Distinct response kinetics of gamma interferon and interleukin-4 in bovine tuberculosis

This study shows that gamma interferon (IFN-gamma) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected with Mycobacterium bovis. The different kinetics of the IFN-gamma and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection with M. bovis are described. An early increase in IFN-gamma was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose of M. bovis which did not result in the development of lesions, positive DTH skin test, or substantial IFN-gamma responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infected M. bovis field reactors showed increased IFN-gamma and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of the M. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematode Onchocerca ochengi, further supporting the presence of this type 2 cytokine in bovine tuberculosis.     

Development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle

As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at ～90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improving as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing.     

Diagnostic implications of antigen-induced gamma interferon,nitric oxide,and tumor necrosis factor alpha production by peripheral blood mononuclear cells from Mycobacterium bovis-infected cattle

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-gamma), nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) responses by blood mononuclear cells from Mycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains of M. bovis (95-1315 and HC-2045T) were used to induce a range of IFN-gamma, NO, and TNF-alpha responses. Infection-specific increases in NO, but not in IFN-gamma or TNF-alpha, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose of M. bovis organisms also influenced responses. At 34 days postinfection, IFN-gamma, NO, and TNF-alpha responses in antigen-stimulated cells from cattle receiving 10(5) CFU of M. bovis organisms were greater than responses of cells from cattle infected with 10(3) CFU of M. bovis organisms. The NO response, but not the IFN-gamma and TNF-alpha responses, was influenced by infective strains of M. bovis. The TNF-alpha, NO, and IFN-gamma responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-alpha, like IFN-gamma, may prove useful as indices for the diagnosis of bovine tuberculosis.     

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.     

Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen.

Development of a serodiagnostic test for bovine tuberculosis necessitates an understanding of the humoral immune responses of animals following infection with Mycobacterium bovis. The antibody responses in groups of calves challenged intranasally with different doses of M. bovis (approximately 10(2), 10(4), and 10(6) CFU) or placed in contact with the infected animals were analyzed by immunoelectrophoretic blotting in which a whole-cell sonicate of M. bovis was utilized as an antigen. Antibody responses were evident early in infections in which calves were exposed to high doses of M. bovis, while in groups exposed to lower doses, the time until antibody was detected increased as the challenge dose decreased. In cattle exposed to M. bovis, immunoblot analysis showed antibody responses to three main antigens of 26, 22, and 16 kDa. It was further demonstrated that antibody responses to the 26-kDa antigen appeared earliest in the course of infection. Preliminary investigations in this study have identified a 26-kDa antigen for potential use in improved serodiagnosis by enzyme-linked immunosorbent assays.     

Flow cytometric detection of gamma interferon can effectively discriminate Mycobacterium bovis BCG-vaccinated cattle from M. bovis-infected cattle

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-gamma) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-gamma-producing lymphocytes by flow cytometric analysis of intracellular IFN-gamma expression would provide a more accurate discrimination of M. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-gamma-expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG vaccinates, with purified protein derived from M. bovis (PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+ T lymphocytes expressing IFN-gamma, compared with 21/22 M. bovis-infected animals. This assay could allow an accurate diagnosis of M. bovis and allow the discrimination of BCG-vaccinated cattle from infected cattle.     

Cattle husbandry in Ethiopia is a predominant factor affecting the pathology of bovine tuberculosis and gamma interferon responses to mycobacterial antigens

