Advies 12-2016 van het Wetenschappelijk Comité van het FAVV · Annex I of opinion 12‐2016...

Annex I of opinion 12‐2016

Subject:

Evaluation of the Belgian bovine tuberculosis control program dossier SciCom 2015/11

Opinion approved by the Scientific Committee on 17 June 2016

Key terms: Mycobacterium bovis – bovine tuberculosis – surveillance – diagnostic tests – epidemiology

Sleutelwoorden: Mycobacterium bovis – boviene tuberculose – bewaking – diagnostische testen – epidemiologie

Mots clés: Mycobacterium bovis – tuberculose bovine – surveillance – examens diagnostiques – épidémiologie

SCIENTIFIC COMMITTEEof the Belgian federal Agency for

the Safety of the Food Chain

Annex I of Opinion 12‐2016 bovine tuberculosis

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Contents Executive summary ................................................................................................................................. 7 1 Definitions & Abbreviations .......................................................................................................... 10 2 Terms of reference ........................................................................................................................ 11 2.1 Question ...................................................................................................................................... 11 2.2 Methodology / Rationale ............................................................................................................ 11

3 Introduction ................................................................................................................................... 11 3.1 Bovine tuberculosis ..................................................................................................................... 11 3.2 Epidemiologic context of bTB in Belgium and the EU ................................................................. 13 3.2.1 Overview of bTB in EU ..................................................................................................... 13 3.2.2 History of bTB outbreaks in Belgium ............................................................................... 14

3.3 Legal framework ......................................................................................................................... 14 3.3.1 EU Legal framework ......................................................................................................... 15 3.3.2 National legal framework ................................................................................................ 16

3.4 Current measures concerning monitoring and surveillance ....................................................... 16 4 Evaluation of the current diagnostic techniques for the detection of bTB ................................... 17 4.1 Intradermal skin test ‐ Delayed hypersensitivity test ................................................................. 17 4.1.1 Principle ........................................................................................................................... 17 4.1.2 Injection site .................................................................................................................... 17 4.1.3 Tuberculin ........................................................................................................................ 18 4.1.4 Sensitivity and specificity and influencing factors ........................................................... 18 4.1.5 Non‐bovine animals ......................................................................................................... 20

4.2 Identification of the agent .......................................................................................................... 20 4.2.1 Microscopic examination ................................................................................................ 20 4.2.2 Bacterial culture............................................................................................................... 21 4.2.3 Molecular methods ......................................................................................................... 21

4.3 Interferon gamma test ................................................................................................................ 22 4.3.1 Principle of the test ......................................................................................................... 22 4.3.2 IFN‐γ test based on PPDs ................................................................................................. 23 4.3.3 IFN‐γ test based on specific antigens .............................................................................. 24 4.3.4 Use of IFN‐γ test as serial or parallel confirmation test .................................................. 25 4.3.5 Influence of skin test on serial use of IFN‐γ ..................................................................... 26 4.3.6 Non‐bovine animals ......................................................................................................... 27 4.3.7 Conclusion ....................................................................................................................... 28

4.4 Serological tests .......................................................................................................................... 29 4.4.1 Introduction ..................................................................................................................... 29 4.4.2 ELISA Ab test IDEXX (MPB70 and MPB83) ....................................................................... 29 4.4.3 EnferplexTM TB assay (chemiluminescent assay): .......................................................... 32 4.4.4 Non‐bovine animals ......................................................................................................... 34 4.4.5 Applicability of an ELISA in the Belgian cattle population ............................................... 34 4.4.6 Conclusions ...................................................................................................................... 35

4.5 Combination of diagnostic tests ................................................................................................. 36 4.6 General conclusion ...................................................................................................................... 37

5 Evaluation of the current Belgian bTB surveillance program ........................................................ 38 5.1 Facts and figures ......................................................................................................................... 38 5.2 Scenario tree analysis ................................................................................................................. 39 5.3 Expected true and false positive reactions ................................................................................. 39 5.4 Estimation of the direct and indirect costs ................................................................................. 41 5.5 Conclusion ................................................................................................................................... 42

6 Risk factors of infection and spread to be considered in bTB surveillance ................................... 42


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7 Recommendations ........................................................................................................................ 44 7.1 Data collection and data warehouse .......................................................................................... 44 7.2 Risk based surveillance and proportionate measures ................................................................ 44 7.3 Raising awareness of actors in the field ...................................................................................... 45 7.4 Surveillance components ............................................................................................................ 45 7.4.1 Purchase surveillance ...................................................................................................... 45 7.4.2 Winter screening ............................................................................................................. 45 7.4.3 Surveillance in suspected / outbreak herds .................................................................... 46 7.4.4 Tracing analysis of outbreaks .......................................................................................... 46 7.4.5 Slaughterhouse surveillance ............................................................................................ 46

7.5 Diagnostic tests ........................................................................................................................... 46 7.6 Molecular typing of bTB isolates ................................................................................................. 46 7.7 Domestic non‐bovine animals ..................................................................................................... 47 7.8 Wildlife ........................................................................................................................................ 47 7.9 Biosecurity measures .................................................................................................................. 47

8 Conclusions .................................................................................................................................... 47 9 Answer to specific questions ......................................................................................................... 48 9.1 Is it, under the current field conditions with growing numbers of cattle on farms which are often not (well) fixated, still feasible to perform a ‘secundem artem’ intradermal skin test? Is there modern equipment to allow a sufficient fixation of cattle in order to correctly perform and read an intradermal skin test?........................................................................................................................ 48 9.2 Can the proposed decision tree (including the use of tuberculination and gamma‐interferon test) be validated? ............................................................................................................................. 49 9.3 Evaluation of proposals to modify the current royal decree of 17 October 2002 regarding the control of bovine tuberculosis........................................................................................................... 49 9.3.1 To introduce the control against Mycobacterium spp. other than M. bovis which is currently the only Mycobacterium species mentioned in the royal decree: M. caprae, M. tuberculosis,… ............................................................................................................................... 49 9.3.2 To install a mandatory notification of M. bovis and M. tuberculosis for animal species other than cattle. Are measures necessary if tuberculosis is diagnosed in other animal species (dogs, cats, sheep, goats, exotic animals, wild animals, zoo animals, other domestic animals, …), also if cattle are held on the same farm? ...................................................................................... 49 9.3.3 To merge the definitions ‘suspected of being affected’ and ‘suspected of being contaminated’ ............................................................................................................................... 50 9.3.4 Adjustment and clarification of the minimum age for intradermal skin test at purchase and at complete herd testing ........................................................................................................ 50

9.4 Evaluation of the Tuberculosis Action plan ................................................................................. 51 References ............................................................................................................................................. 52 Members of the Scientific Committee .................................................................................................. 64 Conflict of interest ................................................................................................................................. 64 Acknowledgements ............................................................................................................................... 64 Composition of the working group ....................................................................................................... 64 Legal framework .................................................................................................................................... 65 Disclaimer .............................................................................................................................................. 65 Appendix I: Specific questions in the request of opinion ...................................................................... 66 Appendix II: Procedure for the intradermal skin test according to the OIE (2009) .............................. 68 Appendix III: Compliance of Belgian field veterinarians to the testing procedures as recommended by USDA (2015) .......................................................................................................................................... 70 Appendix IV: Advantages and disadvantages of equipment which is used by Belgian field veterinarians for the injection of tuberculin during the intradermal skin test ..................................... 72 Appendix V: On which species, other than cattle, can the SIT be applied? .......................................... 73 Appendix VI: Diagnostics strategies for bTB in other countries ............................................................ 85


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Appendix VII: The use of the IFN‐y test in non‐bovine animals ............................................................ 89 Appendix VIII: Serological diagnostic tests to be used in non‐bovine animals ..................................... 99 Appendix IX: Occurrence of M. bovis in domestic and wild animals worldwide – literature overview. ............................................................................................................................................................. 104

Tables Table 1. Parameters affecting the intradermal skin test ................................................................... 19 Table 2. value of sensitivity and specificity of diagnostic test (Courcoul et al., 2014) ...................... 22 Table 3. Summary of meta‐analysis results for sensitivity and specificity of diagnostic tests for bovine TB on cattle from AHVLA systematic review (EFSA, 2012(a)) ................................................... 24 Table 4. Sensitivity and specificity of the IFN‐y assay (BOVIGAM) in different studies in cattle using different recombinant proteins or peptides or a combination of them (Bezos et al., 2014) ............... 25 Table 5. Studies between 2008‐2012 describing the effect of CFT on IFN‐y test (Schiller et al., 2010b) ............................................................................................................................................. 26 Table 6. Studies between 1994‐2010 describing the effect of CCT on IFN‐g (Schiller et al., 2010b) 27 Table 7. Sensitivity of IDEXX M. bovis antibody ELISA with sera collected from naturally infected cattle (Waters et al., 2011). ................................................................................................................... 30 Table 8. Specificity of IDEXX M. bovis antibody ELISA with sera collected from non‐infected cattle from various geographic regions (Waters et al., 2011).. ....................................................................... 30 Table 9. Summarized results of the study of Buddle et al. (2013) .................................................... 30 Table 10. Number of animals detected as positive within herds A and B using the different diagnostic techniques (Casal et al., 2014). ............................................................................................ 31 Table 11. Cohorts of samples tested by Enfer multiplex immunoassay and individual responses to ESAT‐6, CFP‐10, and MPB83 antigens. .................................................................................................. 33 Table 12. Performance of the Enferplex TB multiplex immunoassay on field animals. .................. 33 Table 13. Number of animals detected as positive in herds A and B using the different diagnostic techniques (Casal et al., 2014). ............................................................................................................. 34 Table 14. Added value in terms of sensitivity and specificity for single and a combination of different diagnostic tests under conditional independent events ........................................................ 37 Table 15. Odds of detection of different surveillance components in Belgium using historical data ......................................................................................................................................... 39 Table 16. Input parameters for the benchmarking study according to Welby et al. (2015)........... 39 Table 17. Expected true, false and total positive reactors for tested beef categories in the different surveillance components (purchase (PUR) and slaughterhouse (SLGH)) – adapted from Welby et al. (2015) ................................................................................................................................ 40 Table 18. Summary of direct and indirect cost obtained from available data in Belgium .............. 41 Table 19. Major risk factors described in scientific literature (adapted from Humblet et al., 2009) . ......................................................................................................................................... 42 Table 20. Mycobacterium spp. identified as responsible for bTB ................................................... 49 Table 21. Sensitivity and specificity of diagnostic assays based on CMI and antibody production (ELISA) in some studies performed in goats (from (Bezos et al., 2012)).. ............................................. 74 Table 22. Sensitivity and specificity of diagnostic assays based on CMI and antibody production (ELISA) in some studies performed in goats (from (Bezos et al., 2012)).. ............................................. 74 Table 23. Summary of the number of positive reactors (R+), sensitivity (Se, Wilson CI 95%) detected in the 7 dairy caprine flocks subjected to depopulation using single and comparative intradermal tuberculin test (SIT and SCIT tests respectively (Bezos et al., 2014b). .............................. 75 Table 24. Sensitivity and specificity of skin test on camelid (Alvarez et al., 2012). ........................ 78 Table 25. Summary of CMI assays employed in different non‐bovid wildlife since 2009 (Chambers, 2009, 2013). ......................................................................................................................................... 80 Table 26. Summary of Key Features of Bovine Tuberculosis in Wildlife Reservoirs (Fitzgerald and Kaneene, 2013). ..................................................................................................................................... 82


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Table 27. Examples of free living wildlife or captive wildlife reported with M. bovis (Cousins and Florisson, 2005). .................................................................................................................................... 83 Table 28. Brief overview of bTB status and diagnostic strategies in the USA, in various European countries and in New Zealand (Schiller et al., 2010a). .......................................................................... 87 Table 29. Summary of the application of IFN‐g test internationally (Strain et al., 2012). .............. 88 Table 30. Summary of different Se and Sp estimates for the use of the IFN‐y test in goats .......... 89 Table 31. Results of diagnostic test for TB in suspicious sheep cohabiting with TB‐infected cattle and/or goats (Munoz‐Mendoza et al., 2015) ........................................................................................ 90 Table 32. Evaluation of diagnostic techniques in TB‐infected sheep. Concordance (Cohen’s Kappa), sensitivity and specificity of techniques performed using both culturen and histopthology as ‘gold standards’ (Munoz‐Mendoza et al., 2015) ................................................................................... 90 Table 33. Criteria for interpretation of the IFN‐y assay in pigs (Pesciarolli et al., 2012) ................ 91 Table 34. Comparison between the results of the IFN‐y assay and the results of post mortem inspection (lesions) and bacterial culture (microbiology) (Pesciaroli et al., 2012) ............................... 91 Table 35. title ................................................................................................................................... 91 Table 36. title ................................................................................................................................... 92 Table 37. IFN‐γ ELISA results for a total of 17 cats (numbered sequentially) submitted to the TB Diagnostic section of the VLA plus five healthy control cats (Rhodes et al., 2008) .............................. 93 Table 38. IFN‐γ response rates (%) in study groups using different test interpretation criteria. Percentage of cats in each group that were IFN‐γ test‐positive to mycobacterial antigens (Rhodes et al., 2011). 94 Table 39. title ................................................................................................................................... 95 Table 40. Sensitivity and Specificity of the skin test in Lions (Keet et al., 2010) ............................. 96 Table 41. Mean OD450 values of whole blood samples of 11 lions from BTB‐free areas (Maas et al., 2012) 96 Table 42. Se and Sp of serological test performed in camelids with known infectious status (Alvarez et al., 2012). ........................................................................................................................... 100 Table 43. Reported Se and Sp estimates for currently available serological test for the diagnosis of TB in non‐bovines species (Broughan et al., 2013). ............................................................................ 101 Table 44. Summary of serological tests employed in different non‐bovid wildlife published since 2009. 101 Table 45. Commercial availability of tests referred to by Chambers, 2013 .................................. 102

Figures Figure 1. Situation of Bovine tuberculosis in Europe. .................................................................... 13 Figure 2. Number of bTB outbreaks during the last 15 years in Belgium. ...................................... 14 Figure 3. Principle of the IFN‐y test (https://www.thermofisher.com/content/dam/LifeTech/global/applied‐sciences/pdfs/animal‐health/prionics_literature_tb_link4.pdf). ............................................................................................. 22 Figure 4. Usefulness of different diagnostic test in relation with the progression of bTB infection (Vordermeier et al., 2004). .................................................................................................................... 29 Figure 5. Antibody responses of the two non‐infected bulls (ID numbers 1964 and 1954) and the four M. bovis infected animals (ID numbers 3174, 2148, 2149 and 1977) using IDEXX Mycobacterium bovis antibody kit (cut‐off value >30% indicated by red line). SIT was perfomed at 115 days post‐infection. ......................................................................................................................................... 32 Figure 6. Principle of EnferplexTM TB assay in 96 well plates. ...................................................... 32 Figure 7. Experimental serial testing scheme based on the combination of SICT and gamma‐interferon (IFN‐γ) tests according to Praud et al. (2015). ..................................................................... 86 Figure 8. Genetic sequence of lion and cheetay IFN‐y (Maas et al., 2010) .................................... 96 Figure 9. Results of the IFN‐γ responses for the 51 rhinoceroses (1 excluded), expressed as OD490 nm or as ng/ml (Morar et al., 2013). ......................................................................................... 97


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Figure 10. title ................................................................................................................................... 98 Figure 11. Rapid test called also Stat‐Pak assay by Chembio Diagnostics Systems, Inc ................... 99 Figure 12. DPP (Dual‐path platform) system. ................................................................................... 99


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Executive summary

Background & Terms of reference Bovine tuberculosis (bTB) is an infectious zoonotic disease caused by Mycobacterium bovis that may affect cattle, other domesticated animals and wildlife. People get mostly exposed via the consumption of raw milk and raw milk products or via animal contact (aerosol). Bovine tuberculosis is an officially notifiable disease. Despite the fact that Belgium is officially free of bTB since 2003, its surveillance still remains important because almost every year one or more outbreaks are detected. From an economical perspective, it is very important for Belgium to maintain its officially tuberculosis free status in order to facilitate intracommunity trade. The epidemiological evolution in a number of neighboring countries has demonstrated that, when awareness for bTB in domesticated animals decreases, bTB can reemerge.This decreased awareness in domesticated animals can lead to a spill‐over of bTB to wild animals making the control of bTB even more difficult. Recently, stakeholders have expressed a number of problems and constraints about their role within the current bTB surveillance program. Given these circumstances, the Scientific Committee was asked to perform a thorough evaluation of the current bTB surveillance and control program in Belgium. Next to a general evaluation, a great number of specific questions were asked. Methodology In this opinion, a thorough evaluation of the bTB surveillance in Belgium has been executed. This evaluation is based on simulation exercises (scenario tree analysis to evaluate the sensitivity of each surveillance component), a benchmarking study comparing the expected number of false positive reactors with the actual notified reactors and an estimation of the direct and indirect costs of surveillance. Furthermore, this opinion contains a review of current diagnostic techniques for bTB and risk factors to be considered in bTB surveillance. This review is based on the available knowledge in scientific literature and on expert opinion. Finally, a number of recommendations regarding bTB surveillance are proposed. Their relevance is evaluated by experts. Literature review: diagnostic techniques and risk factors The Scientific Committee has performed a comprehensive literature review to characterize and evaluate different diagnostic techniques for bTB which can be used in bTB surveillance in Belgium. The intradermal skin test or delayed hypersensitivity test involves the intradermal injection of bovine tuberculin purified protein derivative (PPD) and the subsequent detection of swelling (delayed hypersensitivity) at the site of injection 72 hours later. This may be performed using bovine tuberculin alone (single intradermal test, SIT) or as a comparative test using avian and bovine tuberculins (single intradermal comparative test, SICT) to differentiate between an infection with M. avium and M. bovis respectively. The test should be performed in the anterior neck area to render its sensitivity as high as possible. In literature, a wide range of sensitivity (Se) and specificity (Sp) values are reported: Se between 53% (27.3‐81.5, 95% CI) and 69.4% (40.1‐92.2, 95% CI); Sp between 55.1% and more than 99% showing a median value over 95%. Indeed, a lot of technical and socio‐economical parameters can affect the results of the intradermal skin test. The isolation of M. bovis by bacterial culture is the gold standard method for the diagnosis of bTB. According to the Belgian legislation, the solely isolation of the bacteria remains the definitive proof for the confirmation of an outbreak. However, bacterial culture is time consuming and may last from 8 to 12 weeks. Methods which allow a faster result (in 2‐3 weeks) exist, but are not validated in veterinary medicine. A single recommended protocol for bacterial culture of Mycobacteria has not


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been described in literature or in reference manuals. Therefore, it is necessary that the culture protocol is validated in each laboratory. The development of new bacterial media or methods needs to be put in perspective with the development of molecular diagnostic methods (RT‐PCR). Molecular tests always surpass bacteriology in terms of rapidity in results. There are various molecular methods to detect M. bovis. However, the best molecular method in term of Se and Sp is the real‐time PCR (RT‐PCR): Se 87% and Sp 97%. The method is a good option to obtain rapid and good quality diagnosis of bTB. Therefore, the RT‐PCR should be considered as an official tool for the diagnosis of bTB. However, the isolation of M. bovis remains relevant to allow molecular typing and epidemiology. The interferon gamma test measures the release of the gamma interferon (IFN‐γ) lymphokine in a whole‐blood culture system. The assay is based on the release of IFN‐γ from lymphocytes sensitized during a 16–24‐hour incubation period with a specific recall antigen (like PPD‐tuberculin). The detection of bovine IFN‐γ is carried out with a sandwich ELISA that uses two monoclonal antibodies to bovine gamma‐interferon. Because the assay makes use of viable blood cells, it is recommended that the blood samples are transported to the laboratory and the assay set up as soon as practical, but not later than the day of blood collection. Based on a meta‐analysis of 15 field studies conducted between 1991 and 2006, an estimated median Se of 87.6% (with a range between 73% and 100%) and a Sp of 96.6% (with a range of 85% and 99.6%) is reported. Also, a possible boost of IFN‐y production after skin test is reported in literature. Moreover, infected animals are detected sooner with the IFN‐y test than with the skin test. To conclude, the IFN‐y test is a very promising test but validation of the different kits and antigens must be performed under Belgian field conditions. There are several serological tests for bTB. The ELISA test appears to be the most suitable of the antibody‐detection tests and can be complementary to tests based on cellular immunity notably to detect animals that are anergic and do not react to the skin test and IFN‐γ anymore. The test is an easy, fast, objective, and cost‐effective option for bTB surveillance. However, the Se of the ELISA is low (estimated at 63% with a range between 30% and 97%) while the Sp is estimated at 98% (with a range between 88 and 100%). A boost of antibodies after the skin test (2 weeks) is reported in literature. A simulation exercise has been performed to calculate the theoretical Se and Sp of different combinations of diagnostic tests. These results can be used by risk managers to obtain the desired Se en Sp for bTB surveillance in the future. The use of every diagnostic test in non‐bovine animals has also been evaluated. In general, it can be concluded that these studies are based on a small number of animals and herds for estimation of the values for Se and Sp and for some species data are still lacking. Hence, the practical use of these tests in other species is currently not advisable. Finally, all known risk factors for bTB infection in scientific literature are described. These risk factors should be taken into account to allow a risk based surveillance of bTB. Evaluation of the current bTB surveillance program A scenario tree analysis has been performed in order to evaluate the sensitivity of the different components of the Belgian bTB surveillance program to prove the official freedom of disease. The outcome of the model (scenarios) was mostly influenced by the following parameters: the number of tested animals per component as well as the used Se for each of the test(s). External validation of the output using a logistic regression model showed that herd tuberculinations (during winter screening) and slaughterhouse post‐mortem inspections were significant components to detect outbreaks in Belgium. Considering the low number of positive results, this was not the case for the purchase testing, despite it has been identified as a risk factor for introduction and dissemination of bTB.


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Furthermore, a benchmarking study in which the expected number of positive reactors was compared with the actual number of notified reactors clearly shows underreporting during purchase surveillance of bTB. Considering the relatively low declaration of positive or doubtful reactions during tuberculin testing at purchase, its effectiveness in the surveillance program can be questioned. In addition, given the estimated costs of purchase testing of 1.177.461,59 € for 2014, the cost‐benefit of the present strategy is questionable. In conclusion, the efficacy of the current surveillance program, which especially makes use of SIT as first line screening test, can be questioned. Recommendations It is important to raise awareness of all the actors involved in the bTB surveillance program by regular information and training. Basic biosecurity measures (e.g. quarantine) are too often not (well) applied by cattle farmers in Belgium, although they are very important for bTB prevention. Therefore, farmers and veterinarians must be stimulated to respect these biosecurity measures. It is recommended to adapt the surveillance program by:

- replacing the SIT test by a combined IFN‐y and a serological test (in parallel) at purchase. In case of positive/doubtful result, it is recommended that the competent authorities execute themselves a SIT as confirmation test;

- maintaining the SIT test performed by the private veterinarian during winter screening. In case of positive/doubtful result, it is recommended that the competent authorities execute themselves an IFN‐y test as confirmation test;

- executing both a SIT (or SICT), IFN‐y and serological tests on all cattle present on the farm in suspected/outbreak herds;

- maintaining the SIT test performed by the private veterinarian during tracing analysis. In case of a positive/doubtful result it is recommended that the competent authorities execute themselves an IFN‐y test as a confirmation test;

- classification of bovine herds and/or individual animals according to their risk for bTB allowing a more targeted surveillance of high risk herds/animals at slaughter.

It is recommended to store epidemiologic data in a centralized FASFC database consultable for all actors involved in the surveillance network and allowing to perform a risk based surveillance based on bTB history. It is recommended to adapt the control measures and their duration (i.e. blocking of farms) based on indicators allowing to allocate a risk profile to animals and/or herds. It is recommended to adapt legislation in relation to current and future available diagnostics (e.g. inclusion of nucleic acid recognition methods (RT‐PCR), IFN‐y test and serological tests or any other new method). RT‐PCR should be used as first line diagnostic (in parallel with bacteriological culture) to allow a faster confirmation of a positive case and to reduce the time a farm is blocked after a positive or doubtful test. The recent bTB case in an imported alpaca in Belgium shows that vigilance for bTB in non‐bovine domesticated animals is very important as they can be a source of introduction. Moreover, there have been some recent bTB in badger, deer and wild boar in France, close to the border with Belgium. Therefore, it is strongly recommended to install a continuous surveillance program in wildlife in Belgium based on known risk factors. For camelids, a surveillance program is also recommended. Furthermore, it is recommended to stimulate the development and validation of diagnostic tests which can be applied in non‐bovine domesticated species and wildlife and which can be useful in case these animals are kept on the same farms or in close vicinity to bovines.


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1 Definitions & Abbreviations

bTB bovine tuberculosis CFT caudal fold test: single intradermal skin test performed at the caudal fold CI Confidence Interval CMI cell‐mediated immunity DPP dual‐path platform FASFC Federal Agency for the Safety of the Food Chain M. bovis Mycobacterium bovis MAP Mycobacterium avium subspecies paratuberculosis MAPIA multiantigen print immunoassay MTC Mycobacterium tuberculosis complex NOTF not officially tuberculosis free NRL National Reference Laboratory OTF officially tuberculosis free PPD purified protein derivative PPDB/PPDA protein purified derivative from M. bovis (B) or from M. avium (A) PUR purchase RT rapid test RT‐PCR real‐time PCR Se Sensitivity SICT single intradermal comparative test SIT single intradermal test SLGH slaughterhouse Sp Specificity IFN‐γ Interferon‐gamma


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2 Terms of reference

2.1 Question

Despite the fact that Belgium is officially free of bTB since 2003, surveillance against bTB still remains an important task because almost every year one or more outbreaks are detected. From an economical point of view, it is very important for Belgium to maintain its OTF status to allow easy intracommunity trade. The epidemiological evolution in a number of neighboring countries demonstrates that, if awareness for bTB in domesticated animals decreases, bTB can reemerge. This lowered awareness in domesticated animals can lead to a spill‐over of bTB into wild animals making the control of bTB even more difficult. Recently, stakeholders have expressed a number of problems and constraints about their role within the current bTB surveillance program. Given these circumstances, the Scientific Committee was asked to perform a thorough evaluation of the current bTB surveillance and control program in Belgium. Besides a general evaluation, a number of specific questions were asked. All specific questions are listed in Appendix I. For readability purposes, only a limited number of specific questions are answered directly in this opinion. The answers to the other questions can be found throughout the text. The final opinion should serve as a basis for future adaptations of the bTB surveillance and control program.

