1994-2001 A/IND/395/88* (AF390645) A22/IRQ/24/64 (AJ251474) A22/Mahmatli/TUR/65 (TUR/3/69)(EU553879) A28/Polatli/68 (TUR/2/69)(EU553880) A/IND/17/77* (AF204108)

Iran-96 A/IND/57/79 (1978) (EF120385) A/IND/2/93* (AF390635) A/IND/21/90* (AF390620) A/NEP/21/84 A/SAU/23/86 (EU414536) A/IND/256/98* (AF390628) A/BHU/41/2002 (EU414525) A/BHU/7/2003 OS A/BHU/27/2003 A/IND/270/96* (AF390631) A/IND/84/97* (AF390669) A/IND/287/96* (AF390633) A/ALB/1/96 (EU553850) A/IND/148/93* (AF390606) A/IND/299/94* (AF390639) A/SAU/16/95 (EU553874) A/IND/163/97* (AF390611) A/IND/156/97* (AF390607) A/IND/161/97* (AF390609)

India 2000-01

Waheed et al., 2011

0.02

264

ACKNOWLEDGEMENTS FADDL, PIADC, USDA, USA (Dr Michael T. McIntosh and his team) NIBGE, Faisalabad, Pakistan (Dr Qaiser M. Khan PROMOTING A RISK BASED STRATEGIC PLAN FOR FMD CONTROL: IMPROVEMENT OF RISK ASSESSMENT THROUGH REAL TIME TRAINING

and his team) EuFMD Team and FAO, Rome University of Veterinary & Animal Sciences,

Bishnu Adhikari1, Chris Bartels2, Jenny Maud2, Fabrizio Rosso2 1Regional

Support Unit for South Asia Food and Agriculture Organisation (FAO), Kathmandu, Nepal Commission for the Control of FMD (EUFMD), Food and Agriculture Organisation, Rome, Italy

2European

Lahore, Pakistan (Dr Tallat Naseer Pasha, Vice Chancellor and his team) 19

PROMOTING A RISK BASED STRATEGIC PLAN FOR FMD CONTROL: IMPROVEMENT OF RISK ASSESSMENT THROUGH REAL TIME TRAINING Training on clinical FMD, including transect walks: visiting households that had and had not suffered FMD

PCP-FMD workshops to progress FMD control request for long-term FMD strategy, including development of riskbased strategy plan

Considering that Nepal is an extremely diverse country 1] assessing risk factors 2] economic impact 3] attitude towards clinical FMD 4] local organisation

National strategy at local level raising awareness with -

Farmers Service providers (inseminators, AHW and private vet

EuFMD in collaboration with FAO, the Department of Livestock Services and the government of Australia, to train about FMD and promote FMD control in Nepal

265

1Regional

2European

Support Unit for South Asia Food and Agriculture Organisation (FAO), Kathmandu, Nepal Commission for the Control of FMD (EUFMD), Food and Agriculture Organisation, Rome, Italy

PROMOTING A RISK BASED STRATEGIC PLAN FOR FMD CONTROL: IMPROVEMENT OF RISK ASSESSMENT THROUGH REAL TIME TRAINING BB Adhikari1, CJM Bartels2,JL Maud2, F Rosso2

EuFMD under a contract between the FAO, the Departement of Livestock Services and the government of Australia, conducts Sofar, there have been 13 RT courses. Additionally, it includes support on developing a risk-based strategy plan for FMD control

Nepal is very diverse in terms of terrain types, cultures and hence farming practices. Thus, FMD control requires adaptation to different productions systems and regions

As part of the RT course, transect walks are made through villages that had suffered clinical FMD recently. Between 30 to 50 households were visited per RT course a total of .510 households visited. Some of these had suffered clinical FMD (cases), some had not (non-cases).

2. Impact on Milk Production 60-80% milk drop during FMD (2-3 weeks) 10-20% after clinical FMD for extended period of time Probability of mortality (1 10%) Treatment costs Overall, for 1-year period: 15-20% income loss 3. Attitude towards clinical FMD Comparing cases and non-cases indicated that some farmers were taking their own precautions such as: Selecting other places for grazing or keeping livestock on the premises Not visiting other premises and not allowing others to visit theirs Leading to observing clusters of FMD affected households

Information was collected about farm practices and history of recent FMD through structured questionnaire and by non-structured interview: 1] assessing risk factors for clinical FMD 2] economic impact of clinical FMD 3] attitude towards clinical FMD 4] local organisation of small holders Results are summarized in the 4 orange blocks left

Three workshops with the Directorate of Animal Health were conducted in Kathmandu, Biratnagar and Jhapa. The results of workshops have resulted into a long-term nationial control plan for FMD control (2014-2025. ). Key points are: Preparatory phase to develop a risk-based strategy plan, for Nepal to progress to PCP-FMD Stage 2 Planning of implementation phase for FMD control, staring in some pilot areas (East and West), see map

Epidemiological analysis of FMD outbreaks in South-East Asia and China (2010-2014)

In addition to nationwide control measures (use of vaccination, movement restrictions and strengthening the veterinary services) local control of FMD is to become a key component. Based on the experiences from the Real-Time training course (see orange boxes), the following local control measures refer to Training local vets, village animal health workers, inseminators to take FMD control serious and to demonstrate this very clearly to the livestock owners, thus lead by example Raising awareness with livestock owners on risks for livestock catching FMD how to prevent FMD Supportive care of FMD-affected livestock, reducing mortality and production losses. Extension materials were developed during the RT courses and follow-up to reach out is planned through existing dairy cooperatives and farmer groups 4] Organisation of small holders Local dairy cooperatives Farmer groups

For vets, AHW, inseminators Lead by example

Ronello Abila1 and Karan Kukreja2 1Sub-Regional Representative 2Project Officer OIE Sub-Regional Representation for South-East Asia

For livestock owners How to keep your livestock healthy

There are some small steps you can take to keep your anima ls hea lthy and free of disease.

3 2

4 5

Use the 5 steps to lower the risk

Keep livesto ck healthy 1. W hen introduc ing livestock , insp ec t it to make sure it is h ealthy and keep it separate for 5 days 2. Vac cinate you r livestock 3. W a sh you r hands and feet after handling liv estock 4. Ensure any visito rs use g ood biosecurity 5. If y our animal becomes sick separate it, provide treatment and care, inc lud ing w ater and feed.

What can YOU do about FMD?

1] Risk factors Direct animal-animal contact through common grazing and sharing drinking sources Introduction of livestock goats for religious celebrations Unrestricted access of villagers and service providers on premises no biosecurity measures applied

for veterinarians and associated stakeholders.

Contact your local DLS O V eterinarian or technician for further advic e

1Regional

2European

Support Unit for South Asia Food and Agriculture Organisation (FAO), Kathmandu, Nepal Commission for the Control of FMD (EUFMD), Food and Agriculture Organisation, Rome, Italy

FMD viruses in Pool 1 Serotype O Virus Pool 1

Myanmar 98 and Cambodia 94; endemic in SE Asia: reported in China, Korea and Japan in 2010 detected SE Asia in late 1990s

South East Asia

Indigenous in SE Asia; reported in China in 2009 and Korea in 2010

Asia 1

Asian

Last reported in Vietnam in 2007 and in China in 2009

SEA Panasia Cathay

Asia 1

266

Remarks

Pan Asia Cathay

Topotype South East Asia

1st detected in Hong Kong in early 1990s

SE Asia FMD Outbreaks (2000-2014)

SE Asia FMD Outbreaks peaks in 2006 and 2010/11

FMD Outbreaks in SE Asia 1n 2006

FMD Outbreaks in SE Asia 2010/11 Type O Panasia in 2010/11

Type O Mya/98 in 2006

Mya98

Cathay

267

2010 Type O outbreaks

2010 Mya 98 and O/Panasia

2011 serotype O

2011 O/Mya 98 and O/PanAsia

268

2012 serotype O

2012 O/Mya 98 and O/PanAsia

2013 serotype O

2013 O/Mya 98 and O/PanAsia

269

2014 all O

2014 O/Mya 98 No O/Panasia has been detected so far in 2014

2010 serotype A

Serotype A

270

2010 serotype A/India

2011 serotype A

2012 serotype A

2013 serotype A

271

Vaccine matching or r-value of FMDV type A in SEA region

2014 serotype A

(specimens received in 2011-2013) Country

Year

Total sample

A118/87 0.2-0.39

Thailand

Range of r-value by LP ELISA test A/Sakolnakorn/97 A/Lopburi/2012

0.4-1.0

2011

2012

No binding reaction by antigen titration

(Jan-Feb)

2012 (Mar- Dec)

2013

No binding reaction by antigen titration

0.2-0.39

0.4-1.0

39*

0.2-0.39

0.4-1.0

No binding reaction by antigen titration No binding reaction by antigen titration

Source: OIE Ref Lab, Pakchong, Thailand

Vaccine matching or r-value of FMDV type A in SEA region

Molecular epidemiological analysis

(specimens received in 2012-2014) A/Sea-97 strain, located on the other genetic branch(named G2) no genetic relationship with the virus(named G1) found in China in 2009: about 91% homology new virus found in China where did the virus come from? Shared very close relationship with viruses from Thailand and other SEA nations: over 97% homology

Range of r-value by LP ELISA test Country Thailand

Year 2012

Total sample

(Jan-Feb)

2012

A118/87 0.2-0.39

0.4-1.0

No binding reaction by antigen titration No binding reaction by antigen titration

A/Sakolnakorn/97 0.2-0.39

0.4-1.0

No binding reaction by antigen titration

A/Lopburi/2012 0.2-0.39

0.4-1.0

No binding reaction by antigen titration

(Mar- Dec)

Lao PDR Vietnam

2013

2014

2014 2013

3 3

No binding reaction by antigen titration

No binding reaction by antigen titration No binding reaction by antigen titration

Type A FMDV in 2009

type A FMDV in pigs recently G2

The relationship with different FMDV found in China in 2009 and 2013 Source: OIE Ref Lab, Lanzhou, China

Source: OIE Ref Lab, Pakchong, Thailand 26

272

2014 Serotypes

2011 27 2%

32 2%

Increasing number of serotype A compared to serotype O

2012

1429 96%

11 8%

90 63%

41 29%

94 39%

2013

79 33%

67 28%

Summary From 2010-13, serotype O/Mya 98 is predominant in Myanmar, Thailand and Malaysia. However, increasing number is observed in 2014 A resurgence of Serotype O/Panasia was observed since 2010/11 affected Cambodia, Lao PDR, Vietnam and China until 2013. Serotype A increased during this period with a peak in 2013. New lineage of serotype A is observed. No Asia 1 detected during this period. 30

Thank you for your attention!

273

LE AR FMD future: responding to change in the global landscape

274

Areas to Cover 1. FAO-OIE global FMD control strategy 2. Instrument and mechanism for implementation

Global Landscape of FMD Control Samia Metwally, PhD, DVM

3. Regional progress using PCP approach, OIE official status recognition and national control programme endorsement

Animal Production and Health Division Food and Agriculture Organization of UN Rome, Italy Co-authors (GF-TADs FMD Working Group): Joseph Domenech, Giancarlo Ferrari, Nadège Leboucq, Laure Weber-Vintzel, Julio Pinto

4. Challenges and the next steps 1

OIE Recognition of Official Disease Status and Endorsement of Official Control Programmes

FAO Strategic Objectives in context of FMD Control SO1: Contribute to the eradication of hunger, food insecurity and malnutrition SO2: Increase and improve provision of goods and services from agriculture, forestry and fisheries in a sustainable manner SO 5: Increase the resilience of livelihoods to threats and crises

275

Global Strategy Instrument for Implementation

FAO- OIE Global FMD Control Strategy, 2012

Progressive Control Pathway (PCP)

- Developed in consultation with FMD experts and representatives of key countries and regional organizations - Fifteen-year plan with five year increments Components Improve VS

Understand understanding of FMD epidemiology Identify hotspots Mapping value and market chains Understanding socio economic impacts Formulate control options

Control livestock diseases

FMD Control

Risk Assessment Objective Assessment of Progress of PCP for FMD

Reduce Impact

Eliminate virus circulation Implement aggressive control policy Continue routine surveillance Early detection and rapid response Vaccination Eliminate FMD in zones or country OIE endorsement of national control plan at end of this stage

Implement risk-based control measures Stakeholder consultation and engagement Targeted vaccination Monitor and evaluate

Sector level Risk Management

Population level Risk Management Objective Assessment of Progress of PCP for FMD

276

Objective Assessment of Progress of PCP for FMD

Global Strategy Mechanism for Implementation

Composition of PCP stages 0-3 Countries by PCP Stage per Region (87 countries) (as of 2012)

1. Regional platform (virus pools):

CVOs and FMD national specialists

Share information on FMD virus circulation Review progress along the FMD Regional Roadmap

30 Africa

Assist countries preparing national control programmes and project proposals for increased investment in FMD control

East and Southeast Asia

20 15

Eurasia

South America

South Asia

0 Initial PCP 0

Initial PCP 1

Initial PCP 2

Initial PCP 3

Acceptance process for PCP stages (roadmap)

Global Strategy

Self-assessed PCP questionnaires submitted to FMD WG Evidence gathered for the claimed stage through

2. Regional advisory group (RAG):

Country report Control plan PVS evaluations of the Veterinary Services Country interview by FMD WG and PCP/PVS experts

Elected three CVOs and leads of regional laboratory and epidemiology network (Voting members) Representative(s) of FMD WG, PCP/PVS experts and a representative of regional organizations (non-voting members) Responsibilities are: review and assess PCP-FMD stages, guide FMD training for the region and enhance establishing enabling environment for FMD control

Review all above by the RAG in a closed session Feedback for final acceptance

277

Feedback to Country (example) Country

Assess ment 2012

Kenya

Review (SAQ, Presentation, Interview) Well detailed RBSP Sero-surveys Econ impact studies

RAG Evolution Actions for country agreed by RAG assessment since 2012 2014 2 PVS-PCP 7 critical competencies including small ruminants (Re-)analyse sero surveys for riskfactors Update value chain analysis and socioeconomic studies Revise RBSP - Risk hotspots risk estimation - Coherence

Virus pools and FMD Control Programs

Actions expected from RAG Follow-up on provisional Stage 2

SEAC FMD CP-FA PHEFA

West Eurasia Roadmap (virus pool 3) April 2014

SAARC Roadmap (virus pool 2) October 2013 First roadmap 2011

First roadmap in 2008 14 countries

5 countries country

2011

Countries' Progression (2012-2017) from PCP 1

2012 2013 2014 2015 2016 2017 2018 2019 2020 100

Bangladesh Bhutan India Nepal Sri Lanka

1 1 3 1 1

2 1 3 1 2

2 1 4 1 2

2 2 4 2 2

3 2 4 2 3

3 3 4 2 3

3 3 4 2 4

4 3 4 3 4

80 60 40 20 0 1to1

Countries Progression (2012-2017) from PCP 2 100

1to2

1to3

Countries Progression (2012-2017) from PCP 3 100 80 60 40 20 0

80 60 40 20 0 2to2

2to3

3to3

100 90 80 70 60 50 40 30 20 10 0

3to4

278

Westeurasia provisional progression from PCP 1 (2012-2017)

Westeurasia provisional progression from PCP 2 (2012-2017) 100 90 80 70 60 50 40 30 20 10 0 2to2

1to1

1to2

2to3

1to3 16

East Africa Roadmap (pool 4)

Near East and North Africa Roadmap (virus pools 3-5), March 2014 Country Bahrain Egypt Iraq Jordan Palestine Kuwait Lebanon Oman Qatar Saudi Arabia Syria UAE Yemen

2012 1 1 2 1 2 1 2 2 1 2 1 1

2013 2* 1 2 1 2* 1 2 2* 1* 2 1 1

2014 2 1 2* 2* 1 3 2* 2* 3 1 2* 2 1*

NENA (2014) Provisional Progression from PCP 1 (2012-2017) 100

2015 3 1 2 2 3 2 3 3 1 3 3 1

2016 3 2 2 3 3 3 3 3 2 4 3 2

2017 3 2 2 3 4 3 4 4 2 4 3 2

2018 4 2 2 4

2019 4 2 3 4

4 3 4 4 3 4 4 2

4 4 4 4 3 4 4 2

2020 4 3 3 4 4 4 4 4 3 5 4 3

2021 4 3 3 4 4 4 4 4 4 5 4 3

October 2014

First roadmap in 2012

15-19 countries

12 countries

Country Burundi Djibouti DRC Eritrea Ethiopia Kenya Rwanda Somalia South Sudan Sudan Tanzania Uganda

2014

2015

2016

2017

2018

2019

2020

2021

2022

2023

2024

1 1 1 1

2 2 1 1

2 2 2 2

3 3 2 3

3 3 3 3

4 4 4 4

2 3 2 1 3 2 2

2 4 2 1 3 2 3

3 4 2 1 3 3 3

4 4 2 2 4 4 4

1 2 2 2 2 2 3 3 1 1 1 1 1* 1 1 1 2* 2 2 2 1 1 2 2 1 1 2 2 *Provisionally accepted

NENA (2014) Provisional Progression from PCP 2 (2012-2017) 100 80

40 20

0 1to1

1to2

1to3

0 2to2

2to3

2to4

Provisional PCP Stages vs. Global Strategy Prediction (2012-2017) (Virus Pools 2-4, 42 Countries)

Global PCP-FMD Map

100

PCP 1 Provisional Progression from 2012 to 2017.

PCP 0 Provisional Progression from 2012 to 2017. 1 100

20 2014 data

0 0 to 0

0 to 1

GS Y5

2014 data

0 1 to 1

PCP 2 Provisional Progression from 2012 to 2017. 100

100

GS Y5

PCP 3 Provisional Progression from 2012 to 2017. 1

20 2014 data

0 2 to 2

279

1 to 3

60 40

1 to 2

2 to 3

2 to 4

GS Y5

2014 data

0 3 to 3

3 to 4

3 to 5

GS Y5

Support for Implementation of PCP-FMD

PCP-FMD Training for FAO HQ staff July, 2014

Guidelines, post vaccination monitoring is in final stage of development Design of serosurveillance plan Guidelines, formulation of control plans (PCP stages 1 and 2) Guidelines, socioeconomic impact studies FAO technical cooperation program (TCP) :

1. 2.

- TCP for Tunisia - TCP for Uganda - Regional TCP for North Africa

Two-day workshop for 20 participants Objectives: 1. share knowledge and experience on implementing PCP in the field with main focus on stages 1-2 2. Learned how organize and implement a Risk-Based Strategic Plan (RBSP) and how to find the risk hotspots through a simulation exercise EuFMD supported this training with PCP-experts

OIE SCAD and AHG work: endorsement of NCP, Official free statuses, SCAD FMD country missions, OIE articles revision OIE Mission in Tunisia, with FAO collaboration OIE programmes in South East Asia (SEACFMD) and East Asia ExCom88

Ongoing Support

PCP-FMD training for FAO staff in Africa

Training on PCP principles and implementation at national level

October 15-17

Outcomes: Continue to engage countries in FMD control

support national veterinary services in assessing their PCP-FMD status support countries to enter Stage 1 (assessment plan) and countries to enter Stage 2 (Risk based strategic plan)

roadmap meetings Training on PCP principles and implementation Assistance in development control plans

conduct training at national and regional levels on the PCP principles

Resource mobilization for regional/country support

assist in conducting a Regional Roadmap meeting

280

FAO OpenFMD database for sequence exchange- in development 24

Characteristic of Countries per PCP Stage 2012 Agricultural value in GDP by PCP stage 30 25

Challenges

20 15 10 5

Gross National income per capita by PCP stage

25000 0

4, 5

Free HIST Free OIE 20000

PCP stages

15000 10000 5000

Source: UN COMTRADE System, WDI, World Bank

Cost Benefits ( Model) for livestock disease in a country with high start-up cost Benefit Function Total Cost ($)

0 0

Challenge ahead

200000 Cost Shortage in supply 150000 Maintenance of the cold chain Relatively short term immunity 100000 Quality in some regions 50000

- Start up cost for is

prohibitively high to implement the program - Poorest countries

Free HIST Free OIE

Cost of global strategy 5y: Cost of vaccination:

Vaccination represent 93% of global strategy cost 250000 Issues :

4, 5

Estimated costs of vaccination by region (x $1,000) for 5 years

Variable Expenditure ($)

Source; world bank FMD global strategy costing 2012

0 Africa

Initial PCP 0

281

East and Southeast Asia

Initial PCP 1

Eurasia

South America

Initial PCP 2

South Asia

Initial PCP 3

Conclusions

Acknowledgments

Global FMD control is feasible and can be a driver to improve the animal health system

FAO Decentralized offices and ECTAD teams OIE HQs and regional and sub regional offices EuFMD secretariat Continental-Regional organizations: AU-IBAR, IGAD, EU Italian government for funding the FMD secretariat, 2013-2015

PCP-FMD approach and reinforcement of vet services are gradually gaining acceptance. Sixty countries are engaged in several regions including SEA Political will and engagement of international and regional organizations and development partners are crucial to the startup and sustainability of FMD control PCP principles could be adopted and used for control of other TADs as an optional possibility and after regional and national discussions Funding is needed to support the global strategy, particularly those countries at lower PCP stages Vaccination issues need to be addressed by the government authority and research community 29

Thank you for your attention

What do we know about the economic impact of FMD in smallholder production? Summary of the evidence

Jonathan Rushton Professor of Animal Health Economics, Norbrook Endowed Chair in Veterinary Business Management Royal Veterinary College, London, UK

Theo Knight-Jones Epidemiologist

International Livestock Research Institute, Zambia

282

Introduction

Methods

FMD is most prevalent in regions where households are most dependent upon livestock This raises the question of what is known about the burden of the disease on smallholder producer. The paper will attempt to answer this question in order to resolve if FMD is simply a problem for large scale cattle producers and their associated milk and beef value chains Or that more logically that FMD affects all people across a society

Online search was carried out searching for papers in Pubmed, google scholar and google web for terms -benefit In addition 14 expert groups working in the field of the epidemiology and economics of FMD were consulted for additional publications

Methods

FMD Impact

References identified were reviewed in order to develop an understanding of the level of knowledge of FMD impacts in a smallholder system

Losses

Articles written in English or Spanish were included. Articles were retained if they reported either original research or reviewed aspects of FMD economic impact.

Visible Losses

Expenditure and reactions

Invisible Losses

Additional Costs

Lost Foregone

Vaccines Vaccine delivery Movement control Diagnostic tests

Use of suboptimal breeds Denied access to markets both local and international

Impact Impact caused by caused by FMD human reaction

283

Loss of milk production Loss of draught power Lower weight gains Dead animals

Lower fertility Change in the herd or flock structure

Results - FMD and smallholders

Density of susceptible species

The results are broken down into different levels: What can we say from a national and regional level in terms of where FMD is endemic and where are the smallholders? What data and information do we have on the impacts of the disease in smallholder systems? What biological information is available on FMD in smallholder systems?

Density of poor people dependent on livestock

Studies on household impact of FMD

Source: 1Young et al., 2012; Shankar et al 2012; 2Rast et al., 2010 ; 3Barasa et al., 2008; 4 Ferrari et al 2012 8

284

Impact of diseases for pastoralists in Kenya (Onono,

Frequency of outbreaks Based on observations of outbreaks and reports from case studies, pastoralists in Eastern Africa often experienced more than one outbreak a year (67% of herds affected each year) Such high incidences are not unusual in endemic countries across Africa and Asia, (McLaws, 2012) In a cross-sectional survey in rural Tanzania, 80-90% of households reported that they had experienced an outbreak within the last year and FMD was ranked as the second most important livestock disease

(OIE) 25

quantitative evidence of impact in dairy systems Abubakar Suleiman, working on CBPP in Nigeria, has also picked up FMD as being of importance in Fulani systems in the north Ongoing work in SE Asia coordinated by OIE Plus a range of other anecdotes and data in the process of being analysed 20

And what can we say about the gaps on impact?

