fmd-nsp 3 report final · eu fmd-nsp workshop 2 contents page abstract 3 eu fmd-nsp workshop...

EU FMD-NSP workshop 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, 2007

Report

VAR-CODA-CERVA Leuvensesteenweg 17

B-3080 Tervuren Belgium

VAR Belgium

EUFMD Belgian Food Agency

DG RESEARCH DG SANCO


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 10 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 2 102 Annex D: presentation of scenario 2 by group 3 108 Annex E: presentation of scenario 2 by group 4 116 Annex F: presentation of scenario 3 by group 5 122 Annex G: presentation of scenario 3 by group 6 129 Annex H: Post NSP-workshop thoughts – Dr. David Paton 144


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;


4. The current EU Directive (2003/85/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. 3.8.7. of the TAHC);

5. The follow-up of herds with seroreactors by serological investigation has to be based on NSP assays (Paton et al., 2006) 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 2003/85/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 2003/85/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. 10. 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. 3.8.7. 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;


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; 10. 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. 10. 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 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.


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 10. 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 post-vaccination 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


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 “vaccinate-to-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


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!


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 non-structural 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


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.


ELISA

StructuralProteins

Non-StructuralProteins

+ +

+ -

- -

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.

ELISA

StructuralProteins


+ +

+ -

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…

NSPs in the vaccine.

ELISA

StructuralProteins


+ +

+ -

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.


Validation and comparison of NSP tests, Dr. Donal Sammin, Ireland

Comparative evaluation and validation of NSPEs

Dónal Sammin, CVRL-DAF, Ireland

Introduction

• 2003/85/EC: vaccination to live and post-vaccination surveillance

• DIVA testing => use of NSP-based tests• OIE-approved method ex. Panaftosa• 5 other NSPEs in Europe• Evaluate comparative performance• Validate for purpose

Studies

• FMD-ImproCon multinational workshop; IZS-Brescia, May 2004; cattle ( �sheep/pigs); Brocchi et al., 2006 [App 01] + Dekker et al. [App 08]

• EUFMD/WRL field study; Zimbabwe, April 2004; cattle, SAT-type infection; Sammin et al., VR, 2007

• EUFMD/WRL field study; HK-SAR, March 2005; (+ experimental study, IRL, 2006); pigs; Paton et al.

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

FMD_ImproCon WS; IZS, Brescia; May 2004Brocchi et al., 2006 [App 01]

MATERIALS & METHODS• 3551 sera [BE, DE, DK, ISR, IT, NL, TK, UK]• 2579 (67%) bovine; 703 ovine; 269 pig• Different vaccination and infection status• All sera tested x6 NSPE in parallel• Panaftosa, IZS-B, Ceditest, Svanovir, Chekit

and UBI ELISAs

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

• Experimental (n = 1037 sera)425 V+ I-

62 [54] V- I+ 21 [17] C+

550 [285] V+ I+ 225 [67] C+

• Field (n = 1542 sera)672 V- I-

867 V+ I�� (ISR/ZIM)

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

NOINDIRECT; coated

3BUBI FMDV NS ELISA

YESINDIRECT; coated

3ABCCHEKIT-FMD-3ABC

NOINDIRECT; coated

3ABCSVANOVIR FMDV 3ABC-Ab ELISA

NOBLOCKING; trapping

3ABCCeditest FMDV-NS

YESINDIRECT; trapping

3ABCIZS-Brescia

YESINDIRECT; coated

3ABCPanaftosa

“Grey zone”FormatAntigenELISA


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

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

WORKSHOP; IZS, Brescia; May 2004RESULTS: detection rates

• CATTLE V- I+ (n = 54) & V+ I+ (n = 285)

• Sub-categories of V+ I+ cattle: • (i) infection demonstrated (n = 164); • (ii) carrier status demonstrated (n = 67); • (iii) not carriers (n = 26); • (iv) no evidence of infection (n = 17)

• 7-14, 15-27, 28-100 and >100 days p.i.• Subcategories (i) and (ii), 14-27dpi: Panaftosa,

IZS-B and Ceditest NSPEs >60% seropositive• Carrier detection rate, 28-100dpi; Panaftosa

93.9%, IZS-B 86.4% and Ceditest 86.4%

EUFMD/WRL field study; Zimbabwe

• Objective: evaluate NSPEs for SAT-type FMD• 403 cattle; 6 herds; April-May 2004• SAT1/SAT2, 5 herds, 1-5 months pi• 12 - 35% of herds were “carriers” (probangs; VI & PCR)

• Overall seroprevalence of 56% - 75% with NSPEs; 81% with SPCE and 91% with VNT

• Carrier detection rate of 70% - 90%

• Sammin et al., Veterinary Record (2007) 160: 647-654

EUFMD/WRL field study; HK-SAR& exptl study on vaccinated pigs, IRL

Objective: evaluate 3 x NSPEs for use in pigsCeditest, UBI and Chekit ELISAs

Field study• 405 pigs; 4 vaccinated herds; Feb-March 2005• Type O infection, 2 herds, 1-2 months pi• All 3 NSPEs detected infection in pigs; different dSE and dSP• Cedi was both sensitive and specific; UBI was less specific

and Chekit was less sensitiveExperimental study• 99 pigs; vaccination x2; sampling 28 dpv• dSP: 100% 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 2007/early 2008


Sensitivity and specificity of SP and NSP tests – relevance and use, Dr. Aldo Dekker, The Netherlands

Sensitivity and specificity of SP and NSP tests; relevance and use

Aldo Dekker

Outline

� 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 2001

Validation SP tests

� Neutralisation tests considered as gold standard� Sensitivity close to 100% (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

Specificity first ELISA- 675 97.3% 97.3% 97.2% 98.7% 98.2% 99.0% 0.065+ 425 96.9% 97.4% 99.5% 98.1% 96.7% 97.9% 0.035-/+ 1100 97.2% 97.4% 98.1% 98.5% 97.6% 98.5% 0.122

Specificity after retest positives- 675 98.5% 99.9% 99.0% 99.1% 98.8% 99.4% 0.087+ 425 97.4% 99.5% 99.5% 98.8% 98.8% 98.4% 0.067-/+ 1100 98.1% 99.7% 99.2% 99.0% 98.8% 99.0% 0.007

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Detection rate in exposed cattle

- 7 - 14 5 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 1.000- 15 - 27 27 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 1.000- 28 - 100 26 100.0% 100.0% 100.0% 96.2% 92.3% 100.0% 0.434- > 100 2 100.0% 100.0% 50.0% 50.0% 50.0% 50.0% 1.000

+ 7 - 14 180 - 181 48.6% 52.2% 48.6% 40.9% 50.0% 32.0% 0.001+ 15 - 27 131 60.3% 55.7% 52.7% 49.6% 52.7% 38.2% 0.012+ 28 - 100 107 - 108 69.4% 64.8% 62.6% 58.3% 50.0% 56.1% 0.059+ > 100 47 72.3% 63.8% 74.5% 57.4% 38.3% 46.8% 0.001

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Detection rate in exposed and infected cattle

- 7 - 14 5 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 1.000- 15 - 27 26 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 1.000- 28 - 100 26 100.0% 100.0% 100.0% 96.2% 92.3% 100.0% 0.434- > 100 2 100.0% 100.0% 50.0% 50.0% 50.0% 50.0% 1.000

+ 7 - 14 89 56.2% 55.1% 53.9% 47.2% 57.3% 46.1% 0.545+ 15 - 27 97 70.1% 66.0% 61.9% 56.7% 61.9% 47.4% 0.028+ 28 - 100 91 - 92 75.0% 67.4% 68.1% 57.6% 53.3% 61.5% 0.029+ > 100 47 72.3% 63.8% 74.5% 57.4% 38.3% 46.8% 0.001

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Detection rate in carrier cattle

- 15 - 27 13 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 1.000- 28 - 100 7 100.0% 100.0% 100.0% 100.0% 71.4% 100.0% 0.146- > 100 2 100.0% 100.0% 50.0% 50.0% 50.0% 50.0% 1.000

+ 7 - 14 31 54.8% 54.8% 54.8% 51.6% 54.8% 38.7% 0.781+ 15 - 27 36 72.2% 66.7% 63.9% 55.6% 58.3% 58.3% 0.695+ 28 - 100 66 93.9% 86.4% 86.4% 71.2% 68.2% 77.3% 0.001+ > 100 37 89.2% 78.4% 89.2% 70.3% 48.6% 59.5% 0.000

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Conclusion validation at producer cut-off

� NCPanaftosa, IZS-Brescia and Ceditest ELISA superior

� 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)


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

1-Specificity0.0 0.2 0.4 0.6 0.8 1.0

Sen

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0.4

0.6

0.8

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Checkit

Ceditest

IZS-Brescia

UBI

NCPanaftosa

Svanovir

1-Specificity0.0 0.2 0.4 0.6 0.8 1.0

Sen

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0.2

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CheckitCheckit

CeditestCeditest

IZS-BresciaIZS-Brescia

UBIUBI

NCPanaftosaNCPanaftosa

SvanovirSvanovir

Pairwise comparison

Vaccinated cattle Non-vaccinated cattle

NC

Pan

afto

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NC

Pan

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IZS-Brescia - IZS

-Bre

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IZS-Brescia - IZS

-Bre

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Ceditest - - C

edite

st

Ceditest - - Ced

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Svanovir - - - S

van

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Svanovir - - - Sva

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Chekit ** ** ** ** C

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Chekit - - - - Ch

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UBI ** ** * ** - UBI - - - - -

ROC curve

1-Specificity0.0 0.2 0.4 0.6 0.8 1.0

Sen

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0.2

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0.6

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Checkit

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IZS-Brescia

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Svanovir

1-Specificity0.0 0.2 0.4 0.6 0.8 1.0

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Comparison at specified specificity Vaccinated cattle Non-vaccinated cattle

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69.4% NCPanaftosa IZS

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89.1% IZS-Brescia - C

ed

itest

68.8% IZS-Brescia - Ce

dite

st

87.5% Ceditest - - S

vano

vir

68.6% Ceditest - - Sva

novi

r

87.5% Svanovir - - - C

heki

t

64.2% Svanovir - - - Che

kit

85.9% Chekit ** ** ** - 56.7% Chekit - - - - 85.9%

97.5

% s

peci

ficity

UBI ** ** ** ** - 50.9% UBI - - - - - 87.5%

NC

Pa

nafto

sa

NC

Pa

nafto

sa

NCPanaftosa IZS

-Bre

scia

60.1% NCPanaftosa IZS

-Bre

scia

87.5% IZS-Brescia ** C

edi

test

68.2% IZS-Brescia - Ce

dite

st

85.9% Ceditest * - S

vano

vir

67.4% Ceditest - - Sva

novi

r

85.9% Svanovir - ** ** C

heki

t

53.2% Svanovir - - - Che

kit

85.9% Chekit ** ** ** - 50.3% Chekit - - - - 84.4%

99%

spe

cific

ity

UBI ** ** ** ** ** 36.4% UBI - - - - - 85.9%

Likelihood ratio dichotomized results

� 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 a b

c d

+

-

+ -true

test


Likelihood ratio on continuous data

-20 -10 0 10 20 30 40 50 60 70 80 90 100

Percentage inhibition

0

20

40

60

80

100

0

20

40

60

80

100

NON-EXPOSED CATTLE

EXPOSED CATTLE

Num

ber

of c

attle

Num

ber

of c

attle

Percentage inhibition Ceditest ELISA

Likelihood ratio analysis Ceditest ELISA

0

5

10

15

20

25

30

0 20 40 60 80 100

Percentage inhibition

Like

lihoo

d ra

tio

Cut-off

� 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 (= R0>1)

Orange : '1 km culling' sufficient

Green: 'no ring culling' already sufficient (= EU-measures)

On the basis of slowest decreasing kernel and immediate culling


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. MintiensCo-ordination Centre for Veterinary

Diagnostics

Contents• Different approaches for ‘substantiating’

freedom from disease• Two-stage sampling• Discussion

Claiming freedom from Claiming freedom from Claiming freedom from Claiming freedom from diseasediseasediseasedisease