2.2 Methodology / Rationale

A thorough evaluation of the bTB surveillance in Belgium has been executed. It is based on simulation exercises (scenario tree analysis to evaluate the Se of each surveillance component), on a benchmarking study comparing the expected number of false positive reactors with the actual notified reactors and an estimation of the direct and indirect costs of surveillance. This opinion contains also a review of current diagnostic techniques for bTB and a list of risk factors that need to be considered in bTB surveillance. This review is based on the available knowledge in scientific literature and on expert opinion. Finally, a number of recommendations regarding bTB surveillance are proposed, the relevance of which is evaluated by experts. Considering the discussions during the workgroup meetings on 26/08/2015, 01/10/2015, 22/10/2015, 16/11/2015, 05/01/2016, 16/02/2016 and during the plenary sessions of the Scientific Committee on 23/10/2015, 18/03/2016 and 22/04/2016;

the Scientific Committee gives the following advice:

3 Introduction

3.1 Bovine tuberculosis

Bovine tuberculosis is caused by M. bovis which belongs to the Mycobacterium tuberculosis complex (MTC). Members of this complex can induce tuberculosis in animals and humans. The most important species are M. bovis, M. tuberculosis, M. caprae and M.canetti. M. bovis is an intracellular pathogen. The target cells are macrophages and other monocytic cells. According to OIE (2009) bTB is an infectious disease caused by M. bovis (and some other members of MTC) that affects cattle, other domesticated animals and certain free or captive wildlife species. It is


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usually characterised by the formation of nodular granulomas, known as tubercles. Although commonly defined as a chronic debilitating disease, bovine tuberculosis can occasionally assume a more progressive course. Any body tissue can be affected, but lesions are most frequently observed in the lymph nodes (particularly of the head and thorax), lungs, intestines, liver, spleen, pleura, and peritoneum. In many cases, the course of the infection is chronic and signs may be lacking, even in advanced cases when many organs may be involved. When present, clinical signs vary; lung involvement may be manifested by a cough, which can be induced by changes in temperature or manual pressure on the trachea. Dyspnea and other signs of low‐grade pneumonia are also an evidence of lung infection. In advanced cases, lymph nodes are often greatly enlarged and may obstruct air passages, alimentary tract, or blood vessels. Lymph nodes of the head and neck may become visibly affected and sometimes rupture and drain. Involvement of the digestive tract is manifested by intermittent diarrhoea and constipation in some instances. Extreme emaciation and acute respiratory distress may occur during the terminal stages of tuberculosis. Lesions involving the female genitalia may occur. Male genitalia are seldom involved. At necropsy, tubercles are most frequently seen in bronchial, mediastinal, retropharyngeal and portal lymph nodes and may be the only tissue affected. In addition, the lung, liver, spleen and the surfaces of body cavities are commonly affected. Early nodular pulmonary lesions can often be detected by palpation. The lesions are usually non‐odoriferous. Other anatomical sites can be infected and should be examined. bTB is an important zoonosis and human infection mainly occurs through consumption of raw milk and products of raw milk, but the disease is also transferable through the air during close contact with infected animals. bTB has been identified in humans in most countries where isolates of mycobacteria from human patients have been fully characterised. The incidence of pulmonary tuberculosis caused by M. bovis is higher in farm and slaughterhouse workers than in urban inhabitants (occupational disease). The transmission of M. bovis to humans via milk and its products is avoided by the pasteurisation of milk. One of the results of the bovine tuberculosis eradication programs has been a reduction in disease and death caused by bovine tuberculosis in the human population. Although cattle are considered to be the true hosts of M. bovis, the disease has been reported in many domesticated and non‐domesticated animals. Isolations have been made from buffaloes, bison, sheep, goats, equines, camels, pigs, wild boars, deer, antelopes, dogs, cats, foxes, mink, badgers, ferrets, rats, primates, New World camelids (llamas, alpacas, vicuñas, guanacos), kudus, elands, tapirs, elks, elephants, sitatungas, oryxes, addaxes, rhinoceroses, possums, ground squirrels, otters, seals, hares, moles, raccoons, coyotes and several predatory felines including lions, tigers, leopards and lynx (De Lisle et al., 2001; O’Reilly & Daborn, 1995). Bovine tuberculosis in wildlife was first reported in 1929 in greater kudu (Tragelaphus strepsiceros) and common duiker (Sylvicapra grimmi) in South Africa and by the 1940s, the disease was found to be endemic in greater kudu. In 1982 in Uganda, a prevalence of 10% in African buffalo and 9% in warthog (Phacochoerus aethiopicus) was found, and in Zambia, M. bovis infection has been reported in Kafue lechwe (Kobus leche kafuensis) and in a single eland (Traurotragus oryx). An outbreak of tuberculosis in wild olive baboons (Papio cynocephalus anubis) was reported in Kenya. Mycobacterium bovis infection has also been diagnosed in African buffalo in the Kruger National Park in South Africa (Bengis et al., 1996), and more recently spill over to other species such as chacma baboon (Papio ursinus), lion (Panthera leo) and cheetah (Acynonyx jubatus) as well as greater kudu has occurred. The rigorous application of tuberculin testing and culling of reactor cattle has eliminated M. bovis infection from farmed bovine populations in some countries, but this “test & slaughter” strategy has not been universally successful. Extensive investigations of sporadic M. bovis reoccurrence have


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shown that wildlife reservoirs exist in some countries and can act as a source of infection for cattle, deer and other livestock. The risk that these reservoirs of infection constitute for domestic animals and humans is quite variable depending on the specific epidemiological situation for the species and the environment (Corner, 2006; Morris et al., 1994). The detection of infection in a wildlife population requires bacteriological investigation or the use of a valid testing method for the species involved (the tuberculin test is not effective in all species) together with epidemiological analysis of information. The badger (Meles meles) in the United Kingdom (Wilesmith, 1991) and the Republic of Ireland (O’Reilly & Daborn, 1995), wild boar (Sus scrofa) in Spain (Naranjo, 2008), the brush‐tail possum (Trichosurus vulpecula) in New Zealand (Animal Health Division (New Zealand), 1986), and several wild living species in Africa have been shown to be capable of maintaining M. bovis infection. Control of transmission from the wildlife population to farmed species is complex and, up till now has relied on the reduction or eradication of the infected wildlife population. The use of vaccination to control the disease in some species continues to be investigated.

3.2 Epidemiologic context of bTB in Belgium and the EU

3.2.1 Overview of bTB in EU

Figure 1 gives an EU update (14 January 2014) of the bTB situation in Europe with relation to the status (OTF (officially tuberculosis free) versus NOTF (not officially tuberculosis free)). To obtain and maintain the OTF, a country cannot have more than 0.1% positive bTB cattle herds per year. The herd prevalence found in UK, Ireland, Spain, Portugal, regions of Italy, and Greece are respectively 10.4%, 4.4%, 1.2%, 0.2%, 0.3%, 0.4% (EFSA, 2013). Neighboring countries of Belgium all have an OTF status: The Netherlands (OTF since last outbreaks in 1999), Germany (OTF since 1997) and France (OTF since 2001). However, in France there are some problematic areas such as Dordogne, Cote d’Or and Camargue, Pyrenees and the border region of France with the South of Germany and Switzerland. In these regions, other members of the Mycobacterium tuberculosis complex (e.g. M. caprae) are found in wild life (e.g. chamois and Ibex goat) and domestic animals. Figure 1. Situation of Bovine tuberculosis in Europe.


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3.2.2 History of bTB outbreaks in Belgium

In accordance with the European legislation (Directive 64/432/EEC and Decision 2003/467/EC) Belgium has obtained the OTF status (<0.1% positive bTB cattle herds per year) since 2003. During the last years, there were sporadic outbreaks of bTB with a higher number in 2008 and 2013 (consecutive to the tracing of positive herds) (Figure 2). In order to maintain the officially free bTB status, Belgium –like other MS‐ is allowed to have a maximum of 0.1% outbreaks (positive bTB cattle herds) per year on the total of all registered cattle herds. For instance, in 2012, only 1 outbreak was detected although theoretically up to 29 outbreak herds on a total of 28470 herds would have been acceptable to maintain the OTF status (EFSA 2012: EN‐288). Figure 2. Number of bTB outbreaks during the last 15 years in Belgium.

In 2008, the first outbreak was detected during a ‘Herd tuberculination’ campaign, while the 2nd and 3rd were detected via slaughterhouse inspection. From the 2nd and 3rd outbreak, 8 additional outbreaks were detected through SIT following tracing back testing from the primary outbreak. The 12th outbreak in 2008 was detected after the follow up by SIT testing in consequence of a notification from the German authorities (cattle had been imported and purchased but detected following tracing on testing and not by SIT at purchase). In 2009, one outbreak was found via the slaughterhouse surveillance; the second via tracing (herd tuberculination). In 2011, one case was detected via slaughterhouse inspection and resulted in a tracing of 23 contact herds. No further cases were found. In 2012, suspicious lesions were found at slaughter in The Netherlands on an animal born in Belgium. This animal was fattened in The Netherlands. More than 90% (12 out of 13) of the animals in the farm reacted positive by SIT. Tracing back led to the identification of 10 Belgian contact herds. Only 1 out of the 10 contact herds showed positive SIT reactors: 95 positive reactions and 34 doubtful reactions on a total of 190 cattle. A total stamping‐out was realized in this herd (53 out of 190 slaughtered animals presented lesions typical for bTB). A thorough examination of all findings at previous slaughters in Belgian slaughterhouses (before 2012) did not show any suspicious lesions. In 2013, 9 outbreaks were detected. The epidemiologic investigation showed that several of them were contact herds. No outbreaks were detected in 2014. In 2015, three outbreaks were detected. There was also the detection of a bTB positive alpaca in 2015, which was imported from an endemic bTB region in the UK.

3.3 Legal framework


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3.3.1 EU Legal framework

Since 1964, the eradication and the monitoring of bTB and some other animal diseases became important in the EU. The Commission established a legal framework and incorporated rules regarding the health status in relation to some animal diseases, and provisions for tests to detect these diseases, aligned with the health standards of the World Organisation for Animal Health (OIE). The Community legal framework on TB is based on the legislation on trade of bovine animals, the legislation on animal products for human consumption and the legislation regarding Community co‐financing of eradication programs. The first legal initiatives on TB at Community level were aimed at facilitating intra‐community trade among the EEC Member States by establishing comparable health requirements. Council Directive 64/432/EEC1 defined specific requirements for the trade of cattle in relation to TB and defined the officially tuberculosis‐free (OTF) status for bovine herds. Council Directive 64/432/EEC establishes that bovine animals consigned from one Member State to another must originate from an OTF herd and have been submitted to a pre‐movement tuberculin test. The pre‐movement test is exempted for animals send directly for slaughter. The procedures for gaining, maintaining, suspending, withdrawing or re‐gaining the OTF status are laid down in annex A of Council Directive 64/432/EEC and are based on the results of tuberculin tests at herd level. A Member State or a part of a Member State may be declared OTF if certain requirements are fulfilled. Annex B deals with the diagnosis of TB; this annex has been regularly reviewed to incorporate new methods and to align more with the OIE health standards. Commission Decision 2003/467/EC2 establishes the official tuberculosis, brucellosis and enzootic bovine leucosis free status of certain Member States or regions of Member States as regards bovine herds in compliance with certain conditions set out in Council Directive 64/432/EEC. Regulation (EC) N° 852/20043 and Regulation (EC) N° 853/20044 establishes the procedures for the post‐mortem inspection at slaughterhouse. Some of these measures are specially aimed at the detection of lesions of TB. Meat from animals with generalized TB cannot be declared fit for human consumption and strict conditions are laid down for the inspection of carcasses of animals that have shown a positive or inconclusive reaction to the tuberculin test. Community measures regarding milk hygiene are laid down in Council Directive 92/46/EEC5 and are essentially the same in Regulation (EC) N° 853/2004. Only milk from TB free herds can be used for human consumption without heat treatment. Milk from animals that have shown a positive or inconclusive reaction to the tuberculin test may not be used for human consumption.

1 COUNCIL DIRECTIVE of 26 June 1964 on animal health problems affecting intra‐Community trade in bovine animals and swine (64/432/EEC) 2 COMMISSION DECISION of 23 June 2003 establishing the official tuberculosis, brucellosis, and enzootic bovine‐leukosis‐free status of certain Member States and regions of Member States as regards bovine herds (2003/467/EC) 3 REGULATION (EC) No 852/2004 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 29 April 2004 on the hygiene of foodstuffs 4 REGULATION (EC) No 853/2004 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 29 April 2004 laying down specific hygiene rules for on the hygiene of foodstuffs 5 COUNCIL DIRECTIVE of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heat‐treated milk and milk‐based products (92/46/EEC)


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Council Directive 82/894/EEC6 establishes a list of diseases (Annex I list A.2 bovine tuberculosis) which are subject to notification within the Community. An important further step was Council Directive 77/391/EEC7 which introduced Community measures for the eradication of brucellosis, tuberculosis and leucosis in cattle. Member States were obliged to draft eradication programs in order to accelerate, intensify or carry out the eradication of TB. Financial support from the Community for these programs was also foreseen. This legislation provides a legal framework for the TB eradication programs. Council Decision 90/424/EEC8 on expenditure in the veterinary field lays down Community financial resources on eradication and monitoring programs aimed at progressively eliminating animal diseases that are endemic in certain areas of the Community. Detailed rules for reporting on the progress of eradication programs are contained in Commission Decision 2002/677/EC9 while Commission Decision 2004/450/EC10 provides a standard format for Community financed programs.

3.3.2 National legal framework

The control and surveillance of tuberculosis in Belgium is based on Council Directive 64/432/EEC, which was implemented in national legislation since 1963 by Royal Decree of 10 May 1963 and lastly amended by Royal Decree of 17 October 2002 concerning the eradication of bovine tuberculosis.

3.4 Current measures concerning monitoring and surveillance

The surveillance of bTB consists mainly of passive clinical surveillance and routine testing using the following four main pillars of surveillance:

- Post‐mortem inspection of all slaughtered animals at slaughterhouse (visual inspection and palpation/incisions of organs/area): suspected lesions must be sent to the NRL for culture and identification of bTB.

- Tracing analysis of outbreaks: this tracing analysis is based on a thorough tracing back and forward of all animals and herds that came into contact with a confirmed positive animal or herd (outbreak). Tracing forward component detects mostly secondary outbreaks of bTB. The objective of the tracing back is the determination of the origin of outbreaks. A whole herd tuberculination (SIT) by a veterinary practitioner is followed by a SICT performed by an official veterinarian of the FASFC 6 weeks after each non‐negative SIT reactor.

- Most herd tuberculinations are realized during the winterscreening: a) If a herd is identified as a contact herd via tracing‐back and ‐on, these herds will be

followed up using the ‘Herd tuberculination’ each winter (November‐March) during 5 consecutive years using a SIT of all animals above 6 weeks of age. Also the identified outbreak herds (with partial or total stamping‐out) are followed up for 5 years during this ‘herd tuberculination’ campaign (Tracing outbreak).

b) During the Herd tuberculination campaign also dairy farms (female animals >24 months) that sell directly to the consumer raw milk and/or raw milk products are

6 COUNCIL DIRECTIVE of 21 December 1982 on the notification of animal diseases within the Community (82/894/EEC) 7 COUNCIL DIRECTIVE of 17 May 1977 introducing Community measures for the eradication of brucellosis, tuberculosis and leucosis in cattle (77/391/EEC) 8 COUNCIL DECISION of 26 June 1990 on expenditure in the veterinary field (90/424/EEC) 9 COMMISSION DECISION of 22 August 2002 laying down standard reporting requirements for programs of eradication and control of animal diseases co‐financed by the Community and repealing Decision 2000/322/EC (2002/677/EC) 10 COMMISSION DECISION of 29 April 2004 laying down standard requirements for the content of applications for Community financing for programs for the eradication, monitoring and control of animal diseases (2004/450/EC)


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yearly tested via SIT. This component is meant to minimize the zoonotic aspect of bTB and potential risk towards public health (Direct selling)

c) During the Herd tuberculination campaign also imported cattle from Non Officially Tuberculosis Free (NOTF) regions/member states are followed up for 3 consecutive years. The testing is done via SIT of all animals > 12 months of age. (Tracing import)

- Mandatory testing at Purchase: each animal that is purchased/commercialized by national trade must be tested at the farm of destination (= buyer; after movement of the animal between ‘seller’ and ‘buyer’) via SIT by the veterinarian practitioner. This component is important for early detection of infected animals (early warning) at the moment of introduction of animals into a new herd and before contact of new animals with the other livestock of a herd.

4 Evaluation of the current diagnostic techniques for the detection of bTB

4.1 Intradermal skin test ‐ Delayed hypersensitivity test

4.1.1 Principle

For many years the standard method for detection of bovine tuberculosis was the tuberculin test, which involves the intradermal injection of bovine tuberculin purified protein derivative (PPD) and the subsequent detection of swelling (delayed hypersensitivity) at the site of injection 72 hours later. This may be performed using bovine tuberculin alone (SIT) or as a comparative test using avian and bovine tuberculins (SICT) to differentiate between an infection with M. avium and M. bovis respectively (OIE, 2009). Delayed hypersensitivity may not develop before a period of 3–6 weeks following infection. Thus, if a herd/animal is suspected to have recently been in contact with infected animals, later testing should be considered in order to reduce the probability of false‐negatives. As the Se of the test is less than 100%, it is unlikely that eradication of tuberculosis from a herd will be achieved with only a single tuberculination test. Therefore, most eradication programs are not exclusively based on skin testing. Also, in an advanced state of infection, it appears that chronically infected animals with severe pathology, might not respond to the tuberculin test due to anergy (OIE, 2009). The comparative intradermal tuberculin test (SICT) is used to differentiate between animals infected with M. bovis and responding to bovine tuberculin and those exposed to other mycobacteria. This sensitisation can be attributed to the antigenic cross‐reactivity among mycobacterial species and related genera. SICT involves the intradermal injection of bovine tuberculin and avian tuberculin at different cervical sites, usually on the same side of the neck, and measuring of the dermal response 3 days later. The correct procedure for the intradermal skin test according to the OIE can be found in Appendix II.

4.1.2 Injection site

Traditionally, the intradermal skin test is performed either in the neck area or in the caudal fold. Nowadays the caudal fold is typically used in USA, Canada, and New Zealand due to the facility and practice for performing the intradermal inoculations (Casal et al., 2015) whereas the neck is the preferred location in Europe (Council Directive 64/432/EEC). However, it is described in literature that the caudal fold results in a lower sensitivity of the test compared to the neck (Francis et al., 1978; Whipple et al., 1995; Norby et al., 2004; Farnham et al., 2012). Similar results were reported by Baisden et al. (1951) in experimentally M. bovis sensitized cattle, suggesting that the highest sensitivity was obtained after inoculation of the antigen in the skin of the neck compared with the dorsal area of the back, and the upper and lower flanks. Moreover, Casal et al. (2015) demonstrated in a field trial that the probability of detecting a reactor was largely affected by the position in which the skin test was performed; being highest in the anterior neck area compared with the other more caudal neck areas studied. A possible explanation for this finding might be the closer proximity of the anterior neck location to regional lymph nodes (submandibular,


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retropharyngeal, parotid and cervical), which could provoke a larger cellular recruitment to the inoculation site leading to an increased local reaction (Kindt et al., 2006; Flynn and Chan, 2001). In conclusion, the intradermal skin test should be performed in the anterior neck area in order to render the sensitivity of the test as high as possible.

4.1.3 Tuberculin

Tuberculin, developed by Koch in 1890, is a concentrated sterile culture filtrate of tubercle bacilli grown on glycerinated beef broth and, more recently, on synthetic media. Initially, this original tuberculin has been used in intradermal skin tests. However, due to higher specificity and easier standardization, purified protein derivative (PPD) products have replaced heat‐concentrated synthetic medium tuberculins (OIE, 2009). Several studies have proved that the PPD currently used, which is still based on historic isolates, is still adapted to detect latent tuberculosis: Recently a proteomic analysis of PPD was realized by mass spectrometry on PPD‐CT68 and PPD‐S2 (PPD generated from M. tuberculosis) (Prasad, 2013; Cho, 2012) and by LC‐MS/MS on PPD generated from M. bovis/M. avium (Borsuk, 2009). These studies showed that PPDs contain a high number of proteins and that the different PPDs, currently used, share a large number of these proteins. The functional analysis of the proteins common among all PPDs showed that they are implicated in causing infection and protecting the pathogen against various metabolic stresses (Prada, 2013). These proteins are therefore important for the survival of the mycobacteria and will likely be conserved. For the control of bTB, PPDB (protein purified derivative from M. bovis) which is used in the skin test is produced from the M. bovis AN5, a strain that was originally isolated in Great Britain (~1948). M. bovis AN5 had not suffered from any significant loss of genetic information during in vitro culture compared to recent field isolates (contrary to M. bovis BCG) (Inwald, 2003). A recent study defined the global gene expression profile of this strain and showed that its profile is similar to that of field strains (Garcia Pelayo, 2009). This study supports the use of M. bovis AN5 to produce PPD for use in control programs (Garcia Pelayo, 2009). In addition, during the comparative skin test, PPDA (protein purified derivative from M. avium) produced from M. avium D4 was also used in order to differentiate cattle infected by M. bovis versus cattle sensitized by environmental mycobacteria (e.g. differences in constitution and antigenicity of PPDA and PPDB are sufficient to distinguish between both). Moreover, the genome of the M. tuberculosis complex members is 99.95% identical at the nucleotide level, showing a very stable genome (Lu et al., 1987; Garnier, 2003), with the global distribution of M. tuberculosis lineages, sub‐lineages and clones being much more complex than the geographical location and diversity of the clonal complexes among M. bovis isolates (Smith et al., 2012). In 2000, Villarino et al. published the results of a clinical trial comparing skin tests results obtained with PPD‐S1 and PPD‐S2. Indeed, PPD‐S1 was used since 1951 to standardize commercial PPD reagents and perform special tuberculin surveys. At the end of the nineties, PPD‐S1 was in short supply and a new standard (PPD‐S2) was manufactured. The study showed that PPD‐S2 was equivalent and can replace PPD‐S1. In conclusion, the “current” PPDs are still adapted to detect a contact with the M. tuberculosis complex. The main concern should be the quality of the PPD production more than its constitution. Indeed, studies showed that a high‐quality control of the PPD production is essential (Rangel‐Frausto, 2001; Schiller, 2010).

4.1.4 Sensitivity and specificity and influencing factors

In literature, a wide range of Se and Sp values are reported for the intradermal skin test (Schiller et al., 2010; Bezos et al., 2014):


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- Se range: Between 53% (27.3‐81.5, 95% CI) and 69.4% (40.1‐92.2, 95% CI) depending on cattle type (fighting bulls versus other)

- Sp range: Between 55.1% and more than 99% showing a median value over 95% Actually, a lot of parameters can affect the results of the intradermal skin test and they are summarized in the table below: Table 1. Parameters affecting the intradermal skin test

Parameters affecting skin test results Reference

A. Potential causes of false positive reactions

Cross‐reaction in animals exposed to other bacteria (other Mycobacteria spp., Nocardia spp., Actinobacillus spp., Actinomyces spp.)

Bezos et al., 2014 ; Casal et al., 2015; de la Rua Domenech et al., 2006 adopted by Humblet et al., 2011a ; Schiller et al., 2010

Skin contamination, improper test procedure

Skin lesions, e.g., dermatitis

Vaccination for Johne's disease (paratuberculosis – not applied in Belgium)

B. Potential causes of false negative reactions Factors related to the animal being tested:

Desensitisation to bovine tuberculin (test administered too soon after a previous tuberculin test)

Bezos et al., 2014 ; Casal et al., 2015; de la Rua Domenech et al., 2006 adopted by Humblet et al., 2011a ; Schiller et al., 2010

Unreactive (pre‐allergic) period (test administered too soon after infection)

Overwhelming or generalised infection with Mycobacterium bovis (anergy)

Co‐infection with (or pre‐exposure to) an environmental mycobacterium (e.g. M. avium‐intracellular complex) resulting in hypersensitivity to avian tuberculin in the SICT and γ‐IFN tests

Vaccination against Mycobacterium avium subsp. paratuberculosis (Johne’s disease)

Concurrent infection with viruses that depress the immune system, e.g. bovine viral diarrhoea (BVD) virus in acute infection

Drugs (e.g. corticosteroids and other immunosuppressive agents)

Immunodepression during early post‐partum

Nutritional and transport stress

Strain (molecular type) of M. bovis

Factors related to the tuberculin used (use of a sub‐potent product)

Expired product Bezos et al., 2014 ; Casal et al., 2015; de la Rua Domenech et al., 2006 adopted by Humblet et al., 2011a ; Schiller et al., 2010

Improperly stored product (exposed to light and heat for long periods)

Tuberculin manufacturing errors (use of inadequate M. bovis strain, incorrect calibration of batch potency, etc.)

Factors related to the tuberculin used (use of a sub‐potent product)

Factors related to the method of administration, reading and recording of the test (tester errors due to inexperience, lack of attention, poor cattle restraining facilities, fractious animals, poorly maintained testing equipment, etc.)

Injection of a too low dose of bovine tuberculin Bezos et al., 2014 ; Casal et al., 2015; Subcutaneous (rather than intradermal) injection of bovine tuberculin


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Incorrect injection site of tuberculins de la Rua Domenech et al., 2006 adopted by Humblet et al., 2011 ; Schiller et al., 2010

Injecting bovine tuberculin in the avian injection site and vice‐versa (SICT test)

Reading of results too early or too late (not within the prescribed 72 h ± 4–6 h post‐tuberculin injection)

Error in recording the skin readings

Error in identifying the reactor animal

Conscious or unconscious tester bias

Furthermore, field veterinarians may not always comply with the testing procedure following ‘good veterinary practices’ (see recommendations by USDA (2015)), which can have substantial impact on the results of the intradermal test. Possible pitfalls are summarized in Appendix III. Lastly, also the injection device may have an impact on the result of the intradermal skin test. However, only one study in literature was found on this subject (Bénet et al., 2013) in which three injection materials were compared: Mac Lintock, Synthena, (classical) Multo seringes. The conclusions were that Mac Lintock compares similarly in terms of performance to Synthena (the latter one is currently no longer available due to the presence of Bisphenol A in the carpule containing the tuberculin) and both perform better than Multo seringes. However, the robust needle of the Mc Lintock device is hard to change and makes it difficult to clean in contrast to Multo seringes. The advantages and disadvantages of the 2 devices which are used in Belgium by field veterinarians (classical syringe and dermojet) are discussed in Appendix IV. The Scientific Committee concludes that the sensitivity and specificity of the intradermal tuberculin test is not influenced by the type of instrument used (classical syringe versus dermojet). Other factors, such as the compliance of veterinarians to ‘good veterinary practices’ while performing an intradermal skin test, could have a substantially higher importance.

4.1.5 Non‐bovine animals

The Scientific Committee has performed an extensive literature research regarding the use of SIT in other species than cattle. In general, there are rather few publications on the use of SIT in non‐bovine animals and the animal numbers in these studies are relatively limited. More details can be found in Appendix V. This literature research showed that the performance of the SIT can vary considerably depending on the circumstances of its use and the population of animals used for study (Chambers, 2013). All these variations –as observed in cattle– can be explained by parameters such as the quality of the tuberculin, the materials used for injecting tuberculin, the inoculation site, etc. Therefore, the fact that the skin test was first developed and interpreted according to the “bovine standards”, without adaptation to non‐bovine species could explain that estimated Se and Sp were generally lower than in cattle. Interference of MAP infection as observed in cattle could also partially explain the unreliable results of the SIT in these species. In conclusion, although the skin test appears to work well in goats and sheep and also in some wildlife species such as deer, it appeared unreliable as diagnostic test in other species infected with M. bovis, such as Eurasian badgers, possums, swine, wild boar, oryx and large zoo mammals, and is impractical for free‐ranging animals.

4.2 Identification of the agent

4.2.1 Microscopic examination

M. bovis can be demonstrated microscopically on direct smears from clinical samples and from tissue materials. The acid‐fastness of M. bovis is normally demonstrated using the classic Ziehl–Neelsen


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stain, but a fluorescent acid‐fast stain may also be used. Immunoperoxidase techniques may also give satisfactory results. The presumptive diagnosis of mycobacteriosis can be performed if the tissue has characteristic histological lesions (caseous necrosis, mineralisation, epithelioid cells, multinucleated giant cells and macrophages). As lesions are often paucibacillary, the presence of acid‐fast organisms in histological sections may not be detected, although M. bovis can be isolated in culture. However, large numbers of acid‐fast organisms can be seen in lesions in primates, felids, mustelids (badgers) and marsupials (brush‐tailed possums) (OIE, 2009).

4.2.2 Bacterial culture

Isolation of M. bovis remains the gold standard method for the diagnosis of bovine tuberculosis. According to the current Belgian legislation, only the isolation of the bacteria remains the definitive proof for the confirmation of an outbreak. One big drawback of this method is the time needed to grow M. bovis. The OIE manual recommends a minimal culture‐time of 8 weeks (and preferably 10–12 weeks). Technically, the long culture time is a major limiting factor because it allows the development of possible contaminating flora. Isolation protocols therefore need to consider two factors: 1) the use of appropriate techniques for decontamination of the initial sample; 2) the use of appropriate media for Mycobacteria (enriched with specific nutrients) to maximize the chance of isolation. Three decontamination protocols exist: with detergents, with alkali or acid treatment. Decontamination protocols are per se toxic for mycobacteria; the time of contact between the sample and the decontaminant needs to be finely optimized to obtain effective decontamination without inhibition of the mycobacteria. Two types of media for growing Mycobacteria are described: egg‐ (Stonebrink’s and Lowenstein‐Jensen) or agar‐based (Middlebrook 7h10, 7h11 and B83). Egg‐based media are better suited to limit the growth of contaminants while agar‐based media are favorable for the growth of Mycobacteria. Due to these differences, Gormley et al. (2014) suggest that the use of more than one medium for isolation is more appropriate. Isolation in liquid media is also possible. In 1995, the Becton Dickinson Company launched the MGIT system (Mycobacterial growth indicator tube). This system uses liquid media and monitors the bacterial growth by the O2 concentration in the culture tube. This efficient system is well employed in human medicine to provide rapid diagnosis and can be used for antibiotic resistance tests as well. In veterinary medicine, MGIT system is also used as it allows a response in 2 or 3 weeks (Robbe‐Austerman et al., 2013). However, compared with human medicine, this medium and the associated decontamination procedures need to be validated for use in veterinary medicine. In conclusion, the isolation method is until now the gold standard test. The existence of a single recommended protocol is not present in literature or in reference manuals. Therefore, validation of the culturing protocol in each laboratory is required. The development of new bacterial media or methods needs to be put in perspective with the development of molecular diagnostic methods (RT‐PCR). Molecular tests always surpass bacteriology in terms of rapidity in results. Concerning the mycobacterial typing, classical biochemical methods are nowadays replaced by molecular tests, which are more specific, sensitive and standardized between laboratories.

4.2.3 Molecular methods

According to the OIE (2009), there are various molecular methods to detect M. bovis. However, the best molecular method in term of sensitivity and specificity is the real‐time PCR (RT‐PCR). For the diagnosis of bacterial infections, PCR amplification generally targets insertion sequences. For detection of mycobacteria of the M. tuberculosis complex, two insertion sequences are used: IS6110 (Thacker et al., 2011) and IS1081 (Taylor et al., 2007). To date, only one single study has evaluated the added value of the RT‐PCR in the context of the veterinary diagnosis of bTB (Courcoul et al., 2014). This study, based on a Bayesian approach, compares the sensitivity and specificity of three methods: histology, bacteriology and RT‐PCR. It has been realized on samples derived from 5211 animals. Results are as follows (Table 2).


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Table 2. value of sensitivity and specificity of diagnostic test (Courcoul et al., 2014) Test Se confidence interval Sp confidence interval

PCR 87% [82.5‐92.3] 97% [94‐99]

Histology 93.6% [89.9‐96.9] 83,3% [78.7‐87.6]

Bacteriology 78% [72.9‐82.8] 99,1% [97.1‐100]

The authors conclude that RT‐PCR is as predictable as the bacteriological test and therefore it can replace the latter. However, the authors emphasize the importance of isolation to obtain field strains for epidemiological purposes (molecular typing). The NRL is currently validating an RT‐PCR based on the insertion sequence IS6110. In 2015, on 360 samples submitted for isolation, a very good correlation between bacteriology and RT‐PCR was observed. The 11 bacteriologically positive samples were also positive in RT‐PCR. The conclusions of the NRL therefore agree with the results of the study mentioned above. In conclusion, RT‐PCR is a good option to obtain rapid and good quality diagnosis of M. bovis. Therefore, the RT‐PCR could be considered as an official tool for the diagnosis of bTB. The isolation of M. bovis remains relevant to allow molecular typing and epidemiology.