Major gap As far as we are aware there have been no published studies on the economic impacts of FMD on: Small ruminants Pigs

Sero surveillance studies indicates the levels of infections in small ruminants, there appears to be no such data on pigs

285

Why is this important we have uninformed debates

Conclusions

FMD control in endemic countries is often deemed a lesser priority by governments and international funding agencies Some have argued that, in Southern Africa, only wealthy farms with export potential benefit from FMD free status (Scoones et al., 2010) Others have stated that a significant proportion of the benefits of international export filter down through the supply chain to low-income households (Perry et al., 2003)

There are limited data and studies on FMD impact in smallholder settings for cattle in Africa and Asia there are major gaps in our knowledge Everything currently available indicates that FMD is important to smallholders Yet the narrative is weak due to the lack of consistency and frequency of studies Given the disease is highly infectious and causes health problems, plus the people who are smallholders depend on markets for their existence it is logical that it would be important in endemic countries

Recommedations

There is a need for improved scientific measurement of the existence of FMD in smallholder systems, and of the measures taken by these farmers to prevent the disease and to treat animals affected. There is also a need to detail how these farmers manage with regards market access during outbreaks and if their livestock activities are impacted by trade restrictions.

These data need to be collected, captured and analysed a systematic way We need to move from uninformed debates on the importance of FMD towards reasoned agreements of rationalised use of resource to reduce the FMD burdens We as scientists need to give smallholder farmers clarity and guidance on what value we can add to their lives

286

Acknowledgement of Experts consulted

Impact of FMD on milk yield, mastitis, fertility and culling on a large-scale dairy farm in Kenya Nick Lyons

London School of Hygiene and Tropical Medicine

Background FMD Economics

Background Objective: to quantify the impact of FMD on a largescale dairy farm in Kenya focussing on:-

Lack of objective field data looking at FMD impact particularly in endemic settings Tendency to rely upon expert opinion and assumptions More data needed to inform cost-benefit analyses of control measures (e.g. vaccination strategies, culling and compensation measures) Need data from different people involved in the system as outlined in the PCP stage 1

Milk yield Clinical Mastitis Culling Fertility 2

287

Outbreak

Farm background

KENYA

Dairy Herd: 650 mainly Holstein-Friesian Milking around 250 cows Calving all year around Artificial insemination only All cows uniquely identified Record daily milk yields, health and fertility events, sales etc in InterHerd (InterAgri, School of Agriculture, University of Reading, UK).

NAKURU COUNTY 4

Outbreak August/September 2012

Milk yield overall impact Outbreak period

Serotype SAT2, lasting 29 days Case definition: Hypersalivation with any other sign indicative of FMD: decreased milk yield, decreased feed intake, oral/interdigital/teat lesions, pyrexia

Vaccine: Limited/no vaccine effect in preventing clinical disease. Overall Attack rate: 400/644 (62.1%)

288

Milk yield Reported FMD cases versus non-cases

Milk yield No difference? Possible reasons: 1. Poor/inaccurate recording of cases 2. Insensitive case definition 3. Subclinical infection

Outbreak period

Next approach: Predict yield for all individuals based on historic farm records accounting for parity, days in milk, and season (GEE model with a AR1 autocorrelation matrix)

Compare production from beginning of outbreak to end of 305 day lactation irrespective of disease status 8

Milk yield Actual vs Predicted

Clinical mastitis and culling Survival analysis Follow up: 12 months from beginning of outbreak Statistics: Cox proportional hazard regression Adjusted for any non-proportional hazards by incorporating time varying effects Largest impact

Study population: Culling - All animals Mastitis -

Impact dependent on parity and lactation stage when diseased 10

289

Clinical mastitis

Unadjusted

Adjusted Hazard Ratio (first month) = 2.9, 95%CI 0.97-8.9, P=0.057

Study population restricted to animals over the age of 18 months at start of outbreak 12

Culling

Fertility Submission rate, Pregnancy rate Unadjusted

Outbreak

Adjusted Hazard ratio: HR=1.7, 95% CI 0.90-3.4, P=0.10

Culling is defined as exiting the herd for any reason associated with a adverse health event 14

290

Submission rate decreased, but pregnancy rate not affected 15

Fertility Abortion and Early Embryonic Death

Summary - overall

Outbreak

Milk yield Depends on parity and lactation stage Clinical mastitis 3 times the hazard in first month Culling 1.7 times the hazard over 12 months Fertility impact on submission rate, returns to service Data may be used in developing cost analyses Limitations Generalisability Lack of statistical power Vaccination mitigating impact

No obvious effect on abortion, but increased returns to service 16

Conclusions - summary

Acknowledgements

Great need for rigorous evaluations of disease impact There needs to be investment in data collection on disease losses and costs so that we can move away from relying on expert opinion and assumptions Essential to reliably quantify impact for allocating limited resources in animal disease control More need for field data from different farming systems in different settings 18

291

Hamish Grant and his workers at Gogar Farm, Rongai Co-authors of research: Neal Alexander, Paul Fine (LSHTM) Jonathan Rushton, Katharina St rk (RVC) Andrew James (University of Reading) Keith Sumption (EuFMD), Thomas Dulu (Kenyan DVS) Funders: Bloomsbury Colleges, University of London EuFMD MSD Animal Health, Royal Veterinary College (London) 19

292

293

294

295

296

297

298

299

EMERGING MASSIVE FMDV OUTBREAKS IN UGANDA AND POSSIBLE IMPACT ON PC Dr. Ayebazibwe Chrisostom Senior Veterinary Officer, National Animal Disease Diagnostics and Epidemiology Centre (NADDEC), Ministry of Agriculture Animal Industry and Fisheries, Entebbe, Uganda

Introduction

Materials and Methods FMD outbreaks were reported in the districts of Kotido and Napak. During field investigations, outbreaks had spread over the entire Karamoja region and many other surrounding districts: Moroto, Soroti, Ngora and Mbale. Epithelial samples were picked from symptomatic animals (31/150) and stored in PBS and liquid nitrogen.

Unusual massive occurrence of FMD outbreaks was encountered between May and July, 2014 hence the need to study the pattern of occurrence and the serotypes involved.

300

Map of Uganda showing the distribution of serotype O investigate d outbreaks (14th July, 2014)

As part of preliminary serotyping, 23 samples were subjected to antigen ELISA. One or two farms were selected per district to maintain biosecurity. Information on outbreaks was collected through interaction with the DVO´s, farmers, reports and field observations.

Results

Discussion

A total of 9/23 samples tested positive for FMDV antigen ELISA (Serotype O): Kaabong (5/12), Napak (2/3), Mbale (1/3), Moroto (1/4), and Ngora (0/4). All the FMDV outbreaks were blamed on suspected animal movements especially due to cattle restocking programmes and communal farming.

301

Compared to annual FMD outbreaks in 2011 (22), 2012 (15) and 2013 (8), Uganda by 14th of July 2014 registered 23 outbreaks in two months (Kotido, Nakapiripirit, Abim, Kasese, Kween, Bugiri, Napak, Nwoya, Mbale, Alebtong, Ngora, Moyo, Amudat, Kaabong, Moroto, Sironko, Bukedea, Kumi, Soroti, Kapchorwa, Pallisa, Bukwa, Lamwo district). More investigations are required to serotype all outbreaks. Detailed virological and epidemiological information can be obtained through collaboration with reference labs 7

Conclusion/Recommendation

Acknowledgement

The fact that Uganda is surrounded by 5 other countries and FMD is Transboundary, massive outbreaks can be a regional problem with potential to reverse the progress achieved by East African countries along PCP. Regional Approach is the solution for FMD control

1. National Animal Disease Diagnostics and Epidemiology Centre, Ministry of Agriculture Animal Industry and Fisheries, P.O. Box 513, Entebbe, Uganda; 2. Food and Agriculture Organization, Kampala, Uganda 3. Key Persons: Ayebazibwe Chrisostom1, Ruhweza Simon Peter1, Martin Esau1, Okori Edward2, Kauta Nicholas1

END 8

The situation Foot-and-mouth disease (FMD) is endemic in Kenya despite control PCP-FMD Road map for East Africa The country is in stage1 Activities identified at the 2012 roadmap meeting include o Understanding the epidemiology of FMD and develop a risk-based approach to reduce the impact of FMD FMD sero-survey was undertaken in 2012 and 2013.

Sero-survey to understand transmission pathways for foot-and-mouth disease spread between and within different regions of Kenya Kibore B, Gitao C.G, Sangula A, Kitala P

Benson Kibore Kenya Veterinary Vaccines Production Institute (KEVEVAPI-KENYA). benson.kibore@kevevapi.org 1

302

Kenya PCP-FMD Roadmap

Results

NSP prevalence

The Kenya/Uganda border- 95%; north rift DFZ 97.5%. Mt Elgon national park100%; Turkana-PokotTrans Nzoia-Uasin Gishu-Nakuru-Nairobi stock route; 80.5%. The Southern pastoral regions-58.2%; Northern- 49.4%. SP Antibody prevalence All the four serotypes O, A, SAT1 and SAT2-in circulation

The sera samples were collected from counties within international border, proposed disease free zones, trade and stock routes and within national parks and game reserves (3709 samples). The samples analyzed using NSP ELISA. The NSP positive samples were then assayed for SP serotype - specific antibodies using the LPBE

FMDV seroprevalence in Kenyan borderlands 100 90 80 70

FMDV seroprevalence

Material and methods

60 50 40 30 20 10 0 Borderlands Kenya/Somalia border

SAT 2 Kenya/Ethiopia border

SAT 1

TypeA

Kenya/Uganda border

Type O Kenya/Tanzania border

Conclusion The study contributes to identifying the FMD hot spots in the country. Helps in the development of the RiskBased Control Plan for FMD.

Vaccine evaluation on large-scale dairy farms using routine prophylactic schedules for FMD

Acknowledgements Director, Veterinary Services (DVS-Kenya) Director, Kenya Veterinary Vaccines Production Institute (KEVEVAPI) Staff, FMD and KEVEVAPI laboratories SERECU team and VEEU Kabete.

Nick Lyons

London School of Hygiene and Tropical Medicine

303

Background Vaccine effectiveness

Farm A -Background

Vaccine effectiveness: % reduction in incidence among vaccinated individuals attributable to vaccination, measured under field conditions

Kenya, Nakuru County Last known outbreaks: March 2004 (SAT2), December 2010 (NVR) FMD Vaccination Vaccinates animals every 4 months with a locally available quadrivalent (A, O, SAT1, SAT2) vaccine Only vaccinates animals over 6 months old

Reasons for poor FMD vaccine performance in the field: 1. 2. 3. 4. 5. 6.

Poor potency Lack of vaccine match Break in the cold chain Sub-optimal coverage Interference by maternally derived antibody (MDA) Incorrect schedule

Aqueous-adjuvanted, Non-NSP purified vaccine. 50 vaccines are approved for use. 2

Farm A - Outbreak

Farm A - Vaccine

Serotype O October-December 2013 Last dose 3 months before Probable source: Farm workers

Incidence risk

304

Number of lifetime doses

Farm A - Vaccine Incidence risk

Farm A - Vaccine Number of lifetime doses

Incidence risk

Number of lifetime doses Declining incidence implies some vaccine effectiveness

Maternal antibody?

Possible reasons for incidence pattern on Farm A

Farm B - Background Kenya, Nakuru County

40% incidence in multiply vaccinated clearly reveals a

-Friesian

Potency? Match? Cold chain?

Last known outbreak in 2004 (unknown serotype)

Suboptimal schedules as well?

Can have multiple reasons for poor VE! 8

305

FMD vaccination Vaccinates animals every 4-6 months with a locally available quadrivalent (A, O, SAT1, SAT2) vaccine Vaccinates all animals irrespective of age 9

Farm B - Outbreak

Farm B - Vaccine Incidence risk

SAT2 August September 2012 Last vaccination 3 months before Probable source: Farm workers

Number of lifetime doses

Farm B - Vaccine Incidence risk

Number of lifetime doses

Incidence plateau among

Lower incidence in

306

Possible reasons for incidence pattern on Farm B

Farm C - Background Iran, Shahriar County, Tehran Province Dairy herd: 3,500 cattle, Holstein-Friesian

Low incidence in youngstock poorer reporting? less severe disease?, less transmission/exposure? (like Netherlands, 2001?) Vaccine match? (SAT2 VP1 sequence 13% difference to that reported by Sangula et al, 2010) Vaccine potency? SAT2 known to be less stable antigen requires higher antigen dose than other serotypes Cold chain?

Last known outbreak 4 years previously (Not Asia-1) FMD Vaccination Every four months with trivalent (Asia-1 Shamir, O, A). Calves >2 months old get two doses one month apart as a primary course. High potency, NSP purified vaccine.

Farm C - Outbreak

Farm C - Vaccine

Asia-1 January-March 2011 Last dose 11 weeks before Probable source: Local semi- nomadic sheep/goat herds

Incidence risk

Number of lifetime doses

Vaccine Matching 0.23 0.17 0.19 0.28

307

Farm C - Vaccine Incidence risk

Farm C - Vaccine Number of lifetime doses

Incidence risk

Number of lifetime doses

Vaccine Matching

0.23

Most disease

0.17

Another peak

0.19 0.28

0.17 0.19 0.28

Low incidence

Maternal antibody?

Maternal antibody? 18

Summary and conclusions

Vaccine protection and coverage Does appear to be evidence of some cumulative protection with number of doses (Farms A and C) Cannot rule out other age related effects (although not due to exposure in these outbreaks) Age distribution in the herd affects coverage! Farm A (O)

Farm B (SAT2)

Generalisability? Field-based vaccine performance assessments provide additional information on the effectiveness of a control policy Conventional laboratory-based evaluations should be performed alongside field evaluations Standardised protocols for monitoring vaccine performance and investigating apparent low effectiveness are needed

Farm C (Asia-1)

308

Acknowledgements Vets, Farmers and their workers at study farms Co-authors/collaborators of research: Paul Fine (LSHTM), Theo Knight-Jones (ILRI) Katharina St rk (RVC), Thomas Dulu (Kenyan DVS) Chris Bartels, Keith Sumption (EuFMD), Funders: Bloomsbury Colleges, University of London MSD Animal Health Royal Veterinary College, EuFMD

Influence of colostrum in the immune responses of calves to current FMD vaccines Alejandra Capozzo Applied Veterinary Immunology Lab. Institute of Virology. INTA. Buenos Aires, Argentina 22

Danilo Bucafusco Sebastián Di Giácomo Rodrigo Pereyra Juan Pega Darío Malacari María Sol Juncos Juan Schammas Mariano Pérez Filgueira Alejandra Capozzo

FMD IN ARGENTINA

Authors

EPIDEMIOLOGICAL STATUS: - Free with vaccination - Free without vaccination

OIL-based vaccines, local producers Four strains: O1/Campos, A24 Cruzeiro, A/Arg/01 and C3 Indaial

VACCINATION CAMPAIGN: - Adults: once a year

- Calves <2 years old: twice a year 309

VACCINES AND PASSIVE MATERNAL IMMUNITY

Serum IgM levels anti-FMDV in vaccinated calves increased as Mat. Abs titers decreased

Inactivated vaccines are usually incapable of inducing a protective immune response in in the presence of maternal antibodies (Mat-Abs).

IgM

DIFFERENT CONCLUSIONS! Some publications demonstrated inhibition of the immunogenicity of vaccines in calves born to vaccinated dams while others agreed that the vaccine effectiveness was not affected by the presence of Mat-Abs (mostly in the 80´) 4

Animals that did not respond to the boost had higher VNT titers at the time of the first vaccination than those that responded 4

COLOSTRUM HAS IMMUNE CELLS 13 ml isotonic Percoll. 4000 x g for 30 min.

Percoll 43% over Percoll 70%. 5000 x g- 20 min

5 p<0.05

3 3 2

Titers measured at 0 dpv (calves with Mat Abs)

Outcome of booster vaccination 6

310

Experimental design: the idea! Colostrum has immune cells and antibodies Antibodies are efficiently transferred through the gut during the first 12h of birth (mostly IgG1) Some reports suggest immune cells are also transferred

Colostrum without cells

30 Days after birth

Colostrum with cells / whole

VACCINATION Colostrum from

Do immune cells transferred by colostrum influence the immune reponse to vaccination?

different dams /Whole

Colostrum deprived 9

Methods

Methods Experimental design: real life! 4 groups

Group CAL-: colostrum deprived calves (n=3) Day 1 ol life

CAL-: COLOSTRUM DEPRIVED Two liter bottles Different colostrum CSC: COLOSTRUM WITHOUT CELLS preparations CCC: WHOLE COLOSTRUM (WITH CELLS) CMIX: COLOSTRUM FROM DIFFERENT DAMS

ONE VACCINATION DAY 30 AFTER BIRTH

DAYS AFTER VACCINATION 0 7 21 30 35 60

6 hs:milk substitute (2 L) 12 hs:milk substitute(2 L) Gentamicin 8 mg/kg Ceftiofur 6 mg/kg

Formula 4 L/día (3 semanas) Pellets Gentamicin 8 mg/kg (5 días) Ceftiofur 6 mg/kg (10 días) Boxes BSL-2 CICVyA INTA

120

Whole blood and serum samples

311

Group Cmix (n=6) Pool of Colostrum

Group CSC (n=5) Colostrum without cells

Transference of colostral Antibodies Results

Group CCC (n=4) Calostrum with cells

Day 1 post.colostrum intake CALCCC CSC CMix

30 L of pooled colostrum

Pool of colostrum of different dams

-20 C 48 hs

3000 xG 20 min.

-20 C

2 L before 6h of life 2 L before 12hs Formula up to 48 hs

LPB-ELISA

VNT

* IgG1

* IgG2

IgM

(P<0.05)Mann Whitney

No differences were found between calves fed with different colostrum preparations 12

Neutralizing Abs elicited in vaccinated calves that received different colostrum treatments (VNT) 5

Interferon-gamma responses in stimulated plasma pre- and post-vaccination

Colostrum deprived Cal Mix Acellular colostrum Cellular colostrum Non vaccinated (colostred)

Vaccination

4 3

Stimulated with O1 Campos 2.0

Colostrum deprived Whole immune colostrum Acellular colostrum Colostrum mix

Post- vaccination

1.5 1.0

EPP75% 0.5

1 0

Pre- vaccination

0 10 20 30

0.0

Days after birth

The presence of immune cells transferred by colostrum at the time of vaccination did not modify the lack of neutralizing Ab response to the vaccine. 14

Days after birth

312

Days post vaccination

Responses previous to vaccination were observed only in calves that received colostrum

5 4

CAL -

CCC

Edad (días) 5 4

Functional immune cells are transferred from colostrum to the calves

Results

IFN- recall responses

Edad (días) 5

CSC

Stimulated with O1 Campos

Responses previous to vaccination were observed only in calves that received colostrum with cells

Colostrum deprived Whole immune colostrum Acellular colostrum Colostrum mix

Non vaccinated

Responses after vaccination were comparable between all groups and for all the virus strains included in the vaccine

CMix

Lympho-proliferation assay

10 5 0

Days post vaccination Edad (días)

O1C

Edad (días)

A24

A2001

The presence of FMDV-specific immune cells transferred by colostrum at the time of vaccination did not modify the specific CMI induced by the vaccine.

C3I

FINAL CONCLUSIONS Functional immune cells, able to proliferate upon FMDV ex-vivo stimulation are transferred through colostrum The presence or absence of cells in the colostrum did not influence the immune response elicited after vaccination Colostrum-transferred immunity interferes with the induction of humoral neutralizing responses but not with cell-mediated immune responses

313

THE POPE

MESSI

MARADONA

THANK YOU!

MAXIMA

The Applied Veterinary Immunology Lab. INTA- Buenos Aires, ARGENTINA

FMD control in free countries Border controls Contingency plans Vaccine banks Limited number of strains Often limited info on vaccine efficacy with heterologous challenge Challenge to producers: when to develop new vaccine strains

Serotype O vaccine efficacy and challenge with different viruses from South East Asia in various species Wilna Vosloo, Jacquelyn Horsington, Jacqui Morris, Nagendra Singanallur CSIRO-Australian Animal Health Laboratory, Geelong, Australia

BIOSECURITY Flagship www.csiro.au

314

Phylogeny of O-SKR-2010 (Mya-98 strain) by Bayesian inference. Numbers in percentage indicate the posterior probability values and scale bar represents year of divergence

Correlates of protection In vitro assays relatively good predictions of homologous protection Less accurate when looking at heterologous challenge R1-values IgG1/IgG2 ratios Antibody avidity Titres: VNT Titres: ELISA Cytokine responses

Most accurate results with in vivo challenge

Comparing O1 Manisa and O/SKR/2010 in cattle against challenge with O/SKR/2010 2010 outbreak in South Korea

Full dose O1 Manisa vaccine

Full dose O/SKR/2010 vaccine

1/4 dose O1 Manisa vaccine

1/4 dose O/SKR/2010 vaccine

High potency O1 Manisa used (r1 = 0.42) Some reports of vaccine failure

Vaccines provided by Merial O1 Manisa ME-SA topotype O/SKR/2010 Mya 98 lineage All vaccines > 6PD50

Challenge virus provided by Merial (cattle derived O/SKR/2010) Vaccines tested and compared in cattle Cattle: potency tests using 15 cattle and tested at full, ¼ and 1/16 dose

All challenged with O/SKR/2010 10 000 BID50 IDL 21 dpv

Unvaccinated controls

315

1/16 dose1 O1 Manisa vaccine

1/16 dose O/SKR/2010 vaccine

Results

Results Viraemia and probangs

Full 1/4 1/16 dose dose dose PD50

Group O1 Manisa vs. O/SKR challenge Heterologous

5/5

O/SKR vaccine vs. O/SKR challenge Homologous

2/5

5/5

0/5

3/5

2/5

There was no significant difference between the two vaccine groups in the levels of RNA detected in the samples (P>0.05) There was a significant difference between the vaccine groups and the control groups (P<0.05)

NSP sero-conversion

3.47

All cattle, 5-7 dpc

7.94

4-7 dpc One 1/4 dose: no conversion One full dose: transient reaction

Saliva swabs Mean at 1 dpc significantly lower in the O/SKR/2010 full dose group compared to all other groups RNA was less frequently detected in group that was vaccinated with ¼ dose of O/SKR/2010

Comparing O1 Manisa and O/SKR/2010 in pigs against challenge with O/SKR/2010

Testing the early protection of O1 Manisa vaccine against challenge with O/SKR/2010 Two groups of 10 cattle each, full dose >6PD50 vaccine Challenged IDL: 10 000 BID50 Challenged 4 dpv: 0% protection Challenged 7 dpv: 50% protection R.H.

7 dpv

4 dpv

Lesions (10dpc) L.H R.F.

L.F.

R.H.

+ +

Full dose O1 Manisa vaccine

L.F.