• SPS agreement of World Trade Organization (WTO):

– Current animal health-related rules for international trade;

– Countries need to provide science-based 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

• hypergeometricdistribution:

−−

==+

n

N

xn

dN

x

d

xTP )(

Principle• When aiming to substantiate freedom from

disease, x=0:

• Factorial formulae with large numbers are difficult to compute

• Approximations of formula have been developed together with IT capacity

( ) ( )!)!(

!!)0(

NndN

nNdNTp

−−−−==+


• Methods for detecting the presence of a disease

• Assumptions:

– Design prevalence

– Perfect diagnostic tests

• Convenient approximation:

n

nN

dTP

−−−==+

21

1)0(

First approach: Cannon & Roe, 1982


• Sample size:

• Confidence level α (probability of finding at least one positive in the sample):

( )( ) 12

111

+

−×−−≈ dNn dα

n

nN

d

−−−≈

−2

)1(11α

• Tables with standard numbers available in epidemiological text books

• Software tools, e.g. WinEpiscope:

http://www.clive.ed.ac.uk/winepiscope /


Illustration of sample size calculationIllustration of sample size calculationIllustration of sample size calculationIllustration of sample size calculation:

– FMD outbreak in previously free area

– Outbreak controlled and disease no longer present

– Design prevalence: 30%

– Herd with 265 animals

– Confidence level (1-α)=0.95

=>Output from WinEpiscope: n=9


Improvements: Cameron & Baldock, 1998

• Diagnostic tests are not perfect:

• Thus the hypergeometric distribution extends:

SppSepTP

SpppSeTP

)1()1()(

)1)(1()(

−+−=

−−+=−

+

∑∑=

+−−−−

=

+ −×

−−

−

−−

==),min(

00

)1()1()(yx

j

jyxnjxjyjd

y

SpSpjx

ynSeSe

j

y

n

N

yn

dN

y

d

xTP

• Equation is very complex (only small population sizes can be computed)

• Approximation based on binomial distribution

• Considers formal hypothesis testing:– H0: prevalence ≥ design prevalence– Ha: prevalence < design prevalence– Type I error ά: falsely rejecting H0

– Type II error β: falsely accepting H0



• Sample sizes are calculated by iterative process based on probability distributions

• Survey results can be analysed providing a confidence level for rejecting H0

• Available in FreeCalc module of the Survey Toolbox

http://www.ausvet.com.au/content.php?page=res_software


Illustration of sample size calculation:Illustration of sample size calculation:Illustration of sample size calculation:Illustration of sample size calculation:

– Design prevalence: 30%


– Type I error (α )= type II error (β)=0.05

•Confidence level (1-α)=0.95

– ELISA Se=0.95 & Sp=0.98

=> FreeCalc: n= 14 with 1 reactor


• 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., 2004

• Procedure also available as freeware: BayesFreeCalc

http://www.epi.ucdavis.edu/diagnostictests/


Illustration of sample size calculation:– Design prevalence: > 30%


– Type I error = type II error = 0.05

– ELISA Se>0.90 & Sp>0.95

=> 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


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 non-diseased 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 non-

diseased • one minus the probability that a non-diseased herd will be

classified as diseased = 1- β

Two-stage samplingSample size• Number of herds:

– Preset Type I and type II error @ 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 samplingAnalysis of results• Classification of herds

– Each herd is classified as diseased or non-diseased, based on• number of reactors• Se + Sp of animal test• Na, na, 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• Nh, nh, type I and type II error

Two-stage samplingHypothetical example• Survey in 8532 herds to substantiate freedom from

disease• Screening test: Se=0.94, Sp=0.90• 95% confidence and power• Minimal expected herd-p*= 5%• Minimal expected within-herd-p*=20%• Logistical constrain: max 200 herds to be tested


Two-stage samplingHypothetical example: FreeCalc• Trail and error: 193 herds to be tested if:

– Type I = Type II =0.05– Herd-p= 5%– HSe= 90%– HSp= 98.4%– 6 reactors allowed

• Within herd testing sample size:– Type I error= 10%– Type II error= 1.6%– Within-herd-p= 20%– Sample size can be calculated for each herd

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

DiscussionNew approach (Mintiens et al, 2005)• Bayes Theorem:

– The posterior distribution of the probability of freedom from disease (F) given the observed test results (T)

)(

)()()(

TP

FPFTPTFP =

)(

),,,(),,,(),,,(

TP

SpSeprevFPSpSeprevFTPTSpSeprevFP =

Discussion

New approach (Mintiens et al, 2005)• Probability for positive test result:

• 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

)1())1)(1()(1(),,,( SpFSpprevSeprevFSpSeprevFTP −+−−+×−=

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 two-stage sampling

Discussion


Other issues• Surveys in small herds • Surveys with very small design

prevalence• Substantiating freedom from disease

using multiple complex data sources

Discussion

ReferencesCameron, A.R. & Baldock, F.C., 1998, A new probability formula for surveys to substantiate

freedom from disease. Prev.Vet.Med. 34, 1-17.Cameron, A.R. & Baldock, F.C., 1998, Two-stage sampling in surveys to substantiate

freedom from disease. Prev.Vet.Med. 34, 19-30.Cannon, R.M., 2002, Demonstrating disease freedom-combining confidence levels.

Prev.Vet.Med. 52, 227-249.Greiner, M. & Dekker, A., 2005, On the surveillance for animal diseases in small herds.

Prev.Vet.Med. 70, 223-234.Huzurbazar, S., et al., 2004, Sample size calculations for bayesian prediction of bovine viral-

diarrhoea-virus infection in beef herds. Prev.Vet.Med. 62, 217-232.Johnson, W.O., et al., 2004, Sample size calculations for surveys to substantiate freedom of

populations from infectious agents. Biometr. 60, 165-171.Martin, P.A. et al., 2007, Demonstrating freedom from disease using multiple complex data

sources 1: A new methodology based on scenario trees. Prev.Vet.Med. 79, 71-97.Martin, P.A. et al., 2007, Demonstrating freedom from disease using multiple complex data

sources 2: Case study--classical swine fever in denmark. Prev.Vet.Med. 79, 98-115.Mintiens, K. et al., 2005, Estimating the probability of freedom of classical swine fever virus of

the east-belgium wild-boar population. Prev.Vet.Med. 70, 211-222.Ziller, M. et al., 2002, Analysis of sampling strategies to substantiate freedom from disease in

large areas. Prev.Vet.Med. 52, 333-343.

Thank you


Statistical computer tools: theoretical and practical approach. B. Practice, Dr. Koen Mintiens, Belgium

Statistical computer tools: theoretical and practical approach.

B. Practice

K. MintiensCo-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

– 10 km surveillance zone around 6 infected premises:• All infected premises are stamped out• All susceptible animals within 500m radius are culled• All cattle within 10,000m radius are vaccinated

– Density:• Cattle: 252/km²• Pigs: 307/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=0.864, Sp=0.995

– Design prevalence:• Herd-p*: 0.01• Within-herd p*: 0.05

– Type I and II error: 0.05

Exercise– Sample size calculation:

• One stage approach• Two stage approach

– Analysis of results:• One stage approach

• Two stage approach


Sample size one stage approach

– Consider only the animals

– Plug parameters into FreeCalc:• Population: 47,899 animals• Design prevalence: 5%

• CEDI NSP test: Se=0.864, Sp=0.995• Type I and II error= 0.05


– Consider only the animals

– Plug parameters into FreeCalc:• Population: 47,899 animals• Design prevalence: 5%

• CEDI NSP test: Se=0.864, Sp=0.995• Type I and II error= 0.05

Result: 130 animals, 2 reactors


Move on to higher confidence level

– Plug parameters into FreeCalc:• Population: 47,899 animals• Design prevalence: 5%• CEDI NSP test: Se=0.864, Sp=0.995• Type I and II error= 0.01


Move on to higher confidence level

– Plug parameters into FreeCalc:• Population: 47,899 animals• Design prevalence: 5%• CEDI NSP test: Se=0.864, Sp=0.995• Type I and II error= 0.01



Important remarks:• One stage sample requires random

sample of animals– List of all cattle (sampling frame)

– High logistic impact (sampling one animal per herd)

=> Two stage approach

Sample size two stage approach

– First consider the herds

– Plug parameters into FreeCalc:• Population: 1,321 herds• Design prevalence: 1%• HSe=0.95, HSp=0.95• Type I and II error= 0.05



– First consider the herds

– Plug parameters into FreeCalc:• Population: 1,321 herds• Design prevalence: 1%• HSe=0.95, HSp=0.95• Type I and II error= 0.05

Unable to achieve the desired accuracy by sampling every unit


Decrease uncertainty: increase HSe and HSp:– Plug parameters into FreeCalc:

• Population: 1,321 herds

• Design prevalence: 1%• HSe=0.99, HSp=0.99• Type I and II error= 0.05


Decrease uncertainty: increase HSe and HSp:– Plug parameters into FreeCalc:

• Population: 1,321 herds• Design prevalence: 1%• HSe=0.99, HSp=0.99• Type I and II error= 0.05

Result: 1230 herds, 18 reactors


– Then consider the animals within herds

– Plug parameters into FreeCalc:• Population: largest herd: 592 animals• Design prevalence: 5%• Se=0.864, Sp=0.995• Type I and II error= 0.01


– Then consider the animals within herds– Plug parameters into FreeCalc:

• Population: largest herd: 592 animals• Design prevalence: 5%• Se=0.864, Sp=0.995• Type I and II error= 0.01



– Then consider the animals within herds:

– Plug parameters into FreeCalc:• Population: smaller herd: 131 animals• Design prevalence: 5%

• Se=0.864, Sp=0.995• Type I and II error= 0.01



– Then consider the animals within herds

– Plug parameters into FreeCalc:• Population: smaller herd: 131 animals• Design prevalence: 5%

• Se=0.864, Sp=0.995• Type I and II error= 0.01



Below 131: Unable to achieve the desired accuracy by sampling every unit

Results of the survey• 1,230 herds were randomly selected from

the total of 1,321• All cattle within selected herds were

sampled and tested (n=29,105; N= 47,899 )

• 142 animals tested positive with CEDI NSP test

Analysis of resultsOne stage approach

Looking at animal results:

• N=47,899

• n=29,105

• 142 reactors

• Se= 86.4, Sp= 99.5

• Considering the Sp: 146 (0.5%) false positives are allowed


Using Freecalc

• N=47,899

• n=29,105

• 142 reactors

• Se= 86.4, Sp= 99.5

• Type I and type II error is 0.05

• Design prevalence is 5%


Using FreeCalc:• N=47,899• n=29,105• 142 reactors• Se= 86.4, Sp= 99.5• Type I and type II error is 0.05• Design prevalence is 5%

• Result are adequate to reject to H0 and to conclude that the population is free from disease at 5% design prevalence and 100% confidence level


Analysis of resultsTwo stage approach

Classification of herds• All 1230 sampled herds were evaluated• Type I = Type II= 0.01• Se=86.4%, Sp=99.5%• Specific herd size and positive reactors

per herd


For 21 herds H0 could not be rejected

animals reactors animals reactors animals reactors

207 6 48 1 203 6

252 10 13 1 198 6

527 20 45 1 84 279 2 200 6 50 1

103 2 165 5 45 1

97 2 85 2 27 1

60 1 21 1 98 2


Classification of population• Type I = Type II= 0.05• HSe=99%, HSp=99%• Design prevalence= 1%• N=2188• n=1325• Positive reactors= 21


Classification of population

Results are not adequate to conclude that population is free from disease (design prevalence 1%). The confidence level is only 75.815%. We may conclude that population is diseased at confidence level 98.564%.