4.3 Interferon gamma test

4.3.1 Principle of the test

In this test, the release of the gamma interferon (IFN‐γ) lymphokine is measured in a whole‐blood culture system. The assay is based on the release of IFN‐γ from lymphocytes sensitized during a 16–24‐hour incubation period with a specific recall antigen (like PPD‐tuberculin). The test makes use of the comparison of IFN‐γ production following stimulation with avian and bovine PPD. The detection of bovine IFN‐γ is carried out with a sandwich ELISA that uses two monoclonal antibodies to bovine gamma‐interferon. Because the assay uses viable blood cells, it is recommended that the blood samples are transported to the laboratory and the assay is set up as soon as possible(usually within 8 h of collection to ensure optimal assay performance), but not later than the day of blood collection (OIE, 2009). Figure 3. Principle of the IFN‐y test (https://www.thermofisher.com/content/dam/LifeTech/global/applied‐sciences/pdfs/animal‐health/prionics_literature_tb_link4.pdf).

The in vitro IFN‐γ test was developed in Australia in the late 1980s and is recommended by the OIE since 1996 (OIE Terrestrial Manual) as ancillary laboratory‐based test to the tuberculin intradermal test. Most of the bovine TB control programs rely on the use of BOVIGAM® (Prionics, Switzerland) as parallel test to the intradermal test in order to maximize the detection of TB‐infected animals. The assay is accepted for use as ancillary test to the intradermal test by the EU since 2002 [Council Directive 64/432/EEC, amended by (EC) 1226/2002](Bezos et al., 2014). EFSA assessed whether IFN‐γ release assay (IFN‐γ test) could be added to the official armatorium and as a stand‐alone test for demonstration of bovine TB‐free herd status and testing for intra‐


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Community trade. Actually, in order to allow IFN‐γ as stand‐alone test, it should have a sensitivity equivalent or superior to the standard test currently used (SICT) in the EU and have a specificity not lower than the standard test with the lowest specificity (SIT) (EFSA, 2012a).

4.3.2 IFN‐γ test based on PPDs

Purified Protein Derivative (PPD) antigens are derived from M. bovis AN5 and M. avium D4. PPD has been used for ante‐mortem diagnosis of latent and active TB in humans and animals for more than 100 years (Bezos et al., 2014). Numerous field studies, conducted worldwide since 1991 compared the diagnostic performance of the tuberculin intradermal test and IFN‐γ test based on PPD (protein purified derivatives) from M. bovis and/or M. avium (from Prionics, BOVIGAM). These studies showed that:

- Se of IFN‐γ is as sensitive as the SICT (= Se similar); - Sp SICT is higher than IFN‐γ based on PPD; - Sp IFN‐γ based on PPD is similar to SIT (PPD of M. bovis alone) (EFSA, 2012a).

Based on a meta‐analysis of 15 field studies conducted between 1991 and 2006, an estimated median Se of 87.6% (with a range between 73% and 100%) and a Sp of 96.6% (with a range of 85% and 99.6%) were reported for the BOVIGAM® IFN‐γ assay (Bezos et al., 2013; de la Rua‐Domenech et al., 2006) According to the definition of suitability given above, PPD based IFN‐γ test can be included amongst the official tests for the purpose of granting and retaining an officially tuberculosis free herd status (EFSA, 2012a). The apparent higher Se of the IFN‐γ test compared to the SICT in some cases is likely due to the fact that the IFN‐γ test detects TB infected animals as early as 14 days following infection and 60–120 days earlier than the SICT test (Lilenbaum et al., 1999). More importantly, several studies in the UK (Coad et al., 2008) and Ireland (Gormley et al., 2006) have shown that animals negative for the intradermal test but IFN‐γ positive are more likely to be infected with M. bovis than cattle negative for both intradermal test and IFN‐γ and that removal of all animals reacting positive to one of the two tests is critical to controlling bovine TB outbreaks (Bezos et al., 2014). However, the report of EFSA highlights the fact that the Sp of IFN‐γ based PPD may not always be as high as the SIT test (depending on circumstances), the test with the lower Sp currently used in the EU. The Sp of the IFN‐γ test based on PPD could be influenced by factors such as the presence of environmental mycobacteria, prevalence of M. bovis infection in the herd, the age, the type of bovine, the bovine TB history of the animals, these factors could also influence the SIT and in less extent the SICT. A meta‐analysis has been realized by the Veterinary‐Laboratories‐Agency (VLA) in 2011. They have shown that the sensitivities of IFN‐γ based PPDB, PPDA and ESAT‐6/CFP10 were not significantly different from the sensitivities of SIT and SCIT (VLA, 2011). EFSA made an update of this meta‐analysis. They observed that the existing differences in the protocols used in the performance of the IFN‐γ test based on PPDB/A complicate the joint interpretation of the results of the studies reviewed. The effect of the different cut‐off values, as well as other differences in the protocols used in each study (different gold standards, collection of blood samples for the assay 3–10 days after a SCIT test), may explain the width of the confidence intervals reported. The variability in the specificity, ranging from 84% to 99%, is most likely also affected by the cut‐off value applied.


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Table 3. Summary of meta‐analysis results for sensitivity and specificity of diagnostic tests for bovine TB on cattle from AHVLA systematic review (EFSA, 2012(a))

4.3.3 IFN‐γ test based on specific antigens

The development of more specific antigens that can be used for blood stimulation in the IFN‐γ assay has been the “holy grail” of research efforts for improved diagnostic tools for bovine TB. Indeed, identification of specific antigens that are present only in M. bovis but are absent from environmental mycobacteria can significantly increase the Sp of diagnostic tests (Bezos et al., 2014). Besides the tuberculin, two antigens encoded in the region of difference 1 (RD1) and absent from environmental mycobacteria are becoming more and more actively used. These antigens are ESAT‐6 (early secretory antigenic target‐6KDa) and CFP‐10 (culture filtrate protein 10), and are absent from many Bacillus Calmette‐Guérin (BCG) vaccine strains. Research continues to identify other specific antigen, always with the aim to develop DIVA vaccines (differentiating infected from vaccinated animals) and at the end to replace PPD. Among them there are (reviewed by Bezos et al; 2014 and summarized in Table 4):

- Rv3615c (Mb3645c) is very promising as it allows the detection of animal negative with ESAT‐6/CFP‐10 and a combination of these 3 antigens increase the Se.

- Rv3020c: improves diagnostic Se without compromising of the Sp. - RV0899 (outer membrane protein OmpATB): complementary of ESAT‐6 and CFP‐10 - MPB70. - Combination of ESAT‐6/CFP‐10/Rv3019c/Rv0288/Rv3879c/Rv3873.


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Table 4. Sensitivity and specificity of the IFN‐y assay (BOVIGAM) in different studies in cattle using different recombinant proteins or peptides or a combination of them (Bezos et al., 2014)

For all the studies summarized in the table above, no antigen when tested singularly gave equivalent Se compared to the PPD. However, the use of a single antigen minimized the amount of false positive reactors in uninfected animasl (single antigen have a higher Sp compared to PPD). The combination of epitopes of different antigens increases the diagnostic Se without compromising the Sp (Cf. Table 4).

4.3.4 Use of IFN‐γ test as serial or parallel confirmation test

EFSA recommends to use the IFN‐γ test either concurrently or in parallel with the skin test. In general, a parallel implementation of tests increases the sensitivity of the diagnostic protocol (although it can also cause a decrease in diagnostic specificity). In certain countries/areas (usually free of disease the IFN‐γ test is used in the surveillance programs following non negative results to skin tests (serial use of the tests), in order to increase the specificity of the overall diagnostic protocol. The EU regulation, however, does not include this serial use of the IFN‐γ test (EFSA, 2012a). The use of the IFN‐γ assay in parallel with the SIT in infected herds results in a considerable increase in Se and this allows the earlier removal of a considerable number of infected animals that would have given a false negative reaction to the SIT and would otherwise have remained unidentified in the herd for an undetermined period of time. Parallel testing slightly reduces test specificity but may accelerate eradication of infection from the herd. Furthermore, in herds already deemed TB‐positive, the IFN‐γ assay should be considered for use at least for the first retest in parallel with the SIT to obtain the maximum Se (Strain et al., 2012; Task‐force, 2013). The IFN‐γ test is used in serial testing when there are concerns that the routine ante‐mortem test lacks specificity e.g. where the SIT is performed or in areas where there is a low likelihood of animals being truly infected (like in Belgium where relative high prevalence of MAP infection is observed (Van der Stede et al., 2010; Boelaert et al., 2000)). The IFN‐γ test is used in parallel in converse situations i.e. where there is a higher likelihood of infection and where there is concern that infected animals might be missed by one test and where reduced specificity is deemed acceptable (Strain et al., 2012).


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Diagnostic strategies including requirements for removal of individual animals testing positive, whole herd depopulation, and pre‐movement testing differ largely between countries and regions (EFSA, 2012b) and are summarized in Appendix VI. Also, there is a large variation on the test and testing protocols used in Europe. These variations refer to the use of the skin test and the ancillary test (IFN‐y test) but also, and more importantly, on the objectives to be achieved (EFSA, 2012b).

4.3.5 Influence of skin test on serial use of IFN‐γ

The performance of the IFN‐γ test can be influenced by various factors including a recent skin test and the delay between collection and processing of blood samples (Gormley et al., 2004). Schiller et al. (2010b) published a minireview (summarized in Tables 5 & 6) on the effect of the tuberculin test (SIT and SICT) on in vitro IFN‐y responses.

Effect of SIT on IFN‐γ in field studies PPD administrations neither boost nor depress PPD‐specific IFN‐γ production. More importantly, in naturally infected cattle, skin test related boosting increased specifically the in vitro M. bovis PPD (PPD‐B) reaction in comparison to PPDA 3 days after the skin test (Schiller et al., 2010b). Two studies analyzed the impact of the skin test on the IFN‐γ test:

- Coad et al. (2010) performed 4 skin tests at approximately 60‐days intervals: there was a boost of the relative PPD responses since 3 days post skin test; at 10 days, level of IFN‐γ returns to pre‐test. Repeated comparative skin test (SICT) lead to the desensitization of the reaction size but not of the IFN‐γ responses (Cf. Table 5 below, line n°5).

- Ryan et al. (2000) have estimated the Se and the Sp of the IFN‐γ test. The sensitivity was estimated as 85% (163 cattle infected with M. bovis from 21 herds). The specificity was estimated as 93% (213 cattle which had reacted to CFT (single intradermal skin test performed at the caudal fold) from 82 herds with no evidence of M. bovis infection). IFN‐γ was performed at 8 and 28 days post‐CFT.

This study supports the use of the IFN‐y test as a practical serial test (3 day interval) that can be used to complement the CFT (Ryan et al., 2000). However, CFT is not recommended in Belgium due to its lower sensitivity (see higher).

Effect of SIT on IFN‐γ in experimental infection Specific IFN‐γ responses were observed without any effect on the test interpretation in cattle experimentally infected with M. bovis.

- Boost of IFN‐y responses (PPDs) between 7 and 59 days post CFT, (n=4) (Rothel et al., 1992); - Sensitization with killed M. bovis AN5 (n=20), boost observed between 3 and 28 days

(Whipple et al., 2001); - Boost of the IFN‐γ responses (PPD‐B, PPD‐A, ESAT‐6:CFP‐10) between 3 and 7 days after CFT

in M. bovis‐infected cattle, (n=19) (Palmer et al., 2006); - Boost of the IFN‐γ responses (PPD‐B, PPD‐A, ESAT‐6:CFP‐10) 7 days after CFT in M. bovis‐

infected cattle; - Boost of the IFN‐γ responses (PPD‐B, PPD‐A, ESAT‐6:CFP‐10) between 5 days and 17 post‐SIT

(end of infection) n=4 (Roupie et al., 2015).

Effect of SIT on IFN‐γ in non‐infected animals It cannot be excluded that a mild boost of PPD responses occurs particularly in animals sensitized to environmental mycobacteria (i.e. M. paratuberculosis and others).

- No effect on non‐infected animal: low number of false negative (0‐2 animals) (Rothel et al., 1992);

- Effect on one non‐infected animal: false positive reaction (PPD‐B, PPD‐A, ESAT‐6:CFP‐10) between 5 to 17 days post‐infection, (n =2)(Roupie et al., 2015).

Table 5. Studies between 2008‐2012 describing the effect of CFT on IFN‐y test (Schiller et al., 2010b)


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Effect of SICT on IFN‐y No significant effect of SICT on the IFN‐g test was observed (at 3 days post‐SICT (Cf. Table 6). Table 6. Studies between 1994‐2010 describing the effect of CCT on IFN‐g (Schiller et al., 2010b)


Domesticated animals which are cohabiting with bTB‐positive cattle in similar conditions should be tested for their bTB status, as they may represent a potential risk to other susceptible species (Munoz‐Mendoza et al., 2015). In this context, the IFN‐y assay can represent a valuable tool in areas where surveillance plans in livestock, other than cattle, are strongly envisaged. The use of the IFN‐y test has been described in several domestic and wild species. For some species, an adaptation of the protocol used for bovines is necessary. More details about the use of the IFN‐y test in other species can be found in Appendix VII. For some animal species, in particular for goats, the IFN‐y test shows promising results. However, it must be emphasized that the number of studies which estimated the Se and Sp in non‐bovine animals are limited and that these studies generally make use of a small number of animals.


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Therefore, the practical use of the IFN‐y test in non‐bovine animals is currently not advisable and must be evaluated on a large scale under field circumstances before being used in eradication programs.

4.3.7 Conclusion

In the Belgian context, the use of the IFN‐γ release assay should be evaluated, in a first step in parallel with SICT or SIT in order to adjust the interpretation criteria (e.g. in low‐incidence areas the interpretation could be adapted to maximize the specificity of the test). The IFN‐γ release assay could be a very promising test provided a preliminary validation of the different kits and antigens is recommended. In addition, given the Belgian OTF status, the epidemiological context (trading, importing, cattle industry) should be considered in order to optimize the use of these tests (serially or in parallel) in order to be the most cost efficient. Besides tuberculin (PPDB and PPDA) or ESAT‐6 and CFP‐10 antigens, other specific antigens are under development and are currently evaluated under field conditions. Although none of the antigens gives equivalent Se compared to the PPD when used as single test, their combination will probably increase Se. However, the low amount of false positive reactors in uninfected cattle using specific antigens offers definitely promising perspectives to increase specificity. Improving specificity by the replacement of PPDs by defined antigens for stimulation, their application as an in‐tube/in‐plate stimulation device, in combination with a modified interpretation/cut‐off and cost reduction may represent useful developments for the IFN‐γ assay. Thus, the assay may be adapted to provide a highly specific and sensitive screening test for use as a stand‐alone test or in conjunction with other screening tests. In addition, a multispecies IFN‐γ assay for non‐bovine species, such as camelids, cervids, dogs and cats, would be a welcome tool for bTB screening and control in those species and for overall bTB control (Schiller et al., 2010a). Primary screening for bTB in live cattle is performed using one of the variants of the skin test: the caudal fold test (CFT), the (mid) cervical intradermal test (SIT), or the comparative cervical test (SICT). In addition, the interferon gamma (IFN‐γ) assay is applied either as a confirmatory test of reactors to the CFT or SIT (serial testing), or in alongside skin test (CFT, SIT or SICT) to increase diagnostic sensitivity (parallel testing) (Schiller et al., 2010a).

- The serial use of the IFN‐γ assay serves to increase the specificity of the overall testing protocol, i.e. an animal is deemed positive if it tests positive to another test (skin test) and to the IFN‐γ test.

- The parallel use of the IFN‐γ assay serves to increase the sensitivity, i.e. if an animal tests positive to another test or IFN‐γ test (Strain et al., 2012) in infected herds.

The application of the IFN‐γ assay is strongly influenced by the prevalence of infection in the animal population. Typically it is used as a serial test in circumstances where there is doubt over the specificity of the test routinely being used and as a parallel test in areas or herds with high disease prevalence in order to maximize the likelihood of disease detection (Strain et al., 2012). Invariably this leads to false positive animals although this will be to some extent mitigated by the moderately high positive cut‐off used by most countries. The second point to consider before using the IFN‐γ test in series with either SIT or/and SICT is the effect on injection of tuberculins during the skin test. Some authors suggest a boost of IFN‐γ production after SIT but not after SICT (Schiller et al., 2010a). However, this effect is poorly known up till now and further studies, including field studies, are necessary. These studies should also focus on non‐infected M. bovis cattle sensitized by environmental mycobacteria (e.g. MAP). Published field studies using both the CFT and the IFN‐y assay have been designed to focus solely on a general evaluation of the use of Bovigam® in cattle subjected to the caudal fold test, but not on the possible boosting or depression effects of CFT on the IFN‐γ response (Schiller et al., 2010b).


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In summary, based on data from studies with naturally infected animals, there is no evidence for the SICT to boost or depress mycobacterial‐specific IFN‐γ production. Furthermore, disparate results obtained in some studies with experimentally infected cattle could not be verified in naturally infected animals, thus emphasizing the importance to confirm findings in cattle naturally infected with bTB. Similarly to the CFT (not recommended in Belgium), there is a lack of data on the effect of SICT on the IFN‐assay in weak responders or in non‐infected cattle (Schiller et al., 2010b).

4.4 Serological tests

4.4.1 Introduction

There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic tuberculosis tests. ELISA appears to be the most suitable of the antibody‐detection tests and can be a complement, rather than an alternative, to tests based on cellular immunity notably to detect animals that are anergic and do not react to the skin test and IFN‐γ anymore (Cf. Figure 6). Figure 4. Usefulness of different diagnostic test in relation with the progression of bTB infection (Vordermeier et al., 2004).

Serological tests are in practice an easy, fast, reliable and cost‐effective option. Standard serum and plasma collected for other surveillance purpose are easy to handle, store and run. When high numbers of animals are tested within a herd, a reasonable confidence level in the serological test can be obtained.

4.4.2 ELISA Ab test IDEXX (MPB70 and MPB83)

ELISA Ab test IDEXX: literature review Waters and al. (2011) tested the performance of the IDEXX ELISA and found an apparent Se of 63% (range 30‐97, n=478) and a Sp of 98% (range 88‐100% n =1473), Cf. tables 7, 8 and 9 below. No cross‐reaction with MAP was observed. Indication of time sampling before SIT was not always precisely available. The Se increased as the disease severity increased (Waters et al., 2011).


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Table 7. Sensitivity of IDEXX M. bovis antibody ELISA with sera collected from naturally infected cattle (Waters et al., 2011). A) Infection status determined by histopathology with IS6110 PCR and/or culture. B) Infection status determined by culture or presence of gross lesions and/or from a tuberculosis‐affected herd. C) ID, identification. NVSL, National Veterinary Service Laboratory. D) n, number of animals. Three manufacturing scale lots were evaluated with each sample set (Lot 1, 2 and 3).

Table 8. Specificity of IDEXX M. bovis antibody ELISA with sera collected from non‐infected cattle from various geographic regions (Waters et al., 2011).a) Samples obtained from cattle from tuberculosis‐free herds. AHVLA, Animal Health and Veterinary Laboratories Agency; UCD, University College Dublin; DAFF, Department of Agriculture, Fisheries and Food. B) n: number of animals.

Source of noninfected seraa

nb No. of herds

Specificity (%)

Lot 1 Lot 2 Lot 3

Maine 126 2 99.2 98.4 99.2 Maine 126 2 99 98.4 99.2 Pennsylvania 79 1 88.6 92.4 98.7 Arkansas 39 1 100 100 97.4 New York 84 1 98.8 100 98.8 North Dakota 110 1 96.3 97.2 99.1 Washington 84 2 98.8 98.8 98.8 South Dakota 84 1 98.8 100 100 Missouri 92 >2 94.5 95.7 98.9 Texas 96 >2 93.7 93.8 95.8 Michigan 92 2 100 100 100 Iowa 8 1 100 100 100 Colorado 121 11 99.2 100 99.2 Great Britain (AHVLA) 50 >5 94 98 96 Ireland (UCD/DAFF) 92 16 100 100 95.7 Austria 316 >10 97.5 99.1 98.1

Overall value 1,473 >58 97.4 98.2 98.4

Buddle et al. (2013) evaluated the Se and the Sp of the IDEXX ELISA on milk samples. Se amounted 50% (n=22/44) (95% confidence limits, 35.8% and 64.2%) and Sp 97.5% (n=356) (95% confidence limits, 95.2% and 98.7%). There were 9 false‐positive responses out of 356 animals. The median S/P ratio response for these 9 positive responders was 0.41 (range, 0.30 to 2.20). Both milk and serum samples were collected from 38 of the 44 M. bovis‐infected animals and there was a strong positive correlation (Spearman’s rank test, rho= 0.89, P<0.001) between the responses in milk and serum samples from the same animals (Cf. Table 12). Table 9. Summarized results of the study of Buddle et al. (2013)

Milk ELISA result No. of animals with a serum ELISA result of: Total no. of

results Positive Negative Positive 17 3 20 Negative 3 15 18 Total 20 18 38 Dilution of 21 positive test milk samples in milk from non‐infected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Milk

M. bovis‐infected serum type Skin test nd No. of

herds Sensitivity (%)

Source ID or characterizationc Lot 1 Lot 2 Lot 3

Great Britain AHVLA‐2a 134 31 74.6 72.4 73.1

AHVLA‐1b 50 >5 86 88 86

Ireland No visible lesionsb 50 >5 48 44 46

With visible lesionsb 50 >5 72 70 70

Skin test positive, Bovigam positive, with visible lesions

b 30 22 96.7 86.7 90.0

New Zealand AgResearchb 2‐4 weeks after the CFT 42 7 42.9 40.5 35.7

USA Coloradoa 60 days before SIT 81 1 44.4 45.7 49.4

NVSL serum banka 31 12 48.4 48.4 48.4

Michigana 60 days before SIT 10 1 30 30 30

Overall value 478 >89 63.6 61.9 62.6


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samples can be substituted for serum samples for screening individual cows for M. bovis infection, because pooling of milk samples from 10 to 20 animals will result in a reduction in the sensitivity by approximately 50%. However, screening of milk tank samples is unlikely to be useful in countries with low prevalence of M. bovis in cattle and large herd sizes (Buddle et al., 2013) Casal et al. (2014), evaluated the use of ELISA Ab and Enferplex (Cf. below for Enferplex) before and after the skin test, alone or in combination with other tests on a limited panel of serum (Skin test and IFN‐γ) (Casal et al., 2014).

- Prior to the skin test: The Sp of M. bovis Ab Test (IDEXX) was 100% (n=60). These 60 animals were originated from tuberculosis free herds which were negative to the SIT test and the IFN‐γ assay. The Se was 23.9% (11/46).

- At 15 days post‐SIT: Cf Table 10. The M. bovis Ab Test (IDEXX) classified as positive 10.7% (6/56) of animals with samples collected prior to intradermal tests (Herd A), 7.1% (4/56) of animals with samples taken 72 h post‐intradermal tests, and the number of reactors increased to 57.1% (32/56) when samples were collected 15 days post‐intradermal test. The Se increased to 70.4% in confirmed infected animal and 92.3 % in animals with lesions.

Table 10. Number of animals detected as positive within herds A and B using the different diagnostic techniques (Casal et al., 2014). Serological techniques with samples collected 15 days post‐intradermal tests. a) Animals with M. bovis positive culture and/or presence of lesions compatible with tuberculosis. b) SIT, single intradermal tuberculin test (severe interpretation). c) Wilson's 95% confidence intervals. d) SICT, single intradermal comparative cervical tuberculin test (severe interpretation). e) IFN‐γ assay with samples collected in parallel with intradermal tests. f) Serological techniques with samples collected 15 days post‐intradermal tests. g) Inconclusive animals considered as positive. h) Inconclusive animals considered as negative.

Diagnostic techniques Herd A Herd B

Whole herd (n = 56)

Confirmed infected animals

a (n = 27)

Animals with lesions (n = 13)

Whole herd (n = 21)

Confirmed infected animals (n = 6)

Animals withlesions (n = 5)

Intradermal tests

SITb

28/56 (50%) (37%–63%)

c 16/27 (59.3%) (41%–75%)

9/13 (69.2%) (42%–87%)

11/21 (52.4%)

(32%–72%)

5/6 (83.3%) (44%–97%)

5/5 (100%) (56%–100%)

SICTd

13/56 (23.2%)

(14%–36%)

10/27 (37%) (21%–55%)

6/13 (46.1%) (23%–71%)

11/21 (52.4%)

(32%–72%)

5/6 (83.3%) (44%–97%)

5/5 (100%) (56%–100%)

IFN‐γ assaye

Cutoff 0.05 37/56 (66.1%)

(53%–77%)

25/27 (92.6%) (77%–98%)

12/13 (92.3%) (67%–99%)

10/21 (47.6%)

(28%–68%)

4/6 (66.7%) (30%–90%)

3/5 (60%) (23%–88%)

Cutoff 0.1 34/56 (60.7%)

(48%–72%)

23/27 (85.2%) (67%–94%)

11/13 (84.6%) (58%–96%)

8/21 (38.1%)(21%–59%)

3/6 (50%) (19%–81%)

3/5 (60%) (23%–88%)

Serological techniquesf

M. bovis Ab Test (IDEXX) 32/56 (57.1%)

(44%–69%)

19/27 (70.4%) (51%–84%)

12/13 (92.3%) (67%–99%)

9/21 (42.9%)(24%–63%)

4/6 (66.7%) (30%–90%)

3/5 (60%) (23%–88%)

Enferplex TB assay (Enfer)

g

38/56 (67.9%)

(55%–79%)

23/27 (85.2%) (67%–94%)

12/13 (92.3%) (67%–99%)

11/21 (52.4%)

(32%–72%)

5/6 (83.3%) (44%–97%)

4/5 (80%) (37%–96%)


h

36/56 (64.3%)

(51%–75%)

22/27 (81.5%) (63%–92%)

12/13 (92.3%) (67%–99%)

‐ ‐ ‐

ELISA Ab test IDEXX: Belgian experimental infection The diagnostic potential of IFN‐γ release assays and M. bovis specific serology was assessed by Roupie et al. (in preparation) in six naturally M. paratuberculosis (Map)‐exposed bulls: four of them were infected intratracheally with a Belgian field strain of M. bovis and two were kept as control


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animals. 17 blood samples were collected at several time points, SIT was performed on day 115 and animals were euthanized on day 132. Organs were collected and stored for histopathological examination, Map and M. bovis culture and Ziehl Nielsen analysis. ELISA Ab showed a weak but positive response in a bull between 56 and 84 dpi (days post‐infection) and in another bull at 42 dpi. After SIT, a dramatic increase in antibodies was observed, which resulted in a positive response in all four M. bovis infected bulls. In the two control animals, SIT also increased the MPB70/MPB83 specific antibodies, but to a clearly lesser extent than in the four experimentally infected bulls. Figure 5. Antibody responses of the two non‐infected bulls (ID numbers 1964 and 1954) and the four M. bovis infected animals (ID numbers 3174, 2148, 2149 and 1977) using IDEXX Mycobacterium bovis antibody kit (cut‐off value >30% indicated by red line). SIT was perfomed at 115 days post‐infection.

0 14 28 42 56 70 84 98 112

0

100

200

300

400

500

600

800

1,000

115 120 125 130 135

19541964 3174

2148

21491977

30

Times (days post-infection)

E/P

%

SIT also increased initially negative antibody responses against MPB70 and MPB83 to values above the positivity threshold, confirming previous findings (Casal et al., 2014; Waters et al., 2011). One animal non‐infected with M. bovis, was also positive in MPB70/83 specific serology, but the antibody titer was lower, indicating that a higher cut‐off value would be warranted to optimize specificity. These results indicated that, under experimental conditions, the sensitivity of MPB83/MBP70 serology was dramatically increased by prior skin testing. However this must still be investigated under field conditions (Roupie et al., in preparation).

4.4.3 EnferplexTM TB assay (chemiluminescent assay):

The Enferplex TB assay is a chemiluminescent multiplex system that can simultaneously detect and analyze antibody responses to multiple antigens (MPB70 recombinant, MPB83, ESAT6, CFP‐10, Rv3616c, α‐christallin 2 and MPB70 peptide) (Whelan et al., 2008) which are spotted in a single well of a 96‐well plate (Whelan et al., 2008; Whelan et al., 2010). Figure 6. Principle of EnferplexTM TB assay in 96 well plates.


