Poster Abstracts - Diagnosis

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1 - Diagnosis Diagnosis Of Tuberculosis In New World Camelids Infected By Mycobacterium bovis Bezos, J.,, Casal, C., Álvarez, J., Alende, T., Viñolo, C., Sáez, J.L. 4, Domínguez, L., de Juan L., MAEVA SERVET SL. Alameda del Valle, Madrid, Spain VISAVET Health Surveillance Centre. Universidad Complutense Madrid Madrid. Spain Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN, USA 4 Subdirección General de Sanidad e Higiene Animal y Trazabilidad, Ministerio de Agricultura, Alimentación y Medio Ambiente, Madrid, Spain Camelids; tuberculosis; intradermal test; serology Tuberculosis (TB) in llamas and alpacas has gained importance in recent years since they are imported into the European Union mainly for serving as pets and for production of natural fibre. The intradermal tuberculin test has been widely used for diagnosis of TB in these species showing lack of sensitivity (Se) although little information has been previously reported evaluating the effect on its performance in different PPD inoculation sites and time of readings. Moreover, different cost-effective serological assays have been developed in the recent years for TB diagnosis in camelids obtaining a variety of results and, for this reason, new assays still being developed. The main objectives of this study were: () to evaluate the performance of the single and comparative intradermal tuberculin (SIT and SCIT respectively) tests using different inoculation sites and times of reading and () to test a novel serological assay based on MPB8 antigen in a M. bovis infected alpaca herd in Spain. SIT test at the prescapular site and reading at 7h showed the highest proportion of test positiveculture positive animals among all culture positive animals (T+/C+), ranging from 5.8% (95% CI, ) to 80% (95% CI, ) using a more stringent interpretation than typically prescribed. T+/C+ of the serological assay increased using samples collected 5 0 days after PPD injection [76.9% (95% CI, )-00% (95% CI, )]. The best results of T+/C+ were obtained applying in parallel the most sensitive SIT test and serology using samples collected 5 0 days after PPD inoculation [90% (95% CI, )-00% (95% CI, )]. Therefore, implementation of serology in parallel with the most sensitive skin test could maximise the detection of TB infected animals. Acknowledgements This research was funded by the European Union Reference Laboratory for Bovine Tuberculosis (EU-RL). We would like to thank Paloma Rueda and Lissette López (Ingenasa, Madrid, Spain) for the technical assistance. Assessment Of Different Commercial IFN-gamma Detection Assays For Diagnosis Of Bovine Tuberculosis Casal, C., Romero, B., Bezos, J.,, Álvarez, J., de la Cruz, M.L., Viñolo, C., Moya, N., Sáez, J.L. 4, Domínguez, L., de Juan, L., VISAVET Health Surveillance Centre. Universidad Complutense Madrid Madrid. Spain MAEVA SERVET SL. C/ de la Fragua, 8749, Alameda del Valle, Madrid, Spain 77

2 Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN, USA 4 Subdirección General de Sanidad e Higiene Animal y Trazabilidad, Ministerio de Agricultura, Alimentación y Medio Ambiente, Madrid, Spain Bovine tuberculosis, IFN-γ detection, diagnostic tests The use of commercial assays for the detection of bovine interferon-gamma (IFN-γ) was implemented in 00 by the EU Council Directive 64/4/CEE to enable detection of the maximum number of infected and diseased animals in addition to the tuberculin test. Several commercial assays are available for the detection of bovine IFN-γ although, differences regarding validations protocols of the results (using standard optical density read-outs, calculating a sample/positive ratio, or using a typical standard curve with different dilutions of bovine IFN-γ) and the type of required sample (whole blood or peripheral blood mononuclear cells, PBMCs) have been reported. In the present work the sensitivity (Se) of five commercial assays for the detection of bovine IFN-γ testing a set of samples (n = 9) collected from naturally infected herds was analysed. The results obtained with each assay were analysed following different interpretations criteria including the one specified in the manufacturer s instructions. The assays that required a previous step of PBMCs isolation from whole blood yielded poor Se results; moreover, those assays are time consuming and have high logistical requirements, being not practical for a standard laboratory routine work. The assays that use whole blood samples showed a wide range of Se results: 74.9% (95% CI, ) % (95% CI, ). The commercial assay that reported the highest