TAIEX, EUFMD (FAO), EPIZONE & EU COORDINATION ACTION FMD-CSF

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1 TAIEX, EUFMD (FAO), EPIZONE & EU COORDINATION ACTION FMD-CSF Workshop on the design and interpretation of post Foot-and-Mouth Disease (FMD)-vaccination serosurveillance by NSP tests Part III (Scandinavian and Baltic region) October 23-25, 27 Report VAR-CODA-CERVA Leuvensesteenweg 17 B-38 Tervuren Belgium VAR Belgium EUFMD Belgian Food Agency DG RESEARCH DG SANCO EU FMD-NSP workshop 1

2 Contents Page Abstract 3 EU FMD-NSP workshop organising committee 6 EU FMD-NSP workshop supervisory team 6 Participants 6 Observers 6 Sponsors 6 Objectives 7 Report 8 Opening session 8 Session I: Review on FMD vaccination and NSP testing 1 Session II: Review on statistical computer tools, OIE guidelines, EU Directives and carrier problem 19 Session III: Serosurveillance post-vaccination: theory and practice 36 Session IV and V: Results from working groups 71 Session VI: Discussion of results and models for serosurveillance Summary, Conclusions, Recommendations and Observations 84 Acknowledgments 91 Annex A: FMD-NSP workshop program 92 Annex B: presentation of scenario 1 by group 1 93 Annex C: presentation of scenario 1 by group 2 12 Annex D: presentation of scenario 2 by group 3 18 Annex E: presentation of scenario 2 by group Annex F: presentation of scenario 3 by group Annex G: presentation of scenario 3 by group Annex H: Post NSP-workshop thoughts Dr. David Paton 144 EU FMD-NSP workshop 2

3 Abstract Given the current FMD-free status without vaccination in Europe and the possibility of a future outbreak with vaccinate-to-live used as an emergency measure, followed-up by a post-vaccination serosurveillance, to return to the status free from infection without vaccination, this workshop, for countries from the Scandinavian, Baltic and neighbouring area, had two main objectives, viz.: I. on day 1, to make participants familiar with: 1. NSP tests available, other relevant tests and their use 2. computer tools for the calculation of the required number of samples to be taken 3. the legislation relevant for NSP testing 4. the detection of carrier animals: relevant potential control measures 5. the requirements to personnel (field workers) and lab resources (tests and personnel). II. and on day 2 and 3, concentrate on: 1. The design and implementation of a survey to substantiate free from infection with a certain degree of confidence after vaccination has been performed. 2. The guidance to the interpretation on the follow-up on seropositive animals/herds/flocks; 3. The guidance to the use of laboratory test results in decision-making; 4. The identification of the resources (laboratory, veterinarians) required. Three different exercise scenarios were written, including: (a) a scenario with a limited number of outbreaks where only bovine and pigs were vaccinated, (b) a second scenario with a limited number of outbreaks, one in a very peculiar and big pig holding, where only bovine, and pigs were vaccinated, and (c) a third scenario with a large number of outbreaks where only bovine were vaccinated. Seventeen invited countries, including Belarus, Czech Republic, Estonia, Finland, Iceland, Israel, Latvia, Lithuania, Luxemburg, Moldova, Norway, Poland, Russian Federation, Slovakia, Sweden, Switzerland and Ukraine (of which Belarus, Iceland, Lithuania and Russian Federation did not participate) and observers of OIE, EFSA and EPIZONE were divided in 6 groups, each group having to make the best possible survey design (which was not necessarily the design proposed by OIE Guidelines or EU Directives), to follow-up the seropositive herds/animals/flocks and to do an identification of the necessary resources for their own scenario. Summary of workshop conclusions: The approaches taken by the different working groups showed a clear degree of similarity. Summary of conclusions: 1. The vaccination-to-live policy with subsequently substantiating freedom from infection by a survey system including NSP testing is a realistic and achievable option in FMD control. However, stamping out will always remain part of the control policy; 2. Because NSP assays are not sensitive enough (especially for carriers), conclusions on the infection status of the vaccinated herds can only be based on a combination of clinical and serological surveys and epidemiological investigations such as cluster analysis; 3. Proving freedom from infection for vaccinated animals is impossible, in contrast to substantiating freedom from virus circulation or freedom from infection in non-vaccinated animals; EU FMD-NSP workshop 3

4 4. The current EU Directive (23/85/EC) for the control of FMD mentions two surveys, i.e.: (a) A survey for detecting the presence of FMD virus (Article 56) in the vaccination zone, which should be a combination of clinical, epidemiological and serological investigations with high overall system sensitivity, which includes: a survey of the non-vaccinated animals; a serosurveillance of all herds with vaccinated animals (NSP tests). Within herds sampling of all vaccinated ruminants and their non-vaccinated off-spring; For large numbers of other species, sampling should be based on a 5% prevalence with 95% confidence. (b) A survey, to regain freedom from infection after emergency vaccination(article 61), which must have a high specificity. This survey might include a second serosurveillance, but the first serosurvey could serve the purpose of this one (cf. OIE Guidelines in App of the TAHC); 5. The follow-up of herds with seroreactors by serological investigation has to be based on NSP assays (Paton et al., 26) with well-defined performance characteristics. 6. If specificity of the serological test system were known, only seroreactor rates above the Herd Cut Point could be considered, but this is not compatible with EU directive 23/85/EC. 7. A clinical surveillance combined with paired serology can detect holdings where virus circulation is ongoing. Since there is no possibility of detecting each and every carrier within sub-clinically infected herds, all ruminants should be tested. Evidence of virus circulation would lead to herd slaughter, but evidence of carriers would lead to slaughter of these reactor animals only; 8. Testing all animals in the vaccinated population as prescribed in the EU Directive 23/85/EC, is considered as not achievable in areas with a dense pig population or within big pig herds, if such pigs have been vaccinated. 9. Vaccination of small herds remains a controversial item. Two possible options were discussed: (a) a non-vaccination policy for small herds with integration in the survey system as sentinels or (b) a vaccination policy for small herds because of their contribution in achieving the necessary level for population protection and because of political reasons. 1. This kind of workshop should be done for other veterinary diseases, like CSF and AI also (for all EU members, EUFMD countries and EU neighbours). Summary of recommendations: 1. Conclusions on the infection status of the herds after FMD outbreaks in a vaccinated population should only be based on a survey system, including at least clinical, serological and epidemiological investigations; 2. The performance characteristics of the survey system should be determined; 3. The term demonstrate absence should be replaced by substantiate absence ; 4. Contingency plans should include a clear flow chart for the follow-up of seropositive herds (minimum requirements of App of TAHC) 5. All large ruminants should be tested to substantiate freedom from infection in a vaccinated population after FMD outbreaks. While evidence of virus circulation must lead to the declaration of an outbreak, consensus should be sought on the slaughter of reactor animals only, in case it is evident that these animals are carriers; 6. A change in the definition of an outbreak in OIE guidelines and EU Directives is needed where carriers are concerned; EU FMD-NSP workshop 4

5 7. The relative confidence attainable with herd-based and individual certification needs to be explored for different herd sizes and prevalence; 8. Consideration should be given to an amendment of the Directive in order to allow a within-herd sampling scheme based on a 5% prevalence and 95% confidence for vaccinated pigs; 9. The vaccination of small herds should be further discussed; 1. To refine the application of NSP tests, more work could be done in predicting the expected prevalence of infection within and amongst vaccinated herds; 11. Functional FMD expert groups should be created in every country; Summary of workshop observations: Following points were specifically highlighted during the second and third workshop: 1. The different species analysed in the survey system should be taken into account to determine the performance characteristics of the survey system. 2. Containment of an infection region is possible since the change of the OIE code. 3. Training for the set-up of an information system for field data is necessary for some countries. 4. The whole scenario assumes a perfect movement control, which is not a real life situation. 5. Probang testing is not the method of choice to substantiate freedom from infection. 6. The introduction of negative animals as sentinels in a vaccinated herd is of limited value due to the low transmission rate in these herds, as well for cattle as for pigs. 7. Vaccination is not a major option in countries with a lot of small herds 8. The organisation of a workshop on vaccination: how and when, after or in face of an outbreak should be considered. 9. SP-tests can also be used to check the efficiency of and coverage obtained through vaccination. 1. If possible, slaughter for consumption should be considered. 11. Sub-clinical transmission in cattle and pigs is unlikely. 12. Surveillance in the surveillance zone could be of help to substantiate absence of infection. 13. It is essential to have a decision-scheme on the follow-up before the start of the outbreak. 14. If the 95% confidence cannot be achieved for a survey then it is still worth doing it. EU FMD-NSP workshop 5

6 EU FMD-NSP Workshop Organizing Committee See Annex A: FMD-NSP workshop program EU FMD-NSP Workshop Supervisory Team See Annex A: FMD-NSP workshop program Participants Seventeen countries, including Belarus, Czech Republic, Estonia, Finland, Iceland, Israel, Latvia, Lithuania, Luxemburg, Moldova, Norway, Poland, Russian Federation, Slovakia, Sweden, Switzerland and Ukraine were invited. Except for Belarus, Iceland, Lithuania and Russian Federation, every invited country has sent a team to participate to the workshop. Participant list: see Annex A: FMD-NSP workshop program Observers See Annex A: FMD-NSP workshop program Sponsors The workshop was kindly sponsored financially by TAIEX, FAO EUFMD, the EU Coordination action FMD-CSF project, the EU EPIZONE project Work Package 4.3 (DIVA) and logistically by the Veterinary Administration of the Republic of Slovenia, the Federal Agency for the Safety of the Food Chain in Belgium and the Veterinary and Agrochemical Research Centre in Belgium. EU FMD-NSP workshop 6

7 Objectives The objectives of the third FMD-NSP workshop were two-fold: I. To make participants familiar with: 6. NSP tests available, other relevant tests and their use 7. computer tools for the calculation of the required number of samples to be taken 8. the legislation relevant for NSP testing 9. the detection of carrier animals: relevant potential control measures 1. the requirements to personnel (field workers) and lab resources (tests and personnel). II. Given the current FMD-free status without vaccination in Europe and the possibility of a future outbreak with vaccinate-to-live used as an emergency measure, being followed-up by postvaccination serosurveillance, to return to the favoured status of free from infection without vaccination : 1. Design and implementation of a survey to substantiate free from infection (i.e. free from infection with a certain degree of confidence, according to the EU directives and the OIE general and specific serosurveillance guidelines). So a country has an outbreak (or different outbreaks), vaccination of the surrounding herds is performed, the outbreaks are stopped, what will this country do to regain the free from FMD without vaccination status? 2. Guidance to the interpretation on follow-up on seropositive animals/herds/flocks, for example by using 3 different exercise scenario s, where we will have 3 different conclusions on the last day. 3. Guidance to the use of laboratory test results in decision-making 4. Identification of the resources required EU FMD-NSP workshop 7

8 Report Opening session: Wellcome word by Dr. Lea Knopf, Officer in charge of the recognition of countries animal disease status, OIE Dear participants, dear organizers, observers and guests It is a great honour for me that I was invited to address a few words to all of you on the occasion of the opening of this third EUFMD workshop on NSP testing. First of all I would like to thank the organizers and sponsors who made it possible to launch this third workshop on NSP testing. Today, I would like to share with you some general thoughts related to the importance of the present workshop: This workshop series is quite a unique event that brings together different people involved in FMD control around a common goal or challenge - to address emergency vaccination also called vaccinateto-live strategy and follow up activities to regain the FMD free status without vaccination in a European context. This workshop on FMD-NSP testing emphasizes hopefully again that creative interdisciplinary problem-solving, hands-on training and the active involvement of all the participants are a fruitful strategy. As officer in charge of countries official disease status recognition at OIE I am in continuous contact with countries around the globe which either suffer from an acute FMD outbreak or which seek to achieve or re-instate an FMD free status after a FMD epidemic. In all cases the appropriate design of surveys and the interpretation of serological survey results play a crucial role, as does the choice of a thoroughly planned and adapted vaccination strategy, if applicable. During the last years major achievements have been made on a scientific level driven by highly dedicated end experienced colleges. Also the regulatory framework has been continuously updated to the newest scientific evidence, both on EU level as on an international level. It should not be omitted to mention that changes in legislation may also be driven by the public perception or economic constraints in the animal health and animal welfare sector. Despite the hard and excellent work of numerous scientists, CVOs and other officers in the veterinary science field, legislation and guidelines still limp a little bit behind the reality in the field. Amongst other factors, this might be due to the fact that some of the recommended implementations are based on experiences in the past or that the legislative body, as well as scientists, could simply not consider all the amazing eventualities that happen to occur in the real world. For these reasons I was several times facing the challenge of having to stick to guidelines or legislations and to try to adjust for the complexity of the real world that goes far beyond any recommendation or legislation laid out on paper! Because when it comes to practical implementation, the real world occasionally and relentlessly strikes back, thereby pin pointing both, a partial lack of available options to implement prescribed procedures and the difficult task of veterinary services to plan and conduct most efficiently vaccination campaigns and serological surveys under emergency conditions. In the best case this reveals open questions to be addressed in future revisions of guidelines or research, in the worst case this leads e.g. to heavy economic losses in an affected country due to extended waiting periods, additional outbreaks, culling of animals that would have been avoidable, lack of veterinary or laboratory staff and simply distress. It made me think a lot about the problems that the attendees of the last NSP EU FMD-NSP workshop 8

9 workshop had to struggle with during their simulation exercise and emphasises again the importance of such practical exercises well before the real world problems occur! Based on the great experience of the last workshop on NSP-FMD-testing and being a non-native English speaking person I came further to the conclusion that workshop might also means work to shop. Let me explain this daily life association: Having the opportunity to intensively work together for a few days across countries, across disciplines and different levels of expertise, has the wonderful potential to create a huge shopping centre. During the workshop this shopping centre will be continuously filled with well tried (the theoretical background) and new products (your creative synthesis) that will draw the attention of the participants. To shop could therefore be interpreted as shopping around for the products that correspond to your specific needs or to increase your stock of problem solving tools in the context of your country. Its is most likely that in view of the number of products at your sight you may get lost in the labyrinth of this shopping centre, you may not have the chance to check all the shelves or you may be simply spoilt for choice I encourage all of you to catch this opportunity to actively engage in the simulation exercises, YOUR questions, YOUR discussions can lead to alternative approaches to be considered or in other terms they may lead to a brand new products on the shelve of this FMD- shopping centre! I wish you a very successful shopping tour at VAR! EU FMD-NSP workshop 9

10 Session I: Review on FMD vaccination and NSP testing The Science behind Non-Structural Protein (NSP) testing, Dr. Kris De Clercq, Belgium Differentiation of infected vaccinated animals Kris De Clercq A virus infects a cell, the RNA is read by the cell system and produces structural proteins (SPs) to make the structure of the virus; other parts of the RNA are decoded in proteins to help to make the structure of the virus or help to duplicate the RNA: these are the nonstructural proteins (NSPs) The virus cycle goes on and more and more virus is made but also more and more NSPs. When the cell is destroyed, virus is liberated as well as the NSPs. Part comes in the blood, so the animal will make antibodies against the SPs and the NSPs EU FMD-NSP workshop 1

11 A vaccine producer mimics in fact what happens in nature by adding virus to a cell culture. The virus cycle goes exactly in the same way but then he filters the vaccine to have a pure virus culture. The virus is inactivated so that a virus cycle (and the formation of NSPs) becomes impossible. So, when the animal is vaccinated it will make only antibodies against the SPs. Summary of the reaction of the animal after infection or vaccination or no infection/vaccination. EU FMD-NSP workshop 11

12 Structural Proteins ELISA + + Non-Structural Proteins Structural Proteins ELISA + + Non-Structural Proteins Translation to the laboratory using two different ELISAs: one classical ELISA detecting antibodies against SPs and one NSP- ELISA, showing how you can differentiate between infected and/or vaccinated animals. If an animal gets infected after the vaccination, normally the infected animals will react but however some animals will not react, causing a false negative. If a vaccine is not well purified you will have Structural Proteins ELISA + + Non-Structural Proteins NSPs in the vaccine. So, after one injection you will normally get no reaction against NSPs but after several vaccinations with this kind of vaccine you will get false positives. + - EU FMD-NSP workshop 12

13 Validation and comparison of NSP tests, Dr. Donal Sammin, Ireland Introduction Comparative evaluation and validation of NSPEs Dónal Sammin, CVRL-DAF, Ireland 23/85/EC: vaccination to live and post-vaccination surveillance DIVA testing => use of NSP-based tests OIE-approved method ex. Panaftosa 5 other NSPEs in Europe Evaluate comparative performance Validate for purpose Studies FMD-ImproCon multinational workshop; IZS-Brescia, May 24; cattle ( sheep/pigs); Brocchi et al., 26 [App 1] + Dekker et al. [App 8] EUFMD/WRL field study; Zimbabwe, April 24; cattle, SAT-type infection; Sammin et al., VR, 27 EUFMD/WRL field study; HK-SAR, March 25; (+ experimental study, IRL, 26); pigs; Paton et al. FMD_ImproCon WS; IZS, Brescia; May 24 Brocchi et al., 26 [App 1] MATERIALS & METHODS 3551 sera [BE, DE, DK, ISR, IT, NL, TK, UK] 2579 (67%) bovine; 73 ovine; 269 pig Different vaccination and infection status All sera tested x6 NSPE in parallel Panaftosa, IZS-B, Ceditest, Svanovir, Chekit and UBI ELISAs Proposed field and exptl studies on sheep, 27/8 WORKSHOP; IZS, Brescia; May 24 MATERIALS & METHODS: BOVINE SERA Experimental (n = 137 sera) 425 V + I - 62 [54] V - I + 21 [17] C + 55 [285] V + I [67] C + Field (n = 1542 sera) 672 V - I V + I (ISR/ZIM) WORKSHOP; IZS, Brescia; May 24 MATERIALS & METHODS: NSP ELISAs ELISA Panaftosa IZS-Brescia Ceditest FMDV-NS SVANOVIR FMDV 3ABC-Ab ELISA CHEKIT-FMD-3ABC UBI FMDV NS ELISA Antigen 3ABC 3ABC 3ABC 3ABC 3ABC 3B Format INDIRECT; coated INDIRECT; trapping BLOCKING; trapping INDIRECT; coated INDIRECT; coated INDIRECT; coated Grey zone YES YES NO NO YES NO EU FMD-NSP workshop 13

