Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle

The development of novel vaccine strategies supplementing Mycobacterium bovis BCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identified Mycobacterium tuberculosis antigens in M. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized in M. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.     

Evaluation of gamma interferon release assays using Mycobacterium tuberculosis antigens for diagnosis of latent and active tuberculosis in Mycobacterium bovis BCG-vaccinated populations

T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.     

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.     

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.     

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.     

Simultaneous measurement of antigen-stimulated interleukin-1 beta and gamma interferon production enhances test sensitivity for the detection of Mycobacterium bovis infection in cattle

In order to identify cytokines that may be useful as candidates for inclusion in diagnostic tests for Mycobacterium bovis infection in cattle, we compared the levels of gamma interferon (IFN-γ), interleukin 1β (IL-1β), IL-4, IL-10, IL-12, macrophage inflammatory protein 1β (MIP-1β), and tumor necrosis factor alpha (TNF-α) in whole-blood cultures from tuberculosis (TB) reactor animals or TB-free controls following stimulation with M. bovis-specific antigens (purified protein derivative from M. bovis [PPD-B] or ESAT-6/CFP-10). In addition to IFN-γ responses, the production of IL-1β and TNF-α was also statistically significantly elevated in TB reactor cattle over that in uninfected controls following stimulation with PPD-B or ESAT-6/CFP-10 peptides. Thus, we evaluated whether the use of these two additional readouts could disclose further animals not detected by measuring IFN-γ alone. To this end, receiver operating characteristic (ROC) analyses were performed to define diagnostic cutoffs for positivity for TNF-α and IL-1β. These results revealed that for ESAT-6/CFP-10-induced responses, the use of all three readouts (IFN-γ, TNF-α, and IL-1β) in parallel increased the sensitivity of detection of M. bovis-infected animals by 11% but also resulted in a specificity decrease of 14%. However, applying only IFN-γ and IL-1β in parallel resulted in a 5% increase in sensitivity without the corresponding loss of specificity. The results for PPD-B-induced responses were similar, although the loss of specificity was more pronounced, even when only IFN-γ and IL-1β were used as readout systems. In conclusion, we have demonstrated that the use of an additional readout system, such as IL-1β, can potentially complement IFN-γ by increasing overall test sensitivity for the 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.     

Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes.

Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.     

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.     

Mycobacterium bovis BCG-induced human T-cell clones from BCG-vaccinated healthy subjects: antigen specificity and lymphokine production.

A total of 121 human T-cell clones were raised from nine Mycobacterium bovis BCG-vaccinated healthy individuals. Three clones were autoreactive, 74 responded to BCG in the presence of antigen-presenting cells, and the others required in addition exogenous interleukin 2. Only one clone was CD8+ CD4-, and the rest were CD4+ CD8-. Testing with a panel of mycobacteria suggested that the clones were recognizing epitopes of varied specificity. Out of 44 clones tested, 15 were specific to BCG and Mycobacterium tuberculosis, 22 showed limited cross-reactivity, and 8 were broadly cross-reactive. None of the 22 BCG responder clones could differentiate between Danish, French, Prague, and Moreau strains of BCG. BCG and M. tuberculosis H37Rv also paralleled very closely; however, 6 of 18 BCG- and M. tuberculosis H37Rv-responding clones did not proliferate to Mycobacterium africanum. BCG- and M. tuberculosis H37Rv-specific as well as cross-reactive T-cell clones could be induced to produce interleukin 2, gamma interferon, and granulocyte-macrophage colony-stimulating factor activity.     

Chemiluminescent responses of alveolar macrophages from normal and Mycobacterium bovis BCG-vaccinated rabbits as a function of age.

Luminol-dependent chemiluminescence (CL) responses of alveolar macrophages (AM) from normal and Mycobacterium bovis BCG-vaccinated infant and adult rabbits were compared. AM from 1-, 7-, and 14-day-old normal rabbits exhibited much lower peak CL responses than did AM from 28- and 42-day-old normal animals as well as rabbits 2 to 3 or 5 to 6 months and 1 to 2 years of age. The most striking differences among AM from infant and adult rabbits were noted when AM were obtained from 28-day-old and 5- to 6-month old rabbits 21 days after the rabbits were immunized with 200 micrograms of BCG intravenously. In this case, AM from 5- to 6-month-old animals gave peak counts per minute of 400,000 to 500,000 whereas AM from 28-day-old rabbits vaccinated with BCG (harvested at 49 days of age) gave peak counts per minute of only 40,715 +/- 2,688. These data reveal that AM from neonatal animals are grossly deficient as responders to phorbol myristate acetate-induced CL. This deficiency, which improved with age, is still apparent in AM from 28-day-old animals. The data also reveal that BCG vaccination of 28-day-old animals yields AM that are poor responders to phorbol myristate acetate compared with AM from BCG-vaccinated animals 2 to 3 and 5 to 6 months of age. AM from animals vaccinated with BCG at 28 days of age contained fewer and smaller electron-dense lysosomelike structures than did AM from adult rabbits similarly vaccinated. These findings provide an explanation for the difficulties infants have in developing effective cell-mediated immune responses against intracellular parasites.     