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Whelan et al, 2008 reported a sensitivity and specificity of the Enferplex TB assay of respectively 93.1% and 98.4% in a non‐blinded Irish study. Table 11. Cohorts of samples tested by Enfer multiplex immunoassay and individual responses to ESAT‐6, CFP‐10, and MPB83 antigens. A) Data were extracted from the multiplex assay for these individual antigens

Test TB‐positive animals (no.) Sensitivity (%) TB‐negative animals (no.) Specificity (%) Enfer 522 93.1 1,489 98.4 ESAT‐6 522 40.6a 1,489 86.6a CFP‐10 522 82.6a 1,489 69.7a MPB83 522 78.5a 1,489 99.1a

In a second blind study with serum samples from cattle from Great Britain with and without bTB Whelan et al. (2010) compared the relative Se and Sp of the Enferplex TB serum assay with the SICT and the Bovigam IFN‐y test. Sera from 189 animals (96 SICT reactor animals with visible tuberculous lesions and culture‐confirmed bTB; 93 bTB free animals) were analyzed. The Se was significantly lower than in the previous study but the Sp was comparable (Cf. table below) (Whelan et al., 2010). Table 12. Performance of the Enferplex TB multiplex immunoassay on field animals. A) Measured for 96 SICT reactor animals, with visible lesions and positive M. bovis cultures. Both sensitivity and specificity are expressed as percentages, with 95% confidence intervals in parentheses. B) Measured for 93 skin test‐negative cattle from TB‐free herds

Enferplex TB cutoff level Sensitivitya Specificityb 5 77.1 (67.4, 85.0) 100 (96.1, 100) 4 79.2 (69.7, 86.8) 97.9 (92.4, 99.7) 3 81.3 (72.0, 88.5) 94.6 (87.9, 98.2) 2 82.3 (73.1, 89.3) 91.4 (83.8, 96.2) 1 86.5 (78.0, 92.6) 79.6 (70.0, 87.23)

Casal et al. (2014) evaluated the use of ELISA Ab (Cf. before) and Enferplex on a limited panel of serum before and after the skin test and alone or in combination with other tests (Skin test and IFN‐γ). The authors used two different cut‐offs: one for high Sp interpretation (a response to at least four antigens above the individuals’ thresholds) and another one for high Se interpretation (a response to at least two antigens above the individuals’ thresholds)

- Prior to the skin test: The Sp of Enferplex was 98.3% (59/60) and the Se was 32.6% (15/46). However the Se increases significantly at 15 days post‐SIT and reached 85.2%.(Casal et al., 2014).

- At 15 days post‐SIT: The Enferplex TB assay performed with samples collected prior to the intradermal tests (high Se criteria) yielded 12.5% positive animals (7/56; two inconclusive animals were considered as positive. When samples were drawn 72 h post‐intradermal tests the assay detected 25% (14/56) positive cattle. With samples collected at 15 days post‐intradermal tests 67.9% (38/56) positive cattle were detected.


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Table 13. Number of animals detected as positive in herds A and B using the different diagnostic techniques (Casal et al., 2014). Serological techniques with samples collected 15 days post‐intradermal tests. a) Animals with M. bovis positive culture and/or presence of lesions compatible with tuberculosis. b) SIT, single intradermal tuberculin test (severe interpretation). c) Wilson's 95% confidence intervals. d) SICT, single intradermal comparative cervical tuberculin test (severe interpretation). e) IFN‐γ assay with samples collected in parallel with intradermal tests. f) Serological techniques with samples collected 15 days post‐intradermal tests. g) Inconclusive animals considered as positive. h) Inconclusive animals considered as negative.

Diagnostic techniques Herd A Herd B

Whole herd (n = 56)

Confirmed infected animals

a (n = 27)


Whole herd (n = 21)

Confirmed infected animals (n = 6)


Intradermal tests

SITb

28/56 (50%) (37%–63%)

c 16/27 (59.3%) (41%–75%)

9/13 (69.2%) (42%–87%)

11/21 (52.4%)

(32%–72%)

5/6 (83.3%) (44%–97%)

5/5 (100%) (56%–100%)

SICTd

13/56 (23.2%)

(14%–36%)

10/27 (37%) (21%–55%)

6/13 (46.1%) (23%–71%)

11/21 (52.4%)

(32%–72%)

5/6 (83.3%) (44%–97%)

5/5 (100%) (56%–100%)

IFN‐γ assaye

Cutoff 0.05 37/56 (66.1%)

(53%–77%)

25/27 (92.6%) (77%–98%)

12/13 (92.3%) (67%–99%)

10/21 (47.6%)

(28%–68%)

4/6 (66.7%) (30%–90%)

3/5 (60%) (23%–88%)

Cutoff 0.1 34/56 (60.7%)

(48%–72%)

23/27 (85.2%) (67%–94%)

11/13 (84.6%) (58%–96%)

8/21 (38.1%)(21%–59%)

3/6 (50%) (19%–81%)

3/5 (60%) (23%–88%)

Serological techniquesf

M. bovis Ab Test (IDEXX) 32/56 (57.1%)

(44%–69%)

19/27 (70.4%) (51%–84%)

12/13 (92.3%) (67%–99%)

9/21 (42.9%)(24%–63%)

4/6 (66.7%) (30%–90%)

3/5 (60%) (23%–88%)


g

38/56 (67.9%)

(55%–79%)

23/27 (85.2%) (67%–94%)

12/13 (92.3%) (67%–99%)

11/21 (52.4%)

(32%–72%)

5/6 (83.3%) (44%–97%)

4/5 (80%) (37%–96%)


h

36/56 (64.3%)

(51%–75%)

22/27 (81.5%) (63%–92%)

12/13 (92.3%) (67%–99%)

‐ ‐ ‐


A number of serologic diagnostic tests have been developed for use in other species than bovines. More details about these tests can be found in Appendix VIII. In general, it can be concluded that these studies are based on a too small number of animals and herds on order to estimate reliable Se and Sp values. Hence, the practical use of these tests in other species then bovines is currently not recommended.

4.4.5 Applicability of an ELISA in the Belgian cattle population

Applicability of an ELISA in the Belgian cattle population was evaluated by Roelandt et al. (2011). Commercial serological tests for diagnosis of bTB based on specific recombinant antigens (MPB70 and MPB83) (M. bovis Ab indirect Ab ELISA test, Idexx Laboratories) approved by OIE were evaluated on serum samples from cattle herds. The populations were defined as following: “….The “Positive” population = 500 plasma samples obtained between 01/02/06 and 31/01/11 from confirmed bTB outbreak herds based on positive bacterial culture. Animals from these herds were sampled within the 3 weeks preceding and 7 months following the time the ‘official BTB‐free (T4) status’ of the herd was suspended. The “Negative” population = 1000 plasma samples obtained from herds that remained officially BTB‐free since 2005. All samples had been tested with the Pourquier paratuberculosis ELISA (PTB). Some of


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the samples were simultaneously tested for BTB in the IFN assay, but none had been tested with the SIT or culture diagnostic tests, given their BTB‐free status. 25.6% “suspect” animals from positive outbreak herds tested negative for all index tests (SIT, IFN‐y and culture), and since each of these imperfect index tests may underestimate the true prevalence, the true bTB‐infected prevalence within the population was estimated to be between 75% and 100% and suitable for determination of DSe of the ELISA and for the other index tests ...” Results of this Bayesian analysis demonstrated a low sensitivity of the ELISA (12.22‐16.19%). Despite the robust method used for evaluating this test, caution should be made about the study population and sampling design. Indeed, due to unavailability of more reliable data, inference about the population status had to be made, but this approach, does generate bias and hampers the extrapolation of the study results. The selection bias linked to the estimated “positive population” arises from the fact that only cattle that underwent a SICT in a outbreak herd were considered. However, due to the fact that this SICT depends on the results of a beforehand realized SIT and both tuberculin tests have imperfect sensitivity and specificity, this will generate an underestimation of the true ‘animal’ status and thereby will impact all prevalence estimations. This under‐over‐estimation must be accounted for. A within herd prevalence of 75‐100% is unrealistic when looking at outbreak data between 2005 and 2015. Furthermore, because only cattle positive by SICT and sent for culture to the NRL were considered, and not the whole herd, in turn, will disable to correct the within herd prevalence. Therefore the individual status of cattle not tested within those herds cannot be estimated. Finally, considering the imperfect sensitivity and specificity of visual post‐mortem inspection and SIT at purchase, the true status of the “negative population” might be questioned since these herds were not tested during the study. Also as negative populations did not undergo the same testing protocol, positive and negative populations might not be totally comparable. Therefore inference about test sensitivity and specificity is limited.

4.4.6 Conclusions

Several tests for serological diagnosis of bovine tuberculosis exist and a commercial test based on MPB83/MBP70 (M. bovis Ab test, IDEXX Laboratories) is available and approved by OIE. This test could be used as complementary test to confirm bTB infection or to detect M. bovis infected cattle which are anergic to the skin test and/or the IFN‐γ release assay. In order to increase the sensibility of this antibody assay, it is actually recommended to perform serological testing after the SIT (2 weeks) as this could be useful to detect a subpopulation of infected animals that may not react anymore to the skin tests (Casal et al., 2014). The skin test boosts the responses of certain antigens (i.e. MPB83, MPB70), while it does not influence the response to other antigens (e.g. ESAT‐6, CFP‐10, MPB59, MPB64). This boost effect is not the same for all antigens considered. The quality of the response (i.e. avidity) is also increased (Waters et al., 2015). As the specificity is high, antibody ELISA is, in practice, an easy, fast, reliable, and cost‐effective option. Standard serum and plasma (or individual milk) collected for other surveillance purposes are easy to handle, store and run. The greatest sensitivity of TB detection has been achieved for ELISA / Enferplex after skin test for bovines (Casal et al., 2014). A lot of serological tests have been developed beside the IDEXX ELISA like Enferplex, rapid test (RT)/Stak‐Pak, dual‐path platform (DPP) and multiantigen print immunoassay (MAPIA) and are actively used in non‐bovine animals. Among those, the RT and the DPP are the most used, probably due to their simplicity. The greatest sensitivity of TB detection has been achieved when a combination of tests was used, such as a combination of RT or DPP with the skin test for deer (Boadella et al., 2011) or IFN‐y test and serology combined for alpacas (Chambers, 2013; Rhodes et al., 2012). The species for which data have been published has been extended considerably to include a variety of different species during a survey of bovine TB in Ethiopian wildlife, New World


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camelids, black rhinoceros, lions, elephant and a single pygmy hippopotamus, (Cf. Appendix VIII (Chambers, 2013)). The main limitation of these studies (bovine and non‐bovine animals) is the small number of animals/herds used for estimating accurate values of the sensitivity and specificity. For some species data are still lacking. Furthermore, it would be unwise to rely on estimates solely obtained from experimentally infected animals (Chambers, 2013).

4.5 Combination of diagnostic tests

The use of multiple tests is often used in screening programs to improve the overall diagnostic process. In case of serial testing; only samples that react positive to the first test are further tested with a confirmation test. When a sample reacts positive on both tests, the sample is considered positive. In case of parallel testing each sample is tested by two tests and is considered positive if a positive reaction on one or both of the tests is observed. Serial testing increases specificity but may decrease sensitivity, whereas parallel testing increases sensitivity but may decreases specificity. In a serial process, the test with the highest sensitivity should be carried out first as this will drive the whole process; this guarantees that a minimum of infected animals / herds are missed (false negatives). Tests are considered conditionally independent if the probability of getting positive at one test does not depend on the result of the first test (i.e. non infected animal will have the same probability of testing SICT positive regardless whether the animal tested positive or negative at SIT). If events are conditional and nondependent then the sensitivity and specificity of serial and parallel testing will be the following (Dohoo et al., 2009) (Table 18): Parallel: Sep= Se1 + Se2‐(Se1*Se2) Spp=Sp1*Sp2 Serial : Ses=Se1*Se2 Sps=Sp1+Sp2‐(Sp1*Sp2) Se=Sensitivity of the process considering parallel testing (p) or serial testing (s) with first test sensitivity Se1 or second test Se2 Sp=Specificity of the process considering parallel testing (p) or serial testing (s) with first test specificity Sp1 or second test Sp2 Note that if animals tested negative in test 1 then it is most likely to test negative as well in test 2. These events become conditionally dependent and the expected overall sensitivity or specificity might be different from observed, depending on the correlation between both tests. The covariance would have to be estimated first to enable corrections of the estimations obtained from the formulae above. In Table 14, the theoretical Se and Sp of different combinations of diagnostic tests can be found. These results can be used by risk managers to obtain the desired Se en Sp for future bTB surveillance.


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Table 14. Added value in terms of sensitivity and specificity for single and a combination of different diagnostic tests under conditional independent events

Test Se Sp

min max mean min max mean

ELISA* ;*** 41,00% 99,00% 81,00% 53,00% 100,00% 89,00%

IFNg* 36,00% 100,00% 78,00% 70,00% 100,00% 94,00%

SIT* 27,00% 82,00% 53,00% 55,00% 95,00% 99,00%

SICT*; ** 17,63% 63,33% 31,01% 77,79% 100,00% 99,50%

SIT//SICT 39,87% 93,40% 67,58% 42,78% 95,00% 98,51%

SIT‐>SICT 4,76% 51,93% 16,44% 90,00% 100,00% 100,00%

SIT//ELISA 56,93% 99,82% 91,07% 29,15% 95,00% 88,11%

SIT‐>ELISA 11,07% 81,18% 42,93% 78,85% 100,00% 99,89%

SIT//IFNg 53,28% 100,00% 89,66% 38,50% 95,00% 93,06%

SIT‐>IFNg 9,72% 82,00% 41,34% 86,50% 100,00% 99,94%

IFNg//ELISA 62,24% 100,00% 95,82% 37,10% 100,00% 83,66%

“//”parallel testing “‐>”Serial testing *Test sensitivity and specificity values (min, mean, max) are those obtained from the table published in Bezos et al., 2014 **SICT is known to have lower Se values then the SIT and in contrast higher specificity then SIT (Bezos et al., 2014; EFSA, 2012) ***Very few publications on demonstration of a ‘booster effect’ in naturally infected cattle exist =>not

accounted for this priming effect.

4.6 General conclusion

The Scientific Committee has performed a literature review to characterize and evaluate different diagnostic techniques for bTB in bovines which can be used in bTB surveillance in Belgium. The intradermal skin test or delayed hypersensitivity test involves the intradermal injection of bovine tuberculin purified protein derivative (PPD) and the subsequent detection of swelling (delayed hypersensitivity) at the site of injection 72 hours later. This may be performed using bovine tuberculin alone (SIT) or as a comparative test using avian and bovine tuberculins (SICT) to differentiate between an infection with M. avium and M. bovis respectively. The test should be performed in the anterior neck area to render the sensitivity of the test as high as possible. In literature, a wide range of sensitivity (Se) and specificity (Sp) values are reported: Se between 53% (27.3‐81.5, 95% CI) and 69.4% (40.1‐92.2, 95% CI) depending on cattle type; Sp between 55.1% and more than 99% showing a median value over 95%. Indeed, a lot of technical and socio‐economical parameters can affect the results of the intradermal skin test. The bacterial culture is the gold standard method for the diagnosis of bTB and, according to the current Belgian legislation, the solely isolation of the bacteria remains the definitive proof for the confirmation of an outbreak. However, bacterial culture is time consuming (8‐12 weeks). Methods which allow a result in 2‐3 weeks exist, but are not validated in veterinary medicine. The existence of a single recommended protocol for bacterial culture of Mycobacteria is not present in literature or in reference manuals. Therefore, validation of the culturing protocol in each laboratory is necessary. The development of new bacterial media or methods needs to be put in perspective with the development of molecular methods (RT‐PCR). Molecular tests always surpass bacteriology in terms of rapidity in results.


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There are various molecular methods to detect M. bovis. However, the best molecular method in term of Se and Sp is the real‐time PCR (RT‐PCR) with a Se of 87% and a Sp of 97%. The method is a good option to obtain rapid and good quality diagnosis of bTB. Therefore, the real‐time PCR should be considered as an official tool for the diagnosis of bTB. However, the isolation of M. bovis remains relevant to allow molecular typing and epidemiology. The interferon gamma test measures the release of the gamma interferon (IFN‐γ) lymphokine in a whole‐blood culture system. The assay is based on the release of IFN‐γ from lymphocytes sensitized during a 16–24‐hour incubation period with a specific recall antigen (like PPD‐tuberculin). The detection of bovine IFN‐γ is carried out with a sandwich ELISA that uses two monoclonal antibodies to bovine gamma‐interferon. Because the assay makes use of viable blood cells, it is recommended that the blood samples are transported to the laboratory and the assay set up as soon as possible (usually within 8 h of collection to ensure optimal assay performance), but not later than the day of blood collection. Based on a meta‐analysis of 15 field studies conducted between 1991 and 2006, an estimated median Se of 87.6% (with a range between 73% and 100%) and a Sp of 96.6% (with a range of 85% and 99.6%) is reported. Also, a possible boost of IFN‐y production after skin test is reported in literature. Moreover, infected animals are faster positive in the IFN‐y test than in the skin test. To conclude, the IFN‐y test is a very promising test but validation of the different kits and antigens must be performed under Belgian field conditions. There are several serological tests for bTB. The ELISA test appears to be the most suitable of the antibody‐detection tests and can be a complement, rather than an alternative, to tests based on cellular immunity notably to detect animals that are anergic and do not react to the skin test and IFN‐γ anymore. The test is an easy, fast, objective, and cost‐effective option for bTB surveillance. However, the Se of the ELISA is low (estimated at 63% with a range between 30% and 97%) while the Sp is estimated at 98% (with a range between 88 and 100%). A boost of antibodies after the skin test (2 weeks) is reported in literature. Furthermore, a simulation exercise has been performed to calculate the theoretical Se and Sp of different combinations of diagnostic tests. These results can be used by risk managers to obtain the desired Se en Sp for future bTB surveillance. Finally, the use of every diagnostic test in non‐bovine animals has been evaluated. In general, it can be concluded that these studies are based on small numbers of animals and herds for estimation of the values for Se and Sp and, for some animal species, data are still lacking. Hence, the practical use of these tests in other animal species is currently not advisable.

5 Evaluation of the current Belgian bTB surveillance program

5.1 Facts and figures

- Annual number of purchased cattle (SANITEL): ± 650 000 (± 48% fattening calves (315 000) and ± 52% other cattle (335 000)): The annual % of positive and doubtful reactions in SIT at purchase in the ‘other cattle’ category varied in 2011 and 2014 respectively between 0.0020%‐0.0025% and 0.0005%‐0.0043%. The ‘fattening calves’ category is not subjected to mandatory tuberculination at purchase.

- Annual number of slaughtered cattle (SANITEL): ± 837 470 animals (± 40 % fattening calves (336 281) and ± 60% other cattle (± 335 000)). The annual % of suspected lesions sent to the NRL varied between 0.0013% and 0.0081% (respectively in 2011 and 2014).

- Since 2008, all outbreaks in Belgium have been detected by slaughterhouse surveillance and by tracing on and back. No outbreaks were detected via tuberculination at purchase of animals issued from an infected herd.


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5.2 Scenario tree analysis

Welby et al. (2012) performed a scenario tree analysis in order to evaluate the sensitivity of the different bTB components of the Belgian surveillance program to prove freedom of disease at the 0,1% maximum herd level prevalence (as prescribed by Directive 64/432/EEC). In this study, the confidence in freedom of disease obtained was 83%, 85%, 99% and 99% for respectively the following surveillance components i) only herd testing by tuberculination during winter of outbreak herds, contact herds and suspected herds ii) only testing of imported cattle iii) testing of all purchased animals and iv) testing of all slaughtered animals. The outcome of the model (scenarios) was mostly influenced by the following parameters: the number of tested animals per component (the more the better) as well as the used sensitivity (Se) for each of the test(s) (decreasing test Se at animal level significantly reduces the component Se). In addition, within that same study, external validation of the output using a logistic regression model (using data over the last 5 years) showed that herd tuberculinations (during winter) and slaughterhouse post‐mortem inspections were significant components to detect outbreaks in Belgium. This was not the case for the purchase component, despite purchase has been identified as a risk factor for infection (Table 15). Table 15. Odds of detection of different surveillance components in Belgium using historical data

Surveillance component Odds of detection

Purchase 0,39 (p‐value=0,91)

Slaughter 1,69 (p‐value =0,01)

Tracing on and back 1,34 (p‐value=<0,0001)

Welby et al. (2012) concluded that assumptions used for the scenario tree analysis (all tests and all animals are performed/tested according to appropriate guidelines (Good Veterinary Practices)) might not be valid in ‘reality’. Indeed, findings over the last years indicated that the purchase component (in its current application) does not seem to be helpful in (early) detection of outbreaks in Belgium. In general, the purchase component should be an adequate ‘early detection’ surveillance component for animal health surveillance in general and bTB in particular (Humblet et al., 2010). Any bTB test performed in an appropriate way at purchase can be informative about a possible route of introduction for bTB as trade/movement is considered as an important risk factor (Welby et al;, 2012; Humblet et al., 2009; Gilbert et al., 2005).

5.3 Expected true and false positive reactions

Another way to determine the efficacy of a surveillance system is to estimate the expected number of false positive reactors given a free status by taking into account the sensitivity and specificity of the diagnostic tests used in the surveillance system. Welby et al. (2015) performed such a benchmarking study for the different components of the Belgian bTB surveillance program. The aim of this study was to investigate the number of true and false positive reactors that could be expected given predefined design prevalence and taking into account the diagnostic tests characteristics of sensitivity (Se) and specificity (Sp) of the SIT at purchase, as well as post mortem inspection at slaughter house. The input parameters for this study are summarized in Table 16, while the results are summarized in Table 17. Table 16. Input parameters for the benchmarking study according to Welby et al. (2015)

Parameters Value Sources

Intradermal skin test (SIT) Se 0.94 (0.49 ‐1.00) EFSA scientific opinion 2012 Intradermal skin test (SIT) Sp 0.91 (0.70 ‐1.00) EFSA scientific opinion 2012 Inspection at Slaughterhouse Se 0.71 (0.38 ‐0.92) EFSA scientific opinion 2012


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Inspection at Slaughterhouse Sp 1.00 (0.99‐1.00) EFSA scientific opinion 2012 Prevalence herd level 0.1% Directive 64/432/EEC Prevalence animal level 0.1%‐0.01%‐0.001% Simulations

Table 17. Expected true, false and total positive reactors for tested beef categories in the different surveillance components (purchase (PUR) and slaughterhouse (SLGH)) – adapted from Welby et al. (2015)

Simulated animal prevalence

False positives True positives

PUR SLGH PUR SLGH

0.001%

Mean 1.33215% 0.02465% 0.00011% 0.00010%

Minimum 0.00602% 0.00000% 0.00006% 0.00006%

Maximum 3.37939% 0.12777% 0.00012% 0.00014%

0.010%

Mean 1.31266% 0.02504% 0.00105% 0.00104%

Minimum 0.02712% 0.00000% 0.00064% 0.00058%

Maximum 3.51198% 0.12532% 0.00120% 0.00138%

0.100%

Mean 1.31448% 0.02489% 0.01049% 0.01035%

Minimum 0.02203% 0.00000% 0.00651% 0.00594%

Maximum 3.26768% 0.11891% 0.01200% 0.01374%

The comparison of these results with the actual percentage of declared reactors in Belgium (see Facts and figures) highly suggests an underreporting during purchase surveillance of bTB in contrast to the mean expected reactor rate of 1.3 %. USDA (2015) has published a minimum expected reactor rate of 1% (USDA, 2015) and thereby corroborates the study of Welby et al. (2015). The apparent underreporting at purchase surveillance is further corroborated by the fact that, during the winterscreening, a reactor rate of 1.8 % was observed in a comparable population. If the model is run again with the latest Se and Sp values of Visavet (2015), the number of false positive reactors would be as high as 2.5% instead of 1.3% calculated with Se and Sp values according to EFSA (2012).


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5.4 Estimation of the direct and indirect costs

Direct and indirect costs of the bTB program are depicted in the Table 18. Table 18. Summary of direct and indirect cost obtained from available data in Belgium

Sanitary Fund : total costs bovine tuberculosis surveillance 2014: tuberculinations

Winterscreening Sanitary Fund veterinary fees € 382.452,59

Sanitary Fund compensations for

slaughtered animals of outbreak herds € 971.663,00

SIT at purchase Farmer cost for purchase tuberculin test € 519.331,59

FASFC Tuberculins (bovine & avian) 84.387,73

Total € 1.957.834,91

TUBERCULINS average yearly cost (paid by FASFC)

Average per year/2002‐2014 Number Price/unit Total

Bovituber 2ml flacon 20doses 13468 € 3,01 € 40.538,68

Bovituber 5ml flacon 50doses 5296 € 6,75 € 35.748,00

Avituber 2 ml flacon 20doses 795 € 10,19 € 8.101,05

Total € 84.387,73

Estimation costs of the SIT at purchase paid by farmer in 2014

Number Price/unit Total

SIT at purchase 310.977 € 1,67 € 519.331,59

Visits for SIT at purchase* 31.000 €21,23 € 658.130€

Total € 1.177.462

*Visits tuberculinations at purchase = 31.000 x21,23€ = 658.130€ (if we take into account that on average 10 animals are purchased per

year per holding)

Considering the relatively low declaration rate of positive or doubtful reactions during the tuberculin testing at purchase, its effectiveness in the current surveillance program can be questioned. In addition, based on the estimated costs of purchase testing of 1.177.462 € for 2014, the cost‐benefit analysis is questionable. It must be reminded that during the period 2008‐2015, no outbreaks were detected following tuberculin testing at purchase. Yet, most outbreak herds are detected at slaughterhouse or following tracing‐on or ‐back of contact herds in which there has been introduction of purchased cattle subjected to a SIT that turned out “presumably” negative (no individual data recording). In addition, the sometimes high within‐herd prevalence of reactors in a newly detected outbreak combined with the rather chronic stage of infection of infected cattle (generalized lesions on organs and carcass and latent infected cattle) raises serious doubts about the “early warning” efficacy of purchase testing in its current field application. Furthermore, the overall indirect costs generated by the outbreak herds (1,500,000€) discovered only at late stage of infection by slaughterhouse inspection together with the fact that slaughterhouse surveillance is not 100% sensitive, emphasizes the need to adapt the surveillance program in order to reduce costs and to increase the effectiveness and awareness.


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New diagnostic tools have become available and should be considered in a changing epidemiological context of larger herd sizes and increased animal trade within holdings:

- Considering the estimated cost of the IFN‐γ test (25€/test), it is clear that using the test as first line screening tool will lead to significant increased costs for the surveillance. However, the IFN‐y test could definitely be used as a confirmation ancillary test of positive or doubtful results at SIT or SICT (cf. Table 18 further on), as already applied in many other countries (Schiller et al., 2010).

- Considering the estimated cost of an Ab ELISA (about 8€/test), this test could be of added value as general screening test (cf. Table 18 further on) providing sufficient specificity, and allowing detection of an outbreak before slaughterhouse surveillance and/or SIT at purchase in its current form.

5.5 Conclusion

Despite the fact that Belgium has the OTF status for bTB, almost every year some outbreaks are detected. The findings above indicate that, based on animal movements and slaughterhouse data and simulations of different diagnostic tests characteristics (sensitivity and specificity), the number of positive reactors notified at purchase and the number of suspected lesions submitted from slaughterhouse are estimated as too low even considering the low bTb prevalence. This was further corroborated by field observations (Humblet et al., 2011a; Welby et al., 2015). Hence, these results highly suggest that the efficacy of the current surveillance program, which especially makes use of SIT as first line screening test, could be improved.

6 Risk factors of infection and spread to be considered in bTB surveillance

Biological variation, such as stage of infection, past bTB status of the herd but also non‐biological causes such as socio‐economic drivers can provide explanations for differences between similar eradication and surveillance programs applied in different countries. To tackle this problem, national and international regulations have evolved allowing risk managers to adapt the testing scheme (test, sample size and sampling frame) taking into account the heterogeneity in local risk factors (EFSA, 2012, 2013, 2014; More et al., 2009; Welby et al., 2012). This approach for surveillance has the potential to provide higher confidence of freedom of disease and is more cost effective than surveillance based on a prescribed testing scheme (Cameron, 2012). The different possible risks factors of infection/spread to account for during monitoring & surveillance, can be summarized according to a 3 level approach (animal, herd and global) as described by Humblet et al., 2009. In addition, it is recommended to consider risk factors in an anchor model that looks at the interaction between hosts, pathogen and environment including risk of spread linked to social, political, educational or economic incentives as introduced by EFSA (2014). For readability purposes, the issues regarding risk factors and surveillance are all summarized in a single table (Table 19). Risk factors indicated by an asterix * were significant risk factors for bTB in Belgium (Welby et al., 2012; Humblet et al., 2010). Table 19. Major risk factors described in scientific literature (adapted from Humblet et al., 2009)

Level Risk factor of infection/spread Source

Animal Breed/ Gene csˆ

Genderˆ

Age

Feeding of milk/colostrum

Malnutrition

Immunosuppression/drugs

Limita on of diagnos cs tests*ˆ

Bekara et al., 2016; Guta S, et al., 2014; Humblet et al., 2009; Schiller et al., 2010; Welby et al., 2015


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Herd History of bTB*ˆ

Climate (which may influence the environmental persistence of Mycobacteria)

Contact with infected domestic reservoir (cattle, goats, sheep, dog, cat)ˆ

Contact with wildlife (fox, badger, dear, wildboar, opossums)ˆ

Shared pasture

Import of camelids for which diagnostic test efficacy are very poor

Density *ˆ

Herd size*ˆ

Management/Biosecurityˆ

Reduced human contact

Type of ca le industry*ˆ

Contact between cattle

Movements: Purchase*ˆ

Reduced veterinary service, testing*

Limited control measures (partial vs total depopulation)

False negative reactions (anergy or stress)

Infected cattle exempted of testing schemes (i.e. <6weeks old, etc.) lower test frequency

Atkin et al., in press; Bekara et al., 2016; Conlan et al., 2012; Guta S, et al., 2014; Humblet et al., 2009, 2010; Welby et al., 2012

Country Lack of data at individual animal level*ˆ

Poor data recording & centralization & sharing of information of data constitute the main limitations for effective follow up of outbreak herds and cattle (sensitivity & specificity)

Constraints containment of infected herd/area*ˆ

Fear of repressive measures contributes the apparent poor disease awareness and notification amongst farmers and therefore lack of compliance to surveillance (also been demonstrated in other EU member states)

EFSA, 2014; Elbers et al., 2010; Guta et al., 2014; Jansen et al., 2012; Lupo et al., in press; More et al. 2015

Global Bovine tuberculosis prevalence

International, interregional movements: intracommunity trade, import/export third countries

Translocation of wildlife animals

Humblet et al., 2009

* Risk factors specifically identified in Belgium ˆMost significant risk factors identified in Belgium and confirmed elsewhere (Adkin et al., in press; Békara et al., 2016; Humblet et al., 2009; Schiller et al., 2010; Welby et al;, 2012 )

Exploring the animal movement registration data (SANITEL) and merging it with the historical surveillance data provided insights on the main risk factors for bTB in Belgium. History of bTB is a major risk factor in Belgium (Humblet et al., 2010; Welby et al., 2012). This has also been demonstrated elsewhere (Conlan et al., 2012; Guta et al., 2014). However, purchase of cattle constitute a significant risk factor (Humblet et al., 2010), investigation of the surveillance component performance for proving freedom of disease and cross validation using historical data revealed on the contrary that testing ‘at purchase’ (in its current application) was not at all effective in detecting cases in contrast to tracing on and back as well as slaughterhouse visual inspection (Welby et al., 2012), as explained before.


