Enzyme-linked immunosorbent assay of bovine tuberculosis by crude mycobacterial protein 70

MPB70 (mycobacterial protein of BCG 70) as T-cell stimulator has been tried with an intradermal skin test (IST) and enzyme-linked immunosorbent assay (ELISA) of bovine tuberculosis (BTB). In this study, crude mycobacterial protein 70 (CMP70) was prepared by anion exchange chromatography from the culture supernatant of Mycobacterium bovis AN5 and CMP70 ELISA was compared with purified protein derivative (PPD) ELISA. PPD and CMP70 ELISA have shown a positive reaction to the sera of M. bovis infected cattle and IST positive reactors. One of three IST negative cattle showed the nonspecific reaction in PPD ELISA, whereas all of the IST negative cattle (n=3) were did not show the nonspecific reaction in CMP70 ELISA. When each ELISA was applied to sixty-two IST positive cattle, ELISA positive reactors were eighty four per cent to CMP70 antigen and fifty-two per cent to PPD. CMP70 has been shown to be more specific and sensitive than PPD in ELISA.     

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.     

Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.

The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.     

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

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

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.     

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.     

Biological Activity in Sensitized Guinea Pigs of MPB 70, a Protein Specific for Some Strains of Mycobacterium bovis BCG

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.     

Cloning of a species-specific antigen of Mycobacterium bovis.

A DNA library from a virulent strain of Mycobacterium bovis was constructed in the expression vector lambda gt11, and the library was probed with antisera to M. bovis. Clones expressing M. bovis antigens were isolated and characterized by using M. bovis-specific monoclonal antibodies that recognize a 22,000-molecular-weight protein (MPB70). MPB70 is a major protein antigen of the vaccine strain of M. bovis BCG and of virulent M. bovis, the causative agent of bovine tuberculosis. Of 32 clones selected by using polyclonal affinity-purified anti-M. bovis sera, 5 were recognized by the anti-MPB-70 monoclonal antibodies, and one monoclonal antibody, SB10, recognized all 5 clones. Characterization of these clones showed that one clone containing a 253-base-pair insert expressed a polypeptide bound by all of the MPB70-specific monoclonal antibodies. Western blots (immunoblots) showed that this cloned protein was recognized by sera from M. bovis-infected cattle, although not all cattle with bovine tuberculosis produced antibodies reactive to this clone. DNA sequencing of the clone showed that it coded for 84 amino acids from positions 17 to 114 of the 161-amino-acid protein, with a 16-peptide deletion between positions 79 and 94. Apart from this deletion, there were seven other variations between the cloned sequence and that deduced from M. bovis BCG MPB70.     

Purification of MPB70 and production of specific monoclonal antibodies.

MPB70 is a protein secreted into the culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172), which is able to induce a delayed-type hypersensitivity (DTH) skin reaction in guinea pigs immunized with BCG-Tokyo. By high-pressure chromatofocusing and size-exclusion high performance liquid chromatography, a further purified MPB70 protein was obtained, which was visualized as a single band with a molecular mass of 22 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A series of hybridoma cell lines that produced monoclonal antibodies (MAbs) against the purified MPB70 protein was prepared, and three MAbs, Bov-1, Bov-2, and Bov-3, with strong antigen-binding capacities were established. Bov-1 was the most potent MAb among them and binds to only a 22 kDa protein band in culture filtrates of M. bovis, but not to bands in those of M. tuberculosis by Western immunoblotting analysis, suggesting that Bov-1 recognize different epitope of MPB70 from MAbs that have been shown previously to recognize several species of molecules in culture filtrates of M. bovis. The purified MPB70 protein elicited a strong DTH skin reaction in guinea pigs sensitized with BCG-Tokyo vaccine. Bov-1 had no inhibitory effect on generation of the DTH skin reaction, showing that MAb bound to an epitope distinct from that inducing the skin reaction. All of the three MAbs were specific to MPB70 and each recognized a different epitope on MPB70. MPB70 was not detected in the culture filtrate of M. tuberculosis H37Rv. Thus, these MAbs may be useful for detecting MPB70 in studies on discriminating infection with M. bovis in domestic animals or in distinguishing vaccination with BCG-Tokyo from other mycobacterial infections in humans.     

