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Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis-infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB-endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB-endemic ecosystems and evaluate serological and other diagnostic tools in this species.

The present study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current antemortem testing protocols to identify TB-free alpaca herds and individuals for exportation. The tuberculin skin test (TST) failed to identify Mycobacterium bovis-infected animals prior to movement from the United Kingdom (UK) to Poland. This study describes the use of four serological assays [Enferplex Camelid TB, dual-path platform (DPP) VetTB and BovidTB assays, and multi-antigen print immunoassays (MAPIAs)] to detect TB in an alpaca herd with negative TST results. The breeding in Poland purchased alpacas for several years from the UK with the last group arriving in May 2018. In July 2018, two sick alpacas from the centre were hospitalized in a veterinary clinic and both died of TB a few weeks later. In November 2018, 20 alpacas remaining in this M. bovis-affected herd were euthanized and samples were collected. The study population included 20 M. bovis-infected and 20 uninfected alpacas, but only 15 infected animals were tested by all serology tests. The DPP VetTB and DPP BovidTB assays detected antibodies in 14 of the 20 infected alpacas, with results confirmed by MAPIA, and in none (MAPIA and DPP BovidTB) or one (DPP VetTB) of the 20 uninfected animals. None of the infected alpacas tested positive using the Enferplex assay. In addition, the group included three orphans and two cria-dam pairs, which provided an opportunity to analyse immune aspects of cria-mother relationships in this herd. The results suggest high susceptibility of this host species to M. bovis infection and rapid progression to disease. The serological tests used in this study offer useful tools for the detection of M. bovis infection in TST and Enferplex test non-reactive alpacas. These tests should be further evaluated for implementation into TB management and control strategies for camelid species.

A multiantigen print immunoassay (MAPIA) and rapid test (RT) developed and validated for detection of mycobacterial antibodies in elephants (

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.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, infects humans and animals causing lesions and disease like that of Mycobacterium bovis. The aim of this study was to evaluate antibody responses in European Bison (EB, Bison bonasus; a vulnerable species) naturally infected with M. caprae using dual path platform (DPP) BovidTB test and multi-antigen print immunoassay (MAPIA). Study cohorts consisted of naturally M. caprae-infected EB (n = 4), M. caprae-exposed but uninfected (n = 3), EB infected with non-tuberculous mycobacteria or other respiratory pathogens (n = 3), and negative controls (n = 19). M. caprae-infected EB were seropositive by both DPP and MAPIA; 3/4 were seropositive by DPP; and 4/4 were seropositive by MAPIA. One M. caprae-infected animal that developed generalized disease with most advanced gross lesions in the group produced the most robust antibody response. All 25 EB with no culture-confirmed M. caprae infection, including three animals exposed to M. caprae and three other animals infected with non-tuberculous pathogens, were seronegative on both tests. Antibody responses to M. caprae infection included IgM antibodies against MPB70/MPB83 and IgG antibodies to both MPB70/MPB83 and CFP10/ESAT-6. This study demonstrates the potential for use of serological assays in the ante-mortem diagnosis of M. caprae infection in EB.

Using multi-antigen print immunoassay and DPP VetTB Assay approved in the United States for testing captive cervids and elephants, we analyzed antibody recognition of MPB83 and CFP10/ESAT-6 antigens in Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis and in white-tailed deer (Odocoileus virginianus), fallow deer (Dama dama), elk (Cervus elaphus), and cattle (Bos taurus) infected with Mycobacterium bovis. Serum IgG reactivity to MPB83 was found in the vast majority of tuberculous cattle and cervid species among which white-tailed deer and elk also showed significant CFP10/ESAT-6 recognition rates with added serodiagnostic value. In contrast, the infected elephants developed antibody responses mainly to CFP10/ESAT-6 with MPB83 reactivity being relatively low. The findings demonstrate distinct patterns of predominant antigen recognition by different animal hosts in tuberculosis., This work was partially supported by the Small Business Innovation Research Program of the USDA National Institute of Food and Agriculture (Award No. 2016-33610-25688).