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7 Recommendations

7.1 Data collection and data warehouse

There is a need for increased standardized data sharing between the actors of the TB surveillance network. Preferably outbreak field data, results of post‐mortem inspections, results of individual and herd SIT testing, molecular typing of isolates, … should be stored in a centralized database by the FASFC and should be consultable for all actors involved in the network to be able to perform a risk based surveillance taking into account bTB history (see below). Surveillance should be adapted in function of the herd risk score. It is emphasized that good quality and reliable information is a crucial back bone to correctly identify risk score level. Lack of data and poor data recording & centralization or share point are main limitations for effective follow up of outbreak herds and cattle. By extension, such a database can also be be used for other diseases. Medical laboratories (human medicine) should notify detection of M. bovis in humans to the FASFC and vice versa because of the zoonotic aspect of the disease. Inspection results of slaughterhouses and private veterinarians regarding their functioning within the bTB surveillance programs should be collected and used for evaluation purposes.

7.2 Risk based surveillance and proportionate measures

If a well‐managed databank exists, it is strongly recommended to adapt the control measures and their duration (i.e. blocking of farms) based on indicators allowing to allocate a risk profile to animals and/or herds (Adkin et al., in press; More et al., 2015). Key indicators of such a risk profile can be inspired by known risk factors already published and recognized to constitute a major risk of either infection or detection (Table 19). It must be borne in mind that risk is the result of the product of the probability of occurrence with consequence. Both notions must be accounted for (Cameron, 2012). Also it is recommended to consider indicators at herd level rather than at individual level. Indeed, to avoid possible false negative results that could occur at individual level either due to epidemiology of infection (latent versus residual infection) or desensitization (linked to frequent testing which decreases the sensitivity of animal detection). The latter explains why it is also important to extend intervals between testing (minimum >6month) at herd level to allow the progress of diseases and to permit the detection of infected animals in a herd (Guta et al., 2013, 2014; More et al., 2015). Lack of data, lack of centralization or share point and poor data recording are the main limitations for an effective follow up of outbreak herds and cattle which have resided on previous outbreak herds. Surveillance should be adapted as a function of a risk profile at herd level (inspired from known risk factors). To allow this, access to good quality information is required. In summary, proportional measures at herd or animal level should be taken based on the following key elements: For herds with higher risk probability of infection:

- Past bTB status at herd and animal levels (negative and positive results) (SANITEL) - Imports (SANITEL, TRACES) - Trade (SANITEL, TRACES) - Herds that were not screened for more than 5 years - Doubtful reactions - Herds with poor biosecurity measures - Herd density or size


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- Herds exempted of testing scheme (i.e. Herds that have not been tested for more than 5 years)

7.3 Raising awareness of actors in the field

As explained above the performance of the Belgian bTB surveillance program, especially during purchase testing, should be improved. To remediate this situation it is recommended to raise awareness of all actors involved. Veterinarians and hunters (wildlife surveillance) should be stimulated to declare suspected cases by insisting on their primordial role in bTB surveillance and by emphasizing on the zoonotic potential of the disease. It is also recommended that private veterinarians and slaughterhouses are controlled more often by the authorities to stimulate their functioning in the bTB surveillance. Furthermore, data on notifications of suspected or positive cases must be used in a benchmarking exercise on a regular basis to stimulate slaughterhouses and veterinarians in notifying suspected cases.

7.4 Surveillance components

7.4.1 Purchase surveillance

The theoretical test characteristics of the SIT still make it a very good and practical test for the bTB surveillance if performed according to ‘good veterinary practices’. However, as explained above the performance of the Belgian bTB surveillance program, especially during purchase SIT testing, is low due to various reasons. Therefore, the Scientific Committee strongly recommends to replace the SIT test by a combined IFN‐y and serological test (in parallel). In case of a positive/doubtful result it is recommended that the competent authorities execute a SIT as a confirmation test. This approach holds several advantages. The veterinarian has to visit the farm only once and does not have to read the SIT test three days after injection. Furthermore, a blood sample might be more convenient and practical under the current farming conditions with growing numbers of cattle which are often not (well) fixated. Also, in case of suspicion, veterinarians often have a conflict of interest to declare to the competent authorities. In case of a blood sample, although it is still taken by the veterinarian, the analysis and first interpretation of the result is performed by the laboratory. Finally, combination of the IFN‐y test with a serological test allows as well early detection of positive animals (IFN‐y test) as detection of animals during the late stages of disease (serological test) which might pass undetected in a IFN‐y test due to anergy (see Figure 6). On the other hand the replacement of the SIT test by a combined IFN‐y and serological test might be challenging for the following reasons: the IFN‐y test is a relative expensive test and the blood must be processed at the laboratory not later than the day of the collection (usually within 8 h of collection to ensure optimal assay performance). Furthermore, this approach will increase the number of false positive animals. Nevertheless, the Scientific Committee is convinced that this adaptation will greatly improve the Se of purchase surveillance which is very important given the fact that purchase is a main risk factor for bTB propagation.

7.4.2 Winter screening

It is recommended to continue the winter screening using a SIT test performed by the private veterinarian. However, it is strongly recommended to select the herds based on a risk scoring using the risk factors listed in Table 19 (risk based surveillance) and as explained above. Although the evaluation of the surveillance during winter screening indicated that the performance of this surveillance component is relatively good, it is recommended that private veterinarians are controlled more often by the authorities to stimulate their good functioning in this surveillance component.


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In case of a positive/doubtful reaction, it is recommended that the competent authorities execute an IFN‐y test as confirmation test.

7.4.3 Surveillance in suspected / outbreak herds

In case of suspicion or confirmed outbreak, it is very important to follow and assess the spread of the infection within the herd. Therefore, it is recommended that the competent authorities execute a SIT (or SICT), IFN‐y and serological tests on all cattle present in the farm. As explained above, it might be recommended to perform these tests in serial to allow a booster effect in positive animals following a SIT or SICT tests. Furthermore, given the long incubation period of bTB (often several months) it is recommended to test the herd several times and to wait for a sufficient period of time between consecutive herd tests.

7.4.4 Tracing analysis of outbreaks

It is recommended to continue the tracing analysis using a SIT test performed by the private veterinarian. Again, private veterinarians should be controlled more regularly by the authorities to stimulate their good functioning in this surveillance component. Furthermore, given the long incubation period of bTB, it is important to wait for a sufficient period of time before testing cattle in case of a recent purchase or to perform two consecutive tests in case of a first negative test. In case of a positive/doubtful reaction, it is recommended that the competent authorities execute an IFN‐y test as confirmation test.

7.4.5 Slaughterhouse surveillance

As explained above, the fact that slaughterhouse surveillance has detected nearly all recent bTB outbreaks in Belgium indicates the importance of this surveillance component. However, the performance of this surveillance component can still be improved by a risk classification of bovine herds and/or individual animals allowing a more targeted surveillance of high risk herds/animals. If animals are originating from a ‘high risk’ herd, slaughterhouse inspectors must be stimulated to take a pooled sample of organs even if there are no visible lesions. Furthermore, slaughterhouse inspectors must receive education (e.g. pictures of lesions,…) on a regular basis because, given the low prevalence of bTB in Belgium, typical lesions are relatively rare. Finally, data on notifications of suspected or positive cases must be used in a benchmarking exercise (comparing number of declared suspected cases) on a regular basis to stimulate slaughterhouse inspectors in notifying suspected cases.

7.5 Diagnostic tests

It is recommended to adapt the legislation in relation to current and future available diagnostics (e.g. inclusion of nucleic acid recognition methods, IFN‐y test and serological tests or other new methods) and measures taken at herd level should be taken into account the results of new diagnostic tools. Molecular methods such as RT‐PCR should be used in first line diagnostics (in parallel with bacteriological culture) to allow a faster confirmation of a positive case and to reduce the time a herd is blocked after a positive or doubtful test. Furthermore, it is recommended to mitigate or adapt the blocking of herds after a doubtful test or post‐mortem inspection in order to increase the willingness of actors in the field to notify. Although this contains some risks in case of a true positive animal, the benefits (higher sensitivity of surveillance) will probably outweigh the risks.

7.6 Molecular typing of bTB isolates

It is strongly recommended to perform a molecular typing of each bTB isolate and to share this information between animal (including wildlife) and human surveillance networks in a continuous way and, if possible, at an international level. Typing of these isolates and sharing of this information is of great importance to facilitate fast epidemiological investigation.


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7.7 Domestic non‐bovine animals

Although a lot of species are susceptible for bTB (see Appendix IX), their possible role in the infection cycle is not fully understood. Nevertheless, the recent bTB case in an imported alpaca from the UK in Belgium shows that vigilance for bTB in non‐bovine domesticated animals is very important. Especially for camelids, the installation of a surveillance program is strongly recommended. It is also recommended to stimulate the development and validation of diagnostic tests which can be applied in non‐bovine species and which can be useful in case these animals are kept on the same farms as bovines.

7.8 Wildlife

In Belgium, there has never been a detected case of bTB in wildlife. However, the epidemiological evolution in a number of neighboring countries has demonstrated that, if there is a spill‐over of bTB from farmed animals to wildlife, the control of bTB in cattle becomes extremely difficult. Moreover, there have been some recent bTB cases in deer and wild boar in France, close to the border with Belgium. Therefore, it is strongly recommended to install a continuous surveillance program in wildlife in Belgium. This surveillance must be risk based and the following risk factors should be taken into account:

- Possible contact between wildlife and domesticated animals (overlap on density maps of wildlife and domesticated animals)

- Identified regions in close proximity with regions with confirmed bTB in wildlife (e.g. close to the border with France).

It is recommended to stimulate the development and validation of diagnostic tests applicable to wildlife. Finally, it is strongly recommended to bring the intestines of shot wildlife to game‐handling establishments or to let them be collected by a rendering plant, as these intestines pose significant risks for spread in case of bTB infected animals.

7.9 Biosecurity measures

According to Sarrazin et al. (2014) basic biosecurity measures (e.g. quarantine) are often not (well) applied by cattle farmers in Belgium, thereby exposing themselves to the risk of disease transmission within and between farms. Also, for bTB, these basic measures, together with testing at purchase, are of primordial importance for disease prevention. Therefore, farmers and veterinarians should be stimulated to respect these biosecurity measures.

8 Conclusions

Despite the fact that Belgium has an OTF status for bTB, almost every year some outbreaks occur. The Scientific Committee has performed a thorough evaluation of the current surveillance indicating that, based on animal movements and slaughterhouse data and simulations of different diagnostic tests characteristics (sensitivity and specificity), the number of (false) positive reactors notified at purchase and the number of suspected lesions submitted from slaughterhouse are estimated too low, even considering the low bTb prevalence. Hence, the efficacy of the current surveillance program, which especially makes use of SIT as first line screening test, is questioned. Therefore, it is important to raise awareness of all actors involved in the bTB surveillance program to stimulate their functioning It is recommended to adapt the surveillance program by:


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- replacing of the SIT test by a combined IFN‐y and a serological test (in parallel) performed by the private veterinarian at purchase testing. In case of positive/doubtful result, it is recommended that the competent authorities execute a SIT or SICT as confirmation test.

- maintaining the SIT test performed by the private veterinarian during winter screening. In case of positive/doubtful result, it is recommended that the competent authorities execute an IFN‐y test as confirmation test.

- executing both a SIT (or SICT), IFN‐y and serological tests on all cattle present on the farm in suspected/outbreak herds .

- maintaining the SIT test performed by the private veterinarian during tracing analysis. In case of a positive/doubtful result it is recommended that the competent authorities execute an IFN‐y test as a confirmation test.

- Risk classification of bovine herds and/or individual animals allowing a more targeted surveillance of high risk herds/animals at slaughterhouse inspections.

Epidemiological data should be standardized and stored in a centralized database by the FASFC and should be consultable for all actors involved in the surveillance network to be able to perform a risk based surveillance based on bTB history. It is recommended to adapt the control measures and their duration (i.e. blocking of farms) based on indicators allowing to allocate a risk profile to animals and/or herds. It is recommended to adapt legislation in relation to current and future available diagnostics (e.g. inclusion of molecular methods, IFN‐y test and serological tests or other new methods). Molecular methods such as RT‐PCR should be used as first line diagnostics (in parallel with bacteriological culture) to allow a faster confirmation of a positive case and to reduce the time a farm is blocked after a positive or doubtful test. The recent bTB case in an imported alpaca in Belgium shows that vigilance for bTB in non‐bovine domesticated animals is very important. Moreover, there have been some recent bTB cases in deer and wild boar in France, close to the border with Belgium. Therefore, it is strongly recommended to install a continuous surveillance program in wildlife in Belgium based on known risk factors. For camelids, a surveillance program is also recommended. Furthermore, it is recommended to stimulate the development and validation of diagnostic tests which can be applied to non‐bovine domesticated species and wildlife and which can be useful in case these animals are kept on the same farms with bovines. Finally, basic biosecurity measures (e.g. quarantine) are often not (well) applied by cattle farmers in Belgium, although they are very important for bTB prevention. Therefore, farmers and veterinarians must be stimulated to respect these biosecurity measures.

9 Answer to specific questions

9.1 Is it, under the current field conditions with growing numbers of cattle on farms which are often not (well) fixated, still feasible to perform a ‘secundem artem’ intradermal skin test? Is there modern equipment to allow a sufficient fixation of cattle in order to correctly perform and read an intradermal skin test?

The Scientific Committee endorses the practical difficulties that professional workers encounter nowadays to implement the bTB diagnostic protocol in not (well) fixated cattle and especially in regard to the application of the SIT and SICT testing and controlling protocol. However, there is sufficient equipment available on the market to adequately restrain cattle (for some examples see Dudouet (2015)). Cattle farmers must be encouraged to provide such equipment to allow the veterinarian to work in safe and practical conditions.


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Nevertheless, the recommendation of the Scientific Committee to replace the SIT at purchase by an IFN‐y test and ELISA test (see above) will make sampling at purchase, which represents a substantial amount of bTB diagnostic tests, much easier as the blood sample can be taken at the base of the tail.

9.2 Can the proposed decision tree (including the use of tuberculination and gamma‐interferon test) be validated?

The Scientific Committee cannot validate the proposed decision tree as the recommendations (see above) demand substantial changes to the decision tree.

9.3 Evaluation of proposals to modify the current royal decree of 17 October 2002 regarding the control of bovine tuberculosis

9.3.1 To introduce the control against Mycobacterium spp. other than M. bovis which is currently the only Mycobacterium species mentioned in the royal decree: M. caprae, M. tuberculosis,…

The members of the Mycobacterium tuberculosis complex (containing M. tuberculosis, M.bovis, M. bovis BCG, M. africanum, M. microti, M. canettii, M. pinnipedii, M. caprae, M. orygis and M. mungi) are genetically closely related (99.9% similarity at the nucleotide level) and are all able to provoke tuberculosis in humans and animals (Michelet et al., 2016; Rodriguez‐Campos, 2014; Alexander, 2010; Van Ingen, 2012). Concerning bTB, several publications have reported the implication of the different members of the M. tuberculosis complex, mainly M. bovis, M. tuberculosis and M. caprae. Table 20. Mycobacterium spp. identified as responsible for bTB

Species identified as responsible for bTB

References

M. bovis Smith et al, 2006. Nature Reviews Microbiology 4, 670-681. M. tuberculosis Romero et al, 2011. Emerging Infectious Diseases 17, 2393-

2395. Ameni et al, 2011. Clinical and Vaccine Immunology 14, 1356-1361. Prasad et al, 2005 Tuberculosis 85, 421-428

M. africanum Cadmus et al, 2010. Tropical Animal Health and Production 42, 1047-1048. Rahim et al, 2007. The Southeast Asian Journal of Tropical Medicine and Public Health 38, 706-713

M. caprae Prodinger et al, 2005. Journal of Clinical Microbiology 43, 4984-4992. Duarte et al, 2008. Veterinary Microbiology 130, 415-421

M. orygis Van Ingen t al, 2012. Emerging Infectious Diseases 18(4):653-5.

Given these evidences, the Scientific Committee supports the proposition to modify the royal decree of the 17 October 2002 and to replace Mycobacterium bovis by Mycobacterium tuberculosis complex. This extension to the M. tuberculosis complex as causal agents of bovine tuberculosis was previously adopted in other countries (Austria, Croatia, the Netherlands, Portugal, Ireland and Spain) (Rodriguez‐Campos, 2014).

9.3.2 To install a mandatory notification of M. bovis and M. tuberculosis for animal species other than cattle. Are measures necessary if tuberculosis is diagnosed in other animal species (dogs, cats, sheep, goats, exotic animals, wild animals, zoo animals, other domestic animals, …), also if cattle are held on the same farm?

A literature overview of animal species (domestic and wild animals) that are susceptible and/or receptive to M. bovis around the world has been performed. The results of this review can be found in Appendix IX.


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In conclusion, M. bovis is capable of infecting a whole range of animal species. Therefore, it is strongly recommended to install a mandatory notification for non‐bovine animals not only for M. bovis but also for other species of the M. tuberculosis complex. Furthermore, these animal species should be included in sanitary measures, in particular if they might consist a source of infection for cattle.

9.3.3 To merge the definitions ‘suspected of being affected’ and ‘suspected of being contaminated’

The Scientific Committee is not in favor of merging both definitions as they represent two different situations. However, this terminology is confusing. Therefore, it is recommended to adapt the terminology in legislation. The Scientific Committee proposes to use the following definitions:

1) Free of tuberculosis: an animal belonging to a herd which is officially free of tuberculosis (to be defined in the royal decree)

2) Suspected of being infected with tuberculosis: an animal which belongs to one of the following categories:

- An animal which displays typical macroscopic lesions of tuberculosis during slaughterhouse inspection or during autopsy

- An animal which displays typical histological lesions of tuberculosis during laboratory examination

- An animal with a positive result for a diagnostic test for tuberculosis (SIT, SICT, serological test or IFN‐y test) whatever the circumstances in which the test is performed

3) Infected with tuberculosis: in the following cases: - An animal which displays clinical symptoms of tuberculosis together with a positive

result of a diagnostic test for tuberculosis (SIT, SICT, serological test or IFN‐y test) - An animal after isolation and identification of a member of the Mycobacterium

tuberculosis complex in an accredited laboratory - An animal after detection by (RT‐)PCR of a member of the Mycobacterium

tuberculosis complex in an accredited laboratory - An animal with a positive result for a diagnostic test for tuberculosis (SIT, SICT,

serological test or IFN‐y test) together with the isolation and identification of a member of the Mycobacterium tuberculosis complex in an accredited laboratory

- An animal with a positive result for a diagnostic test for tuberculosis (SIT, SICT, serological test or IFN‐y test) together with the detection of typical histological lesions of tuberculosis during laboratory examination

- An animal belonging to a herd which is declared infected with tuberculosis and which belong to one of categories under 2) above.

4) In contact with tuberculosis: an animal belonging to a herd which is declared infected with tuberculosis, but does not belong to one of categories under 3) above.

9.3.4 Adjustment and clarification of the minimum age for intradermal skin test at purchase and at complete herd testing

Fattening calves were traditionally exempted from the purchase testing scheme as they were considered as minor risk of being infected (considered as dead‐end reservoir sent directly to slaughter). Recent data (outbreak 2015) reveals interesting features regarding the age of infection. Testing those fattening calves could potentially provide interesting insight about the status of the source farm. Therefore, it is strongly recommended to include all ages for purchase surveillance. For whole herd testing during winter screening and tracing surveillance, the minimum age of 6 weeks can be maintained. In case of herd testing during a (suspected) outbreak, again all ages should be included in the testing scheme. However, for cattle younger than 6 months it is recommended to perform a SIT test and not an IFN‐y test because of nonspecific responses. Although the reasons for this phenomenon are not fully


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understood, γδ11 immune T cells play a role in this effect. These cells play a critical role in the early response to M. bovis. These cells, from either infected or uninfected cattle could proliferate and produce interferon‐γ when stimulated with M. bovis antigens and be therefore responsible for non specific response in the IFN‐y test (Plattner et al., 2009; Plattner and Hostetter, 2011; Welsh et al., 2002).

9.4 Evaluation of the Tuberculosis Action plan

The Scientific Committee appreciates that some of its recommendations (e.g. raising awareness and education of actors in the bTB surveillance) are already implemented by the authorities. However, it is not possible to fully evaluate this action plan as the recommendations (see above) request substantial modifications.

For the Scientific Committee, The Chair,

Prof. Dr. E. Thiry (Sgd.) Brussels, <date>

11 Gamma delta T cells represent a small fraction (1 ‐ 5 %) of the overall T cell population but are enriched (more than 50 % of the T cell population) in epithelial cell‐rich compartments like skin, the digestive tract, and reproductive organ mucosa


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Members of the Scientific Committee

The Scientific Committee is composed of the following members: D. Berkvens, A. Clinquart, G. Daube, P. Delahaut, B. De Meulenaer, S. De Saeger, L. De Zutter, J. Dewulf, P. Gustin, L. Herman, P. Hoet, H. Imberechts, A. Legrève, C. Matthys, C. Saegerman, M.‐L. Scippo, M. Sindic, N. Speybroeck, W. Steurbaut, E. Thiry, M. Uyttendaele, T. van den Berg

Conflict of interest

T. van den Berg, D. Fretin and V. Mathys have declared a conflict of interest because of their responsibilities in the national reference laboratory regarding tuberculosis. Consequently, these persons were consulted by the working group as ‘heared experts’ in the parts of the opinion related to diagnostic techniques.

Acknowledgements

The Scientific Committee acknowledges the Staff Department for Risk Assessment and the members of the working group for preparing the draft opinion.

Composition of the working group

The working group was composed of: Members of the Scientific Committee: C. Saegerman (reporter), D. Berkvens, J. Dewulf, H.

Imberechts, T. van den Berg External experts: D. Fretin (CODA‐CERVA), K. Huygen (WIV‐ISP), J.

Laureyns (Ghent University), A. Linden (Ulg), V. Mathys (WIV‐ISP), L. Rigouts (ITM), V. Roupie (CODA‐CERVA), S. Welby (CODA‐CERVA)

File manager: P. Depoorter

The activities of the working group were attended by the following members of the administration (as observers): J. Evers (FASFC), J. Hooyberghs (FASFC), E. Stoop (FASFC), L. Vanholme (FASFC).

Presentation of the Scientific Committee of the FASFC The Scientific Committee is an advisory body of the Belgian Agency for the Safety of the Food Chain (FASFC) that provides independent scientific opinions on risk assessment and risk management in the food chain, and this at the request of the Chief Executive Officer of the FASFC, the Minister competent for food safety or at its own initiative. The Scientific Committee is administratively and scientifically supported by a secretariat managed by the Directorate for Risk Assessment of the Agency. The Scientific Committee consists of 22 members who are appointed by Royal Decree on the basis of their scientific expertise in areas related to the safety of the food chain. When preparing an opinion, the Scientific Committee can call on external experts who are not a member of the Scientific


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Committee. Similar to the members of the Scientific Committee, they must be able to work independently and impartially. To ensure the independence of the opinions, potential conflicts of interest are managed transparently. The opinions are based on a scientific assessment of the question. They express the view of the Scientific Committee which is taken in consensus on the basis of a risk assessment and the existing knowledge on the subject. The opinions of the Scientific Committee may contain recommendations for food chain control policy or for the stakeholders. The follow‐up of these recommendations for control policy is the responsibility of the risk managers. Questions on an opinion can be directed to the secretariat of the Scientific Committee: [email protected].

Legal framework

Law of 4 February 2000, on the creation of the Federal Agency for the Safety of the Food Chain, in particular article 8; The Royal Decree of 19 May 2000, on the composition and operating procedures of the Scientific Committee, as established within the Federal Agency for the Safety of the Food Chain; The Internal Rules as mentioned in Article 3 of the Royal Decree of 19 May 2000, on the composition and operating procedures of the Scientific Committee, as established within the Federal Agency for the Safety of the Food Chain, approved by the Minister on 9 June 2011.

Disclaimer

The Scientific Committee at all times reserves the right to modify the opinion by mutual consent, should new information and data become available after the publication of this version.


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Appendix I: Specific questions in the request of opinion

1. Evaluation of the current surveillance program a. Which risk factors can be taken into account during the systematic post‐mortem

examination of cattle? b. Which risk factors (age, animal category, farm type,…) can be taken into account

during intradermal skin tests at purchase and does the intradermal skin test at purchase, as actually performed by veterinary practitioners, still makes sense for the bTB surveillance?

c. Which risk factors should be taken into account in order to take proportionate measures in case of a suspicion of bTB infection?

d. Is the surveillance of milk producing animals on farms with direct sell of raw milk still useful under the current epidemiologic situation?

e. Which risk factors (age, animal category, farm type,…) can be taken into account for the follow‐up via whole herd tuberculinations of previous herds, contact herds or suspected herd and can this surveillance pillar be optimized?

f. Is the risk for bTB introduction via import of animals from officially free member states with a considerable number of outbreaks larger than the risk via purchase of Belgian cattle?

2. Evaluation of the current diagnostic methods for the detection of bovine tuberculosis a. Intradermal skin test

i. Should the intradermal skin test be maintained as the reference test or are there other diagnostic methods that could replace it?

ii. It is currently mandatory to perform the intradermal skin test in the neck area. Could it also be performed in other areas on the animal in order to make the test more feasible under field conditions (e.g. caudal fold, gluteal cleft,…)?

iii. Is it, under the current field conditions with growing numbers of cattle on farms which are often not (well) fixated, still feasible to perform a ‘secundem artem’ intradermal skin test? Is there modern equipment to allow a sufficient fixation of cattle in order to correctly perform and read an intradermal skin test?

iv. Are the sensitivity and specificity of the intradermal skin test influenced by the type of syringe/device (classic syringe, dermojet,…)?

v. Is the currently used tuberculin, which is produced based on a historic isolate, still adequate to detect the current Mycobacteria?

vi. On which species, other than cattle, can the intradermal skin test be applied? b. Can the current culture method of M. bovis be optimized in order to speed up the

growth of M. bovis and to obtain a earlier denial or confirmation of bacterial growth (including isolation and identification)?

c. Are the characteristics of the current PCR tests adequate enough in order to use these tests for screening or confirmation purposes without having to wait for the result of the bacterial culture?

d. Gamma‐interferon test i. Which specific antigen(s) have to be used? ii. Are the characteristics of the gamma‐interferon test adequate enough to

replace the intradermal skin test in routine testing under Belgian field conditions?

iii. Could this test be used as a serial or parallel confirmation test after a positive intradermal skin test?