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


Discussion• How to increase confidence level and power:

– Increase accuracy: serial testing?– Look at geographical clusters?– Look at non-vaccinated populations, sentinels, buffer zone, …– …

• 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

Thank you


EU Directives for FMD control, Dr. Alf Füssel, EU DG SANCO, Head of Trade and Zootechnics Sector

11

FMDFMD

NSP Tests inNSP Tests in

PostPost--VaccinationVaccination SurveillanceSurveillance

Dr. Alf-Eckbert FüsselEuropean Commission - DG Health and Consumer Protection

Directorate D - Animal health and welfareUnit D1 - Animal health and Standing Committees

This presentation does not necessarily represent the views of the Commission 2

EmergencyEmergency vaccinationvaccination

►► CouncilCouncil DirectiveDirective 2003/85/EC 2003/85/EC providesprovides forforemergencyemergency vaccinationvaccination in in casecase of of moremore thanthan oneoneoutbreakoutbreak of FMDof FMD

►► TypesTypes of of vaccinationvaccination�� ProtectiveProtective ((vaccinatevaccinate--toto--livelive))

�� Suppressive (Suppressive (vaccinatevaccinate--toto--killkill))

►► VaccinationVaccination isis carriedcarried out in out in accordanceaccordance withwith�� ArticleArticle 52(1) 52(1) -- vaccinationvaccination zonezone

�� ArticleArticle 52(2) 52(2) -- surveillancesurveillance areaarea surroundingsurrounding thethevaccinationvaccination zonezone

3

3 3 PhasesPhases of of PostPost--vaccinationvaccination

MeasuresMeasures

1. from the beginning of emergency vaccination until at least 30 days have elapsed following the completionof such vaccination (Article 54)

2. from emergency vaccination until the survey and theclassification 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 theclassification of holdings until FMD free status isrecovered (Article 58)

4

Sheep and cattle notvaccinated

Sheep not vaccinated

Herd vaccinated

5

Clinical and serological survey

►Aim : identification of herds within the vaccination zone with contact to the FMDV without showing overt clinical signs

►Content: 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

6

Classification of holdingsClassification 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


7

RecoveryRecovery of FMD of FMD freefree statusstatus

followingfollowing emergencyemergency vaccinationvaccination

► 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

8

ModifiedModified recoveryrecovery of of freefree statusstatus

Following emergency vaccination, and by way of derogation, it may be decided byComitology, to withdraw the restrictionsapplied in the vaccination zone after theclinical and serological survey and theclassification of herds have been completedand confirmed the absence of foot-and-mouthdisease virus infection

Risk assessment taking into account different test strategies

9

Objectives Objectives

of postof post--vaccination measuresvaccination measures►►Risks from products from vaccinated Risks from products from vaccinated

animals can be mitigatedanimals can be mitigated

butbut

►►PostPost--vaccination surveillance is carried out vaccination surveillance is carried out to ensure that after the recovery of the to ensure that after the recovery of the status vaccinated ruminants can be moved status vaccinated ruminants can be moved safely out of the previous vaccination zone safely out of the previous vaccination zone and continue their productive life within the and continue their productive life within the vaccinating Member Statevaccinating Member State

10

ConclusionConclusion

►►When the free status is recovered there When the free status is recovered there

should only be herds which are should only be herds which are ►►either either serosero--negative negative

OrOr

►►SeroSero--positive exclusively from the administration of positive exclusively from the administration of

an inactivated vaccine an inactivated vaccine

►►No danger of carriers or undetected disease No danger of carriers or undetected disease


Combining multiple sources of evidence to demonstrate disease freedom, Dr. Tom Murray, APO FAO EUFMD

03 July 200703 July 2007 FAOFAO--EUFMD CommissionEUFMD Commission 11

Combining Multiple Sources of Combining Multiple Sources of Evidence to Demonstrate FMD Evidence to Demonstrate FMD

FreedomFreedom

Tom Murray, Associate Tom Murray, Associate Professional Officer, FAOProfessional Officer, FAO--

EUFMD CommissionEUFMD Commission


Key participants in the development of Key participants in the development of work are Ausvet, The International Epilab work are Ausvet, The International Epilab in the Danish Institute for Food and in the Danish Institute for Food and Veterinary Research and The Australian Veterinary Research and The Australian Biosecurity CRC Biosecurity CRC

The objective is to develop a system to The objective is to develop a system to quantitatively evaluate confidence in quantitatively evaluate confidence in Disease FreedomDisease Freedom

Details of methodology available at Details of methodology available at www.ausvet.com.au/freedomwww.ausvet.com.au/freedom


Summary of PresentationSummary of Presentation

1.1. Overview of The use of the stochastic Overview of The use of the stochastic Scenario Tree ApproachScenario Tree Approach

2.2. Review of various quantitative inputs and Review of various quantitative inputs and outputs in the context of an overall surveillance outputs in the context of an overall surveillance systemsystem

3.3. Example: Evaluation of Random SeroExample: Evaluation of Random Sero--survey survey using the stochastic Scenario Tree approachusing the stochastic Scenario Tree approach



Overview of Scenario Tree Overview of Scenario Tree ApproachApproach


Surveillance System Surveillance System Component(SSCComponent(SSC))



ExplanationsExplanations

The minimum proportion The minimum proportion of animals within a herd of animals within a herd that you would expect to that you would expect to have FMD if it was have FMD if it was presentpresent

Design Unit PrevalenceDesign Unit Prevalence

The minimum proportion The minimum proportion of herds you would of herds you would expect to have FMD if expect to have FMD if FMD is present in a FMD is present in a regionregion

Design Herd PrevalenceDesign Herd Prevalence



Given that surveillance Given that surveillance systems have not detected systems have not detected FMD, how confident are we FMD, how confident are we that this is a true reflection of that this is a true reflection of the situationthe situation

Negative Negative predictive value predictive value (NPV)(NPV)

The probability that the The probability that the surveillance systems will surveillance systems will detect FMD if it is present in detect FMD if it is present in the regionthe region

Surveillance Surveillance System SensitivitySystem Sensitivity



The estimate of the probability The estimate of the probability of FMD based on prior of FMD based on prior probability and the survey probability and the survey resultsresults

Posterior/PostPosterior/Post--test test probability of probability of diseasedisease

The estimate of the probability The estimate of the probability of FMD presence before the of FMD presence before the survey is carried outsurvey is carried out

Prior/Pretest Prior/Pretest probability of probability of diseasedisease


SpecificitySpecificity

Objective: To demonstrate disease Objective: To demonstrate disease freedomfreedom

Implies all tests have lead to a negative Implies all tests have lead to a negative outcomeoutcome

Specificity = 1Specificity = 1


Bayes TheoremBayes Theorem

Inputs and Outputs are based on Inputs and Outputs are based on distributions rather than point values. This distributions rather than point values. This reflects the uncertainty and variability reflects the uncertainty and variability associated with biological dataassociated with biological data

Prior probability of diseases and the actual Prior probability of diseases and the actual survey results are combined to give a survey results are combined to give a posterior probability of diseaseposterior probability of disease


Example: Evaluation of a Simple Random Example: Evaluation of a Simple Random SeroSero--SurveySurvey(Using Point Values)(Using Point Values)

Followed by combination of various SSCFollowed by combination of various SSC’’s s



Step 1: Calculate the probability Step 1: Calculate the probability that any one sample tests positivethat any one sample tests positive


Step 2:Calculate Sensitivity of Step 2:Calculate Sensitivity of Surveillance System Component.Surveillance System Component.If If ““aa”” is the probability that any sample is the probability that any sample tests positivetests positive

Then the probability that the surveillance Then the probability that the surveillance system component detects disease if it is system component detects disease if it is present is present is 11--(1(1--a)a)n n

(where (where ““nn”” is the number of samples taken)is the number of samples taken)


Step 3: Step 3: Calculate Negative Predictive Value of Calculate Negative Predictive Value of Surveillance System and the Posterior probability of FMDSurveillance System and the Posterior probability of FMD


Step 4: Carry out similar procedure Step 4: Carry out similar procedure for other surveillance system for other surveillance system

componentscomponentsOther SSCOther SSC’’s may require more s may require more assumptions and the uncertainty assumptions and the uncertainty associated with inputs would be greaterassociated with inputs would be greater

The degree of uncertainty associated with The degree of uncertainty associated with inputs can be reduced with time through inputs can be reduced with time through increased knowledge and the use of increased knowledge and the use of expertiseexpertise


Step 5:Combine the various SSCStep 5:Combine the various SSC’’s s together to get an overall value of together to get an overall value of

the surveillance systemthe surveillance system

Lack of independence between various Lack of independence between various surveillance system components needs to surveillance system components needs to be accounted forbe accounted for


Key PointsKey Points

This is a simplified overview of the processThis is a simplified overview of the processThe quantitative evaluation of surveillance systems can The quantitative evaluation of surveillance systems can provide for comparisons between systemsprovide for comparisons between systemsThis work requires the close involvement of expertise This work requires the close involvement of expertise and experience from various fieldsand experience from various fieldsThis work emphasizes the value of current over historical This work emphasizes the value of current over historical data.data.The use of this approach can identify gaps in knowledge The use of this approach can identify gaps in knowledge for further researchfor further researchAn effective system for demonstration of FMD freedom An effective system for demonstration of FMD freedom also means a system which is more likely to detect new also means a system which is more likely to detect new incursions earlierincursions earlier


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

7/3/2007 2

OIE Terrestrial Animal Health Code

• Chapter 1.3.5.1. Zoning & Compartmentalisation• Chapter 2.2.10. FMD• Appendix 3.8.1. General guidelines for animal

health surveillance• Appendix 3.8.7. Guidelines for the surveillance

of FMD

OIE Diagnostic Manual

7/3/2007 3

OIE Code FMD Chapter 2.2.10.7: Recovery of FMD-free status following outbreaks

(1)

Outbreaks in a country or zone that is “FMD-free with

vaccination”.

Can regain this status by:

– Vaccination, slaughter of infected, serosurveillance to detect virus circulation rather than infection, 6 month

minimum waiting period

– OIE provides guidelines for serosurveillance

– Do not need to detect carriers at all

– Risk of carriers still being present is mitigated by restrictionon trade in live animals if “free with vaccination”, rather than

“free without vaccination”

7/3/2007 4

Outbreaks in a “FMD-free country without vaccination”

Can regain this status by:

– Slaughter of infected, no vaccination, serosurveillance to

demonstrate absence of infection, 3 months minimum wait

– Slaughter of infected, vaccinate-to-kill, serosurveillance to

demonstrate absence of infection, 3 months minimum

– Slaughter of infected animals, vaccinate-to-live, serosurveillance to demonstrate absence of infection, 6 months minimum

OIE Code FMD Chapter 2.2.10.7: Recovery of FMD-free status following outbreaks

(2)

7/3/2007 5

OIE Code FMD Chapter 2.2.10.Article 2.2.10.1. 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

7/3/2007 6

OIE Code Appendix 3.8.1.General Guidelines for Surveillance

• Article 3.8.1.6. Surveillance to demonstrate freedom from disease/infection

– Demonstration of freedom from infection

• Implies absence of the pathogenic agent

• Complete absence cannot be proven with 100% 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.


7/3/2007 7

OIE Code Appendix 3.8.7.

Guidelines for FMD Surveillance

• Article 3.8.7.7. 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

7/3/2007 8

• Article 3.8.7.7. 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

7/3/2007 9

Fig. 1. Schematic representation of laboratory testsfor determining evidence of FMDV infection

through or following serological surveys

7/3/2007 10

OIE Code FMD Chapter 2.2.10.Article 2.2.10.6 (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 2.2.10.7.

