Control of bovine tuberculosis in a farmed red deer herd in England.

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.

Is targeted removal a suitable means for tuberculosis control in wild boar?

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 60km2 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.

Testing Eurasian wild boar piglets for serum antibodies against Mycobacterium bovis.

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.

Virulence of two strains of mycobacterium bovis in cattle following aerosol infection.

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.

Performance of immunochromatographic and ELISA tests for detecting fallow deer infected with Mycobacterium bovis.

Fallow deer (Dama dama) are widely distributed as natural or naturalised populations, as well as in game parks and deer farms. We used 157 fallow deer sampled in populations considered to be Mycobacterium tuberculosis complex (MTC) free and 73 Mycobacterium bovis-infected fallow deer confirmed postmortem by culture to evaluate the diagnostic performance of two tests for the detection of anti-mycobacterial antibodies: the dual path platform (DPP) VetTB assay and the bovine purified protein derivative (bPPD) ELISA. We also compared their sensitivity with that of the skin test, analyzed the effect of haemolysis degree on the antibody detection and described the relationship between the test readings and presence/absence of gross tuberculosis (TB) compatible lesions. Sensitivity of bPPD ELISA was 51% at a specificity of 96%. Depending on the cut-off value selected, the sensitivity of DPP VetTB ranged from 62 to 71%, while its specificity was 88-95%. In the subgroup of M. bovis-infected deer for which the skin test data were available (33 of 73); this method detected 76% of culture-positive animals, although the specificity of the intradermal test was not determined in this study. When the DPP VetTB and skin test data were combined, the resulting sensitivity obtained in this sub-group of M. bovis-infected deer increased to 97%. Gross pathology identified TB compatible lesions (TBL) in 89% culture-confirmed fallow deer. The infected animals with visible lesions had significantly higher readings in the DPP VetTB, but not in the bPPD ELISA. Only high levels of haemolysis decreased antibody test sensitivity and this effect was more evident for the bPPD ELISA. The results allowed inferring a number of management recommendations for rapid detection of MTC infection in live fallow deer and in surveys on hunter-harvested cervids.

Bovine tuberculosis: a review of current and emerging diagnostic techniques in view of their relevance for disease control and eradication.

Existing strategies for long-term bovine tuberculosis (bTB) control/eradication campaigns are being reconsidered in many countries because of the development of new testing technologies, increased global trade, continued struggle with wildlife reservoirs of bTB, redistribution of international trading partners/agreements, and emerging financial and animal welfare constraints on herd depopulation. Changes under consideration or newly implemented include additional control measures to limit risks with imported animals, enhanced programs to mitigate wildlife reservoir risks, re-evaluation of options to manage bTB-affected herds/regions, modernization of regulatory framework(s) to re-focus control efforts, and consideration of emerging testing technologies (i.e. improved or new tests) for use in bTB control/eradication programs. Traditional slaughter surveillance and test/removal strategies will likely be augmented by incorporation of new technologies and more targeted control efforts. The present review provides an overview of current and emerging bTB testing strategies/tools and a vision for incorporation of emerging technologies into the current control/eradication programs.

Immune responses in cattle inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii.

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.

Evaluation of the CervidTB STAT-PAK for the detection of Mycobacterium bovis infection in wild deer in Great Britain.

Deer are acknowledged as hosts of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), and determining the prevalence of infection in deer species is one of the key steps in understanding the epidemiological role played by cervids in the transmission and maintenance of bTB in the United Kingdom. This study evaluated a rapid lateral-flow test for the detection of bTB in samples from wild deer species in the United Kingdom. Fallow deer (Dama dama), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) from areas in Wales, the Cotswolds, and southwestern England were necropsied for a bTB survey. Serum samples from individual deer were tested with the CervidTB STAT-PAK, and the results were evaluated against the culture of M. bovis from tissues (n = 432). Sensitivity and specificity were 85.7% (95% confidence interval [CI], 42.1 to 99.6%) and 94.8% (95% CI, 92.3 to 96.7%), respectively, with an odds ratio of 109.9 (95% CI, 12.7 to 953.6%) for a positive STAT-PAK result among culture-positive deer. The low prevalence of infection (3.8%, n = 860) affected the confidence of the sensitivity estimate of the test, but all culture-positive fallow deer (n = 6) were detected by the test. In addition, antibodies to M. bovis could be detected in poor-quality serum samples. The results suggest that the CervidTB STAT-PAK could be deployed as a field test for further evaluation.