14 WORKSHOP; IZS, Brescia; May 24 RESULTS: diagnostic specificities 11 bovine sera [675 V + I - & 425 V + I - ] 97.2% % on first screening test 98.3% % on retesting positives WORKSHOP; IZS, Brescia; May 24 RESULTS: detection rates CATTLE V - I + (n = 54) & V + I + (n = 285) Sub-categories of V + I + cattle: (i) infection demonstrated (n = 164); (ii) carrier status demonstrated (n = 67); (iii) not carriers (n = 26); (iv) no evidence of infection (n = 17) 7-14, 15-27, 28-1 and >1 days p.i. Subcategories (i) and (ii), 14-27dpi: Panaftosa, IZS-B and Ceditest NSPEs >6% seropositive Carrier detection rate, 28-1dpi; Panaftosa 93.9%, IZS-B 86.4% and Ceditest 86.4% EUFMD/WRL field study; Zimbabwe Objective: evaluate NSPEs for SAT-type FMD 43 cattle; 6 herds; April-May 24 SAT1/SAT2, 5 herds, 1-5 months pi 12-35% of herds were carriers (probangs; VI & PCR) Overall seroprevalence of 56% - 75% with NSPEs; 81% with SPCE and 91% with VNT Carrier detection rate of 7% - 9% Sammin et al., Veterinary Record (27) 16: EUFMD/WRL field study; HK-SAR & exptl study on vaccinated pigs, IRL Objective: evaluate 3 x NSPEs for use in pigs Ceditest, UBI and Chekit ELISAs Field study 45 pigs; 4 vaccinated herds; Feb-March 25 Type O infection, 2 herds, 1-2 months pi All 3 NSPEs detected infection in pigs; different dse and dsp Cedi was both sensitive and specific; UBI was less specific and Chekit was less sensitive Experimental study 99 pigs; vaccination x2; sampling 28 dpv dsp: 1% for Cedi and UBI; 98% for Chekit Still to do Publish paper on HK & IRL pig studies Publish paper on LRs and ROC analysis of results from Brescia WS Perform field and experimental studies on sheep during late 27/early 28 EU FMD-NSP workshop 14

15 Sensitivity and specificity of SP and NSP tests relevance and use, Dr. Aldo Dekker, The Netherlands Outline Sensitivity and specificity of SP and NSP tests; relevance and use Aldo Dekker Objective Validation of SP tests Results validation study NS ELISA's in Brescia Validation using dichotomised results Validation using continuous results Validation SP tests Neutralisation tests considered as gold standard No differentiation between vaccinated and infected animals (DIVA) Large variation in results when using different cell lines No true validation study available LPB and SPC ELISA Very well validated Different cut-offs for different purposes Definition of FAO cut-off sera unclear since UK outbreak 21 Validation SP tests Neutralisation tests considered as gold standard Sensitivity close to 1% (by definition) Very high specificity (> 98% when using one serotype) LPB and SPC ELISA Sensitivity and specificity similar to neutralisation test Objective Brescia workshop Compare different DIVA tests for FMDV Provide estimates that can be used for surveillance Specificity in cattle Vaccinated n Panaftosa IZS Cedi Specificity first ELISA % 97.3% 97.2% 98.7% 98.2% 99.% % 97.4% 99.5% 98.1% 96.7% 97.9%.35 -/ % 97.4% 98.1% 98.5% 97.6% 98.5%.122 Specificity after retest positives % 99.9% 99.% 99.1% 98.8% 99.4% % 99.5% 99.5% 98.8% 98.8% 98.4%.67 -/ % 99.7% 99.2% 99.% 98.8% 99.%.7 Svanova Bommeli UBI p Fischerexact test EU FMD-NSP workshop 15

16 Vaccinated Detection rate in exposed cattle DPI n Panaftosa IZS % 1.% 1.% 1.% 1.% 1.% % 1.% 1.% 1.% 1.% 1.% % 1.% 1.% 96.2% 92.3% 1.% > % 1.% 5.% 5.% 5.% 5.% % 52.2% 48.6% 4.9% 5.% 32.% % 55.7% 52.7% 49.6% 52.7% 38.2% % 64.8% 62.6% 58.3% 5.% 56.1%.59 + > % 63.8% 74.5% 57.4% 38.3% 46.8%.1 Cedi Svanova Bommeli UBI p Fischerexact test Vaccinated Detection rate in exposed and infected cattle DPI n Panaftosa IZS % 1.% 1.% 1.% 1.% 1.% % 1.% 1.% 1.% 1.% 1.% % 1.% 1.% 96.2% 92.3% 1.% > % 1.% 5.% 5.% 5.% 5.% % 55.1% 53.9% 47.2% 57.3% 46.1% % 66.% 61.9% 56.7% 61.9% 47.4% % 67.4% 68.1% 57.6% 53.3% 61.5%.29 + > % 63.8% 74.5% 57.4% 38.3% 46.8%.1 Cedi Svanova Bommeli UBI p Fischerexact test Detection rate in carrier cattle Conclusion validation at producer cut-off Vaccinated DPI n Panaftosa IZS Cedi Svanova Bommeli UBI p Fischerexact test NCPanaftosa, IZS-Brescia and Ceditest ELISA superior % 1.% 1.% 1.% 1.% 1.% % 1.% 1.% 1.% 71.4% 1.% > % 1.% 5.% 5.% 5.% 5.% % 54.8% 54.8% 51.6% 54.8% 38.7% % 66.7% 63.9% 55.6% 58.3% 58.3% % 86.4% 86.4% 71.2% 68.2% 77.3%.1 + > % 78.4% 89.2% 7.3% 48.6% 59.5%. Good specificity for most tests in cattle, pigs and small ruminants Very high sensitivity in non-vaccinated cattle Good sensitivity in carrier cattle More data from infected pigs and small ruminants are needed Comparison of ELISAs without cut-off ROC curves Non-vaccinated cattle Vaccinated cattle Conclusion similar to comparison using cut-off Materials Continuous results NS ELISA validation in Brescia Sera from non-exposed cattle (non-endemic regions) Non-vaccinated (n=425) Vaccinated (n=675) Sera from experimentally exposed cattle Unique serum per cow randomly selected 21 dpi Non-vaccinated (n=64) Vaccinated (n=173) EU FMD-NSP workshop 16

17 Methods ROC analysis S-Plus library developed by Beth Atkinson and Doug Mahoney (Mayo clinic) McNemar's Chi squared test at 97.5% and 99% specificity level Likelihood Ratio calculation Using logistic regression ROC curve Sensitivity NCPanaftosa IZS-Brescia Ceditest Svanovir Checkit UBI Specificity Pair wise comparison of the area under the curve Pairwise comparison Vaccinated cattle NCPanaftosa IZS-Brescia Ceditest Non-vaccinated cattle IZS-Brescia - IZS-Brescia - Ceditest - - Ceditest - - Svanovir Svanovir Chekit ** ** ** ** Chekit UBI ** ** * ** - UBI Svanovir Chekit NCPanaftosa IZS-Brescia Ceditest Svanovir Chekit ROC curve Sensitivity NCPanaftosa IZS-Brescia Ceditest Svanovir Checkit UBI Specificity Comparison at specified specificity Vaccinated cattle Non-vaccinated cattle Likelihood ratio dichotomized results 97.5% specificity 99% specificity NCPanaftosa IZS-Brescia Ceditest NCPanaftosa IZS-Brescia NCPanaftosa 69.4% NCPanaftosa 89.1% IZS-Brescia % IZS-Brescia % Ceditest % Ceditest % Svanovir % Svanovir % Chekit ** ** ** % Chekit % UBI ** ** ** ** - 5.9% UBI % NCPanaftosa IZS-Brescia Ceditest Svanovir Chekit NCPanaftosa IZS-Brescia NCPanaftosa 6.1% NCPanaftosa 87.5% IZS-Brescia ** 68.2% IZS-Brescia % Ceditest * % Ceditest % Svanovir - ** ** 53.2% Svanovir % Chekit ** ** ** - 5.3% Chekit % UBI ** ** ** ** ** 36.4% UBI % Svanovir Chekit Ceditest Ceditest Svanovir Svanovir Chekit Chekit Post test odds = pre test odds x LR Independent of prevalence in target population Pre test odds = (a+c)/(b+d) Post test odds = a/b LR+ = sens/(1-spec) =# true /# false positives + test - true + - a b c d EU FMD-NSP workshop 17

18 Likelihood ratio on continuous data Likelihood ratio analysis Ceditest ELISA Number of cattle NON-EXPOSED CATTLE EXPOSED CATTLE Percentage Percentage inhibition inhibition Ceditest ELISA Number of cattle Likelihood ratio Cut-off Percentage inhibition At cut-off already high likelihood for infection Many sera from exposed cattle with low response Result can be used in decision scheme With the same sera different tests produce a different LR Conclusion ROC analysis similar to results obtained previously The estimation of the Likelihood ratios will help diagnosticians and decision makers Recommendation: The LR should be implemented in decision scheme and used when ELISA's for FMD virus non-structural proteins are used after an FMD emergency vaccination. Risk maps with 3 colours: Red: '1 km culling' not sufficient to avoid large outbreaks (= R >1) Orange: '1 km culling' sufficient Green: 'no ring culling' already sufficient (= EU-measures) On the basis of slowest decreasing kernel and immediate culling EU FMD-NSP workshop 18

19 Session II: Review on statistical computer tools, EU Directives and carrier problem Statistical computer tools: theoretical and practical approach. A. Theory, Dr. Koen Mintiens, Belgium Statistical computer tools: theoretical and practical approach. A. Theory K. Mintiens Co-ordination Centre for Veterinary Diagnostics Contents Different approaches for substantiating freedom from disease Two-stage sampling Discussion Claiming freedom from disease SPS agreement of World Trade Organization (WTO): Current animal health-related rules for international trade; Countries need to provide sciencebased evidence to support their claims to freedom from livestock diseases; One way is using survey samples. Starting point Population in which a disease may be present or absent If disease is present, then: minimal expected prevalence = design prevalence (p*) Diagnostic test which can identify the presence of the disease Survey sample which is sufficient to substantiate presence/absence of disease with a certain confidence level Principle Pick enough balls without replacement to have at least one red from N balls hypergeometric distribution: d N d + x n x P ( T = x) = N n Principle When aiming to substantiate freedom from disease, x=: ( ) ( ) + N d! N n! p( T = ) = ( N d n)! N! Factorial formulae with large numbers are difficult to compute Approximations of formula have been developed together with IT capacity EU FMD-NSP workshop 19

20 First approach: Cannon & Roe, 1982 Methods for detecting the presence of a disease Assumptions: Design prevalence Perfect diagnostic tests Convenient approximation: P( T + d = ) = 1 n 1 N 2 n First approach: Cannon & Roe, 1982 Sample size: n 1 d d ( 1 ( 1 α ) ) N Confidence level α (probability of finding at least one positive in the sample): n d α 1 1 N ( n 1) 2 First approach: Cannon & Roe, 1982 Tables with standard numbers available in epidemiological text books Software tools, e.g. WinEpiscope: First approach: Cannon & Roe, 1982 Illustration of sample size calculation: FMD outbreak in previously free area Outbreak controlled and disease no longer present Design prevalence: 3% Herd with 265 animals Confidence level (1-α)=.95 =>Output from WinEpiscope: n=9 Improvements: Cameron & Baldock, 1998 Diagnostic tests are not perfect: P( T P( T + ) = pse + (1 p)(1 Sp) ) = p(1 Se) + (1 p) Sp Thus the hypergeometric distribution extends: P( T + = x) = d y= d N d y n y N n min( x, y j= ) y j Se (1 Se) j y j n y (1 Sp) x j x j Sp n x y+ j Improvements: Cameron & Baldock, 1998 Equation is very complex (only small population sizes can be computed) Approximation based on binomial distribution Considers formal hypothesis testing: H : prevalence design prevalence H a : prevalence < design prevalence Type I error ά: falsely rejecting H Type II error β: falsely accepting H EU FMD-NSP workshop 2

21 Improvements: Cameron & Baldock, 1998 Sample sizes are calculated by iterative process based on probability distributions Survey results can be analysed providing a confidence level for rejecting H Available in FreeCalc module of the Survey Toolbox software Improvements: Cameron & Baldock, 1998 Illustration of sample size calculation: Design prevalence: 3% Herd with 265 animals Type I error (α )= type II error (β)=.5 Confidence level (1-α)=.95 ELISA Se=.95 & Sp=.98 => FreeCalc: n= 14 with 1 reactor Further extension: Johnson et al., 24 Addresses the uncertainty in sensitivity, specificity and design prevalence Uses hypothesis testing Bayesian approach Prior distributions for Se, Sp and p* Focus on the posterior probability that p is below a threshold Further extension: Johnson et al., 24 Procedure also available as freeware: BayesFreeCalc Further extension: Johnson et al., 24 Illustration of sample size calculation: Design prevalence: > 3% Herd with 265 animals Type I error = type II error =.5 ELISA Se>.9 & Sp>.95 => BayesFreeCalc: n= 18, 2 reactor Two-stage sampling Survey can be used at any level: herd, region, nation, etc Available statistical tools can provide confidence levels for rejecting the hypothesis of disease presence, given: Population size N Sample size n Design prevalence p* Se and Sp of diagnostic test Allowable Type I and II error EU FMD-NSP workshop 21

22 Two-stage sampling For practical reasons often two-stage sampling strategy Diseases tend to cluster in the population (e.g. within farms) Two-levels for substantiating disease freedom: Each herd is classified as diseases or non-diseased: test results of individual animals Population of herds is classified as diseased or nondiseased based on results of individual herds Two-stage sampling Sensitivity (HSe) and specificity (HSp) of herd classification is influenced by: Se + Sp of individual-animal test # animals tested per herd Interpretation of individual test results Two-stage sampling In this context: HSe = probability that a diseased herd will be classified as diseased one minus the probability that a diseased herd will be classified as non-diseased = 1-α Similarly HSp = probability that a non-diseased herd will be classified as nondiseased one minus the probability that a non-diseased herd will be classified as diseased = 1- β Two-stage sampling Sample size Number of herds: Preset Type I and type II herd level Desired HSe and HSp Minimal expected herd prevalence Number of animals per herds Type I error = 1-HSe Type II error = 1-HSp Se and Sp of assay for animal testing Minimal expected within-herd prevalence Two-stage sampling Analysis of results Classification of herds Each herd is classified as diseased or non-diseased, based on number of reactors Se + Sp of animal test N a, n a, type I and type II error Result: number of reactor herds Classification of population Based on number of reactor herds, and Se + Sp of animal test N h, n h, type I and type II error Two-stage sampling Hypothetical example Survey in 8532 herds to substantiate freedom from disease Screening test: Se=.94, Sp=.9 95% confidence and power Minimal expected herd-p*= 5% Minimal expected within-herd-p*=2% Logistical constrain: max 2 herds to be tested EU FMD-NSP workshop 22

23 Two-stage sampling Hypothetical example: FreeCalc Trail and error: 193 herds to be tested if: Type I = Type II =.5 Herd-p= 5% HSe= 9% HSp= 98.4% 6 reactors allowed Within herd testing sample size: Type I error= 1% Type II error= 1.6% Within-herd-p= 2% Sample size can be calculated for each herd Discussion Design prevalence Either minimal expected prevalence Or, small enough to be considered negligible Can often not be defined: Wildlife populations or emerging diseases Vaccinated populations Small design prevalence implies large sample size The application of design prevalence does not allow zero prevalence Discussion New approach (Mintiens et al, 25) Bayes Theorem: The posterior distribution of the probability of freedom from disease (F) given the observed test results (T) P( T F ) P( F) P ( F T ) = P( T ) P( T F, prev, Se, Sp) P( F, prev, Se, Sp) P ( F, prev, Se, Sp T ) = P( T ) Discussion New approach (Mintiens et al, 25) Probability for positive test result: P( T F, prev, Se, Sp) = (1 F)( prev Se + (1 prev)(1 Sp)) + F(1 Sp) The likelihood function takes into account that the observed prevalence only occurs when the population is not free from disease. Function can be solved using Bayesian methods Discussion Statistical tools: Have probabilities and confidence as outcome Allow for positive reactors Quantify uncertainty Competent authorities need yes/no answers Competent authorities acknowledge uncertainty and finally have to ignore it Discussion Sample size: Larger sample size when accounting for more uncertainty Sample size calculations provide guidance to confident results Important impact on the costs Determine optimal sample size in twostage sampling EU FMD-NSP workshop 23

