ASSESSMENT OF MULTIANTIGEN PRINT IMMUNOASSAY AND RAPID LATERAL-FLOW TEST FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN MALAYAN TAPIR (TAPIRUS INDICUS).

A multiantigen print immunoassay (MAPIA) and rapid test (RT) developed and validated for detection of mycobacterial antibodies in elephants (Elephas maximus and Loxodonta africana) was assessed in Malayan tapir (Tapirus indicus). Retrospective analysis of banked serum from one Mycobacterium bovis infected and seven presumably uninfected tapir was performed by MAPIA and RT. A sample collected 2 mon prior to the death of a culture-confirmed M. bovis-infected tapir served as a positive control. Seroreactivity of this sample was demonstrated via both MAPIA and RT testing. Seven uninfected animals, including four without postmortem evidence of mycobacterial disease and three that remain healthy, were negative controls; none demonstrated seroreactivity to key antigens with either test. These results suggest that MAPIA and RT have potential utility for rapid detection of M. bovis infection in Malayan tapir.

Potential for rapid antibody detection to identify tuberculous cattle with non-reactive tuberculin skin test results.

BACKGROUND: Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. RESULTS: Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. CONCLUSIONS: Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.

Mycobacterium bovis infection in humans and cats in same household, Texas, USA, 2012.

Mycobacterium bovis infection of cats is exceedingly rare in regions where bovine tuberculosis is not endemic. We describe the diagnosis and clinical management of pulmonary M. bovis infection in 2 indoor-housed cats and their association with at least 1 M. bovis-infected human in Texas, USA, in September 2012.

Potential for serodiagnosis of tuberculosis in black rhinoceros (Diceros bicornis).

A case of fatal Mycobacterium tuberculosis infection was diagnosed postmortem in a captive 33-yr-old male black rhinoceros (Diceros bicornis) after a nonspecific illness in April 2013. Retrospective testing of sera from this individual revealed that it had been seroreactive by ElephantTB STAT-PAK, dual-path platform VetTB, and multi-antigen print immunoassay for over 12 yr prior to death. Although samples collected at the time of intradermal tuberculin test performed in October 2000 were nonreactive in all three serologic assays, the animal appeared to seroconvert approximately 2.5 wk after the skin test administration. The antibody response remained detectable for the duration of the animal's life (12+ yr), indicating ongoing immunologic stimulation. The current case report supports the use of serologic assays for diagnosis of TB in black rhinoceros and may provide information for earlier detection. However, further research is needed to develop tools for recognition of mycobacterial infections in rhinoceros.

Retrospective serological investigation of bovine tuberculosis in two gemsbok (Oryx gazelle gazelle) and an onager (Equus hemionus onager).

In 1997 a 26-yr-old gemsbok (Oryx gazelle gazelle) died of bovine tuberculosis in a zoo. Three remaining gemsbok were administered the comparative tuberculin skin test repeatedly over a period of 5 mo. Two animals showed inconclusive results on the second test. All three gemsbok were euthanatized. Mycobacterium bovis was isolated from one of those with an inconclusive skin test result, whereas Mycobacterium fortuitum was detected in the other gemsbok. Eight years later, an onager (Equus hemionus onager) died of bovine tuberculosis. This animal had been kept in the same building as the gemsbok. Three herd mates were culled after administering the comparative tuberculin skin test. They were all nonreactors and produced no evidence of tuberculosis at postmortem examination. Retrospectively, using plasma samples collected from the gemsbok and onagers, three antibody tests, Elephant TB STAT-PAK, multiantigen print immunoassay (MAPIA), and dual-path platform (DPP) VetTB (Chembio Diagnostic Systems Inc., Medford, New York, 11763, USA), were used to assess their diagnostic value for these species. The M. bovis-infected gemsbok tested strongly positive by Elephant TB STAT-PAK at the time of euthanasia and 5 mo earlier when the skin test was negative. This animal was not antibody reactive in MAPIA and DPP VetTB. No M. bovis-specific antibody was detected in the other two gemsboks by any of the immunoassays. Among the onagers, Elephant TB STAT-PAK, MAPIA, and DPP VetTB revealed gradually increasing antibody response in the animal that died of bovine tuberculosis, but not in the three disease-free herd mates euthanatized. Seroconversion in the M. bovis-infected onager was first noticed 5 yr before death when the tuberculin skin test was negative.