7/3/2007 11

FMD modelling and NSP testing

Mark Arnold – VLA

John Wilesmith – Defra

David Paton, Eoin Ryan, Sarah Cox – IAH Pirbright

7/3/2007 12

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


7/3/2007 13

• Transmission depends

on

– Number of animals in

IP

– Distance from IP

– Number of animals in

susceptible farm

Transmission model

7/3/2007 14

Transmission vs distance from IP

7/3/2007 15

Animal number Infectiousness/susceptibility

Susceptibility=0.13 0.1336 ( ) ( )CATTLE SHEEPN N× +

Infectiousness= 0.13 0.131.4 ( ) ( )CATTLE SHEEPN N× +

7/3/2007 16

Susceptibility to infection post-vaccination

Animal (and farm) susceptibility reduced

according to days between exposure and

vaccination

7/3/2007 17

Likelihood of disease post vaccination

Infectiousness assumed to correlate with

probability of disease

7/3/2007 18

Probability of carriers in vaccinated farms

Estimate initial infs

Calculate clinicals

Determine if IP

Likelihood of disease vs days post vaccinationexperiment

Efficacy of vaccinationmequation or simulation

Estimates from 2001 epidemic stil into ke

<5 days post vaccination OR

>0 clinicals – dairy; >1 clinical -suckler

50% probability for each infectedCalculate if carriers

IF NOT AN IP


7/3/2007 19

Number of initial infections

Small dependence on number of

cattle:Mean infs=

4.1+0.005Ncattle

7/3/2007 20

Carrier probability vs herd size and no of clinicals for detection

7/3/2007 21

Rate of vaccination

• Commences 7 days after initial IP identified

• 50 vaccination teams, vaccinating up to 250 animals per day

• Clinical inspection of sheep and pigs also performed

• 10k vaccination ring around each IP, nearest farms vaccinated first

7/3/2007 22

Other assumptions

• Time to detection for infected herds taken from 2001 epidemic

• All animals in each IP are culled, time to slaughter taken from 2001 epidemic

7/3/2007 23

Outline of simulations

• Population at risk=Devon• 5 initial infections• 200 runs of each of the following

scenarios:– No vaccination– Vaccinate farms>50 cattle, <10k from IP

– Vaccinate all cattle farms < 10k from IP• 44% of herds with cattle have <50 in Devon

7/3/2007 24

Post epidemic testing

• Commences 30 days after last IP• Cattle tested on all vaccinated farms with

Cedi screen/Cedi retest/Svanova confirm• Sensitivity=67%, specificity=99.99%.• Sufficient animals tested to detect 5%

prevalence with 95% probability• Have also looked at testing all animals


7/3/2007 25

Epidemic size distribution – 200 runs

7/3/2007 26

Mean no of IPs/day

�Vaccinating small farms

– 66 vaccinated

farms/IP, 71% reduction in cases

�Vaccinating farms >50

cattle – 35 vaccinated farms/IP, 68% reduction

in cases

7/3/2007 27

Distribution of carrier farms by herd size

� Reducing number

of farms as herd

size increases

� But higher

incidence of those farms

7/3/2007 28

Within herd prevalence

� Farms with many initial infs->IPs

� Farms infected late after vaccination have few initial infs

� Therefore carriers occur at low prevalence

7/3/2007 29

Sensitivity of testing (at farm level)

Strategy True positives

Detected Sensitivity

Vacc >50 only

335 118 35%

Vacc all 325 140 43%

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

7/3/2007 30

Specificity of testing (at farm level)

Strategy True negatives

False positives

Specificity

Vacc >50 only

233,000 1,562 99.32%

Vacc all 397,000 1,890 99.52%

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


7/3/2007 31

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

7/3/2007 32

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


Presentation of adopted FMD vaccination plans - scenarios prepared for the workshop, Dr. Phillippe Houdart, Belgium

Federal Agency for the Safety of the Food Chain

INTRODUCTION TO THE EXERCISE

Federal Agency for the Safetyof the Food Chain


Aim of 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


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 km2

• density: – cattle = medium density (37 / km2)– pigs = high density (300 / km2)

– sheep/goat = low density (2 / km2)



• 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



• PZ and SZ surrounding 2 neighbouring outbreaks

• size = 380 km2

• FMDV introduced through movement of infected sheep (outbreak 5)

• density:

– cattle = medium density (40 / km2)– pigs = medium density (124 / km2)– sheep/goat = low density (4 / km2)



• culling: susceptible animals in 2 outbreaks

• vaccination of cattle and pigs in protection zone

• particularities:

– one of outbreaks = large pig farm (>40.000 pigs)– only infected pigs in a quarantine unit separated

from the other buildings– rest of holding is vaccinated






• vaccination area surrounding 23 outbreaks

• size = 1.440 km2

• FMDV introduced through movement of infected sheep (outbreak 5)

• density:

– cattle = high density (160 / km2)– pigs = high density (>1.000 / km2)– sheep/goat = low density (13 / km2)



• 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


0,0 2,5 5,0 7,5 10,0

Kilometers


0,0 2,5 5,0 7,5 10,0

Kilometers



What is expected of you?

• each of the scenarios is approached by 2 groups of2 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 11. Moldova + Ukraine Per Have + David Paton2. Norway + Latvia Tom Murray

• scenario 23. Slovakia + Estonia Åse Uttenthal + Philippe Vanier4. Finland + Poland Aldo Dekker

• scenario 3: 5. Czech Rep. + Sweden Dónal Sammin6. Switzerland + Israel Lea Knopf

room

308333

309333

265138


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

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.


- 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 10 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. Characteristics of cluster 2 Cluster 2 is comprised of the 10 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: 380 km2;

- 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 10 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: 1.440 km2; - Composition: see tables.


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 10 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.


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 3.000 m radius around the outbreaks (the former protection zone). The size of the vaccination area is 57 km2. 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 3.000 m radius around the outbreaks (the former protection zone). The size of the vaccination area is 32 km2. 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 706 km2. 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 (10 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 10 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


OUTBREAK 1 = pig holder

ID-number 100332373

cluster 1

link to = index case

source of infection contaminated products from Turkey, brought home by owner’s son

date of infection around 13-02

date of confirmation 27-02

localisation municipality of Ljutomer

type of holding fattening pig holding

structure/capacity different small sheds

type of rearing Indoors

number of animals present 84 pigs: - 9 sows - 12 piglets - 60 fattening pigs of 80 kg - 2 gelts - 1 boar

Particularities

- On 20-02 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 25-02 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 26-02 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.


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 27-02 FMD is diagnosed in the lab. - On 28-02 the animals in the outbreak are killed and destroyed.

OUTBREAK 2 = brother of the index

ID-number 100344771

cluster 1

link to outbreak 1

source of infection indirect contact due to owner’s contact with infected pig from index case

date of infection 20-02


localisation municipality of Ljutomer, 140 m from index

type of holding cattle and sheep

structure/capacity 1 stable

type of rearing indoors

number of animals present 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 26-02 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 27-02 FMD is diagnosed in the lab. - On 28-02 the animals in the outbreak are killed and destroyed


OUTBREAK 3 = neighbour of the index

ID-number 100322348

cluster 1

link to outbreak 1

source of infection indirect contact due to probably airborne spread



localisation municipality of Ljutomer, 50 m from index

type of holding cattle

structure/capacity 1 stable


number of animals present 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 27-02 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 28-02 FMD is diagnosed in the lab. - On 29-02 the animals in the outbreak are killed and destroyed.


OUTBREAK 4 = first case in cluster 2

ID-number 100337021

cluster 2

link to outbreak 5

source of infection direct contact due to infected sheep brought by the sheep trader of outbreak 5



localisation municipality of Domzale

type of holding mixed holding

structure/capacity several sheds for maximal 200 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 24-02 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 01-03 following the notification by the veterinarian of

the farm of a suspicion of FMD in the pigs of the farm. - On 02-03 in the morning FMD is diagnosed in the lab. - On 02-03 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.


OUTBREAK 5 = sheep trader – source of spread towards clusters 2 and 3

ID-number 100474259

cluster 1

link to outbreak 1

source of infection indirect contact due to the butcher who slaughtered the first infected pig



localisation municipality of Ljutomer, other part of the village 450 m from index

type of holding sheep trader

structure/capacity several sheds for maximal 200 sheep


number of animals present 30 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 (20-02), 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 02-03 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 02-03 in the evening FMD is diagnosed in the lab. - On 03-03 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.


OUTBREAK 6 = big pig farm

ID-number 100295700

cluster 2

link to outbreak 4

source of infection 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

date of infection around 28-02/01-03


localisation municipality of Domzale, 30 m from outbreak 4

type of holding big commercial pig farm

structure/capacity closed holding with 9 stables for sows/piglets and 36 stables for fattening pigs; separate quarantaine unit


number of animals present 39.844 in total: 3916 sows, 72 boars, 16444 piglets, 18.885 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 03-03, 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 04-03.

- The same day (03-03), 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


not spread to the rest of the farm. As a precautionary measure, on 05-03, vaccination is started immediately in the rest of the farm, progressing outwards beginning at the stables closest to the quarantaine stable.


OUTBREAK 7 = sheep farm

ID-number 40000672

cluster 3

link to outbreak 9

source of infection direct contact due to infected sheep bought from outbreak 9



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 09-03 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 10-03 and samples are taken and sent to the laboratory. - On 11-03 FMD is diagnosed in the lab. - On 10-03 the animals in the outbreak are killed and destroyed.


OUTBREAK 8 = sheep farm

ID-number 30030867

cluster 3

link to outbreak 9

source of infection direct contact due to infected sheep bought from outbreak 9



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


number of animals present 125 dairy cattle, 1.031 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 10-03 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 11-03 FMD is diagnosed in the lab. - On 10-03 and 11-03 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.


OUTBREAK 9 = sheep trader

ID-number 100357660

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)



localisation municipality of Aalter

type of holding sheep and cattle trader

structure/capacity different pens for maximal 230 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 24-02 a group of 30 sheep, some of which are infected, at the trader of outbreak 5. These sheep are put in a stable at outbreak 9 with some 150 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 10-03 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 11-03 FMD is diagnosed in the lab. - On 10-03 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

20 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.


OUTBREAK 10 OUTBREAK 11 OUTBREAK 12

ID-number 30031138 31089581 40023141

cluster 3 3 3

link to outbreak 8 outbreak 8, 9 and 10 unknown

source of infection neigbouring contact neigbouring contact of outbreaks 8 and 10; contact of outbreak 9

unknown

date of infection around 10-03 around 10-03 around 05-03

date of confirmation 14-03 15-03 15-03

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 30 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 10 and direct contact of outbreak 9 following the purchase of 4 sheep. The 12 sheep were kept on a pasture. Suspicion is raised on 14-03 and confirmed on 15-03 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 15-03 by the farm veterinarian. The cattle show 10-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.


OUTBREAK 13 OUTBREAK 14 OUBTREAK 15

ID-number 30021906 40134423 40038270

cluster 3 3 3

link to unknown outbreak 9 unknown

source of infection unknown neighouring holding of outbreak 9 unknown



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 1019 pigs 156 dairy cattle, 973 fattening pigs

particularities The suspicion is anonymously raised to the veterinary service on 14-03. 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 15-03 in the framework of the monitoring in the protection zone around outbreak 9. The farm is situated at 600 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 16-03 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 10 to 12,5 km from the nearest outbreaks (7, 9 and 12). The first infected animal on the farm (a calve that died on 09-03 with, in retrospect, very mild symptoms of FMD) has probably been missed. No links with existing outbreaks exist.



ID-number 40001504 30882006 40038531

cluster 3 3 3

link to outbreak 9 unknown outbreak 15 (at 2,5 km)

source of infection neighouring holding of outbreak 9 unknown unknown



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, 1.734 pigs 178 cattle 10 fattening cattle

particularities The suspicion is raised by the veterinarian on 16-03 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 17-03 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 17-03 and 18-03.

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.



ID-number 40038343 30081003 30082050

cluster 3 3 3

link to outbreak 15 (at 4 km) outbreak 17 outbreak 17

source of infection unknown neighbouring holding of outbreak 17 (at 1,6 km)

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, 1.116 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.



ID-number 30880641 40193195 40038841

cluster 3 3 3

link to outbreak 17 unknown outbreaks 15, 18 and 19

source of infection neighbouring holding of outbreak 17 (at 1,1 km)

unknown 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.



ID-number 31098406 30053190 40038580

cluster 3 3 3

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)

neighbouring holding of outbreak 18 (at 400 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 550 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


OUTBREAK 28 OUTBREAK 29

ID-number 400038262 31027381

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)

neighbouring holding of outbreak 25 (at 1 km)

date of infection around 18-03 around 23-03

date of confirmation 27-03 28-03

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.