24 Discussion Other issues Surveys in small herds Surveys with very small design prevalence Substantiating freedom from disease using multiple complex data sources References Cameron, A.R. & Baldock, F.C., 1998, A new probability formula for surveys to substantiate freedom from disease. Prev.Vet.Med. 34, Cameron, A.R. & Baldock, F.C., 1998, Two-stage sampling in surveys to substantiate freedom from disease. Prev.Vet.Med. 34, Cannon, R.M., 22, Demonstrating disease freedom-combining confidence levels. Prev.Vet.Med. 52, Greiner, M. & Dekker, A., 25, On the surveillance for animal diseases in small herds. Prev.Vet.Med. 7, Huzurbazar, S., et al., 24, Sample size calculations for bayesian prediction of bovine viraldiarrhoea-virus infection in beef herds. Prev.Vet.Med. 62, Johnson, W.O., et al., 24, Sample size calculations for surveys to substantiate freedom of populations from infectious agents. Biometr. 6, Martin, P.A. et al., 27, Demonstrating freedom from disease using multiple complex data sources 1: A new methodology based on scenario trees. Prev.Vet.Med. 79, Martin, P.A. et al., 27, Demonstrating freedom from disease using multiple complex data sources 2: Case study--classical swine fever in denmark. Prev.Vet.Med. 79, Mintiens, K. et al., 25, Estimating the probability of freedom of classical swine fever virus of the east-belgium wild-boar population. Prev.Vet.Med. 7, Ziller, M. et al., 22, Analysis of sampling strategies to substantiate freedom from disease in large areas. Prev.Vet.Med. 52, Thank you EU FMD-NSP workshop 24

25 Statistical computer tools: theoretical and practical approach. B. Practice, Dr. Koen Mintiens, Belgium Statistical computer tools: theoretical and practical approach. B. Practice K. Mintiens Co-ordination Centre for Veterinary Diagnostics Aim To illustrate a few of the statistical approaches to substantiate disease freedom Use of a simulated FMD outbreak in Belgium Provide guidance to the workshops Case FMD outbreak in Belgium 1 km surveillance zone around 6 infected premises: All infected premises are stamped out All susceptible animals within 5m radius are culled All cattle within 1,m radius are vaccinated Density: Cattle: 252/km² Pigs: 37/km² Sheep/goats: 24/km² Vaccination area of 313 km² Case (ctn.) Survey Survey in vaccinated cattle: Using CEDI NSP test in vaccinated cattle: Se=.864, Sp=.995 Design prevalence: Herd-p*:.1 Within-herd p*:.5 Type I and II error:.5 Exercise Sample size calculation: One stage approach Two stage approach Analysis of results: One stage approach Two stage approach EU FMD-NSP workshop 25

26 Sample size one stage approach Consider only the animals Plug parameters into FreeCalc: Population: 47,899 animals Design prevalence: 5% CEDI NSP test: Se=.864, Sp=.995 Type I and II error=.5 Sample size one stage approach Consider only the animals Plug parameters into FreeCalc: Population: 47,899 animals Design prevalence: 5% CEDI NSP test: Se=.864, Sp=.995 Type I and II error=.5 Result: 13 animals, 2 reactors Sample size one stage approach Move on to higher confidence level Plug parameters into FreeCalc: Population: 47,899 animals Design prevalence: 5% CEDI NSP test: Se=.864, Sp=.995 Type I and II error=.1 Sample size one stage approach Move on to higher confidence level Plug parameters into FreeCalc: Population: 47,899 animals Design prevalence: 5% CEDI NSP test: Se=.864, Sp=.995 Type I and II error=.1 Result: 238 animals, 4 reactors Sample size one stage approach Important remarks: One stage sample requires random sample of animals List of all cattle (sampling frame) High logistic impact (sampling one animal per herd) Sample size two stage approach First consider the herds Plug parameters into FreeCalc: Population: 1,321 herds Design prevalence: 1% HSe=.95, HSp=.95 Type I and II error=.5 => Two stage approach EU FMD-NSP workshop 26

27 Sample size two stage approach First consider the herds Plug parameters into FreeCalc: Population: 1,321 herds Design prevalence: 1% HSe=.95, HSp=.95 Type I and II error=.5 Unable to achieve the desired accuracy by sampling every unit Sample size two stage approach Decrease uncertainty: increase HSe and HSp: Plug parameters into FreeCalc: Population: 1,321 herds Design prevalence: 1% HSe=.99, HSp=.99 Type I and II error=.5 Sample size two stage approach Decrease uncertainty: increase HSe and HSp: Plug parameters into FreeCalc: Population: 1,321 herds Design prevalence: 1% HSe=.99, HSp=.99 Type I and II error=.5 Sample size two stage approach Then consider the animals within herds Plug parameters into FreeCalc: Population: largest herd: 592 animals Design prevalence: 5% Se=.864, Sp=.995 Type I and II error=.1 Result: 123 herds, 18 reactors Sample size two stage approach Then consider the animals within herds Plug parameters into FreeCalc: Population: largest herd: 592 animals Design prevalence: 5% Se=.864, Sp=.995 Type I and II error=.1 Result: 213 animals, 4 reactors Sample size two stage approach Then consider the animals within herds: Plug parameters into FreeCalc: Population: smaller herd: 131 animals Design prevalence: 5% Se=.864, Sp=.995 Type I and II error=.1 EU FMD-NSP workshop 27

28 Sample size two stage approach Then consider the animals within herds Plug parameters into FreeCalc: Population: smaller herd: 131 animals Design prevalence: 5% Se=.864, Sp=.995 Type I and II error=.1 Result: 127 animals, 3 reactors Sample size two stage approach Below 131: Unable to achieve the desired accuracy by sampling every unit Results of the survey 1,23 herds were randomly selected from the total of 1,321 All cattle within selected herds were sampled and tested (n=29,15; N= 47,899 ) 142 animals tested positive with CEDI NSP test Analysis of results One stage approach Looking at animal results: N=47,899 n=29, reactors Se= 86.4, Sp= 99.5 Considering the Sp: 146 (.5%) false positives are allowed Using Freecalc N=47,899 n=29, reactors Analysis of results One stage approach Se= 86.4, Sp= 99.5 Type I and type II error is.5 Design prevalence is 5% Analysis of results One stage approach Using FreeCalc: N=47,899 n=29, reactors Se= 86.4, Sp= 99.5 Type I and type II error is.5 Design prevalence is 5% Result are adequate to reject to H and to conclude that the population is free from disease at 5% design prevalence and 1% confidence level EU FMD-NSP workshop 28

29 Analysis of results Two stage approach Classification of herds All 123 sampled herds were evaluated Type I = Type II=.1 Se=86.4%, Sp=99.5% Specific herd size and positive reactors per herd Analysis of results Two stage approach For 21 herds H could not be rejected animals reactors animals reactors animals reactors Analysis of results Two stage approach Classification of population Type I = Type II=.5 HSe=99%, HSp=99% Design prevalence= 1% N=2188 n=1325 Positive reactors= 21 Analysis of results Two stage approach Classification of population Results are not adequate to conclude that population is free from disease (design prevalence 1%). The confidence level is only %. We may conclude that population is diseased at confidence level %. Discussion Two-stage sampling: More practical Increases sample size More confident results (accounts for clustering) Positive results are marginal, but this can be expected in a vaccinated population Sometimes these marginal positive result borderline or not acceptable at herd level EC directive requires testing of each vaccinated animal Discussion Can t we assume Sp=1 at animal level? Reduces sample size All positives (in the sample) in a herd are culled Does that mean that the infection is eliminated from the herd? What about stamping out of positive herds? Does that mean that all infected herds are eliminated? Sp=1 can be assumed if all efforts are made to exclude false positive results EU FMD-NSP workshop 29

30 Discussion How to increase confidence level and power: Increase accuracy: serial testing? Look at geographical clusters? Look at non-vaccinated populations, sentinels, buffer zone, Thank you What about decreasing the power (increasing type II error)? Decreases sample size At herd level: less reactors are allowed More (false) positive herds are stamped out Cost of taking more samples per herd vs. stamping out of herds EU FMD-NSP workshop 3

31 EU Directives for FMD control, Dr. Alf Füssel, EU DG SANCO, Head of Trade and Zootechnics Sector Emergency vaccination FMD NSP Tests in Post-Vaccination Surveillance Dr. Alf-Eckbert Füssel European Commission - DG Health and Consumer Protection Directorate D - Animal health and welfare Unit D1 - Animal health and Standing Committees Council Directive 23/85/EC provides for emergency vaccination in case of more than one outbreak of FMD Types of vaccination Protective (vaccinate-to-live) Suppressive (vaccinate( vaccinate-to-kill) Vaccination is carried out in accordance with Article 52(1) - vaccination zone Article 52(2) - surveillance area surrounding the vaccination zone This presentation does not necessarily represent the views of the Commission Phases of Post-vaccination Measures 1. from the beginning of emergency vaccination until at least 3 days have elapsed following the completion of such vaccination (Article 54) 2. from emergency vaccination until the survey and the classification of holdings are completed (Article 55) 1. Clinical and serological survey in the zone (Article 56) 2. Classification of herds in the zone (Article 57) 3. after the completion of the survey and the classification of holdings until FMD free status is recovered (Article 58) Sheep and cattle not vaccinated Sheep not vaccinated Herd vaccinated 3 4 Aim : Clinical and serological survey Content: identification of herds within the vaccination zone with contact to the FMDV without showing overt clinical signs clinical inspection of all animals in all herds and laboratory testing Sampling: herds not vaccinated with species not showing clinical signs 5 % prevalence, 95 % confidence and all vaccinated animals and their non- vaccinated offspring in all vaccinated herds Classification of holdings holdings with at least 1 animal positive in the NSP test Presence of FMDV = outbreak Previous contact with FMDV, no actual virus circulation all animals are killed and the carcasses processed, or animals are classified: the animals positive to at least one of the approved tests are killed and their carcasses processed, and the remaining animals are slaughtered under conditions authorised by the competent authorities; fresh meat from ruminants de-boning and maturation, meat from porcine animals - heat treated products 5 6 EU FMD-NSP workshop 31

32 Recovery of FMD free status following emergency vaccination all the measures provided for in Articles 36, 44, 54, 55, 56 and 57 have been completed, at least one of the following conditions applies: OIE rules as in force, or 3 months after slaughter of vaccinated animals + serological surveillance in accordance with OIE guidelines, or 6 months after the last outbreak and last vaccination + serological survey for NSP demonstrated absence of infection in vaccinated animals a Decision has been adopted to re-establish the free status Modified recovery of free status Following emergency vaccination, and by way of derogation, it may be decided by Comitology, to withdraw the restrictions applied in the vaccination zone after the clinical and serological survey and the classification of herds have been completed and confirmed the absence of foot-and-mouth disease virus infection 7 Risk assessment taking into account different test strategies 8 Objectives of post-vaccination measures Risks from products from vaccinated animals can be mitigated but Post-vaccination surveillance is carried out to ensure that after the recovery of the status vaccinated ruminants can be moved safely out of the previous vaccination zone and continue their productive life within the vaccinating Member State Conclusion When the free status is recovered there should only be herds which are either sero-negative Or Sero-positive exclusively from the administration of an inactivated vaccine No danger of carriers or undetected disease 9 1 EU FMD-NSP workshop 32

33 Combining multiple sources of evidence to demonstrate disease freedom, Dr. Tom Murray, APO FAO EUFMD Combining Multiple Sources of Evidence to Demonstrate FMD Freedom Tom Murray, Associate Professional Officer, FAO- EUFMD Commission Key participants in the development of work are Ausvet, The International Epilab in the Danish Institute for Food and Veterinary Research and The Australian Biosecurity CRC The objective is to develop a system to quantitatively evaluate confidence in Disease Freedom Details of methodology available at 3 July 27 FAO-EUFMD Commission 1 3 July 27 FAO-EUFMD Commission 2 Summary of Presentation 1. Overview of The use of the stochastic Scenario Tree Approach 2. Review of various quantitative inputs and outputs in the context of an overall surveillance system 3. Example: Evaluation of Random Sero-survey using the stochastic Scenario Tree approach 3 July 27 FAO-EUFMD Commission 3 3 July 27 FAO-EUFMD Commission 4 Overview of Scenario Tree Approach Surveillance System Component(SSC) 3 July 27 FAO-EUFMD Commission 5 3 July 27 FAO-EUFMD Commission 6 EU FMD-NSP workshop 33

34 Design Herd Prevalence Design Unit Prevalence Explanations The minimum proportion of herds you would expect to have FMD if FMD is present in a region The minimum proportion of animals within a herd that you would expect to have FMD if it was present 3 July 27 FAO-EUFMD Commission 7 Surveillance System Sensitivity Negative predictive value (NPV) Explanations The probability that the surveillance systems will detect FMD if it is present in the region Given that surveillance systems have not detected FMD, how confident are we that this is a true reflection of the situation 3 July 27 FAO-EUFMD Commission 8 Explanations Specificity Prior/Pretest probability of disease Posterior/Post-test test probability of disease The estimate of the probability of FMD presence before the survey is carried out The estimate of the probability of FMD based on prior probability and the survey results Objective: To demonstrate disease freedom Implies all tests have lead to a negative outcome Specificity = 1 3 July 27 FAO-EUFMD Commission 9 3 July 27 FAO-EUFMD Commission 1 Bayes Theorem Inputs and Outputs are based on distributions rather than point values. This reflects the uncertainty and variability associated with biological data Prior probability of diseases and the actual survey results are combined to give a posterior probability of disease Example: Evaluation of a Simple Random Sero-Survey Survey (Using Point Values) Followed by combination of various SSC s 3 July 27 FAO-EUFMD Commission 11 3 July 27 FAO-EUFMD Commission 12 EU FMD-NSP workshop 34

35 Step 1: Calculate the probability that any one sample tests positive Step 2:Calculate Sensitivity of Surveillance System Component. If a is the probability that any sample tests positive Then the probability that the surveillance system component detects disease if it is present is 1-(1-a) n (where n is the number of samples taken) 3 July 27 FAO-EUFMD Commission 13 3 July 27 FAO-EUFMD Commission 14 Step 3: Calculate Negative Predictive Value of Surveillance System and the Posterior probability of FMD Step 4: Carry out similar procedure for other surveillance system components Other SSC s s may require more assumptions and the uncertainty associated with inputs would be greater The degree of uncertainty associated with inputs can be reduced with time through increased knowledge and the use of expertise 3 July 27 FAO-EUFMD Commission 15 3 July 27 FAO-EUFMD Commission 16 Step 5:Combine the various SSC s together to get an overall value of the surveillance system Lack of independence between various surveillance system components needs to be accounted for Key Points This is a simplified overview of the process The quantitative evaluation of surveillance systems can provide for comparisons between systems This work requires the close involvement of expertise and experience from various fields This work emphasizes the value of current over historical data. The use of this approach can identify gaps in knowledge for further research An effective system for demonstration of FMD freedom also means a system which is more likely to detect new incursions earlier 3 July 27 FAO-EUFMD Commission 17 3 July 27 FAO-EUFMD Commission 18 EU FMD-NSP workshop 35

36 Session III: OIE guidelines and serosurveillance post-vaccination: practice (1) OIE rules and guidelines on use of serosurveillance to demonstrate freedom from infection with FMDV and (2) use of a model to predict expected prevalence of carriers and to design serosurveillance for their detection. Dr. David Paton, United Kingdom 1) OIE rules and guidelines on use of serosurveillance to demonstrate freedom from infection with FMDV 2) Use of a model to predict expected prevalence of carriers and to design serosurveillance for their detection OIE Diagnostic Manual OIE Terrestrial Animal Health Code Chapter Zoning & Compartmentalisation Chapter FMD Appendix General guidelines for animal health surveillance Appendix Guidelines for the surveillance 7/3/27 of FMD 2 OIE Code FMD Chapter : Recovery of FMD-free status following outbreaks (1) Outbreaks in a country or zone that is FMD-free with Outbreaks in a FMD-free country without vaccination vaccination. Can regain this status by: Can regain this status by: Slaughter of infected, no vaccination, serosurveillance to Vaccination, slaughter of infected, serosurveillance to demonstrate absence of infection, 3 months minimum wait detect virus circulation rather than infection, 6 month Slaughter of infected, vaccinate-to-kill, serosurveillance to minimum waiting period demonstrate absence of infection, 3 months minimum OIE provides guidelines for serosurveillance Slaughter of infected animals, vaccinate-to-live, Do not need to detect carriers at all serosurveillance to demonstrate absence of Risk of carriers still being present is mitigated by restriction infection, 6 months minimum on trade in live animals if free with vaccination, rather than free without vaccination 7/3/27 4 7/3/27 3 OIE Code FMD Chapter : Recovery of FMD-free status following outbreaks (2) OIE Code FMD Chapter Article Definitions Occurrence of FMDV infection FMD virus isolated or viral RNA or virus antigen demonstrated in samples from an animal NSP antibodies not due to vaccination in one or more animals showing signs of FMD or linked to a confirmed or suspected outbreak, or giving cause for suspicion of previous association or contact with FMDV 7/3/27 5 OIE Code Appendix General Guidelines for Surveillance Article Surveillance to demonstrate freedom from disease/infection Demonstration of freedom from infection Implies absence of the pathogenic agent Complete absence cannot be proven with 1% confidence rather provide adequate evidence to an acceptable level of confidence that infection is at less than a certain acceptable prevalence (however, none of these acceptance criteria are defined for FMD within Code) However, finding evidence of infection at ANY level automatically invalidates a claim for freedom. 7/3/27 6 EU FMD-NSP workshop 36