Detection of antibodies to tuberculosis antigens in free-ranging lions (Panthera leo) infected with Mycobacterium bovis in Kruger National Park, South Africa.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become established in Kruger National Park, South Africa, in the cape buffalo (Syncerus caffer) population and in other species. TB in prey species has resulted in infection and morbidity in the resident lion (Panthera leo) prides. The only validated live animal test currently available for lions is the intradermal tuberculin test. Because this test requires capture twice, 72 hr apart, of free-ranging lions to read results, it is logistically difficult to administer in a large ecosystem. Therefore, development of a rapid animal-side screening assay would be ideal in providing information for wildlife managers, veterinarians, and researchers working with free-living lion prides. This study reports preliminary descriptive results from an ongoing project evaluating two serologic tests for M. bovis (ElephantTB Stat-Pak and dual path platform VetTB). Disease status was determined by postmortem culture and presence of pathologic lesions in 14 free-ranging lions. Seropositivity was found to be associated with M. bovis infection. Extended field studies are underway to validate these rapid animal-side immunoassays for antemortem screening tests for TB in lions.

Pinniped tuberculosis in Malayan tapirs (Tapirus indicus) and its transmission to other terrestrial mammals.

In the last 7 yr, three different species of terrestrial mammals were diagnosed with Mycobacterium pinnipedii either within one collection or through the introduction of an infected animal from another zoo. The affected species included the Malayan tapir (Tapirus indicus), Bactrian camel (Camelus bactrianus bactrianus), and crested porcupine (Hystrix cristata). In the first zoo, all of these were living in exhibits adjacent to a group of South American sea lions (Otariaflavescens) and were cared for by the same keeper. One infected tapir was transferred to a different zoo and transmitted M. pinnipedii infection to three other Malayan tapirs. The tapirs were tested with various diagnostic methods, including comparative intradermal tuberculin test, PCR and culture of sputum samples, Rapid Test (RT), and multiantigen print immunoassay (MAPIA). The M. pinnipedii infection was confirmed at postmortem examination in all animals. RT and MAPIA showed the diagnostic potential for rapid antemortem detection of this important zoonotic disease.

Mycobacterium kansasii infection in a bontebok (Damaliscus pygaragus dorcas) herd: diagnostic challenges in differentiating from the Mycobacterium tuberculosis complex.

Two adult female bontebok (Damaliscus pygarus dorcas) were euthanized because of signs of pneumonia and weakness (case 1), and a nonresponsive lameness with draining fistula (case 2). Necropsy findings were similar in both cases and consisted of disseminated granulomatous lesions in the liver, kidneys, spleen, lungs, pleural surfaces, and multiple lymph nodes. Mycobacterium kansasii was isolated from both cases after multiple attempts on a variety of samples by two laboratories. The remaining four animals in the herd were tested for antibody responses using the Chembio ElephantTB STAT-PAK, DPP VetTB kits, and multi-antigen print immunoassay (MAPIA), for immune reaction using the intradermal tuberculin test, and by tracheal wash cultures, and thoracic radiographs. Banked serum samples collected in 2005 and obtained from the original institution, revealed 1/9 (11.11%) seropositive animals using the three immunoassays. Retesting the current herd in 2008 showed 2/6 (33.33%) seropositive animals by the three tests, with MAPIA demonstrating antibody reactivity to MPB83 and MPB70 proteins. Inconsistent intradermal tuberculin test results, cross-reactivity in serologic assays designed for tuberculosis detection, difficulty in obtaining definitive identification by culture, and inability to identify a source of infection created challenges in distinguishing the atypical mycobacteriosis due to M. kansasii from the initially suspected tuberculous infection in this herd. Owing to regulatory considerations, differences in host-to-host transmission, and source of infection between Mycobacterium tuberculosis complex and nontuberculous mycobacteria, correct diagnosis is crucial for management of these diseases in wildlife species.

Use of blood matrices and alternative biological fluids for antibody detection in animal tuberculosis.

Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97-0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.