Full dose O/SKR/2010 vaccine

Challenged with 103 TCID50 O/SKR/2010, heel bulb 21 DPV

Unvaccinated controls

316

Testing O1 Manisa vaccine against O/VIT/2010 in pigs Serotype O (Mya98 lineage) is predominant in SEA Vaccinate pigs with high potency vaccine (>6PD50) Three groups of 5 pigs each, challenged with 105 TCID50, pig derived virus via heel bulb Full dose O1 Manisa vaccine

Vaccinated 7 days prior to challenge Vaccinated 4 days prior to challenge Unvaccinated pigs

Full dose O/SKR/2010 vaccine Heterologous challenge

Homologous challenge

# with VNT titres at 21 dpv

3/5

5/5

# protected

1/5

3/5

Viraemia

4/5 viraemic at 1 dpc (5th pig neg)

4/5 viraemic 2-5 dpc

Saliva swabs

Saliva positive 1-3 dpc

3-10 dpc

Nasal swabs

Nasal swabs positive 2-3 dpc

2-10 dpc

Groups

Testing vaccine efficacy of O1 Manisa against challenge with O/SKR/2010 in sheep

Protection RNA nasal swabs RNA in saliva

Sero-conversion to NSP

7 dpv

80%

1-14 dpc

2/5 transient (neg by 14 dpc)

4 dpv

60%

2-14 dpc

20%

Controls

1-14 dpc

100%

Testing vaccine efficacy of O1 Manisa against challenge with O/SKR/2010 in sheep Direct coronary band challenge: 106 BID50 (talk and poster by N. Singanallur)

Two different routes of challenge Direct coronary band injection Contact with infected sheep

Contact with infected sheep (talk and poster by J. Horsington) All sheep were clinically protected 4 dpv No RNA detected in swabs and no sero-conversion to NSP 317

Recommended dose 1ml

7 dpv

0% protection

Recommended dose 1ml

14 dpc

57% protection

Double dose 2ml

7 dpv

43% protection

Double dose 2 ml

14 dpv

57% protection

Summary

Cattle

Vaccine efficacy and pathogenesis

O1 Manisa protected cattle against challenge with O/SKR/2010 at full dose 21 dpv, but provided only partial protection 7 dpv Vaccination did not prevent virus replication when challenged at 21 dpv

Differences observed in virulence of various FMDV isolates Vaccination decreased the amount of virus excreted Vaccine efficacy is dependant on the challenge strains even within lineages O/SKR/2010 is a concern if introduced into pigs in Australia

Pigs Neither O1 Manisa nor O/SKR fully protected pigs against challenge with O/SKR at 21 dpv (O1 Manisa protected 20% O/SKR/2010 protected 60%) O1 Manisa was partially protective 4 and 7 dpv against challenge with O/VIT/2010 (60 and 80% protection respectively)

South Korea experience showed that other risk measures are equally important Caused clinical disease in sheep

Sheep Route of challenge resulted in different results Contact challenge - 100% protection 4 dpc Coronary band challenge - 2ml vaccine at 14 dpv not sufficient to protect all sheep

NAVETCO

Different routes of challenge lead to differences in vaccine protection outcomes

Pirbright Institute

Regional Animal Health Office 6 Coordinación de Virología, DILAB-SENASA Merial Plum Island Animal Diseases Centre

Kevin de Witte - AHA Johann Schroder - MLA

Simultaneous immunization of cattle with FMD and live anthrax vaccines

National Centre for Foreign Animal Diseases

Thank you

Alejandra Capozzo

Head of the Applied Veterinary Immunology Lab. Institute of Virology, Buenos Aires. Argentina

BIOSECURITY FLAGSHIP

318

In collaboration with: Universidad del Centro de la Provincia de Buenos Aires 1

FMD IN ARGENTINA

Authors Juan Lahore Myrian Trotta Nancy Cardoso Osvaldo Melucci María Catena Fernando Fernández Mariano Pérez Filgueira Alejandra Capozzo

EPIDEMIOLOGICAL STATUS: - Free with vaccination

ARGENTINA

- Free without vaccination

OIL-based vaccines, local producers Four strains: O1/Campos, A24/Cruzeiro, A/Arg/01 and C3 Indaial

VACCINATION CAMPAIGN: - Adults: once a year

- Calves <2 years old: twice a year 2

Bovine anthrax: a global issue

FMD vaccination in Argentina SCENARIO: Compulsory and rigorously controlled FMD vaccination campaign Vaccine efficacy as well as surveillance of vaccine immunity is performed by serology. Antibody titers obtained with Liquid Phase Blocking ELISA (LPBE) have

Hyper-Endemic-epidemic

been correlated to in vivo protection to assess vaccine potency and herd immunity (EPP 75%). 4

319

Endemic

Free

Sporadic

Unknown

Bovine Anthrax

Vaccination against bovine anthrax

Buenos Aires Province

Sterne-strain live anthrax vaccine Applied exclusively to adult animals Boosters needed annually

1977-2013

Adult cattle population has received at least three FMD vaccinations Buenos Aires Province represents 32% of farming land and 28% of the livestock stock: 49% of the farms have had at least one outbreak of bovine Bacillus Anthracis between 1977 and 2013 6

Combining FMD with other vaccines: background

Why to combine vaccination schedules? FMD-vaccines are applied to the whole cattle population on fixed schedules

Immunization of young calves (sc) against FMD and against infectious bovine IBR- adhenovirus/PI-3 (in) did not interfere with the serological response against the FMD (de Clercq et al., 1989). Interference between FMD-V and a VSV-live vaccine

The combination or co-administration of vaccines together with the FMD vaccine appears as a practical and efficient option for immunizing livestock, as long as this practice does not interfere with the immunogenicity conferred by those vaccines applied.

(Castaneda et al., 1976).

The simultaneous application of FMD vaccines, particularly with live vaccines, needs to be evaluated.

320

Experimental design

Current and novel assays

LPB-ELISA titers: 3.97 3.96

n=16

Aimed to evaluate different parameters of anti FMDV specific Abs

n=16

INFORMATION

LPB-ELISA

Total Abs

VNT

Neutralizing Abs

Avidity ELISA

Avidity index of Abs

IgG1 ELISA

IgG1 titer

IgG1/IgG2 ELISA

Ratio between IgG1 and IgG2

CURRENT

FMD-V + ABV

FMD-V

NAME

(each side of the neck)

Serum samples obtained at 0, 25, 55 and 90 days post-vaccination

NOVEL

SAFETY FIRST! no adverse effects were observed along the whole experiment

Total specific antibodies A O1/Campos

FMD-V 5

Total specific antibodies

FMD-V + ABV #

A O1/Campos

FMD-V 5

p=0.004

FMD-V + ABV 6

FMD-V + ABV *#

25 DPV

p=0.019

3 2

EPP=75% 0

100

DPV

A24/Cruzeiro B

0 26/11/13

100

DPV B A24/Cruzeiro

25 DPV

28/02/14

FMD-V

21/12/13

p=0.003

5 &

EPP=75%2

21/12/13

FMD-V + BAV

4 2

3 0

26/11/13 0

EPP=75% 0

DPV

21/12/13 25

DPV

100 12

321

EPP=75% 0

100

DPV 13

Avidity of specific antibodies

IgG isotypes induced by vaccination

100

5 4

IgG1

FMD-V

20 0

FMD-V + ABV

2 1

0 0

100

DPV

100

FMD-V

FMD-V + ABV

100

FMD-V

FMD-V + ABV p=0.035

6 5 4

IgG2

3 2

20 0

1 0

100

FMD-V

DPV 0

FMD-V + ABV Vaccine

100

DPV

25 DPV

Beef comsuption in Argentina is over 60 Kg/person/year

CONCLUSIONS

FMD

The Sterne-strain live anthrax vaccine can be coadministered with an oil-based tetravalent FMD vaccine in adult cattle, producing no impact in the humoral responses against FMDV booster responses. This is the first study on the FMD responses elicited in cattle simultaneously immunized with FMD and anthrax vaccines. The information provided here can be particularly useful in endemic regions with compulsory FMD vaccine programs

THANK YOU

322

The Applied Veterinary Immunology Lab. INTA- Buenos Aires, ARGENTINA 17

Study Design Study Aim: To test if vaccination 4 days prior to challenge with high potency O1 Manisa (ME-SA) vaccine will protect sheep from heterologous challenge with O/SKR/2010 (SEA) virus

24 x Rideau Arcott/Ile de France sheep, 6 12 m/o

Early Protection in Sheep Against Heterologous Challenge with Serotype O Foot-and-mouth Disease Virus Using High Potency Vaccine

6 rooms

2 donor and 2 contact sheep per room direct-contact transmission 4 rooms with vaccinated sheep 2 rooms with control sheep

Jacquelyn Horsington1, Zhidong Zhang2, Soren Alexandersen2, Nagendra Singanallur1, Wilna Vosloo1 1Australian 2National

Animal Health Laboratory-CSIRO, Geelong, Australia Centres for Animal Disease, Winnipeg, Canada

BIOSECURITY Flagship www.csiro.au

Days post infection -1

Donors

Viraemia in 10 of 12 sheep Nasal and oral swabs positive in most sheep, mainly early in infection Probangs positive in 10 of 12 sheep

10/12 0/8 2/4

In 6 sheep at 28 and/or 35 dpi

Sheep 9 not infected?

Antibody Response (ELISA) Vaccinated

Unvaccinated Controls

Structural Proteins

11/12

8/8

3/4

Non-structural Proteins

11/12

0/8

2/4

Oral Swab

Donors

323

Probang

Donors Vaccinated Unvaccinated Controls

Nasal Swab

Clinical Signs

Blood

FMDV RNA

Sheep 9 Sheep 10 Sheep 11 Sheep 12 Sheep 13 Sheep 14 Sheep 15 Sheep 16 Sheep 21 Sheep 22 Sheep 23 Sheep 24 Sheep 9 Sheep 10 Sheep 11 Sheep 12 Sheep 13 Sheep 14 Sheep 15 Sheep 16 Sheep 21 Sheep 22 Sheep 23 Sheep 24 Sheep 9 Sheep 10 Sheep 11 Sheep 12 Sheep 13 Sheep 14 Sheep 15 Sheep 16 Sheep 21 Sheep 22 Sheep 23 Sheep 24 Sheep 9 Sheep 10 Sheep 11 Sheep 12 Sheep 13 Sheep 14 Sheep 15 Sheep 16 Sheep 21 Sheep 22 Sheep 23 Sheep 24

10 14 21 28 35

FMDV RNA

Donors

Days post contact

Unvaccinated Controls

7 10 14 21 28 35

Viraemia in 10 of 12 sheep Nasal and oral swabs positive in most sheep, mainly early in infection Probangs positive in 10 of 12 sheep

Sheep 18 Sheep 19 Sheep 20 Sheep 17

In 6 sheep at 28 and/or 35 dpi

Sheep 18

Sheep 9 not infected?

Sheep 19 Sheep 20

Unvaccinated Controls

Probang

Oral Swab

Sheep 17

Viraemia only detected in sheep 19, 4 dpc Nasal and oral swabs 3 of 4 sheep positive Probangs positive in 2 of 4 sheep Up to 35 dpc

Sheep 18

Viraemia only detected in sheep 19, 4 dpc Nasal and oral swabs 3 of 4 sheep positive Probangs positive in 2 of 4 sheep

Sheep 19 Sheep 20 Sheep 17 Sheep 18 Sheep 19 Sheep 20

Up to 35 dpc

Vaccinated Viral RNA was not detected in any sample from the vaccinated sheep

Nasal Swab

In 6 sheep at 28 and/or 35 dpi

Sheep 17

Oral Swab

Blood

Sheep 9 not infected?

Nasal Swab

-8 -1

Viraemia in 10 of 12 sheep Nasal and oral swabs positive in most sheep, mainly early in infection Probangs positive in 10 of 12 sheep

Probang

Days post contact

Blood

-8 -1

10 14 21 28 35

Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6 Sheep 7 Sheep 8 Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6 Sheep 7 Sheep 8 Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6 Sheep 7 Sheep 8 Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6 Sheep 7 Sheep 8

Two exceptions - Sheep 4 Day 6 NS and 7 Day 4 NS

Thank you

Summary

Acknowledgements

Vaccination with high potency O1 Manisa monovalent vaccine is effective at protecting sheep from challenge with FMDV O/SKR/2010 as early as 4 dpv using a directcontact challenge model

Charles Nfon Kate Hole Hilary Bittner Melissa Goolia Tim Salo

FMD Risk Management Project Jacquelyn Horsington (PhD, MSc, BSc) Research Scientist t +61 3 5227 5127 e jacquelyn.horsington@csiro.au w www.csiro.au BIOSECURITY FLAGSHIP

324

Kurtis Swekla Jaime Bernstein Margaret Forbes Marlee Phair Cory Nakamura

Overview on the performance of FMD vaccines in South America: a manufacturer perspective Otto Mozzer, PhD Director Technology VALLÉE S/A - BRAZIL

325

Defining Requirements and Execution of International Field Trials for Next Generation FMD Vaccines and Diagnostics

Overview Introduction to DHS S&T

M. Colby1, P. Hullinger, T. Beckham, and D. Brake

Overview of DHS S&T Vaccine and Diagnostic projects

EU FMD Open Session 31 Oct 2014

Overview of approaches to proposed International Field Trials for FMD Vaccines and Diagnostics

1Michelle

Colby, DVM, MS Branch Chief, Agricultural Defense Chemical and Biological Defense Division Homeland Security Advanced Research Projects Agency Science and Technology Directorate

Agricultural Defense Mission The Agricultural Defense mission is to enhance current capabilities and develop state-of-the-art countermeasures for high-priority foreign animal diseases (FAD). This includes near- and long-term research and development for vaccines and diagnostics, in coordination with internal and external stakeholders. HSPD-9 Paragraph 23: The Secretaries of [DHS, USDA, of Science and Technology Policy, will accelerate and expand development of current and new countermeasures against the intentional introduction or natural occurrence of catastrophic animal, plant, and zoonotic diseases. The Secretary of Homeland Security will coordinate these activities.

326

FAD Vaccines and Diagnostics Project

the entire outbreak spectrum

FMD Vaccines (initiated 2005) Tools to support planning and response, drive requirements for countermeasures development and inform postoutbreak response activities by creating scalable (local to national) simulation and modeling tools to analyze potential responses and control options to minimize FAD spread.

Vaccines (VX) to rapidly prevent disease in healthy animals prevents disease spread among healthy herd, maintaining business continuity.

Near term - Enhanced characterization and import permits -manufactured, inactivated FMD vaccines *Import permit July 2011 Agricultural Screening Tools (AST) to verify disease free status so uninfected animals and products can continue to move maintaining business continuity w/in U.S.

Mid term - New serotype- and subtype-specific, marked, molecular vaccines (pipeline) *Conditional License May 2012 Long term - Broad spectrum countermeasures: multi/panvalent vaccines, biotherapeutics and immunomodulators *Ongoing Projects

Enhanced Passive Surveillance (EPS) includes diagnostic tests, surveillance tools and data integration procedures to identify infected animals prior to overt symptoms and improve our ability to detect diseases that threaten the U.S. agricultural critical infrastructure.

Diagnostics (DX) to distinguish vaccinated from infected animals may allow more rapid return to trade status, enhancing business continuity and minimizing economic impact. High throughput diagnostics (DX) allow more rapid confirmation of disease status and increased sample processing capabilities enhancing our ability to contain outbreaks.

Countermeasures for Other FAD

Develop more effective vaccines and diagnostic countermeasures for high priority FADs, in partnership with the USDA and industry

Livestock Decontamination, Disposal and Depopulation (3D): New methodologies and decision support tools for depopulation, disposal and decontamination that facilitate rapid response and prevent disease spread in a manner that minimizes waste, environmental impact and negative public perception.

Prioritized agents identified by key customer (USDA-APHIS Emergency Management NVS) and interagency working groups (FADT) Includes near term, mid term and long term R&D funding projects for African Swine Fever and Classical Swine Fever

Diagnostics Differentiating infected from vaccinated animals (DIVA) companion assays for vaccines in development 6

FMD Virus Vaccine, Serotype A24, Live Adenovirus Vector (USDA Code 1FM1.R0)

Ad5FMD Vaccine Candidate Pipeline

Conditional license granted for use in cattle on May 31, 2012 Recently renewed to May 6, 2016 Met the expiration potency after three years in storage at -18+/-5oC.

Included U.S. field safety study with 500 dairy and beef cattle Included pregnant dairy

14 AdFMD monovalent vaccine candidates efficacy tested

9 AdFMD vaccine candidates have been identified for master seed virus production 5 MSVs completed to date (CVB approvals in process)

AdFMD candidates for 6 of 7 major serotypes Multiple, serotype A candidates Multiple serotype O candidates Includes vaccine candidates for most recent outbreaks in S. Korea (O/South Korea/2010) and Egypt (SAT2/Egypt/2012)

Monovalent vaccine Public-private partnership between DHS, USDA and industry Replication deficient viral vector Does not contain full FMDv genome, so can be produced at BSL-2 Recent PIADC studies Cattle - Demonstrated 6 mo. duration of immunity(experimental challenge) Swine - Demonstrated proof-of-concept efficacy (experimental challenge at 2 7 weeks post-vaccination)

327

AdFMD Construction In Progress

Pre-Master Seeds Made/Tested

4 Master Seeds Planned

5 Master Seeds Made

3 Master Seeds Submitted for Approval

Additional FMD DIVA Vaccine Platform Research Investments

3B Foot and Mouth Disease ELISA Kit

Replicon Particle (Harrisvaccines; SirraVax Collaborative project

swine vaccine products (influenza, PRRS, PEDV) Only veterinary platform that currently qualifies as a CVB conditionally licensed products (VS Memo 800.213) costs and timelines to licensure

Strong academic (Texas A&M) and government (USDA APHIS and ARS) partnership

significantly reduces R&D

Leveraging diagnostic industry (BIOO, VMRD) fee-for-service expertise

Single dose FMD vaccine candidate shown to be 100% efficacious in both cattle and swine proof-of-concept efficacy studies (experimental challenge at 2 weeks postvaccination

Develop a new FMD serology Dx test based on the FMDV non-structural protein 3B with performance equivalent to/better than commercially available diagnostic assays used for distinguishing vaccinated from infected animals

Modified Vaccinia Ankara (Bavarian-Nordic; MVA-BN) )

Develop a DIVA test to differentiate cattle vaccinated with next generation FMD molecular vaccines

Replication deficient in livestock 2-dose FMD vaccine candidate shown to be 100% efficacious in recently completed cattle proof-of-concept study (experimental challenge at 21 days post-boost)

to policy to differentiate vaccinated from infected animals, based on the detection of antibodies to FMDV non structural proteins (NSP) Shorter assay result time (hours vs. day) Enables manufacturing of kits and reagents in U.S 9

Why International FMD Vaccine Trials? Two-year, multi-herd study to compare DHS-funded Ad5 FMD vaccines and new 3B ELISA test kit to traditional FMD vaccines and diagnostics in an endemic country. Cannot be done in field conditions in the U.S. due to absence of FMD Head-to-head natural challenge with traditional vaccines provides data supporting vaccine The use of DHS-funded 3B ELISA test kit in conjunction with both vaccines provides data to support use in a -tostrategy (vs. stamping out)

OVERVIEW OF APPROACH TO THE PROPOSED INTERNATIONAL FIELD TRIALS FOR FMD VACCINES AND DIAGNOSTICS

Establish relationships with the FAO to facilitate future trials of DHS products in transboundary disease endemic countries. Critical to establishing efficacy in a production setting and promoting commercial potential.

328

Technical Approach and Timeline

Vaccines and Diagnostics Trial Metrics (1)

Objectives: Demonstrate Ad5 vectored FMD vaccine is as effective as traditional killed vaccine Demonstrate effectiveness of Ad5 vectored FMD vaccine in an FMD outbreak/endemic situation Demonstrate process for using vaccine and companion Differentiating Infected from Vaccinated Animals (DIVA) diagnostic in a disease situation Build international partnerships to facilitate future trials in endemic settings 1 - Workshop(s) to identify partner countries and detailed plan for casecontrol study and validating 3ABC DIVA diagnostic assay in endemic countries 2 - Production of additional master seed viruses needed for the bi- or tri-valent vaccine Procurement of required vaccine and diagnostic kits

Year 1 Begin in-country planning for vaccine trial

If vaccine efficacious at 6 months, continue study to 12 and 18 mo. time points to obtain information related to duration of immunity, effect of boostering, and serological response to vaccine

Year 2

Year 3

Begin field study, initial study period of 6 months

Comparable or better performance against traditional killed vaccines in an outbreak/endemic field situation at 6, 12 and 18 months post-vaccination Number of animals diagnosed with FMD clinical disease at 6, 12 and 18 months post-vaccination Number, severity and duration of post-vaccination injection site reactions Serum virus neutralization (SVN) titers to each FMDV fraction in multivalent vaccine at 3 days, 1, 2 and 3 weeks and 3, 6, 12 and 18 months Onset of functional antibody response (mean days to 50%, 80% and > 90% herd seropositivity) Assess viral load and shed by qPCR on sera, nasal swabs and probang samples

Future possibility of additional trials with African or Classical Swine Fever vaccines, or additional FMD vector platforms in other countries

Demonstrate duration of immunity of at least 6 months (threshold), ideally 18 months (objective)

Year 4 Analysis of data and final report

Duration of response (number of animals that require re-vaccination at 6, 12 and 18 months) 13

Vaccine and Diagnostic Trial Approach

Vaccines and Diagnostics Trial Metrics (2) 3B cELISA DIVA assay complementary to Ad5 FMD vaccine

A series of meetings/workshops to establish a common understanding of DHS goals and objectives

Supports a differentiating infected from vaccinated animals strategy (DIVA) Sensitivity and Specificity equivalent or better than commercially available diagnostic assays used for distinguishing vaccinated from infected animals Successful Repeatability and Reproducibility

Discuss logistical considerations and how to best succeed at the execution of an international trial of the FMD Virus Vaccine, Live Adenovirus Vector and the companion 3B ELISA (short term) Establish general recommendations for international field trials

respectively) Cost lower by 33-50% Time points evaluated; 3 days, 1, 2 and 3 weeks and 3, 6, 12 and 18 months

Identify potential field trial locations and partners for current and future collaborations respond to once the study requirements and design are established

*3B is the immunodominant B-cell epitope of the 3ABC recombinant protein

329

Overarching Goal Identify and foster the development of collaborative opportunities for international partnerships to further advance the state of the art for FAD vaccines and diagnostics

If interested in additional information please contact;

Build enduring partnerships for the future

Michelle Colby, DVM, MS Branch Chief, Agricultural Defense Chemical and Biological Defense Division Homeland Security Advanced Research Projects Agency Science and Technology Directorate Michelle.Colby@HQ.DHS.GOV 17

FMD Virus Vaccine, Serotype A24, Live Adenovirus Vector: USDA CVB Licensing Studies (Safety Related)

Back-up

1. 10X dose (calf) Acceptable - no local or systemic reactions

2. Backpassage/Reversion to Virulence (cattle) Inoculation of master seed virus (MSV) into 10 cattle showed no reversion to virulence Only a small amount of vaccine virus inoculum was isolated from nasal swabs at 1-2 days post-administration in 4/10 animals No amplification of vaccine virus was evident

3. Shed-spread (cattle, swine)

330

MSV could not be isolated from either 10 cattle or 10 swine after intramuscular administration. Naive cattle and swine co-mingled with vaccinated cattle or swine do not seroconvert to adenovirus or FMD seroconvert. Portion of study published in Grubman, MJ, et. al. 2010. Future Virol. 5(1):51-64. 20

FMD Virus Vaccine, Serotype A24, Live Adenovirus Vector: USDA CVB Licensing Studies (Safety Related)

5. Supplemental Safety Studies

4. Field Safety (beef, dairy)

Milk safety

500 total

10 lactating cows and nursing calves; milk collected daily on days 0-4 and days 7, 10, and 14 None of the milk samples from any of the vaccinated cattle tested positive for the adenovirus vector Study conclusion: Vaccine is not shed in the mammary tissue of cattle after IM inoculation

4 geographic, commercial sites Both sexes Young (19-22 weeks) and older (> 2yrs) Includes 100 lactating dairy; 58 pregnant (all trimesters)

96% experienced no recorded adverse events of any kind during the 3 week post-vaccination observation period. In the remaining 4%, there were no recorded instances of serious, permanent or systemic adverse reaction attributable to vaccination. Study conclusion: Vaccine is safe for use in healthy cattle when used per proposed label recommendations

Vaccine biodistribution and persistence in cattle Very low level of vaccine vector detected at inoculation site and draining lymph node up to 72 hrs postinoculation Results published: Montiel, NA. et. al. 2013. Vet. Immunol. Immunopathol. 151(1-2):37-48 Montiel, NA. et al. 2012. Vaccine. 30(9):1690-1701. 22

3B Development

FMD Virus Vaccine, Serotype A24, Live Adenovirus Vector: USDA CVB Licensing Studies (Efficacy Related) Minimum Protective Dose (MPD) Studies:

Good separation of positive and negative results

14 days post single dose vaccination (n=34 vaccinates) 94 % SVN positive 97% protection against generalized disease 91% protection against plasma viremia (VI, rRT-PCR)

Enables diagnostic serological testing of FMDV susceptible species

7 days post single dose vaccination (n=10 vaccinates) 50% SVN positive 100% protection against generalized disease 90% protection against plasma virema (VI, rRT-PCR)

Enables U.S. manufacturing and production of critical reagent for protecting the homeland

Supplemental Information: 100% (12/12) protected against generalized disease and viremia following direct contact challenge at 1 week post-single dose vaccination Grubman, MJ, et. al. 2010. Future Virol. 5(1):51-64.

88% (91/104) of the vaccinated animals tested from the Field Safety Study had positive FMDV SVN titers (>0.9 log10) at approximately 12 months postsingle dose vaccine administration 23

331

i.e. PrioCHECK® FMDV NS test (Thermo Fisher Scientific; formerly Prionics) is not currently licensed by USDA CVB for import, distribution, and use

BACKGROUND FDCs and FMDV: FMDV genome and protein were detected in the light zone in germinal centres (GCs) of lymphoid tissues and maybe associated with FDC [Cattle, sheep and buffalo] *

Follicular dendritic cells: a key player in the pathogenesis of foot-and-mouth disease virus?