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

Tuberculosis (TB) is a pathogenic disease that affects a range of wildlife species, including African elephants (

Domestic pigs and wild suids are susceptible to Mycobacterium bovis (M. bovis) infection and may even serve as reservoir hosts in some situations. Therefore, detection of infected animals is important for understanding their role in the epidemiology of the disease as well as for management and control of bovine tuberculosis. Infected suids develop strong humoral responses, making serological screening a feasible approach to disease surveillance. However, to optimize sensitivity of the antibody assays, it is necessary to identify and incorporate immunodominant antigens recognized by the target species. The objective of this study was to characterize the antigen recognition by three suid species in a commercially available serological test, DPP VetTB Assay. Serum samples from naturally M. bovis-infected domestic pigs, wild boar and common warthogs were tested. MPB83 protein appeared to be the immunodominant antigen recognized by antibodies in all three species. Overall test sensitivity was increased in wild suids when seroreactivity to CFP10/ESAT-6 antigen was included. Infected animals with visible lesions showed more robust antibody responses than those without gross lesions. The high sensitivity and specificity of the DPP VetTB Assay demonstrated in the present study supports the utility of antibody tests employing these antigens in serological screening of the suid species for M. bovis infection., Financial support (MAM, EOR) was provided by the South African Medical Research Council, NRF South African Research Chair Initiative (SARChI, grant 86949) and the American Association of Zoological Medicine Wild Animal Health Fund (#7-2016).

We assessed the suitability of targeted removal as a means for tuberculosis (TB) control on an intensely managed Eurasian wild boar (Sus scrofa) hunting estate. The 60 km large study area included one capture (treatment) site, one control site, and one release site. Each site was fenced. In the summers of 2012, 2013 and 2014, 929 wild boar were live-captured on the treatment site. All wild boar were micro-chipped and tested using an animal side lateral flow test immediately after capture in order to detect antibodies to the Mycobacterium tuberculosis complex (MTC). The wild boar were released according to their TB status: Seropositive individuals onto the release site (hunted after summer), and seronegative individuals back onto the treatment site. The annual summer seroprevalence of antibodies to the MTC declined significantly in live-captured wild boar piglets from the treatment site, from 44% in 2012 to 27% in 2013 (a reduction of 39%). However, no significant further reduction was recorded in 2014, during the third capture season. Fall-winter MTC infection prevalence was calculated on the basis of the culture results obtained for hunter-harvested wild boar. No significant changes between hunting seasons were recorded on either the treatment site or the control site, and prevalence trends over time were similar on both sites. The fall-winter MTC infection prevalence on the release site increased significantly from 40% in 2011–2012 to 64% in 2012–2013 and 2013–2014 (60% increase). Recaptures indicated a persistently high infection pressure. This experiment, the first attempt to control TB in wild boar through targeted removal, failed to reduce TB prevalence when compared to the control site. However, it generated valuable knowledge on infection pressure and on the consequences of translocating TB-infected wild boar., This is a contribution to Spanish Government MINECO Plan Nacional grant AGL2014-56305 and FEDER, to CDTI, and to the EU FP7 WIldTBvac#613779 grant. Azlan Che’Amat has a PhD grant from the Malaysian Government.

Immunological memory is a central feature of adaptive immunity. Memory B cells are generated upon stimulation with antigen presented by follicular dendritic cells in the peripheral lymphoid tissues. This process typically involves class-switch recombination and somatic hypermutation and it can be dependent or independent on germinal centers or T cell help. The mature B cell memory pool is generally characterized by remarkable heterogeneity of functionally and phenotypically distinct sub-populations supporting multi-layer immune plasticity. Memory B cells found in human patients infected with Mycobacterium tuberculosis include IgD+ CD27+ and IgM+ CD27+ subsets. In addition, expansion of atypical memory B cells characterized by the lack of CD27 expression and by inability to respond to antigen-induced re-activation is documented in human tuberculosis. These functionally impaired memory B cells are believed to have adverse effects on host immunity. Human and animal studies demonstrate recruitment of antigen-activated B cells to the infection sites and their presence in lung granulomas where proliferating B cells are organized into discrete clusters resembling germinal centers of secondary lymphoid organs. Cattle studies show development of IgM+, IgG+, and IgA+ memory B cells in M. bovis infection with the ability to rapidly differentiate into antibody-producing plasma cells upon antigen re-exposure. This review discusses recent advances in research on generation, re-activation, heterogeneity, and immunobiological functions of memory B cells in tuberculosis. The role of memory B cells in post-skin test recall antibody responses in bovine tuberculosis and implications for development of improved immunodiagnostics are also reviewed.