Comparison of three immunodiagnostic assays for antemortem detection of Mycobacterium bovis stimulation in domestic cats.

Mycobacterium bovis causes disease in numerous mammalian species including humans, thus making research, surveillance, and control important in the eradication of tuberculosis. Domestic cats are susceptible to multiple mycobacterial species including Mycobacterium bovis; however, their role in the epidemiology of bovine tuberculosis is not fully documented. The current study was an evaluation of the immune response in specific pathogen-free (SPF) cats stimulated with sensitinogen, a heat-killed M. bovis product, using the rapid test, multiple antigen print immunoassay (MAPIA), and bovine-purified protein derivative (bPPD) single skin test. Six cats were inoculated with sensitinogen subcutaneously on days 0 and 24; 2 noninoculated cats and 49 non-SPF cats were controls. Serial serum samples were collected during 135 days and assayed for M. bovis antibodies by rapid test and MAPIA. On day 123, bPPD skin test was performed and read at 48 and 72 hr. The bPPD test at 72 hr had a mean skin thickness of 0.3 mm for stimulated cats and 0.1 mm for controls. Rapid test identified 4 of 6 stimulated cats after bPPD injection. The MAPIA detected antibody against MPB83, 16/83, 16 kDa, and M. bovis culture filtrate (MBCF) antigens. All assays differentiated between stimulated and control cats; however, 7 of 49 non-SPF control cats had a reaction for either antigen MBCF or 16/83. These preliminary studies show potential for antemortem detection of M. bovis among domestic cats. Additional studies to better characterize virulent M. bovis infection in cats would be of value.

Immunological responses following experimental endobronchial infection of badgers (Meles meles) with different doses of Mycobacterium bovis.

The Eurasian badger (Meles meles) is a wildlife reservoir for Mycobacterium bovis infection in Ireland and Great Britain and has been implicated in the transmission of tuberculosis to cattle. Vaccination of badgers is an option that could be used as part of a strategy to control the disease. In this study we used an endobronchial infection procedure to inoculate groups of badgers with three different doses (3x10(3), 2x10(2) and <10 Colony Forming Units (CFUs)) of M. bovis. After 17 weeks the disease status of each animal was determined by post-mortem pathology and culture for M. bovis. Each of the inoculum doses resulted in establishment of infection in the badgers. The cell-mediated immune (CMI) responses were measured by lymphocyte transformation assay (LTA) of peripheral blood mononuclear cells (PBMCs) cultured with bovine tuberculin (PPD-B). In each infected group the CMI responses increased with a kinetic profile corresponding to the delivered dose and the post-mortem pathology. The serological responses were measured by ELISA and a multi-antigen print immunoassay (MAPIA) in order to investigate any changes in the antigenic repertoire associated with different infective doses. In contrast to the CMI responses, the ELISA and MAPIA showed that the recognition of antigens by the badgers was intermittent and not strongly influenced by the dose of M. bovis.

Accuracy of three diagnostic tests for determining Mycobacterium bovis infection status in live-sampled wild meerkats (Suricata suricatta).

Accurate diagnosis of Mycobacterium bovis infection (bovine tuberculosis [bTB]) in live animals is notoriously problematic. The aim of the present study was to evaluate the diagnostic potential of 2 new serologic tests (multiantigen print immunoassay [MAPIA] and lateral flow immunoassay rapid test [RT]) in comparison with mycobacterial culture of tracheal washes for determining M. bovis infection status in a free-ranging population of wild meerkats (Suricata suricatta). During a longitudinal study lasting 2.5 years, 240 individually identifiable meerkats were each sampled up to 8 times under anesthesia every 3 months. Diagnostic accuracy was determined through Bayesian and maximum likelihood estimations of sensitivity, specificity, and likelihood ratios for each diagnostic test when used independently and in parallel to classify the disease status of individual meerkats in the absence of a gold standard. Culture of tracheal washes was highly specific (0.99; 95% confidence interval [CI] = 0.77, 1.00) but of low sensitivity (0.36; 95% CI = 0.24, 0.50) for diagnosing M. bovis-infected individuals. The longitudinal nature of the study with repeated sampling of the same individual animals served simultaneously to improve chances of detecting infection and increase confidence in a negative result in individual animals repeatedly testing negative. Although MAPIA and RT were individually of limited diagnostic use, interpreting the results of these 2 tests in parallel produced estimates of sensitivity (0.83; 95% CI = 0.67, 0.93) and specificity (0.73; 95% CI = 0.62, 0.82) high enough to usefully inform decision making when determining exposure to bTB in wild meerkats and potentially other species in which bTB poses a diagnostic challenge.