PROJECT AIM Using mouse as a model: Assess the FDC network, FMDV antigen retention and T-dependent immune responses in the generation and maintenance of long-term antibody responses to FMDV infection and vaccination, using recombinant -Ig fusion protein treatment

Mohammed H. A. Doudo PhD Student Department of Veterinary Medicine - University of Cambridge Viral Immunology Group - The Pirbright institute

* Juleff et al, 2008

Determine the impact of depletion of FDC on the response to FMDV and in the maintenance of longterm antibody responses

CONCLUSIONS We have detected FMDV genome and capsid proteins in GCs of mouse spleen in association with FDCs up to 46 days post-infection No infectious FMDV was recovered from the purified FDC populations FDCs may play a role in the persistence and pathogenesis of FMDV

Hu-Ig treated (+ve)

LTBR-Ig treated

Naïve

332

ACKNOWLEDGMENTS Viral immunology group (Pirbright) James Wood (Cambridge) Bryan Charleston (Pirbright) Miriam Windsor Eric Lefevre Eva Perez-Martin (Pirbright) Julian Seago Donald King (Pirbright) Marti Cortey Nicholas Juleff (Pirbright)* Clare Grant Veronica carr Fuquan Zhang Liz Reid Animal Isolation Ben Jackson Supervisors

Unit (Pirbright) Paul Smith Dave Selby Beckie Evans

THANK YOU

Ryan Waters Graham Belsham Francisco J. Salguero John Bashiruddin Valerie Mioulet Antonello Di-Nardo Pip Hamblin Jennifer Simpson CSU Staff

Development of a Novel, Recombinant, Potent, and Safe DIVA Vaccine for Foot and Mouth Disease Virus (FMDV)

HAVE A LOOK AT OUR POSTER...

Shela Aman, Diya Raghavan, and Masarrat Ali* Alpha Diagnostic Intl. Inc., San Antonio, Texas, USA

www.4adi.com

FMDV Vaccines (Whole Virus/Inactivated) Virus Culture - BSL3 facility Expensive and tedious Safety issues Short term immunity Weak vaccines Presence of NSPs in vaccines No reliable DIVA tests

www.4adi.com

333

www.4adi.com

Recombinant FMDO VP1 - Tetanus Toxoid Vaccine

TeLC alone or VP1-TeLC produced high titer antibodies in rabbits

His-tag

FMDO VP1 (213aa) Weak antigen & Weak vaccine

Tetanus Toxin (1-457aa): Most lethal toxin and super antigen

VP1-TeLC produced 400% more antibodies than FMDVX vaccine (killed virus) in rabbits

-437aa): Non-Toxic and super antigen Recombinant VP1-TeLC Designer Protein Non-Toxic and Highly Effective FMD+Tetanus Vaccine TeLC antibodies also serve as DIVA test

1=VP1 ~25 Kda

3=TeLC ~52Kda

4=VP1-TeLC ~75Kda

www.4adi.com

334

ARALLEL FMD future: responding to change in the global landscape

335

Background Rapid and accurate detection of FMDV Disease control policy

Effective control

The level of detection

Development of probe-based real time RTPCR assays for detection and serotyping of FMDVs circulating in West EurAsia

specific tests

Syed M. Jamal & Graham J. Belsham

Region specific ELISA kits for serotyping

Stamping out vs. vaccination

Heterogeneity of FMDVs in different areas

Department of Biotechnology, University of Malakand, Pakistan National Veterinary Institute, Lindholm, DTU, Denmark

geographical region

Region specific rRT-PCR assays 1

Available rRT-PCR Assays

Strategies for rRT-PCR assays

PanFMDV rRT-PCR assays: Reid et al., 2002 Serotype-specific Assays

Callahan et al., 2002

Serotype-specific rRT-PCR assays: Region specific

5 UTR

VP4

Ahmed et al., 2012 SAT2 (G-VII)

VPg

IRES

VP2

VP3

VP1

3 UTR

Reid et al., 2014 (O, A, Asia1; Middle East) Reid et al., 2002

Bachanek-Bankowska et al., 2014 (O, A, SAT1, SAT2; East Africa) Knowles et al., O/Ind-2001 (in press)

Callahan et al., 2002 Pan FMDV Assays

336

Results: Diagnostic sensitivity and specificity of serotype A specific rRT-PCR assay

Methodology

FMDV sequences generated from samples from West Eurasian region were aligned to design serotype-specific primers and TaqMan probes. Assays were evaluated using field samples originated from Pakistan, Afghanistan, (Iran & Bulgaria, O-ANT10), belonging to different sublineages of O-PanAsia, A-Iran05 and Asia 1 (Group II and VII/Sindh-08) Samples were analysed, using primers and probes as described by Reid et

The Ct values obtained were compared.

4 8 9 1495 2813 2816 1364 2809 P/P

-------------------------------------------------------------------------------------------------------------------------A------------------A-------------------------------

----------------------------------------------------------------------------------------------------------------A-----------------A-------------ACGACCATCCACGAGCTYC Forward

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

------------------------------------------------------------T-----------T----------------------------------------------------------------------------CGTGCGCATGAAACGTGCCG Probe

As-Reid et al

No Ct

18.4

No Ct

1576

17.8

16.6

No Ct

19.1

17.9

No Ct

1430

20.1

No Ct

21.5

20.3

No Ct

1428

21.3

23.5

No Ct

24.5

23.6

No Ct

1411

21.7

No Ct

23.4

No Ct

1491

22.1

22.9

No Ct

24.6

No Ct

2808

30.8

No Ct

29.5

No Ct

1355

31.2

No Ct

30.8

29.7

No Ct

2819

27.7

30.5

No Ct

31.7

31.4

No Ct

3641

19.8

18.8

No Ct

20.7

No Ct

I-711

18.6

17.2

No Ct

19.9

19.1

No Ct

1421

19.6

18.9

No Ct

19.2

No Ct

629

26.5

No Ct

26.7

25.4

No Ct

21.9

27.8

No Ct

24.4

33.1

No Ct

23.2

28.8

No Ct

25.7

34.2

No Ct

693

25.1

27.8

No Ct

28.8

27.9

No Ct

1435

22.7

24.5

No Ct

27.1

26.4

No Ct

1486

28.5

26.9

No Ct

28.1

28.5

No Ct

1495

19.3

No Ct

22.8

No Ct

2813

21.8

27.2

No Ct

33.6

No Ct

2816

26.4

27.5

No Ct

28.6

36.8

No Ct

1364

31.4

33.4

No Ct

2809

25.1

28.6

No Ct

A22

32.3

37.1

No Ct

----------------------------------------------------------------------C------------------------------------------------------G---A--A-A----------G---A--A-A--------------------------

---T -C--------C------T -C--------C------T -C--------C----------------------------C -T -A------------C -T -A------T-----C--T-----A---T-----C--T-----A---CTACTGTCCCAGGCCTCTGC Reverse (c)

O-Reid et AA-Reid Asia1al Jamal et al Jamal

17.3

Diagnostic sensitivity and specificity of serotype O specific rRT-PCR assay

Type A FMDVs and Reid et al., 2014 primers/probe having differences in Ct values between the two assays 4 8 9 1495 2813 2816 1364 2809 P/P

5` UTR O- Jamal

1575

337

5`UTR O-Jamal

O-Reid et al

A-Jamal

A-Reid et al

Asia-1 Jamal

As-Reid et al

1347

22.47

22.45

24.16

33.17

No Ct

1346

23.44

25.26

25.54

34.92

No Ct

1343

35.13

36.42

35.99

No Ct

37.06

No Ct

1413

22.66

24.26

25.81

34.27

No Ct

1497

20.65

20.4

21.96

30.83

No Ct

1494

26.08

28.59

29.98

38.97

No Ct

1499

21.73

23.23

23.53

32.25

No Ct

1412

21.15

23.63

25.24

29.67

No Ct

643

30.19

32.61

31.85

38.13

No Ct

3B-1-1

21.65

31.81

23.74

26.26

No Ct

3B-3-1

19.83

30.15

22.56

No Ct

Nzm-5

18.36

18.28

20.46

22.52

No Ct

1370

25.99

24.36

25.06

25.09

No Ct

1414

24.44

27.03

26.4

27.3

No Ct

1417

29.82

32.18

32.24

32.65

No Ct

2826

35.24

29.39

28.62

28.72

No Ct

I-709

28.17

17.91

18.36

No Ct

(38.2)

Diagnostic sensitivity and specificity of serotype Asia-1 specific rRT-PCR assay

Type O FMDVs and Reid et al., 2014 primers/probe having differences in Ct values between the two assays 1347 1343 1346 1412 1413 643 14941 497 1499 BUL1 BUL3 Nzm5 P/P

-A-------------------A-------------------A-------------------A-------------------A-------------------A-------------------A-------------------A-------------------A------------------------------------------------------------------------------CCGAGACAGCGTTGGATAACA Forward

---G-----G----------------G-----G----------------G-----G----------------G-----G----------------G-----G----------------G-----G----------------G-----G----------------G-----G----------------G-----G-------------T--T-------------------T--T-------------------T--T-------------------CCGACTTGCACTGCCTTACACGGC Probe

--------G-T--------C---------G-T--------C---------G-T--------C-----------T--------C---------G-T--------C-----------T--------C---------G-T--------C---------G-T--------C---------G-T--------C---------G----------CA--------G----------CA--------G----------C-ACAACGGGAACTGCAAGTATGG

5`UTR O-Jamal

O-Reid A-Reid Asia-1 As-Reid et al A-Jamal et al Jamal et al

639

26.4

30.6

No Ct

30.6

29.0

590

17.8

18.1

No Ct

23.5

21.0

30.3

34.3

No Ct

34.6

32.3

2824

21.7

26.4

No Ct

26.4

26.1

2823

18.8

22.7

No Ct

22.7

22.6

2825

27.6

30.1

No Ct

28.8

29.0

8 (A/As)

25.5

26.8

No Ct

34.84

No Ct

25.6

24.6

2810

28.2

35.1

No Ct

31.1

32.8

No significant difference was noted between the CT values obtained with the two serotype-specific assays.

Reverse (c)

Sample No. 8, also positive for serotype A, could be tested positive for type A in the second generation assay but negative in the first generation assay.

Conclusions We developed serotype-specific rRT-PCR assays for detection and serotyping of FMDVs circulating in West EurAsian region (pool 3 viruses) using primers and probes designed for the VP1 coding region.

All the three primers/probe sets detected RNAs from homotypic viruses

The second generation serotype-specific assay has some advantages compared to the first generation assay in terms of the sensitivity and specificity for viruses in West Eurasia.

Comparable Ct values with serotype-independent assays Lower or comparable Ct values with serotype-specific assays by

The second generation assays can help in early detection and typing of FMDVs circulating in West Eurasia.

Reid et al., 2014 No cross reactivity with heterotypic viruses circulating in the region.

338

Acknowledgements

Thanks for your attention EUFMD and interest of Keith Sumption Giancarlo Ferrari, Project Leader, GTFS/907/ITA Tina Frederiksen Preben Normann

Context FMD is endemic in West Africa: Continuous FMDV circulation Lack of diagnostic capacities & under-reporting: Lack of information on FMD dynamics

DEVELOPMENT AND EVALUATION OF A MULTIPLEX CONVENTIONAL RT-PCR FOR SIMULTANEOUS DETECTION AND TYPING OF FMDV IN WEST AFRICA

Difficulties for implementation of control strategies in this sub-region and analysis of risk of FMDV incursion

Kamila Gorna & Sandra Blaise-Boisseau

Knowledge required on circulating strains in WA

ANSES, Animal Health Laboratory Virology Unit (ANSES-INRA-ENVA joint research unit)

339

Context

Context RT-PCR method: Pool 3 O, A, Asia1 Pool 2 O, A, Asia1

Reliable, fast, sensitive

Pool 1 O, A, Asia1

Pool 5 O, A, SAT1-2

Allows detection from a wide range of clinical samples Implemented in most laboratories

Pool 4 O, A, SAT1-2

Pool 7 O, A

Pool 6 SAT1-2-3

Modified from: Paton et al., 2009, pool of serotypes according to the final report EUFMD (August 2014)

Four serotypes reported in the last few decades: O, A, SAT1 & SAT2 i.e. pool 5 3

Aim

Primers design and selection

To develop:

Bioinformatics: analysis of available FMDV sequences from WA P1/P2 alignment

a one-step multiplex conventional RT-PCR for simultaneous detection & typing of FMDV circulating in West Africa

Pan-FMDV primers (target:3D gene) Primers specific for each serotype (target: VP1) O typing

Pan-FMDV

A typing SAT1 typing SAT2 typing

340

internal control ( -actin gene) 6

Primers design and selection

Description of the test

Expected amplicon sizes (100-600 bp, 2% agarose gel) 600bp

Type A

550bp

Type O

400bp

Type SAT1

250bp

Type SAT2

186bp 100bp

-actin 3D

Sample treatment

Set of primers (Type A) Set of primers (Type O)

RNA Extraction

Multiplex RT-PCR

Electrophoresis Interpretation

Set of primers (Type SAT1) Set of primers (Type SAT2) Set of primers (B-actin / sample quality CTRL) Set of primers (Pan-FMDV, 3D target)

Development steps

6 plex RT-PCR Prototype (O/A/SAT1/SAT2/3D/ -actin) Developed using reference strains & field isolates

Primer sets assessed in simplex

A O SAT1 SAT2

Optimisation of gel electrophoresis conditions

-actin 3D

-PCR protocols 6plex prototype

1- O/BEN/1/2010 2- O/BEN/26/2010 3- A/BEN/19/2010 4- A/BEN/36/2010 5- Negative Epithelium 6- Positive CTRL (O/A/SAT1/SAT2) 7- NTC

341

1- SAT1/KEN/2/2011 2- SAT1 BOT 1/68 3- SAT2/LIB40/2012 4- SAT2/EGY3/2012 5- Negative Epithelium 6- Positive CTRL (O/A/SAT1/SAT2) 7- NTC

Satisfactory results for this prototype on the tested panel 10

6 plex evaluation on a panel of field samples from WA and the corresponding isolates SEROTYPE O

6 plex prototype specificity

Assassed on a larger panel of reference strains or field isolates Strains tested

Typing

7/7

West Africa

2/2

Other topotypes

5/5

8/8

5/8

West Africa

2/2

Other topotypes

6/6

3/6

serotype SAT1 strains (Ken/Bot)

2/2

serotype SAT2 strains (Lib/Egy/Zim)

3/3

serotype SAT3 strain (Zim)

1/1

0/1

serotype C strain

1/1

0/1

serotype Asia strains

2/2

0/2

serotype O strains

serotype A strains

« + » Positive « - » Negative « d » Doubtful

Specific amplification of each serotype Negative samples (n=24) & SVDV strains (n=3): no amplification => OK 11

6 plex evaluation on a panel of field samples from WA and the corresponding isolates SEROTYPE A

Conclusions & Perspectives Development of a 6plex RT-PCR prototype allowing both detection and typing of FMDV in West Africa Satisfactory results on panels tested Flexibility of the test (triplex, 4plex,... as required) What remains to be done ... To test this prototype on a larger panel of WA samples To determine the sensibility of the test To assemble reagents as custom-designed mastermixes To evaluate protocols under field conditions Further developments ... To apply the same approach for the other FMDV pools

« + » Positive « - » Negative « d » Doubtful 13

342

Aknowledgments « BIOPIC » Team

Anthony Relmy

Kamila Gorna

Aurore Romey

Aude Allemandou

Evelyne Houndje

Donald King Nick Knowles

Labib Bakkali Kassimi

Stephan Zientara & All members of the Virology Unit 15

FMDV distribution pattern

Development of tailored specific real-time RT-PCR assays for detection of FMDV serotypes circulating in East Africa Kasia Bankowska, PhD The Pirbright Institute, UK

Vesicular Disease Reference Laboratory Group

343

FMDV RT-PCR target

Serotype-specific RT-PCR: East Africa * putative functions

Protease

VPG

Capsid 1A VP4

1B VP2

Membrane-binding Genome-linked (VPg)

Carboxy-terminal self-cleaving

1C VP3

1D VP1

NTP binding*

Protease

Polymerase

AAA

Poly(C) Primary cleavages

Secondary cleavages

1B/RNA?

(n)

8 kbp

Variable VP1 region target for tailored assays Conserved IRES and 3D regions targets for pan-serotype reactive assays

East Africa Pool 4 Tanzania, Uganda and Kenya FMDV types: O, A, SAT1 and SAT2 3

Primer design

FMDV type O topotype EA-2 & EA-4

VIII IX X

XII

EA-2

EA-4

FMDV type SAT2 topotype IV or VIII IX X XII

344

SAT2/IV/TAN/ SAT2/IV/TAN/ SAT2/IV/TAN/ SAT2/IV/TAN/ SAT2/IV/U267 SAT2/IV/K183 SAT2/IV/KEN/ SAT2/IV/K81/ SAT2/IV/K46/ SAT2/IV/K65/ SAT2/IV/K70/ SAT2/IV/K151 SAT2/IV/KEN/ SAT2/IV/K34/ SAT2/IV/K52/ SAT2/IV/K52/ SAT2/IV/K52/ SAT2/IV/KEN/ SAT2/IV/K37/ SAT2/IV/KEN/ SAT2/IV/KEN/ SAT2/IV/K13/ SAT2/IV/KEN/ SAT2/IV/K37/ SAT2/IV/KEN/ SAT2/IV/KEN/ SAT2/IV/KEN/ SAT2/IV/KEN/ SAT2/IV/KEN/ SAT2/IV/K49/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/K40/ SAT2/IX/KEN/ SAT2/IX/K65/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/K3/9 SAT2/IX/KEN/ SAT2/IX/K14/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/K32/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/K5/9 SAT2/IX/K25/ SAT2/IX/KEN/ SAT2/IX/K37/ SAT2/IX/K39/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/KEN/ SAT2/IX/K77/ SAT2/IX/KEN/ SAT2/IX/UGA/ SAT2/IX/UGA/ SAT2/VIII/RW SAT2/VIII/RW SAT2/VIII/BU SAT2/VIII/RW SAT2/X/Uga_2 SAT2/X/Uga_1 SAT2/X/UGA_B SAT2/X/UGA_B SAT2/X/UGA/1 SAT2/X/UGA/2 SAT2/XII/UGA SAT2/XII/UGA SAT2/XII/UGA

420 430 440 450 460 470 480 490 500 510 520 530 540 550 |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| CGGGTTTCCGCAATCCGCGGTGACCGAGCTGTGCTGGCAGCCAAGTACGCCGACACCAGGCACACGCTTCCGTCCACGTTCAACTTTGGACACGTGACCGCTGACCAACCGGTCGACGTTTACTACAGAATGAAGCGGGCAGAG .....................................................................................................C.......................................... ..........................C......T....................T....A...........A....................T........C.....G.....T..............G...........G... AA....A.......T.....C.....T...........G...............T....A...G.......A..............C.....T........C........A........C........G...........G... .....C....................C......T....G...............T....A...........A....................T........C...A.G..A.................G............... ..A...A...................C......T.................AG.T....A...G.......A...........T.................C..........................G............... ..A...A...................C......T.................AG.T....A...G.......A...........T.................C..........................G...........T... .....C.................T..C......T....................T....A...........A....................T........C.....G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C.....G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A..............C.....T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... .....C.................T..C......T....................T....A...........A....................T........C...A.T..A.................G............... .....C....................T....................T......T................A...........T.................C...A....A........C........G............... .....C.................T..C......T....................T....A...........A....................T........C.....G..A.................G............... .....C.................T..C......T.....A..............T....A...........A....G...............T........C.....G..A.................G............... .....C....................T....................T......T................A...........T.................C...A....A........C........G............... .....C....................C..........................AT................A....................T........C...........T..............G........A...... .....C.................T..C......T....................T....A...........A....................T........C...A.G..A.................G............... ..A..C....................C......T.................AG.T....A...G.......A...........T......T..........C..........................G............... .....C....................C..........................AT................A....................T........C...........T..............G........A...... .....C....................CCTGC......................AT................A....................T........C...........T..............G........A...... .....C....................CA.........................AT................A....................T........C...........T..............G........A...... .....C....................C..........................AT................A....................T........C...........T..............G........A...... .A...C....................T...........................T................A.............................C...A....A........C........G............... ACA...A.......T........T....AG...T....GCAG..A...T.TTC.G...A....T.T..A..A.....C........C..GTTT........C...A.G..A...............C.G........T...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... ACA..CA.......T........TA...AG...T....GCGG..A...T.TTC.G...A....T.T..G..A.....C........C..GTTT........C...G.G..A...............C.G........T...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........G...A.G..A..G............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...C...........C.......A......... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... AA.ACCAT...C..T..T..C..TA.G............CAA.....T..TTC.....AA..TG.T..G..A.....T.....T..C..TTTT........C...A.G..A...............C.......A..C...... ACT.....T..T........A...A....G...T....T..A........GTCTGG..AA.....A..A..C.....C........C...TT......G..A...A....A...A....C......C..........T...... GCT........C........A...A....G...T....T..A........GTCTGG..AA.....A..A..C.....C.....T..C...TT......G..A...G.G..A........C......C..........T...... ACT........C........A...A.G..G..A.....T...........GTC...TC.....G.A..G..T.....C........C..GTTT..A.....C...A....A........C......C.G........T..T... ACT........C..T..T..A...A....G...T....T..A......AG.TCTGGTG.C...G....A..T.....C...........GTTT........C...A.G..A...............C.G........T..C... AAA..CA....C..T..T......A.GCAG..T.....TCAG........GTCA....AA...G..T.G..A..............C...TTT........C........C...............C.G.....A..T...... AAA..CG....C.....T..C...A.GCAG..TT.A..TCAA........GTCGG.T.AA...G..T.A..A..............C..GTTT........C...AGG..A...............C.G.....A..T..G... AAA...A....T..T..T......A.GCAA.....A..TCAG........GTCA....A....G..T.A.................C..GTTT........C...GC...A...............C.G.....A..T..G... A.A...G.T.....T..T..C...A..CAG.C.T.A..TCAG........GTC.CA...A...G..T.G..A..A.....T.....C..TTT.........C...G....A...............C.G.....A..T..T... AA...GA....C.....T..C...A..CAA..T...CAGCAA........GTCG.....A...G..T.G..T.....T........C..GTT.........C...A....A...............C.C.....A..T..T... AA...GA....C.....T..C...A..CAA..T...CAGCAA........GTCG.....A...G..T.G..T.....T........C..GTT.........C...A....A...............C.C.....A..T..T... .CT..GAG.........T..C...A.GCAA.C...C..G..A.........TCTGGT........A..G..C..G..C........C..GTT.........C...A.G..A...............C.G........A..G... .CT..GAG.........T..C...A.GCAA.C...C..G..A..A......TCTGGTG.......A..A..C..G..C........C..GTT.........C...A....A...............C.G........A..G... .CT..GAG...G.....T..C...A.GCAA.C...C..G..A.........TCTGGT........A..G..C..G..C........C..GTT.........C...A.G..A...............C.G........A..G...