Tuberculosis (TB) was diagnosed in four Asian elephants ( Elephas maximus) in a zoo in the United States. The first case was detected by isolation of Mycobacterium tuberculosis during routine trunk wash (TW) culture testing of a herd of eight elephants. Retrospective antibody analyses revealed seroconversion 1 yr before diagnosis. Serological testing of the whole elephant herd identified two additional suspect bulls with detectable antibody, but which remained culture-negative and had no clinical signs of disease. In the following months, M. tuberculosis, identical to the isolate from the index case, was isolated from TW samples of these two elephants. A fourth elephant seroconverted nearly 4 yr after the first TB case was detected, and M. tuberculosis was isolated from a TW sample collected 1 mo later. All four infected elephants received anti-TB therapy. Two treated elephants were eventually euthanized for reasons unrelated to M. tuberculosis and found to be culture-negative on necropsy, although one of them had PCR-positive lung lesions. One infected animal had to be euthanized due to development of a drug-resistant strain of M. tuberculosis; this animal did not undergo postmortem examination due to risk of staff exposure. The fourth animal is currently on treatment. Serial serological and culture results of the other four herd mates have remained negative.

A. Che’ Amat et al., Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (. Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (. n=. 53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account., This is a contribution to Spanish Government MINECO Plan Nacional I+D+I grant AGL2014-56305 and FEDER, to a contract between CDTI and Glenton, and to the EU FP7 grant WildTBvac #613779. Azlan Che Amat has a PhD grant from the Malaysian Government, and José Angel Barasona and Iratxe Diéz-Delgado acknowledge PhD grants from the Spanish Government.

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.

Bovine tuberculosis (TB), caused by Mycobacterium bovis , remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (∼95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.

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.

Over the past two decades, highly virulent strains of Mycobacterium tuberculosis have emerged and spread rapidly in man, suggesting a selective advantage based on virulence. A similar scenario has not been described for Mycobacterium bovis infection in cattle (i.e. bovine tuberculosis). An epidemiological investigation of a recent outbreak of bovine tuberculosis in a USA dairy indicated that the causative strain of M. bovis (strain 10-7428) was particularly virulent, with rapid spread within the herd. In the present study, the virulence of this strain (10-7428) was directly compared in the target host with a well-characterized strain (95-1315) of relevance to the USA bovine tuberculosis eradication programme. Aerosol inoculation of 10(4) colony forming units of M. bovis 95-1315 (n = 8) or 10-7428 (n = 8) resulted in a similar distribution and severity of gross and microscopical lesions of tuberculosis as well as mycobacterial colonization, primarily affecting the lungs and lung-associated lymph nodes. Specific cell-mediated and antibody responses, including kinetics of the response, as well as antigen recognition profiles, were also comparable between the two treatment groups. Present findings demonstrate that M. bovis strains 95-1315 and 10-7428 have similar virulence when administered to cattle via aerosol inoculation. Other factors such as livestock management practices likely affected the severity of the outbreak in the dairy.

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.

Bovine tuberculosis (TB) in cervids remains a significant problem affecting farmed herds and wild populations. Traditional skin testing has serious limitations in certain species, whereas emerging serological assays showed promising diagnostic performance. The recently developed immunochromatographic dual-path platform (DPP) VetTB assay has two antigen bands, T1 (MPB83 protein) and T2 (CFP10/ESAT-6 fusion protein), for antibody detection. We evaluated the diagnostic accuracy of this test by using serum samples collected from groups of white-tailed deer experimentally inoculated with Mycobacterium bovis , M. avium subsp. paratuberculosis , or M. bovis BCG Pasteur. In addition, we used serum samples from farmed white-tailed deer in herds with no history of TB, as well as from free-ranging white-tailed deer culled during field surveillance studies performed in Michigan known to have bovine TB in the wild deer population. The DPP VetTB assay detected antibody responses in 58.1% of experimentally infected animals within 8 to 16 weeks postinoculation and in 71.9% of naturally infected deer, resulting in an estimated test sensitivity of 65.1% and a specificity of 97.8%. The higher seroreactivity found in deer with naturally acquired M. bovis infection was associated with an increased frequency of antibody responses to the ESAT-6 and CFP10 proteins, resulting in a greater contribution of these antigens, in addition to MPB83, to the detection of seropositive animals, compared with experimental M. bovis infection. Deer experimentally inoculated with either M. avium subsp. paratuberculosis or M. bovis BCG Pasteur did not produce cross-reactive antibodies that could be detected by the DPP VetTB assay. The present findings demonstrate the relatively high diagnostic accuracy of the DPP VetTB test for white-tailed deer, especially in the detection of naturally infected animals.