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iv. Could this test be used as a screening test in case of repeated herd testing with the intradermal skin test with unfavorable results that cannot be confirmed by bacteriological culture (possible infection of environmental Mycobacteria)?

v. In which animal species, other than cattle, can this test be used? e. Could the commercially available Ab‐ELISA be of use in Belgium? f. Can the proposed decision tree (including the use of tuberculination and gamma‐

interferon test) be validated? 3. Evaluation of propositions to modify the current Royal Decree of 17 October 2002 regarding the

control of bovine tuberculosis a. To introduce the combat against Mycobacterium spp. other than M. bovis which is

currently the only Mycobacterium species mentioned in the Royal Decree: M. caprae, M. tuberculosis,…

b. To install a mandatory notification of Mycobacterium bovis and M. tuberculosis for animal species other than cattle. Are measures necessary if tuberculosis is diagnosed in other animal species (dogs, cats, sheep, goats, exotic animals, wild animals, zoo animals, other domestic animals, …), also if cattle are held on the same farm?

c. To merge the definitions ‘suspected of being affected’ and ‘suspected of being contaminated’

d. To propose proportional measures in case of a suspicion on animal level as well as on herd level which are based on the epidemiologic investigation and on risk assessment

e. Diagnostics: to provide in legislation the use of the gamma-interferon test, PCR and other molecular techniques.

f. Adjustment and clarification of the minimum age for intradermal skin test at purchase and at complete herd testing

g. Adjustment of the pillars of surveillance with special attention to intradermal skin testing at purchase

4. Evaluation of the Tuberculosis Action plan


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Appendix II: Procedure for the intradermal skin test according to the OIE (2009)

i) A correct injection technique is important. The injection sites must be clipped and cleaned. A fold of skin within each clipped area is measured with callipers and the site marked prior to injection. A short needle, bevel edge outwards and graduated syringe charged with tuberculin attached, is inserted obliquely into the deeper layers of the skin. The dose of tuberculin is then injected. A multi‐dose syringe or multiple injection gun may be used provided that delivery of the volume and safety are assured. The dose of tuberculin injected must be no lower than 2000 International Units (IU) of bovine or avian tuberculin. A correct injection is confirmed by palpating a small pea‐like swelling at each site of injection. The distance between the two injections should be approximately 12–15 cm. In young animals in which there is no room to separate the sites sufficiently on one side of the neck, one injection must be made on each side of the neck at identical sites in the centre of the middle third of the neck. The skin‐fold thickness of each injection site is re‐measured 72 hours after injection. The same person should measure the skin before the injection and when the test is read.

ii) A number of alternative methods of interpreting the skin test responses have been adopted, recognising that false‐positive reactions may be caused by sensitisation by other mycobacteria and by local inflammation. It is important to recognise that there is a balance between sensitivity and specificity and achieving high concurrent values may not be possible. Appropriate policies need to be in place depending on disease prevalence and according to risk (e.g. where a wildlife reservoir is present). The interpretation is based on observation and the recorded increases in skin‐fold thickness. In the single intradermal test (which requires a single injection of bovine tuberculin), the reaction is commonly considered to be negative if only limited swelling is observed, with an increase of no more than 2 mm and without clinical signs, such as diffuse or extensive oedema, exudation, necrosis, pain or inflammation of the lymphatic ducts in that region or of the lymph nodes. The reaction is considered to be inconclusive if none of these clinical signs is observed and if the increase in skin‐fold thickness is more than 2 mm and less than 4 mm. The reaction is considered to be positive if clinical signs, as mentioned above, are observed or if there is an increase of 4 mm more in skin‐fold thickness. Moreover, in M.‐bovis‐infected herds, any palpable or visible swelling should be considered to be positive. Sometimes a more severe interpretation is used, particularly in a high risk population or in‐contact animals. Animals that are inconclusive by the single intradermal test should be subjected to another test after an interval of 42 days to allow desensitisation to wane (in some areas 60 days for cattle and 120 days for deer are used). Animals that are not negative to this second test should be deemed to be positive to the test. Animals that are positive to the single intradermal test may be subjected to a comparative intradermal test or blood test. Any retest should be performed in accordance with the local or national control programmes standard.

iii) In the interpretation of the intradermal comparative test, a reaction is usually considered to be positive if the increase in skin thickness at the bovine site of injection is more than 4 mm greater than the reaction shown at the site of the avian injection. The reaction is considered to be inconclusive if the increase in skin thickness at the bovine site of injection is from 1 to 4 mm greater than the avian reaction. The reaction is considered to be negative if the increase in skin thickness at the bovine site of injection is less than or equal to the increase in the skin reaction at the avian site of injection. This interpretation scheme is used in European Union (EU) countries and is recommended in Council Directive 64/432/EEC (22). Sometimes a more stringent interpretation is used.

iv) In the caudal fold test, a short needle, bevel edge outwards, is inserted obliquely into the deeper layers of the skin on the lateral aspect of the caudal fold, midway along the fold and


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midway between the hairline and the ventral aspect of the fold. The standard interpretation is that any palpable or visible change is deemed to be a reaction. A modified interpretation is also in use: a positive test is any palpable or visible swelling at the site of the injection that has a caudal fold thickness difference of 4 mm when compared with the thickness of the opposite caudal fold. If an animal has only one caudal fold, it is considered to be test positive if the caudal fold thickness is 8 mm or more.


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Appendix III: Compliance of Belgian field veterinarians to the testing procedures as recommended by USDA (2015)

Testing procedures as recommended by USDA Compliance in Belgium

Use a new needle for each animal in order to minimize transmission, or the appearance of transmission of blood‐borne infectious agents between animals. All cattle and bison tested must be sufficiently restrained to permit careful application of the tuberculin injection(s), correct reading of animal identification, and careful observation and palpation of the injection sites. No test should be applied or observed without having the animal restrained in a satisfactory manner.

Sick cattle are not to be injected.

Animals to be tested should not have vaccines, or be treated with drugs, pharmaceuticals, or anthelmintics administered in conjunction with tuberculin injections.

Ensure the injection site is free of manure, debris, and hair. If necessary, clean the site to make a sanitary injection.

Check the syringe and needle for cleanliness, leakage, and proper needle gauge and length of needle.

All cattle and bison tested must be sufficiently restrained to permit careful application of the tuberculin injection(s), correct reading of animal identification, and careful observation and palpation of the injection sites. No test should be applied or observed without having the animal restrained in a satisfactory manner.

Very stringent criteria are used to produce tuberculin for the tuberculosis eradication program. Proper handling and storage are essential to maintain potency and ensure consistently accurate testing. Keep these considerations in mind when using tuberculins:

Refrigeration: Store tuberculins between 35°F and 46°F (2°C and 8°C); do not allow tuberculin to freeze. Tuberculous protein can precipitate with freezing and result in inadequate responses. Tuberculin that has been frozen should be discarded and not

No standard procedure in Belgium (Humblet et al., 2011b)



Rather well applied in Belgium (Humblet et al., 2011b)


No literature found

No standard procedure in

Belgium, in particular car

storage insufficient (Humblet et

al., 2011b)

Not a standard in Belgium, as


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used to perform testing. Room temperature: Tuberculins are

relatively stable at room temperature for up to 8 hours. Using tuberculins at room temperature during the work day is acceptable, while performing injections. Return the tuberculin to refrigeration temperatures once injections are completed.

Expiration: Tuberculin is not to be used after the expiration date. Expired tuberculin should be discarded.

Exposure to light: Store in a dark location as light denatures the tuberculous protein; tuberculin stored in clear syringes must be placed in a dark container.

Oxidation: Tuberculin in 10cc amber colored glass containers must be discarded 2 weeks after initial usage. Tuberculous protein denatures slowly in air and can completely oxidize when stored in partially filled containers for 4 weeks

Absorption to containers: Do not store dose amounts longer than 12 hours in containers other than original packaging; tuberculous protein can adsorb into inner surfaces of plastic containers (such as syringes) causing loss of potency.

tuberculin is not always stored cool in the veterinarian’s car (Humblet et al., 2011b)

No literature found


Belgium (Humblet et al., 2011b)





Other important procedures, not mentioned by USDA

Scientific literature

Post‐injection verification (formation of a pea‐size swollen area after tuberculin injection)

Reading the result of the skin reaction within the prescribed 72 h ± 4–6 h post tuberculin injection. This is a potential cause of false negative reactions, as mentioned by de la Rua‐Domenech et al (2006).


Rather well applied in Belgium (Humblet et al., 2011b)


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Appendix IV: Advantages and disadvantages of equipment which is used by Belgian field veterinarians for the injection of tuberculin during the intradermal skin test

Classical syringe Dermojet

More risk of spreading infections

Less maintenance

Tuberculin remains intact (particularly when using carpules)1

Invites the veterinarian to check if the tuberculin is situated intradermally

Less spread of infections

Needs regular, strict maintenance

Has to be emptied and washed after each administration

Gives the impression that the tuberculin always is situated intradermally, although this is not always the case2

1 Carpules can be removed from the syringe after administration, stored in the refrigerator and used again (over a short period). However, carpules are currently not available on the market. 2Experience from veterinary practice: when injecting several animals, one can feel a small swelling on the injection spot of the cattle first injected. In contrast, after serial injection of 5‐6 cattle, this swelling is not present anymore. This observation might suggest that the tuberculin is not situated intradermally in those animals. Nevertheless, it is the responsibility of the veterinarian to check if the tuberculin is situated intradermally and if not to repeat the injection. It can be concluded that the dermojet might offer a false sense of security. Because,

o veterinarians do not check if the tuberculin is situated intradermally, as they trust the dermojet more than a syringe.

o the dermojet is not always emptied and washed after use o the dermojet often is not well maintained

In theory, the dermojet is more reliable. In practice, the instrument is often not properly used and therefore, it loses its advantage over the syringe.


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Appendix V: On which species, other than cattle, can the SIT be applied?

Introduction Several diagnostic tests are used to ascertain individual and flock bTB status. Amongst others, diagnostic tests measuring the CMI (cell‐mediated immunity) using the single intradermal tuberculin test (SIT) or the single intradermal comparative tuberculin test (SICT) and/or the IFN‐γ test. Serology detection is also available. In cattle, the cell mediated immunity tests have usually higher sensitivity and specificity than the antibody detection although Ab ELISAs have the capacity to detect anergic animals in advanced stages of infection. Sensitivity of the Intradermal Skin Test was evaluated between 53% (27.3‐81.5, 95% CI) and 69.4% (40.1‐92.2, 95% CI) depending on cattle (fighting bulls vs other), and the specificity between 55.1% and more than 99% showing a median value over 95% (Bezos et al., 2014a; Schiller et al., 2010). Tuberculosis is usually a chronic debilitating disease in cattle, but it can occasionally be acute and rapidly progressive. Early infections are often asymptomatic. In countries with eradication programs, most infected cattle are identified early and symptomatic infections are uncommon. In the late stages, common symptoms include progressive emaciation, a low–grade fluctuating fever, weakness and inappetence which are not pathognomonic and depend on the organs affected. Animal with pulmonary disease usually have a moist cough and may have dyspnea or tachypnea. In the terminal stages, animals may become extremely emaciated and develop acute respiratory distress. bTb in the veterinary species is primarily a respiratory disease and transmission is mainly by the airborne route. Depending on animal species, clinical signs could be absent (ex. elephant), subacute or chronic disease with very few symptoms (ex. cervids) or disseminated with rapid and fulminating course (http://www.cfsph.iastate.edu/Factsheets/pdfs/bovine_tuberculosis.pdf). Moreover the distribution of tuberculous lesions of the various wildlife species reviewed indicates that M. bovis may spread through infected aerosol droplet inhalation, but contamination of feed sources and pastures, as well as omnivores feeding on infected carcasses, have been reported as important routes of BTB transmission (Fitzgerald and Kaneene, 2013).

Domesticated animals Goat Tuberculosis in goat is mainly caused by M. bovis and M. caprae (Pesciaroli et al., 2014). The transmission between cattle and goats can occur as watering and grazing points are often shared (Bezos et al., 2012). In some European countries, including Greece, Italy, Spain and Portugal, which have high small ruminant census figures and are not officially TB‐free (OTF), the transmission risk is present and surveillance of tuberculosis in goats in these countries is therefore important (Zanardi et al., 2013). Given its zoonotic potential, goats used for raw milk production living in mixed cattle‐goat herds must be tested for TB by the official tuberculin test in these not‐OTF countries (Regulation (EC) 853/2004). In Spain, goats co‐existing with cattle are subjected to the official tuberculin test. Pathology/ Clinical Signs Tuberculosis clinic signs in goat are not very specific and could induce chronic loss and appetite, reduced milk yield and debiliting disease with or without respiratory signs (DEFRA, access 2015) occasional diarrhoea and death (Pesciaroli et al., 2014) A chronic cough can be a sign of tuberculosis even more if goat when an antibiotic treatment for a respiratory infection fails to response (DEFRA, access 2015). Post‐mortem studies have shown that in the affected tuberculosis goats in the 2008 outbreak (many of which were subjected to detailed post mortem examination), tubercle lesions did not develop, and instead large abscesses were produced with more liquid pus, which often eroded quickly into the


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airways, in such way that they were quickly able to “cough up” and “breath out” M. bovis (or M. caprae) organisms into the environment (http://www.goatvetsoc.co.uk/goat‐health/tuberculosis/). Post‐mortem examination of goats infected with M. bovis frequently reveals circumscribed pale yellow, white, caseous or caseo‐calcareus lesions of various sizes, often encapsulated, especially in the lungs and mediastinal lymph nodes, or in the mesenteric lymph nodes (Bezos et al., 2012). Tuberculin skin test The Se and Sp known for the skin test in goat were calculated on a limited panel of animals (Cf. tables 21 and 22 below). The sensibility of SIT was evaluated as between 54.2 ‐ 93.8% and the specificity as 87.5% (Cf. Table 21). The sensibility of SICT was evaluated as between 29.2‐83.7% and the specificity 100% (Cf. Table 22). However, studies use a small sample number for the estimation of Sp in comparison of Se. Table 21. Sensitivity and specificity of diagnostic assays based on CMI and antibody production (ELISA) in some studies performed in goats (from (Bezos et al., 2012)). Standard interpretation of SIT: Negative if reaction <2 mm; Inconclusive: If reaction >2 and <4 mm; Positive if reaction ≥4 mm. Severe interpretation: Considering inconclusive result (>2 and <4 mm) as positive result.

Table 22. Sensitivity and specificity of diagnostic assays based on CMI and antibody production (ELISA) in some studies performed in goats (from (Bezos et al., 2012)). SICT, Standard interpretation: Negative if no reaction with PPDB or if it is positive or inconclusive but equal or lower than a positive or inconclusive reaction with PPDA; Inconclusive: Positive or inconclusive reaction with PPDB >1 and <4 mm greater than the reaction with PPDA. Positive: Positive reaction with PPDB >4 mm higher than the reaction with PPDA. Severe interpretation: Considering inconclusive result as positive result.

More recently a study was performed using data from 17,450 goats (54 different flocks) classified as Tb‐infected in the control programmes (2010‐2011). Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold‐standard. The overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4–47.4) and decreased to 38.8% (CI 95%, 35.5–42.3) using the standard interpretation. The overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6–25.4) and 7% (CI

Test Number of goats

True status (bacterial species) Sensitivity Specificity Comments Reference

SIT a 97 M. bovis (natural infection) 93.8 87.5 – (García Marín,

1993)

87 M. bovis (natural infection) 44.6/53.2 – Standard/severe interpretation

(Liebana et al.,1998)

131 M. caprae (natural infection) 71 – Severe interpretation (Alvarez et al.,2008) 24 M. caprae and M. avium subsp.

paratuberculosis (natural infection) 54.2

45 M. caprae and M. avium subsp. paratuberculosis (natural infection)

88.8 – Severe interpretation (Bezos et al.,2009)

Test Number of goats

True status (bacterial species) Sensitivity Specificity Comments Reference

SICT b

49 M. bovis (natural infection) or M.avium subsp. paratuberculosis (natural infection)

83.7 – Severe interpretation (Gutierrez etal., 1998)

25 Culture negative goats ND 100 Specificity value was calculated using culture negative animals (n = 12) from the positive herd

131 M. caprae (natural infection) 42.7 – Severe interpretation (Alvarez etal., 2008) 24 M. caprae and M. avium subsp.

paratuberculosis (natural infection)

29.2


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95%, 4.9–9.8) depending of the interpretation criteria (Cf. Table 23). There is no indication of the specificity in this paper. Results from this study yielded, in general, low Se values than previously described probably due to (Bezos et al., 2014b).:

i) The systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years, meaning that less positive goats were available.

ii) And/or the presence of factors that may interfere in the diagnosis. For example the SICT is not recommended when there is mixed infection with Mycobacterium avium paratuberculosis (MAP) due to decreased sensitivity (Alvarez et al., 2008).

Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings Among ancillary diagnostic tools, the IFN‐γ assay, although not included as routinely diagnostic tool in caprine eradication programmes, show promising results (Cf. next question). Ab ELISA could also be used but it is evaluation was performed on a limited number of animals (Cf. serology question) (Bezos et al., 2014b). Table 23. Summary of the number of positive reactors (R+), sensitivity (Se, Wilson CI 95%) detected in the 7 dairy caprine flocks subjected to depopulation using single and comparative intradermal tuberculin test (SIT and SCIT tests respectively (Bezos et al., 2014b).

Conclusion: Tuberculosis Skin test can be applied in goats and help to control Tb disease. However, there is a lack of standardization of SICT and SICTT in this species, and certain aspects like the site of injection (neck or shoulder) or the interpretation of the results vary between the studies and are usually applied just following the standards developed in cattle. SICT should be used with caution on herd with presence of MAP.

Sheep Tuberculosis in sheep is mainly caused by M. bovis and M. caprae, and TB transmission to sheep seems to occur through aerosols. Tuberculosis in sheep is infrequently diagnosed, however, several outbreaks in Spain have been reported with epidemiological links with TB‐infected cattle herds (Munoz‐Mendoza et al., 2015). The infection has also been described in UK, New Zealand and Ethiopia (Pesciaroli et al., 2014). Single cases or outbreaks have been reported and M. bovis infection was typically detected at slaughter or at a diagnostic post‐mortem examination where the lesions have often been incidental findings (van der Burgt et al., 2013).


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Pathology/ Clinical Signs In the UK sheep outbreaks, detected on post‐mortem examination, M. bovis cause no specific clinical disease as gradual weight loss in adult sheep of variable ages and bruxism, no coughing was observed in this outbreak and diarrhoea was not a feature. Infected flock reported that some ewes had showed chronic ill thrift for 4 year before diagnostic (2004‐2008) (van der Burgt et al., 2013). However, coughing and dyspnoea were observed in another outbreak in Spain (Munoz Mendoza et al., 2012). Necropsy of ewes showed enlarged purulent mediastinal lymph nodes, and enlarged mesenteric nodes in one ewe. Post‐mortem findings included multiple lung abscesses and caseous/purulent lesions in liver, spleen, hepatic, inguinal and mesenteric nodes (van der Burgt et al., 2013) TB lesions were mostly confined to the respiratory tract, no lesion extended to the gastrointestinal tract Sensibility and Specificity of skin test Spain: 897 suspected of infected goat belonging to 23 flocks (Munoz‐Mendoza et al., 2015)

SIT: out of 606 sheep, 151 were positive (24.92%; 95% CI: 21.52–28.56%) and the presence of clinical signs such as oedema and necrosis was recorded in 135 sheep. In this study they also compared the Skin test with other diagnostic tests. The SIT shows a low sensitivity and depends on the type of lesion observed (Table 6).

Two other studies have shown higher sensitivity results but this was based on a small sample size (Cordes et al., 1981; van der Burgt et al., 2013) Conclusion There is few data on the efficacy of the skin test in sheep. However, the SIT and SICT are regarded as first option in case of suspicion. The SIT and ELISA are recommended as the simplest and most cost‐effective initial approaches for the diagnosis of TB in sheep under field conditions (Munoz‐Mendoza et al., 2015).

Pig/ wild boar


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Before implementation of control and eradication of tuberculosis in cattle, M. bovis was commonly found in pigs and prevalence in pig population was usually correlated to that in cattle. Nowadays tuberculosis in pig is mainly related to feral pigs and wild boars (Pesciaroli et al., 2014). Pigs/ wild board are susceptible to M. bovis infection and the oral route is considered as the most important way of infection (ingestion of milk, milk product or offal from infected cows) (Pesciaroli et al., 2014). The European wild boar (Sus scofa) is widely distributed throughout Eurasia and Africa and subsequently has been introduced by humans to many other areas, as well as giving rise to our domestic pigs. Wild boar is a significant wildlife reservoir of BTB (Fitzgerald and Kaneene, 2013). Pathology/ Clinical Signs As observed in other animal species the proportion of M. bovis infected pigs and pigs with gross lesions increases with age, as already observed in wild boars. Lesions in feral pigs can be localized in the mandibular lymph nodes or may be generalized with involvement of mandibular, retropharyngeal and thoracic lymph nodes. The involvement of those lymph nodes, whose afferents drain the nasal cavities, the nasopharynx, the auditory tubes and the lung, suggests that both respiratory and food‐borne transmission may occur (Pesciaroli et al., 2014). Tuberculin skin test There is only few data on the feasibility and the performance of the skin test in pig, only two studies have been published on this topic on a limited number of animals: Nugent et al.,: tested the feasibility of using a more wide ranging species, feral pig (Sus scrofa), as an alternative sentinel capable of indicating TB presence. Before the 17 pigs were release, SIT was performed to the pinna and tested negative as expected (Nugent et al., 2002). Joroso et al., have tested the specificity of the skin test, in Spain, in Eurasian wild boar (Sus scrofa). They found the Sp at 77.4% (24/31), using the broadest criteria (PPDB response larger than 2mm and PPDB response larger than PPDA response). In this experiment, animals were injected intradermally in the inguinal region, separated 10 cm from each other (Jaroso et al., 2010). Conclusion Although the ante‐mortem test currently used in pigs in GB is the comparative intradermal tuberculin test applied at the base of the pinna, few data exist on its diagnostic accuracy. The diagnosis of pig is mainly base on the post mortem examination and the bacterial isolation of M. bovis coupled with the detection by histology or/and PCR (Broughan et al., 2013). Ab ELISA or IFN‐γ have been suggested for the diagnosis although the specificity of these tests may be compromised by the high degree of exposure to nontuberculous mycobacteria (Pesciaroli et al., 2014), for example M. hominissuis (Barandiaran et al., 2015; Vyt et al., 2013).

Camelids/llama/alpaca Tuberculosis is mainly caused by M. bovis and M. microtti and gained greater importance in Europe in the last decades, because of the growing number of animals (mainly llamas – Lama glama – and alpacas – Vicugna pacos) being imported into several European countries to serve as pets, pack animals or for production purposes. Risk factors for the transmission of tuberculosis consist in close contact with infected cattle or sharing pasture combined to the intensive condition management, the introduction of undiagnosed M. bovis‐infected alpaca (clearly demonstrated in 3 alpaca herds) (Alvarez et al., 2012). Pathology/ Clinical Signs Tuberculosis is a chronic debilitating disease. The clinical signs in camelids include wasting, anorexia, and respiratory distress, enlargement of superficial lymph nodes, recumbency and eventually death. Clinical signs are often associated with extensive respiratory pathology, and it is surprising that overt


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respiratory distress is sometimes not observed in animals with severe lung lesions. Animals are occasionally found dead with no previous clinical observations (Wernery and Kinne, 2012). “Lesions are mainly located in lung, pleura and associated lymph nodes, usually in the form of small multifocal white‐yellowish caseous nodules, although larger abscesses have been also described. However, generalized disease is not uncommon, and additional locations of macroscopical pathology include the pericardial sac, liver and bronchial and mediastinal lymph nodes” (Alvarez et al., 2012). Tuberculin Skin test In the UK, surveillance for TB in camelids is primarily done by post‐mortem examination of routine casework or carcasses of suspect clinical cases. The disease is notifiable to AHVLA; however, no regular control program is applied. Ante mortem TB testing of camelid usually takes place only for export certification purposes and in response to TB outbreaks that are confirmed by positive culture of M. bovis (Rhodes and Vordermeier, 2012). In UK, as for the rest of the EU and many other countries, the tuberculin skin test remains the primary official test for TB and is recommended for the trading between Member States of the EU (Directive 92/65/EEC). The skin test, although specific, lacks sensitivity (Cf. table below: studies summarized by (Alvarez et al., 2012)). However, the sensitivity and specificity in this summary were based on studies where animals have a known infection status, limiting their accuracy. Large scale evaluation should be necessary to evaluate the Se and Sp of skin test (Cf. Table below). Table 24. Sensitivity and specificity of skin test on camelid (Alvarez et al., 2012).

Animal species tested (n)

True status (bacterial species)

Sensitivity (95% CI)

Specificity (95% CI)

Comments Reference

Single intrad

erm

al tuberculin

(SIT) test

Alpaca (16) Experimentally infected (M. bovis)

100 (79.4–100)

ND No data on site of inoculation and interpretation criteria

R. de la Rua‐Domenech, personal communication (cited in Cousins and Florisson, 2005)

Llama (5) Experimentally infected (M. bovis)

80 (28.4–99.5)

ND Axillary site, day 80 post‐challenge, readings at 96 h post‐inoculation

Stevens et al., 1998


100 (29.2–100)



Dromedary (2) Naturally infected (M. bovis)

100 (15.8–100)

ND Axillary site, reading 5 days post‐inoculation, standard interpretation

Wernery et al., 2007

Alpaca (12) Non‐infected ND 100 (73.5–100)

No data on site of inoculation and interpretation criteria


Llama (2) Non‐infected ND 100 (15.8–100)

Axillary site, performed twice in both animals


Single comparative intraderm

al tuberculin

(SCIT) test Alpaca (2) Naturally

infected (M. bovis)

0 (0–84.2) ND Test performed in the month before the onset of clinical signs.

Garcia‐Bocanegra et al., 2010

Alpaca (21) Experimentally infected (M. bovis)

76.2 (52.8–91.8)



Llama (14) Naturally infected (M. bovis)

14.3 (1.8–42.8)

ND Axillary site, no data on interpretation criteria (standard?)

Dean et al., 2009

Llama (7) Naturally infected (M. microti)

0 (0–41) ND Axillary site, standard interpretation

Lyashchenko et al., 2007


87.5 (67.6–97.3)


F. Stuart, personal communication (cited in Cousins and Florisson, 2005)

Alpaca (5) Naturally 0 (0–52.18) ND Axillary and cervical sites Ryan et al., 2008


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Animal species tested (n)

True status (bacterial species)

Sensitivity (95% CI)

Specificity (95% CI)

Comments Reference

infected (M. bovis)

assayed. No data on site of inoculation

Dromedary (2) Naturally infected (M. bovis)

100 (15.8–100)


Wernery et al., 2007

Alpaca (12) Non‐infected ND 100 (73.5–100)



Llama (12) Non‐infected ND 100 (73.5–100)


F. Stuart, personal communication (cited in Cousins and Florisson, 2005)

Conclusion There is a clear difference of the skin test performances between experimentally vs naturally infected alpaca. Se and Sp in experimental infection were higher than those obtained in natural infection. There is a lack of records in naturally and non‐infected camelid. Skin test is usually performed following cattle protocols, and further evaluation is needed to define the best dosage and time interval of reading. For example, a better performance was observed when the skin test is performed at the axillary sites (Broughan et al., 2013; Wernery and Kinne, 2012) and read after 5 days, but this was only performed on two animal (Wernery et al., 2007). However it is recommended to perform SIT in order to increase the production of antibody and to combine then skin test with Ab ELISA after 2 weeks (Rhodes et al., 2012) (Cf. Point 5). Despite the poor sensitivity of skin tests in camelids, two consecutive negative skin‐test results 90 days apart remains the minimum requirement for the removal of movement restrictions in camelid herds in GB. However, this approach is typically complemented by the voluntary use of in vitro tests (Broughan et al., 2013). Horses Pathology/ Clinical Signs Tuberculosis in horses relies on histopathological examination and culture, as the disease can manifest as a range of nonspecific clinical signs. The clinical signs of TB in horses could be, as in other animals, weight loss, anorexia, pyrexia, diarrhoea or chronic cough. Ultimately, the infected horse could die (Keck et al., 2010; Sarradell et al., 2015). Bovine TB infection in horses is assumed to be acquired by ingestion, leading to the development of a primary complex in the mesenteric lymph nodes. However, primary respiratory infection could also occur in situations where animals are exposed to a high infection pressure (Keck et al., 2010; Sarradell et al., 2015). Tuberculin skin test / Conclusion The intradermal tuberculin test is considered as unreliable in horses and false positive results are common (Broughan et al., 2013; Pesciaroli et al., 2014).