Sample name serotype origin topotype O-Ct value A-Ct value SAT1-Ct value SAT2t4-Ct value SAT2 Ct value ETH/04/2013 O T/C EA-4 18.23 23.43 KEN/11/2011 O clinical EA-2 27.51 27.38 KEN/15/2011 O clinical EA-2 31.09 30.81 KEN/16/2011 O clinical EA-2 27.42 27.8 KEN/137/2010 O clinical EA-2 36.53 39.27 23.09 23.27 KEN/145/2010 O clinical EA-2 17.74 18.14 KEN/146/2010 O clinical EA-2 13.84 13.87 37.67 KEN/148/2010 O clinical EA-2 16.55 17.21 KEN/151/2010 O clinical EA-2 23.53 23.49 KEN/152/2010 O clinical EA-2 14.16 14.74 37.98 38 KEN/154/2010 O clinical EA-2 22.01 20.26 KEN/1/2011 O clinical EA-2 16.26 16.41 38.87 KEN/10/2009 O clinical EA-2 35.36 34.58 TAN/39/2012 O clinical EA-2 16.45 16.56 TAN/16/2008 O clinical EA-2 20.95 21.2 TAN/5/2009 O clinical EA-2 15.8 15.69 TAN/49/2013 SAT1 clinical I 13.73 13.81 36.38 36.29 TAN/50/2013 SAT1 clinical I 13.59 13.48 KEN/12/2009 SAT1 clinical I 18.27 18.35 KEN/139/2010 SAT1 clinical I 17.72 17.75 KEN/140/2010 SAT1 clinical I 21.99 21.33 KEN/121/2009 SAT1 clinical I 36.11 37.66 23.19 23.59 KEN/123/2009 SAT1 clinical I 38.31 20.21 19.46 KEN/1/2010 SAT1 clinical I 23.78 23.74 KEN/71/2010 SAT1 clinical I 21.6 20.75 KEN/72/2010 SAT1 clinical I 16.12 16.2 KEN/16/2009 SAT1 clinical I 28.2 29.14 16.44 15.88 KEN/26/2008 SAT1 clinical I 15.46 15.32 KEN/9/2009 SAT1 clinical I 37.46 37.96 17.69 17.25 TAN/01/2013 A T/C Africa/G-I 14.92 14.64 TAN/61/2012 A T/C Africa/G-I 18.83 18.9 TAN/71/2012 A T/C Africa/G-I 16.04 15.93 TAN/42/2009 A clinical Africa/G-I 18.9 18.82 TAN/45/2009 A clinical Africa/G-I 15.74 16.19 KEN/28/2008 A clinical Africa/G-I 18.96 18.37 KEN/22/2009 A clinical Africa/G-I 21.89 21.71 ETH/12/2009 A clinical Africa/G-VII 19.4 19.23 KEN/5/2012 A T/C Africa/G-I 17.1 16.78 KEN/6/2012 A T/C Africa/G-I 14.16 14.68 TAN/64/2012 SAT2 clinical IV 20.22 19.93 KEN/12/2011 SAT2 clinical IV 17.93 17.85 KEN/21/2011 SAT2 clinical IV 35.98 35.21 15.59 15.78 TAN/14/2012 SAT2 clinical IV 15.49 14.86 TAN/16/2012 SAT2 clinical IV 18.21 18.43 TAN/19/2012 SAT2 clinical IV 16.13 15.5 TAN/3/2011 SAT2 clinical IV 18.73 19.5 TAN/6/2011 SAT2 clinical IV 22.31 21.5 TAN/7/2011 SAT2 clinical IV 21.98 22.16 KEN/2/2007 SAT2 clinical IV 25.32 25.49 KEN/11/2007 SAT2 clinical IV 18.99 20.84 KEN/2/2008 SAT2 clinical IV 16.51 15.95 29.3 29.26 FMDV topotypes uncharacteristic to the geografic area of interest ETH/16/2012 O T/C EA-3 39.09 39.52 ETH/17/2012 O T/C EA-3 25.54 25.73 ETH/18/2012 O T/C EA-3 29.16 29.76 ETH/19/2012 O T/C EA-3 28.62 28.96 NIG/39/2009 A clinical Africa/G-IV 38.61 ETH/70/2009 SAT2 clinical XIII 34.46 33.73 ETH/76/2009 SAT2 clinical XIII 35.79 34.74

Method

RNA extraction

Pan specific FMDV detection

Tailored FMDV typing

the same assay composition and thermal profile

3D-Ct value 15.78 15.5 24.98 25.38 31.88 30.79 24.94 25.1 24.23 23.43 15.49 15.63 12.43 12.3 15.48 15.43 14.56 14.35 13.52 13.87 19.97 19.82 15.26 15.52 20.55 20.27 13.44 13.55 18.42 18.52 16.14 16.31 12.46 12.42 12.59 12.68 16.48 16.4 20.88 21.26 20.31 19.63 21.58 21.95 18.89 44.41 22.7 22.96 18.71 18.71 14.1 14.25 15 15.25 13.54 14.14 16.05 15.86 13.26 13.21 17.86 18.63 15.79 15.68 18.56 18.33 15.87 16.19 18.39 18.34 23.22 23.24 19.12 19.1 16.03 16.07 13.96 14.34 17.84 17.28 15.42 15.23 13.33 13.22 13.91 14.22 18.21 17.72 15.86 16.04 18.86 18.99 20.21 20.07 19.78 19.93 20.86 21.23 17.74 17.67 15.27 14.88

8.72 8.66 13.35 11.49 23.98 15.53 15.78

7 Sample name serotype origin topotype O-Ct value A-Ct value SAT1-Ct value SAT2t4-Ct value SAT2 Ct value ETH/04/2013 O T/C EA-4 18.23 23.43 KEN/11/2011 O clinical EA-2 27.51 27.38 KEN/15/2011 O clinical EA-2 31.09 30.81 KEN/16/2011 O clinical EA-2 27.42 27.8 KEN/137/2010 O clinical EA-2 36.53 39.27 23.09 23.27 KEN/145/2010 O clinical EA-2 17.74 18.14 KEN/146/2010 O clinical EA-2 13.84 13.87 37.67 KEN/148/2010 O clinical EA-2 16.55 17.21 KEN/151/2010 O clinical EA-2 23.53 23.49 KEN/152/2010 O clinical EA-2 14.16 14.74 37.98 38 KEN/154/2010 O clinical EA-2 22.01 20.26 KEN/1/2011 O clinical EA-2 16.26 16.41 38.87 KEN/10/2009 O clinical EA-2 35.36 34.58 TAN/39/2012 O clinical EA-2 16.45 16.56 TAN/16/2008 O clinical EA-2 20.95 21.2 TAN/5/2009 O clinical EA-2 15.8 15.69 TAN/49/2013 SAT1 clinical I 13.73 13.81 36.38 36.29 TAN/50/2013 SAT1 clinical I 13.59 13.48 KEN/12/2009 SAT1 clinical I 18.27 18.35 KEN/139/2010 SAT1 clinical I 17.72 17.75 KEN/140/2010 SAT1 clinical I 21.99 21.33 KEN/121/2009 SAT1 clinical I 36.11 37.66 23.19 23.59 KEN/123/2009 SAT1 clinical I 38.31 20.21 19.46 KEN/1/2010 SAT1 clinical I 23.78 23.74 KEN/71/2010 SAT1 clinical I 21.6 20.75 KEN/72/2010 SAT1 clinical I 16.12 16.2 KEN/16/2009 SAT1 clinical I 28.2 29.14 16.44 15.88 KEN/26/2008 SAT1 clinical I 15.46 15.32 KEN/9/2009 SAT1 clinical I 37.46 37.96 17.69 17.25 TAN/01/2013 A T/C Africa/G-I 14.92 14.64 TAN/61/2012 A T/C Africa/G-I 18.83 18.9 TAN/71/2012 A T/C Africa/G-I 16.04 15.93 TAN/42/2009 A clinical Africa/G-I 18.9 18.82 TAN/45/2009 A clinical Africa/G-I 15.74 16.19 KEN/28/2008 A clinical Africa/G-I 18.96 18.37 KEN/22/2009 A clinical Africa/G-I 21.89 21.71 ETH/12/2009 A clinical Africa/G-VII 19.4 19.23 KEN/5/2012 A T/C Africa/G-I 17.1 16.78 KEN/6/2012 A T/C Africa/G-I 14.16 14.68 TAN/64/2012 SAT2 clinical IV 20.22 19.93 KEN/12/2011 SAT2 clinical IV 17.93 17.85 KEN/21/2011 SAT2 clinical IV 35.98 35.21 15.59 15.78 TAN/14/2012 SAT2 clinical IV 15.49 14.86 TAN/16/2012 SAT2 clinical IV 18.21 18.43 TAN/19/2012 SAT2 clinical IV 16.13 15.5 TAN/3/2011 SAT2 clinical IV 18.73 19.5 TAN/6/2011 SAT2 clinical IV 22.31 21.5 TAN/7/2011 SAT2 clinical IV 21.98 22.16 KEN/2/2007 SAT2 clinical IV 25.32 25.49 KEN/11/2007 SAT2 clinical IV 18.99 20.84 KEN/2/2008 SAT2 clinical IV 16.51 15.95 29.3 29.26 FMDV topotypes uncharacteristic to the geografic area of interest ETH/16/2012 O T/C EA-3 39.09 39.52 ETH/17/2012 O T/C EA-3 25.54 25.73 ETH/18/2012 O T/C EA-3 29.16 29.76 ETH/19/2012 O T/C EA-3 28.62 28.96 NIG/39/2009 A clinical Africa/G-IV 38.61 ETH/70/2009 SAT2 clinical XIII 34.46 33.73 ETH/76/2009 SAT2 clinical XIII 35.79 34.74

8.72 8.66 13.35 11.49 23.98 15.53 15.78

8.38 9.31 13.39 11.4 24.57 15 16.63

Validation A panel of 50 FMDV RNA samples of O, A, SAT1 and SAT2 types Collected in Kenya and Tanzania between 2007 and 2013 Tested in duplicates with homologues and heterologous assays In direct comparison to the 3D assay All samples were identified by homologous serotype-specific assays with Ct values comparable to the 3D assay. Samples uncharacteristic to the region either did not produce a Ct value or were amplified much later in comparison to 3D.

FMDV-A FMDV-O FMDV-SAT1 FMDV-SAT2 (T4) No Ct 8

Limit of detection

Increase in fluorescence was recorded with more than one of these serotype-specific RT-PCR assays indicating mixed infection laboratory contamination poor specificity of these tests

FMDV-O

FMDV-3D

FMDV-A FMDV-SAT1

FMDV-3D FMDV-3D

KEN/137/2010 FMDV-SAT2

KEN/9/2009

FMDV-A FMDV-O FMDV-SAT1 FMDV-SAT2 (T4) No Ct 9

345

FMDV-3D

Diagnostic sensitivity similar to 3D assay At least six points of 10-fold dilution series

Efficiency rates close to 100% (92102%)

animal no

Transfer of technology

Sample name 37 7 450 451 452 106 54 116 48 59 66 32 359 360 355 354 358 76

FMDV- FMDVS1 S2T4 16.04 14.78 27.16 No Ct 18.88 13.95 32.38 37.19 16.16 16.37 32.32 No Ct 27.79 27.87 33.34 No Ct 25.69 26.5 34.94 No Ct No Ct 25.33 21.67 No Ct No Ct 22.43 19.99 No Ct 27.4 26.88 34.19 No Ct No Ct 29.77 29.07 No Ct 30.66 No Ct 27.66 No Ct 25.72 30.94 24.19 No Ct 34.58 40.64 29.74 No Ct 18.85 35.57 13.82 No Ct 15.35 34.76 13.77 No Ct 17.34 No Ct 13.62 No Ct 22.8 33.87 16.96 No Ct 20.04 33.81 15.11 34.55 28.95 32.79 32.17 23.78 3D FMDV-O

27.76

822

29.84

30.31

32.98

31.94

29.61

839

27.09

24.77

32.16

26.61 25.52

33.35

802

26.53

38.39

29.21

26.93

26.92

27.61

29.22

36.17 28.87 31.95

29.12

26.54

28.61

29.03

38.22

893

29.42

39.3

34.03

27.53

885

25.97

31.18 24.59 40.06 31.83

100

30.68

839

27.08

809

33.54

27.83 35.48 30.38 39.14

26.9 31.91

26.26

45.05 36.44

29.8 27.62

885

31.88

30.53

837

31.93

34.94 35.43

38.6

850

30.22

835

30.96

28.83

36.18

873

20.83

20.72

36.76 37.66

36.76

34.43

841

29.25

37.98

871

27.21

26.02

848

22.47

22.27

848

29.63

850

28.1

25.4

808

34.87

850

28.87

892

21.44

808

25.95

866

22.95

871

25.69 22.29

31.24

28.12

23.47

877

31.82

38.72

841

34.37

860

27.71

29.68 31.42 33.28 30.84

854

33.15

852

27.81

32.79

32.98

892

28.08

802

48.45

848

27.46

850

26.88

841

37.7

Feb-Nov 2013

Probang samples in lysis buffer shipped to Pirbright for diagnosis Identification of FMDV type possible

30.59

848 893

853

MCF herd in Tanzania maintained and monitored over a long period of time (CIDLID project)

29.18

827

Aug 2012

32.82

808

885

29.54

29.87

860

802 No Ct

27 26.82

Mar 2012

31.71 42.41

830

873

SAT2

28.75

886

890

SAT2 (T4)

SAT1

43.55

32.98 29.16 28.68 20.63

25.17

19.39

29.25 28.94

18.56

29.3

827

44.5

830

35.21

891

40.44

876

35.04

24.43

34.33

25.99

28.5 31.68 28.55

FMDV-A FMDV-O FMDV-SAT1 FMDV-SAT2 (T4) No Ct No type results

A molecular toolbox?

Development of type specific real-time RT-PCR assays for endemic settings

SAT2 viruses in Egypt - Pool 4 Ahmed et al., 2012

FMDV-O, A and Asia1 in the Middle East Pool 3

Reid et al., 2014

FMDV-O/ME-SA/Ind-2001 Knowles et al., In press

kasia.bankowska@pirbright.ac.uk donald.king@pirbright.ac.uk FMDV-O, A, SAT1 and SAT2 in East Africa Pool 4

346

Acknowledgements The Pirbright Institute: Don King Nick Knowles Valerie Mioulet and the WRLFMD team Veronica Fowler Miki Madi Technical University of Denmark: Graham Belsham Preben Normann

Real-time RT-PCR for the detection of FMDV in Milk

Makerere University, Uganda: Shelia Balinda

Bryony Armson

Sokoine University of Agriculture, Tanzania: Christopher Kasanga Raphael Sallu

The Pirbright Institute

University of Glasgow : Sarah Cleaveland Tiziana Lembo Miriam Casey

Foot-and-mouth disease

Objectives

There is a recognized need for rapid diagnostics and surveillance to enable early diagnosis of foot-and-mouth disease in cattle.

Multi-centre collaboration between: - Institute for Infectious Animal Diseases (IIAD) (previously FAZD) - Foreign Animal Disease Diagnostic Laboratory (FADDL) - The Pirbright Institute

FMDV detection in milk presents unique opportunities for surveillance and early detection both prior to and during an outbreak. Screening milk would involve non-invasive sampling, on material that is already collected on a regular basis. At present there is a lack of a high-throughput screening tool and bulk tank milk surveillance plan. Previous studies have shown that: Mammary gland = site for FMDV replication. (104 TCID50 per ml). Studies have observed various first detection points in milk, both preceding the onset of clinical signs and at the same time. (Burrows, 1968; Reid et al., 2006, Thurmond and Perez, 2006).

347

To evaluate the effectiveness of preclinical indicators of FMDV infection. To evaluate a high-throughput screening tool using real-time(r)RTPCR which could be scaled up for use with bulk tank milk. To compare real-time PCR protocols developed in the US with the routine diagnostic assay currently used in the UK. Both assays based on the pan-serotypic rRT-PCR targeting 3D. To obtain data suitable for the development of a national surveillance plan for screening milk for FMDV. Need to be suited to high-throughput screening in the event of, or during recovery from an outbreak.

Samples collected

Experiment Protocol Dpi -1

Whole milk: - skimmed milk - cell fraction - cream

Dpi 5

Dpi 0

Blood (serum and whole blood) Mouth and nasal swabs Probangs Temperature

Sample processing

108 Whole Milk

Two parts:

- Virus Isolation - US rRT-PCR

PIRBRIGHT qRT-PCR

BTY cells (whole and skimmed milk) IB-RS2 cells

US RT-PCR

-2

25 PIRBRIGHT qRT-PCR US RT-PCR

40 45

50 2

DPC

825 Skimmed Milk

PIRBRIGHT qRT-PCR

-2

Ct 10

348

US RT-PCR

- Pirbright rRT-PCR MagNa Pure LC Extraction Robot & Stratagene Mx3005.

DPC

825 Whole Milk

BTY cells

PIRBRIGHT qRT-PCR

35 40

-2

MagMax Express-96 & ABI 7500 Fast Real Time PCR System.

Quantitative tests: - Virus titrations

Real time diagnostic tests (on the day of collection):

108 Skimmed Milk

US RT-PCR

50 -2

DPC

867 Whole Milk

10 15

PIRBRIGHT qRT-PCR

US RT-PCR

DPC

8 10 12 14 16 18 20 22 24 26 28

951 Whole Milk

-2 0

PIRBRIGHT qRT-PCR

US RT-PCR

DPC

8 10 12 14 16 18 20 22 24 26 28

US RT-PCR-Serum Virus Isolation-Serum

DPC -1

-2 0

8 10 12 14 16 18 20 22 24 26 28

1 - 200017 (c27) 2 - 261883 300105 3 - 261883 700158 4 - 2616477 00036 5 - 20027 261883 6 - SY003000369 7 - SY003000341 8 - 26164750069 9 - 261647 10030 10 - 261883 300161 11 - 261883 400092 12 - SG0704 01241 13 - 261883 500093

Log TCID50

ID 108 ID 825

ID 867

ID 951

0 15

DPC

Skimmed Milk 6 Log TCID50

5 4

ID 108

ID 825

ID 867

ID 951

0 5

DPC

Sample

Positive Cohort

-5

Virus Isolation-Milk

PCR-Milk

Foot lesions

Whole Milk

PIRBRIGHT qRT-PCR

BTY Titration Results

-5

US RT-PCR

45 50

DPC

951 Skimmed Milk

35 40

6 8 10 12 14 16 18 20 22 24 26 28

2 4

Ct 10

Cows mixed

Pirbright qRT-PCR-Serum

-2 0

867 PIRBRIGHT qRT-PCR

FMDV Detection Points

-2 0

867 Skimmed Milk

DPC

349

TaqMan EZ Path-ID Pirbright 3D Average Ct Average Ct Ct 34.44 28.31 17.22 32.04 21.89 18.60 * Indicates sample testing NEG 36.78 NEG was repeated 32.5* 27.18 22.71 33.78 26.00 26.28 36.23 24.37 21.16 29.49 25.12 25.37 25.59 23.93 25.74 NEG* 28.85 22.51 31.81 28.77 27.81 NEG* 34.00 29.22 22.64 19.17 19.02 NEG* 23.87 23.99

Milk samples collected from field cases from the UK 2007 outbreak were used as a positive cohort to evaluate diagnostic sensitivity.

Thank you!

Conclusions FMDV can be detected in whole milk, skimmed milk and the cell fraction, by all methods tested. FMDV was detected in milk before the onset of characteristic clinical signs. The greatest window for virus detection was by rRT-PCR in the milk up to 21 days post contact Both rRT-PCRs detected virus for a longer period than seen in virus isolation. Data not included in this talk suggest that rRT-PCR of milk from a bulk tank in a large herd could detect a single infected cow in the early stages of infection.

Acknowledgements: Collaborators - Mangkey Bounpheng - Karissa Lemire - Amaresh Das - Diane Holder - Michael McIntosh Valerie Mioulet Don King Claudia Doel Miki Madi Satya Parida

Milk could be an excellent sample type for the detection of FMDV and could be used for the development of a national FMD surveillance plan in the event of an outbreak. Further Work Test samples from dairies in endemic countries.

Isolation Unit Staff Sheila Wilsden Mandy Swan IIAD, FADDL, DEFRA

qPCR Results

Potential Virus Detection in Bulk Tank Milk

Whole Milk 1.00E+08

3.00E+08

1.00E+07

Samples with high medium and low titers of FMDV were tested in serial dilutions. Jersey milk revealing positive Ct at 250 fold to 30,000 fold dilutions Inconclusive Ct at >1,000 fold dilution for the sample with the lowest titer and >700,000 fold dilution for the sample with the highest titer.

2.50E+08

1.00E+06 2.00E+08

1.00E+05 108

1.00E+04

825

1.00E+03

951

1.50E+08

867

1.00E+08

1.00E+02 5.00E+07

1.00E+01

Detection from one study sample also yielded positive Ct up to a 100,000 fold dilution.

1.00E+00 -2 0

Milk production was reduced by no more than 50% on a given day.

3.00E+07

0.00E+00 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

8 10 12 14 16 18 20

1.00E+08 9.00E+07 8.00E+07 7.00E+07 6.00E+07 5.00E+07 4.00E+07 3.00E+07 2.00E+07 1.00E+07 0.00E+00 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Skimmed Milk 2.50E+07 2.00E+07

Taken together this suggests that early in infection, rRT-PCR may reasonably detect a single infected cow from a herd size of 100 to 1,000 milking into a bulk tank.

108

1.50E+07

825

1.00E+07

951

5.00E+06

350

0.00E+00 -2 0

867

qPCR Results Serum 3.50E+06

4.50E+07

3.00E+06

4.00E+07 3.50E+07

2.50E+06

3.00E+07 825 Serum

2.50E+07

1.50E+06

951

2.00E+07

108

1.50E+07

Copy number

2.00E+06

1.00E+06

867 Serum

1.00E+07

5.00E+05

5.00E+06

0.00E+00 -2 0

Realising the potential of simple isothermal molecular tools for field diagnosis of FMD

0.00E+00 2

8 Dpi 10

Probangs 1.20E+07

4.00E+05 3.50E+05

1.00E+07

3.00E+05 8.00E+06

2.50E+05

108 Probangs

2.00E+05

867

1.50E+05

951

1.00E+05

6.00E+06

Blood

Emma Howson

-virus/genome -antibody 825 probangs

Research Scientist, Vesicular Disease Reference Laboratory Group, The Pirbright Institute

4.00E+06 2.00E+06 Foot lesions

5.00E+04

0.00E+00 -virus in0 epithelium 0.5 1 1.5

0.00E+00 0 2 4 6 8 101214 1618202224 2628

2.5 1

The Problem:

The Solution:

Field deployment of RT-LAMP

Lyophilised reagents

Thermostable reagents

Requirements for in situ diagnostics: Three elements of a molecular test (sample preparation, amplification, detection) -toReagents compatible with field deployment 2

351

Experimental or field use

rRT-LAMP

Field use

RT-LAMP-LFD 3

Lyophilised singleplex RT-LAMP:

Molecular LFDs:

Singleplex RT-LAMP-LFD format

Biotin

Analytical sensitivity comparable to rRT-PCR

Laboratory concordance (rRT-PCR/Wet/Dry) Dilution series of RNA standard Comparison with the gold-standard Callahan rRT-PCR

Anti-biotin antibody linked to latex beads

Flc

Copy number of RNA standard

TEST LINE Anti-Flc antibody

CONTROL LINE* Anti-Ig antibody

RT-LAMP (RNA standard)*

106

105

104

103

102

101

100

Negative

rRT-PCR

+/-

Wet reagents

RT-LAMP-LFD rRT-LAMP RT-LAMP-LFD Test --Control ---

Lyophilised reagents

rRT-LAMP

*RNA standard supplied by Graham Freimanis

*control represents a read line indicating LFD has run correctly 4

Expansion of simple sample preparation:

Detection of FMDV in clinical animals:

From the laboratory to the field

Cattle samples from experimental infection

Serum samples

Two days after the appearance of clinical signs

Animal ear tag

OP* fluid samples

0dpc

1dpc

2dpc

3dpc

0dpc

1dpc

2dpc

3dpc

Extracted RNA

Neat

1 in 5 dilution

rRT-PCR

Foot Epithelium

7803

7804

7805

7806

Negative**

Mobile rRT-PCR*

rRT-LAMP

RT-LAMP-LFD

rRT-LAMP

1 in 10 dilution

rRT-LAMP RT-LAMP-LFD

*oesophageal-pharyngeal (probang) **Non-specific amplification

OP fluid

rRT-LAMP RT-LAMP-LFD

Test --Control ---

From Jose Gonzales

Serum

Epithelial samples positive by rRT-PCR were also positive by Ag-LFD

352

*mobile rRT-PCR is ~one log less sensitive than the OIE laboratory gold standard **Negative water control King et al., 2012 (data from Alexandersen et al. 2003) 7

Detection of FMDV in late infection:

Detection of FMDV in clinically normal cattle:

Ten days after the appearance of clinical signs

One month after the appearance of clinical signs (Serengeti)

Animal ear tag 7808

7809

7810

7812

Negative

Mobile rRT-PCR* Mouth Epithelium

Animal ear tag

7807

rRT-LAMP

Serum

OP fluid

rRT-LAMP

7735

7737

7739

7741

7742

7645

7648

7649

7650

rRT-LAMP

RT-LAMP-LFD

RT-LAMP-LFD OP fluid

7734

Test --Control ---

rRT-LAMP

7733

RT-LAMP-LFD

RT-LAMP-LFD Serum

rRT-LAMP

7732

previously displayed clinical symptoms

never displayed clinical symptoms

Test ---

Control ---

RT-LAMP-LFD

Epithelial samples were all negative on Ag-LFD *mobile rRT-PCR is ~one log less sensitive than the OIE laboratory gold standard King et al., 2012 (data from Alexandersen et al. 2003) 8

Detection of FMDV in clinically normal cattle:

Field observations:

~Six weeks after the report of clinical signs (Morogoro)

looking to the future

Design of field protocols

Animal number

OP fluid

rRT-LAMP

RT-LAMP-LFD

N/A

rRT-LAMP

+ *

RT-LAMP-LFD

*confirmed by mobile rRT-PCR

Test --Control ---

Serum

Farm 1

- separation of animal sampling and pre / post LAMP procedures - disposable on site - use of closed systems

Use in different FMD situations

Farm 2

- outbreaks in non-endemic vs endemic regions - species, serotypes, strains, environments

Operator characteristics and sample quality - samples affecting results vs

353

Source The Pirbright Institute

Source EuFMD

Co-authors

Summary

Veronica Fowler (Pirbright) Sarah Cleaveland (University of Glasgow) Bryony Armson (Pirbright) Donald King (Pirbright) Valerie Mioulet (Pirbright) Miki Madi (Pirbright) Christopher Kasanga (Sokoine University) Sengiyumva Kandusi (Sokoine University)

RT-LAMP and RT-LAMP-LFD - simple sample preparation protocols - simple amplification and detection methods - performed in situ on OP fluid, serum and epithelium - lyophilised reagents suitable for field deployment

Acknowledgements

Sample collection to result calling in < 30 minutes

Jose Gonzales (Pirbright) Graham Freimanis (Pirbright) WRLFMD staff Raphael Sallu (TVLA) Raphael Mahemba Shabani Magumu field staff Optigene Ltd.