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 2,09 35 0,18 2 5,40 1.423 cluster 1 - surveillance zone (including vaccination zone)

433 2,31 37 0,23 2 6,09 300

cluster 2 - vaccination zone 32 3,28 48 0,06 1 0,16 1.441 cluster 2 - surveillance zone (including vaccination zone)

380 3,15 40 0,18 4 0,18 124

cluster 3 - vaccination zone 770 3 188 2 15 1 1.187

cluster 3 - surveillance zone 1440 2 160 2 13 1 1.010

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 119 1.989 10 88 308 81.111

surveillance zone 883 13.909 88 925 2.328 48.920

1.002 15.898 98 1.013 2.636 130.031


zone number of

cattle holdings

total number of

cattle


holdings


number of pig

holdings

total number of

pigs

vaccination zone 105 1.549 2 19 5 46.098

surveillance zone 1.092 13.697 66 1.367 63 894

1.197 15.246 68 1.386 68 46.992



zone number of

cattle holdings

total number of

cattle


holdings


number of pig

holdings

total number of

pigs

vaccination zone 2.188 144.872 1.365 11.780 1.075 914.155

surveillance zone 1.372 85.007 885 6.771 677 540.289

3.560 229.879 2.250 18.551 1.752 1.454.444

Number of mixed farms:

cluster mixed cattle & pigs

cattle & sheep

sheep & pigs

cluster 1 - vaccination zone 2 96 1 5

cluster 1 - surveillance zone 21 731 1 48





Cluster 1 – 0 to 3000m = vaccination zone – size of holdings:

size number of cattle holdings

total number of cattle

average number of cattle


holdings


average number of sheep/goat

number of pig

holdings

total number of

pigs

average number of

pigs

1 to 4 20 47 2 4 6 2 96 269 3

5 to 10 33 238 7 3 21 7 63 455 7

11 to 25 35 608 17 2 32 16 64 1.128 18

26 to 50 29 914 32 1 29 29 32 1.104 35

51 to 100 2 182 91 0 0 - 28 2.020 72

101 to 250 0 0 - 0 0 - 18 2.594 144

251 to 500 0 0 - 0 0 - 4 1.257 314

501 to 1000 0 0 - 0 0 - 0 0 -

1001 to 0 0 - 0 0 - 3 72.284 24.095

119 1.989 10 88 308 81.111


Cluster 1 – 3000 to 10000m = surveillance zone = non-vaccinated zone – size of holdings





holdings



number of pig

holdings

total number of

pigs

average number of

pigs

1 to 4 252 570 2 41 78 2 1.108 2.927 3

5 to 10 230 1.676 7 13 98 8 528 3.636 7

11 to 25 258 4.273 17 27 444 16 350 5.634 16

26 to 50 102 3.364 33 4 124 31 161 5.619 35

51 to 100 31 2.057 66 3 181 60 82 6.071 74

101 to 250 8 982 123 0 0 - 70 10.735 153

251 to 500 1 276 276 0 0 - 22 8.158 371

501 to 1000 1 711 711 0 0 - 5 3.038 608

1001 to 0 0 - 0 0 - 2 3.102 1.551

883 13.909 88 925 2.328 48.920

Cluster 2 – 0 to 3000m = vaccination zone – size of holdings





holdings



number of pig

holdings

total number of

pigs

average number of

pigs

1 to 4 46 114 2 1 3 3 4 7 -

5 to 10 25 166 7 0 0 - 0 0 -

11 to 25 12 201 17 1 16 16 0 0 -

26 to 50 15 553 37 0 0 - 0 0 -

51 to 100 6 411 69 0 0 - 0 0 -

101 to 250 1 104 104 0 0 - 0 0 -

251 to 500 0 0 - 0 0 - 0 0 -

501 to 1000 0 0 - 0 0 - 0 0 -

1001 to 0 0 - 0 0 - 1 46.091 46.091

105 1.549 2 19 5 46.098


Cluster 2 – 3000 to 10000m = surveillance zone = non-vaccinated zone – size of holdings





holdings



number of pig

holdings

total number of

pigs

average number of

pigs

1 to 4 382 1.023 3 23 55 2 41 98 2

5 to 10 330 2.302 7 11 74 7 14 96 7

11 to 25 258 4.086 16 17 269 16 4 63 16

26 to 50 85 2.907 34 9 318 35 0 0 -

51 to 100 31 2.099 68 3 185 62 0 0 -

101 to 250 5 685 137 3 466 155 4 637 159

251 to 500 0 0 - 0 0 - 0 0 -

501 to 1000 1 595 595 0 0 - 0 0 -

1001 to 0 0 - 0 0 - 0 0 -

1.092 13.697 66 1.367 63 894

Cluster 3 – vaccination zone = inner fused 5 km of surveillance zone – size of holdings





holdings



number of pig

holdings

total number of

pigs

average number of

pigs

1 to 4 331 746 2 748 1.949 3 19 44 2

5 to 10 215 1.594 7 395 2.675 7 9 60 7

11 to 25 280 4.663 17 158 2.433 15 14 225 16

26 to 50 278 10.599 38 38 1.281 34 14 497 36

51 to 100 564 42.031 75 17 1.251 74 49 3.839 78

101 to 250 471 68.997 146 8 991 124 134 23.970 179

251 to 500 45 14.054 312 0 0 - 198 73.245 370

501 to 1000 4 2.188 547 0 0 - 289 214.091 741

1001 to 0 0 - 1 1.200 1.200 349 598.184 1.714

2.188 144.872 1.365 11.780 1.075 914.155


Cluster 3 – non-vaccinated zone = outer fused 5 km surveillance zone – size of holdings





holdings



number of pig

holdings

total number of

pigs

average number of

pigs

1 to 4 225 519 2 456 1.252 3 33 76 2

5 to 10 147 1.054 7 279 1.870 7 9 63 7

11 to 25 202 3.517 17 123 1.961 16 13 223 17

26 to 50 207 7.861 38 15 534 36 17 623 37

51 to 100 294 22.116 75 9 640 71 22 1.714 78

101 to 250 261 38.891 149 3 514 171 91 16.519 182

251 to 500 36 11.049 307 0 0 - 144 54.444 378

501 to 1000 0 0 - 0 0 - 157 115.983 739

1001 to 0 0 - 0 0 - 191 350.644 1.836

1.372 85.007 885 6.771 677 540.289


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 = Moldova + Ukraine + Per Have + David Paton

(annex B) Group 2 = Norway + Latvia + Tom Murray

(annex C) Vaccination/protection zone (VZ/PZ) • Vaccination of bovine/pigs:

• bovine: 119 herds – 1,989 animals • Pigs: 308 herds – 81,111 animals

• No vaccination of small ruminants: 10 herds – 88 animals • Vaccination zone = protection zone Design: G1 G2

Bovine • CS(1) all herds • Sample all herds / all animals • All animals marked for follow-up sampling

• Sample all herds / all animals

Small ruminants

• CS(1) all herds • Sample all herds / all animals • All animals marked for follow-up sampling

• Sample all herds / all animals

Pigs • 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

• Sample all herds ≤ 1000/ all animals • Sample all herds >1000/(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.

(1)CS: clinical surveillance Figures: G1 G2 Bovine 119 herds / 1,989 samples 119 herds/1,989 samples

Small ruminants

10 herds / 88 animals 10 herds / 88 animals

Pigs 308 herds / 8,990 samples 308 herds/12,377 samples


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 0.70 / Sp 0.99), 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 14,454 + 2,185.

Surveillance zone (SZ) • No vaccination of animals in 3 km – 10 km zone:

• Bovine: 883 herds – 13,909 animals • Small ruminants: 88 herds – 2,328 animals • Pigs: 2,328 herds – 48,920 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

• CS all herds/all animals Sample all herds/all animals

• All animals marked for follow-up sampling

Sample all herds / all animals

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/13,909 animals CS 883 herds/13,909 animals

+ ~ 280 samples

Small ruminants 88 herds/925 samples 88 herds/925 samples

Pigs CS 2,328 herds/48,920 animals CS 2,328 herds/48,920 animals + ~ 980 samples

Resources: G1 G2 Vet teams

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

Lab Cedi-NS + confirmatory VNT


Initial laboratory results (given by the supervisory team, based on the design made by the group) Cluster 1:

G1 G2 • VZ/PZ: 2 big pig herds (ID 327, 325) have 31

(25) and 20 (17) positive results • VZ/PZ: 5 medium pig herds (ID 171, 263, 33,

178, 257) have 16 (10) positive results • VZ/PZ: 2 small pig herds (ID 312, 295) have 5

(1) positive results • VZ/PZ: 1 cattle herd (ID 103) 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

• VZ/PZ: 70 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 103 • VZ/PZ: 1 bovine reactor in herds 99, 270 • VZ/PZ: no reactors in small ruminants • SZ: 3 sheep reactors in herd 1317

Interpretation and follow-up

G1 G2 • Analysis of test results: o No. of reactors higher than expected from

design? Yes in farm 327, 325, 312, 263, 2364, 379, 1317, 103. 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)

• 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 (60,000 pigs) – 20 strong positive results (all sows and in one section of 600 sows) + 50 weak positives distributed all over

o SZ: positive sheep herd: collect epidemiological information from the herd, sample all animals for virology, cull the flock


Field follow-up

G1 G2 • 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

• 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


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: 105 herds – 1,549 animals � Pigs: 5 herds – 46,098 animals

• No vaccination of small ruminants : 2 herds – 19 animals • Vaccination zone = protection zone Design: G3 G4 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 10% within herd prevalence, dependant on the size of the herds. If herds <26, clinical examination only.

Sample all herds/all animals

Small ruminants Cull all animals or individual sampling + clinical examination

Sample all herds/all animals

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

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 524 + 264 + 256 samples 105 herds/1,549 samples

Small ruminants

2 herds/19 samples 2 herds/19 samples

Pigs 465 sows + 130 fattening pigs + 130 piglets 4 herds/7 samples 1 herd/2,700 samples CS 36 stables/ 4270 animals, check 4270 samples at slaughter


Resources: G3 G4 Vet teams / • Examine farms were sampling would be carried

out first • 20 vet. Days for sampling cattle herds + 13 vet.

Days for sampling sows (2700 in 9 stables) in large pig farm + 18 vet. Days for CS of 36 stables with 19,000 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 – 10 km zone:

� Bovine: 1,092 herds – 13,697 animals � Small ruminants: 66 herds – 1,367 animals � Pigs: 63 herds – 894 animals

Design: G3 G4 Bovine Clin. surv. all herds/all animals

Herds >26: 10% within herd prevalence Clin. surv. all herds/all animals

Small ruminants

Herd <35: sample all animals Herd>35: 5% within herd prevalence

Clin. surv. all herds/all animals + Sample all herds/ all animals in sheep farms without other ruminants

Pigs Clin. surv. small herds/all animals 4 big holdings: 5% within herd prevalence

Clin. surv. all herds/all animals

Figures: G3 G4 Bovine CS 1,092 herds/13,697 animals

Sample 122 herds/4237 samples CS 1,092 herds/13,697 animals

Small ruminants

Herd <35: 60 herds/716 samples Herd >35: 6 herds/124 samples

CS + sample 22 herds/951 animals

Pigs CS 59 herds/894 animals Sample 4 herds of ±159 pigs: 70 pigs per herd.