37 OIE Code Appendix Guidelines for FMD Surveillance Article The use and interpretation of serological tests Reference to Manual for recommended tests Option to use NSP tests in vacc/unvacc animals and NSP tests in vacc animals Need diagnostic follow-up on presumptive positive serological test results All herds with seropositive reactors should be investigated including clinical and epidemiological evaluation and supplementary lab tests High Sp and Se equivalent to screening assays Information needed on performance characteristics and validation of tests used 7/3/27 7 Article The use and interpretation of serological tests (cont) Follow-up if no vaccination Where possible virological investigations should be made Reactor animal assumed positive unless suspicion ruled out Follow-up if vaccination Procedures described are aimed at detection of virus circulation only and not detection of carriers Resample and test initially surveyed animals in reactor unit Sample and test additional contacts Sample and test other epidemiologically linked units Sentinels can be used Relate lab results to epidemiological situation 7/3/27 8 Fig. 1. Schematic representation of laboratory tests for determining evidence of FMDV infection through or following serological surveys 7/3/27 9 OIE Code FMD Chapter Article (bis). New proposals for containment zones within an FMD free country or zone Reduces impact on non-affected regions Only applicable to limited outbreak that has been already brought under control and surveillance demonstrates no undetected cases Increased passive and targeted surveillance in rest of free zone/country Recovery of status within containment zone as in Article /3/27 1 FMD modelling and NSP testing Mark Arnold VLA John Wilesmith Defra David Paton, Eoin Ryan, Sarah Cox IAH Pirbright Outline of approach Simulation of epidemic with/without vaccination Include simple model of the probability of farm being a carrier Model post epidemic testing using NSP and record status of each herd Examine sensitivity and specificity of NSP testing at herd level 7/3/ /3/27 12 EU FMD-NSP workshop 37

38 Transmission model Transmission depends on Number of animals in IP Distance from IP Number of animals in susceptible farm Transmission vs distance from IP 7/3/ /3/27 14 Animal number Infectiousness/susceptibility Susceptibility to infection postvaccination Susceptibility= 36 ( NCATTLE ) + ( NSHEEP ) Infectiousness= ( N ) + ( N ) CATTLE SHEEP Animal (and farm) susceptibility reduced according to days between exposure and vaccination 7/3/ /3/27 16 Likelihood of disease post vaccination Infectiousness assumed to correlate with probability of disease Probability of carriers in vaccinated farms Estimate initial infs Calculate clinicals Estimates from 21 epidemic stil into ke Efficacy of vaccinationm equation or simulation Likelihood of disease vs days post vaccinationexperiment Determine if IP IF NOT AN IP <5 days post vaccination OR > clinicals dairy; >1 clinical -suckler Calculate if carriers 5% probability for each infected 7/3/ /3/27 18 EU FMD-NSP workshop 38

39 Number of initial infections Carrier probability vs herd size and no of clinicals for detection Small dependence on number of cattle: Mean infs= N cattle 7/3/ /3/27 2 Rate of vaccination Commences 7 days after initial IP identified 5 vaccination teams, vaccinating up to 25 animals per day Clinical inspection of sheep and pigs also performed 1k vaccination ring around each IP, nearest farms vaccinated first 7/3/27 21 Other assumptions Time to detection for infected herds taken from 21 epidemic All animals in each IP are culled, time to slaughter taken from 21 epidemic 7/3/27 22 Outline of simulations Population at risk=devon 5 initial infections 2 runs of each of the following scenarios: No vaccination Vaccinate farms>5 cattle, <1k from IP Vaccinate all cattle farms < 1k from IP 44% of herds with cattle have <5 in Devon 7/3/27 23 Post epidemic testing Commences 3 days after last IP Cattle tested on all vaccinated farms with Cedi screen/cedi retest/svanova confirm Sensitivity=67%, specificity=99.99%. Sufficient animals tested to detect 5% prevalence with 95% probability Have also looked at testing all animals 7/3/27 24 EU FMD-NSP workshop 39

40 Epidemic size distribution 2 runs Mean no of IPs/day Vaccinating small farms 66 vaccinated farms/ip, 71% reduction in cases Vaccinating farms >5 cattle 35 vaccinated farms/ip, 68% reduction in cases 7/3/ /3/27 26 Distribution of carrier farms by herd size Reducing number of farms as herd size increases But higher incidence of those farms Within herd prevalence Farms with many initial infs->ips Farms infected late after vaccination have few initial infs Therefore carriers occur at low prevalence 7/3/ /3/27 28 Sensitivity of testing (at farm level) Strategy True Detected Sensitivity positives Vacc > % only Vacc all % 75% sensitivity if all animals tested (for vacc>5) Specificity of testing (at farm level) Strategy True False Specificity negatives positives Vacc >5 233, 1, % only Vacc all 397, 1, % 98.4% specificity if all animals tested (for vacc>5) 7/3/ /3/27 3 EU FMD-NSP workshop 4

41 Main results (1) Little benefit for vaccinating small herds in terms of total cases Carrier farms distributed across all herd sizes fewer large herds but higher incidence of large herds Small number of carriers in positive herds means low herd-level sensitivity (with 5% design prevalence) therefore need to test all animals in vaccinated herds 7/3/27 31 Main results (2) Vaccinating small herds Smaller average herd size of positives Higher herd level sensitivity larger proportion of animals tested Higher herd level specificity But far more vaccinated farms to test Same conclusions from results of NSP testing in Cumbria 7/3/27 32 EU FMD-NSP workshop 41

42 Presentation of adopted FMD vaccination plans - scenarios prepared for the workshop, Dr. Phillippe Houdart, Belgium Aim of the exercise Federal Agency for the Safety of the Food Chain INTRODUCTION TO THE EXERCISE design and interpret a post-vaccination FMD surveillance scheme including the use of NSP tests, based on EU and OIE guidelines objective: substantiate a claim to return to the status free of infection starting point: FMD epizootic with 3 clusters of outbreaks each cluster = one scenario vaccination to live policy vaccinated animals are not killed Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain Approach different control strategy in each of the clusters, but always with vaccination of one or more species: cluster 1 = index case + 3 neighbouring outbreaks cluster 2 = 2 outbreaks cluster 3 = 23 outbreaks in different subclusters outbreaks between end of February and end of March start of the surveillance programme = end of April beginning of May, > 1 month after last outbreak Scenario 1 = cluster 1 PZ and SZ surrounding index case + 3 neighbouring outbreaks size = 433 km 2 density: cattle = medium density (37 / km 2 ) pigs = high density (3 / km 2 ) sheep/goat = low density (2 / km 2 ) Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain Scenario 1 = cluster 1 culling: susceptible animals in 4 outbreaks vaccination of cattle and pigs in protection zone particularities high number of pig farms: 3 very large pig farms 85% are small farms very low number of sheep in the north of the SZ the farms are grouped (villages) Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain EU FMD-NSP workshop 42

43 Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain Scenario 2 = cluster 2 PZ and SZ surrounding 2 neighbouring outbreaks size = 38 km 2 FMDV introduced through movement of infected sheep (outbreak 5) density: cattle = medium density (4 / km 2 ) pigs = medium density (124 / km 2 ) sheep/goat = low density (4 / km 2 ) Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain Scenario 2 = cluster 2 culling: susceptible animals in 2 outbreaks vaccination of cattle and pigs in protection zone particularities: one of outbreaks = large pig farm (>4. pigs) only infected pigs in a quarantine unit separated from the other buildings rest of holding is vaccinated Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain EU FMD-NSP workshop 43

44 Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain Scenario 3 = cluster 3 vaccination area surrounding 23 outbreaks size = 1.44 km 2 FMDV introduced through movement of infected sheep (outbreak 5) density: cattle = high density (16 / km 2 ) pigs = high density (>1. / km 2 ) sheep/goat = low density (13 / km 2 ) Scenario 3 = cluster 3 culling: susceptible animals in all outbreaks vaccination of all cattle in vaccination area particularities: high density area for pigs and cattle outbreaks in grouped into subclusters several outbreaks with no or unclear epidemiological links Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain, 2,5 5, 7,5 1, Kilom eters, 2,5 5, 7,5 1 Kilometers Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain EU FMD-NSP workshop 44

45 What is expected of you? each of the scenarios is approached by 2 groups of 2 countries tasks: design surveillance scheme for one of the given clusters (both vaccinated and non vaccinated animals) interpret set of results of your surveillance scheme given by the supervisory team design follow up for positive results present results to the audience tomorrow (powerpoint presentation) Assignment of the groups scenario 1 1. Moldova + Ukraine Per Have + David Paton 2. Norway + Latvia Tom Murray scenario 2 3. Slovakia + Estonia Åse Uttenthal + Philippe Vanier 4. Finland + Poland Aldo Dekker scenario 3: 5. Czech Rep. + Sweden Dónal Sammin 6. Switzerland + Israel Lea Knopf room Federal Agency for the Safety of the Food Chain Federal Agency for the Safety of the Food Chain documentation publications about FMD testing and in particular NSP testing regarding scenarios: background information tables with composition of the different clusters timeline of the outbreaks short information about each of the outbreaks maps Federal Agency for the Safety of the Food Chain Background information about the epizootic At the end of February an outbreak of FMD strain A Iran 96 is observed in a pig holding. The FMD virus has most likely been introduced onto the holding through contaminated leftovers brought back from Turkey by the son of the owner around mid February. The initial clinical signs are ignored by the owner, resulting in a rather late detection of the disease by the farm veterinarian. This outbreak is the first case detected and is the index case of the epizootic. The infected farm is situated at the centre of cluster 1. Three major routes are responsible for the subsequent spread of the disease: - A diseased pig is slaughtered in the initial stages of the outbreak. The village butcher who kills the diseased pig subsequently spreads the virus to a sheep trader on the opposite site of the village. The brother of owner of the first outbreak assists at the slaughtering and spreads the virus to his own farm in the same village. - Subsequently, a sheep trader whose farm is infected via the butcher spreads the virus to clusters 2 and 3 through the selling of infected sheep. The trader is very reluctant to disclose information about his comings and goings, amongst others about the movement of infected animals to his colleague trader who is at the origin of cluster 3. EU FMD-NSP workshop 45

46 - Where in cluster 2 the infection is detected in an early stage, in cluster 3 the disease is detected only after the virus has already spread to several farms. The common link in the initial spread in this cluster is a second sheep trader that has bought infected animals from the sheep trader in cluster 1. The infected animals have stayed in this trader s stable in a larger group of sheep. Sheep of this group are sold to several known and unknown clients in the region. In the weeks following the detection of the first outbreak, a total of 29 outbreaks are recorded grouped into 3 distinct clusters, namely 4 outbreaks in cluster 1 (including the index case), 2 in cluster 2 and 23 in cluster 3. Cluster 3 is the largest of the 3 clusters; the 23 outbreaks in this cluster are for the greater part grouped into smaller clusters of outbreaks. The last outbreak is detected at the end of March, almost 5 weeks after the detection of the index case. All outbreaks are finally managed by a combination of culling and preventive vaccination. The vaccination is always performed in either cattle or pigs or both, but never in small ruminants. Characteristics of cluster 1 Cluster 1 is comprised of the 1 km surveillance zones surrounding the index case and 3 other outbreaks in the same village: - general composition: high density area for pigs, medium density area for cattle, low density area for small ruminants; - size: 433 km 2 ; - composition: see tables. The index case is not immediately detected; the 3 other outbreaks in the cluster pop up within a week of the detection of the index case. Characteristics of cluster 2 Cluster 2 is comprised of the 1 km surveillance zone surrounding 2 neighbouring outbreaks: - general composition: medium density area for cattle, medium density area for pigs, low density area for small ruminants; - size: 38 km 2 ; - composition: see tables. Both outbreaks are detected in an early stage and subsequently no more outbreaks are recorded in this area. The medium density area for pigs is misleading: there is only one big pig holding (one of the outbreaks) in the area and a few very small backyard holdings Characteristics of cluster 3 Cluster 3 is comprised of the fused 1 km surveillance zones surrounding a total of 23 outbreaks in cattle, pigs or small ruminants. Vaccination is performed throughout the inner part of this zone, but is not performed in a 5 km band on the edge; the latter acts as a buffer zone with the non-infected areas. The main characteristics of cluster 3 are: - general composition: high density area for pigs, high density area for cattle and low density area for small ruminants; - size: 1.44 km 2 ; - Composition: see tables. EU FMD-NSP workshop 46

47 The outbreaks in cluster 3 are grouped into 2 larger sub clusters (7 and 6 outbreaks), 2 smaller sub clusters (3 and 3 outbreaks) and 4 separate outbreaks. Several of the outbreaks have no clear link with any other outbreak or are recorded after vaccination in this cluster has started. Control strategy and vaccination The authorities have opted for a combination of culling, zoning, restriction measures and vaccination since: - the FMDV has spread rapidly from the orginal cluster to other parts of the country; - the epidemiological circumstances of the spread are not clear; - the number of outbreaks and the fact that large professional farms are involved put an enormous strain on the capacity for depopulation. In each of the scenarios all susceptible animals in the outbreaks are culled. Only in the large pig farm (outbreak 6) in cluster 2, solely the pigs in the affected quarantaine stable are culled. In all outbreaks, the classic 3 km protection zone and 1 km surveillance zone are originally delimited. Since the exercise is situated at least one month after the last outbreak, the protection zones no longer exists; only vaccination zones and the non-vaccinated remainders of the surveillance zones remain. The details of the vaccination strategy in each of the clusters are summarized under scenarios. Scenarios of the exercise The time setting for the exercise is the end of April, at least 1 month after the last outbreak has occurred. Since the last outbreak, the surveillance has been based on clinical surveillance. Apart from the holdings that are culled, no serological surveillance has been performed yet. The aim of the exercise is to device and to interpret a post-vaccination FMD surveillance scheme that includes the use of NSP tests. The surveillance scheme must substantiate a claim to return to the status free of infection (i.e. freedom of infection with a certain degree of confidence, according to the EU guidelines and the OIE general and specific serosurveillance guidelines) taking into account the characteristics of the laboratory test or tests chosen. The participants are divided into working groups that will device the surveillance scheme for one of the scenarios. - Since the exercise focuses on the use of NSP test, the scheme for dealing with both the vaccinated and non-vaccinated animals in the vaccination areas is the principal assignment. - Secondly, the scheme for the rest of the surveillance zone must also be elaborated. - Finally, based on the schemes presented, the groups will be presented with results of the serosurveillance scheme chosen. They will have to interpret these results and present a suitable follow up of the seropositive animals/herds/flocks. A sample is considered positive when it will be twice positive in a double, successive testing in the chosen analysis scheme. EU FMD-NSP workshop 47

48 Scenario 1 Scenario 1 focuses on cluster 1 that surrounds the 4 outbreaks around the index case. It takes into account the vaccination of all cattle and pigs in a 3. m radius around the outbreaks (the former protection zone). The size of the vaccination area is 57 km 2. Scenario 2 Scenario 2 focuses on cluster 2 surrounding the large pig farm and a second outbreak. All cattle and small ruminants in the second outbreak and the pigs kept in the affected stable on the large pig farm are culled. The vast majority of the pigs in the affected pig farm are not culled (separate production units). This scenario takes into account the vaccination of all cattle and pigs in a 3. m radius around the outbreaks (the former protection zone). The size of the vaccination area is 32 km 2. Scenario 3 Scenario 3 focuses on cluster 3 that surrounds 23 outbreaks. It takes into account the vaccination of all all cattle in the whole of the fused surveillance zones around the outbreaks, except an outermost 5 km band that acts as a buffer area around the vaccinated zone. The size of the vaccination area is 76 km 2. Annexes The tables in annexe reproduce: - the surface of the different zones, - the composition of the various clusters with a stratification by radius, - the composition of the various clusters with a stratification by size. Several maps visualise the three clusters. General time setting - The outbreaks occur between the end of February and the end of March. - The vaccination campaign is performed: - in the first week of March for clusters 1 and 2, starting on 4 respectively 5 March; - in between 14 and 21 March for cluster 3. - The final monitoring begins at the end of April. Standard policy applied t control the outbreaks - Delimitation of the classical protection zone (3 km) and surveillance zone (1 km). - Culling of infected herds. - At the detection of the first outbreak, on 27 February, a standstill is imposed in the part of the country where clusters 1 and 2 are situated. A standstill is also imposed on 1 March in the part of the country comprising cluster 3 after the detection of the first outbreak in this cluster (outbreak 7). - Vaccination is performed according to the above mentionned dates and the schemes mentionned in the background document. Time schedule of the outbreaks See timeline EU FMD-NSP workshop 48

49 OUTBREAK 1 = pig holder ID-number cluster 1 link to source of infection = index case date of infection around 13-2 date of confirmation 27-2 localisation type of holding structure/capacity type of rearing number of animals present contaminated products from Turkey, brought home by owner s son municipality of Ljutomer fattening pig holding different small sheds Indoors 84 pigs: - 9 sows - 12 piglets - 6 fattening pigs of 8 kg - 2 gelts - 1 boar Particularities - On 2-2 one of the heavier pigs is showing symptoms of inappetite. Since this pig is destined to be slaughtered soon, the owner decides to slaughter it immediately at home. The slaughter is done by the local butcher. The owners s brother participates in the slaughter and takes some of the meat home. - On 25-2 the owner notices inappetite in one of the pigpens. The same day, after a quick investigation, the veterinarian suspects traumatic injuries due to the feed the animals have received, allthough some animals have fever. - On 26-2 in the morning, the veterinarian is recalled since more pigs show the same symptoms of inappetite, salivation, marked fever and lameness as well. On detailed clinical examination of the animals, he establishes changes in the tongue and mucous tissue of the oral cavity, and additionally in the skin of the feet between the claws in the interdigital space, characteristic of foot-and-mouth disease. Consequently, he suspects the presence of foot-and-mouth disease virus. The veterinary service is notified. EU FMD-NSP workshop 49