Se was to one currently in use in most European countries. This research was funded by the European Union Reference Laboratory for Bovine Tuberculosis (EU-RL). Validation Of Stimulants and Interferon Gamma ELISA For Bovine Tuberculosis Detection Anstaett, O., Olagnon, L., Pourquier, P., Comtet, L., IDVET 0 rue Louis Pasteur 4790 Grabels, France bovine tuberculosis, diagnosis, IGRA Introduction IGRA (Interferon Gamma Release Assay) is widely used to detect the cellular response to pathogens such as Mycobacterium bovis by measuring the difference between activated and non-activated whole blood interferon gamma (IFNg) signals. IDvet offers a complete solution for the detection of bovine tuberculosis. It includes stimulation antigens and an ELISA kit for detection. The aim of this study is to assess the sensitivity and specificity of this solution. Materials and methods PPDa and PPDb tuberculins (IDvet) were used as stimulation antigens. IFNg level was then determined using the ID Screen Ruminant Interferon Gamma ELISA (IDvet). All ELISA results were standardised and expressed as sample/positive control ratios (S/P). Different freeze-dried IFNg reference materials were created from natural IFNg from different species (bovine, ovine, caprine or buffalo). The stability, repeatability, reproducibility and robustness of the ELISA itself was studied. Specificity for bovine tuberculosis diagnosis was evaluated on cattle from different breeds and countries (n=077). Sensitivity was evaluated in comparison with confirmatory techniques (PCR, single and comparative intradermal tuberculin test). 78

3 Results The ELISA reagents showed excellent stability. Repeatability and reproductibility were excellent, with CV% between and 8%. Using the reference material, analytical sensitivity was found to be constant and equivalent between runs. Measured specificity was 99.0% (IC95 98,4 99,6%) and sensitivity was 88.% (IC95 8, 95,5%). Discussion/Conclusion The use of the IDvet PPDs with the IDvet IFNg ELISA gave a measured test sensitivity comparable with published values thanks to the use of tuberculins with appropriate potencies, as well as well-defined interpretation criteria, test specificity was high. The performance of the IDvet PPDs and IFNg ELISA meets bovine TB eradication program requirements. Non Tuberculous Mycobacteria Generating False Positive Results In TB Diagnostics in France De Cruz, K., Karoui, C., Bulach, T., Hénault, S., Moyen, J.L., Boschiroli, M.L., Bacterial Zoonosis Unit, Animal Health Laboratory, ANSES, Maisons-Alfort, France b Regional Analysis and Research Laboratory of Dordogne (LDAR4), 6 Av. Churchill, 4660 Coulounieix-Chamiers, France In cattle, non-tuberculous mycobacteria (NTM) widely distributed in soil and water, can lead not only to non-specific sensitivity to the skin test but also to cause lesions, most of which are indistinguishable from those caused by M. bovis. Interference of NTM-infection with bovine tuberculosis (btb) surveillance in officially btb-free countries like France is of important concern. Indeed, lack of specificity of the diagnostic system leads to a general decline of confidence by field actors and in turn to under-declaration of btb. In order to assess which mycobacterial agents lead to diagnostic interferences in France, we analysed NTM strains isolated from cattle in the last 5 years. The studied animals were either positive reactors to the skin-test/ifng-test and/or presented btb-like lesions at the abattoir. We used a PCR-based system that allows discriminating M. bovis, M. avium (subsp avium and hominissuis), M. nonchromogenicum and Mycobacterium sp, further characterised by hsp65 sequencing. An important increase of isolated mycobacteria was observed with time. This is the consequence of an intensification of btb surveillance leading to a higher number of animals being diagnostic-culled and an increase of analysed samples from suspected animals. At least 45 different species of NTM could be identified although M. avium and M. nonchromogenicum are predominant, representing 0% of the strain-collection respectively. This study supports previous findings of M. nonchromogenicum as a potentially predominant NTM in cattle. Further work on assessing its effect on the skin-test and IFNg-test as well as the means to limit these interferences would be worth pursuing. How Useful Are Combinations Of Diagnostic Tests For Detecting M. bovis Infection In Groups Of Live Badgers? Drewe, J.A.¹, Buzdugan, S.¹, Chambers, M.A., Delahay, R.J., ¹Royal Veterinary College, London, UK ²Animal Health and Veterinary Laboratories Agency, Weybridge, UK National Wildlife Management Centre, Animal Health and Veterinary Laboratories Agency, Woodchester Park, Gloucestershire, UK Badger, diagnosis, infection, protocol, test 79