Novel monoclonal antibodies against major antigens of Mycobacterium bovis

MPB70 and MPB83 are among the most characteristically exported proteins defining a strongly expressed phenotype of Mycobacterium bovis. These proteins are known to be homologous to osteoblast-specific factor 2. By in vitro culture of mycobacteria they appear to have a limited species distribution and to be relatively specific for M. bovis. Virtually identical genes are however, present in Mycobacterium tuberculosis. In order to facilitate further research into the immunobiology of these proteins and their potential application for differential diagnosis of tuberculosis as a result of M. bovis, we describe the reactivities of 20 monoclonal antibodies (MoAbs) to these proteins. Immunizing with bovine PPD generated 10 MoAbs. These antibodies reacted preferentially with the soluble MPB70 antigen using reducing conditions in SDS-PAGE with western blotting. Ten MoAbs were generated by immunizing mice with fractions derived from a whole cell sonic extract of M. bovis. These antibodies reacted preferentially with the surface exposed MPB83 lipoglycoprotein.     

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.     

Characterization of the early antibody response in bovine tuberculosis: MPB83 is an early target with diagnostic potential

A 26-kDa antigen has been shown to be a dominant antibody target in Mycobacterium bovis-infected cattle. In this study, that antigen was used as an immunogen to raise a panel of mouse monoclonal antibodies. The majority of those bound to native protein with a molecular mass of 26 kDa and to recombinant MPB83, strongly suggesting that MPB83 is an important B-cell antigenic target in bovine tuberculosis. In order to provide assessment of the potential of measuring antibody responses to the native protein, one monoclonal antibody, 1F11, was incorporated into an enzyme-linked immunosorbant assay format to trap antigen from a crude bacterial extract. Despite some disadvantages of this format, serum samples from cattle which had been infected experimentally with M. bovis, and from tuberculin skin-test-negative and -positive field cattle were tested for the presence of antibodies. Data from the skin-test-negative cattle allowed an arbitrary cut-off value to be established and, under these conditions, test sensitivity and specificity were estimated at 37.5 and 89%, respectively. These results indicate potential for MPB83 in the development of assays for serological diagnosis of bovine tuberculosis.     

Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG.

The immunogenic proteins MPB64 and MPB80 of Mycobacterium bovis BCG were purified to homogeneity and compared with MPB70. MPB70 and MPB80 showed a similar distribution in substrains of BCG, both being present in high concentrations in culture fluids of BCG substrain Tokyo, BCG Moreau, BCG Russia, and BCG Sweden and in only very small amounts in BCG Glaxo, BCG Tice, BCG Copenhagen, and BCG Pasteur. In various physicochemical properties MPB70 and MPB80 were closely similar, but MPB80 had a distinctly lower pI value. The N-terminal amino acid sequence was identical for the first 30 residues. In reactions with anti-MPB70 antibodies and delayed-type hypersensitivity skin reactions, MPB70 and MPB80 also had very similar properties. These results show that MPB70 and MPB80 are two closely similar forms of the same gene product, and postsynthetic changes probably explain the observed differences. By contrast, MPB64 had a higher molecular weight. The N-terminal amino acid sequence showed no homology with MPB70, and these two proteins showed no immunologic similarity. MPB64 and MPB70 showed only very restricted cross-reactivity with other species of mycobacteria but cross-reacted with Nocardia asteroides. The similar occurrence in eight different substrains of BCG indicated that the two proteins are influenced by similar control mechanisms, but in contrast to MPB70, MPB64 occurred in sufficient concentration in two strains of Mycobacterium tuberculosis to give a distinct spot in two-dimensional polyacrylamide gel electrophoresis of their culture fluids.     

Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy,non-mycobacterium bovis BCG-vaccinated Malawian young adults

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.     

Antigen specificity in experimental bovine tuberculosis

This report describes the kinetics of T-cell responses to a panel of mycobacterial antigens (PPD-M, PPD-A, ESAT-6, Ag85, 38kD, MPB64, MPB70, MPB83, hsp16.1, hsp65, and hsp70) following experimental infection of cattle with Mycobacterium bovis. Increased antigen-specific lymphocyte proliferation, gamma interferon, and interleukin-2 responses were observed in all calves following infection. Positive lymphocyte proliferation and cytokine responses to PPD-M and ESAT-6 were observed throughout the infection period studied. In contrast, responses to all other antigens were more variable and were not constantly present, suggesting that antigen cocktails rather than individual antigens should be used for immunodiagnosis. The detection of cytokine responses in the absence of lymphocyte proliferation, particularly during the early stages of infection, suggests a role for antigen-specific cytokine readout systems in the early identification of M. bovis infection in cattle.     