50 The veterinary service visits the farm, takes the necessary samples and sends the samples immediately to the laboratory. In view of the suspicion, the holding is blocked and all farms in the same village are put under surveillance and cannot move cattle, small ruminants and pigs any more. - On 27-2 FMD is diagnosed in the lab. - On 28-2 the animals in the outbreak are killed and destroyed. ID-number cluster 1 link to outbreak 1 source of infection date of infection 2-2 date of confirmation 27-2 localisation type of holding structure/capacity type of rearing number of animals present OUTBREAK 2 = brother of the index indirect contact due to owner s contact with infected pig from index case municipality of Ljutomer, 14 m from index cattle and sheep 1 stable indoors 13 cattle, 3 pigs Particularities - The FMD virus is brought on the farm by the owner who has helped slaughter the first infected pig at the index case. - The infection in this farm is detected by the veterinary service that visits the farm on 26-2 based on the inquiry at the index case. Two cattle in this farm show few mild clinical signs of FMD: changes in the tongue and mucous tissue of the oral cavity and fever. Samples are taken and sent to the laboratory. - On 27-2 FMD is diagnosed in the lab. - On 28-2 the animals in the outbreak are killed and destroyed EU FMD-NSP workshop 5

51 ID-number cluster 1 link to outbreak 1 source of infection OUTBREAK 3 = neighbour of the index date of infection around 2-2 date of confirmation 28-2 localisation type of holding structure/capacity type of rearing number of animals present indirect contact due to probably airborne spread municipality of Ljutomer, 5 m from index cattle 1 stable indoors 16 cattle Particularities - The introduction of the FMD virus is probably due to airborne spread from the index case. - The infection in this farm is detected by the veterinary service that visits the farm on 27-2 after being allerted by the owner that some of his cattle in the shed next to the index case show fever. Samples are taken and sent to the laboratory. - On 28-2 FMD is diagnosed in the lab. - On 29-2 the animals in the outbreak are killed and destroyed. EU FMD-NSP workshop 51

52 OUTBREAK 4 = first case in cluster 2 ID-number cluster 2 link to outbreak 5 source of infection direct contact due to infected sheep brought by the sheep trader of outbreak 5 date of infection 24-2 date of confirmation 2-3 localisation municipality of Domzale type of holding mixed holding structure/capacity several sheds for maximal 2 sheep type of rearing outdoors, next to the stables and the quarantaine stable of outbreak 6 number of animals present 8 cattle, 4 sheep, 3 pigs Particularities - The introduction of the FMD virus is due to direct contact on 24-2 when the trader of outbreak 5 takes infected sheep to the farm. - The sheep are placed in a shed on a pasture next to large pig farm (outbreak 6) where the cattle and pigs from the same owner are kept. - The infection in this farm is detected by the veterinary service who visits the farm on 1-3 following the notification by the veterinarian of the farm of a suspicion of FMD in the pigs of the farm. - On 2-3 in the morning FMD is diagnosed in the lab. - On 2-3 the animals in the outbreak are killed and destroyed. - The infected animals are not supposed to be on this pasture so close to the big commercial pig farm, but the owner has decided to ignore this rule and has temporarily housed the animals on the pasture because of the bad state of his stable. EU FMD-NSP workshop 52

53 OUTBREAK 5 = sheep trader source of spread towards clusters 2 and 3 ID-number cluster 1 link to outbreak 1 source of infection indirect contact due to the butcher who slaughtered the first infected pig date of infection around 2-2 date of confirmation 2-3 localisation municipality of Ljutomer, other part of the village 45 m from index type of holding sheep trader structure/capacity several sheds for maximal 2 sheep type of rearing indoors number of animals present 3 sheep, 2 pigs Particularities - The introduction of the FMD virus is due to indirect contact through the butcher that slaughtered the first infected pig in the index case. On the same day (2-2), the butcher goes slaughter a sheep at the trader s. - The infection in this farm is detected by the veterinary service that visits the farm on 2-3 following the inquiry at the first case in the second cluster. Some of the sheep at the trader s show some changes in the tongue and mucous tissue of the oral cavity and some fever. Samples are taken and sent to the laboratory. - On 2-3 in the evening FMD is diagnosed in the lab. - On 3-3 the animals in the outbreak are killed and destroyed. - The trader is very reluctant to disclose information about his activities in the last 2 weeks. He does not disclose the selling of sheep to a sheep trader that later on is declared outbreak 9, the first outbreak in cluster 3. EU FMD-NSP workshop 53

54 OUTBREAK 6 = big pig farm ID-number cluster 2 link to outbreak 4 source of infection date of infection around 28-2/1-3 date of confirmation 4-3 indirect contact due to airborne spread of FMD from the pigs of outbreak 4 kept in a pasture; the ventilation fan of the infected quarantaine stable sucks air coming from the pasture localisation municipality of Domzale, 3 m from outbreak 4 type of holding structure/capacity type of rearing number of animals present big commercial pig farm closed holding with 9 stables for sows/piglets and 36 stables for fattening pigs; separate quarantaine unit indoors in total: 3916 sows, 72 boars, piglets, fattening pigs of different age Particularities - The introduction of the FMD virus is due to airborne spread from the pigs kept on the pasture next to the quarantaine stable of the farm. The ventilator of the stable sucks air passing over the pasture. - The quarantaine stable is separated from the other stables on the farm. The animals are only cared for as the last duty in the daily routine. Before entering or leaving the quarantaine stable, staff have to go through strict biosecurite procedures. - The farm veterinarian raises the suspicion on mild clinical symptomes in the pigs in the quarantaine stable. As a result of outbreak 4, the pigs in this stable (that is the closest to the infected pasture) are inspected twice a day. At the morning inspection on 3-3, 4 out of 69 pigs (all gelts brought in the week before) in the stable show a temperature and reddening in the mouth. The other animals in the stable show no clinical signs of disease. - The authorities are called in, the suspected pigs in the stable are killed and samples are sent to the laboratory, where FMD is diagnosed on The same day (3-3), the owner decides to kill the 67 remaining gelts in the quarantaine unit. - It is believed that because of the very early detection of the outbreak, the fact that it is a quarantaine stable with strict biosecurity measures and the fact that the stables next to the quarantaine stable and the pasture with the infected pigs of outbreak 4 are empty, the disease will EU FMD-NSP workshop 54

55 not spread to the rest of the farm. As a precautionary measure, on 5-3, vaccination is started immediately in the rest of the farm, progressing outwards beginning at the stables closest to the quarantaine stable. EU FMD-NSP workshop 55

56 OUTBREAK 7 = sheep farm ID-number 4672 cluster 3 link to outbreak 9 source of infection direct contact due to infected sheep bought from outbreak 9 date of infection 5-3 date of confirmation 11-3 localisation municipality of Lotenhulle type of holding backyard sheep holding structure/capacity - type of rearing outdoors number of animals present 3 Particularities - The introduction of the FMD virus is due to the purchase of 3 sheep from outbreak 9. - The infection is detected by the farm veterinarian who visits the farm on 9-3 after the owner has observed some inappetite in the purchased sheep. The veterinarian does not suspect FMD at first but reviews his opinion after visiting the next day outbreak 8 where he observes some clearer symptoms of FMD, also in sheep bought from outbreak 9. - The veterinary service is called in on 1-3 and samples are taken and sent to the laboratory. - On 11-3 FMD is diagnosed in the lab. - On 1-3 the animals in the outbreak are killed and destroyed. EU FMD-NSP workshop 56

57 OUTBREAK 8 = sheep farm ID-number cluster 3 link to outbreak 9 source of infection direct contact due to infected sheep bought from outbreak 9 date of infection 5-3 date of confirmation 11-3 localisation municipality of Beernem type of holding mixed pig and cattle farm with a few hobby sheep structure/capacity fattening pig in closed stables and dairy cattle type of rearing indoors number of animals present 125 dairy cattle, 1.31 pigs of 12 and 18 weeks, 9 sheep Particularities - The introduction of the FMD virus is due to the purchase of 2 sheep from outbreak 9. - The farm veterinarian is called in on 1-3 after the owner has observed some inappetite and salivation in the 2 purchased sheep. Since the day before, the veterinarian has seen similar symptoms in sheep of outbreak 7 and the sheep are coming from the same trader, he investigates the affected animals more closely this time. Based on the changes he finds in the tongue and mucous tissue of the oral cavity and a slight fever in the affected animals, he suspects FMD and calls in the veterinary service. - Samples are taken and sent to the laboratory. - On 11-3 FMD is diagnosed in the lab. - On 1-3 and 11-3 the animals in the outbreak are killed and destroyed. - At close inspection at culling, a limited number of pigs begin to show the first symptoms of FMD. The samples taken at culling reveal infected animals. Neither clinical signs nor unfavourable analysis results are observed in the cattle. EU FMD-NSP workshop 57

58 OUTBREAK 9 = sheep trader ID-number cluster 3 link to outbreak 5 source of infection direct contact due to the purchase of infected sheep at outbreak 5 (sheep trader in cluster 1) date of infection 24-2 date of confirmation 11-3 localisation municipality of Aalter type of holding sheep and cattle trader structure/capacity different pens for maximal 23 sheep and 25 cattle under one roof type of rearing indoors and outdoors number of animals present 122 sheep, 9 cattle Particularities - The FMD virus is brought into the premises by purchasing on 24-2 a group of 3 sheep, some of which are infected, at the trader of outbreak 5. These sheep are put in a stable at outbreak 9 with some 15 other sheep. Most of the animals are sold to sheep farmers in the surrounding villages in the 2 weeks following the arrival of the infected group. - The infection in this farm is detected by the veterinary service that visits the farm on 1-3 following the inquiry at outbreaks 7 and 8, the first detected outbreaks in cluster 3. Only very obscure changes in the tongue and mucous tissue of the oral cavity and some slight fever are observed in some of the sheep present. Samples are taken and sent to the laboratory. - On 11-3 FMD is diagnosed in the lab. - On 1-3 the animals in the outbreak are killed and destroyed. - The inquiry at the trader s reveales the link to outbreak 5 (sheep trader in cluster 1) and the selling of animals (cattle and sheep) to some 2 clients in the region since the arrival of the infected animals from cluster 1. Most of the clients are one-time buyers who have bought and immediatley taken away their sheep, without the trader recording their coordinates. The subsequent epidemiological inquiry into these contact herds does not succeed in retracing all the animals sold. EU FMD-NSP workshop 58

59 OUTBREAK 1 OUTBREAK 11 OUTBREAK 12 ID-number cluster link to outbreak 8 outbreak 8, 9 and 1 unknown source of infection neigbouring contact neigbouring contact of outbreaks 8 and 1; contact of outbreak 9 unknown date of infection around 1-3 around 1-3 around 5-3 date of confirmation localisation municipality of Beernem municipality of Beernem municipality of Nevele type of holding dairy cattle holding backyard sheep holding cattle holding type of rearing indoors outdoors indoors number of animals present 125 cattle 12 sheep 3 fattening cattle particularities Neighbouring holding of outbreak 8. Family ties and important professional contacts exist between the two premises. The outbreak is detected in its early stages. Neighouring holding of outbreaks 8 and 1 and direct contact of outbreak 9 following the purchase of 4 sheep. The 12 sheep were kept on a pasture. Suspicion is raised on 14-3 and confirmed on 15-3 by the veterinary service in the framework of inquiry into oubreak 9. The outbreak is detected in its early stages The suspicion is raised on 15-3 by the farm veterinarian. The cattle show 1-to-12-day-old lesions. The owners, a 75-year-old couple has only observed limping the previous day. The premise is situated within the surveillance zone surrounding outbreak 7. The outbreak is an isolated case: no epidemiological link with any of the existing outbreaks is revealed. EU FMD-NSP workshop 59

60 OUTBREAK 13 OUTBREAK 14 OUBTREAK 15 ID-number cluster link to unknown outbreak 9 unknown source of infection unknown neighouring holding of outbreak 9 unknown date of infection around 5-3 around 1-3 around 5-3 date of confirmation localisation municipality of Oostkamp municipality of Aalter municipality of Waarschoot type of holding fattening cattle and pigs closed pig farm cattle + pig type of rearing indoors + outdoors indoors Indoors + outdoors number of animals present 57 cattle, 11 pigs 119 pigs 156 dairy cattle, 973 fattening pigs particularities The suspicion is anonymously raised to the veterinary service on A visit on the next day reveals indeed very mild symptoms in some of the pigs. These symptomes are confirmed by laboratory analysis on the same day. The subsequent indeep inquiry reveals that the owner has been trying to hide the infection: meat (in the freezer) and offal (in the dungheap) of 2 pigs are discovered and turn out to be positive in the laboratory analysis. There are no links to previous outbreaks. The suspicion is raised by the veterinarian on 15-3 in the framework of the monitoring in the protection zone around outbreak 9. The farm is situated at 6 m of outbreak 9. The infection is detected in its early stages. It might be the result of the culling activities at outbreak 9. The owner raises the suspicion on 16-3 following the appearance of clinical signs in both pigs (housed indoors) and the calves (housed in a semi-open stable). The premises are situated outside of the existing surveillance zone at about 1 to 12,5 km from the nearest outbreaks (7, 9 and 12). The first infected animal on the farm (a calve that died on 9-3 with, in retrospect, very mild symptoms of FMD) has probably been missed. No links with existing outbreaks exist. EU FMD-NSP workshop 6

61 OUTBREAK 16 OUTBREAK 17 OUBTREAK 18 ID-number cluster link to outbreak 9 unknown outbreak 15 (at 2,5 km) source of infection neighouring holding of outbreak 9 unknown unknown date of infection around 1-3 around 11-3 around 9-3 date of confirmation localisation municipality of Aalter municipality of Schuiferskapelle municipality of Waarschoot type of holding fattening cattle + closed pig holding dairy cattle cattle farm type of rearing indoors indoors outdoors (shed on pasture) number of animals present 45 cattle, pigs 178 cattle 1 fattening cattle particularities The suspicion is raised by the veterinarian on 16-3 in the framework of the monitoring in the protection zone around outbreak 9. The farm is situated in between the outbreaks 9 and 14. The infection is detected in its early stages. It might be the result of the culling activities at outbreak 9. The owner raises the suspicion on 17-3 following the appearance of clinical signs in the cattle. The holding is situated on the outskirts of the existing surveillance zones around outbreaks 9 and 7. There is no link with existing outbreaks. The outbreak is detected in its early stages. The premises is culled on 17-3 and Suspicion is raised by the veterinarian following a visit of the pasture in the framework of the clinical surveillance in the protection zone around outbreak 15. There are no known direct or indirect contacts with any of the other outbreaks. The outbreak is detected in its early stages. EU FMD-NSP workshop 61

62 OUTBREAK 19 OUTBREAK 2 OUBTREAK 21 ID-number cluster link to outbreak 15 (at 4 km) outbreak 17 outbreak 17 source of infection unknown neighbouring holding of outbreak 17 (at 1,6 km) date of infection around 12-3 around 18-3 around 18-3 date of confirmation neighbouring holding of outbreak 17 (at 3 km) localisation municipality of Waarschoot municipality of Ruiselede municipality of Ruiselede type of holding cattle cattle + pigs diary cattle and closed pig farm type of rearing indoors indoors indoors number of animals present 145 cattle 83 fattening cattle, 763 fattening pigs 84 dairy cattle, pigs particularities Suspicion is raised by the veterinary service following a visit of the premises in the framework of the intensified clinical monitoring just outside of the protection zone around outbreak 15. There are no known direct or indirect contacts with any of the other outbreaks. The outbreak is detected in its early stages. Suspicion is raised by the farm veterinarian following his visit of the premises in the framework of the clinical surveillance of the protection/ vaccination zone around outbreak 17. There are no known contacts with any other outbreak. The outbreak is detected in its early stages with a few pigs showing recent lesions of FMD. The outbreak might be the result of the culling activities at outbreak 17. The farm is situated on the outskirts of the protection zone around outbreak 17. Suspicion is raised by the farm veterinarian in the framework of his clinical surveillance of the protection/ vaccination zone around outbreak 17. There are no known contacts with any other outbreak. The outbreak is detected in its early stages with 2 dairy cows showing the initial symptoms of FMD. EU FMD-NSP workshop 62

63 OUTBREAK 22 OUTBREAK 23 OUBTREAK 24 ID-number cluster link to outbreak 17 unknown outbreaks 15, 18 and 19 source of infection neighbouring holding of outbreak 17 (at 1,1 km) unknown date of infection around 18-3 around 1-3 around 18-3 date of confirmation neighbouring holding of outbreaks 15, 18 and 19 (at 1,3 to 2,5 km) localisation municipality of Schuiferskapelle municipality of Lovendegem municipality of Waarschoot type of holding cattle hobby sheep breeding cattle type of rearing outdoors outdoors indoors number of animals present 6 fattening cattle 5 sheep 59 cattle particularities Suspicion is raised by the farm veterinarian following his visit of the premises in the framework of the clinical surveillance of the protection/ vaccination zone around outbreak 17. There are no known contacts with any other outbreak. The outbreak is detected in its early stages. The outbreak might be the result of the culling activities at outbreak 17. Suspicion is raised by the owner following the dead of 2 of his sheep on their pasture. All 5 sheep turn out to have lesions consistent with a 2- weeks-old FMD infection. The owner has neglected the compulsory control visits and thus has missed the first symptoms. There are no links with any other outbreak. Suspicion is raised by the farm veterinarian following his visit of the premises in the framework of the clinical surveillance of the protection/ vaccination zone around outbreak 15. There are no known contacts with any other outbreak. The outbreak is detected in its early stages. EU FMD-NSP workshop 63