4 Introduction Accurate detection of M. bovis in live badgers is necessary for targeted disease control. We compared a range of sampling strategies for optimum detection of infection at the badger group level using existing diagnostic tests. Our focus was on the ability to correctly detect infection in live badgers. Materials and methods Individual badger test results from a longitudinal study using serology (Stat-Pak), gamma interferon (IFNg) and culture of clinical samples from a naturally-infected badger population in southwest UK were analysed. We modelled the impact of variations in trapping efficiency, infection prevalence, and use of each diagnostic test (singly and in combination with one another), on the ability to correctly classify the infection status of badgers at a sett or social group level. We examined the impact of different thresholds (number of badgers that were required to test positive) for classifying badger groups as infected on the accuracy of group-level diagnosis of infection. Results Our model suggests that StatPak and INFg appear able to identify infected social groups without the need to capture all individuals. Culture of clinical samples added little to the accuracy of infection detection. When used singly, both StatPak and INFg were able to detect infection in a badger group, even if a minority of the group were tested. However, using a threshold of a minimum of two badgers needing to test positive to consider the group infected meant that approximately 80% of the group needed to be tested when using Stat-Pak, or 50% when using IFNg. Using Stat-Pak and IFNg together improved the outcome by identifying infected social groups when fewer animals were tested. Discussion/Conclusion Our results suggest some practical implications for the diagnosis of TB in badger populations in the field. Encouragingly, according to our results, levels of trapping efficiency that have been observed in previous field studies appear to be sufficient to usefully employ a combination of these tests (or others of similar accuracy) to identify infected groups. Multiple Gene Sequence Analysis Of Non Tuberculous Mycobacterium (NTM) Isolates in South Africa Reveals Novel Species Gcebe, N.,, Jenkins, A., Rutten, V.,, Michel, A., Tuberculosis Laboratory, Agricultural Research Council - Onderstepoort Veterinary Institute, Onderstepoort, South Africa Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South Africa Division of Immunology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands The potency of non tuberculous mycobacteria (NTM) to induce anti-mycobacterial immune responses cross reactive with mycobacteria pathogenic in humans and/or animal species, amongst which M. bovis, has repeatedly been recognised. A group of NTM isolates showing 95%-98% homology in their 6S rrna sequences to M. moriokaense has been shown to be among the most abundant NTM in the environments of cattle and African buffaloes in South Africa. Preliminary results for assessment of cross reactivity induced by the isolates showed that a purified protein derivative (PPD) derived from this M. moriokaense like isolate (PPD-M) initiated immune responses (OD 0.5) against M. bovis positive and positive SICCT animals in a higher proportion compared to the routinely used PPD derived from M. fortuitum. Furthermore, PPD -M very rarely showed cross reactivity with M. avium reactor animals, thus indicating its bias towards M. bovis infection. Hence our study aimed at in depth characterisation of these NTM isolates. To do so, we performed multiple gene sequence analysis targeting the 6S rrna, and in addition rpob, hsp65, soda 80

5 genes, phenotypic characterisation and antibiotic susceptibility tests. This approach revealed the existence of principal phylogenetically distinct groups representing novel NTM species. This study confirms the importance of the use of multiple genes for identification of new species. It also reveals the occurrence of very closely related NTM species in their environments that animals may be exposed to, induction of cross reactive immune responsiveness, and hence potential implications to diagnosis of infection with pathogenic mycobacteria. Bovine Micro-RNA Profiling In Bovine TB Golby, P., Villarreal-Ramos, B., Dean, G.D., Jones, G.J., Vordermeier, H.M., Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Weybridge, UK micro-rna, bovine tuberculosis, pathology, DIVA, BCG There is an urgent need to identify additional bovine TB diagnostic