Cats and Dogs In Great Britain, isolations of M. bovis, M. microti, and M. avium in cats appear to have discrete, almost entirely non‐overlapping geographical distributions, with M. bovis isolations concentrated in areas where the bTB is endemic in cattle (Chomel, 2014; Roberts et al., 2014). When bovine tuberculosis is uncontrolled in cattle, a high incidence of disease may be seen in cats; up to 50% of the cats may be infected on affected farms. (http://www.cfsph.iastate.edu/Factsheets/pdfs/bovine_ tuberculosis.pdf) Few cases of infection of dogs with M. bovis and M. tuberculosis are reported throughout the world. For example, in UK, only 7 cases of infected dog have been reported between 2004‐2010 (Broughan


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et al., 2013). There are no documented cases of M. tuberculosis infection spreading from dogs to humans, thus this disease is known to be an anthropo‐zoonosis (Engelmann et al., 2014). Pathology/ Clinical Signs In cats and dogs, the clinical signs of TB infection are non‐specific and diagnosis is difficult and based primarily on mycobacterial culture (Broughan et al., 2013). Clinical signs in cat include dyspnea, tachypnea, hyporexia, and lethargy (Ramdas et al., 2015). Cats commonly present single or multiple cutaneous lesions (74%), which were sometimes ulcerated or discharging, located most frequently on the head (54%). Lymph nodes were usually involved (47%), typically the submandibular nodes. Systemic or pulmonary signs and/or respiratory infection were rarely seen (10%–16%) and (Broughan et al., 2013; Chomel, 2014). In dogs, all reported cases of infections with M. tuberculosis were localized in the pulmonary system however some other localisation could be observed as for example abdominal (Engelmann et al., 2014). At the end, animal could presented progressive weight loss, anorexia, nonproductive cough, melena, hematemesis, epistaxis, mild diarrhea, and dyspnea (Martinho et al., 2013). Tuberculin skin test In cat, the SIT is considered as unreliable, however others tests like IFN‐g and serology could be considered (Broughan et al., 2013; Fenton et al., 2010; Gunn‐Moore, 2014; Ramdas et al., 2015). In dogs the use of SIT has produced inconsistent results and to date, there does not appear to be any published assessment of the use of the IFN‐γ or of antibody tests to detect bTB in dogs (Broughan et al., 2013). Conclusion Skin test in Cat and Dog is not applied. Wild and zoo animals Introduction Mycobacterium bovis has an extraordinary host range, especially when compared to other M. tuberculosis complex species. The list of animals susceptible to M. bovis is extensive: domesticated animals (see part 1) and a variety of wildlife species, both in the wild and in captivity (Cousins and Florisson, 2005). (Cf. Table Annex) Skin test in wildlife species The SIT test appears to work well in some wildlife species, such as deer but is unreliable as a diagnostic test in other species infected with M. bovis, such as Eurasian badgers possums, oryx and large zoo mammals, and is impractical for free‐ranging wildlife because of the need to measure the cutaneous reaction some 24‐72h after the injection of tuberculin (Cf. table 5) (Chambers, 2009, 2013). Table 25. Summary of CMI assays employed in different non‐bovid wildlife since 2009 (Chambers, 2009, 2013). Species Reference(s) (E)xperimentally

or (N)aturally infected

Mycobacteriumspecies

Method employed

Stimulatory antigen(s)

Eurasian wild boar (Sus scrofa)

Jaroso et al. (2010a)

N M. bovis SICT PPD‐B, PPD‐A

Fallow Deer (Dama dama)

Boadella et al. (2012) and Waters et al. (2011b)

N M. bovis SIT/SICT PPD‐B, PPD‐A

Red/Elk Deer (Cervus elaphus)

Palmer et al. (2011) and Waters et al. (2011b)

N M. bovis SIT PPD‐B


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Buddle et al. (2010)

N/E M. bovis (MAP) SIT/SICT PPD‐B, PPD‐A

Pygmy hippopotamus (Hexaprotodon liberiensis)

Bouts et al. (2009)

Unconfirmed M. interjectum SIT PPD‐B, PPD‐A

Lion (Panthera leo) Keet et al. (2010) N M. bovis SIT/SICT PPD‐B, PPD‐A

Non‐human primates Rhesus macaque (Macaca mulatta)

Parsons et al. (2010)

N M. tuberculosis, M. kansasii

SIT, QFG‐IT PPD‐B Proprietary peptides

Cynomolgus macaque (Macaca fascicularis)

Panarella and Bimes (2010)

N M. bovis SIT MOT

Silvered langur (Trachypithecus cristatus ultima)

Georoff et al. (2010)

NT SIT/SICT MOT, AOT, PPD‐B, PPD‐A

Chacma baboon (Papio ursinus)

Parsons et al. (2009)

N M. tuberculosis SIT, QFG‐ITELISA

PRO

PPD‐B Proprietary peptides Proprietary peptides

Elephant (Elaphus maximus)

Mikota et al. (2001)

N M.tb SICT PPDA‐PPDB

Black rhinoceros (Diceros bicornis)

Mann et al. (1981)

N M. bovis SICT PPDA‐PPDB

Seal (Phocidae) Cousins (1987) N M. bovis SICT PPDA‐PPDB

Arabian oryx (Oryx leucoryx)

Flamand et al. (1994)

N M. bovis SICT PPDA‐PPDB

Elephant Tuberculosis in elephant is mainly caused by M. tuberculosis followed by M. bovis. Due to the close contact between human and elephant, M. tuberculosis is the major cause of tuberculosis in elephant. For example, US reported 34 cases of tuberculosis between 1994 and 2005 and 33 were caused by M. tuberculosis and only one by M. bovis (Mikota and Miller, 2005). Pathology/ clinical signs M. tuberculosis is the main causative agent of tuberculosis and diagnosis is limited: clinical signs are typically absent until the disease is well advanced and chest radiographs are not feasible in adult elephants. On post‐mortem, some elephants have significant abscess formation and caseation of the lungs, thoracic and abdominal lymph nodes, and liver. (Mikota et al., 2006). Zoo Elephant The trunk wash, the elephant equivalent of a sputum sample, has poor sensitivity due to inefficient sample collection, contamination of samples by microbial contaminants in the trunk, or low and/or intermittent bacterial shedding. In one outbreak in Sweden, only 7 of 189 trunk wash samples sequentially collected from 5 elephants diagnosed with TB at the end by post‐mortem analysis were culture positive (very poor sensitivity method) (Maslow and Mikota, 2015; Mikota and Maslow, 2011). However, serology works very well, better than in another mammalian host. The particularly striking performance of serodetection of TB in elephants may in part be due to the fact that most elephants with active TB display no clinical signs and therefore are likely to progress undetected to advanced stages of diseases with high bacterial burden and commensurate circulating antibodies in the blood (Chambers, 2013). Lyanchchenko et al, have demonstrated that elephants diagnosed with M. tuberculosis produce robust antibody responses to multiple antigens long before positive cultures can be detected from trunk washes (Lyashchenko et al., 2006). The ElephantTB STAT‐PAK, Dual Path Platform [DPP] VetTB, and multiantigen print immunoassay [MAPIA], have thus proven valuable in the diagnosis of TB in elephants (Cf. Question on serology) (Maslow and Mikota, 2015; Mikota and Maslow, 2011)(ANSES, 2013). Conclusion


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One study evaluated the Se to 16.7 % (6 elephants affected) and the Sp to 74.2% (8/31 healthy elephants) (Mikota et al., 2001). The intradermal tuberculin test is not unreliable as a screening test and correlation between the intradermal tuberculin test (skin test) and culture results (excretion of M. tuberculosis) has not been established. The Skin test is not recommended in elephant (ANSES, 2013; Mikota et al., 2006).

Badgers Tuberculosis in badgers is a chronic infection and in a naturally‐infected population the severity of disease can vary widely, from latent infection (infection without clinical signs and no visible lesions) or severe disease with generalized pathology through mild disease with small pulmonary and extra‐pulmonary lesions, to severe disease with generalized pathology, cachexia and death (Corner et al., 2011; Fitzgerald and Kaneene, 2013). Tuberculosis is present in badgers in parts of England and can be transmitted among cattle, among badgers, and between the 2 species. Skin test in badgers was not used because of handle animals twice over 2‐3 days interval. In addition badgers produce unreliable delayed type hypersensitivity (DTH) responses to skin test (Corner et al., 2011). Other tests, IFN‐γ test and serology were used (Cf. next questions) (Corner et al., 2011).

Conclusion Skin test both the single (SIT) and comparative intradermal tuberculin (SICT) tests have been first developed to control and eradicate bovine tuberculosis in domestic ruminants (mainly cattle). Although the skin test appears to work in goats and sheeps, and also in some wildlife species such as deer, it appeared unreliable as diagnostic test in other species infected with M. bovis, such as Eurasian badgers possums, swine, wild board, oryx and large zoo mammals, and is impractical for free‐ranging animals. The performance of the skin test can vary considerably depending on the circumstances of its use and the population of animals used for study (Chambers, 2013). All the variations observed in cattle depends on quality of tuberculin, materials used for injecting tuberculin, localization….and have also an impact on the quality of this test in non‐bovine animals. Therefore, the fact that the skin test was first developed and interpreted according to the “bovine standards”, without adaptation to non‐bovine species could explain that estimated Se and Sp were generally lower than in cattle. Interference of MAP infection observed in cattle could also have a strongest negative effect on the skin test as observed in goat.

Annex Table 26. Summary of Key Features of Bovine Tuberculosis in Wildlife Reservoirs (Fitzgerald and Kaneene, 2013).


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Table 27. Examples of free living wildlife or captive wildlife reported with M. bovis (Cousins and Florisson, 2005).


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Appendix VI: Diagnostics strategies for bTB in other countries

The Netherlands (OTF) No routine test performed for bTB surveillance SIT is used on known infected herds and the IFN‐γ is performed after the skin test in positive herds in order to increase the specificity. Problem of possible cross reaction with presence of MAP has been reported (EFSA, 2012b).

Poland (OTF) SIT are performed every 5 years. If positive, animals are culled and if inconclusive the SICT is performed 42 days later. The IFN‐γ BA is used in a serial way in case of inconclusive results with the tuberculin skin test (SIT) (EFSA, 2012b).

Germany (OTF) In Germany no routine testing is performed. Disease monitoring is performed at the slaughterhouse with the clinical ante‐mortem and postmortem inspection. When positive cases are identified, skin tests are performed in the corresponding herds. To avoid false positive results, SICT is the test choice IFN‐γ BA tests are performed in some regions in herds where positive cases were identified. IFN‐ γ BA test has been used as an ancillary test while performing comparative skin test on suspect animals, parallel use of the test (EFSA, 2012b).

Hungary (Non‐OTF) Routine tests are performed once a year, SIT test for all animals older than 3 months and SICT test for confirmation in officially free herds. Since 2010, the SICT test is recommended as a first test in herds with a high number of non‐confirmed reactors. IFN‐γ poorly used due to its high cost supported by farmer and lack of agreement with the SICT (EFSA, 2012b).

UK (non‐OTF) Scotland is an officially tuberculosis free region since October 2009. England and Wales are not free. The SICT skin test, with avian and bovine tuberculins from Prionics is the primary screening test for bovine TB (EFSA, 2012b). The test IFN‐γ test is routinely used in parallel to the comparative skin test (SICT) in specified circumstances e.g. where there is a suspicion of spread of disease to a new area and on a discretionary basis where there are high disease incidences. Only animal over 6 month were tested. On occasion the IFN‐γ test is also used as a serial test to the SICT to improve test specificity where there are suspicions of non‐specific skin test reactions or where there is suspicion of skin test interference. The test is also used in two other circumstances using defined antigens.

- The first is in apparent chronic herd infections which fail to have TB confirmed either by bacteriology or by histopathology. In these cases an IFNg test can be applied using ESAT6 and CFP10 antigens to maximise the specificity of the test. Results from this are used by the competent authority to assess whether there is likely to truly be infection in the herd and if so to identify any animals apparently infected but not detected by the SCIT.

- The second circumstance is where there is suspicion that positive SCIT results are due to interference with the test (Coad et al., 2008; Strain et al., 2012).

Republic of Ireland (non‐OTF) In Ireland routines SICT tests are performed. The IFNg test is applied in parallel to the SICT in high prevalence herds, typically on the first day of the follow‐up skin test following infection disclosure (a minimum of 60 days after the first skin test, SIT). Sampling is also permitted 10 days following the initial skin test (SIT) to allow for the more rapid removal of test positive animals (EFSA, 2012b; Strain et al., 2012).


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New Zealand Situation: presence of M. bovis in wild life and livestock, but under a strong control program (http://www.tbfree.org.nz/bovine‐tuberculosis‐information.aspx). The IFN‐γ test is available to be used as both a serial and a parallel test of skin test.

- The IFN‐γ test can be used as parallel with the SIT still animals were tested negative to the CFT (based on history of infection). All animals testing IFNg positive are declared reactors and slaughtered.

- As a serial test it is only used in animals positive to the caudal fold test (CFT) in order to confirm positivity. In these cases it is used 13 to 33 days following the skin test.

The SICT is available to be used as a primary or a serial test but only following approval of the National Disease Control Manager (Strain et al., 2012).

Spain (non‐OTF) Spain performs routine tests (SIT) on live animals, complemented by monitoring at slaughterhouse. SICT test can be used only in those herds in low prevalence regions and where there could be some cross‐reactions and low risk of bovine TB. The IFN‐γ is used as a parallel test for the detection of the maximum number of infected animals.. It is usually applied in regions with a perceived high incidence of TB infection (herd prevalence of greater than 1%) although it can be applied in herds in other regions at the discretion of the competent authority. Only animal over 6 month were tested (EFSA, 2012b; Strain et al., 2012).

France Situation: OTF but high prevalence in some regions including Côte d’Or, Dordogne, Landes, Pyrénées Atlantique and Camargue (Fediaevsky et al., 2011). The general bovine TB testing protocol includes initially a SIT test, after 6 weeks, if positive; a SICT test is also performed. If the result is also positive the animal is culled and after 3 month histology and culture results are also available. A PCR test was also introduced for direct diagnosis. IFN‐γ BA test was introduced in parallel to skin test for increasing diagnosis sensitivity in detecting positive animals in infected herds and in a serial manner to discriminate within positive skin tests (EFSA, 2012b) Bloods are taken no later than at the time of the skin test reading. It is applied as a serial test following either a single or a comparative intradermal test. The IFN‐γ test is used to clarify the status of all SIT inconclusive animals and any positive SIT test results where there is a query over the validity of the skin test result after considering the epidemiological context of the result. If the animal tests positive the herd’s official TB status is classified as withdrawn. If the animal tests negative the herd’s status is suspended and all animals within the herd are required to undertake one comparative skin test. Where the comparative skin test is applied, animals with inconclusive reactions are subjected to a follow‐up interferon‐gamma test (Strain et al., 2012). In Côte d’or (France) an experimental serial testing scheme based on the combination of SICT and gamma‐interferon (IFN‐γ) tests have been initiated in order to shorten the interval between suspicion and its invalidation in herds with false‐positive results to skin tests. Figure 7. Experimental serial testing scheme based on the combination of SICT and gamma‐interferon (IFN‐γ) tests according to Praud et al. (2015).


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This study includes 1768 animals and the Se and Sp were estimated by a Bayesian approach in a French area with low bTB prevalence but numerous false‐positive results to skin tests. In farm where non‐negative results (i.e. positive or doubtful results) to SICT were observed, an IFN‐γ test was performed between 74h and 5 days after the tuberculin injections, on a batch of cattle including animals showing non‐negative results by SICT, in order to confirm or invalidate the suspicion. No significant difference could be demonstrated between the sensitivities of the serial testing scheme used in Côte d'Or (73•1%, 95% CrI 41•1‐100) and the European Union serial testing scheme using SIT and SICT (70•1%, 95% CrI 31•5‐100•0) (Praud et al., 2015).

Italy The distribution of bTB is highly regionalized. SIT tests are performed with different frequency according to the regions situation: no routine tests (Friuli Venezia Giulia), every year, every 2‐3 or 4 years. In Lombardia and Valle D’Aosta the test is applied in parallel to herds where there is confirmed presence of bTB (i.e. culture positive) and any test positive animals are slaughtered. In some region, farmer applied private IFNg and slaughtering or selling positive test animals before an official test can be undertaken (Strain et al., 2012). Table 28. Brief overview of bTB status and diagnostic strategies in the USA, in various European countries and in New Zealand (Schiller et al., 2010a).

1Herd prevalence numbers of bTB in 2007 (Anon, 2009c,d; Wilson et al., 2009; P. Livingstone, Animal Health Board, personal communication, 2009). 2 Maintenance hosts, ‘‘in brackets’’ spillover or sentinel hosts (Corner, 2006). 3 A serial IFN‐γ test is used for re‐testing TST‐positive cattle, and a parallel test is used for testing TST (= SIT) ‐negative animals.


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Table 29. Summary of the application of IFN‐g test internationally (Strain et al., 2012).


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Appendix VII: The use of the IFN‐y test in non‐bovine animals

IFN‐γ is a cytokine playing a major function in the innate and adaptive immunity against viral, some bacterial and protozoal infections and (Savan et al., 2009). The principles used to individuate infected bovines have been translated also to other animal species as cervids, African buffalo (Syncerus caffer), goat, etc. and proved to be valuable in detecting Eurasian wild boar (Sus scrofa scrofa) infected with M. bovis in experimental settings (Pesciaroli et al., 2012). Domesticated animal

Goat The few studies on IFN‐γ test in goats have yielded a wide range of data on sensitivity and specificity use in goat. The BOVIGAM IFN‐γ kits was used in these studies and the cut‐off used for the interpretation is the same as in cattle. Table 30. Summary of different Se and Sp estimates for the use of the IFN‐y test in goats

Number of goats

True status (bacterial species) Se Sp Comments Reference

49 M. bovis (natural infection) or M. avium subsp. paratuberculosis (natural infection)

83.7 – PPD CZ Veterinaria. Goat positive if bovine PPD OD was ⩾1.5 times to the no antigen sample (Nil) OD and ⩾avian PPD OD

Gutierrez et al., 1998

25 Culture negative goats – 96 Specificity value was calculated using culture negative animals (n = 12) from a positive herd

ND

M. bovis (natural infection) Remark: 97 randomly selected goats from 19 infected herds

91.6 100 Blood sampling before and 5–10 d following intradermal test

García Marín, 1993

48 M. bovis (natural infection) 87.2 – Goat positive if bovine PPD OD minus no antigen sample OD ⩾ 0.05 and bovine PPD OD > avian PPD OD

Liébana et al. (1998)

131 M. caprae (natural infection) 58/71 – Standard/severe interpretation Álvarez et al. (2008)

24 M. caprae/M. avium subsp. paratuberculosis (natural infection)

58.3/66.7

45 M. caprae/M. avium subsp. paratuberculosis (natural infection)

92.9 – Severe interpretation Bezos et al. (2009)

35 M. caprae (natural infection) 85.7/82.9 – Standard/severe interpretation Bezos et al. (2010a)

Few data exists regarding the specificity of diagnostic assays for Tb in goats based on the CMI response, since this parameter has been estimated mostly in animals from positive herds or with low numbers of animals. There is a need for further studies using larger numbers of animals from herds that are historically Tb negative, with confirmation of their Tb‐free status by culture. The sensitivity of the IFN‐γ assay in goats is similar to or higher than that obtained with the SIT test (Bezos et al., 2012).

Sheep


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Although rarely applied to sheep, the IFN‐γ assay yielded results in agreement with the SICT, being able to recognize 4/4 skin reactors from which 3/4 had visible TB lesions at necroscopy (Pesciaroli et al., 2014). A recent report an evaluation of IFN‐γ test in Spain outbreaks. In this study, 453 sheep that were tested by the IFN‐γ test, 22 were positive (4.86%; 95% CI: 3.07–7.26%, Table 31, below). Interpretation of the test: all animals in which mean optical density (OD) of the sample stimulated with bovine PPD minus, the mean OD of nil antigens was >0.05 and greater than the OD of the avian PPD‐stimulated sample were considered positive (Munoz‐Mendoza et al., 2015). Table 31. Results of diagnostic test for TB in suspicious sheep cohabiting with TB‐infected cattle and/or goats (Munoz‐Mendoza et al., 2015)

In this study, only 23 sheep were also subjected to all the diagnostic technique (ELISA, SIT, culture and histopathology). Two type of ELISA were used: one for the detection of bovine TB (use of bovine PPD) and PPA‐3 (for detection of Johnes disease). In this small group, ELISA and the IFN‐γ test provided moderate or good sensitivity whereas the SIT provide low sensitivity. The poor result of SIT and the moderate result of ELISA results are influenced by the presence of MAP infection. In the IFN‐γ test, the use of the two PPDs (avium and bovine) explained the results of IFN‐γ test (Table 32, below) (Munoz‐Mendoza et al., 2015). Table 32. Evaluation of diagnostic techniques in TB‐infected sheep. Concordance (Cohen’s Kappa), sensitivity and specificity of techniques performed using both culturen and histopthology as ‘gold standards’ (Munoz‐Mendoza et al., 2015)

As for goat, few data exits on Se and Sp values of the IFN‐γ test in sheep. However, the single intradermal tuberculin test and ELISA were the simplest and most cost‐effective initial approaches for the diagnosis of TB in flocks of sheep under field conditions. However, when possible, IFN‐γ should be applied to increase sensitivity (Munoz‐Mendoza et al., 2015)

Pig/wild boar Pesciaroli and al., were the one to make an evaluation on the capability of the interferon‐gamma (IFN‐γ) assay to identify pigs infected with M. bovis (Pesciaroli et al., 2012) In pig, the criteria for interpretation of IFN‐y assay was describe below:


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Table 33. Criteria for interpretation of the IFN‐y assay in pigs (Pesciarolli et al., 2012)

The IFN‐γ assay correctly identified all the 31 healthy pigs enrolled in the study as negative, giving a specificity of 100%. That Sp value is higher than observed in others animal including cattle. The Se was evaluated on 100 pigs at risk of infection. The IFN‐γ assay identified 15 out of 19 animals positive to the bacterial culture and 22 out of 26 animals with tuberculosis lesions. The IFN‐γ assay displayed, therefore, a sensitivity of 84.6% (CI 95% 52.6–100%) and 78.9% (CI 95% 44.8–100%) when compared to data from post mortem inspection and bacterial culture, respectively. Table 34. Comparison between the results of the IFN‐y assay and the results of post mortem inspection (lesions) and bacterial culture (microbiology) (Pesciaroli et al., 2012)

Camelids/llama/alpaca In camelids, the IFN‐γ test is considered unsuitable when whole heparinized blood (as used for cattle IFN‐γ testing) is used: production of IFN‐γ obtained in these conditions is too low to be in a diagnostic assay (Rhodes et al., 2012; Rhodes and Vordermeier, 2012; Wernery and Kinne, 2012). Rhodes and Vordermeier have developed and evaluated a adapted method of the IFN‐γ test in camelids (Rhodes et al., 2012; Rhodes and Vordermeier, 2012): To perform the IFN‐γ test in camelids, a preparation of PBMC (peripheral blood mononuclear cells) from the whole blood sample should be applied. The principle is to separate the PBMC out of the whole blood using a density gradient (i.e.: histopaque), and then to wash the cells and to resuspend them in complete culture medium at a concentration of 10e6 Cells/ml. In opposite to cattle, the time of incubation should be increase to 3 days at 37°C in 5% CO2 in a humidified incubator. In this study, the ELISA used to quantify the production of IFN‐γ is homemade and based on a commercial kit with cross reagents for bovine, ovine, or equine IFN‐γ (from Mabtech, cat 3115, Sweden). Table 35. title

Test resulta

VL alpacas (n = 55) TB‐free alpacas (n = 257)

n/55 % sensitivity 95% CI n/257 % specificity 95% CI

PPD+ 35 63.6 50.9–76.3 28 89.1 85.3–92.9

EC+ 27 49.1 35.9–62.3 26 89.9 86.2–93.6

EC+ PPD− 8 14.5 5.2–23.8 21 91.8 88.4–95.2

PPD+ EC+ 19 34.6 22.0–47.2 5 98.1 96.4–99.8

PPD+ or EC+ 44 80.0 69.4–90.6 49 80.9 76.1–85.7


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aPPD, PPDB − PPDA; EC, ESAT6‐CFP10 peptide cocktail.

VL: with typical gross visible lesions.

The results show that while the Se value of the IFN‐γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN‐γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN‐γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB‐free cohort, which stimulates a positive purified protein derivative (PPD) response. Remark: M. microti is an another member of the TB complex that causes clinical TB in camelids In a confirmed TB outbreak situation, data of this study provide options for combinations of tests to maximize the detection of infected animals, to accelerate the resolution of TB outbreaks, and thereby to substantially reduce or remove the risk of residual infection, recurrent outbreaks, and further spread. Combining one antibody test with the IFN‐γ test offered significant increase in the sensitivity of VL detection over the use of either the IFN‐γ test alone or an antibody test alone (Rhodes et al., 2012; Rhodes and Vordermeier, 2012). Table 36. title

Test combination

VL alpacas (n = 48) TB‐free alpacas (n = 257)

n/48 % sensitivity 95% CI n/257 % specificity 95% CI

IFN‐γ + Stat‐Pak 45 93.8 87.0–100 52 79.2 74.2–84.2

IFN‐γ + Idexx 44 91.7 83.9–99.5 55 78.6 73.6–83.6

IFN‐γ + DPP 44 91.7 83.9–99.5 58 77.4 72.3–82.5

IFN‐γ + Enferplex 45 93.8 87–100 58 77.4 72.3–82.5

IFN‐γ + Stat‐Pak + Idexx 48 100 92.6–100 57 77.8 72.7–82.9

IFN‐γ + Stat‐Pak + DPP 48 100 92.6–100 60 76.7 71.5–81.9

IFN‐γ + Stat‐Pak + Enferplex 47 97.9 93.8–100 60 76.7 71.5–81.9

IFN‐γ + Idexx + DPP 45 93.8 87.0–100 62 75.9 70.0–81.2

IFN‐γ + Idexx + Enferplex 44 91.7 83.9–99.5 62 75.9 70.0–81.2

IFN‐γ + Enferplex + DPP 47 97.9 93.8–100 65 74.7 69.4–80

aIFN‐γ positive, PPDB‐PPDA > 0.1 or EC‐nil > 0.1.

Horses Some IFN‐γ kits for use in horses are available on the market as for example from Sigma‐Aldrich, and R&D Systems. However, no data has been published about the use of IFN‐γ test in a TB outbreak context.

Cats/ feline tuberculosis Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for M. bovis, and rodents for M. microti (Rhodes et al., 2008). Rhodes et al. (2008) have investigated the application of antigen‐specific IFN‐γ production as a potential diagnostic test for feline tuberculosis using both ELISA and ELISPOT techniques (not discussed here). As for camelids, they work with PBMC at 2 × 106 cells/ml in tissue culture medium and with incubation for 4 days. ELISA homemade with commercial AB:


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- ELISA plates (Meso Scale Discovery, U.S.A.) were coated with 25 μl/well of 1 μg/ml capture antibody (AF764, R&D Systems Europe Ltd., U.K.) diluted in PBS and incubated overnight at 4 °C.

- A recombinant feline IFNγ (764‐FG/CF, R&D Systems) - cocktail of biotinylated secondary antibody at 1 μg/ml (BAF764, R&D Systems) plus 1 μg/ml

streptavidin‐alkaline phosphatase (0369, DAKO, U.K.) - Plates were washed, the colour developed using 1‐step™PNPP (Pierce, USA), and results

determined at 405 nm on an ELISA reader

Table 37. IFN‐γ ELISA results for a total of 17 cats (numbered sequentially) submitted to the TB Diagnostic section of the VLA plus five healthy control cats (Rhodes et al., 2008)

Cat no. IFN‐γ response [mean ng/ml (±S.D.)]

Un‐stimulated PPDA PPDB E6/CFP PMA/Ca

M. bovis‐positive

1 0.3(0) 0.3(0) 1.7 (0.9) 0.3 (0) 5.2 (2.8)

2 0.6(0) 9(3.8) 9.8(1) 18.8 (7.9) 9.1(0)

3 0(0) 26.2 (1.4) 28.5 (5.5) 18.9 (1.1) 37(0.1)

5 0(0) 0.3(0) 1.2 (0.2) 1.1(0) 6.4 (1.1)

M. microti‐positive

6 0(0) 0(0) 0.3 (0.1) 0(0) 8.7 (2.5)

7 0(0) 0(0) 1.1(0) 0(0) 1.1(0)

8 0(0) 2.5(0) 6.2 (0.5) 0(0) 7.4 (0.6)

M. avium‐positive

9 1.0 (0.3) 0.8(0) 0.8(0) 0.6 (0.3) 29.4(9.9)

10 0(0) 0(0) 0(0) 0(0) 47.9(5.2)

Culture‐negative

11 0(0) 0(0) 0(0) 0(0) 44.4(0.6)

12 0(0) 0(0) 0(0) 0(0) 7.6(6.2)

13 0(0) 0(0) 0(0) 0(0) 3.5(0.9)

14 0(0) 0(0) 0(0) 0(0) 26.7(7.3)

15 0.8(0.6) 4(0) 1.9(0.2) 1(0.3) 3.6(0.6)

16 0(0) 2.2(0.1) 2.5(0) 0.3(0.2) 0.6(0.1)

17 0(0) 0(0) 3.9(0.9) 0(0) 33.1(7.4)

18 0(0) 0.3(0.2) 2.1(0.1) 0(0) 0.75(0)

Control cats

19 0(0) 1.5(0.6) 0(0) 0(0) 48(2)

20 0(0) 0.5(0.4) 0(0) 0(0) 160(24.5)

21 0(0) 0(0) 0(0) 0(0) 20(15)

22 0(0) 0(0) 0(0) 0(0) 29.2(6.4)

23 0(0) 0(0) 0(0) 0(0) 26(7.2) Cats are divided into sections depending upon the mycobacterial culture result of the co‐submitted biopsy. The mean

concentration of IFN‐γ in ng/ml of PBMC culture supernatant is shown plus the standard deviation (S.D.) of duplicate

samples for each culture condition, i.e. un‐stimulated, PPDA‐stimulated, PPDB‐stimulated and PMA/Ca‐stimulated PBMC.

Results in bold type (cats 16–18) highlight IFN‐γ results that suggested M. bovis or M. microti infection, but nothing was

isolated from the co‐submitted biopsy material. A positive response was considered as a mean IFN‐γ concentration greater

than the mean plus 3S.D. of the un‐stimulated PBMC control.


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This test could distinguish M. bovis from M. microti infection when positive responses to the specific proteins ESAT6/CFP10 (present only by M. bovis) were detected (Rhodes et al., 2008). ELISA Made home with commercial AB:

- ELISA plates (Meso Scale Discovery, U.S.A.) were coated with 25 μl/well of 1 μg/ml capture antibody (AF764, R&D Systems Europe Ltd., U.K.) diluted in PBS and incubated overnight at 4 °C.

- A recombinant feline IFNγ (764‐FG/CF, R&D Systems) - a cocktail of biotinylated secondary antibody at 1 μg/ml (BAF764, R&D Systems) plus 1 μg/ml

Streptavidin‐Sulfo‐Tag (MSD, U.S.A.) - 150 μl/well of MSD Read Buffer T (diluted 1:1 in dH2O) - Plates were read on the MSD electro‐chemiluminescence platform and data analysed using

MSD software. Table 38. IFN‐γ response rates (%) in study groups using different test interpretation criteria. Percentage of cats in each group that were IFN‐γ test‐positive to mycobacterial antigens (Rhodes et al., 2011).