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.

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas fromMycobacterium bovisculture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities 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 undisclosedMycobacterium microtiinfection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a “test package,” although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.

Three serologic methods for antibody detection in elephant tuberculosis (TB), the multiantigen print immunoassay (MAPIA), ElephantTB STAT-PAK kit, and DPP VetTB test, were evaluated using serial serum samples from 14 captive elephants infected withMycobacterium tuberculosisin 5 countries. In all cases, serological testing was performed prior to the diagnosis of TB by mycobacterial culture of trunk wash or tissue samples collected at necropsy. All elephants produced antibody responses toM. tuberculosisantigens, with 13/14 recognizing ESAT-6 and/or CFP10 proteins. The findings supported the high serodiagnostic test accuracy in detecting infections months to years beforeM. tuberculosiscould be isolated from elephants. The MAPIA and/or DPP VetTB assay demonstrated the potential for monitoring antimycobacterial therapy and predicting TB relapse in treated elephants when continuously used in the posttreatment period. History of exposure to TB and past treatment information should be taken into consideration for proper interpretation of the antibody test results. Data suggest that the more frequent trunk wash culture testing of seropositive elephants may enhance the efficiency of the TB diagnostic algorithm, leading to earlier treatment with improved outcomes.

This study describes the comparison of the cell-based interferon-gamma (IFNγ) test with serological rapid antibody tests (STAT-PAK and DPP VetTB) for the ante mortem testing of tuberculosis in domestic cats. The antibody specificities of rapid antibody test-positive cats were further discerned using multi-antigen print immunoassay. A total of 62 cats with culture-confirmed Mycobacterium bovis, Mycobacterium microti, Mycobacterium avium and Mycobacterium malmoense, as well as negative controls and dangerous-contact cats were tested. Tests were also applied longitudinally to one further cat undergoing TB chemotherapy for suspected M. bovis infection. Our data from this small study show excellent test specificity (100% for all tests) and encouraging levels of test sensitivity for M. bovis and TB Complex infections (IFNγ 70–100% depending upon test interpretation criteria; rapid tests both 90% for M. bovis infection and up to 46.2% for M. microti infection). The differential diagnosis of very pathogenic TB Complex (M. bovis, Mycobacterium tuberculosis), as opposed to less-pathogenic TB Complex (M. microti) was possible where positive responses to the protein cocktail ESAT6/CFP10 were observed (80% of M. bovis-infected cats in this study showed positive IFNγ responses to ESAT6/CFP10, while 20% had antibody responses to ESAT6/CFP10 using MAPIA). Finally, preliminary data from a longitudinal study of one M. bovis-exposed cat with a positive IFNγ test pre-treatment suggest that a decrease in bacterial burden may be reflected in the IFNγ response, and thus the IFNγ test may provide a monitor for TB chemotherapy.

This study aimed to maximize the sensitivity of bovine tuberculosis detection in living wild fallow deer (Dama dama) under field conditions. We evaluated the rapid test (RT; CervidTB STAT-PAK Assay, Chembio Diagnostic Systems, Inc., USA) in comparison with the comparative cervical skin test (CCT). A total of 134 fallow deer were captured between January and March 2008. At time 0, 0.1 ml of avian purified protein derivative (avian PPD; Cooper-Zeltia, Spain), 0.1 ml bovine PPD (Cooper-Zeltia, Spain), 0.1 ml negative control PBS and 0.1 ml of a positive control (the mitogen phytohaemagglutinin, PHA; containing 250 mg PHA, diluted in PBS) were injected intradermally at four shaved sites in the neck. The skin fold thickness at each injection site was measured at time 0 and 72 h (3 repeats each time). Animals with a skin test response of 2 mm or more at the bovine PPD injection site and animals with any visible reactivity in the RT were necropsied and tissues submitted for culture and for histopathology. A total of 36 fallow deer were considered reactors to bovine PPD or to the RT (apparent prevalence 27%). Regarding both bovine PPD reactivity and the skin fold increase at the PHA injection site, we found significant effects of age and sex by age interaction. Adult males had the largest responses. Mycobacterium bovis was isolated from lymphoid tissues of 21 fallow deer. Skin test sensitivity, as compared to M. bovis culture confirmed deer, was 80.1% (17/21). But, the CCT alone would have missed 4 of 21 culture confirmed animals. RT sensitivity, based on culture confirmed deer, was also 80.1% (17/21). Similarly, the RT alone would have missed another 4 of 21 culture confirmed deer. However, combining the CCT and the RT allowed for detecting all 21 culture positive fallow deer. We conclude that the combined application of the RT and the skin testing can maximize the sensitivity of bTB detection in living fallow deer, thus facilitating control programs for wildlife disease surveillance., This study was funded by Patrimonio Nacional (special thanks to M. Moreno and A. Jacoste) through contracts to IREC and TRAGSEGA (A. Rosón). This is a contribution to MCINN Plan Nacional Research grant AGL2008-03875 and FEDER, and to EU grant TB-STEP 212414. Studies on diseases shared between domestic animals and wildlife are also supported by grants and contracts from INIA and Ministerio de Medio Ambiente y Medio Rural y Marino (OAPN and SDGSPP), and Grupo Santander – Fundacion Marcelino Botin. MPM-H acknowledges a post-doctoral contract from Instituto de Salud Carlos III.