Evaluation of blood assays for detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus) in Michigan.

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all >92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Application of Elephant TB STAT-PAK assay and MAPIA (multi-antigen print immunoassay) for detection of tuberculosis and monitoring of treatment in black rhinoceros (Diceros bicornis).

Many wildlife species including rhinos are susceptible to infection with Mycobacterium tuberculosis or M. bovis. Antemortem diagnostic testing in large exotic hoof stock species has been limited by challenges associated with test administration, sample collection, and interpretation. Hence, a simple, rapid, blood-based test is needed. Two confirmed M. tuberculosis-infected black rhinoceros and one exposed suspect were evaluated for antibody responses using a lateral-flow rapid test (ElephantTB STAT-PAK) and multi-antigen print immunoassay (MAPIA). All three animals were seropositive by both tests. MAPIA detected antibodies to ESAT-6, CFP10, and MPB83 antigens. When the rhinos were treated with antitubercular therapeutics, their antibody responses gradually declined. One rhinoceros died approximately 9 mo after initiation of treatment and showed an increase in antibody titer shortly before death. The other two rhinoceros, which were treated for 1 and 2 yr, respectively, had no clinical signs or positive culture for M. tuberculosis at the time of necropsy performed 2 or 6 yr later for unrelated reasons. The antibody levels in these rhinos continued to be significantly decreased. The findings suggest that the ElephantTB STAT-PAK and MAPIA may be useful tools to detect M. tuberculosis infection and monitor treatment in black rhinoceros.

Antigen specific immunological responses of badgers (Meles meles) experimentally infected with Mycobacterium bovis.

European badgers (Meles meles) are considered to be an important reservoir of infection for Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. Accurate tests are required for tuberculosis surveillance in badger populations and to provide a basis for the development of strategies, including vaccination, to reduce the incidence of the infection. In this study, we have developed an endobronchial M. bovis infection model in badgers in which we measured cell-mediated immune and serological responses for up to 24 weeks post-infection. Groups of badgers were subjected to necropsy at 6-week intervals and the gross lesion severity status compared with immune responses measured in blood samples taken throughout the course of the study. The panel of antigens included bovine and avian tuberculins (PPD) as well as single antigens, ESAT-6, CFP-10, MPB70, Rv3019c, Rv3873, Rv3878 and Rv3879, all known to be recognised by the immune system in other animal models of tuberculosis infection. Our results demonstrated that M. bovis infected badgers responded to specific antigens as early as 6 weeks post-infection, consistent with the presence of visible lesions. The data also revealed unique patterns of antigen recognition with high levels of PBMC proliferation in the presence of CFP-10 but low proliferation levels with ESAT-6. Using a multi-antigen print immunoassay (MAPIA), we were able to confirm that MPB83 is the dominant antigen recognised by serum antibodies in infected badgers.

Two cases of atypical mycobacteriosis caused by Mycobacterium szulgai associated with mortality in captive African elephants (Loxodonta africana).

Mycobacterium szulgai was associated with mortality in two captive African elephants (Loxodonta africana) housed at Lincoln Park Zoo. The first elephant presented with severe, acute lameness of the left rear limb. Despite extensive treatments, the animal collapsed and died 13 mo after initial presentation. Necropsy revealed osteomyelitis with loss of the femoral head and acetabulum and pulmonary granulomas with intralesional M. szulgai. The second elephant collapsed during transport to another institution with no premonitory clinical signs. This animal was euthanized because of prolonged recumbency. Granulomatous pneumonia with intralesional M. szulgai was found at necropsy. Two novel immunoassays performed on banked serum samples detected antibody responses to mycobacterial antigens in both infected elephants. It was not possible to determine when the infection was established or how the elephants were infected. When reviewing the epidemiology of this organism in humans, however, transmission between elephants seemed unlikely because human-to-human transmission of this organism has never been reported and a third elephant in the herd was not affected. In addition to Mycobacterium bovis and Mycobacterium tuberculosis, atypical mycobacterial organisms need to be considered potentially pathogenic in elephants.