The Problem FMD OR VS?

Development of a multiplex RT-LAMP for the discrimination of FMD from other vesicular diseases

FMD or VS? (Vesicular lesions on dental pad-cattle)

DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL OBSERVATION = CHALLENGING

FMD or SVD? (Vesicular lesions on snout-pig)

LABORATORY DIAGNOSIS (Local or NRL)

Dr. Veronica Fowler Applied Diagnostics Research Coordinator, The Pirbright Institute

Delays impact upon the potential size and cost of an epidemic -side tests need to be developed that can 1

354

Solutions?

FMDV antigen detection: Lateral-flow devices -

Developed collaboration with international partners Quick and simple to perform Used in the UK (during 2007)

FMDV antigen LFDs

DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL OBSERVATION = POSSIBLE

Rapid (<10 mins) confirmation of FMD in the field Also useful in the Lab for triage of samples

Mobile rRT-PCR

Recognises all seven FMDV serotypes Similar assay performance to lab-based AgELISA (Boehringer Ingelheim) LFD marketed by

RT-LAMP

Ferris et al., 2009: J. Virol. Methods

The Solution

FMDV detection by mobile real-time RT-PCR Non-specialist user 1. Nucleic acid extraction 2. PCR set-up 3. Analysis Location on/or near farms Sample to report < 60 mins Powered by car auxiliary/battery Platform for other diseases Uses mature and established technologies Equivalent to lab-based methods

DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL OBSERVATION = POSSIBLE FMD or VS?

FMD or SVD?

Multiplex RT-Loop Mediated Isothermal Amplification (RT-LAMP) assay Molecular lateral flow devices (LFD)

355

Improved decision algorithm

Control----

FMDV----

VSV or SVD----

Madi et al., 2012: The Vet Journal

Control----

FMDV----

FMDV + VSV or SVD----

VSV/SVD +

Expensive Can only run one sample at a time

6 6

Multiplex RT-LAMP: Rapid with simple detection

Loop mediated isothermal amplification (RT-LAMP) Isothermal autocyling strand-displacement DNA synthesis technique Utilises six Formation of loop structures enables explosive polymerase-based enzymatic amplification Generates double-stranded, multi-sized amplicons Sensitivity equivalent to rRT-PCR Rapid detection of nucleic acid Accommodate reverse transcription (RT-LAMP) Detection of multiple pathogens (multiplexing) Test can be performed using simple heat source

FMDV, VSV and SVD can be detected in less than ten minutes at 108 copies Biotin Flc

FMDV +

Labelled beads

FMDV

CONTROL Anti-Ig antibody

DIG Flc

VSV/SVD +

Control

TEST Anti-Flc antibody

VSV/SVD

(anti-flc antibody linked to gold beads)

Labelled beads

(anti-flc antibody linked to gold beads)

TEST Anti-DIG antibody

CONTROL Anti-Ig antibody

Multiplex RT-LAMP: Analytical sensitivity comparable to rRT-PCR Multiplex RT-LAMP (RNA standard)

100

101

102

103

104

105

106

rRT-PCR

-/+

RT-LAMP

SVD

VSV

RT-LAMP-LFD

RT-LAMP-LFD rRT-PCR

RT-LAMP

RT-LAMP-LFD

rRT-PCR Multiplex RT-LAMP (RNA dilution)

VSV

10-7

Cannot be assayed as different target 10-6 10-5 10-4 10-3 10-2

RT-LAMP can be performed directly on clinical (epithelium) samples (1:10 dilution)

Direct Multiplex RT-LAMP RT-LAMP

rRT-PCR

RT-LAMP

10-1

RT-LAMP

RT-LAMP-LFD

rRT-PCR

+/-

Asia 1

Sat 1

Sat 2

NJ 27405

NJ 27324

NJ 27946

NJ29336

+(36)

FMDV clinical samples

NJ29344 NJ 27775

VSV clinical samples rRT-PCR

UKG 24/72

Control --VSV/SVD --FMDV ---

FMDV

Multiplex RT-LAMP: Simple sample preparation

SVD clinical samples

356

RT-LAMP rRT-PCR

UKG 50/22 UKG 51/72 UKG 63/73

+ 10

Co-authors

Summary and impacts

Emma Howson (Pirbright) Donald King (Pirbright) Valerie Mioulet (Pirbright) Bryony Armson (Pirbright) Miki Madi (Pirbright)

Test provides an improved decision algorithm via: Rapid pathogen detection within 10 minutes. Performance at penside Test confidence via: Comparable limit of detection to rRT-PCR. Test desirability via: Disposability. Assay can be performed using non technical heating systems. Easily adaptability for use as a field kit (e.g. by use of dry down reagents and E Howson talk closed system

Acknowledgements WRLFMD reference laboratory staff Luis Rodriguez(USDA) Steve Pauszek (USDA) Mike McIntosh (APHIS) Fernando Torres (APHIS) Tammy Beckham (IIAD) Melissa Berquist (IIAD) Rapidia-field Optigene Ltd

IP4

IP1

From sequences to prevalence: phylodynamics of foot-and-mouth disease virus

IP2

IP3

(Cottam et al., 2008)

IP4 IP5 IP4

IP1

IP2

IP3

Antonello Di Nardo The Pirbright Institute, Pirbright, Woking, Surrey, UK Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Science, University of Glasgow, Glasgow, UK

IP1

IP2

IP3

IP5

IP5 1

357

A Di Nardo

Can the evolutionary transmission chain be correlated to the effective number of infected epi-units?

Phylodynamics of FMDV

Ne Prevalence Incidence

Effective Population Effective Population Size ( Size ) Generalised Skyline ot (Strimmeret and Bayesian SkylinePl(Drummond al. Pybus 2005) 2001) A Di Nardo

Phylodynamics of FMDV

Individual based-model with disease staged progression in space-time

Evolutionary model based on real data (IP4 FMDV sequence from 2001) A Di Nardo

Phylodynamics of FMDV

A Di Nardo

358

Phylodynamics of FMDV

Effective population size (Skyline plot) reconstructed from BEAST 1.8.0 A Di Nardo

Phylodynamics of FMDV

2.38 10-5 (95%HPD 2.28-2.49) 2.37 10-5 (input - BEAST) 2.26 10-5 (Cottam et al., 2006) 2.08 10-5 (Cottam et al., 2008) A Di Nardo

Phylodynamics of FMDV

A Di Nardo

Phylodynamics of FMDV

359

A Di Nardo

Phylodynamics of FMDV

A Di Nardo

Phylodynamics of FMDV

A Di Nardo

Phylodynamics of FMDV

A Di Nardo

Phylodynamics of FMDV

360

A Di Nardo

Phylodynamics of FMDV

A Di Nardo

Phylodynamics of FMDV

Effective population size (skyline plot) measure of: diversity?!?...prevalence?!?...incidence?!? Correlation behaviour differ between prevalence and incidence data and according to the state of infection

Viral demographic size might be affected by the type and amount of data available (accuracy reduces with the decrease in the sampling proportion) Different effect on the recovery of FMDV population dynamics according to the sample design strategy used (i.e. proportional, cluster, spatial, temporal, spatio-temporal, etc.)?

Epidemic setting (UK 2001, UK 2007, Bulgaria) Endemic setting A Di Nardo

Phylodynamics of FMDV

A Di Nardo

Phylodynamics of FMDV

Dan Haydon Paul Johnson Don King Nick Knowles Simon Gubbins

Beyond the consensus: investigating intra-herd variability of FMDV using the Illumina MiSeq

Samuel Soubeyrand Marco Morelli Nick Taylor

David King

Vesicular Disease Reference Laboratory Group, The Pibight Insitiute

361

Summary

FMDV variability & Next generation sequencing

1. FMDV variability and Next Generation Sequencing (NGS) 2. UK 2007 3. Protocol 4. Results 5. Conclusions

FMDV within a sample exists as a heterogeneous population comprising of related but non-identical genomes Population complexity Large population size High replication rate Poor proof reading

NGS allows high throughput variant analysis on the MiSeq (Illumina) 2

Investigating intra-herd variability from UK 2007

Protocol Extraction

Aim: Use the MiSeq to deep sequence samples from a single herd to discover shared variations

2007 UK outbreak BFS 1860 O1 1967 3rd August to end of September

Normalisation

RNA normalised 105 cp/ul

Superscript III (Invitrogen)

Novel long-PCR Library prep

Consensus level sequences Cottam et al, 2008 Valdazo-gonzalez et al, 2014 submitted for publication

RNeasy mini kit (Qiagen)

Processed in duplicates, 7.6kb (KAPA HiFi) Nextera XT - 300 cycle v2 PE on Miseq 1

8 epithelium samples: IP2B Samples 3 and 6 from same cow 7 animals

7.6 kb fragments

362

Consensus sequencing -TCS network 10 animals Sample 8

NGS analysis pipeline X

X X

Sample 7

150bp short sequence reads

Sample 4

Read trimming and quality control FastQC, prinseq, Sickle

Mapping/alignment BWM-MEM, Samtools

Consensus and coverage calling

Open access tools

Sample 5

Sample 2

Sample 3/6

Sample 1

Samtools, Bedtools

Variant calling

2b/95

2b/92

2b/93

loFreq - 0.5% freq to 49.9%

Genome coverage

Beyond the consensus Number of samples 1 2 3 4 5 6 7 Total number of mutations Total number of shared mutations

Frequency of mutation (above 0.5%) 342 30 12 1 1 1 1 388 46

2 3 4 5 6 7 8 2 3

Average coverage ranged from: 5.8x102 to 104 Average number of reads for each sample: 5.54x105 Average Viral reads: 98% 8

363

5 6

A heatmap showing the number of shared variants. The darker the colour, the higher number of variants which are shared Samples 6 and 7 Nine variants are shared Samples 5 and 3 One variant is shared 9

Beyond the consensus

Variation

Genome Position Base Change Mutation change Amino acid Shared between 2429 C>A Synonymous Val 6 3380 G>A Synonymous Pro 5 4 7566 G>A Non-Synonymous Ala > Thr 1156 G>A Non-Synonymous Gly > Glu 3 1493 A>G Synonymous Ala 3 2960 C>A Synonymous Ala 3 2984 G>A Synonymous Pro 3 2987 C>T Synonymous Pro 3 3296 A>G Synonymous Flu 3 3318 A>G Non-Synonymous Ile > Val 3 3353 T>A Non-Synonymous Ile > Met 3 3728 T>C Synonymous Pro 3 3902 G>C Synonymous Leu 3 7871 G>A Synonymous Met 3

Sample 3 and 6 (same cow) Identical consensus sequence Different swarm structures (Diversity) Only 5 variants shared

Sample 3 16

Genome position 2429 (VP2) Synonymous mutation C > T Amino acid - Val

Change becomes fixed at a consensus level during the rest of the outbreak

Sample 6

Synonymous Non-Synonymous 10

Conclusions 1.

2. 3. 4.

FMDV variability and NGS FMDV exists as a heterogeneous population NGS can allow us to deep sequencing a viral population to detect variants. Protocol A high fidelity polymerase used to deep sequence the L-fragment Revealed a number of shared nonsynonymous and synonymous mutations Future Use of data to reconstruct infection roots within intra-herd transmission dynamics. Link consensus sequences together with variants To deep sequence other viruses, to identify variants which are passed between farms and become fixed in the outbreak.

Acknowledgement Global Phylogeography !((( !( !( !!(( !( !(!( !( !(!!(( !(!( !(!( !(!( !( !(!( !(!(!(!!((!( !(!( !(!(!(!(!(!(!(!(!!((!(!(!(!(!(!(!(!(!(!(!!((!(!(!( !( !(!(!( !(!(!(!(!(!(!!((!(!( !!(( !(!(!( !(!(!(!(!(!!((!(!(!!((!!((!(!((!(!!((!!((!!((!!((!!((!(!!((!!(( !(!( !(!(!(!!(( !(!(!(!( !( !( !(!(!!((!( !( !(!(!(!(!( !!((!!!(((!!!(((!!!!((((!!(((!!!(((!!!(((!(!(!!!(((!!!(((!!((!(!!((!!!(((!!((!!!(((!!!(((!!((!( !( !(!!((!(!(!(!(!!((!(!(!(!!(!(!(!( !( !(!(!( !!((!(!!((!(!!((!(!(!(!(!(!!((!!((!((!(!!((!(!!((!!((!(!(!( (! !(!( !(!(!( !(!(!(!(!!((!!((!!((!!((!!((!!((!!((!(!( !(!!(( !!!(((!!!((!!(((!!!(((!!((!!!((!!((!(!(!( !( !(!( !( !(!(!!((!!(!(!!!(((!!((!!((!!!(((!(!(!(!(!( !(!( !(!(!( !(!( !(!(!(!(!(!( !( !(!( !( !(!(!(!( !(!( !(!(!(!(!!((!( !( !(!(!(!(!!((!( !(!( !( !(!!((!( !( !(!!(( !( !( !(!(!(!!((!(!(!(!(!( !( !( !( !!((!!!(((!!((!!!(((!(!!((!!((!(!(!(!(!(!( !( !( !(!( !(

!(!(!(!(

!(!( !(!(!( !( !(!( !( !( !(!(!( !( !( !( !(!(!(!!(( !( !(!(!( !( !( !( !( !!((!( !(!(!(!!(( !( !!((!(!(!(!(!(!!((!!((!!(( !( !(!(!( !( !(!( !(!(!( !( !(!( !(!( !(!!((!(!(!( !( !( !(!(!( !(!(!!((!( !(!(!(!( !!((!!((!(!( !(!(!( !(!(!(!( !(!(!(!(!(!( !(!( !(!!((!( !(!!((!(!!((!(!(!(!!((!!((!!((!!((!!((!!((!!((!(!(!(!(!!((!!((!(!( !(!( !( !( !(!(!(!(!(!( !( !( !( !( !(!(!(!(!(!(!(!(!(!( !(!( !( !( !(!( !((!!(

!( !(!(!( !(!!((!( !(!!((!!((!!((!!!(((!(!!((!!!(((!(!!((!!!(((!!((!!(( !( !(!(!!((!( !( !( !!((!!((!(!(!(!(!!((!!((!(!(!(!(!( !( !( !(!(!!((!!((!!!(((!!!(((!(!(!( !(!( !(!( !(!(!( !( !(

!( !( !(!( !(!( !(!( !(!(!(

!(!(

The Pirbright Institute Graham Freimanis Nick Knowles Begona Valdazo-Gonzalez Donald King

Outbreak epidemiology Farm-to-farm spread Animal-animal transmission Within host pathways

The University of Glasgow Richard Orton Daniel Haydon

Cell-to-cell infection Population diversity Intra-cellular dynamics 12

364

Minimum dose to cause infection?

Probability of infection of cattle, sheep and pigs exposed to FMDV aerosols Jose L Gonzales

Gibson and Donaldson 1986

Gibson 1985

What is the relationship between virus dose and the likelihood of infection and disease? (French et al 2002)

365

Objectives

Methods Published and unpublished data

Quantify (update) the virus-aerosol dose-response relationship for cattle, sheep and pigs Evaluate the effect of serotype Evaluate the relationship between dose and incubation period

Analysis

Host

Virus

# experiments

# animals

Cattle

O1 BFS 1860

SAT2

A22

O UKG 2001

Sheep

O1 BFS 1860

Pigs

O1 Lausanne

O SKR 1/2000

Cattle

Recalculated inhaled dose for sheep

Rate of infection per FMDV infectious (TCID50) aerosol LCL UCL Rate

Dose-response: exponential model were the probability of infection/disease is a function of: Rate of infection/disease per TCID50 Challenge dose in TCID50

Infection

0.027

0.016

0.045

Disease

0.005

0.003

0.009

50% cow infectious dose (TCID50)

Incubation period: Parametric survival analysis

Dose

LCL

UCL

Infection

1.4

1.1

1.6

Disease

2.1

1.8

2.3

No relationship between FMDV serotype (OBFS, OUK, A22,SAT2) and doseresponse was observed

366

Sheep

Pigs Rate of infection per FMDV infectious (TCID50) aerosol LCL UCL Rate

Rate of infection per FMDV infectious (TCID50) aerosol LCL UCL Rate

Infection

0.024

0.012

0.044

Infection

0.0003

0.0001

0.0007

Disease

0.006

0.003

0.011

Disease

0.0001

0.0000

0.0003

50% sheep infectious dose (TCID50) Dose LCL UCL

50% pig infectious dose (TCID50) Dose LCL UCL

Infection

1.5

1.2

1.8

Infection

3.3

3.0

3.7

Disease

2.0

1.8

2.4

Disease

3.9

3.5

4.7

infection rate of a virus particle (TCID50)

Rates of infection/disease

Incubation period

0.05 0.045

Host

FMDV

Sheep

O1 BFS 1860a

Cattle

SAT2

2.1

5.9 (2.2 - 9.6)

A22

2.1

6.8 (2.5 - 11.0)

0.005

O UKG 2001

2.1

7.4 (2.7 - 12.0)

O1 BFS 1860

2.1

9.3 (3.4 - 15.2)

0.04

log10 ID50

IP (95% CI) in days

0.035 0.03 0.025 0.02 0.015 0.01

367

5.2 (2.3 - 8.1)

Conclusions

Acknowledgements

The estimated rates of infection can be used to improve the accuracy of predictions of infection when using windborne spread models

Satya Parida Noel Nelson Aravindh Babu David Paton

This estimated rates will be used as priors for the analysis of transmission (aerosol) experiments. Combined information on dose, incubation period and shedding patters can guide the implementation of aerosol challenge for experimental studies including potency tests

Thank you The Pirbright campus is being redeveloped

Next Generation Sequencing (NGS) dsDNA Fragmentation

WHOLE GENOME SEQUENCING OF FMDV: DEVELOPMENT OF PROTOCOLS AND APPLICATION

Amplification

TO A LARGE OUTBREAK Sequencing

Graham Freimanis Non-Vesicular Reference Lab, The Pirbright Institute

Alignment to reference genome

368

Why Use NGS?

Lesion

qRT-PCR Genomic DNA digest

Criteria for inclusion in protocol development:

1st Strand Synthesis

Reduce Bias: Whole genome coverage: S and L fragments (polyC tract) Applicable to other viruses Consensus-level or deeper Potential for high-throughput

Fastq files

Sequence on illumina MiSeq 2nd Strand Synthesis Purify dscDNA

Analysis Pipeline (R.Orton, UoG)

Sickle

DiversiTools (Java) Genome Data File (Cov, A, C, G, T, Site Quality, Site Entropy, Site dNdS) Sample Data File Sample entropy, dNdS Mutation Spectrum

Samtools

R plots

Genome Coverage All Variants File

True Variants File (Model output)

Bam files/ Visualisation

Consensus

Library Preparation

Protocol sensitivity: viral loads >106 copies/µl

Consensus Sequence

BWA-Mem

Protocol

RNA extraction

Previous strategies (i.e. Sanger sequencing) subject to: limited throughput/labour intensive Subject to a priori knowledge for sequencing i.e. primers, variation

1. 2. 3. 4. 5.

Tissue Homogenisation

MULTI SAMPLE COMPARISON Shared Variant Heatmap Sample similarity Heatmap LoFreq Variants

369

FMDV Serotype Coverage

Depletion of host genomic DNA increases the proportion of viral reads

Genome coverage of 5 UK 2001 field isolates UKG/1450 UKG/1558 UKG/1734

Other Applications

Genome coverage of 7 FMDV serotypes

UKG/14597 UKG/4998

Serotype O Serotype A Asia 1

Serotype C SAT1 SAT2

Summary I Limit of Detection: >106 copies/µl Able to sequence all 7 serotypes of FMDV Able to sequence other viruses (non-FMDV) Able to sequence viruses without amplification (culture)

WGS of viruses unable to grow in culture (FMDV O/ISR/2/2013)

370

SAT3

Application: UK 2001 Outbreak Economic impact: > £2billion Aim: Apply WGS to give a fine point resolution to outbreak tracing Adapt protocol to high-throughput format (i.e. 96 samples) @consensus level 88 samples, 8 controls 24 samples repeated 24 format FMDV: SAT1/2/3/UKG/Prev Pos Non-FMDV : ERAV/EMCV/VESV Picture source: Telegraph: http://www.telegraph.co.uk/earth/agriculture/8335705/Farmers-risefrom-the-ashes-of-foot-and-mouth.html

termini recovery - 24 and 96 sample format

Cross-reactivity between samples EMCV

Validation with Sanger Sequencing 4 Samples previously sequenced using Sanger sequencing (gold standard):

VESV

3/4 consensus identical with Sanger sequences 1/4 differed by one position (ambiguous in Sanger)

8 controls on run validated 24 and 96 sample formats: Both gave identical consensus Cross reactivity between FMDV samples against EMCV and VESV reference genome

371

Application II: Consensus Similarity

Applications I: Outbreak Tracing

96 Sample run Repeats of previously published (Sanger) Previously published sequences (NCBI)

Summary II

Acknowledgements The Pirbright Institute: Grace Logan David King Kasia Bankowska Begona Valdazo-Gonzalez Nick Sanderson Nick Knowles Donald King Eleanor Cottam Valerie Mioulet & WRLFMD

Protocol successfully generates consensus level (or deeper) sequence for whole FMDV genomes (BMC Genomics, 2014) dependent upon viral load No detectable cross-reactivity Adaptable for high-throughput format (96 samples) application for larger outbreaks Sequencing of UK 2001 outbreak ongoing

University of Glasgow: Richard Orton The Epi-Seq Consortium

372

Background Transboundary animal diseases role of wild boar FMD incursion to Bulgaria (2011) CSF outbreaks in Latvia and Lithuania and other European countries (2011-2013) ASF epidemic in the Caucasus, Russian Federation, Lithuania, Latvia and Poland (2007-2014) still ongoing remarkable increase of wild boar populations in Europe

Non-invasive sampling systems for the detecion of FMDV in wild boar Susan Mouchantat*, B. Haas, A. Globig, W. Böhle, K. Depner *Junior Research Group Wildlife Diseases Friedrich-Loeffler-Institut Greifswald - Insel Riems Germany

Current sampling approaches Wildlife surveillance mostly linked to hunting or trapping Sampling rather irregular Hunting seasonally limited Rarely adequate number of samples collected Mostly serological tests performed only retrospective analysis

pSWAB

Animal Trials in Wild Boar Mohamed et al., 2011; Breithaupt et al., 2012

p athogen Wild A nimals with B aits

clinical Phase

incubation period

S ampling of

antibody detection viral RNA detection (saliva)

virus shedding (saliva)

Cotton rope

viremia

raw Ø 0,8 cm

L 10 cm

pSWAB ???

Cereal-based bait matrix (same as for CSF oral vaccine bait)

373

4 weeks p.i.