Tuberculosis caused by Mycobacterium microti in South American camelids.

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.

Humoral immune responses of white-tailed deer (Odocoileus virginianus) to Mycobacterium bovis BCG vaccination and experimental challenge with M. bovis.

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n=5), orally in liquid form (n=5), and subcutaneously (n=6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n=6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife.

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.

Mycobacterium pinnipedii: transmission from South American sea lion (Otaria byronia) to Bactrian camel (Camelus bactrianus bactrianus) and Malayan tapirs (Tapirus indicus).

Tuberculosis infections caused by Mycobacterium (M.) pinnipedii in a South American sea lion, Bactrian camel, and Malayan tapirs kept in two zoological gardens spanning a time period of 5 years are reported. The zoos were linked by the transfer of one tapir. Conventional bacteriological and molecular methods were applied to detect the pathogen. Spoligotyping and MIRU/VNTR-typing performed to assess the genetic similarity revealed identical molecular characteristics of the isolates from all animals involved. Anti-tuberculosis antibodies were detected using ELISA and a recently developed serological rapid test. The study shows that: (i) using molecular methods, the assessment of the genetic relationship of infectious agents helps to confirm the routes of infection, and that (ii) immunological tests may help to detect tuberculosis infections ante mortem more reliably and early. This would prevent the transfer of tuberculosis by asymptomatic animals.

Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti.

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.

Tuberculosis outbreak in a dromedary racing herd and rapid serological detection of infected camels.

A recent outbreak of tuberculosis (TB) in a dromedary racing herd of 58 animals involved 3 infected animals. Disease was confirmed at necropsy by finding gross lesions from which Mycobacterium bovis (antelope type) was isolated. Sera collected from the camels in this herd were used to evaluate two new serological methods, Multiantigen Print Immunoassay (MAPIA) and rapid test (RT) developed using the lateral-flow technology, in comparison with the intradermal tuberculin tests. Antibodies were found in all three infected dromedaries by both RT and MAPIA, but not in the remaining 55 animals in the herd. With the limited number of animals tested in this study, the serological assays showed the potential for convenient, rapid, and accurate diagnosis of TB in live camels.

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii.

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

Early antibody responses to experimental Mycobacterium bovis infection of cattle.

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.

Effects of different tuberculin skin-testing regimens on gamma interferon and antibody responses in cattle experimentally infected with Mycobacterium bovis.

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-gamma) has been developed. The IFN-gamma 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-gamma 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-gamma 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-gamma 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-gamma or antibody production were not seen in noninoculated cattle. Measurement of both IFN-gamma 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.

Antibody responses in reindeer (Rangifer tarandus) infected with Mycobacterium bovis.

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.

Antigen recognition by serum antibodies in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis.

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 approximately 24 to 26 kDa, approximately 33 kDa, approximately 42 kDa, and approximately 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.

Immunological characterization of antigens encoded by the RD1 region of the Mycobacterium tuberculosis genome.

Development of immunoassays specific for the diagnosis of tuberculosis requires antigens unique to Mycobacterium tuberculosis. In a search for such antigens we tested six proteins encoded by RD1, a region present in M. tuberculosis and virulent M. bovis genomes but missing from the DNA of all substrains of M. bovis Bacillus Calmette-Guerin (BCG). The six proteins (Rv3871, Rv3872, Rv3873, MTSA-10, ESAT-6 and Rv3878) were purified to near-homogeneity from recombinant Escherichia coli. When tested for the ability to elicit antibody responses and delayed type hypersensitivity in tuberculous guinea pigs, only two of six antigens, ESAT-6 and MTSA-10, elicited strong skin reactions, while vigorous antibody responses were observed to all six proteins. When antibody responses to RD1 antigens were evaluated in sera from patients having pulmonary tuberculosis and from control subjects (patients having mycobacterioses other than tuberculosis, and healthy persons), a sizeable proportion (25%) of tuberculosis patients but none of the control subjects, had antibodies against MTSA-10 and/or ESAT-6. We conclude that MTSA-10 and ESAT-6 are promising candidates for immunodiagnostic assays specific for tuberculosis.