CS 63 herds/894 animals

Resources: G3 G4 Vet teams / • Examine farms were sampling would be carried out first

• 100 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 /


Initial laboratory results (given by the supervisory team, based on the design made by the group)

G3 G4 • VZ: 4 NSP+ sows, 1 NSP+ pig, 1 NSP+ piglet • SZ: 4 positive cattle, 2 reactors in 2 large pig

herds

• 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


G3 G4 • 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 130 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 130 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 108 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

• 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 + 50% of penmates and pigs in adjacent pens

• VZ: small bovine farms (16-104 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


Field follow-up

G3 G4 / • For big pig farm: 23 reactors in the retesting of

the positive sows + 12 reactors out of 2107 samples in the group of penmates and adjacent pens → option for decision-making:

o Culling and disinfection of infected premises (46,000 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


Scenario 3: 25 outbreaks – 3 clusters Only bovine vaccinated All bovine/sheep/goats/pigs culled 0 - 500 m

Group 5 = Czech Republic + Sweden + Dònal Sammin (see annex F) Group 6 = Israel + Switzerland + Lea Knopf (see annex G)

Vaccination zone (VZ) • Vaccination of all bovine in 500m – 5 km:

� Bovine: 2,188 herds – 144,872 animals • No vaccination of pigs/small ruminants:

� Small ruminants: 1,365 herds – 11,780 animals � Pigs: 1,075 herds – 914,155 animals

Design: G5 G6-EU approach G6-Israeli-Swiss approach Bovine Large survey: sample all

herds/all animals CS all herds /all animals Sample all herds /all animals

CS (95:5) herds and sample 10% of inspected holdings + Two-stage sampling Hse 80%, Hsp 99.5%, (95:5) herds, if herd >=60 and if herd ≤75 animals / all animals, if herd >75 animals/75 animals per herd. (within herd prevalence of 5%)

Small ruminants

Preliminary survey: Sample (95:5) herds/(95:20) animals Large survey: sample (95:2) herds/(95:5) animals, but all animals in flocks <25 animals and 1 large farm (1,200 animals)

CS all herds /all animals Sample in two-stage approach: Hse 90%, Hsp 99%, (95:5) herds, if herd >=10 and if herd ≤30 animals / all animals, if herd >30 animals/30 animals per herd. (within herd prevalence of 10%)

No clinical inspection since no clinical signs Two-stage sampling Hse 90%, Hsp 99%, (95:5) herds, if herd >=10 and if herd ≤30 animals / all animals, if herd >30 animals/30 animals per herd. (within herd prevalence of 10%)

Pigs Preliminary survey: Sample (95:5) herds/(95:20) animals Large survey: CS


CS all herds /all animals and sample 5% of inspected holdings + Two-stage sampling Hse 90%, Hsp 99%, (95:5) herds, if herd >=10 and if herd ≤20 animals / all animals, if herd >20 animals/20 animals per herd. (within herd prevalence of 20%)


Figures: G5 G6-EU approach G6-Israeli-Swiss

approach Bovine Large survey: 2,188

herds/144,872 samples 2,188 herds/144,872 samples CS 523 herds and sample 50

herds/ 250 animals + 181 herds/13,575 animals

Small ruminants

Preliminary survey: 67 herds / 536 samples Large survey: 391 herds/2,920 animals

Pigs Preliminary survey: 67 herds / 1,474 samples

CS 1,365 herds – 11,780 animals + CS 1,075 herds – 914,155 animals 164 herds/4,100 animals 2,315 samples from clinical investigations (463 holdings with 5 samples each)

Sample 164 herds/4,100 animals + Pigs : CS 1075 herds/914,155 animals and sample 55 herds/275 animals

Resources: G5 G6-EU approach G6-Israeli-Swiss approach Vet teams

20 vets for CS of pig holdings

Lab Preliminary survey: SP ELISA (Se 0.99, Sp 0.95) Analysis of 144,000 NSP tests

Bovine: Cedi-test with retesting (Se 98%, Sp 99%) and virus detection (samples from clinical survey)

Cedi test with retesting (Se 98%, Sp 99%) and virus detection (samples from clinical survey)

Surveillance zone (SZ) • No vaccination in 5 km – 10 km zone:

� Bovine: 1,372 herds – 85,007 animals � Small ruminants: 885 herds – 6,771 animals � Pigs: 677 herds – 540,289 animals


Design: G5 G6-EU approach G6-Israeli-Swiss

approach Bovine Large survey: CS all herds/all

animals CS all herds /all animals Sample in two-stage approach: Hse 90%, Hsp 99%, (95:5) herds, if herd >=10 and if herd ≤30 animals / all animals, if herd >30 animals/30 animals per herd. (within herd prevalence of 10%)

CS (95:5) herds with Se 95% and Sp 99%/ Sample in two-stage approach: Hse 90%, Hsp 99%, (95:5) herds, if herd >=10 and if herd ≤20 animals / all animals, if herd >20 animals/20 animals per herd. (within herd prevalence of 20%)

Small ruminants

Preliminary survey: Sample (95:5) herds/(95:20) animals Large survey: sample (95:2) herds/(95:5) animals, but all animals in flocks <25 animals



Pigs Preliminary survey: Sample (95:5) herds/(95:20) animals Large survey: CS



Figures: G5 G6-EU approach G6-Israeli-Swiss

approach Bovine Large survey: CS 1,372

herds/85,007 animals

Small ruminants

Preliminary survey: 43 herds / 344 samples Large survey: 328 herds/2,509 animals

Pigs Preliminary survey: 43 herds / 946 samples Large survey: CS 677 herds/540,289 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/4100 animals

CS 132 herds + sample 31 herds/65 animals Sample 164 herds/4,100 animals

Resources: G5 G6-EU approach G6-Israeli-Swiss

approach Vet teams 2 vets for CS

Lab Preliminary survey: SP ELISA (Se 0.99, Sp 0.95) Analysis of 8,509 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)


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: o 107 pig farms with

reactors o 6 sheep farms with

reactors o Geographical cluster in

Waarschoot • 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

• VZ: 1,159 positive cattle • VZ: 404 positive pigs • VZ: 9 positive sheep • SZ: 140 positive cattle • SZ: 404 positive pigs • SZ: 21 positive small

ruminants

• VZ: 111 positive cattle • VZ: 404 positive pigs • VZ: 9 positive sheep • SZ: 140 positive cattle • SZ: 404 positive pigs • SZ: 21 positive small

ruminants


G5 G6-EU approach G6-Israeli-Swiss approach • Preliminary survey -

Analysis of test results: o 107 pig farms with reactors

(2 expected) o 6 sheep farms with reactors

(0 expected) o Some farms with

unexpectedly high numbers of reactors

• Actions: o Intensified CS in pigs in

entire VZ, but focus on Waarschoot and neighboring municipalities, prioritize large farms (>1,000)

o CS of cattle in buffer zone o Actions on all infected

(confirmed) farms (culling, tracing, ..)

o Perform large survey

• Analysis of test results: o VZ:

- cattle: 1,159 reactors – 1,159 allowable

- pigs: 404 reactors – 404 allowable

- small ruminants 9 reactors – 9 allowable o SZ:

- cattle: 140 reactors – 140 allowable


- small ruminants 21 reactors – 21 allowable

• Analysis of test results: o VZ:



- small ruminants 9 reactors – 9 allowable o SZ:



- small ruminants 21 reactors – 21 allowable


Field follow-up

G5 G6 • 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

• 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 Svanova-

test 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 G6 • Vaccination:

o The group proposes not to vaccinate in herds <50 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

• First approach (following EU guidelines) proposes 156,980 samples for NSP testing and 3,780 samples for virus detection

• Second approach (IL-CH suggestion) proposes 22,365 samples for NSP testing and 590 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 follow-up were found positive = freedom of disease is not demonstrated


Session Vi: Discussion of results and models for serosurveillance: Summary, Conclusions, Recommendations and Observations Summary Vaccination zone: Total no. of samples/herds: G1 G2 Bovine (vacc.) Small ruminants (non-vacc.) Pigs (vacc.)

119/1,989 10/88

308/8,990

119/1,989 10/88

308/12,377 G3 G4 Bovine (vacc.) Small ruminants (non-vacc.) Pigs (vacc.)

1,044 2/19 725

105/1,549 2/19

5/2,707 + check 4,270 animals at slaughter

G5 G6 (IL-CH) Bovine (vacc.) Small ruminants (non-vacc.) Pigs (non-vacc.)

2,188/144,872 391/2,920

CS

50+181/250+13,575 55+164/275+4,100

• 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 2003/85/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 2003/85/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 4,270 fattening pigs at slaughter

• Non-vaccinated sheep and goats: o Groups 1, 2, 3 and 4 agree on sampling following EU Directive 2003/85/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.


• G2 and G4 calculated the number of vet teams necessary to perform the surveys. Surveillance zone: Total no. of samples/herds: G1 G2 Bovine (non-vacc.) Sheep/goats (non-vacc.) Pigs (non-vacc.)

CS

88/925 CS

CS + sampling of animals showing clinical signs

2,534/299 CS + sampling of animals showing

clinical signs G3 G4 Bovine (non-vacc.) Sheep/goats (non-vacc.) Pigs (non-vacc.)

122/4,237 66/840 4/280

CS 22/951

CS G5 G6 Bovine (non-vacc.) Sheep/goats (non-vacc.) Pigs (non-vacc.)

CS 328/2,509

CS

CS + (31+164)/(65+4,100) 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 2003/85/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


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 2.2.10.7 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 (2003/85/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., 20071). 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 off-spring 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 3.8.7 of the Terrestrial Animal Health Code2.

5. The follow-up of herds with seroreactors by serological investigation has to be based on a

combination of NSP assays (Paton et al., 2006) 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 2007 May 16;79(2-4):71-97. Epub 2007 Jan 16. Review. 2 In 2003 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.


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., 20063). However, this is not considered as compatible with the OIE Code and the EU Directive 2003/85/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., submitted4). 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 2003/85/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 2003/85/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. 2006 Oct 30;24(42-43):6503-12. Epub 2006 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.


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 90% 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.

10. 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).


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 3.8.7. 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;

10. 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 2003/85/EC for the EU Member States5), 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


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 system6.

2. As the OIE code has changed in May 2007, 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 < 50 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).

10. 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 2007 May 16;79(2-4):71-97. Epub 2007 Jan 16. Review.


Acknowledgments

DG RESEARCH

DG SANCO

VAR Belgium Food Agency Belgium

EUFMD


Annex A see FMD-NSP workshop program


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


Characteristics of cluster 1

• Cluster 1 is comprised of the 10 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.


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 3.000 m radius around the outbreaks (the former protection zone). The size of the vaccination area is 57 km2.


PZ Census

81.11130888101.989119

24.09572.2843-00-001001 to

-00-00-00501 to 1000

3141.2574-00-00251 to 500

1442.59418-00-00101 to 250

722.02028-0091182251 to 100

351.1043229291329142926 to 50

181.1286416322176083511 to 25

74556372137238335 to 10

326996264247201 to 4

average number of pigs

total number of pigs

number of pig holdings



number of sheep/goat holdings



number of cattle holdings

size

cluster 1 - 0 to 3.000 m = vaccination zone

48.9202.3289258813.909883

1.5513.1022-00-001001 to

6083.0385-007117111501 to 1000

3718.15822-002762761251 to 500

15310.73570-001239828101 to 250

746.07182601813662.0573151 to 100

355.619161311244333.36410226 to 50

165.6343501644427174.27325811 to 25

73.6365288981371.6762305 to 10

32.9271.1082784125702521 to 4

average number of pigs

total number of pigs

number of pig holdings






number of cattle holdings

size

cluster 1 - 3.000 to 10.000 m = surveillance zone = non-vaccinated zone

SZ Census


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 (~2,000)– 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=70% 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


Protection zoneNumber of cattle to be tested (vaccinated) 1989 in 119 holdings Number of sheep and goats (non vaccinated) 88 in 10 holdings Number of pigs (vaccinated) 234 per holding in 3 large holdings

182 per holding in 4 medium holdings 7560 pigs in 301 little holdings (all pigs)

Surveillance zoneNumber of sheep and goats (non vaccinated) 925 in 88 holdings (all animals)