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.     

Immune responses induced in cattle by virulent and attenuated Mycobacterium bovis strains: correlation of delayed-type hypersensitivity with ability of strains to grow in macrophages

Comparison of immune responses induced in cattle by virulent and attenuated strains of Mycobacterium bovis will assist in identifying responses associated with resistance or susceptibility to disease. Four strains of M. bovis, one which is virulent in guinea pigs (WAg201) and three which are attenuated in guinea pigs (an isoniazid-resistant strain [WAg405], ATCC 35721, and BCG) were compared for their abilities to induce immune responses in cattle and to grow in bovine lung alveolar macrophage cultures. Extensive macroscopic lesions were found only in cattle inoculated with the virulent M. bovis strain. Strong antibody responses to M. bovis culture filtrate, as well as persistently high levels of gamma interferon and interleukin-2 released from purified protein derivative (PPD)-stimulated peripheral blood lymphocyte cultures, were observed in the cattle inoculated with the virulent strain compared to those inoculated with the attenuated strains. All cattle inoculated with the virulent strain or two of the attenuated strains (WAg405 and ATCC 35721) elicited strong delayed-type hypersensitivity responses to PPD in skin tests, while animals inoculated with BCG induced only a weak response. The three strains which produced strong skin test responses proliferated well in bovine alveolar macrophages and induced high levels of proinflammatory cytokine mRNAs compared to BCG. Our study showed that skin test responsiveness to PPD correlated with the ability of the strains to grow in alveolar macrophages rather than to their pathogenicity in cattle.     

Heterogenous Expression of the Related MBP70 and MPB83 Proteins Distinguish Various Substrains of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv

MPB70 and MPB83 are homologous cross-reactive secreted mycobacterial proteins with very limited species distribution. The expression of these two proteins was compared between several substrains of Mycobacterium bovis BCG, virulent M. bovis and Mycobacterium tuberculosis H37Rv. A polyclonal antibody specific for MPB70 in Western blotting, and a monoclonal antibody, MBS43, found to be specific for MPB83 in ELISA and Western blotting, were used for the comparison. The previously established pattern of high- and low-producing substrains of BCG for MPB70 is only partially applicable for MPB83. MPB70 low-producing strains are also MPB83 low-producing, but the expression of MPB83 is much more variable than the expression of MPB70 in the MPB70 high-producing strains. Purified MPB83 (23 kDa) was found to be glycosylated. A band in SDS-PAGE at 1–2 kDa lower than that of purified MPB83 may represent unglycosylated MPB83. Furthermore, it was confirmed that purified MPB70 (22 kDa) is unglycosylated. There is cross-reactive antigen at 26 kDa. The MPB83 related antigen at 26 kDa was found to be the most abundant. These findings indicate greater heterogeneity between different substrains of BCG than previously realized. Virulent M. bovis produce and secrete large amounts of MPB70 and MPB83 while both these proteins occur in a far lower concentration in M. tuberculosis     

Comparative studies with various substrains of Mycobacterium bovis BCG on the production of an antigenic protein, MPB70.

A protein, isolated and purified from the unheated culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172) and designated MPB70, elicited a delayed skin reaction in guinea pigs sensitized with viable cells of BCG but not in those sensitized with heat-killed cells. The skin reaction reached the maximum 4 to 8 weeks after the inoculation of the BCG and then decreased gradually, resulting in conversion to negative after 20 weeks, whereas the skin reaction to purified protein derivative (PPD) continued to be positive. Guinea pigs immunized with viable cells of various substrains of BCG were skin tested with MPB70 and PPD. Guinea pigs immunized with the BCG substrain Tokyo 172 and the substrain Moreau (Brazil) showed strong delayed skin reactions to both MPB70 and PPD. On the other hand, guinea pigs immunized with the Pasteur substrain 1173P2, the Glaxo substrain 1077, the Copenhagen substrain 1331, the Tice substrain, or the Beijing substrain 64-42 showed negative skin reactions to MPB70, whereas they were strongly positive to PPD. In a two-dimensional acrylamide gel electrophoretic analysis of proteins from the culture filtrates of the BCG substrains, the culture filtrates of the Tokyo and Moreau substrains showed the spot of MPB70 on the gel slabs, whereas those of the other BCG substrains did not.