64 OUTBREAK 25 OUTBREAK 26 OUBTREAK 27 ID-number cluster link to outbreak 17 outbreak 25 outbreak 18 source of infection neighbouring holding of outbreak 17 (at 1,6 km) neighbouring holding of outbreak 25 (at 1,2 km) date of infection around 18-3 around 23-3 around 18-3 date of confirmation neighbouring holding of outbreak 18 (at 4 m) localisation municipality of Wingene municipality of Wingene municipality of Waarschoot type of holding pigs dairy cattle breeding cattle and breeding pigs type of rearing indoors indoors indoors number of animals present 55 fattening pigs 84 cattle 38 cattle and 443 pigs particularities Suspicion is raised by the farm veterinarian following his visit of the premises in the framework of the clinical surveillance of the protection/ vaccination zone around outbreak 17. There are no known contacts with any other outbreak. About a dozen pigs show clinical signs consistent with a week-old infection of FMD. Suspicion is raised by the veterinary service following a visit of the premises in the framework of the intensified clinical surveillance of the farms around outbreak 25. There are no known contacts with any other outbreak. The infection is detected in its early stages. Suspicion is raised by the farm veterinarian following his visit of the premises in the framework of the clinical surveillance of the protection/ vaccination zone around outbreak 15. There are no known contacts with any other outbreak. The outbreak is detected in vaccinated sows showing little symptoms, some around a week old. It might be the result of the culling activities at outbreak 18 EU FMD-NSP workshop 64

65 OUTBREAK 28 OUTBREAK 29 ID-number cluster 3 3 link to outbreaks 15 and 18 outbreak 25 source of infection neighbouring holding of outbreaks 15 and 18 (at 1 to 1,3 km) date of infection around 18-3 around 23-3 date of confirmation neighbouring holding of outbreak 25 (at 1 km) localisation municipality of Waarschoot municipality of Wingene type of holding fattening calves pigs type of rearing indoors indoors number of animals present 243 fattening calves 425 fattening pigs particularities Suspicion is raised by the farm veterinarian following his visit of the premises in the framework of the clinical surveillance of the protection/ vaccination zone around outbreak 15. There are no known contacts with any other outbreak. The outbreak is detected in 4 calves showing mild symptoms of about a week old. Suspicion is raised by the veterinary service following a visit of the premises in the framework of the intensified clinical surveillance of the farms around outbreak 25. There are no known contacts with any other outbreak. The infection is detected in its early stages with only a few pigs in one pen showing symptoms. EU FMD-NSP workshop 65

66 Size of the clusters: area surface (km2) cattle farm density (per km2) cattle density (per km2) sheep/goat farm density (per km2) sheep/goat density (per km2) pig farm density (per km2) pig density (per km2) cluster 1 - vaccination zone 57 2,9 35,18 2 5, cluster 1 - surveillance zone (including vaccination zone) 433 2,31 37,23 2 6,9 3 cluster 2 - vaccination zone 32 3,28 48,6 1, cluster 2 - surveillance zone (including vaccination zone) 38 3,15 4,18 4, cluster 3 - vaccination zone cluster 3 - surveillance zone high density medium density low density Composition of Cluster 1: zone number of cattle holdings total number of cattle number of sheep/goat holdings total number of sheep/goat number of pig holdings total number of pigs vaccination zone surveillance zone Composition of Cluster 2: zone number of cattle holdings total number of cattle number of sheep/goat holdings total number of sheep/goat number of pig holdings total number of pigs vaccination zone surveillance zone EU FMD-NSP workshop 66

67 Composition of Cluster 3: zone number of cattle holdings total number of cattle number of sheep/goat holdings total number of sheep/goat number of pig holdings total number of pigs vaccination zone surveillance zone Number of mixed farms: cluster mixed cattle & pigs cattle & sheep sheep & pigs cluster 1 - vaccination zone cluster 1 - surveillance zone cluster 2 - vaccination zone cluster 2 - surveillance zone cluster 3 - vaccination zone cluster 3 - surveillance zone Cluster 1 to 3m = vaccination zone size of holdings: size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to EU FMD-NSP workshop 67

68 Cluster 1 3 to 1m = surveillance zone = non-vaccinated zone size of holdings size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to Cluster 2 to 3m = vaccination zone size of holdings size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to EU FMD-NSP workshop 68

69 Cluster 2 3 to 1m = surveillance zone = non-vaccinated zone size of holdings size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to Cluster 3 vaccination zone = inner fused 5 km of surveillance zone size of holdings size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to EU FMD-NSP workshop 69

70 Cluster 3 non-vaccinated zone = outer fused 5 km surveillance zone size of holdings size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to EU FMD-NSP workshop 7

71 Session IV and V: Results from working groups Scenario 1: 4 outbreaks 1 cluster Vaccination of bovine and pigs in 3 km zone around index case All bovine/pigs/small ruminants culled in the 4 outbreak farms Group 1 = Group 2 = Moldova + Ukraine + Per Have + David Paton (annex B) Norway + Latvia + Tom Murray (annex C) Vaccination/protection zone (VZ/PZ) Vaccination of bovine/pigs: bovine: 119 herds 1,989 animals Pigs: 38 herds 81,111 animals No vaccination of small ruminants: 1 herds 88 animals Vaccination zone = protection zone Design: Bovine Small ruminants Pigs (1) CS: clinical surveillance G1 CS (1) all herds Sample all herds / all animals All animals marked for follow-up sampling CS (1) all herds Sample all herds / all animals All animals marked for follow-up sampling CS all herds and Sample all herds (stratified into 3 herd-size groups: small, medium, large)/ (95:5) animals All animals marked for follow-up sampling G2 Sample all herds / all animals Sample all herds / all animals Sample all herds 1/ all animals Sample all herds >1/(95:1) animals: select all animals showing clinical signs, sample a fixed proportion of the animals and ensure that all sections are represented in the sample. Figures: G1 G2 Bovine 119 herds / 1,989 samples 119 herds/1,989 samples Small 1 herds / 88 animals 1 herds / 88 animals ruminants Pigs 38 herds / 8,99 samples 38 herds/12,377 samples EU FMD-NSP workshop 71

72 Resources: G1 G2 Vet teams / VZ/PZ: 8 teams (16 persons) doing 2 visits per day during 3.8 weeks (331 visits taking 14,454 samples) 8 persons supporting sampling Prioritization of sampling in large herds, larger sheep flocks and if closer to positive cases Lab Bovine: Cedi NSP, retest all positives with a second Cedi-NS test Pigs: Cedi NSP (Se.7 / Sp.99), confirmation with UBI test Small ruminants: Cedi NSP, retest all positives with a second Cedi-NS test 12 persons doing lab testing, for a total number of samples of 14, ,185. Surveillance zone (SZ) No vaccination of animals in 3 km 1 km zone: Bovine: 883 herds 13,99 animals Small ruminants: 88 herds 2,328 animals Pigs: 2,328 herds 48,92 animals Design: G1 G2 Bovine CS all herds/all animals CS all herds / all animals Serology/virology of all animals showing clinical signs Small ruminants Sample all herds / all animals CS all herds/all animals Sample all herds/all animals All animals marked for follow-up sampling Pigs CS all herds/all animals CS all herds / all animals Serology/virology of all animals showing clinical signs Figures: G1 G2 Bovine CS 883 herds/13,99 animals CS 883 herds/13,99 animals + ~ 28 samples Small ruminants 88 herds/925 samples 88 herds/925 samples Pigs CS 2,328 herds/48,92 animals CS 2,328 herds/48,92 animals + ~ 98 samples Resources: Vet teams Lab G1 Cedi-NS + confirmatory VNT G2 SZ: 22 teams (22 persons) doing 5 visits per day during 3.5 weeks (2,477 visits taking ~ 1185 samples + CS Prioritization of sampling in large herds, in sheep flocks and take wind direction and other possible routes for spread into consideration EU FMD-NSP workshop 72

73 Initial laboratory results (given by the supervisory team, based on the design made by the group) Cluster 1: G1 VZ/PZ: 2 big pig herds (ID 327, 325) have 31 (25) and 2 (17) positive results VZ/PZ: 5 medium pig herds (ID 171, 263, 33, 178, 257) have 16 (1) positive results VZ/PZ: 2 small pig herds (ID 312, 295) have 5 (1) positive results VZ/PZ: 1 cattle herd (ID 13) has 1 positive result VZ/PZ: no reactors in small ruminants SZ: 9 reactors in small ruminants (ID 2364, 379, 117), distributed over 3 farms G2 VZ/PZ: 7 pig reactors in herd 48 VZ/PZ: 9 pig reactors in herd 325 VZ/PZ: 7 pig reactors in herds 33, 171, 178, 296, 327 VZ/PZ: 8 bovine reactors in herd 13 VZ/PZ: 1 bovine reactor in herds 99, 27 VZ/PZ: no reactors in small ruminants SZ: 3 sheep reactors in herd 1317 Interpretation and follow-up G1 Analysis of test results: o No. of reactors higher than expected from design? Yes in farm 327, 325, 312, 263, 2364, 379, 1317, 13. o Reactors: most pig herds have low level reactors; one pig herd with strong suspicion of infection; one cattle reactor; one sheep reactor Resample and retest groups of animals with reactors to look if positives remain positive and some negatives became positive Cull single cattle and sheep reactors (since no probing samplers or virological confirmation tests available) G2 Analysis of test results: o VZ/PZ bovine: sample all animals for serology and virology, cull cattle herd with 8 positive reactors o VZ/PZ pigs: collect epidemiological information from the herds, sample animals showing clinical signs for virology, if positive PCR: stamping out of the herd o VZ/PZ: special case herd no. 48 (6, pigs) 2 strong positive results (all sows and in one section of 6 sows) + 5 weak positives distributed all over o SZ: positive sheep herd: collect epidemiological information from the herd, sample all animals for virology, cull the flock EU FMD-NSP workshop 73

74 Field follow-up G1 Closer follow-up of pig herd no. 325 (only pigs, strong suspicion): o Pigs not carriers, so if re-sampling and testing shows no circulation, the herd provides low risk o Herd will need to be killed and farm disinfected can be slaughtered for human consumption if not seropositive o Consider this an outbreak, but occurred in February, so does not invalidate freedom claim G2 VZ/PZ pigs: if negative PCR and if no epidemiological links and if no clinical signs in the herds: apply for FMD freedom VZ/PZ special case herd 48: o In suspicious section: clinical examination of all sows, sample animals showing clinical signs for virology and SP-testing, cull all sows o In other units: collect epidemiological information, clinical examination of all pigs, sample animals showing clinical signs for virology o Keep herd under restrictions with additional bio-security measures EU FMD-NSP workshop 74

75 Scenario 2: 2 outbreaks 1 cluster All bovine/small ruminants culled in outbreak herds Only culling of pigs kept in affected stable Vaccination of bovine and pigs Group 3 = Estonia + Slovakia + Åse Uttenthal + Philippe Vanier (Annex D) Group 4 = Poland + Finland + Aldo Dekker (Annex E) Vaccination zone/protection zone (VZ/PZ) Vaccination of all bovine/pigs: Bovine: 15 herds 1,549 animals Pigs: 5 herds 46,98 animals No vaccination of small ruminants : 2 herds 19 animals Vaccination zone = protection zone Design: G3 Bovine Clinical examination; at the herd level in 1 km area around the outbreak, collect all animals in all holdings; outside the 1 km zone, test 1% within herd prevalence, dependant on the size of the herds. If herds <26, clinical examination only. Small ruminants Cull all animals or individual sampling + clinical examination Pigs Clinical examination of small holdings, For big pig farm: Sample sows/ 2% within herd prevalence Sample fattening pigs/5% within herd prevalence Piglets/5% within herd prevalence G4 Sample all herds/all animals Sample all herds/all animals Sample all herds/all animals, but for big pig farm, divide in: 9 separate stables of sows, boars and piglets - sample all units/ (95:5) animals 36 separate stables of fattening pigs: Clinical examination (once per day by farmer, once per week by official vet). Justification: (1) closed holding, (2) FMD is clinical disease and even if not clear signs in vaccinated population, transmission of virus effectively prevented, so no carrier status documented in pigs, (3) fattening pigs going to be slaughtered, so possible to check/sample at slaughter, (4) required a lot of resources Figures: G3 G4 Bovine samples 15 herds/1,549 samples Small 2 herds/19 samples 2 herds/19 samples ruminants Pigs 465 sows + 13 fattening pigs + 13 piglets 4 herds/7 samples 1 herd/2,7 samples CS 36 stables/ 427 animals, check 427 samples at slaughter EU FMD-NSP workshop 75

76 Resources: G3 G4 Vet teams / Examine farms were sampling would be carried out first 2 vet. Days for sampling cattle herds + 13 vet. Days for sampling sows (27 in 9 stables) in large pig farm + 18 vet. Days for CS of 36 stables with 19, fattening pigs 1 veterinary officer for coordination and compilation of field data 1 person from central vet. administration for planning etc. Lab Cedi NSP, 86% Se, 99.5% Sp / Surveillance zone (SZ) No vaccination of animals in 3 km 1 km zone: Bovine: 1,92 herds 13,697 animals Small ruminants: 66 herds 1,367 animals Pigs: 63 herds 894 animals Design: Bovine Small ruminants Pigs Figures: Bovine Small ruminants Pigs G3 Clin. surv. all herds/all animals Herds >26: 1% within herd prevalence Herd <35: sample all animals Herd>35: 5% within herd prevalence Clin. surv. small herds/all animals 4 big holdings: 5% within herd prevalence G3 CS 1,92 herds/13,697 animals Sample 122 herds/4237 samples Herd <35: 6 herds/716 samples Herd >35: 6 herds/124 samples CS 59 herds/894 animals Sample 4 herds of ±159 pigs: 7 pigs per herd. G4 Clin. surv. all herds/all animals Clin. surv. all herds/all animals + Sample all herds/ all animals in sheep farms without other ruminants Clin. surv. all herds/all animals G4 CS 1,92 herds/13,697 animals CS + sample 22 herds/951 animals CS 63 herds/894 animals Resources: G3 G4 Vet teams / Examine farms were sampling would be carried out first 1 vet. Days for CS of cattle and pig farms + 11 vet. Days for sampling sheep farms 1 veterinary officer for coordination and compilation of field data 1 person from central vet. administration for planning etc. Lab test, 98% Se, 99.5% Sp / EU FMD-NSP workshop 76

77 Initial laboratory results (given by the supervisory team, based on the design made by the group) G3 VZ: 4 NSP+ sows, 1 NSP+ pig, 1 NSP+ piglet SZ: 4 positive cattle, 2 reactors in 2 large pig herds G4 VZ: backyard pig farm with 2 reactors VZ: 3 mixed farms with one positive pig each VZ: 5 small bovine farms have 1-3 reactors VZ: 27 reactors in 5 different stables in large pig farm SZ: 1 sheep farm with 3 reactors Interpretation and follow-up G3 VZ: Positive cattle samples: If positive result, retest the same sample, if positive again, collect a sample from the same animal, if positive cull the animal and retest the whole herd + clinical inspection of all animals; if negative results, previous results are considered as false positive, else, cull the herd. VZ: Positive sow samples: (Lower than maximum number of reactors allowed) Retest sample, if positive, cull the 4 sows. Clinical inspect and test the sows around these 4 ones depending on the epidemiological picture in the herd. VZ: Positive fattening pigs: (Lower than maximum number of reactors allowed) Retest sample, if positive, re-sample from the same pig. If positive, sample 13 pigs again. VZ: Positive piglets: (Lower than maximum number of reactors allowed) Retest sample, if positive, re-sample from the same pig. If positive, sample 13 pigs again. SZ: positive cattle: Retest the samples with another test system (f.e. VNT); if possible re-sample the same animals+ the ones surrounding the positive ones (epid. investigation) and collect blood from 18 animals (5% prevalence) + clinical examination of the whole herd SZ: positive pigs: Retest the samples with another test system (f.e. VNT); if positive, clinical examination of the whole herd, including cattle in the mixed herd G4 VZ: backyard pig farm has 2 pigs which are both positive cull (located in same municipality as outbreak no. 4) VZ: one mixed farm with 37 negative cattle and 1 positive pig out of 2 false positive pig VZ: one mixed farm with 5 negative cattle and 1 seropositive pig out of 2 false positive pig VZ: one mixed farm with 1 seropositive pig and 16 negative sheep false positive pig (high specificity of the test in non-vacc. sheep). VZ: large pig farm: retesting of positive stables = (1) positive animals + 5% of penmates and pigs in adjacent pens VZ: small bovine farms (16-14 bovine): probing sampling and PCR-testing to rule out carrier status SZ: 1 sheep farm with 3 reactors all reactors tested by VNT 1 positive in VNT (1:45) = singleton reactor EU FMD-NSP workshop 77

78 Field follow-up G3 G4 / For big pig farm: 23 reactors in the retesting of the positive sows + 12 reactors out of 217 samples in the group of penmates and adjacent pens option for decision-making: o Culling and disinfection of infected premises (46, pigs) + repopulation with vaccinated animals = unrealistic o Culling of 5 positive stables (3988 sows+piglets) and disinfection + repopulation with vaccinated animals usefulness? o Vaccination of piglets from positive stables before transport to empty stables for fattening o Revaccination of all sows (+ some fattening pigs youngest stables) o Do nothing EU FMD-NSP workshop 78