biomarkers to complement existing read-out systems such as interferon-gamma and for predictive markers of vaccine efficacy to accelerate vaccine development. To evaluate the potential of mirnas as such biomarkers, we have analysed their expression in bovine PPD stimulated PBMC isolated from control and BCG vaccinated cattle before and following M. bovis infection. Using a bovine mirna microarray, mir-55 was found to show a significant up-regulation in expression in early and late M. bovis post-infection samples from unvaccinated cattle, while in BCG vaccinated cattle up-regulation was observed only in late samples, and not at all after BCG vaccination but before infection. This suggests that this analyte could be exploited as marker distinguishing vaccinated from infected animals (DIVA). Analysis by TaqMan RT-PCR, verified the up-regulation of mir-55 in unvaccinated cattle post-infection. Significant correlation was found between the degree of pathology and mir-55 induction in the experimentally infected cattle, suggesting mir-55 is a biomarker of disease development. Induction of mir55 expression in cattle sourced from farms with confirmed btb that tested positive in the tuberculin skin or interferon-gamma blood test was found to be significantly higher in cattle presenting with more advanced pathology compared to infected cattle without visible pathology and thus likely of lower infectivity than those with more advanced disease. The data indicate that mir-55 is a potential diagnostic and prognostic biomarker identifying animals with advanced pathology or as well as potential DIVA test read-out. The Use Of Mycobacterium bovis-specific Antigens For The Diagnosis Of Tuberculosis In African Buffaloes (Syncerus Caffer) Goosen, W.J., Miller, M.A., Chegou, N., Cooper, D., Warren, R.M., van Helden, P.D., Parsons Sven, D.C., DST/NRF Centre of Excellence for Biomedical TB Research/MRC Centre for Molecular and Cellular/Biology, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, P.O. Box 906, Tygerberg 7505, South Africa Ezemvelo KZN Wildlife, P.O. Box 5, Mtubatuba 95, South Africa African buffalo, Bovigam, bovine tuberculosis, CFP-0, diagnosis, ESAT-6, interferon-gamma Background The diagnosis of M. bovis-infected African buffaloes is important in preventing the transmission of bovine tuberculosis (BTB) to other species and preventing the geographic spread of BTB through movement of diseased buffaloes. We evaluated the use of M. bovis-specific peptides in tests of cell mediated immunity for the diagnosis of BTB in this species. 8

6 Material and Methods Eighty-four buffaloes were tested using the Bovigam EC (BEC) and HP (BHP) assays,), assays utilising the QuantiFERON TB-Gold (in tube) system using 0h (mqft0 assay) and 0h (mqft0 assay) whole blood incubation periods and the single intradermal comparative tuberculin test (SICTT). Agreement between these tests was calculated as Cohen s kappa co-efficient. Results Agreement between the Bovigam assays was high (κ=0.86, 95% CI ) and these detected the most test-positive animals (4/84). Equally high agreement was observed between the mqft assays (κ=0.88, 95% CI ) although these detected fewer test-positive animals (4/84). All animals with discordant mqft0/mqft0 results (n=6) were mqft0-positiveand mqft0-negative, including confirmed M. bovis-infected animals. Conclusion Our results suggest that the Bovigam assays were the most sensitive assays used in this study and that the mqft0 assay appears more sensitive than the mqft0 assay. The Role Of Tuberculin Assays On Naturally Infected Cattle In The Irish Eradication Programme Duignan, A., Good, M., Department of Agriculture, Food and the Marine, Kildare St, Dublin, Ireland Key words Bovine TB, Intradermal Tuberculin Test, Tuberculin, Potency The Irish Bovine Tuberculosis (btb) eradication programme includes annual Single Intradermal Comparative Tuberculin Test (SICTT) screening of all herds. The SICTT is conducted by comparing the separate cellmediated immune responses to an intra-dermal injection of avian and bovine PPD tuberculins in each animal. Tuberculin PPD standardisation is necessary in order to ensure sufficient diagnostic strength for eradication and also to guarantee animal status for international trade. The potency of a tuberculin is estimated by comparing the size of the reactions, elicited by an intradermal inoculation, to the size of the reactions of a standard tuberculin of known potency. OIE recommends that tuberculins are assayed in the population of animals in which the eradication programme is to be carried out, but due to practical difficulties in performing potency assays in