Sampling & testing numbers

Results of testing in PZ

0<21081GRLAVA4108103

01 (0)<21030KRAPJE1030257

02 (0)<1250VERŽEJ2537295

03 (1)060VERŽEJ60312

03 (1) 41820CVEN3143178

03 (2)41820IŽAKOVCI2521033

04 (3)<21190KRAPJE1190263

05 (4)41820CVEN2800171

020 (17)52340CVEN91150325

031 (25)52340LJUTOMER30270327

Pos_sheep

Pos_pigs

Expected

falsepositive

Numberof samples

Pos_cattle

MUNICIPALITYPIGSCATTLEID


Breakdown of Positive results for 2 large pig herds

Stable weak pos strong pos1 02 03 04 125 06 07 18 29 110 111 212 013 1

Stable weak pos strong pos1 52 23 64 75 56 5

3027 pig herd 9115 pig herd

Results of testing in SZ

2

<165

SATAHOVCI651317

3

<162

GOMILA PRI KOGU62379

14

<154

HOTIZA542364

VNT

Positive shee

p

Expectedfalse

positive

Number of

samples

MUNICIPALITYSHEEP/GOA

TSID


Seropositive findings

PZ• Pig herds with low level reactors• One pig herd with strong suspicion of

infection• One cattle reactorSZ• 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


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 (< 1000)

� Sample in large herds (3027; 9115; 60,142)

Within herd design prevalence 1%;

No. tests: 1150 (1135), 1200 (1172), 1200 (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


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

2,477

88

2,328

883

Visits

2,18563,75414,454331Total

9259258810Sheep

~98048,92012,377308Pigs

~28013,9091,989119Cattle

SamplesExam.SamplesVisits

Non-vaccinated zoneVaccination zone


Human resources

3.55222477Non-vacc. zone

3.828331Vacc. zone

Weeks *Visits /

Team*day

TeamsVisits

* Assuming 6 days working week

Vacc. Zone 22 teams á 1 person 22Non vacc. Zone 8 teams á 2 persons 16Supporting sampling 8Laboratory personell 12Total 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


Results

0300650nv1317

0010074v270

0013044v99

700302700v327

70041100v296

70031403v178

70028000v171

700252010v33

00840108v103

900911500v325

20+50006014200v48

PigsSheepCattlePigsSheepCattleID

No positivesNo animals

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


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


Follow up of herd with 60,000 pigs

● NSP-results

� 20 strong positives, all sows in one section of 600 sows

� 50 weak positives distributed all over

● Action in suspicious section

� Clinical examination of all sows

� Sample animals showing clinical signs for virology and SP-testing

� 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


Annex D

Group 3Cluster 2

Estonia and Slovakia


46.09851921.549105

46.09146.0911-00-001001 to

-00-00-00501 to 1000

-00-00-00251 to 500

-00-001041041101 to 250

-00-0069411651 to 100

-00-00375531526 to 50

-0016161172011211 to 25

-00-007166255 to 10

-743312114461 to 4

average number of

pigs

total number of

pigs

number of pig

holdings




holdings

average number of

cattle

total number of

cattle

number of cattle

holdingssize

cluster 2 - 0 to 3.000 m = vaccination zone


894631.3676613.6971.092

-00-00-001001 to

-00-005955951501 to 1000

-00-00-00251 to 500

159637415546631376855101 to 250

-00621853682.0993151 to 100

-00353189342.9078526 to 50

166341626917164.08625811 to 25

796147741172.3023305 to 10

298412552331.0233821 to 4

average number of

pigs

total number of

pigs

number of pig

holdings




average number of

cattle

total number of

cattle

number of cattle

holdingssize

cluster 2 - 3.000 to 10.000 m = surveillance zone = non-vaccinated zone

Description of outbreak

• One vaccinated zone– Divide into a 1km zone close to outbreak: 0- 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)


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.


Vacc zone outside the 1 Km

• CATTLE: In all the herds, test depending on the size of the herds; the basis is 10% of within herd prevalence:

• 6 herds with 51 to 100: test 44=(6x44)264• 8 herds with 26 to 50: test 37 (8x37)=256• For the rest of herds lower than 26: clinical

examination only.

Pigs vacc zone 0- 3 kmClinical 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.


Surveillance zone• Cattle: Clinical examination in all herds; herds less than 26 only

clinical examination• For the rest 10% within herd prevalence• With tests of 98% of sensitivity and Spe: 99.5%

• Herds of:• 26 to 50: 33 samples in 85 holdings: 2805• 51 to 100: 38 31 1178• 101 to 150: 42 5 210• 595 44 1 44. • 4 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 108 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): 130 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 130 pigs.

• Piglets: 5% within herd prevalence (2 reactors possible): 130 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.


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: 70 tested per herd• All negative. No further action


Pigs surveillance zone

59 small herds: only clinical examination• 4 holdings with average of 159 pigs: 70

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!!


Annex E

1.1.2007alatunnistetiedot

46 992681 3866815 2461 197

894631 3676613 6971 092surveillance zone

46 09851921 549105vaccination zone

totalnumber of pigs

numberof pigholdin

gs

total number of

sheep/goat

number of

sheep/goat

holdings

totalnumber of cattle

numberof

cattleholdin

gs

zone

Cluster 2 by Poland and Finland

• Two outbreaks; – mixed farm culled– large pig farm (n= 46 000 pigs) ->

animals at quarantine unit culled and all the restvaccinated


Vaccination zone

• All cattle and pigs vaccinated• Small ruminants non-vaccinated

• Survey design:

– All cattle sampled (all animals in all farms)-> 1 549 samples from 105 holdings

– All small ruminants sampled->19 animals 2 farms

– All backyard pigs sampled-> 7 pigs from 4 holdings



Vaccination zone

• Large pig farm:

– 9 stables with sows, boars and piglets->sampling to detect 5 % within-stable prevalencewith 95% confidence (appr. 300 samples/stable,altogether 2700 samples)– 36 stables with fattening pigs-> clinical

monitoring only (once per day by the farmer, once per week by the official vet)


Vaccination zone

• Justification for not sampling all the animals in the largepig herd:

1. A closed holding2. FMD is a clinical disease and even if not clear signs

in vaccinated population, transmission of virus effectively prevented-> no carrier status documentedin pigs

3. Fattening pigs going to be slaughtered-> possible to check/sample at slaughter

4. Requires a lot of resources

Altogether 4270 samples from the vaccination zone



Surveillance zone

• A clinical examination carried out in all farms in the surveillance zone

• In addition, those sheep farms with no ruminants(22 holdings),a survey to detect 5 % prevalencewith 95% confidence would be carried out -> in the mixed herds to rely on the clinical signs in cattle orpigs

• A total of 951 samples


Surveillance

• Sampling carried out in 134 farms ( 5220 samples)

• Altogether 1200 farms to be monitored

• Farms were sampling would be carried out wouldbe examined first



Results in the vaccination zone

• Five farms with seropositive animals– One backyard farm with both two pigs

seropositive-> culling (located in the samemunicipality as the outbreak nb 4)

– One mixed farm with 37 cattle (negative) and 1+ve pig out of two -> false positive pig

– One mixed farm with 5 cattle (negative) and 1 seropositive pig out of two-> false positive pig

– One mixed farm with one seropositive pig and 16 negative sheep-> false positive pig (highspecifity of the test in non-vacc. Sheep)


Results in the vaccination zone

• 27 reactors in 5 different stables in the large pigfarm (3-10/stable)

-> retesting of positive stables: positive animals+ 50 % of penmates and pigs in adjacent pens

-> 23 reactors in the retesting of the positivesows + 12 reactors out of 2107 samples in the group of penmates and adjacent pens



Options for the decision-making in the large pig farm

• Culling and desinfection of the infected premises(46 000 pigs) + repopulation with vaccinatedanimals –unrealistic

• Culling of the 5 positive stables (3988 sows + piglets) and desinfection + repopulation withvaccinated animals –usefullness?

• Vaccination of the piglets from positive stablesbefore transport to empty stables for fattening

• Revaccination of all the sows (+ some fatteningpigs-youngest stables)

• Do nothing


Vaccination zone

• In cattle, 1-3 reactors were detected in five smallfarms having 16-104 bovines -> probang-sampledand PCR-tested to rule out the carrier status



Results in the surveillance zone

• Monitoring for clinical signs negative in cattle and pig farms and mixed farms

• Only sheep/goat farms sampled

• One farm of 232 sheep with 3 reactors -> allreactors tested by virus neutralisation-> 1 positivein virus neutralisation (1:45) = singleton reactor


Resources for the design and implementation of the survey

• Central Vet. Adm. : 2 persons for the planning etc.• Province: 2 prov. vet officers for coordination and compilation of the field data

• Field staff: 160 veterinary working days– 20 vet.days for sampling the cattle herds + 13

vet.days for sampling the sows (2700 in 9 stables) in the large pig farm + 18 vet.days for cl. monitoring of the 36 stables with 19 000 fattening pigs

– 100 vet.days for cl. monitoring of cattle and pigherds in the surv. zone + 11 vet. Days for sampling the sheep farms

• Laboratory personell:


Annex F

Scenario 3

• Densely populated area• 3500 cattle holdings, 2000 sheep holdings,

1700 pig holdings• Mainly small farms• Vaccination to control outbreak, all cattle

vaccinated but no other animals

Cluster 3

0,0 2,5 5,0 7,5 10,0 12,5

Kilometers


Holdings

0,0 2,5 5,0 7,5 10,0 12,5

Kilometers

1 month after outbreak

• Preliminary survey to estimate size of remaining problem (check clinicalsurveillance)

• Later, when more confident that outbreakis controlled, large survey to substantiatefreedom from infection


Preliminary survey

• Testing of non-vaccinated animals (pigs & sheep) with SP test (Se 0.99, Sp 0.95)

• Sample to detect 5% herd prevalence, 20% within-herd prevalence

• 110 pig holdings, 110 sheep holdings (22 pigs, 8 sheep)

Results (serology)

• 107 pig farms with reactors (2 expected)

• 6 sheep farms with reactors (0 expected)• Geographical cluster in Waarschoot

• Some farms with unexpectedly high numbersof reactors


Actions

• Intensified clinical surveillance in pigs in entire vacc zone, but focus on Waarschoot and neighbouring municipalities, prioritise large farms (>1000)

• Clinical surveillance of cattle in buffer zone, large farms first (>250 cows)

• Paired serum samples• Surveillance in the buffer zone• Actions on all infected (confirmed) farms (culling,

tracing etc)

Large survey

• Pigs –clinical surveillance only • Sheep SP test to detect 2% infected herds and within-

herd prevalence 5% • Vaccination zone :Random selection of herds (plus 1

large with 1200 animals), all animals in smaller flocks (<25 animals), random samples in larger = 391 flocks, 2920 animals

• Buffer zone: Random selection of herds, all animals in smaller flocks (<25 animals), random samples in larger = 328 flocks, 2509 animals

• Cattle – NSP test in vaccinated, all animals in all herds. Non-vaccinated clinical surveillance only


Results (serology)

• 49 positive sheep holdings in vacc zone (1-2/herd)

• 23 positive sheep holdings in surv zone (1-3/herd)

• 259 positive cattle holdings (1-15/herd)• Herds with >3 pos in 3 municipalities

(Sleidinge, Waarschoot, Lovendegem)

Actions

• Re-test positive sheep samples, send vets to flocks where still positives, sample all animals Prioritise surveillance zone

• Positive cattle herds: Cull all NSP positive cattle, slaughter rest of herd in 3 municipalities, other municipalities only cull NSP pos animals

• Also sample all sheep herds in 3 municipalities


Resources

• Clinical surveillance of 36 cattle holdings (1 visitevery 2nd week, 2 vets)

• Clinical surveillance of 349 pig holdings in VZ (1 visit every 2nd week, 20 vets)

• Culled animals (outbreak) 172 sheep, 9096 pigs, 1842 cattle

• Culled animals (surveillance) 348 cattle• Slaughtered 5229 cattle 7655 pigs 86 sheep• Lab must analyse 8509 SP tests, 144,000 NSP

tests

Hindsight

• Vaccination�No vaccination in herds<50 animals. �Vaccinate pigs on vaccinated cattle holdings• Testing �Clinical surveillance only in non-vaccinated

cattle herds�Random sampling of vaccinated cattle

herds(126 herds), test all animals in each herd Non-vaccinated pigs and sheep same as exercise


Freedom?