79 Scenario 3: 25 outbreaks 3 clusters Only bovine vaccinated All bovine/sheep/goats/pigs culled - 5 m Vaccination zone (VZ) Group 5 = Czech Republic + Sweden + Dònal Sammin (see annex F) Group 6 = Israel + Switzerland + Lea Knopf (see annex G) Vaccination of all bovine in 5m 5 km: Bovine: 2,188 herds 144,872 animals No vaccination of pigs/small ruminants: Small ruminants: 1,365 herds 11,78 animals Pigs: 1,75 herds 914,155 animals Design: Bovine Large survey: sample all herds/all animals Small ruminants Pigs G5 G6-EU approach G6-Israeli-Swiss approach CS all herds /all animals Sample all herds /all animals Preliminary survey: Sample (95:5) herds/(95:2) animals Large survey: sample (95:2) herds/(95:5) animals, but all animals in flocks <25 animals and 1 large farm (1,2 animals) Preliminary survey: Sample (95:5) herds/(95:2) animals Large survey: CS CS all herds /all animals Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 3 animals / all animals, if herd >3 animals/3 animals per herd. (within herd prevalence of 1%) CS all herds /all animals Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 2 animals / all animals, if herd >2 animals/3 animals per herd. (within herd prevalence of 2%) CS (95:5) herds and sample 1% of inspected holdings + Two-stage sampling Hse 8%, Hsp 99.5%, (95:5) herds, if herd >=6 and if herd 75 animals / all animals, if herd >75 animals/75 animals per herd. (within herd prevalence of 5%) No clinical inspection since no clinical signs Two-stage sampling Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 3 animals / all animals, if herd >3 animals/3 animals per herd. (within herd prevalence of 1%) CS all herds /all animals and sample 5% of inspected holdings + Two-stage sampling Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 2 animals / all animals, if herd >2 animals/2 animals per herd. (within herd prevalence of 2%) EU FMD-NSP workshop 79

80 Figures: G5 G6-EU approach G6-Israeli-Swiss approach Bovine Large survey: 2,188 2,188 herds/144,872 samples CS 523 herds and sample 5 herds/144,872 samples herds/ 25 animals herds/13,575 animals Small ruminants Preliminary survey: 67 herds / 536 samples Large survey: 391 herds/2,92 animals Pigs Preliminary survey: 67 herds / 1,474 samples CS 1,365 herds 11,78 animals + CS 1,75 herds 914,155 animals 164 herds/4,1 animals 2,315 samples from clinical investigations (463 holdings with 5 samples each) Sample 164 herds/4,1 animals + Pigs : CS 175 herds/914,155 animals and sample 55 herds/275 animals Resources: G5 G6-EU approach G6-Israeli-Swiss approach Vet teams 2 vets for CS of pig holdings Lab Preliminary survey: SP Bovine: Cedi-test with retesting (Se Cedi test with retesting (Se 98%, ELISA (Se.99, Sp.95) Analysis of 144, NSP tests 98%, Sp 99%) and virus detection (samples from clinical survey) Sp 99%) and virus detection (samples from clinical survey) Surveillance zone (SZ) No vaccination in 5 km 1 km zone: Bovine: 1,372 herds 85,7 animals Small ruminants: 885 herds 6,771 animals Pigs: 677 herds 54,289 animals EU FMD-NSP workshop 8

81 Design: Bovine Small ruminants Pigs G5 G6-EU approach G6-Israeli-Swiss approach CS all herds /all animals Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 3 animals / all animals, if herd >3 animals/3 animals per herd. (within herd prevalence of 1%) Large survey: CS all herds/all animals Preliminary survey: Sample (95:5) herds/(95:2) animals Large survey: sample (95:2) herds/(95:5) animals, but all animals in flocks <25 animals Preliminary survey: Sample (95:5) herds/(95:2) animals Large survey: CS CS all herds /all animals Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 3 animals / all animals, if herd >3 animals/3 animals per herd. (within herd prevalence of 1%) CS all herds /all animals Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 2 animals / all animals, if herd >2 animals/2 animals per herd. (within herd prevalence of 2%) CS (95:5) herds with Se 95% and Sp 99%/ Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 2 animals / all animals, if herd >2 animals/2 animals per herd. (within herd prevalence of 2%) CS (95:5) herds with Se 95% and Sp 99%/ Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 3 animals / all animals, if herd >3 animals/3 animals per herd. (within herd prevalence of 1%) CS (95:5) herds with Se 95% and Sp 99%/ Sample in two-stage approach: Hse 9%, Hsp 99%, (95:5) herds, if herd >=1 and if herd 2 animals / all animals, if herd >2 animals/2 animals per herd. (within herd prevalence of 2%) Figures: G5 G6-EU approach G6-Israeli-Swiss approach Bovine Large survey: CS 1,372 CS 132 herds + sample 31 herds/85,7 animals herds/65 animals Small Sample 164 herds/4,1 ruminants animals Preliminary survey: 43 herds / 344 samples Large survey: 328 herds/2,59 animals Pigs Preliminary survey: 43 herds / 946 samples Large survey: CS 677 herds/54,289 animals CS 884 herds/48,455 animals + CS 885 herds/6,771 animals + CS 137 herds/81,448 animals CS samples from 193 herds/1,465 animals Sample 164 herds/41 animals Resources: Vet teams Lab 2 vets for CS G5 G6-EU approach G6-Israeli-Swiss approach Preliminary survey: SP ELISA (Se.99, Sp.95) Analysis of 8,59 SP tests Bovine: Cedi-test with retesting (Se 98%, Sp 99%) and virus detection (samples from clinical investigations) Serology Cedi-test with retesting Se 98%, Sp 99% and virus detection (samples from clinical investigations) EU FMD-NSP workshop 81

82 Initial laboratory results (given by the supervisory team, based on the design made by the group) G5 G6-EU approach G6-Israeli-Swiss approach After preliminary survey: VZ: 1,159 positive cattle VZ: 111 positive cattle o 17 pig farms with VZ: 44 positive pigs VZ: 44 positive pigs reactors VZ: 9 positive sheep VZ: 9 positive sheep o 6 sheep farms with SZ: 14 positive cattle SZ: 14 positive cattle reactors SZ: 44 positive pigs SZ: 44 positive pigs o Geographical cluster in SZ: 21 positive small SZ: 21 positive small Waarschoot ruminants ruminants After large survey: o VZ: 49 sheep herds with 1 or 2 reactors o VZ: 259 cattle herds with 1 to 15 reactors and herds with >3 positives in 3 municipalities (Sleidinge, Lovendegem and Waarschoot) o SZ: 23 sheep herds with 1 to 3 reactors Interpretation and follow-up G5 G6-EU approach G6-Israeli-Swiss approach Preliminary survey - Analysis of test results: Analysis of test results: Analysis of test results: o VZ: o VZ: o 17 pig farms with reactors (2 expected) - cattle: 1,159 reactors 1,159 allowable - cattle: 111 reactors 111 allowable o 6 sheep farms with reactors ( expected) - pigs: 44 reactors 44 allowable - pigs: 44 reactors 44 allowable o Some farms with - small ruminants 9 reactors - small ruminants 9 reactors unexpectedly high 9 allowable 9 allowable numbers of reactors o SZ: o SZ: Actions: o Intensified CS in pigs in - cattle: 14 reactors 14 allowable - cattle: 14 reactors 14 allowable entire VZ, but focus on - pigs: 275 reactors 44 - pigs: 275 reactors 44 Waarschoot and allowable allowable neighboring municipalities, - small ruminants 21 - small ruminants 21 prioritize large farms reactors 21 allowable reactors 21 allowable (>1,) o CS of cattle in buffer zone o Actions on all infected (confirmed) farms (culling, tracing,..) o Perform large survey EU FMD-NSP workshop 82

83 Field follow-up G5 Large survey analysis of test results: Sheep: retest positive samples, send vets to flocks where still positives, sample all animals and prioritize SZ. Cattle: cull all NSP+ cattle, slaughter rest of herds in 3 municipalities, but only cull NSP+ animals Sample all sheep herds in 3 municipalities G6 Vaccinated animals (cattle): 1. resample all positive animals 2. epidemiological investigation of herds with positive results 3. testing the re-samples with (a) Cedi-test (if test negative = negative) and (b) if Cedi-test positive retesting with Svanova-test 4. If Svanova positive: elimination of positive animals and testing of whole herd Non-vaccinated animals: 1. Resample all positive animals 2. Epidemiological investigation of herds with positive results 3. Testing the resamples: cattle with Svanovatest and pigs, small ruminants with Cedi-test 4. If animals still positive, retest with SPCE 5. If SPCE positive: elimination of positive animals and testing of whole herd Group conclusions (Remarks) G5 Vaccination: o The group proposes not to vaccinate in herds <5 animals o The group proposes to vaccinate pigs on vaccinated cattle holdings Testing: o CS only in non-vaccinated cattle herds o Random sampling of vaccinated cattle herds (126 herds), test all animals in each herd o Non-vaccinated pigs and sheep should be treated as was proposed in the exercise Freedom cannot be substantiated yet G6 First approach (following EU guidelines) proposes 156,98 samples for NSP testing and 3,78 samples for virus detection Second approach (IL-CH suggestion) proposes 22,365 samples for NSP testing and 59 samples for virus detection The confidence of the combined surveys (clinical + serological) needs to be calculated Would the second approach with considerably less testing be accepted by neighboring countries? After follow-up of positive animals (epidemiological investigation + serological survey): (i) all animals could be described as false positive = freedom of disease demonstrated, (ii) some animal in the followup were found positive = freedom of disease is not demonstrated EU FMD-NSP workshop 83

84 Session Vi: Discussion of results and models for serosurveillance: Summary, Conclusions, Recommendations and Observations Summary Vaccination zone: Total no. of samples/herds: Bovine (vacc.) Small ruminants (non-vacc.) Pigs (vacc.) Bovine (vacc.) Small ruminants (non-vacc.) Pigs (vacc.) Bovine (vacc.) Small ruminants (non-vacc.) Pigs (non-vacc.) G1 119/1,989 1/88 38/8,99 G3 1,44 2/ G5 2,188/144, /2,92 CS G2 119/1,989 1/88 38/12,377 G4 15/1,549 2/19 5/2,77 + check 4,27 animals at slaughter G6 (IL-CH) 5+181/25+13, /275+4,1 Vaccinated bovine: o Groups 1, 2, 4 and 5 propose testing all vaccinated bovine (based on the risk of becoming a carrier), although for group 5 this was only proposed for in the second (large) survey. o Groups 3 and 6 propose to do a survey and test part of the population (cf. according to EU Directive 23/85/EC, all herds/all animals have to be sampled). Group 6 makes a distinction between the serological and the clinical survey. Samples taken from the clinical survey will be investigated in serology and virus detection. Non-vaccinated pigs: o Only clinical surveillance (CS) of non-vaccinated pigs proposed by group 5. o Group 6 proposes sampling and CS. They make a distinction between the serological and the clinical survey. Samples taken from the clinical survey will be investigated in serology and virus detection. Vaccinated pigs: o Smaller sample size recommended instead of sampling all animals (as according to EU directive 23/85/EC) proposed by groups 1 and 2, since it is nearly impossible to sample all animals in big pig holdings. o Group 4 proposes to sample all small holdings and sample part of the population in the big pig holding and to check 4,27 fattening pigs at slaughter Non-vaccinated sheep and goats: o Groups 1, 2, 3 and 4 agree on sampling following EU Directive 23/85/EC. o Groups 5 and 6 propose sampling part of the population Most groups calculated the Herd Cut Point to see if SP positive results should be considered as significant. EU FMD-NSP workshop 84

85 G2 and G4 calculated the number of vet teams necessary to perform the surveys. Surveillance zone: Total no. of samples/herds: Bovine (non-vacc.) Sheep/goats (non-vacc.) Pigs (non-vacc.) Bovine (non-vacc.) Sheep/goats (non-vacc.) Pigs (non-vacc.) Bovine (non-vacc.) Sheep/goats (non-vacc.) Pigs (non-vacc.) G1 CS 88/925 CS G3 122/4,237 66/84 4/28 G5 CS 328/2,59 CS G2 CS + sampling of animals showing clinical signs 2,534/299 CS + sampling of animals showing clinical signs G4 CS 22/951 CS G6 CS + (31+164)/(65+4,1) CS CS Non-vaccinated bovine and pigs: o Groups 1, 4, agree on clinical surveillance only, G2 proposes to take samples from animals showing clinical signs as well, G3 proposes to take samples Non-vaccinated sheep and goats: o Group 6 proposes clinical surveillance only o Groups 1, 2 and 3 follow EU Directive 23/85/EC o Group 4 proposes to sample all animals in sheep holdings without other small ruminants o Group 5 proposes to sample part of the population The in advance calculation of necessary lab resources and vet teams to execute the survey was very useful for some groups. Calculation of expected positive herds and expected positives per herd was done by most groups EU FMD-NSP workshop 85

86 Conclusions The approaches taken by the different working groups/countries showed a clear degree of similarity. The results were extensively discussed and the conclusions taken all three workshops were accepted: 1. The vaccination-to-live policy with subsequently substantiating freedom from infection by a survey system including NSP testing is a realistic and achievable option in FMD control. However, it must be clear that in order to regain a free status without vaccination within a reasonable time, i.e. in accordance with Article paragraph (1)(c) of the Terrestrial Animal Health Code, a stamping out policy, as defined by OIE, remains part of the control policy such as in the beginning of an epidemic. 2. NSP assays are not sensitive enough to detect all infected animals (especially carriers) in a vaccinated population and therefore conclusions on the status of the herds can only be on a herd basis and in combination of results from clinical and serological surveys and epidemiological investigations such as cluster analysis. 3. Demonstrating absence of infection, in particular in a vaccinated population, is impossible and therefore the term demonstrate absence should be replaced by substantiate absence. 4. The current EU Directive (23/85/EC) for the control of FMD mentions two surveys: (a) A first survey, for detecting the presence of FMD virus in the vaccination zone (Article 56), should be a combination of clinical, epidemiological and serological investigations with high overall system sensitivity (Martin et al., 27 1 ). Article 56 takes into account that the vaccination status within a vaccination zone may not be homogeneous, i.e. that within the declared vaccination zone there might be vaccinated and non-vaccinated animals. Consequently this survey includes: a survey of non-vaccinated animals for detecting FMD virus infection in the same manner as in a protection zone without vaccination; a serosurveillance of all herds with vaccinated animals for detecting FMD virus infection by use of NSP tests. Within the herds all vaccinated ruminants and their non-vaccinated offspring must be sampled and other species kept in large numbers making individual testing impractical shall be sampled based on a 5% prevalence with 95% confidence. (b) A second survey, to regain the freedom from infection status after emergency vaccination (Article 61), must have a high specificity. This survey might include a second serosurveillance. However, the serosurvey described in Article 56 could serve the purpose of the serosurvey required in Article 61 of the Directive, referring to surveillance guidelines to be laid down and in fact being the OIE Guidelines in Appendix of the Terrestrial Animal Health Code The follow-up of herds with seroreactors by serological investigation has to be based on a combination of NSP assays (Paton et al., 26) with well-defined performance characteristics and 1 Martin PA, Cameron AR, Greiner M. Demonstrating freedom from disease using multiple complex data sources 1: a new methodology based on scenario trees. PrevVet Med 27 May 16;79(2-4): Epub 27 Jan 16. Review. 2 In 23 there were no Guidelines of OIE and it was discussed at OIE not at all to lay down Guidelines. A decision is in preparation to lay down that the OIE Guidelines should be the Guidelines mentioned in Article 61. The current OIE Guidelines also do not specify the period that must elapse between the last vaccination and the beginning of the survey. EU FMD-NSP workshop 86

87 those assays should preferably be conditional independent. The final conclusion must take into account the specificity of the overall test system. 6. If the specificity of the serological test system used were known, then one approach would be to anticipate the rate of false positive test reactions and only consider seroreactor rates above this expected number or proportion as significant (Herd Cut Point - Paton et al., 26 3 ). However, this is not considered as compatible with the OIE Code and the EU Directive 23/85/EC, which require all herds with seroreactors to be followed up and classified as either containing or free from infection. Therefore re-sampling and testing herds with seroreactors should be applied. The latter approach would enable active virus circulation to be confirmed or ruled out by use of paired serology to look for changes in antibody status or titer rises and/or by evaluating results using Likelihood Ratios (Dekker et al., submitted 4 ). Contingency plans should include a clear flow chart for the follow-up of seropositive herds. 7. A clinical surveillance combined with paired serology can detect holdings where virus circulation is ongoing. However, there is no possibility of detecting each and every carrier within sub-clinically infected herds if, as seems likely, they are present in few herds and at a low level. Therefore all ruminants should be tested. Evidence of virus circulation would lead to the declaration of an outbreak and the stamping out of the herd, but evidence of carriers (in particular if at a low number within a herd) should, different to the current requirements, lead to slaughter of these reactor animals only, but not of the whole herd. The advantages of this approach would be: (a) The test specificity can be lowered since the consequence of false positive results is now individual animal rather than whole herd slaughter. (b) This in turn leads to an increased test system sensitivity (c) The small herd problem is decreased (cf. Annex H). 8. Testing all animals in the vaccinated population as prescribed in the EU Directive 23/85/EC, is certainly the way forward in case of vaccinated bovine, it is however considered as not achievable in areas with a dense pig population or within big pig herds, if such pigs have been vaccinated. In addition is the risk of developing a carrier state in pigs considered to be minimal, if not negligible. It is proposed to replace Article 56(3) (b) of Directive 23/85/EC by the following: testing for antibodies against NSP of the FMD virus shall be carried out on samples taken from all vaccinated large ruminants and their non-vaccinated offspring and from all vaccinated pig herds with a within herd sampling based on a 5% prevalence with 95% confidence. 3 DJ Paton et al., Application of non-structural protein antibody tests in substantiating freedom from foot-and-mouth disease virus infection after emergency vaccination of cattle, Vaccine. 26 Oct 3;24(42-43): Epub 26 Jul 5. Review. 4 Dekker A et al., Comparison of ELISAs for antibodies against foot-and-mouth disease virus non-structural proteins in cattle sera based on the continuous results, submitted, see Annex 9 of FMD-NSP program. EU FMD-NSP workshop 87