Cat group PPDB > PPDA ESAT6/CFP10+PPDB > PPDA andESAT6/CFP10+

PPDB > PP‘DA or ESAT6/CFP10+

PPDA ≥ PPDB

M. bovis+ (n = 10) 90.0 80.0 70.0 100.0 10.0

M. microti+ (n = 12) 83.3 0.0 0.0 83.3 16.7

Non‐TBC+ (n = 11) 0.0 0.0 0.0 0.0 54.5

Negative controls (n = 12)

0.0 0.0 0.0 0.0 16.7

Dangerous contacts (n = 3)

100.0 66.7 66.7 100.0 0.0

In this study, the IFN‐γ test (PPDB‐biased responses) detected 90% M. bovis‐confirmed cats and 83.3% M. microti‐confirmed cats as having TB Complex infection. Using the PPDB‐bias‐based interpretation with or without ESAT6/CFP10 responses demonstrated 100% test specificity in this study, as no positive results were obtained in the negative or non‐TBC groups (Rhodes et al., 2011).

Dogs Parsons et al. (2012) have investigate a novel IFN‐γ test to determine the risk of transmission of M. tuberculosis from infectious human TB patients to contact dogs. Twenty‐four dogs at high risk of M. tuberculosis infection (i.e. TB‐exposed animals) were identified as animals living on the same property as a person being treated for sputum smear‐positive pulmonary TB. Within two hours of collection, blood was diluted 1:5 in RPMI 1640 complete medium, and 5 days of incubation. ELISA based on Quantikine® canine IFN‐γ Immunoassay, R&D Systems, Minneapolis, USA). Conversely, an IFN‐γ‐based assay was able to effectively recognize dogs with a previous exposure to TB human patients (Parsons et al., 2012). The IGRA proved a useful test of M. tuberculosis infection in dogs and the high levels of transmission of this pathogen from humans to companion dogs should be considered when assessing the zoonotic risks associated with such animals. IFN‐γ test detects immunological sensitization to M. tuberculosis antigens, they identified a 50% infection rate in dogs in contact with smear‐positive TB patients (Parsons et al., 2012). One of the advantages of IFN‐γ test is that sedation of dogs, which is required to perform the skin test, is not required for the IFN‐γ test. Wild and Animals


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Elephant Angkawanish et al. (2013) have described an IFN‐γ assay applicable for diagnosis of tuberculosis in elephants. Whole blood was diluted 1/2 with complete medium RPMI1640 and incubated in 24‐well tissue culture plates at 37°C, 5% CO2 for 48 h The ELISA was homemade including the specific monoclonal antibodies. Table 39. title

Stimulant Non‐infected elephant 1

Non‐infected elephant 2

TB‐suspected elephant

M. tb‐infected elephant

PWM 1.577 ± 0.24 1.058 ± 0.19 1.867 ± 0.19 2.569 ± 0.43

PMA/I 2.021 ± 0.02 1.976 ± 0.05 2.563 ± 0.12 2.572 ± 0.55

EC 0.103 ± 0.00 0.196 ± 0.03 0.129 ± 0.00 1.208 ± 0.02

PPDB 0.127 ± 0.03 0.164 ± 0.04 0.104 ± 0.03 0.530 ± 0.02

PPDA 0.132 ± 0.00 0.206 ± 0.01 0.123 ± 0.00 1.141 ± 0.10

Medium 0.080 ± 0.00 0.151 ± 0.03 0.062 ± 0.00 0.119 ± 0.00

IFN‐γ, interferon gamma; M. tb, Mycobacterium tuberculosis; MTBC, Mycobacterium tuberculosis complex; PPD‐B PPD‐A,

protein extracts of mycobacteria. (Angkawanish et al., 2013)

Availability of MoAbs specific for rEpIFN‐γ and native elephant IFN‐γ is an important pre‐requisite for the development of an IFN‐γ release assay for the diagnosis of tuberculosis in elephants. Angkawanish et al. (2013) have now developed an IFN‐γ test for both Asian and African elephants, but this test needs further validation for its use in diagnosis, which relies on its application in large populations of non‐infected, suspected and infected elephants.

Lion (Cf. cat) Ingestion of infected prey animals has been commonly accepted to be the main route of infection for lions. However, research is needed to find other possible sources and routes of infection. For example, in South Africa, African buffalo are a maintenance host of M. bovis and lion M. bovis infection has been link to the ingestion of infected buffalo (Viljoen et al., 2015). The diagnosis of M. bovis infection in lions may involve gross post mortem examination with associated histopathology, bacteriological examination of clinical and post mortem samples, and immunological assays (Viljoen et al., 2015) Serology Some serological test as ELISA/EIA and ELISA based on MPB70 have too low sensitivity to serve as reliable antemortem diagnostic tests for individual animals but could complement the SIT (Viljoen et al., 2015) Unfortunately, the IFN‐γ test does not distinguish between infection status (e.g. recent, latent or advanced/diseased). Skin test could be applied with modifications (Keet et al., 2010): 1. use of 0.2 ml tuberculin per injection site (double the volume prescribed for cattle); 2. while both avian and bovine tuberculin were injected at separate sites, they only considered the result of the bovine tuberculin reaction This test identified over 86.5% of lions (n = 52) in which M. bovis infection was confirmed through mycobacterial culture. However, 13.5% of culture positive lions tested negative (false negative) and 18.8% of true negative animals tested positive (false positive)


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Table 40. Sensitivity and Specificity of the skin test in Lions (Keet et al., 2010)

Total Sensitivity

false negative

reactors;

TP: true

positive

reactors.

SIT 52 0.865 7 45

SICT 52 0.808 10 42

Total Specificity

false negative

reactors;

TP: true

positive

reactors.

SIT 44 1 44 0

SICT 44 1 44 0

IFN‐y test (Maas et al., 2012) The genetic sequence of lion, cheetah and domestic IFN‐γ were compared and the sequences are highly conserved between these species: This may suggest that a lion or cat specific IFN‐γ ELISA could be used for other feline species (Maas et al., 2010). Figure 8. Genetic sequence of lion and cheetay IFN‐y (Maas et al., 2010)

Maas et al. (2010) developed antibodies needs to the developpement of IFN‐γ dosage and this test shows potential as a diagnostic assay for bTB in lions (Cf. Table 41). Table 41. Mean OD450 values of whole blood samples of 11 lions from BTB‐free areas (Maas et al., 2012)

Rhinoceros Rhinoceroses are susceptible to infection by M. bovis. However, there is a need for diagnostic assays able to support the monitoring of the bTB‐free status of these animals (Morar et al., 2013). While traditional tests for diagnosing bTB include microscopy, bacterial culture techniques and SIT, these are of little value for screening purposes in rhinoceroses (Morar et al., 2013).


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The SIT is not practical due to difficulties in defining suitable injection sites in pachyderm animals, and culture techniques, although presumably the most reliable and specific, are performed on either post‐mortem or clinical specimens, which require invasive sampling and results are obtained only after 6–8 weeks. The lack of bTB diagnostic tools for use in rhinoceroses led to the development of a rhinoceros‐specific IFN‐γ assay (Morar et al., 2013). Morar et al, developed reagents to set up a rhinoceros IFN‐γ assay (RhIFN‐gamma) assay (Morar et al., 2007). Heparinized bloods of 52 rhinoceroses from bTB‐free areas were used. Whole blood sample were incubated at 37°C in 5% CO2 for 48 h. Results of IFN‐γ responses are shown below (Figure 11) for the 51 rhinoceroses (1 excluded), expressed as OD490 nm or as ng/ml. Figure 9. Results of the IFN‐γ responses for the 51 rhinoceroses (1 excluded), expressed as OD490 nm or as ng/ml (Morar et al., 2013).

Badger They have compared 2 “sandwich ELISA” for a badger IFN‐γ test with a monoclonal antibody pair based on a monoclonal pair (mEIA) or based on a rabbit polyclonal antiserum (pEIA). The Protocol used is the following :whole heparinised blood was mixed in 24 well culture plates, in a 1:1 or 1:5 ratio with RPMI 1640 medium (Invitrogen) and antibiotics. Cultures were incubated at 37 °C plus 5% CO2 for 16–24 h. For the pEIA, a cut‐off of 0.0365 gave a sensitivity of 74.5% (95% CI, 59.6–86.1%) and a specificity of 93.6% (95% CI, 89.1–96.7%). For the mEIA, a cut‐off of 0.044 gave a sensitivity of 80.9% (95% CI, 66.7–90.9%) and a specificity of 93.6% (95% CI, 89.1–96.7%).


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Figure 10. title

Dalley et al. (2008) have developed a sensitive IFN‐γ mEIA that can be used to detect M. bovis infection in badgers in a way analogous to the measurement of IFN‐γ in cattle. With a sensitivity of 80.9% for the mAb‐based EIA and a specificity of 93.6%, this represents a significant improvement in the immunodiagnosis of TB in the badger and was better than a recently developed quantitative real‐time PCR method for measurement of badger IFNγ mRNA (Dalley et al., 2008). Tomlinson and al. (2015) used this IFNγ mEIA test on samples collected from free‐living, wild badgers to investigate whether there was any evidence for correlation of the cell‐mediated immune response at the incident event with the degree of subsequent disease progression. They have shown that the magnitude of the early IFN‐γ responses of badgers to M. bovis arising from naturally acquired infection is positively correlated with subsequent progressive disease. In addition, they have shown that IFN‐γ responses in all badgers reduce over time, for which they offer several hypotheses (Tomlinson et al., 2015). Conclusion To conclude, sheep/goat or others domesticated animals cohabiting with TB‐positive cattle in similar conditions should be tested for their TB status, as they may represent a potential risk to other susceptible species (Munoz‐Mendoza et al., 2015). The IFN‐y assay has the principal advantage to require a single intervention on the animals, it is promptly repeatable and it is able to diagnose M. bovis and M. tuberculosis infection more precociously than SIT. In this context the IFN‐y assay can represent a valuable tool in areas where surveillance plans in livestock, other than cattle, are strongly envisaged. The IFN‐γ assay has also proven useful for the detection of TB in wildlife. For some species, the protocol should be adapted in comparison with the protocol used for bovines ( e.g. camelids, dog, cat, elephant..).


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Appendix VIII: Serological diagnostic tests to be used in non‐bovine animals

RT/Stat Pak Since the first publication in 2003 for the detection of TB in Eurasian badgers (Greenwald et al., 2003), the rapid test (RT) (marketed as a Stat‐Pak assay by Chembio Diagnostics Systems, Inc.) has become an extremely popular tool for the serodiagnosis of TB in a wide variety of wildlife species. The range of species for which data have been published has been extended considerably during a survey of bovine TB in Ethiopian wildlife, South American camelids, black rhinoceros, lions and a single pygmy hippopotamus (Chambers, 2013). Figure 11. Rapid test called also Stat‐Pak assay by Chembio Diagnostics Systems, Inc

The test employs a unique cocktail of M. tuberculosis or M. bovis antigens and a blue latex bead‐based signal detection system (Waters et al., 2006). The Vet‐TB STAT‐PAK has been tested with sera from experimental infection of cattle. In this study, 60% (15/25) of sera from experimentally infected cattle were reactive by the VetTB STAT‐PAK test by 7 weeks after challenge, and 96% (24/25) of the sera were reactive by 18 weeks (Waters et al., 2006).

DPP vetTB Recently, a new‐generation of immunochromatographic tests for rapid serological detection of TB in multiple animal species has been described (Greenwald et al., 2009). Called the DPP VetTB assay (Chembio, Medford, NY, USA), it uses a dual‐path platform (DPP) technology, whereby two nitrocellulose strips are connected in a T‐shape inside the device to allow independent delivery of test sample and antibody‐detecting reagent. The principle advantages of the DPP assay are that it includes two diagnostic targets (MPB83 and CFP10/ESAT‐6) producing 0, 1 or 2 lines in the reading window and that the strength of the positive test result(s) can be quantified by placing the test cassette in a proprietary optical reading device. The device generates a reading in relative light units (RLU) based on the reflectance of the two test and one control reactions. The latter feature means that the RLU corresponding to a positive test result can be determined and optimized for each species and situation (Chambers, 2013; Lyashchenko et al., 2013). Figure 12. DPP (Dual‐path platform) system.


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Published data have demonstrated that antibody detection assays may provide useful ancillary tools for improved bovine TB control in both cattle and cervids (Lyashchenko et al., 2013). The DPP VetTB assay detected antibody responses in 58.1% of experimentally infected animals within 8 to 16 weeks post‐inoculation and in 71.9% of naturally infected deer, resulting in an estimated test sensitivity of 65.1% and a specificity of 97.8%.(Lyashchenko et al., 2013).

MAPIA: Multiantigen print immunoassay The antigen panel consisted of 12 recombinant proteins of M. tuberculosis and two native antigen preparations of M. bovis as follows: the ESAT‐6 and CFP10 proteins, CFP10/ESAT‐6, Acr1/MPB83, MPB59, MPB64, MPB70, MPB83 proteins, PPDB, M. bovis culture filtrate (MBCF), Mtb8 and polyepitope fusion TBF10, alpha‐crystallin (Acr1) and the 38‐kDa protein (Lyashchenko et al., 2013; Waters et al., 2004). The use of multiple antigens enabled a detailed analysis of the antibody profiles observed in infected white‐tailed deer. The figure below shows typical antigen reactivity patterns obtained with representative sera from deer experimentally inoculated with M. bovis as example (Lyashchenko et al., 2013)

Se and Sp in Alpaca for MAPIA and Rapid test Table 42. Se and Sp of serological test performed in camelids with known infectious status (Alvarez et al., 2012).

Cat, goats, pigs and llamas Eighteen out of 743 blood samples tested seropositive (2.4%, CI: 1.5‐3.9%) by ELISA, and the results for 61 animals previously assessed using culture and PCR indicated that this serological test was not 100% specific for M. bovis, cross‐reacting with M. microti. Nevertheless, serology appears to be an appropriate test methodology in the harmonisation of wild boar testing throughout Europe. In accordance with previous findings, the low seroprevalence found in wild boar suggests wildlife is an unlikely source of the M. bovis infections recently detected in cattle in Switzerland. This finding contrasts with the epidemiological situation pertaining in southern Spain (Beerli et al., 2015).


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Table 43. Reported Se and Sp estimates for currently available serological test for the diagnosis of TB in non‐bovines species (Broughan et al., 2013).

Summary of serological tests employed in different non‐bovid wildlife published since 2009 Table 44. Summary of serological tests employed in different non‐bovid wildlife published since 2009. More species tested, but not listed here as belonging to bovid family. NT, not tested; RT, rapid

(immunochromatographic) test (Stat‐Pak, Chembio Diagnostics Systems, Inc.); ELISA, enzyme‐linked immunosorbent

assay; DPP, dual‐path platform (Chembio Diagnostics Systems, Inc.); FPA, flouresence polarization assay; MAPIA,

multiantigen print immunoassay; LAM, lipoarabinomannan; MBCF, M. bovis culture filtrate). From Chambers, 2013

(Chambers, 2013)

Species Reference(s) (E)xperimentallyor (N)aturally infected


Method employed

Target antigen(s)

Eurasian badger (Meles meles)

Drewe et al. (2010) N M. bovis RT CFP10/ESAT‐6, MPB83

Chambers et al. (2010)

RT CFP10/ESAT‐6, MPB83

Chambers et al. (2009)

RT, ELISA MPB83

Boar (Sus scrofa) Boadella et al. (2011b)

N M. bovis DPP, ELISA CFP10/ESAT‐6, MPB83, PPD‐B

Boadella et al. (2011a) MTC ELISA

Deer

Fallow (Dama dama)

Gowtage‐Sequeira et al. (2009), Waters et al. (2011b) and

Boadella et al. (2012)

N M. bovis RT MAPIA, RT,

DPP DPP, ELISA

CFP10/ESAT‐6, MPB83 Multiple

CFP10/ESAT‐6, MPB83 (PPD‐B for

ELISA)

Roe (Capreolus capreolus)

Gowtage‐Sequeira et al. (2009)

N M. bovis RT CFP10/ESAT‐6, MPB83

Red/Elk (Cervus elaphus)

Gowtage‐Sequeira et al. (2009), Waters et al. (2011b) and Buddle et al. (2010)

N/E M. bovis RT CFP10/ESAT‐6,

MPB83

Surujballi et al. (2009) RT, DPP CFP10/ESAT‐6, MPB83

White‐tailed (Odocoileus virginianus)

O'Brien et al. (2009), Nol et al. (2009)

N/E M. bovis FPA RT, MAPIA, immunoblot,

Multiple (including LAM for ELISA)


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Species Reference(s) (E)xperimentallyor (N)aturally infected


Method employed

Target antigen(s)

ELISA

Pygmy hippopotamus (Hexaprotodon liberiensis)

Bouts et al. (2009) N M. interjectum RT, MAPIA MBCF, MPB59, MPB83, CFP‐10

Elephant

African (Loxodonta africana)

Greenwald et al. (2009) and

Lyashchenko et al. (2012)

N M. tuberculosis RT, DPP, MAPIA

Multiple

Tschopp et al. (2010)a NT RT CFP10/ESAT‐6,

MPB83

Asian (Elaphus maximus)

Greenwald et al. (2009) and

Lyashchenko et al. (2012)

N M. tuberculosis RT, DPP, MAPIA

Multiple

Lion (Panthera leo) Miller et al. (2012) N M. bovis RT, DPP CFP10/ESAT‐6, MPB83

Non‐human primates

Cynomolgus macaque (Macaca

fascicularis)

Panarella and Bimes (2010)

N M. bovis RT CFP10/ESAT‐6,

MPB83

Silvered langur (Trachypithecus cristatus ultima)

Georoff et al. (2010) N NT RT CFP10/ESAT‐6,

MPB83

Blick's grass rat (Arvicanthis blicki)

Tschopp et al. (2010)a N NT RT CFP10/ESAT‐6,

MPB83

Black rhinoceros (Diceros bicornis)

Duncan et al. (2009) N M. tuberculosis RT, MAPIA Multiple

South American camelids

Llama (Lama glama) Lyashchenko et al. (2011)

N M. bovis RT, DPP MAPIA, RT Multiple, including

two commercial ELISA tests (Idexx,

Enferplex) Alpaca (Vicugna

pacos) Dean et al. (2009), Rhodes et al. (2012)

N M. bovis RT, DPP

M. microti ELISA

Table 45. Commercial availability of tests referred to by Chambers, 2013

Test Commercial name For use in species Source

RT

ElephantTB STAT‐PAK

Asian and African elephants (licensed by USDA) and other exotic/zoo species (off‐label use) for which supportive data are published (tapirs, sea lions, rhinoceros, camelids, felids, wild boar, etc.)

Chembio Diagnostic Systems, Inc.

CervidTB STAT‐PAK USDA‐approved for use in elk, red deer, white‐tailed deer, fallow deer and reindeer


BrockTB STAT‐PAK Eurasian badger (manufactured by Chembio, marketed by AHVLA under its brand name)

AHVLA

DPP DPP VetTB USDA‐licensed for use in elephants (Asian and African) and cervids (elk, red deer, white‐tailed deer and fallow deer); off‐label use in other cervid species



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Test Commercial name For use in species Source

MAPIA Elephant TB MAPIA Asian and African elephants (confirmatory testing services performed by Chembio laboratory for US customers only)


FPA Not currently available for TB

N/A Diachemix, LLC

ELISAPRO ELISA

PRO kit NHP (rhesus and/or cynomolgus macaque) Mabtech AB

ELISA

Tuberculosis (Myc. bovis) ELISA Kit

Swine Vacunek, S.L.

The Enferplex™ TB assay

Eurasian badgers, goats, deer, cattle, wild boar, llamas, and Alpacas Enfer Group

IDEXX M. bovis Ab Test

Currently only sold for cattlea IDEXX Laboratories, Inc.

Llamas, and alpacas Available through AHVLA in the UK

ETB (IgG1 ELISA for TB in deer)

For National deer herd TB testing in New Zealand DRL

QFG‐IT QuantiFERON

®‐TB

Gold In‐Tube Sold for human testing Cellestis Inc.

IGRA

PRIMAGAM® NHP Prionics AG

N/A Eurasian badgers Antibodies available from AHVLA

South American camelids Antibodies (anti‐bovine) available from Mabtech AB

Validated and certified by the OIE for cattle (registration number 20120107).

Chembio Diagnostics, Inc. – www.chembio.com; AHVLA – www.defra.gov.uk/ahvla‐en/; Diachemix, LLC –

www.diachemix.com; Mabtech AB – www.mabtech.com; Vacunek, S.L. – www.vacunek.com; Enfer Group –

www.enfergroup.com; IDEXX Laboratories, Inc. – www.idexx.co.uk; Disease Research Laboratory, University of Otago, New

Zealand – http://139.80.82.2/; Cellestis Inc. – www.cellestis.com; Prionics AG – www.prionics.com).


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Appendix IX: Occurrence of M. bovis in domestic and wild animals worldwide – literature overview.

For readability purposes a bigger Table will be put on the website of the Scientific Committee.

Continent Country Region Location Official TB free statusj Status Prevalence (%)

Domesticated (farmed) animal

species summaryAlpaca buffalo cattle cats deer dog donkey goats horses lama pigs sheep

wild

boarLatin name Wild animal species summary badger bear black birds bison bobcat buffalo cat feral coyote Deer Axis

Deer

Fallow

Deer

mule

Deer

Red

Deer

Si lka

Deer

Roe

Deer

white‐

tai led

elk ferret fox red hare kafue kudu letchwe lynx mink mole mouflon mouse oppossum rabbit raccoon ratseal

(grey)

squirrel

(grey)

swine

ferralvole warthog

wild

boarwolf Reference article I Reference article II

Austria y Cattle# x

Belgium y Cattle# x

Bulgaria n Wild boar# x

Cyprus y x

Czech Republic y Deerg,h,i x Red deer

g,h,i

Denmark y

Estonia y

Finland y

Francey

Pigs#, Cattle

#, Goats

#, Cats

#,

Horsest

x x x x xRed deer

#, Wild boar

#, Roe

deer#, Red Fox

a, Badger

qx x x x x

Ardennes North‐East Cattle, Badger x

Ariège Middle‐South‐West Cattle, Wild boar x

Côte d’Or Middle‐East Cattle, Badger, Deer, Wild boar x x x

Corsica Island Mediterranean Isle Cattle, Wild boar x

Dordogne & Charente Middle‐West Cattle, Badger, Deer, Wild boar x x x

Dordogne Cattle, Badger, Wild boar x x

Normandy Middle‐North Cattle, Wild boar x

Atlantic Pyrenees South‐West Cattle, Badger, Wild boar x x

Landes South‐West Cattle, Badger x

Lot et Garonne South‐Middle‐West Cattle, Badger x

Germany y Sheep#, Pigs

#, Cats

#, Cattle

#f x x x x

Greece n Cattle#,p x

Hungaryn Deer

#, Cattle

g,h,i, Pigs

# x x xFox

#, Roe deer

#, Fal low Deer

#,

Wild boar#, Red deer

g,h,ix x x x x

Ireland*

n

Cattle#, Cats

#, Donkey

#, Goat

#,

Sheepr, Alpaca

#, Red

s, Fallow

s

and Sika deerm,

x x x x x x Badger#, Red deer

s

x x

Italy*Some provinces y

Cattle#, Buffalo

#, Goats

#, Pigs

sw,

Dogs#, Sheep

#x x x x x x Birds

#, Wild boar

#, Roe deer

v

x x

Latvia y

Lithuania n

Luxembourg y

Malta y

Netherlands y

Norway y

Poland y Cattle#, Pigs

#, Sheep

#, Dog

g,h,i x x x x Bison, Roe deerg,h,i

, Badgerx

x x x

Portugal* n Cattle#, Pigs

#, Sheep

#, Goats

e x x x x Red deere, Wild boar

e, Rabbit

#x x x

Romania n Cattle# x

Slovakia y Cattleg,h,i x Wild boar

g,h,ix

Slovenia y Cattleg,h,i x

Spain*

nCattle

#, Goats

#, Free‐ranging

Iberian Pigso

x x x

Red deer#, Wild boar

#, Fal low

deer#, Lynx

d, Hare

d, Badger

d, Red

foxd, Mouflon

#, Roe deer

Vx x x x x x x x x

Sweden y Deerb x

Switzerland y

U.K.*

n, Scotland yCattle

#, Cats

#, Dogs

k, Llama

l,

Deer#, Pigs

#, Sheep

#, Alpaca

#x x x x x x x x

Wild Boaru, Badger

#, Fallow

#,

Roe#, Sika deer

#, Fox

#, Mink

c,d,

Molec,d, Brown Rat

c,d, Ferret

c,d,

Bank volec,d, Stoat

n, Common

shrewn, Field vole

n, Grey

squirreln, Yellow‐necked

n and

Wood micen, Grey seal

y. x x x x x x x x x x x x x

Endemic <5∙0 Axis deer x

Endemic 20∙0–3∙8 Feral swine x

Endemic 4∙9–0∙2 White‐tailed deer x

Endemic 52–4∙8 Coyote x

Endemic 4∙6–2∙4 Raccoon x

Endemic 3∙3–2∙4 Black bear x

Endemic 12∙5–7∙0 Bobcat x

Endemic 16∙6–10∙0 Red fox x

Endemic 2∙4 Opossum x

Endemic 0∙3 Rocky Mountain elk x

Endemic n.a. Feral cat x

Minnesota Below detection level <1∙2 White‐tailed deer x

Reported 4∙9 Mule deer x

4∙3 Coyote x

New York Reported n.a. White‐tailed deer x

Endemic 5∙5 Manitoban elk x

Endemic 5∙6 Canadian moose x

Endemic 0∙8 Mule deer x

Endemic 49∙0–42∙0 Wood bison x

Endemic 53∙7 Plains bison x

Endemic n.a. Plains bison x

Endemic n.a. Wolf (pups) x

Endemic 3∙6–0∙4 Manitoban elk x

Endemic <0∙5 White‐tailed deer x

Ontario Reported 0∙2 White‐tailed deer x

Tamaulipas Unconfirmed 8∙8 White‐tailed deer x

Nuevo León Unconfirmed 8∙5 White‐tailed deer x

Coahui la Unconfirmed 6∙0–18∙7 White‐tailed deer x

Eastern Cape

Province

Free State Province

Gauteng Province

KwaZulu–Natal

Province

Limpopo Province

Mpumalanga

Province (KNP)

MP (Non‐KNP)

Northern Cape

Province

North West Province

Western Cape

Province

Mozambique

African buffalos (Syncerus

caffer), Kudu, Warthogx x x

UgandaAfrican buffalos (Syncerus

caffer), Kudu, Warthogx x x

Zambia Kafue basin Cattle Kafue lechwe antelopes x

Mycobacterium bovis infection at the

interface between domestic and wild

animals in Zambia (Suzuki et al ., 2012)

ovis from Wildl i fe to Livestock, So

Ethiopia Camels, Cattle, Goats

Pathology of Camel Tuberculosis and

Molecular Characterization of Its

Causative Agents in Pastoral Regions of

Ethiopia (Ameni et al ., 2011)

en Pastoral ists and Their Livestoc

Ratón de campo (Akodon sp.)

Laucha de campo (Calomys sp.)

Ratón col ilargo (Oligoryzomys sp.)

Rata (Rattus norvegicus)

Ratón común (Mus musculus)

Cuis común (Cavia aperea)

Comadreja overa (Didelphys albiventris)

Peludo (Chaetophractus vellosus)

Zorro gris (Lycolapex gimnocercus)

Hurón menor (Galictis cuja)

Liebre europea (Lepus europaeus)

Laos Elephant

Tuberculosis in Laos, who is at risk: the

mahouts or their elephants? (Bouchard et

al ., 2014)

China Northeast China Sika deer

Evaluation of MIRU‐VNTR for typing of

Mycobacterium bovis isolated from Sika

deer in Northeast China (Wang et al .,

2015)

Cattle, Porcine

USA

Canada

Cattle, Eland

Europe

North America

Africa

Hawaii

Michigan

Montana

Alberta

Manitoba

South America

Asia

Mexico

South Africa

Argentinia Santa Fe

Evidence of increasing intra and inter‐

species transmission of Mycobacterium

bovis in South Africa: Are we losing the

battle? (Michel et al ., 2015)

Spi llover of Mycobacterium bovis from

Wildl i fe to Livestock, South Africa (Michel

et al., 2015)

Mycobacterium bovis en fauna si lvestre

de la cuenca lechera de Santa Fe,

Argentina (Tarabla et al ., 2015)

Evaluating the tuberculosis hazard posed

to cattle from wildl ife across Europe

(White et al., 2015)

Genetic Evolution of Mycobacterium bovis

Causing Tuberculosis in Livestock and

Wildl ife in France since 1978 (Boschiroli

et al., 2015)

Evaluating the tuberculosis hazard posed

to cattle from wildl ife across Europe

(White et al., 2015)

Mycobacterium bovis (bovine

tuberculosis) infection in North American

wildl ife: current status and opportunities

for mitigation of risks of further infection

in wildl ife populations (Mil ler &

Sweeney, 2013)

Cattle, Buffalo

Cattle

Cattle

Cattle, Buffalo, Baboon

Cattle, Buffalo, Cheetah, Lion, Nyala, Rhino, Warthog

Buffalo, Baboon, Impala, Kudu, Leopard, Lion, Warthog

Cattle, Buffalo, Bushbuck, Hyena, Impala, Kudu, Lion, Warthog, Waterbuck, Wildebeest

Cattle

Advies 12-2016 van het Wetenschappelijk Comité van het FAVV · Annex I of opinion 12‐2016...

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