Induction of alpha and beta interferons during the hyporeactive state of gamma interferon by Mycobacterium bovis BCG cell wall fraction in Mycobacterium bovis BCG-sensitized mice.

Characterization of interferon (IFN) induced by a second challenge with specific antigen was investigated during the development of hyporeactivity established after challenge of Mycobacterium bovis BCG-sensitized mice with BCG cell wall fraction (BCG-CW). When BCG-sensitized mice were challenged with BCG-CW, IFN-gamma was detected in the circulation 4 h later and rapidly disappeared. After IFN-gamma disappeared from their blood, mice became completely hyporeactive to the second challenge with BCG-CW for 1 day, and thereafter they recovered from this hyporeactive state. However, we always observed that IFN induced at first by the second challenge with BCG-CW during the hyporeactive state was type I IFN-alpha/beta, but thereafter was entirely IFN-gamma. Induction of IFN-alpha/beta by the specific antigen during restoration from the hyporeactive state in BCG-sensitized mice is discussed.     

Role of eosinophils in the pathogenesis of Mycobacterium bovis BCG infection in gamma interferon receptor-deficient mice

A profound eosinophil infiltration of granulomas is observed in the lungs of Mycobacterium bovis bacillus Calmette Guérin-infected gamma interferon receptor-deficient mice. Blockade of eosinophil proliferation and recruitment into the lung by treatment with anti-interleukin-5 monoclonal antibody marginally reduced mycobacterial growth within the lung but did not affect dissemination of the infection to other tissues.     

Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle.

Tuberculosis continues to be a worldwide health problem for both humans and animals. The development of improved vaccines and diagnostic tests requires detailed understanding of the immune responses generated and the antigens recognized during the disease. This study examined the T-cell response which develops in cattle experimentally infected with Mycobacterium bovis. The first significant T-cell response was found 3 weeks after the onset of infection and was characterized by a pronounced gamma interferon (IFN-gamma) response from peripheral blood mononuclear cells directed to antigens in culture filtrates. Short-term culture filtrate (ST-CF) was separated into molecular mass fractions and screened for recognition by T cells from experimentally infected and field cases of bovine tuberculosis. Cattle in the early stages of experimental infection were characterized by strong IFN-gamma responses directed predominantly toward the lowest-mass (<10-kDa) fraction of ST-CF, but cattle in later stages of experimental infection (16 weeks postinfection) exhibited a broader recognition of antigens of various molecular masses. Field cases of bovine tuberculosis, in comparison, preferentially recognized low-mass antigens, characteristic of animals in the early stages of infection. The major T-cell target for this dominant IFN-gamma response was found to be the secreted antigen ESAT-6. This antigen was recognized strongly by the majority of field cases of bovine tuberculosis tested. As ESAT-6 is unique to pathogenic mycobacterial species, our study suggests that ESAT-6 is an antigen with major potential for vaccination against and specific diagnosis of bovine tuberculosis.     

Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis.

Two insertion sequences, IS6110 and IS1081, specific to the tuberculosis complex mycobacteria and a highly reiterated DNA element (pTBN12) cloned from Mycobacterium tuberculosis were systematically used to identify restriction fragment length polymorphism (RFLP) types among bovine isolates of Mycobacterium bovis in Northern Ireland. In a sample of 109 isolates, probes IS6110, IS1081, and pTBN12 identified 10, 2, and 12 distinct patterns, respectively. By combining the patterns generated by the three probes it was possible to identify 28 distinct RFLP types. The standard protocol advocated for RFLP analysis of M. tuberculosis was used and would facilitate computer-based gel documentation and image analysis to establish a database of M. bovis types for large-scale epidemiological studies. These procedures will facilitate interlaboratory comparisons of M. bovis isolates and will help to elucidate the precise epidemiology of bovine tuberculosis in different countries.     

Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.     

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.     

Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG.

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.     

Differential expression of gamma interferon mRNA induced by attenuated and virulent Mycobacterium tuberculosis in guinea pig cells after Mycobacterium bovis BCG vaccination

To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-gamma) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-gamma from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-gamma, respectively. Spleen or lymph node cells from na?ve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a (32)P-labeled probe derived from the cDNA clone. Compared to the IFN-gamma mRNA expression in cells of na?ve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-gamma mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-gamma mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4(+) T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-gamma mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-gamma mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-gamma mRNA accumulation is currently under investigation.     

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.