88 9. Vaccination of small herds remains a controversial item. Two possible options were discussed: (a) A non-vaccination policy for small herds and integration in the survey system as sentinels. This option is based on the fact that the 95% confidence cannot be achieved in small herds (considering a realistic within herd prevalence) and that small herds slow down the emergency vaccination campaign. Spatial cluster analysis should be applied to evaluate the serological survey results of the sentinels. (b) Vaccinate small herds and test all animals in all of these small herds. This option is considered because vaccination of small herds contributes to an increase in vaccination coverage and thus to achieving the necessary level of protection for the population, considering the fact that vaccinating all animals with a homologous vaccine will only protect about 9% of the animals. It might also be politically difficult to deny vaccination to owners of small herds and thus exposing the animals to infection and possible stamping out. 1. This kind of workshop should also be done for other veterinary diseases such as Classical Swine Fever or Avian Influenza (for all EU members, EUFMD countries and EU neighbours). EU FMD-NSP workshop 88

89 Recommendations 1. Conclusions on the infection status of the herds after FMD outbreaks in a vaccinated population should only be based on a survey system including at least clinical, serological and epidemiological investigations; 2. The performance characteristics of the survey system should be determined. When the desired confidence level cannot be reached, spatial clusters analysis should be considered; 3. The term demonstrate absence of infection should be replaced by substantiate absence of infection ; 4. Contingency plans should include a clear flow chart for the follow-up of seropositive herds, which must take into account at least the requirements of Appendix of the Terrestrial Animal Health Code. Serological investigation should be based on a combination of NSP assays with well-defined performance characteristics; 5. All large ruminants should be tested to substantiate freedom from infection in a vaccinated population after FMD outbreaks. While evidence of virus circulation must lead to the declaration of an outbreak, consensus should be sought on the slaughter of reactor animals only, in case there should be evidence that these animals are carriers, instead of the whole herd removal as currently required in Article 57(3). Consideration should be given to finding out if there would be a consensus on the latter; 6. A change in the definition of an outbreak in OIE Guidelines and EU Directives is needed where carriers are concerned; 7. The relative confidence attainable with herd-based and individual certification needs to be explored for different herd sizes and prevalence; 8. Consideration should be given to an amendment of the Directive in order to allow a within-herd sampling scheme based on a 5% prevalence and a 95% confidence for vaccinated pigs; 9. The vaccination of small herds should be further discussed; 1. To refine the application of NSP tests, more work could be done in predicting the expected prevalence of infection within and amongst vaccinated herds; 11. Functional FMD expert groups should be created in every country (cf. article 78 of Council Directive 23/85/EC for the EU Member States 5 ), including in non-eu countries. 5 In article 78 it is stated that the group shall compose of epidemiologists, veterinary scientists and virologists in a balanced way EU FMD-NSP workshop 89

90 Observations of WS2 and WS 3: During the discussions, following points were highlighted: 1. Recommendation 2 specifies that the performance characteristics of the survey system should be determined, but this should take into account the different species analysed in the survey system As the OIE code has changed in May 27, making containment of an infection region possible, the status of the country remains unaffected. 3. Some countries need training for the set-up of an information system for field data. Also, countries with a large population of backyard animals face a lot of problems in the collection, the control and the management of disease information. 4. The scenarios assume a perfect movement control, but it has to be kept in mind that this is not a real life situation. 5. Probang testing will not prove the absence of virus circulation. Therefore it was stated that probang testing brings more problems than solutions to substantiate freedom from infection. 6. It was concluded that the introduction of negative animals as sentinels in a vaccinated herd is of limited value due to the low transmission rate in these herds, as well for cattle as for pigs. 7. Several countries indicated that vaccination will not be their main option, especially if there are a lot of small herds in the country. 8. The organisation of a workshop on vaccination: how and when, after or in face of an outbreak should be considered. It was considered important to have a good vaccination coverage. Also the purpose of vaccination was questioned: is it a disease control measure in a rising epidemic curve in combination with the DIVA principle to try to stop disease spread and to reduce the total outbreak number? But the expected residual infection remains to be detected. The vaccination of herds < 5 animals was questioned as well: the detection of 5% prevalence is easier if these herds are not vaccinated, but vaccination coverage is reduced (coverage of herds is probably more important than coverage of animals). 9. SP test can be used for the survey of non-vaccinated animals. SP-tests can also be used to check the efficiency of and coverage obtained through vaccination programmes (Conclusion 2). 1. Always try to slaughter for consumption, if slaughter should be necessary (Recommendation 5). 11. Sub-clinical transmission in cattle and pigs is unlikely. Virus circulation in pigs can be determined by paired serology, similar to what is done in South America for cattle. Clinical signs in sheep are not absent but very difficult to discern from other diseases and easily missed. 12. It will be easier to prove absence of infection in the surveillance zone and it could be of help to substantiate absence of infection. 13. The WS shows that countries should be prepared for the possible outcomes of the surveillance. Also false positive results can be clustered, so it is essential to have a decision-scheme on the follow-up before the start of the outbreak. 14. If the 95% confidence cannot be achieved for a survey then it is still worth doing it but the confidence level should be determined and specified. The actual confidence level obtained should be taken into account for the final conclusion on the disease status. 15. It should be noted that during the 3 WSs different figures for diagnostic Se and Sp were used as well as different design prevalence although similar situations. 6 Martin PA, Cameron AR, Greiner M. Demonstrating freedom from disease using multiple complex data sources 1: a new methodology based on scenario trees. PrevVet Med 27 May 16;79(2-4): Epub 27 Jan 16. Review. EU FMD-NSP workshop 9

91 Acknowledgments DG RESEARCH DG SANCO VAR Belgium Food Agency Belgium EUFMD EU FMD-NSP workshop 91

92 Annex A see FMD-NSP workshop program EU FMD-NSP workshop 92

93 Annex B SCENARIO 1 First outbreak at end of February due to serotype A The initial clinical signs are ignored by the owner, resulting in a rather late detection of the disease EU FMD-NSP workshop 93

94 Characteristics of cluster 1 Cluster 1 is comprised of the 1 km surveillance zones surrounding the index case and 3 other outbreaks in the same village: general composition: high density area for pigs, medium density area for cattle, low density area for small ruminants; size: 433 km2; composition: see tables. The index case is not immediately detected; the 3 other outbreaks in the cluster pop up within a week of the detection of the index case. EU FMD-NSP workshop 94

95 EU FMD-NSP workshop 95

96 Exercise The time setting for the exercise is at the end of April, at least 1 month after the last outbreak has occurred (2 months for cluster 1). Since the last outbreak, the surveillance has been based on clinical surveillance. Apart from the holdings that are culled, no serological surveillance has been performed yet. The aim of the exercise is to device and to interpret a post-vaccination FMD surveillance scheme that includes the use of NSP tests. Scenario 1 Scenario 1 focuses on cluster 1 that surrounds the 4 outbreaks around the index case. It takes into account the vaccination of all cattle and pigs in a 3. m radius around the outbreaks (the former protection zone). The size of the vaccination area is 57 km2. EU FMD-NSP workshop 96

97 cluster 1 - to 3. m = vaccination zone PZ Census size number of cat tle hol din gs total nu mb er of cat tle average nu mb er of cat tle number of sh ee p/g oat hol din gs total nu mb er of sh ee p/g oat average nu mb er of sh ee p/g oat number of pig hol din gs total nu mb er of pig s average nu mb er of pig s 1 to to to to to to to to to SZ Census cluster 1-3. to 1. m = surveillance zone = non-vaccinated zone size number of cat tle hol din gs total nu mb er of cat tle average nu mb er of cat tle number of sh ee p/g oat hol din gs total nu mb er of sh ee p/g oat average nu mb er of sh ee p/g oat number of pig hol din gs total nu mb er of pig s average nu mb er of pig s 1 to to to to to to to to to EU FMD-NSP workshop 97

98 PZ Surveillance strategy Vaccinated cattle and pigs, unvaccinated sheep/goats Clinical examination of all species on all holdings Blood sampling of all vaccinated cattle on all holdings as numbers not too great (~2,) Pigs to be sampled/tested so as to detect a 5% within herd prevalence of infection with 95% confidence (stratified into three herds-size groups) Bleed and test all unvaccinated sheep & goats (very small numbers, n=88) Selected Cedi-NS for all screening (retesting all positives with a second Cedi-NS test). For pigs assumed Sp=99%, Se=7% to calculate sample numbers with Freecalc. UBI test used for confirmation in pigs All animals to be marked for follow-up sampling SZ Surveillance strategy No vaccination Clinical examination of all species on all holdings Blood sampling of sheep and goats only since clinical signs less obvious Decided to test all animals as numbers relatively small (n=925) Used Cedi-NS and then asked CRL to carry out confirmatory VNT (no SP test available) All animals to be marked for follow-up sampling EU FMD-NSP workshop 98

99 Sampling & testing numbers Protection zone Number of cattle to be tested (vaccinated) 1989 in 119 holdings Number of sheep and goats (non vaccinated) 88 in 1 holdings Number of pigs (vaccinated) 234 per holding in 3 large holdings 182 per holding in 4 medium holdings 756 pigs in 31 little holdings (all pigs) Surveillance zone Number of sheep and goats (non vaccinated) 925 in 88 holdings (all animals) Results of testing in PZ ID CATTLE PIGS MUNICIPALIT Y Pos_cattl e Number of sam ples Expecte d false positive Pos_pig s Pos_shee p LJUTOMER (25) CVEN (17) CVEN (4) KRAPJE 119 <2 4 (3) IŽAKOVCI (2) CVEN (1) VERŽEJ 6 3 (1) VERŽEJ 25 <1 2 () KRAPJE 13 <2 1 () GRLAVA 1 18 <2 EU FMD-NSP workshop 99

100 Breakdown of Positive results for 2 large pig herds 327 pig herd 9115 pig herd Stable weak pos strong pos Stable weak pos strong pos Results of testing in SZ ID SHEEP/GOA TS MUNICIPALITY Number of sam ples Expected false positive Positive shee p VNT 54 < HOTIZA < GOMILA PRI KOGU 3 65 < SATAHOVCI 2 EU FMD-NSP workshop 1

101 PZ Seropositive findings Pig herds with low level reactors One pig herd with strong suspicion of infection One cattle reactor SZ One sheep reactor Follow-up Resample and retest groups of animals with reactors to look if positives remain positive and if some negatives became positive No probang samplers or virological tests available for testing cattle and sheep, so if single reactors remain they should be culled Problem of pig herd with strong suspicion of infection Pigs not carriers, so if resampling and testing shows no circulation, the herd provides low risk Nevertheless, pigs will need to be killed and farm disinfected can be slaughtered for human consumption if not seropositive Consider this an outbreak, but that this occurred in February and therefore does not invalidate freedom claim EU FMD-NSP workshop 11

102 Annex C Testing in the vaccination zone Clinical examination and serological testing by NSP-CEDI test Cattle All herds; all animals Sheep and goats All herds; all animals Pigs All animals in small herds (< 1) Sample in large herds (327; 9115; 6,142) Within herd design prevalence 1%; N o. tests: 115 (1135), 12 (1172), 12 (1192) Selection of pigs in the large pig herds All animals showing clinical signs of disease A systematic sample taking a fixed proportion of the animals prop = (sample size) / (herd size) Ensure that all sections are represented in the sample EU FMD-NSP workshop 12

103 Testing in the non-vaccinated zone Cattle Clinical examination of all herds Serology/virology of animals showing clinical signs Sheep and goats Serology all herds; all sheep and goats Pigs Clinical examination of all herds Serology/virology of animals showing clinical signs Number of visits and samples Vaccination zone Non-vaccinated zone Visits Samples Visits Exam. Samples Cattle 119 1, ,99 ~28 Pigs 38 12,377 2,328 48,92 ~98 Sheep Total ,454 2,477 63,754 2,185 EU FMD-NSP workshop 13

104 Human resources Visits Teams Visits / Team*day Weeks * Vacc. zone Non-vacc. zone * Assuming 6 days working week Vacc. Zone 22 teams á 1 person 22 Non vacc. Zone 8 teams á 2 persons 16 Supporting sampling 8 Laboratory personell 12 Total 58 Prioritisation of sampling In vaccinated zone Large herds Larger sheep flocks (sheep not vaccinated) Closeness to the positive cases In non-vaccinated zone Large herds Sheep flocks Take wind direction and other possible routes for spread into consideration EU FMD-NSP workshop 14

105 Results No animals No positives ID Cattle Sheep Pigs Cattle Sheep Pigs v v v v v v v v v v nv Follow up of NSP-positive samples In non-vaccinated zone (1 sheep flock with 65 animals) Collect epidemiological information from the herd Sample all animals for virology Cull the flock EU FMD-NSP workshop 15

106 Follow up of NSP-positive samples In vaccinated zone Cattle Sample all animals for serology and virology Cull cattle herd with 8 positive reactors Pigs Collect epidemiological information from the herds Sample animals showing clinical signs for virology If positive PCR FMD detected: stamping out If negative PCR Follow up of herds with NSP positive samples If all samples examined for PCR is negative If no epidemiological links and If no clinical signs in the herds Apply for freedom of FMD EU FMD-NSP workshop 16

107 Follow up of herd with 6, pigs NSP-results 2 strong positives, all sows in one section of 6 sows 5 weak positives distributed all over Action in suspicious section Clinical examination of all sows Sample animals showing clinical signs for virology and SPtesting Cull all sows Action in other units Collect epidemiological information Clinical examination of all pigs Sample animals showing clinical signs for virology Keep herd under restrictions with additional bio security measures EU FMD-NSP workshop 17

108 Annex D Group 3 Cluster 2 Estonia and Slovakia EU FMD-NSP workshop 18

109 cluster 2 - to 3. m = vaccination zone size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to EU FMD-NSP workshop 19

110 cluster 2-3. to 1. m = surveillance zone = non-vaccinated zone size number of cattle holdings total number of cattle average number of cattle number of sheep/goat holdings total number of sheep/goat average number of sheep/goat number of pig holdings total number of pigs average number of pigs 1 to to to to to to to to to Description of outbreak One vaccinated zone Divide into a 1km zone close to outbreak: - 1 km 1-3 km vaccination zone CEDI test sens 86%; spec 99.5 % One non vaccinated zone CEDI test sens 98%; spec 99.5 Other confirmatory tests possible (vnt) EU FMD-NSP workshop 11

111 Within 1 km zone Clinical examination of all herds Sheep (19): the best is to cull them (welfare problems?) or individual sampling plus clinical examination. Cattle (1.549): 2% of design prevalence in total animal population collection of 391 sera (FreeCalc minimum no of sera to be tested) At the herd level, in 1 km area around the outbreak, collect all the animals in all the holdings: 524 tests. Precautions on pos samples cattle For positive results, Retest the same sample, if positive again, collect a second time the blood from the same animal; if positive, cull the animal (to test the animals for virus and abs) and retest the whole herd and inspect clinically all the animals. If negative results, the previous results are considered as false positive. If positive, the herd is culled. EU FMD-NSP workshop 111

112 Vacc zone outside the 1 Km CATTLE: In all the herds, test depending on the size of the herds; the basis is 1% of within herd prevalence: 6 herds with 51 to 1: test 44=(6x44)264 8 herds with 26 to 5: test 37 (8x37)=256 For the rest of herds lower than 26: clinical examination only. Pigs vacc zone - 3 km Clinical examination of the 7 pigs in 4 small holdings Blood testing in the big herd: With sub clusters as the transmission of viruses is not the same in different categories of animals: Sows (3916): 2 % within herd prevalence (5 reactors possible): 465 samples: 4 positive results: retest the sample. If positive: cull the 4 sows (to test virus and abs). Clinical inspect and test the sows around these 4 ones depending on the epidemiological picture in the herd. EU FMD-NSP workshop 112

113 Surveillance zone Cattle: Clinical examination in all herds; herds less than 26 only clinical examination For the rest 1% within herd prevalence With tests of 98% of sensitivity and Spe: 99.5% Herds of: 26 to 5: 33 samples in 85 holdings: to 1: to 15: positive; retest the samples with another test system such as neutralization test; if positive, resample the same animals plus the ones surrounding the positive ones (epidemiological investigations) and collect blood from 18 animals (5 % prevalence) plus clinical examination of the whole herd. Pigs vacc zone 3 km continued Fattening pigs: 5% within herd prevalence (2 reactors possible): 13 samples out of 18885; 1 positive, retest the sample; if positive, (normally individually identified at the first sampling): to collect blood from the same pig; if positive, a new sampling of 13 pigs. Piglets: 5% within herd prevalence (2 reactors possible): 13 samples out of 16444; 1 positive: the same as for fattening pigs. Particular attention to the piglets born after the vaccination and not vaccinated and use as sentinels. EU FMD-NSP workshop 113

114 Surveillance zone continued For the other herds, one single reactor in the biggest herds; result expected; so, same samples should be retested with another test system such as neutralization test; if still positive, clinical examination of herds. Sheep Surveillance zone 5% within herd prevalence Herds less than 35 sheep: test all 3 herds of 62 sheep: 54 tested per herd 3 herds of 155 sheep: 7 tested per herd All negative. No further action EU FMD-NSP workshop 114

115 Pigs surveillance zone 59 small herds: only clinical examination 4 holdings with average of 159 pigs: 7 pigs are tested per herd (5% within herd prevalence). 2 reactors in 2 large herds. So, same samples should be retested with another test system such as neutralization test; if still positive, clinical examination of the herds including cattle in the mixed herd. We will survive!! EU FMD-NSP workshop 115