Mycobacterium spp. infection is an important health concern for Asian elephant (Elephas maximus) populations worldwide. The disease is of particular concern considering its potential to affect not only the individual animal but also herd and public health. Although elephant tuberculosis susceptibility is poorly understood, immune function alterations are central to disease pathogenesis in other species and probably affect outcome of mycobacterial infections in elephants. Measurement of immune mediator (cytokine) levels within blood samples can provide information regarding immune function that may elucidate disease susceptibility. For this study, mRNA levels of interleukin (IL)-2, IL-4, IL-10, and IL-12; interferon (IFN)-gamma; tumor necrosis factor (TNF)-alpha; and transforming growth factor (TGF)-beta were measured using elephant-specific, real-time reverse transcription-polymerase chain reaction (RT-PCR) assays in RNA-preserved whole blood samples from 106 Asian elephants, 15% of which were Mycobacterium tuberculosis complex seropositive. The Elephant TB STAT-PAK (Chembio Diagnostics, Inc., Medford, New York 11763, USA), a novel lateral flow antibody detection assay developed for specific use in elephants, was used to determine serologic status for the study. Seropositive animals had higher levels of TNF-alpha and lower levels of TGF-beta than seronegative animals; these differences between groups were statistically significant when levels were analyzed as categorical variables. Trends toward higher levels of IFN-gamma and IL-4 and slightly lower levels of IL-10 and IL-12 were noted in the seropositive group, although differences between groups were not statistically significant. Presence of other inflammatory conditions was found to be a significant confounding variable in the analysis of the relationship between tuberculosis status and TNF-alpha levels, necessitating its inclusion in statistical models. Age and sex were not found to significantly affect the relationship between tuberculosis status and any of the cytokines measured. Interleukin-2 levels were below the sensitivity of the real-time RT-PCR assay irrespective of tuberculosis status. These findings provide a foundation for future research into the immunopathogenesis of elephant tuberculosis.

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.

Between October 2005 and May 2006, a total of 727 badgers found dead in Wales were reported, and 550 were delivered to the Regional Laboratories of the Veterinary Laboratories Agency (VLA). Of the 459 carcasses suitable for examination, 55 were deemed to be infected with Mycobacterium bovis on the basis of culture, spoligotyping, and variable-number tandem repeat typing. Acid-fast bacteria were observed histologically in a further six badgers, but these bacteria were not confirmed as M. bovis by culture. A rapid serological test (BrockTB Stat-Pak) performed on thoracic blood showed a sensitivity of 35% and a specificity of 99%. Presence of M. bovis infection was 45 times more likely to be confirmed postmortem by culture in BrockTB Stat-Pak-reactive animals than in seronegative ones. Using visible carcass lesions as a marker of bovine tuberculosis (bTB) infection had a similar sensitivity (38%) but was significantly less specific (84%) than serology. The overall accuracy of the antibody detection was 93% (346 correct results from 374 tests), whereas the accuracy of regarding visible lesions as a marker for bTB infection was 78% (354 correct from 453 carcasses examined). Culture remains the gold standard method for detecting M. bovis infection in badgers. However, where resources are limited and/or an instant result is preferred, the BrockTB Stat-Pak could be used in field surveillance efforts to identify animals which should be examined further by only submitting test-negative animals to more detailed postmortem examination and culture.