cattle they are usually assayed in guinea pigs. However, ever since inadvertently using sub-potent tuberculin many years ago the routine assay of tuberculins in naturally infected cattle is regarded as imperative to the assurance of potency of tuberculins used in the Irish bovine tuberculosis eradication programme. The purpose of this presentation is to give an account of the procedures, and outline the role of assays, carried out on naturally infected cattle in the quality control on the Irish Bovine Eradication Programme. Ireland is perhaps unique in that it is one of the few, if not the only country conducting routine tuberculin assay in naturally infected bovines as part of the quality control of the national btb eradication programme. Immune Responsiveness To Recombinant Mycobacterial Antigens Of NTM and MTBC With Various Degrees Of Homology In Diagnosis Of Tuberculosis In Cattle Jenkins, A.O,,, Gormley, E., Rutten, V.P.M.G,,, Michel, A.L., Division of Immunology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan, 584 CL, Utrecht, The Netherlands Tb diagnostics and Vaccine Research Laboratory, School Of Veterinary Medicine, Veterinary 8

7 Science Centre, University College Dublin, Dublin 4, Ireland Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 00, South Africa Pre-sensitisation of immune responsiveness to environmental non-tuberculous mycobacteria (NTM) has been shown to confound diagnosis of bovine tuberculosis (BTB) in cattle whereas it has been hypothesised that mycobacterial antigens specific to members of the Mycobacterium tuberculosis complex (MTBC) are capable of improving the sensitivity of current diagnostic assays. Ten recombinant mycobacterial antigens with varying levels of homology (0-00%) between environmental mycobacteria and MTBC were used to test a herd of cattle with a well characterised BTB positive status and a BTB negative herd as the control group using the IFNy ELISpot assay as a read out. Interferon y responses to antigens belonging to the ESX group of antigens namely, Rv65 (ESpC), Rv087 (esxg) and the ESAT6/CFP0 were significantly higher in the M. bovis infected group compared to the uninfected group whilst that of Rv0c (HSpX), a heat shock protein indicated to be of relevance in latent TB infections tended to be higher in the M. bovis infected animals. All the mycobacterial antigens used yielded a higher level of expression in the infected cattle group relative to the uninfected group, indicating that the higher homology of these antigens in M. bovis relative to NTM improves their recognition in infected animals. Our results further support the notion that immune responsiveness to ESX antigens is restricted to M. bovis infected cattle over M. avian sensitised thus indicating the potential benefit of these antigens in improving the diagnosis of bovine tuberculosis in NTM endemic regions. Performance Of The SD Bioline TB Ag MPT64 Rapid Test For Rapid Confirmation Of Mycobacterium bovis Isolates From Animals Ji, M.J., Kim, J., Kang, S., Byeon, H., Kim, G., Cho, S. 4, Jeon, B., Department of Life Science, College of Natural Science, Kyonggi University, Suwon, Korea Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju, Korea Chungbuk Veterinary Service Labratory, Chungju, Korea Mycobacterium bovis, SD MPT64 Rapid Test, animals Mycobacterium bovis, a bacterium in the M. tuberculosis complex is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for bovine tuberculosis diagnosis, but it laborious and time-consuming. In the present study, the performance of the SD Bioline TB Ag MPT4 Rapid Test was evaluated using reference bacterial strains and M. bovis field isolates from animals. Using the SD MPT64 Rapid Test, positive results were 95.5% (6/66) for M. bovis isolates from cattle and 97.9% (46/47) for isolates from deer. The SD MPT64 Rapid Test against M. bovis isolates had sensitivity 96.5% (95% CI: ), specificity 00% (95% CI: ), positive predictive value 00% (95% CI: ), and negative predictive value 9.9% (95% CI: ). In conclusion, the SD MPT64 Rapid Test was simple and might be a useful diagnostic test for confirmation of M. bovis from animals. 8