Early Detection of Circulating Antigen and IgM-Associated Immune Complexes during Experimental Mycobacterium bovis Infection in Cattle.

The presence of circulating antigen in cattle experimentally infected with Mycobacterium bovis was demonstrated using dual-path platform (DPP) technology. The antigen capture immunoassays employed rabbit polyclonal antibody recognizing predominantly M. tuberculosis complex-specific epitopes and were able to detect soluble substances and whole cells of mycobacteria. The antigen found in serum appeared to be mostly bound to IgM, but not to IgG, within the immune complexes formed at early stages of M. bovis infection. The antigen was also detected in bile and urine, indicating possible clearance pathways. The data correlation analyses supported the idea of the role of IgM responses in antigen persistence during M. bovis infection. The antigen was detectable in serum months prior to detectable antibody seroconversion. This proof-of-concept study suggested the potential for improved immunodiagnostics for bovine tuberculosis.

Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle.

Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

Improved serodetection of Mycobacterium bovis infection in badgers (Meles meles) using multiantigen test formats.

Despite attempts to control bovine tuberculosis, the incidence of disease in Great Britain continues to rise. In GB, the European badger (Meles meles) is a reservoir of infection with Mycobacterium bovis. In an effort to improve the serodetection of badger tuberculosis, we examined sera from M. bovis culture-positive and culture-negative badgers for their ability to recognize M. bovis antigens, using a multi-antigen print immunoassay (MAPIA). Depending on the antigens used in the MAPIA, the assay had a sensitivity of 49-59% and a specificity of 84-88% Results from the MAPIA were used to select antigens for the development of a lateral-flow immunoassay. This so-called 'Rapid Test' used 5microl of serum and gave unambiguous results within 10 min. When applied to 178 badger sera, the Rapid Test had a sensitivity of 53% and a specificity of 95%. This represented an improvement over the performance of the existing ELISA Test, which had a sensitivity of 47% and a specificity of 89% on the same sera. This is the first report of a diagnostic test for badger tuberculosis that can be performed alongside the captive animal.

Field evaluation of three blood-based assays for elk (Cervus canadensis) naturally infected with Mycobacterium bovis.

Diagnosis of Mycobacterium bovis in wild populations is very challenging due to complications imposed by the use of traditional skin tests, poor sensitivity of gold standard tests which rely on culture of M. bovis from tissues and wide variations in severity of disease. Various combinations of a lymphocyte stimulation test (LST), fluorescence polarization assay (FPA) and the Cervid TB Stat-Pak were evaluated using two different validation approaches: a latent class analysis and classical statistical approach using culture as a gold standard. A validation subsample consisting of animals culled for population control and mortalities from capture provided an unbiased estimate of test performance for comparison. The sensitivity of the LST (0.83, 95% CI: [0.70-0.97] as a single test was similar to existing tuberculin skin tests, but the sensitivity of the FPA (0.40, 95% CI: [0.22-0.58]) and Cervid TB Stat-Pak (0.62, 95% CI: [0.41-0.83]) were lower in this population. Test performance of the LST and Cervid TB Stat-Pak in parallel was similar to the use of all three tests in parallel and inclusion of the FPA did not greatly enhance test performance. Prevalence of M. bovis in elk varied substantially between the high risk area of southern Manitoba (9.1%, 95% CI: [6.09-12.1%]) and lower risk areas outside this zone (0.76%, 95% CI: [0-2.26%]). Bayesian latent class analysis indicated lack of covariance between the two antibody tests (FPA and Cervid TB Stat-Pak) while the classical two-stage analysis indicated there was conditional dependence between the tests. All three tests when used in parallel resulted in 100% NPV using all three validation methods, indicating few elk were misclassified as false negative by post mortem culture. Similar to previous studies, this study found that combinations of blood tests that utilize cell mediated responses along with humoral antibody responses maximize the sensitivity of tests for diagnosis of M. bovis in wild cervid populations.