In 1996, the Hook Lake Wood Bison Recovery Project was initiated to establish a small, disease-free, captive, bison-breeding herd. Founders originated from wild bison herds in the Slave River Lowlands in northern Canada, which, like other bison herds in and around Wood Buffalo National Park, are endemically infected with bovine tuberculosis (caused by Mycobacterium bovis) and brucellosis (caused by Brucella abortus). After 9 yr of apparent disease freedom, tuberculosis was detected within the captive herd, leading to complete depopulation. This study examined the performance of antemortem tuberculosis diagnostic tests used during the project. Performances of the caudal-fold test, fluorescent polarization assay, multiantigen print immunoassay (MAPIA), and the rapid test (RT) were assessed by estimating sensitivity, specificity, positive predictive value, and negative predictive value for each test. Kappa values measuring agreement between tests were calculated. Overall, the tests did not differ with respect to sensitivities and specificities, which ranged from 50% to 92% and from 34% to 100%, respectively. The MAPIA tended to show high sensitivity, and there was significant agreement only between the MAPIA and RT. Serum collected from infected animals at slaughter produced highly variable results on the different assays, and one infected bison was negative on all antemortem tests. The results of this analysis suggest use of multiple antemortem tests in parallel, particularly those incorporating multiple antigens, to optimize sensitivity in detecting bovine tuberculosis in bison. However, as demonstrated in this herd, even a seemingly optimal antemortem testing regimen can fail to detect M. bovis-infected individuals.

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.

Background: Infection with Mycobacterium microti can cause chronic disease in animals and threaten human health through its zoonotic potential. Objective: To describe clinical findings, diagnostic investigations, necropsy, and epidemiology results in South American camelids (SAC) infected with M. microti, member of the Mycobacterium tuberculosis complex. Animals: Eleven SAC with tuberculous lesions. Methods: Description of 10 llamas and 1 alpaca, aged 4–18 years, from 6 herds with a history of wasting and weakness admitted to the Vetsuisse-Faculty of Berne over 8 years. Results: Clinical signs included weight loss, recumbency, and anorexia in late stages of the disease. Respiratory problems were seen in 6 animals of 11. No consistent hematologic abnormalities were identified. Suspect animals were examined in detail by abdominal ultrasonography and thoracic radiology. Abnormal findings such as enlarged mediastinal, mesenteric, or hepatic lymph nodes were seen only in animals with advanced disease. Single comparative intradermal tuberculin test with bovine protein purified derivate (PPD) and avian PPD was negative in all animals. At necropsy, typical tuberculous lesions were found, and confirmed by bacteriological smear and culture, molecular methods, or both. Conclusions and Clinical Importance: Infection caused by M. microti should be considered a differential diagnosis in chronic debilitating disease with or without respiratory signs in SAC. Antemortem confirmation of the diagnosis remains challenging at any stage of infection. Because cases of M. microti infection have been reported in immunocompromized human patients, the zoonotic potential of the organism should be kept in mind when dealing with this disease in SAC.

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.

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

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n = 1532), white-tailed deer (n = 463), brushtail possums (n = 129), and wild boar (n = 177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock., The authors acknowledge the support of staff from CSL, VLA Starcross, Defra Wildlife Unit, and permission from the Independent Scientific Group for use of sera from the RBCT. Spanish wild boar samples were obtained with support from MEC Plan Nacional AGL2005-07401 and Santander – Fundación M. Botín.

Captive and free-ranging wildlife animals are implicated in the maintenance and transmission of bovine tuberculosis and therefore pose a significant obstacle to eradication of the disease from domestic livestock. The current antemortem diagnostic method, the intradermal tuberculin skin test, is impractical for routine use with many wild animals. Antibody-based assays are particularly attractive because the animals are handled only once and immediate processing of the sample is not required. This report characterizes the antibody responses of red deer-elk hybrids ( Cervus elaphus ) against Mycobacterium bovis and subsequently evaluates the diagnostic performance of select antigens in a rapid-test format. Sequential serum samples were collected from 10 animals experimentally infected with M. bovis and 5 noninfected animals over a 7-month period postinfection (p.i.). Samples were evaluated by enzyme-linked immunosorbent assays, immunoblot analyses, and multiantigen print immunoassays for seroreactivity to mycobacterial antigens. Although all infected animals produced antibodies to M. bovis protein antigens, there was significant animal-to-animal variation in the kinetics and magnitudes of responses and the antigens recognized. The most frequently recognized antigens included MPB83, ESAT-6, CFP10, and MPB70. Responses to some antigens, such as MPB83, were consistently detected as early as 4 weeks after inoculation, whereas other antigens were detected only much later (>140 days p.i.). Antibody responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Comparison of single-antigen (fluorescence polarization assay) with multiantigen (CervidTB STAT-PAK) rapid tests demonstrated that a highly sensitive and specific serodiagnostic test for tuberculosis in cervids will require multiple and carefully selected seroreactive antigens covering a broad spectrum of antibody specificities.