8 Detection and Characterisation of IL- Production in Bovine Tuberculosis Jones, G.J., Steinbach, S., Vordermeier, H.M., Department of Bacteriology, Animal Health and Veterinary Laboratories Agency, Weybridge, Surrey, UK IL-, bovine tuberculosis, flow cytometry Introduction Previous gene transcription studies identified the cytokine IL- as a potential biomarker for vaccine success in bovine tuberculosis. Here we describe the development of an assay to detect IL- protein production in PBMC of M. bovis-infected cattle. Material and Methods Bovine IL- specific bivalent F(ab) antibodies were generated using the Human Combinatorial Antibody Phage Display library. PBMC from M. bovis-infected and non-infected cattle were stimulated with bovine tuberculin (PPD-B), mitogen (PWM) or cell culture medium (negative control), following which lymphocyte sub-populations and intracellular cytokine production were investigated by flow cytometry. Results Several of the novel bovine IL- specific antibodies detected IL- production in cattle PBMC following PPD-B and PWM stimulation. PPD-B stimulated greater IL- production in M. bovis-infected cattle compared to non-infected controls, while PWM-induced IL- levels were similar between the two groups of animals. In M. bovis-infected animals, PPD-B specific IL- responses were observed in both CD4+ and CD4- lymphocyte populations, with a trend for the responses to be more frequently detected in the latter population. Although PPD-B stimulation induced both IL- and IFN-γ production in M. bovis-infected cattle, these cytokines appeared to be differentially produced in distinct sub-populations of lymphocytes. Discussion These results support our previous gene transcription studies and extend the observation of increased IL- responses in M. bovis-infected animals to the level of protein production. This assay will now be used to investigate whether IL- protein levels seen post-bcg vaccination characterise animals subsequently protected from disease following M. bovis challenge. Use Of A Serological Test For Bovine Tuberculosis In France Moyen, J-L., Gueneau, E., Keck, N., Gares, H., Boschiroli, M-L. 4, Laboratoire Départemental d Analyses et de Recherche de Dordogne, 6 Av. Churchill, 4660 Coulounieix-Chamiers, France Laboratoire Départemental de la Côte d Or, rue Hoche, 000 Dijon, France Laboratoire Départemental de l Hérault, 4000 Montpellier, France 4 Unité Zoonoses Bactériennes, Laboratoire de Santé Animale, Anses,, avenue du Général de Gaulle, Maisons-Alfort cedex, France In-vivo diagnosis of bovine tuberculosis (btb) is mainly based on cell-mediated immunity. Skin-tests have good sensitivity and specificity but they can be significantly degraded by field conditions and cross reactions. Interferon-gamma test has very good characteristics although it is expensive and needs a heavy logistics. Serology-based tests, fast and inexpensive, could be ideal in beef or fighting-bull herds as they need only one intervention. However, the first btb serological tests presented low sensitivity, high background and a long delay between infection and seroconversion. New specific antigens for M. tuberculosis-complex allowed the development of promising serological tools with good preliminary results in different countries 84

9 (Se: 60-90%). We tested Idexx btb serological test in France. Specificity and sensitivity tests were conducted in Camargue, Côte d Or and Dordogne. Specificity is good and consistent between different races, regions and laboratories (98 to 99.5%). However, average sensitivity in infected fighting-bull herds is about 50% and in beef and dairy herds between 0 to 0%. The very low btb prevalence in France, generally associated with early detection of outbreaks, could explain the large difference in sensitivity results compared to those in other countries. Other tests on infected animals previously skin-tested were conducted to take advantage of an anamnestic reaction. Preliminary results are quite encouraging (Se: 50-60%) and close to those obtained in other countries for similar purposes. This use of serology could secure the test and cull programme and be used as an ancillary tool to improve detection of anergic animals. Tuberculosis In Captive Elephants In The United States Payeur, J.B., USDA APHIS VS National Veterinary Services Laboratories, 90 Dayton Ave, Ames, IA 5000 Tuberculosis, elephants, diagnosis, elephant TB guidelines There continues to be interest in tuberculosis (TB) in captive elephants in the United States (U.S.) over the past several years. There are approximately 450 elephants - 50 Asian and 00 African in U.S. zoos, circuses and privately owned facilities. Between 994 and 0, Mycobacterium tuberculosis (MTB) was isolated from 56 captive elephants from multiple facilities in different parts of the U.S. The majority of elephants did not show clinical signs and were discovered by antemortem testing using bacterial culture. Mycobacterium bovis has been isolated from one captive African elephant in 000. Clinical signs include: chronic weight loss, diminished appetite, chronic nasal discharge, coughing and intolerance to exercise. The majority