• Cannot be substantiated yet• Delenda Cartago…


Annex G

Workshop on the design and interpretation of post FMD

vaccination serosurveillance by NSP tests

Group 6, Cluster 3Israel, Switzerland

Cluster 3

0,0 2,5 5,0 7,5 10,0 12,5

Kilometers


Cluster 3

0,0 2,5 5,0 7,5 10,0 12,5

Kilometers

Cluster 3

10 km

5 km


Two Approaches

1. Approach ± according EU Regulation (Council Directive 2003/85/EC)

2. Israeli-Swiss Approach

1. EU Approach

1.A. Vaccination Zone1.A.1. Clinical Inspection

– All susceptible animals in all holdings1.A.2. Serological Investigations

– Cattle (all vaccinated): full investigation– Pigs, Goats, Sheep: survey (not compulsory)

1.B. Surveillance (non-vaccination) Zone1.B.1. Clinical Inspection

– All susceptible animals in all holdings1.B.2. Serological Investigations

– Cattle, Pigs, Goats, Sheep: survey (not compulsory)


1.A.2. Serological Investigations in Vaccination Zone EU

• Test: Cedi Test with retesting (Se 98%, Sp 99%)

• Pig, sheep, goat herds (total 2440 holdings)– Two stage approach:

– Hse 90%, Hsp99%– 5% prevalence, 95% confidence level

– =164 herds (4 reactors)– focus on sheep and goats herds

1.A.2. Serological Investigations in Vaccination Zone EU

• sheep/goat herds at least 10 animals herd size(within herd prevalence of 10%)herds <= 30 animals: all animals testedherds > 30 animals: 30 animal tested

• pig herds at least 10 animals herd size(within herd prevalence of 20%)herds <= 20 animals: all animals testedherds > 20 animals: 20 animal tested


1.A. Investigations Vaccination Zone EU

Number of samples :• 2315 samples from clinical investigations (463 holdings

with 5 samples each)

• 4100 samples from serological survey from sheep/goat/pig

• 145000 samples (around 750 reactors) from serological investigation cattle

• Total: 151415 samples for NSP testing (all samples) plus 2315 samples virus detection (clinical survey)

1.B.2. Serological Investigations in Non-Vaccination Zone EU


• Cattle, pig, sheep, goat herds (total 2934 holdings)

– Two stage approach:


– =164 herds (4 reactors)


1.B.2. Serological Investigations in Non-Vaccination Zone EU


• Cattle + pig herds at least 10 animals herd size(within herd prevalence of 20%)herds <= 20 animals: all animals testedherds > 20 animals: 20 animal tested

1.B. Investigations NON-Vaccination Zone EU

Number of samples :

• 1465 samples from clinical investigations (293 holdings with 5 samples each)

• 4100 samples from serological survey from cattle/sheep/goat/pig

• Total: 5565 samples for NSP testing (all samples) plus 1465 samples virus detection(clinical survey)


2. Israeli Swiss approach

2.A. Vaccination Zone2.A.1. Clinical Inspection

– Full investigation pig holdings (1075)– Cattle: clinical survey– Sheep: no clinical inspection (no clin. Signs)

2.A.2. Serological Investigations– Cattle: survey– Pigs, Goats, Sheep: survey

2.B. Surveillance (non-vaccination) Zone2.B.1. Clinical Inspection

– Survey in cattle, pigs, sheep, goats2.B.2. Serological Investigations

– Survey in cattle, pigs, sheep, goats

2.A.1. Clinical Investigations in Vaccination Zone IL-CH

– Pigs : full investigations in 1075 holdingsthere will be sampling for lab from 5% of inspected holdings (around 55)

– Cattle (vaccinated) – Survey :Se 95%, Sp 99% (including laboratory test)prevalence 2%, confidence level 95%523 herdsthere will be sampling from 10% of inspected holdings (around 50)

– Sheep and goat: no clinical inspection(except together with serological survey)


2.A.2. Serological Investigations in Vaccination Zone IL-CH


• Pig, sheep, goat herds (total 2440 holdings)

– Two stage approach:– Hse 90%, Hsp99%– 5% prevalence, 95% confidence level– =164 herds (4 reactors)– focus on sheep and goats herds



• pig herds at least 10 animals herd size(within herd prevalence of 20%)herds <= 20 animals: all animals testedherds > 20 animals: 20 animal tested



• Cattle herds (total 2188 holdings): survey- Two stage approach- Hse 80%, Hsp 99%- 181 herds- Test Se 68%, Sp 99.5%, within herd prevalence:

5%- within cattle herds at least 60 animals herd size- herds <= 75 animals: all animals testedherds > 75 animals: 75 animal tested

2.A. Investigations Vaccination Zone IL-CH

Number of samples :• 275 samples from clinical investigations pig (55 holdings

with 5 samples each)• 250 samples from clinical investigations cattle (50

holdings with 5 samples each)

• 4100 samples from serological survey from sheep/goat/pig

• 13575 samples from serological survey cattle

• Total: 18200 samples for NSP testing (all samples) plus 525 samples virus detection (clinical survey)


2.B.1. Clinical Investigations in Non-Vaccination Zone IL-CH

– Cattle, sheep,gaot,pig (2934 holdings) –survey :Se 95%, Sp 99% (including laboratory testing of clinical suspect cases)

– Prevalence 5%, 95% confidence level– 132 holdings

2.B.2. Serological Investigations in Non-Vaccination Zone IL-CH


• Cattle, pig, sheep, goat herds (total 2934 holdings)

– Two stage approach:


– =164 herds (4 reactors)


2.B.2. Serological Investigations in Non-Vaccination Zone IL-CH


• Cattle + pig herds at least 10 animals herd size(within herd prevalence of 20%)herds <= 20 animals: all animals testedherds > 20 animals: 20 animal tested

2.B. Investigations Non-Vaccination Zone IL-CH

Number of samples :

• 65 samples from clinical investigations (3 holdings with 5 samples each)

• 4100 samples from serological survey from cattle/sheep/goat/pig

• Total: 4165 samples for NSP testing (all samples) plus 65 samples virus detection(clinical survey)


Total Number of Samples both approaches

First approach (following ± 2003/85/EC)• 156,980 Samples NSP

• 3’780 Virus detection

Second approach (IL-CH Suggestion)• 22,365 Samples NSP

• 590 Virus detection

Open Questions

• Confindence of Combined surveys (clinical + serological) needed to be calculated

• Would the second approach with considerably less testing be accepted by neighbouringcountries?


Results from serological survey I

Survey according EU-scheme:• Vaccination zone

– Cattle 1159 pos. (allowable pos. 1159)– Pigs 404 pos. (allowable pos. 404)– Sheep/goat 9 pos. (allowable pos. 9)

• Surveillance zone– Cattle 140 (allowable pos. 140)– Pigs 275 (allowable pos. 404)– Sheep/goat 21 (allowable pos. 21)

Results from serological survey II

Survey according „option of woking group“:• Vaccination zone

– Cattle 111 pos. (allowable pos. 111)– Pigs 404 pos. (allowable pos. 404)– Sheep/goat 9 pos. (allowable pos. 9)

• Surveillance zone– Cattle 140 (allowable pos. 140)– Pigs 275 (allowable pos. 404)– Sheep/goat 21 (allowable pos. 21)


Follow up of positive animals I

Vaccinated animals (cattle):1. Resample all positive animals2. Epidemiological investigation of herds with positive

results3. Testing the resamples:

a) Cedi-test (if test negative = negative)

b) If Cedi-test positive retesting with Svanova-test4. If Svanova positive:

- Elimination of positive animals- Testing of whole herd

Follow up of positive animals II

Non-vaccinated animals:1. Resample of all positive animals2. Epidemiological investigation of herds with positive results3. Testing the resamples:

– Cattle with Svanova-test– Pig, sheep, goat with Cedi-test

4. If animals still positive:– Retest with solid phase competitive ELISA (SPCE)

5. If SPCE positive:- Elimination of positive animals- Testing of whole herd


Conclusions

After follow up of positive animals (epidemiologicalinvestigation + serological survey):

• All animals could be described as false positive

= freedom of disease is demonstrated

• Some animals in the follow up were foundpositive= freedom of disease is not demontrated


Annex H Post NSP Workshop thoughts, after the first FMD-NSP workshop in January 2007. The approach to post-vaccination serosurveillance set out in the EU Directive assumes that serological tests for virus infected animals will have an insufficient sensitivity to certify individual animals as infection-free, but that they could be used for herd-based certification. Accordingly, it is required that all vaccinated animals in all vaccinated herds or flocks should be sampled and tested serologically and that if infection cannot be ruled out, then the entire herds must be slaughtered and animals either disposed of into the food chain (in cases where it is considered that the probability of genuine infection is extremely low) or by destruction (in other cases). Available tests can be used to support this strategy (Paton et al., 2006) subject to certain limitations, namely that:

1. The need to slaughter entire herds where seroreactors are found makes it essential that the test system used has a very high specificity which in turn reduces the sensitivity.

2. The strategy will still lead to slaughter of some seroreactor herds of uncertain infection status. 3. The low test system sensitivity means that a low prevalence of infection cannot be reliably

detected and that small herds cannot be certified as infection free, even if they have a moderate (5%) prevalence of infection.

Another problem is that the strategy espoused by the EU Directive does not distinguish between the two different aims of detecting (1) virus circulation and (2) virus carriers, a concept that has recently been introduced into the OIE Code. Demonstrating absence of virus circulation can be substantiated by paired serology and arguably, is barely necessary if there is a properly conducted campaign of vaccination and clinical surveillance. In contrast, the feasibility of demonstrating the presence of carriers is entirely dependent on their prevalence within the population and poses the main challenge for post-vaccination serosurveillance in countries wishing to substantiate freedom from infection without vaccination, after use of emergency vaccination. There is a lack of certainty about the prevalence of infection (circulating virus or carriers) that is likely to be found after use of emergency vaccination within and amongst vaccinated herds. However, limited experience of outbreaks where emergency vaccination has been used (Balkans and The Netherlands) as well as preliminary findings from modelling studies suggest that the number of infected herds or flocks will be very few and within them the prevalence of infection will also be low – perhaps one or two animals only. This is because either immunity blocks transmission or else, where immunity is insufficient such that extensive infection can occur, then clinical signs are likely to be seen allowing the herd to be diagnosed as an infected holding without serology. In summary, 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. For example, even assuming a prevalence as high as 5% for carriers within herds, the approach outlined by Paton et al (2006) is only designed to detect carrier containing herds with 95% confidence, which means that 5% of herds containing carriers could expect to be missed.


If the prevalence of carriers is so low amongst and between herds, that herd based testing with a relatively insensitive test cannot succeed, than an alternative testing strategy may be one based on individual animal certification. This could be combined with herd-based testing to detect circulating virus, under the assumption that evidence of virus circulation would lead to herd slaughter, but that evidence of carriers (particularly if at a low number within a herd) would lead to slaughter of these reactor animals, but not of the whole herd. This has a number of advantages:

(d) Test specificity can be lowered since the consequence of false positive results is now individual animal rather than whole herd slaughter.

(e) This in turn leads to an increased test system sensitivity (f) There is no longer a “small herd” problem nor a problem of inadequate specificity in very

large herds. For example, in cattle, a test system based on Ceditesting with repeat Ceditesting of seroreactors gives a test system sensitivity of 82% and specificity of 99.2%. If all vaccinated animals in all vaccinated herds were tested, then there is now an 82% probability of detecting all carriers, regardless of prevalence. At a specificity of 99.2%, 80 false positive seroreactors would be expected and have to be wrongly slaughtered for every 10,000 cattle tested. However, the advantage of the system is that the “unnecessary” slaughtering is shared amongst the herds so that each only suffers a little! To investigate this option further:

1. More work is needed to refine models predicting the expected prevalence of infection within and amongst vaccinated herds.

2. The relative confidence attainable with “herd-based” and “individual” certification needs to be explored for different herd sizes and prevalences.

3. The serosurveillance needed to substantiate freedom from virus circulation should be considered. 4. Consideration should be given to finding out if there would be a consensus on not slaughtering

individual carrier animals within vaccinated herds where there was no evidence of virus circulation.

David Paton 6th Feb 2007

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