FLI Animal Trial - FMD Wild Boar

Animal trial FMD I

Mouchantat et al., 2014 (Vet Microbiol 172, 329-333)

Animals

2/4 dpi

5 wild boar (10 months, 65-75 kg)

Inoculation s.c. (bulb of the heel) of 2 donor pigs 106,8 TCID50 FMDV O/BUL/1/2010 Tiletamin/Zolazepam (Zoletil® 100) 2,2 mg/kg

Sedation

Euthanized 29 d.p.i. pSWABs (n=5)

distribution every day collection same or next day

Blood samples/oral swabs 2(1)x per week under sedation Nucleic acid Serology

RTqPCR (3D (OIE-Protocol) + IRES) PrioCHECK FMDV Type O

Animal trial FMD I

Mouchantat et al., 2014 (Vet Microbiol 172, 329-333)

Conclusion

clinical Phase

incubation period

FMD:

antibody detection

pSWAB: feasibility for the detection of FMDV infection in wild boar comparable sensitivity (conventional saliva swabs)

viral RNA detection (oral swab) virus shedding (saliva) viremia 0

pSWAB 1-23 dpi

all animals infected (contact animals 2 d later) incubation period 2 d severe and evident lesions 5-7 dpi all animals recovered

4 weeks p.i.

374

FLI Animal Trial - FMD Domestic Pig Animals

5 pigs (2 months

Inoculation s.c. (bulb of the heel) of 2 donor pigs 106,5 TCID50 FMDV O/BUL/1/2010 Euthanized 2x 2 d.p.i.; 2 x 7 d.p.i; 1x 10 d.p.i pSWABs + Maize ears distribution every day, collection on same day

Field validation work e.g. by Tsviatko Alexandrov

Oral swabs (Salivetten) taken 7 times Nucleic acid Serology

RTqPCR (3D, OIE-Protocol) no antibody found

3 holes on each side of the maize ear, in total 6, for 3 Q-tips cut in half

FLI Animal Trial - FMD Domestic Pig RNA load (each box correlates to one Q-Tip Pool d.p.i

d.p.i

Q-Tip Pools

or one pSWAB)

Stability trials: 8 day, 3 temperatures

pSWABs

5 6

no ct

6 7

9 10

(max. n=6)

FLI Animal Trial - FMD Domestic Pig

no ct

9 10

no ct

Ct values

375

Non-invasive sampling

Field trial

Advantages : pSWAB

Distribution at different feeding points in Germany in progress

Collective sample Early pathogen detection

Acceptance?

Repeated frequent sampling possible

Quantity? Collection?

Applicable where/when hunting is not possible

Quality?

Easy to incorporate into existing wildlife management practices

Influence on testing?

Cost effective and logistically simple

Diversion of pSWABs?

Open Issues

Acknowledgement FLI

Evaluation of the practical use in the field

Junior Research Group WD

Anne Leske

Distribution and collection of the sampling baits

Institute of Immunology

Robert Kammerer

Field studies in FMD/CSF endemic regions

Institute of Diagnostic Virology Anja Schulz, Holger Scholten (NRL FMD)

Use of pSWABs for other investigations (serology, genetics...)

Department of Experimental Animal Facilities and Biorisk Management

Doreen Fiedler, Frank Klipp

Evaluation of multiplex PCR for viral genome detection and quantification of chewing

IDT Biologika

376

Christian Kaiser, Peter Schuster, Ad Vos

Thank you for your attention

Marzipan for pigs

"Saliva is not one of the popular bodily fluids. It lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears." Irwin D. Mandel (1990)

Deer in New Zealand In New Zealand:

PERFORMANCE OF DIAGNOSTIC TESTS FOR FOOT-AND-MOUTH DISEASE IN RED DEER

1.1 Million farmed deer. about 50% of the farmed deer population in the world,

Reinhold Kittelberger, Charles Nfon, Kurtis Swekla, Zhidong Zhang, Kate Hole, Hilary Bittner, Tim Salo,

85% are red deer or red deer crosses,

McFadden, Richard Spence, Soren Alexandersen

after cattle and sheep, largest species of livestock,

EuFMD Open Session, Cavtat, Croatia 31 October 2014

In an FMD incursion, deer would have to be tested with test methods for which the performance characteristics for deer are not known.

Lots of good reasons to evaluate FMD test methods for red deer!

www.mpi.govt.nz

377

FMD in Deer Project Winnipeg Component

FMD in Deer Project Results

10 red deer were experimentally, intra-nasally inoculated with FMDV strain O UKG 11/2001, 106 TCID50

Of the 10 red deer, only one animal developed clinical signs:

Samples were collected over the course of infection over 4 weeks o Nasal swabs o Oral swabs o Probang samples o Whole bloods o Clotted bloods o Lesion samples if present Samples were tested 1 animal euthanized and PM at dpi 5. 6 animals were re-inoculated i.m. 108 TCID50 at dpi 14. 9 animals euthanized and PM at dpi 28, 29 or 30.

FMD in Deer Project Results

Results to date Red deer are difficult to infect only 10% became infected FMDV IRES TagMan rRT-PCR works well FMDV 3D TagMan rRT-PCR works well O-type blocking ELISA (Prionics) works well comparable to VNT NSP competitive ELISA (Prionics) works well not others Rapid FMD NSP Ab test (BioNote) useful Interesting results after i.m. re-inoculation DSP:

378

3D rRT-PCR IRES rRT-PCR SPO-ELISA-PR NSP-ELISA-PR

100% 100% 99.9% 99.8%

n = 200 serum and nasal swabs n = 200 serum and nasal swabs n = 950 n = 950

Thank you! More results at the poster!

Evaluation of air samplers for the detection and quantification of airborne foot and mouth disease Claire Colenutt The Pirbright Institute

7 1

Limits of detection

Sampler sensitivity

qPCR

Virus Isolation

Impinger

104 103

101

AirPort

BioBadge Biosampler

Cyclone

105

104

103

102

Spike concentration

May

101

Filters

3 2

106

Foils

102

100

Impinger

Filters

105

Expected

Foils

TCID50/ml

Relative TCID50/ml

106

AirPort MD8

Expected

107

Cyclone

0 106

105 104 103 102 Spike concentration

101

ELPI+ Samplers used in previous studies: sensitivity for detection of virus aerosols at different stages of infection 2

379

Application of study Quantifying physical and biological efficiency of air sampling devices Application of this knowledge to account for the effect of post sampling processes on samples collected in the field or from experimentation

Evolution of FMDV during persistence in African buffalo (Syncerus caffer) Martí Cortey1, Francois Maree2, Lin-Mari de Klerk-Lorist3, Eva Pérez1, Fuquan Zhang1, Louis van Schalkwyk3, Dave Cooper4, Roy Bengis4, Bryan Charleston1, Nick Juleff1 1Viral Immunology Group, The Pirbright

Institute, UK Veterinary Institute-Transboundary Animal Diseases Programme, Onderstepoort, South Africa Veterinary Services, Skukuza, South Africa 4Ezemvelo KZN Wildlife, St Lucia, South Africa 2Onderstepoort 3State

Previous knowledge (Juleff et al. 2008, 2012)

African buffalo study

FMDV is maintained in the light zone of GCs Likely in association with FDCs Non-replicating state (viral capsid and genome, no NSP) This finding could explain FMDV persistence despite the high level of neutralising antibody Is this a mechanism for FMDV persistence? Immune complexed FMDV is able to infect FcR expressing cells ex vivo and in vitro. Potential for low-level replication (macrophages, B cells, DCs?) 2

380

Virus isolation and real-time qRT-PCR

VNT: homologous and heterologous neutralization

All animals are protected (from day 14 to day 400 pi) against all virus isolates, Ab titers > 45 (log2= 5.5).[ No immune escape] 4

Laser Micro Dissection (LMD) Tissue (PhT, PtT, DSP)

Sanger Sequencing

Cryostat

24 Minipreps per GC/Epi

Staining

Nucleotide Diversity ( : average number of nucleotide differences per site between any two DNA sequences chosen randomly from the sample population

Cloning

Select Germinal Centers and PCR of the ~SAT1-VP1 Epitheliums (Crypts) RNA extraction cDNA PCR of the ~SAT1-VP1

Dilution (Ct~35) qPCR Positive 6

381

MJ Network Consensus

Nucleotide Diversity ( : average number of nucleotide differences per site between any two DNA sequences chosen randomly from the sample population

MJ Network_Buffalo44_400Dpi_Aa

MJ Network_Buffalo19_35Dpi_Aa

382

Population PCA

Analysis of Molecular Variance(AMOVA) Buffalo 19 (PhT/PtT/DSP)

1. Are there significant differences among tissues?

2. Are there significant differences among GC/Epi/Cr within the tissues ? 3. Are there significant differences among GC/Epi/Cr

4. Are there significant differences within GC/Epi/Cr?

Yes

---------------------------------------------------------------------Source of Sum of Variance Percentage variation d.f. squares components of variation ---------------------------------------------------------------------Among groups 2 4.030 -0.00240 Va -0.36 Among populations within groups

32.598

0.07365 Vb

11.03

Pharyngeal Tonsil (GC vs Epi)

---------------------------------------------------------------------Source of Sum of Variance Percentage variation d.f. squares components of variation ---------------------------------------------------------------------Among groups 1 4.621 0.06368 Va 9.55 Among populations within groups

3.005

0.01745 Vb

2.62

Yes 13

Analysis of Molecular Variance(AMOVA) Buffalo 44 (PhT/PtT/DSP)

Pharyngeal Tonsil (GC vs Epi) Palatine Tonsil (GC vs Epi)

Implications

Palatine Tonsil (GC vs Epi)

---------------------------------------------------------------------Source of Sum of Variance Percentage variation d.f. squares components of variation ---------------------------------------------------------------------Among groups 1 2.097 0.00955 Va 1.52 Among populations within groups

5.705

0.03615 Vb

5.74

Within populations 383 228.345 0.59620 Vc 89.32 ---------------------------------------------------------------------Total 399 264.973 0.66746 ---------------------------------------------------------------------Fixation Indices FSC : 0.10996 FST : 0.10676 FCT : -0.00359 ----------------------------------------------------------------------

Within populations 114 66.777 0.58576 Vc 87.83 ---------------------------------------------------------------------Total 118 74.403 0.66690 ---------------------------------------------------------------------Fixation Indices FSC : 0.02893 FST : 0.12166 FCT : 0.09549 ----------------------------------------------------------------------

Within populations 134 78.204 0.58362 Vc 92.74 ---------------------------------------------------------------------Total 139 86.007 0.62932 ---------------------------------------------------------------------Fixation Indices FSC : 0.05833 FST : 0.07263 FCT : 0.01518 ----------------------------------------------------------------------

Significance tests (1023 permutations) ------------------

Vc and FST : P(rand. value < obs. value) = P(rand. value = obs. value) = P-value =

0.00000 0.00000 0.00000+-0.00000

Vc and FST : P(rand. value < obs. value) = P(rand. value = obs. value) = P-value =

0.00000 0.00000 0.00000+-0.00000

Vc and FST : P(rand. value < obs. value) = P(rand. value = obs. value) = P-value =

0.00098 0.00000 0.00098+-0.00098

Vb and FSC : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.00000 0.00000 0.00000+-0.00000

Vb and FSC : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.08798 0.00000 0.08798+-0.00787

Vb and FSC : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.00782 0.00000 0.00782+-0.00242

Va and FCT : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.62952 0.00000 0.62952+-0.01438

Va and FCT : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.09286 0.09971 0.19257+-0.01155

Va and FCT : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.10068 0.09189 0.19257+-0.01322

DSP (GC vs Epi)

---------------------------------------------------------------------Source of Sum of Variance Percentage variation d.f. squares components of variation ---------------------------------------------------------------------Among groups 1 5.727 0.04764 Va 6.27 Among populations within groups

11.442

0.09504 Vb

12.50

Within populations 135 83.363 0.61750 Vc 81.23 ---------------------------------------------------------------------Total 140 100.532 0.76018 ---------------------------------------------------------------------Fixation Indices FSC : 0.13338 FST : 0.18769 FCT : 0.06267 ---------------------------------------------------------------------Significance tests (1023 permutations) -----------------Vc and FST : P(rand. value < obs. value) = P(rand. value = obs. value) = P-value =

0.00000 0.00000 0.00000+-0.00000

Vb and FSC : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.00000 0.00000 0.00000+-0.00000

Va and FCT : P(rand. value > obs. value) = P(rand. value = obs. value) = P-value =

0.14467 0.06256 0.20723+-0.01413

1. Are there significant differences among tissues?

2. Are there significant differences among GC/Epi/Cr within the tissues ?

3. Are there significant differences among GC/Epi/Cr?

Yes

4. Are there significant differences within GC/Epi/Cr?

Yes 14

383

Thank you very much for your attention!!! Acknowledgements The Pirbright Institute Don King Nick Knowles Graham Fermianis

Genetic characterization of circulating foot-andmouth disease viruses from African buffalo (syncerus caffer) and cattle in Kenya: evidence for independent virus populations Sabenzia Wekesa, Abraham Sangula*, Graham Belsham, Kirsten Tjornehoj, Vincent Muwanika, Francis Gakuya, Dominic Mijele, Hans Siegismund

* = Presenter Foot-and-Mouth Disease Lab Embakasi 16

Introduction

Objectives

1. To determine the presence/and characterise FMDV in selected buffalo populations in Kenya

FMD is Endemic in Kenya; Records since 1954 Serotypes O, A, SAT 1 and SAT 2 currently circulating >150 outbreaks (most in cattle) confirmed annually AgELISA/PCR/Sequencing essential in effective disease control Large buffalo populations usually interact with livestock in Kenya What is the impact? The African buffalo (Syncerus caffer) - reservoir for SAT serotypes of FMD viruses 2

384

To investigate the serotypes of FMDV in these buffalo populations

Genetic characterization of buffalo isolates, alongside cattle isolates from Kenya and compare with others in GenBank 3

Materials and Methods

Materials and Methods(2)

In 2012, sera and corresponding probang samples from 102 buffalos collected

(Maasai-Mara ecosystem (MME) (n = 40), Tsavo ecosystem (TSE) (n = 33), and Meru ecosystem (ME) (n=29)

47 cattle tongue epithelia from clinically diseased cattle, collected between 2008 and 2012

Materials and Methods(3) Serological assays on buffalo sera: (NSP-ELISA (Screening), LPBE (SP serotype specific) and VNT (serotype-specific & confirmatory) Virological tests on buffalo probangs and cattle epithelia: Virus isolation (live virus detection) Ag-ELISA (serotype Id) real time RT-qPCR (RNA detection) partial genome (VP1 coding region) sequencing and phylogenetic inferences.

BUFFALO Sampling Sample types; Probangs - for virology Blood- Serum (for serology)

Results - Buffalo Seroprevalence 64% (95% CI = 61 - 74%) with 44/102 and 53/102 also having neutralising antibodies (by VNT) directed against FMDV SAT 1 and SAT 2, respectively FMDV RNA was detected in 42% (95% CI = 38 - 48%) of the buffalo probang (reservoir)

385

Two buffalo probang samples were positive by VI and were identified as FMDV SAT 1 and SAT 2 by AgELISA

Cattle Vs buffalo results AgELISA detected serotypes O (1), A (20), SAT 1 (7) and SAT 2 (19) in the 47 cattle epithelia The VP1 coding sequences were generated for 2 buffalo and 21 cattle samples

Cattle Vs buffalo results(2) SAT 2 virus lineages within buffalo were distinct from those circulating in cattle

SAT 1 TSE buff. Strain 2012

Current SAT 1 vaccine strain

Phylogenetic analyses revealed SAT 1 and SAT 2 virus lineages within buffalo that were distinct from those circulating in cattle

Current SAT 2 vaccine strain

Recommendations

Conclusions

Control efforts should focus primarily on reducing FMDV circulation among livestock and subsequently limiting interaction with buffalo

FMDV O, A, SAT 1 and SAT 2 are constantly circulating among cattle in Kenya and causing disease Only SAT 1 and SAT 2 viruses were isolated from clinically normal buffalo Buffalo viruses were genetically distinct from those found circulating in cattle Both cattle and buffalo viruses were genetically distinct from current vaccine strains Evidence for independent virus cycles (wild and Domestic)

MME 2012 buff strain

386

Comprehensive studies - additional buffalo viruses

Acknowledgement DANIDA TADEA project Govts of Kenya (DVS,KWS), Uganda- MAAIF Makerere Univ. Uganda, University of Copenhagen, Danish Tech. University-Lindholm FMD lab. Embakasi colleagues

Ugandan results from the TADEAFMD-project 2011-2013 multiple serotypes identified. (Transboundary Diseases in East Africa)

- funded by the Danish International Development Agency (DANIDA).

AHSANTE

Presented by: Kirsten Tjørnehøj

END

Department of Virology, Lindholm 12

TADEA-project:

Conclusions:

2011-2014 5 Institutes:

Five FMDV serotypes were isolated from Ugandan cattle during 2011-2013:

Makerere University, Uganda National Animal Disease Diagnostics and Epidemiology Centre, Uganda Kenya FMD laboratory, Embakasi, Kenya DTU National Veterinary Institute, Lindholm, Denmark Copenhagen University, Denmark

2011 outbreaks: Two different O FMDVs: Southern and Eastern regions 2013 outbreaks: One A FMDV: Central region, 2013 One SAT 2 FMDV: Southwestern region, 2013 Serological evidence for: » Serotype O: 2 outbreaks » Serotype SAT 1: 1 outbreak » Serotype SAT 2: 3 outbreaks

3 PhD-students: 2 Ugandan and 1 Kenyan Uganda: -

Kenya:

Chris Ayebazibwe - Abraham Sangula Vincent Muwanika - Sabenzia Wekesa Alice Namatovu Moses Dhikusooka

Denmark: - Hans Siegismund - Graham Belsham - Kirsten Tjørnehøj

2011 random sampling: One SAT 1 FMDV: Southwestern region, 2011 2012 sentinel herd grazing inside national park: One SAT 3 FMDV: Southwestern region, 2012

DTU Vet, Technical University of Denmark

387

DTU Vet, Technical University of Denmark

2011: Serotype O FMDV isolated and VP1 sequenced from four outbreaks : Investigated outbreaks in 7 districts:

Bukedea Kumi Sembambule Rakai.

- 13/80 OPs from 4 districts positive by VI/Ag-ELISA/RT-qPCR

Serological investigation:

- 72/218 sera positive i PrioCHECK FMDV NS®

VNT: 49/72 serotype O

DTU Vet, Technical University of Denmark

Ugandan 2011 serotype O FMDV outbreak isolates.

DTU Vet, Technical University of Denmark

Currently being reviewed.

Characterization of foot-and-mouth disease viruses from Ugandan cattle outbreaks during 2012-2013: Evidence for circulation of multiple serotypes

Alice Namatovu1,2, Kirsten Tjørnehøj3, Graham J. Belsham3*, Moses T. Dhikusooka1, Sabenzia N. Wekesa4,5, Vincent B. Muwanika4, Hans R. Siegismund6 and Chrisostom Ayebazibwe1

2012: investigated outbreaks in 3 districts serologically: - 25/33 sera positive i PrioCHECK FMDV NS®

2013: Investigated outbreaks in 4 districts: - 6/16 tissues + 1/30 OPs + 1/14 oral swabs (OS) from 2 districts positive by VI/Ag-ELISA, RT-qPCR: Ct<25 - 36/46 sera positive i PrioCHECK FMDV NS®

DTU Vet, Technical University of Denmark

388

DTU Vet, Technical University of Denmark

Ugandan 2013 serotype SAT 2 FMDV VP1 sequences.

FMDV isolated and VP1 sequenced from two 2013 outbreaks: - SAT 2: Isingiro - A:

Wakiso

Serological investigation: 2012: VNT: Kiruhura: SAT 2 Kween: O Nwoya: SAT 1 2013: VNT: Isingiro: SAT 2 Ntungamo: SAT 2 Rakai: O 8

DTU Vet, Technical University of Denmark

Ugandan 2013 serotype A FMDV VP1 sequences.

DTU Vet, Technical University of Denmark

Preliminary results from random sampling in Kasese district

Unrecognized circulation of SAT 1 foot-and mouth disease virus in cattle herds around Queen Elizabeth National Park in Uganda Moses Tefula Dhikusooka1, Chrisostom Ayebazibwe1, Alice Namatovu1,2, Graham J. Belsham3, Hans Redlef Siegismund4, Sabenzia Nabalayo Wekesa 5,6, Sheila Nina Balinda6, Vincent B. Muwanika6, and Kirsten Tjørnehøj3**

- 24 settled pastoralists living close to National Park in Kasese district - 247 6-24-month old cattle sampled:

DTU Vet, Technical University of Denmark

389

2 OPs from 1 herd positive by VI/Ag-ELISA, RT-qPCR: Ct 16/19

37 sera from 14 herds positive i PrioCHECK FMDV NS®

DTU Vet, Technical University of Denmark

Ugandan 2011 serotype SAT 1 FMDV VP1 sequences.

Serotype SAT 1 FMDV isolated and VP1 sequenced from two cattle without clinical symptoms of FMD.

Serological investigation (SPBE/VNT): 3 herds: O 2 herds: SAT 1 1 herd:

(O/SAT 2)

DTU Vet, Technical University of Denmark

Conclusions:

Emerg Infect Dis [Internet]. 2015 Jan

Five FMDV serotypes were isolated from Ugandan cattle during 2011-2013: 2011 outbreaks: Two different O FMDVs: Southern and Eastern regions 2013 outbreaks: One A FMDV: Central region, 2013 One SAT 2 FMDV: Southwestern region, 2013 Serological evidence for: » Serotype O: 2 outbreaks » Serotype SAT 1: 1 outbreak » Serotype SAT 2: 3 outbreaks

- As part of sentinel herd survey in national park SAT 3 was isolated from asymptomatic calf.