Bovine tuberculosis is a major economic problem and a potential public health risk. Improved diagnostics like the gamma interferon (IFN-gamma) test with ESAT6 and/or CFP10 could contribute to the control program. We assessed IFN-gamma responses in zebu (Ethiopian Arsi breed) and Holstein cattle kept indoors or in a pasture to tuberculin purified protein derivative (PPD) and an ESAT6-CFP10 protein cocktail. Furthermore, the intensity and distribution of pathology of bovine tuberculosis were compared between the two breeds. Our data demonstrated significantly (all P < 0.02) higher IFN-gamma responses to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail in Holstein than in zebu cattle, while lesion severities in infected animals and tuberculin skin test responses did not differ significantly (P > 0.05) between the two breeds. Holstein cattle that were kept indoors produced significantly (all P < 0.01) higher IFN-gamma levels in response to avian PPD, bovine PPD, and the ESAT6-CFP10 protein cocktail than did Holstein cattle kept in a pasture. Moreover, lesion severity was significantly higher in Holstein cattle kept indoors (P = 0.001) than in those kept in the pasture. Lesions were localized predominantly in the digestive tract in cattle kept in a pasture, while they were localized in the respiratory tract in cattle kept indoors. In conclusion, in Holstein cattle, husbandry was a dominant factor influencing the severity of tuberculosis lesions and IFN-gamma responses to mycobacterial antigens compared to breed. A difference in the cellular immune response between zebu and Holstein cattle was observed, while tuberculosis lesion severities were identical in the two breeds, when both were kept in a pasture.     

Genomic approach to identification of Mycobacterium bovis diagnostic antigens in cattle

Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10(6) CFU) and a low (10(4) CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.     

Association of tuberculin-boosted antibody responses with pathology and cell-mediated immunity in cattle vaccinated with Mycobacterium bovis BCG and infected with M. bovis

Vaccine development and our understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by definition of the immunological correlates of protection and/or pathology. In this study we analyzed humoral immune responses in Mycobacterium bovis BCG-vaccinated and control cattle (in particular, the relationship between the intradermal comparative tuberculin skin test and serum immunoglobulin G [IgG] responses) against a range of mycobacterial antigens (MPB59, MPB64, MPB70, MPB83, ESAT-6, CFP-10, Acr1, and PstS-1) by multiantigen print immunoassay and conventional enzyme-linked immunosorbent assay. Following M. bovis infection, the comparative tuberculin skin test strongly boosted IgG, IgG1, and IgG2 antibody responses, particularly against MPB83 and MPB70, in unvaccinated cattle but failed to boost these responses, or did so only weakly, in BCG-vaccinated calves. In addition, the skin test-induced increases in MPB83-specific IgG responses correlated positively with bacterial loads and ESAT-6-induced in vitro gamma interferon responses. In conclusion, both the negative correlation of skin test-enhanced MPB83-specific antibody responses with BCG-induced protection and their positive correlation with bacterial loads can serve as useful markers for vaccine efficacy after challenge.     

Enhancement of the sensitivity of the whole-blood gamma interferon assay for diagnosis of Mycobacterium bovis infections in cattle

In this study, we determined if the sensitivity of the currently available in vitro test to detect bovine tuberculosis could be enhanced by adding the following immunomodulators: interleukin-2 (IL-2); granulocyte-macrophage colony-stimulating factor (GM-CSF); antibodies neutralizing IL-10 and transforming growth factor beta (TGF-beta); mono-methyl-l-arginine, which blocks nitric oxide production; and l-methyl-tryptophan, which interferes with the indoleamine dioxygenase pathway. Blood was obtained from uninfected control cattle, experimentally infected cattle, cattle responding positively to the skin test in tuberculosis-free areas (false positives), and cattle naturally infected with Mycobacterium bovis from New Zealand and Great Britain. Gamma interferon (IFN-gamma) responses to bovine purified protein derivative (PPD-b), avian purified protein derivative, and a fusion protein of ESAT-6 and CFP-10 were measured. Mono-methyl-l-arginine, l-methyl-tryptophan, or an antibody neutralizing TGF-beta had minimal impact on IFN-gamma production. IL-2 and GM-CSF promoted IFN-gamma release whether antigen was present or not. In contrast, adding an antibody against IL-10 enhanced only antigen-specific responses. In particular, addition of anti-IL-10 to ESAT-6/CFP-10-stimulated blood cultures enhanced the test sensitivity. Furthermore, whole blood cells from field reactors produced substantial amounts of IL-10 upon stimulation with PPD-b or ESAT-6/CFP-10. Testing "false-positive" cattle from tuberculosis-free areas of New Zealand revealed that addition of anti-IL-10 did not compromise the test specificity. Therefore, the use of ESAT-6/CFP-10 with anti-IL-10 could be useful to detect cattle potentially infected with tuberculosis, which are not detected using current procedures.     