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.

Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.

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.

Warthogs (Phacochoerus africanus) have been implicated as potential maintenance hosts of Mycobacterium bovis. Our preliminary investigation of bovine tuberculosis in three warthogs describes pathologic findings and associated positive serologic results in two infected animals. This demonstrates the potential use of serodiagnostic tests for M. bovis infection in this species.

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.

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.

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis -infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis -infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.

Although tuberculin skin testing has been a hallmark of bovine tuberculosis eradication campaigns, it lacks sensitivity, can be confounded by exposure to nontuberculous mycobacteria, and cannot be repeated for 60 days due to desensitization. To overcome these difficulties, an effective whole-blood cellular immunoassay for bovine gamma interferon (IFN-γ) has been developed. The IFN-γ test is commonly used in conjunction with tuberculin skin testing as a confirmatory test following a positive response to the caudal fold test (CFT). The present study was conducted to determine the effect of different tuberculin skin-testing regimens on IFN-γ and antibody production by using calves that were experimentally infected with Mycobacterium bovis . Holstein calves were CFT tested 60 days after inoculation and the comparative cervical test (CCT) was conducted 7 (7-day CCT) or 55 (55-day CCT) days after the CFT. In both the 7-day CCT and 55-day CCT groups, IFN-γ responses increased 3 days after the CFT; this was immediately followed by a decrease to pre-skin test levels 7 days after the CFT. In both groups, the application of the CCT at 7 or 55 days after the CFT resulted in no significant increase in IFN-γ production. The administration of the CFT and the CCT to M. bovis -inoculated cattle boosted antibody responses to M. bovis PPD, rMPB83, ESAT-6, and the fusion protein Acr1-MPB83. The boosting effect was more pronounced in the 55-day CCT group. Increases in either IFN-γ or antibody production were not seen in noninoculated cattle. Measurement of both IFN-γ and antibody responses after skin testing may be useful in identifying M. bovis -infected cattle; however, the timing of collection of such samples may influence interpretation.

White-tailed deer ( Odocoileus virginianus ) have emerged as reservoirs of bovine tuberculosis in northern America. For tuberculosis surveillance of deer, antibody-based assays are particularly attractive because deer are handled only once and immediate processing of the sample is not required. Sera collected sequentially from 25 Mycobacterium bovis -infected and 7 noninfected deer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for immunoglobulin specific to M. bovis antigens. Various routes of experimental M. bovis infection, such as intratonsillar inoculation ( n = 11), aerosol ( n = 6), and exposure to infected deer (in contact, n = 8), were studied. Upon infection, specific bands of reactivity at ∼24 to 26 kDa, ∼33 kDa, ∼42 kDa, and ∼75 kDa to M. bovis whole-cell sonicate were detected by immunoblot. Lipoarabinomannan-specific immunoglobulin was detected as early as 36 days postchallenge, and responses were detected for 94% of intratonsillarly and “in-contact”-infected deer. In MAPIA, sera were tested with 12 native and recombinant antigens coated on nitrocellulose. All in-contact-infected (8 of 8) and 10 of 11 intratonsillarly infected deer produced antibody reactive with one or more of the recombinant/native antigens. Responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Additionally, three of six deer receiving a very low dose of M. bovis via aerosol exposure produced antibody specific to one or more recombinant proteins. M. bovis was isolated from one of three nonresponding aerosol-challenged deer. Of the 12 antigens tested, the most immunodominant protein was MPB83; however, a highly sensitive serodiagnostic test will likely require use of multiple antigens.