2011 random sampling: One SAT 1 FMDV: Southwestern region, 2011 2012 sentinel herd grazing inside national park: One SAT 3 FMDV: Southwestern region, 2012 14

DTU Vet, Technical University of Denmark

390

DTU Vet, Technical University of Denmark

Introduction The South African Territories (SAT) type foot-and-mouth disease viruses (FMDV) are endemic to the greater Kruger National Park (KNP) area in South Africa, where it is maintained through persistent infections of African buffalo The occurrence of FMDV within the KNP constitutes a continual threat to the livestock industry

Emergence of antigenic variants of SAT2 FMDV at the wildlife/livestock interface in South Africa

FMD outbreaks are costly and require extensive logistical efforts SAT types display high genomic and antigenic variation: implications for the control of the disease by vaccination Several outbreaks have occurred in South Africa since the turn of the century emphasising the need for ongoing disease surveillance

Blignaut, B.1, van Heerden, J.1, Reininghaus, B.2, Heath, L.1 & Fosgate, G.T.3

To expand on knowledge of FMDV diversity, the genetic and antigenic relatedness of SAT2 type viruses isolated from cattle in 2013/2014 were investigated

1Transboundary

Animal Diseases Programme, Onderstepoort Veterinary Institute, Agricultural Research Council, Onderstepoort, 0110, South Africa, 2Mpumalanga Veterinary Services, Orpen, Mpumalanga, South Africa, 3Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, 0110, South Africa

Aims

Recent SAT2 FMD outbreaks

Molecular epidemiological relationships of the viruses were determined by sequencing and phylogenetic analysis Genetically disparate viruses were chosen to determine neutralisation titres between outbreak viruses using virus neutralisation tests (VNT) against four reference sera

SADC

Antigenic relatedness (r1-values) of the outbreak viruses and the most suitable vaccine match was determined

South Africa

Zimbabwe Zambia

2001

Botswana Namibia

2003-2004

Mozambique Malawi

2011-2014

South Africa

391

Geographic distribution of the SAT2 types 27

SAT2 outbreak South Africa 2013/2014

KNP/16/93/Capricorn

KNP/5/06/2/Nwambi_block 25

KNP/3/10/Banyini_Pan

KNP/9/03/Ribye-Waterhole

MOZ/1/10/Gaza_Province

SAR/6/04/Poporoga-diptank

KNP/11/07/Bububu KNP/6/96

KNP/1678/98/Bume SAR/01/01/Mhala-district

KNP/12/08/Lower_Sabie

KNP/07/88

KNP/19/89 KNP/31/95

SAR/11/1919/Luphisi_DT 89

KNP/1/11/Impala 95

SAR/12/0050/Huntingdon_DT SAR/15/13/2/Welverdiend A

Topotype 1 South Africa Kruger National Park Mozambique

Kruger National Park

SAR/5/13/2/Hlalakahle DT SAR/11/13/2/Seville B

63 99

SAR/3/13/2/Hlalakahle DT SAR/1/13/2/Hlalakahle DT SAR/2/13/2/Hlalakahle DT

SAR/14/13/2/Welverdiend A

SAR/17/13/2/Welverdiend B SAR/18/13/2/Welverdiend B

SAR/16/13/2/Welverdiend B SAR/2/14/2/Athol

SAR/19/13/2/Eglington 11

SAR/4/14/2/Islington SAR/1/14/2/Athol SAR/3/14/2/Athol

KNP/5/91/Satara 95

SAR/1/03/Masisi

26 42

SAR/1/08/Maruleng/buffalo KNP/04/03/Punda-Maria

SAR/1/10/Limpopo_Safaris/buffalo ZIM/08/94 KNP/141/91

21 99

KNP/32/92/Boyela ZAM/9/93

ZAM/7/96

BOT/31/98/Vumbura 98

NAM/4/07/Caprivi ZIM/16/91/Matusadona

21 24

ZIM/267/98/Chizarira

Topotype 2 Namibia Botswana Zambia Zimbabwe Malawi

ZIM/01/00

ZIM/14/90/DomaSafariarea 66

ZIM/34/91 BOT/13/02

ZIM/01/01/Bulawayo

99 36

ZIM/01/02/Beitbridge

ZIM/7/95/Sengwa 99

NAM/01/92

NAM/3/10/buffalo NAM/286/98

ZIM/05/02/Lupane 99

BOT/18/98

BOT/1/98/Nxaraga ZIM/07/83/Nyamandlovu

ZIM/1/97/Chiredzi

ZIM/1/88/Hwange

99 97 75

0.02

ZIM/4/88

Topotype 3 Namibia Botswana Zimbabwe

ZIM/7/89

Neighbour joining tree depicting SAT2 viruses from topotypes 1, 2 and 3 5

SAT2 antibody titres (ELISA)

Genetic relatedness of SAT2 outbreak viruses KNP/16/93/Capricorn

27 50

KNP/5/06/2/Nwambi_block KNP/3/10/Banyini_Pan

KNP/9/03/Ribye-Waterhole 29

MOZ/1/10/Gaza_Province

2.5

SAR/6/04/Poporoga-diptank

KNP/11/07/Bububu

KNP/6/96 37

KNP/1678/98/Bume

2.0

SAR/01/01/Mhala-district KNP/12/08/Lower_Sabie

Topotype 1 South Africa Kruger National Park Mozambique

KNP/07/88

99 52

KNP/19/89 KNP/31/95

Antibody titre (log 10)

2011 v 2012

SAR/11/1919/Luphisi_DT

KNP/1/11/Impala

SAR/12/0050/Huntingdon_DT

SAR/15/13/2/Welverdiend A SAR/5/13/2/Hlalakahle DT SAR/11/13/2/Seville B 63 99 SAR/3/13/2/Hlalakahle DT

SAR/1/13/2/Hlalakahle DT SAR/2/13/2/Hlalakahle DT 40 SAR/14/13/2/Welverdiend A

2013

1.5

1.0

0.5

SAR/17/13/2/Welverdiend B 45 29

SAR/16/13/2/Welverdiend B

0.0

SAR/2/14/2/Athol

53 11

SAR/18/13/2/Welverdiend B

SAR/19/13/2/Eglington

SAR/4/14/2/Islington SAR/1/14/2/Athol

2014

* * * * *

Animal number

SAR/3/14/2/Athol

* * *

KNP/5/91/Satara 95

SAR/1/03/Masisi

SAR/1/08/Maruleng/buffalo

KNP/04/03/Punda-Maria SAR/1/10/Limpopo_Safaris/buffalo

ZIM/08/94 KNP/141/91

KNP/32/92/Boyela

99 99 98

ZAM/9/93 ZAM/7/96

Neighbour joining tree depicting the SAT2 outbreak strains characterised from cattle, Mpumalanga Province 7

392

SAT2 neutralising antibody titres

Antigenic profiles of SAT2 outbreak viruses

4 SAR/5/13/2/Hlalakahle DT SAR/11/13/2/Seville B 57

SAR/3/13/2/Hlalakahle DT

100%

3.5

SAR/2/13/2/Hlalakahle DT 30 SAR/1/13/2/Hlalakahle DT

90%

SAR/14/13/2/W elverdiend A SAR/16/13/2/W elverdiend B 42 28

SAR/18/13/2/W elverdiend B SAR/2/14/2/Athol 99 53

SAR/19/13/2/Eglington SAR/4/14/2/Islington

46 SAR/1/14/2/Athol

SAR/3/14/2/Athol SAR/15/13/2/W elverdiend A

SAR/3/04/2

SAR/6/04/Poporoga-diptank

KNP/1/10/2

KNP/3/10/Banyini_Pan

51 37

SAR/1/01/2 KNP/19/89/2

SAR/11/1919/Luphisi_DT

KNP/1/11/Impala

75 90

Neutralising antibody titre

SAR/17/13/2/W elverdiend B

ZIM/14/90

80%

ZIM/7/83

70%

2.5

KNP/19/89

60% 50%

SAR/3/04

40% 1.5

30% 20%

10%

SAR/12/0050/Huntingdon_DT

SAR/1/10/Limpopo_Safaris/buffalo

0.5

SAR/1/03/Masisi

SAR/1/08/Maruleng/buffalo ZIM/7/83/2 BOT/4/06/2

ZIM/17/91/2

SAR/1/13

ZIM/14/90/2

0.05

12 SAR 2014

SAR 2013

SAR 2014

SAR 2004

Animal number

SAR 2004

KNP 2010

SAR/13/13

SAR/15/13

SAR/4/14

BOT 2006

Summary

Vaccine match of SAT2 outbreak viruses

Phylogenetic analysis of the recent outbreak viruses revealed their genetic relatedness to other SAT2 isolates from topotype I (South Africa, Zimbabwe and Mozambique)

1.00 0.90

The recent SAT2 outbreak viruses were genetically distinct from previously isolated viruses (2011 and 2012) and form an outgroup within the topotype I viruses

0.80

r1-values

0.70 0.60

Neutralising antibody responses were observed for samples from the outbreak when tested against viruses from South Africa, the KNP and Botswana

0.50 0.40

High neutralisation titres were observed for all outbreak viruses tested against the reference sera representative of viruses from the endemic area in South Africa, as well as Zimbabwe

0.30 0.20 0.10

Cross-neutralisation data for the SAT2 outbreak viruses yielded different antigenic profiles

0.00

SAR/1/13 SAR/3/04

SAR/13/13 KNP/19/89

SAR/15/13

SAR/4/14

ZIM/7/83

ZIM/14/90

Comparison of the outbreak viruses with reference sera indicated a good vaccine match with 75% of r1-values > 0.4, 13% of r1-values between 0.3 and 0.4, and 13% of r1-values < 0.3

393

Acknowledgements Transboundary Animal Diseases Programme (ARC-OVI) Department of Agriculture, Forestry and Fisheries Mpumalanga Veterinary Services

CHALLENGES FOR FMDV DIAGNOSIS outbreak confirmation in Uganda (2011 2013)

National Research Foundation of South Africa (Grant Number 90578)

Chrisostom Ayebazibwe1, Alice Namatovu1,3, Moses Tefula Dhikusooka1, Graham J. Belsham2, Vincent Muwanika4, Hans Redlef Siegismund5 and Kirsten Tjørnehøj2. Transboundary Animal Diseases in East Africa (TADEA) Project, National Animal Diseases Diagnostics and Epidemiology Centre (NADDEC), Ministry of Agriculture Animal Industry and Fisheries, Uganda

Introduction

Results

FMD is endemic in Uganda (FMDV serotypes include: O, A, SAT 1, SAT 2 & SAT 3) Vaccination is largely applied to contain outbreaks Rapid serotype identification is key for decision making on the choice of vaccines to order Ag ELISA and PCR & sequencing have been utilized in Uganda for FMDV serotyping with variable success This abstract analysed FMD outbreak investigation & confirmation in Uganda (2011 2013)& discussed the challenges involved 2

No. of outbreaks investigated (Districts) 7

Stage of outbreaks at sampling Midway

No. of outbreaks confirmed (Districts) 4

Serotype(s) Serotype (s) identified by Ag Confirmed ELISA RT-PCR & Sequencing 4 (O)

2012

Resolving

2013

Onset

A, SAT 2

2 (A), 5 (SAT 2)

Midway

Year

No. of districts affected

2011

2014* 23

*: By 14th July, 2014; -

14th July samples)

More than 50% of the FMDV outbreaks were neither investigated nor confirmed. 394

Discussion

Conclusion/Recommendation

Efficient diagnostic tests & test programmes are necessary for FMD confirmation FMD diagnosis in Uganda is limited by delayed investigations and lack of appropriate samples. Antigen ELISA results can be obtained in less than 1 week compared to RT-PCR and sequencing where the results may take over 1 month due to cell culture and sequencing services abroad. Long term and sustained investments in FMD diagnosis in developing countries inevitable

Availability of affordable, robust pen side serotypespecific tests may offer lasting solutions to FMD diagnosis in endemic countries.

The recently Constructed Biosecurity Level 3 Laboratory at NADDEC to play a big role in control of FMD

Acknowledgement DANIDA for funding TADEA project Collaborators on the project: 1. 2. 3.

4. 5.

395

National Animal Disease Diagnostics and Epidemiology Centre, Ministry of Agriculture Animal Industry and Fisheries, Uganda; National Veterinary Institute, Technical University of Denmark, Denmark; Department of Biotechnical and Diagnostic Sciences, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Uganda; Department of Environmental Management, College of Agricultural and Environmental Sciences, Makerere University, Uganda, Department of Biology, University of Copenhagen, Denmark.

END THANK YOU!!!!

FMDV is endemic in East Africa East Africa includes: Ethiopia Somalia Kenya Uganda Eritrea Tanzania

Antigenic and Genetic Characterization of FMD Serotype O Virus Circulating in East Africa.

5/7 serotypes in East Africa (O, A, SAT-1, SAT-2, SAT-3)

Katie Lloyd-Jones, Mana Mahapatra, Aravindh Babu, Sasmita Upadhyaya, Fufa Bari, David Paton and Satya Parida

Serotype O is the most prominent Control Policy vaccination is in practice but using older vaccines

The Pirbright Institute

(WRL website)

Viruses selected 2005-2012 AIM:

EA-1

To test existing vaccine strains for efficacy and recommend relevant vaccine strains for use in East Africa

Eritrea Ethiopia Somalia Kenya Uganda Tanzania

EA-2

2 2 2

EA-1

EA-2 2

EA-3 4 23 3

EA-4 1 1

ME-SA TOTAL 4 23 3 6 2 2

Approach: Recent FMDV circulating in East Africa were randomly selected 3 new candidate vaccine strains were chosen.

Zambia Sudan Libya Egypt

R1 values calculated by 2D-VNT using the new candidate vaccine sera and existing vaccine strains.

396

EA-3 10 1

EA-4

ME-SA

9 7

TOTAL 2 10 10 7

Genetic characterization of FMD field viruses (full capsid sequence)

O-ETH-03-2009

O-ETH-11-2009

100

O-ETH-44-2009 100

O-ETH-49-2009 O-ETH-39-2009 67

O-ETH-54-2006

100

O-ETH-03-2004

100

Virus neutralization Tests against 4 sera:

O-ETH-01-2007 O-ETH-29-2008

O-ETH-07-2010

EA-3

O-ETH-10-2011

100

O-ETH-05-2011

100 69

O-ETH-42-2011

O-ETH-31-2008 O-SOM-02-2007

99 100

O-SOM-04-2007 O-EA-2010 O/ETH/3/96/EU919240

100

Commonly used vaccine strain: O/EA/1978 (EA-1)

O-SUD-01-2009

O-SUD-01-2010 O-SUD-09-2010

100

O-LIB-54-2012

O-ERI-01-2011

100 68

O-ERI-08-2011

O/K/52/1992/HM625674 O/UGA /5/96/EU919247

O/KEN/10/95/EU919242

O-EA-1978

100

O-KEN-37-2008

EA-1

Chose 3 candidate new vaccine strains: Candidate vaccine strain O/EA/2002 (EA-2) Candidate vaccine strain O/EA/2009 (EA-2) Candidate vaccine strain O/EA/2010 (EA-3)

61 O-KEN-125-2009

O-KEN-100-10

95 99

EA-4

O-ETH-58-2005/FJ798141 O/UGA /6/76/EU919246 60 O-EGY -03-2008 94 O-EGY -07-2008 98

Sh-72

O-EGY -06-2009

O-EGY -17-2009

100

O-EGY -10-2006_

ME-SA

O-EGY -23-2009 71 O-EGY -29-2009 96

O-LIB-04-2010 O-LIB-02-2012 100

38 O-LIB-05-2012 99

PA-2

O-LIB-48-2012

O-LIB-29-2011

O-LIB-33-2011

58 O-LIB-25-2011 68 O-LIB-07-2012

O-TAN-16-2008 O-KEN-01-2011 O/K/117/1999/HM625676

100 53

O/EA/2009

EA-2

O/Ken/109/2000/HM625675 O/K/48/2005/HM625673 100 O-EA-2002

O-UGA-03-2002/DQ165077 O-ZAM-01-2010

O/U/25/2006/HM625677

O/U/312/2006/HM191257

100

0.02

O-ETH-03-2009

Genetic characterization of FMD field viruses (full capsid sequence)

VNT protection with the existing vaccine strain O/EA/1978

O-ETH-11-2009

100

O-ETH-44-2009

100

O-ETH-49-2009 O-ETH-39-2009 67

O-ETH-54-2006

100

100 100

O-ETH-03-2004 O-ETH-01-2007 O-ETH-29-2008

O-ETH-07-2010

EA-3

O-ETH-10-2011

100

O-ETH-05-2011

100 69

O-ETH-42-2011

O-ETH-31-2008 O-SOM-02-2007

99 100

O-SOM-04-2007 O-EA-2010 O/ETH/3/96/EU919240

100

O-SUD-01-2010 O-SUD-09-2010

100

O-LIB-54-2012

O-ERI-01-2011

0.9

O-ERI-08-2011

61 O-KEN-125-2009

EA-4

60 O-EGY -03-2008

EA-1

EA-2

58 O-LIB-25-2011 68 O-LIB-07-2012

O-TAN-16-2008 O-KEN-01-2011

O/Ken/109/2000/HM625675 O/K/48/2005/HM625673 100 O-EA-2002

EA-2

O-UGA-03-2002/DQ165077 O-ZAM-01-2010

O/U/25/2006/HM625677

99 100

0.02

EA-3

EA-2

ME-SA

O/EA/2009

EA-1

O/K/117/1999/HM625676

100

O/U/312/2006/HM191257

397

ME-SA

O/LIB/05/2012

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/ETH/03/2012

O/EGY/10/2006

O/ETH/42/2011

O/SUD/09/2011

O/ETH/29/2011

O/ETH/09/2011

O/ERI/08/2011

O/ETH/05/2011

O/ERI/02/2011

O/SUD/02/2010

O/ETH/07/2010

O/SUD/01/2010

O/ETH11/2009

EA-3

O-LIB-33-2011

O/SUD/01/2009

PA-2

O-LIB-48-2012

O-LIB-29-2011

O/NIG/15/2009

O/ETH/49/2009

38 O-LIB-05-2012 99

O/ETH/44/2009

O-LIB-04-2010 O-LIB-02-2012 100

O/ETH/03/2009

ME-SA

O-EGY -23-2009 71 O-EGY -29-2009 96

O/ETH/31/2008

Sh-72

O-EGY -06-2009 O-EGY -17-2009 O-EGY -10-2006_

O/ETH/29/2008

100

O/SOM/04/2007

O/SOM/02/2007

0.3

94 O-EGY -07-2008 98 49

O/ETH/01/2007

O/UGA /6/76/EU919246

0.6

O/SOM/01/2007

O-ETH-58-2005/FJ798141

O/ETH/54/2006

O-KEN-100-10

O/KEN/01/2011

O-KEN-37-2008

95 99

O/SUD/02/2005

100 O-EA-1978

O/TAN/16/2008

r1 value

EA-1

O/KEN/10/95/EU919242

O/ZAM/01/2010

O/UGA /5/96/EU919247

O/UGA/18/2007

O/KEN/37/2008

100

O/K/52/1992/HM625674

O/KEN/125/2009

1.2

O-SUD-01-2009

O/KEN/37/2008

0.00 EA-1

O/KEN/01/2011

EA-2

EA-1 EA-3

EA-2 ME-SA EA-3

O/LIB/05/2012

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

ME-SA

EA-1

398 EA-2

0.30 0.30

0.00 0.00

O/ETH/01/2007

O/ETH/54/2006

O/SUD/02/2005

EA-3 ME-SA EA-1 EA-2

O/ETH/01/2007

O/ETH/54/2006

EA-3

O/ETH11/2009 O/NIG/15/2009 O/ETH/07/2010

ME-SA

O/LIB/05/2012

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

O/EGY/06/2009

EA-3

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/NIG/15/2009

O/LIB/05/2012

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

O/ETH/07/2010

O/SUD/01/2009

EA-3

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

EA-2

O/SUD/01/2009

O/ETH11/2009

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

O/SOM/02/2007

O/SOM/01/2007

O/ETH/01/2007

O/ETH/54/2006

O/SUD/02/2005

O/KEN/01/2011

O/ZAM/01/2010

O/EA/1978

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

1.20

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

O/SOM/02/2007

O/SOM/01/2007

0.90

O/KEN/01/2011

O/EA/2010

O/SUD/02/2005

EA-1

O/ZAM/01/2010

0.00

O/TAN/16/2008

0.3

O/UGA/18/2007

1.20 O/KEN/37/2008

0.6

O/TAN/16/2008

ME-SA

O/UGA/18/2007

EA-2

O/KEN/125/2009

ME-SA

O/KEN/37/2008

EA-2

O/KEN/125/2009

0.60 r1 value

0.9 r1 value

EA-2

O/LIB/05/2012

EA-3

O/LIB/05/2012

EA-1

O/LIB/04/2010

EA-3

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

O/ETH/07/2010

O/SUD/01/2009

O/NIG/15/2009

O/ETH11/2009

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

EA-1

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

EA-3

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

O/ETH/07/2010

O/SUD/01/2009

EA-2

O/NIG/15/2009

O/ETH11/2009

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

1.20

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

1.2

O/SOM/02/2007

O/SOM/01/2007

O/ETH/01/2007

O/ETH/54/2006

O/SUD/02/2005

O/KEN/01/2011

O/ZAM/01/2010

EA-1

O/SOM/02/2007

0.60

O/SOM/01/2007

EA-1

O/KEN/01/2011

0.90 O/KEN/37/2008

0 O/TAN/16/2008

ME-SA

O/UGA/18/2007

VNT protection with O/EA/2002 O/KEN/125/2009

O/KEN/01/2011

O/LIB/05/2012

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

O/ETH/07/2010

O/SUD/01/2009

O/NIG/15/2009

O/ETH11/2009

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

O/SOM/02/2007

O/SOM/01/2007

O/ETH/01/2007

O/ETH/54/2006

O/SUD/02/2005

O/KEN/01/2011

O/ZAM/01/2010

O/TAN/16/2008

O/UGA/18/2007

O/KEN/125/2009

O/KEN/37/2008

O/LIB/05/2012

O/LIB/04/2010

O/EGY/29/2009

O/EGY/23/2009

O/EGY/17/2009

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

O/ETH/07/2010

O/SUD/01/2009

O/NIG/15/2009

O/ETH11/2009

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

O/SOM/02/2007

O/SOM/01/2007

O/ETH/01/2007

O/ETH/54/2006

O/SUD/02/2005

r1 value

0.60

O/KEN/37/2008

O/TAN/16/2008

0.90

O/TAN/16/2008

0.00

O/ZAM/01/2010

0.90

O/UGA/18/2007

0.00 O/UGA/18/2007

1.20

O/ZAM/01/2010

r1 value

0.30

O/KEN/37/2008

0.30

O/KEN/125/2009

r1 value

1.20

O/KEN/125/2009

0.30 r1 value

EA-3

O/EGY/17/2009

O/EGY/06/2009

O/EGY/03/2008

O/EGY/07/2008

O/EGY/10/2006

O/ETH/03/2012

O/SUD/09/2011

O/ETH/42/2011

O/ETH/29/2011

O/ETH/09/2011

O/ETH/05/2011

O/ERI/08/2011

O/ERI/02/2011

O/SUD/02/2010

O/SUD/01/2010

O/ETH/07/2010

O/SUD/01/2009

EA-2

O/NIG/15/2009

O/ETH11/2009

O/ETH/49/2009

O/ETH/44/2009

O/ETH/03/2009

O/ETH/31/2008

O/ETH/29/2008

O/SOM/04/2007

O/SOM/02/2007

O/SOM/01/2007

O/ETH/01/2007

O/ETH/54/2006

O/SUD/02/2005

EA-1

O/ZAM/01/2010

O/TAN/16/2008

O/UGA/18/2007

O/KEN/125/2009

r1 value

VNT protection with O/EA/2009 VNT protection with O/EA/2010

0.60

ME-SA

All vaccine strains 1.20

O/EA/2009

0.90

0.60

0.30

ME-SA

O/EA/2002

0.90

0.60

O/EA/2002 is protective

SUMMARY

All vaccine strains are broadly protective where protection predicted by R1 values >0.3 If look at R1 values where 1 is most protection the most protective is O/EA/2002

ACKNOWLEDGEMENTS: O/EA/1978 O/KEN/37/2008 O/KEN/125/2009 O/UGA/18/2007 O/TAN/16/2008 O/ZAM/01/2010 O/KEN/01/2011 O/SUD/02/2005 O/ETH/54/2006 O/ETH/01/2007 O/SOM/01/2007 O/SOM/02/2007 O/SOM/04/2007 O/ETH/29/2008 O/ETH/31/2008 O/ETH/03/2009 O/ETH/44/2009 O/ETH/49/2009 O/ETH11/2009 O/NIG/15/2009 O/SUD/01/2009 O/ETH/07/2010 O/SUD/01/2010 O/SUD/02/2010 O/ERI/02/2011 O/ERI/08/2011 O/ETH/05/2011 O/ETH/09/2011 O/ETH/29/2011 O/ETH/42/2011 O/SUD/09/2011 O/ETH/03/2012 O/EGY/10/2006 O/EGY/07/2008 O/EGY/03/2008 O/EGY/06/2009 O/EGY/17/2009 O/EGY/23/2009 O/EGY/29/2009 O/LIB/04/2010 O/LIB/05/2012

Funded by:

399

O/EA/2002

O/EA/2009

O/EA/2010

PROTECT BORDERLI NE FAIL

EA-1 EA-2 EA-3 ME-SA

ANTIGENIC AND GENETIC CHARACTERIZATION OF FOOT-AND-MOUTH DISEASE VIRUS SEROTYPE O CIRCULATING IN EAST AFRICA Katie Lloyd-Jones, Mana Mahapatra, Aravindh Babu, Sasmita Upadhyaya, Fufa Bari , David Paton, and Satya Parida The Pirbright Institute, Pirbright Laboratory, Ash Road, Woking, Surrey, GU24 0NF, UK Introduction: Foot-and-mouth disease (FMD) is one of the most economically important livestock diseases. The disease is endemic across Africa, with five of the seven known FMDV serotypes circulating in East Africa. Despite this, there is no effective control policy except ring vaccinations in selected dairy farms. The vaccine strains used in the region are out of date and do not match. Here we report the genetic and antigenic characterization of serotype O FMD viruses circulating in East Africa with a view to recommending suitable vaccine strains for use in the region. Materials and methods: Two-dimensional virus neutralisation tests (VNT) were carried out using four different bovine post-vaccinal sera including one current vaccine strain and three putative vaccine strains and 50 FMDV serotype O viruses isolated from six East African countries and three neighbouring and livestock-trade-related countries, and results represented as antigenic relationship (r1) values. In addition, full capsid sequence data was generated for all the viruses used in this study. Results: Phylogenetic analysis revealed circulation of mainly East Africa (EA) topotype viruses in East African countries. In addition Middle East and South Asian (ME-SA) topotype viruses are also circulating in Libya and Egypt. Within East Africa topotype of all four sublineages (EA-1 to 4) of FMDV serotype O were detected. Preliminary vaccine matching results indicate all three putative novel vaccine strains were broadly protective with East African serotype O FMD viruses compared to the locally produced O-KEN 78 vaccine strain. There was no linear correlation between r 1 values and no. of capsid amino acid changes. Discussion: The serology and capsid sequence data will now be analysed further to predict the vaccine match. This may lead to identification of sequence motifs contributing to the loss of cross-reactivity with the antisera that can be tested in a reverse genetics system to study their impact on the antigenicity of the virus. 17

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