Use of an electronic nose to diagnose Mycobacterium bovis infection in badgers and cattle

It is estimated that more than 50 million cattle are infected with Mycobacterium bovis worldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs of M. bovis that are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers with M. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection with M. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosing M. bovis infection in cattle and badgers.     

Analysis of possible factors affecting the specificity of the gamma interferon test in tuberculosis-free cattle herds

Bovine tuberculosis (TB) is still a zoonotic problem in the world. Despite the fact that eradication programs for bovine TB are being implemented in many countries, it remains a public health problem. These programs are mainly based on a single intradermal tuberculin test using bovine tuberculin purified protein derivative (PPD), isolation, and slaughtering of infected animals. The aim of this study was to assess the specificity of the gamma interferon (IFN-gamma) test in TB-free cattle herds, by using not only Australian tuberculins but also tuberculins produced at our institute, and to correlate the response with the type of production (beef cattle, dairy cattle, and a dual-purpose breed), the housing system, and the age of the animals. We studied 800 animals selected from 20 TB- and paratuberculosis-free herds. The animals were tested in parallel, after stimulation with Australian tuberculins and tuberculins produced at our institute, by using the skin test and two IFN-gamma assays. The results of this trial showed that the specificity of the IFN-gamma test is higher than that of the skin test (96.8%) and ranges from 97.3% (using only Australian tuberculins) to 98.6% (using tuberculins produced at our institute). We found that different categories of cattle could influence the specificity of the skin test but that these differences tended to be reduced in the IFN-gamma assay, especially when Italian PPDs were used.     

Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle

In the search for improved tools with which to control bovine tuberculosis, the development of enhanced immunodiagnostic reagents is a high priority. Such reagents are required to improve the performance of tuberculin-based reagents and allow the discrimination of vaccinated cattle from those infected with Mycobacterium bovis. In this study, we identified the immunodominant, frequently recognized peptides from Rv3873, Rv3879c, Rv0288, and Rv3019c, which, together with peptides comprising the current lead diagnostic antigens, ESAT-6 and CFP-10, were formulated into a peptide cocktail. In a test of naturally infected cattle, this cocktail was significantly better than tuberculin was for identifying skin test-negative animals with confirmed bovine tuberculosis. In addition, the specificity of this cocktail was not compromised by Mycobacterium bovis BCG vaccination. In summary, our results prioritize this peptide-based, fully synthetic reagent for assessment in larger trials.     

Correlation of ESAT-6-specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis

Vaccine development and the understanding of the pathology of bovine tuberculosis in cattle would be greatly facilitated by the definition of immunological correlates of protection and/or pathology. To address these questions, cattle were vaccinated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and were then challenged with virulent M. bovis. Applying a semiquantitative pathology-scoring system, we were able to demonstrate that BCG vaccination imparted significant protection by reducing the disease severity on average by 75%. Analysis of cellular immune responses following M. bovis challenge demonstrated that proliferative T-cell and gamma interferon (IFN-gamma) responses towards the M. bovis-specific antigen ESAT-6, whose gene is absent from BCG, were generally low in vaccinated animals but were high in all nonvaccinated calves. Importantly, the amount of ESAT-6-specific IFN-gamma measured by enzyme-linked immunosorbent assay after M. bovis challenge, but not the frequency of responding cells, correlated positively with the degree of pathology found 18 weeks after infection. Diagnostic reagents based on antigens not present in BCG, like ESAT-6 and CFP-10, were still able to distinguish BCG-vaccinated, diseased animals from BCG-vaccinated animals without signs of disease. In summary, our results suggest that the determination of ESAT-6-specific IFN-gamma, while not a direct correlate of protection, constitutes nevertheless a useful prognostic immunological marker predicting both vaccine efficacy and disease severity.     