This report describes how Mycobacterium bovis infection was controlled and eventually eradicated in a farmed red deer herd in the north of England, following sustained tuberculin skin testing supplemented with serological (antibody) tests over a period of approximately two years. By taking advantage of the anamnestic antibody response produced by the skin test to detect skin test-negative, antibody-positive infected individuals, a total of 35 additional animals were identified, including 2 with gross visible lesions typical of bovine tuberculosis (BTB). Without detection and removal, these animals would have posed a continued risk of BTB persistence within the herd and potentially contributed to the spread of infection from deer into wildlife and surrounding cattle farms in an area of low BTB incidence. This case supports the use of ancillary diagnostic serological tests to speed up the resolution of incidents of BTB caused by M bovis in captive deer herds.

Pioneer studies on infectious disease and immunology by Jenner, Pasteur, Koch, Von Behring, Nocard, Roux, and Ehrlich forged a path for the dual-purpose with dual benefit approach, demonstrating a profound relevance of veterinary studies for biomedical applications. Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is an exemplary model for the demonstration of this concept. Early studies with cattle were instrumental in the development of the use of Koch's tuberculin as an in vivo measure of cell-mediated immunity for diagnostic purposes. Calmette and Guerin demonstrated the efficacy of an attenuated M. bovis strain (BCG) in cattle prior to use of this vaccine in humans. The interferon-γ release assay, now widely used for TB diagnosis in humans, was developed circa 1990 for use in the Australian bovine TB eradication program. More recently, M. bovis infection and vaccine efficacy studies with cattle have demonstrated a correlation of vaccine-elicited T cell central memory (TCM) responses to vaccine efficacy, correlation of specific antibody to mycobacterial burden and lesion severity, and detection of antigen-specific IL-17 responses to vaccination and infection. Additionally, positive prognostic indicators of bovine TB vaccine efficacy (i.e., responses measured after infection) include: reduced antigen-specific IFN-γ, iNOS, IL-4, and MIP1-α responses; reduced antigen-specific expansion of CD4(+) T cells; and a diminished activation profile on T cells within antigen stimulated cultures. Delayed type hypersensitivity and IFN-γ responses correlate with infection but do not necessarily correlate with lesion severity whereas antibody responses generally correlate with lesion severity. Recently, serologic tests have emerged for the detection of tuberculous animals, particularly elephants, captive cervids, and camelids. B cell aggregates are consistently detected within tuberculous lesions of humans, cattle, mice and various other species, suggesting a role for B cells in the immunopathogenesis of TB. Comparative immunology studies including partnerships of researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in both man and animals.

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research., This study is a contribution to EU FP7 grant 613779 WildTBVac and to MINECO Plan Nacional grant AGL2011-30041 and FEDER.

[Background]: Field vaccination trials with Mycobacterium bovis BCG, an attenuated mutant of M. bovis, are ongoing in Spain, where the Eurasian wild boar (Sus scrofa) is regarded as the main driver of animal tuberculosis (TB). The oral baiting strategy consists in deploying vaccine baits twice each summer, in order to gain access to a high proportion of wild boar piglets. The aim of this study was to assess the response of wild boar to re-vaccination with BCG and to subsequent challenge with an M. bovis field strain. [Results]: BCG re-vaccinated wild boar showed reductions of 75.8% in lesion score and 66.9% in culture score, as compared to unvaccinated controls. Only one of nine vaccinated wild boar had a culture-confirmed lung infection, as compared to seven of eight controls. Serum antibody levels were highly variable and did not differ significantly between BCG re-vaccinated wild boar and controls. Gamma IFN levels differed significantly between BCG re-vaccinated wild boar and controls. The mRNA levels for IL-1b, C3 and MUT were significantly higher in vaccinated wild boar when compared to controls after vaccination and decreased after mycobacterial challenge. [Conclusions]: Oral re-vaccination of wild boar with BCG yields a strong protective response against challenge with a field strain. Moreover, re-vaccination of wild boar with BCG is not counterproductive. These findings are relevant given that re-vaccination is likely to happen under real (field) conditions., Carlos Martín, Universidad de Zaragoza, kindly provided the vaccine BCG strain. Many colleagues at SaBio IREC, NEIKER and VISAVET granted valuable technical help. This is a contribution to EU FP7 grant 613779 WildTBVac and to MINECO Plan Nacional grant AGL2011-30041 and FEDER.

MPT53 is a secreted protein of Mycobacterium tuberculosis . Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.