of TB cases have been diagnosed in Asian elephants although African elephants are susceptible. There has been an increase in the number of multiple drug resistant MTB cases which increases the cost and length of treatment in elephants. Because tuberculosis is a zoonotic disease, there is concern that MTB could spread to other elephants or humans. Elephant handlers are probably at greatest risk due to close daily contact. In November of 997, guidelines were implemented by the U.S. Department of Agriculture s Animal Care staff which requires the annual testing of all elephants owned by licensed exhibitors and outlines diagnostic methods, TB management groups, postmortem procedures, treatment guidelines and employee safety and health guidelines. These guidelines have been updated as new data has become available. The latest version was published in 00. Wales TB Cluster Pilot: Wrexham Cluster - Exploration Of Recurrence Of Bovine Tuberculosis In g-ifn Tested Herds In An Emerging Area Williams, S., Bishop, H., Sainz Dominguez, P., Animal Health and Veterinary Laboratories Agency, AHVLA Cardiff Field Services, Government Buildings, 66 Ty Glas Rd, Llanishen, Cardiff, CF4 5ZB, UK gamma interferon, cluster, btb Between 008 and 0, there were 40 new incidents in this area; % had lesions typical of btb or Mycobacterium bovis cultured at slaughter. In 0, herd level gamma testing was introduced for all new incidents with evidence of btb at slaughter in low incidence areas of Wales. 85

10 Information regarding btb incidents in 0 and 0 for Wrexham and the neighbouring English counties, Cheshire and Shropshire, was obtained from the national TB database and local intelligence. The incidence and probability of a repeat incident was compared between Wrexham and the adjacent English counties. In Wrexham, gamma testing was undertaken in 8 herds in 0 (9) and 0 (9). None of the gamma positive animals had evidence of btb at slaughter. Of the 9 cases in 0, 4 had further incidents in 0. In 0 and 0, the incidence was 5 and 6 incidents per 00 herd years at risk for Wrexham and England respectively (p=0.448). Incidents in Wales had twice the odds of recurring compared to incidents in Cheshire and Shropshire (p=0.05). Gamma interferon detects infection earlier and improved test sensitivity should decrease recurrence rates. However, recurrence is the result of both re-introduction and persistence. Therefore, the high recurrence rate, despite gamma testing, could suggest that the majority of repeat breakdowns result from re-introduction. Further investigation is required to determine the benefits of gamma interferon in the control of btb. Evaluation Of The Single Cervical Skin Test and Interferon Gamma Responses To Detect Mycobacterium bovis Infected Cattle In A Herd Co-infected With Mycobacterium avium subsp. Paratuberculosis Seva, J., Sanes, J.M., Ramis, G., Mas, A., Quereda, J.J., Villarreal-Ramos, B. 4, Villar, D. 5, Pallares, F.J., Depto. Anatomía y Anatomía Patológica Comparadas Depto. Producción Animal. Fac. Vet. Univ. De Murcia. 000 Murcia, Spain Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 8049 Madrid, Spain 4 TB Research Group, Veterinary Laboratories Agency, Addlestone, Surrey, KT5 NB, UK 5 Escuela Med. Vet. Univ. de Antioquia, Medellin, Colombia Key words Bovine TB, Paratuberculosis, IFNγ test, SIT This study reports the performance of the single intradermal tuberculin (SIT) test and IFN-γ assay for M. bovis in a cattle herd with high prevalence of paratuberculosis (PTB). Over years 58/50 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ and/or ELISA to PTB. Infection status was confirmed by post mortem diagnostic tests that included histopathology examination and mycobacterial cultures and PCR for M. bovis and M. avium subsp. paratuberculosis. In 7 of the 58 animals, tuberculosis (TB) primary lesions affecting only the retropharyngeal and/or mediastinal lymph nodes were found. PTB was confirmed in 5 animals, of which 0 were positive by ELISA. Of the 7 confirmed TB cases, SIT was positive in animals. A total of 45 of the 58 animals were positive by IFN-γ using the most stringent criterion (cut-off point= 0.05); however, it was only positive in animals if a higher threshold was used (cut-off point= 0.). Three co-infected animals with PTB confirmed showed extensive TB lesions and diffuse PTB lesion. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines with the most stringent criterion to maximise sensitivity, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of animals identified by IFN-γ with no TB lesion could be due to a level of protection conferred by co-infection with PTB. 86