Influence of pathological progression on the balance between cellular and humoral immune responses in bovine tuberculosis

Studies of tuberculosis have suggested a shift in dominance from a T helper type 1 (Th1) towards a Th2 immune response that is associated with suppressed cell-mediated immune (CMI) responses and increased humoral responses as the disease progresses. In this study a natural host disease model was used to investigate the balance of the evolving immune response towards Mycobacterium bovis infection in cattle with respect to pathogenesis. Cytokine analysis of CD4 T-cell clones derived from M. bovis-infected animals gave some indication that there was a possible relationship between enhanced pathogenesis and an increased ratio of Th0 [interleukin-4-positive/interferon-gamma-positive (IL-4(+)/IFN-gamma(+))] clones to Th1 (IFN-gamma(+)) clones. All animals developed strong antimycobacterial CMI responses, but depressed cellular responses were evident as the disease progressed, with the IFN-gamma test failing to give consistently positive results in the latter stages. Furthermore, a stronger Th0 immune bias, depressed in vitro CMI responses, elevated levels of IL-10 expression and enhanced humoral responses were also associated with increased pathology. In minimal disease, however, a strong Th1 immune bias was maintained and an anti-M. bovis humoral response failed to develop. It was also seen that the level of the anti-M. bovis immunoglobulin G1 (IgG1) isotype antibody responses correlated with the pathology scores, whereas CMI responses did not have as strong a relationship with the development of pathology. Therefore, the development and maintenance of a Th1 IFN-gamma response is associated with a greater control of M. bovis infection. Animals progressing from a Th1-biased to a Th0-biased immune response developed more extensive pathology and performed less well in CMI-based diagnostic tests but developed strong IgG1 humoral responses.     

Use of Antigen-Specific Interleukin-2 To Differentiate between Cattle Vaccinated with Mycobacterium bovis BCG and Cattle Infected with M. bovis

We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.     

Vaccination of guinea pigs with DNA encoding the mycobacterial antigen MPB83 influences pulmonary pathology but not hematogenous spread following aerogenic infection with Mycobacterium bovis

Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistent Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from which M. bovis could be excreted. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolized M. bovis. Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembled M. bovis BCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection with M. bovis.     

Diagnostic implications of antigen-induced gamma interferon production by blood leukocytes from Mycobacterium bovis-infected reindeer (Rangifer tarandus)

The only approved method of tuberculosis (TB) surveillance of reindeer within the United States is tuberculin skin testing; however, skin testing has an apparent lack of specificity, since numerous reindeer are classified as reactors, yet Mycobacterium bovis is not isolated from tissues upon necropsy. The objective of this study was to evaluate the ability of an in vitro assay (the Cervigam assay) to detect gamma interferon (IFN-gamma) produced by blood leukocytes in response to mycobacterial antigens from M. bovis-infected reindeer. Thirteen male reindeer approximately 9 months of age were inoculated with 10(5) CFU M. bovis in their tonsillar crypts. Stimulation of whole-blood cultures with a mitogen resulted in significant production of IFN-gamma compared to that by nonstimulated samples. Responses by infected reindeer to M. bovis purified protein derivative (PPD) were as much as 3.5-fold higher than those by noninfected reindeer (n = 4). Despite differences in responses to PPD by the two groups, reindeer within the noninfected group had responses of >0.1 change in optical density (DeltaOD) (a level generally considered positive) to PPD. Mean responses by infected reindeer to a rESAT-6-CFP-10 fusion protein (Mycobacterium tuberculosis complex specific) were as much as 20-fold higher than respective responses by noninfected reindeer at all time points. Additionally, responses by 3/4 noninfected reindeer were <0.1 DeltaOD (considered negative) at each time point. To further evaluate the specificity of the assay, samples were collected from reindeer in a TB-free herd. All reindeer had responses to mitogen; however, only 1 of 38 had a response to PPD, and none of the reindeer responded to rESAT-6-CFP-10. Together, these findings indicate that IFN-gamma-based tests may prove useful for TB surveillance of reindeer.