Homology Between the MPB70 and MPB83 Proteins of Mycobacterium bovis BCG

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS-PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross-reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.     

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.     

Immunochemical Characterization of the MPB70/80 and MPB83 Proteins of Mycobacterium bovis

MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.     

Macrophage-Dependent Lymphocyte Immunostimulation in Cattle Infected with Mycobacterium bovis and with Mycobacterium avium

Cultures of bovine peripheral blood leukocytes were prepared from calves inoculated with Mycobacterium bovis and with M. avium. Suitable lymphocyte stimulation was obtained with specific and nonspecific mycobacterial mitogens. Leukocyte cultures depleted of adherent, macrophage-type cells did not respond to stimulation with purified protein derivative, although they were responsive to phytohemagglutinin.     

Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70.

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.     

Cloning and Sequencing of an MPB70 Homologue Corresponding to MPB83 from Mycobacterium bovis BCG

MPB70 is secreted in high concentrations by Mycobacterium bovis BCG substrain Tokyo (BCG Tokyo), but little by substrains Pasteur (BCG Pasteur) and M. tuberculosis . The gene encoding a MPB70 homologue secreted by BCG Tokyo was found at the upstream region of the gene encoding MPB70, with approximately 2.3 kilobase pairs (kbp) spacing: the same gene was also found in BCG Pasteur. This gene was cloned and sequenced from BCG Tokyo. The DNA sequence which contained a 663 base pair (bp) open reading frame beginning at position 1 and ending with a TAA codon at position 661 was found. Its theoretical molecular mass was calculated to be 22.068 kDa. This gene was highly homologous to the coding region of mpb70 and the deduced amino acid sequence was very similar to MPB83 reported by Harboe et al . It was speculated that the gene the authors characterized probably corresponded to the mpb 83 gene.     

Antigen Recognition by Serum Antibodies in White-Tailed Deer (Odocoileus virginianus) Experimentally Infected with Mycobacterium bovis

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

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.     

MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

A multitude of different proteins are synthesized by the mycobacterial cell. It is often valuable to consider these proteins in different broad categories based on common characteristics, such as physicochemical properties, localization in the mycobacterial cell, and active secretion during culture. In turn, these distinct properties are closely related to their functional properties and the tendency to interact with the immune system of the host after infection.MPB70 was initially isolated by Nagai et al. (37). This secreted protein is of particular interest since it is highly species specific (13). It is consistently present in virulent Mycobacterium bovis, whereas it is synthesized and secreted in markedly different amounts by various substrains of avirulent M. bovis BCG (13, 32, 35).We recently studied the closely related MPB83 protein and isolated three peptides derived from it after CNBr cleavage, showing that MPB70 and MPB83 are homologous but clearly distinct proteins and are therefore encoded by different genes (15). The heterogeneity between different substrains of BCG in regard to the synthesis of MPB70 and MPB83 proteins is clearly greater than previously realized (53). Another type of heterogeneity has also previously been identified; 26- and 23-kDa fractions of a protein that was presumed to be MPB70 differed markedly in carbohydrate content (9). The available data indicate that MPB83 occurs in 26- and 23-kDa forms, both glycosylated, whereas MPB70 (at 22 kDa) is nonglycosylated.The term MPB was introduced (37) for the designation of a protein purified from M. bovis BCG, with the number after MPB denoting the relative mobility by electrophoresis on a 7.7% polyacrylamide gel run at pH 9.5. MPT is used for similar designations of proteins purified from M. tuberculosis (38). The designations mpb70 and mpt70 denote the corresponding genes.Cloning of mpb70 (43) revealed the sequence of a polypeptide chain preceded by a signal peptide that is typical of secreted proteins. In contrast, both mpb83 from M. bovis BCG (33, 45) and mpt83 from M. tuberculosis (20) revealed a typical lipoprotein consensus element in the signal sequence. The relative concentrations of the 26- and 23-kDa components of these proteins vary markedly between sonicates and culture fluids of BCG bacilli. In sonicates, the 26-kDa component, consisting of MPB83, dominates. In culture fluids, the reverse is observed, with a markedly higher concentration of the 22- to 23-kDa components in BCG Sweden and BCG Russia (18).The purpose of the present work was to investigate these properties at the level of native proteins in relation to the localization of these and other marker proteins in the mycobacterial cell. Using monoclonal antibody (MAb) MBS43, which reacts with MPB83 but not with MPB70 (53), permitted more precise distinctions between these proteins than previously obtained.     

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.     

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     

Recognition of a common mycobacterial T-cell epitope in MPB59 of Mycobacterium bovis.

Bovine tuberculosis, which persists as a residual level of infection in many European countries, has implications not only for the economy of farming communities but also for human health. The aim of this study was to identify a common mycobacterial antigen which was recognized in bovine tuberculosis and to characterize the response to this antigen at the epitope level. A T-cell clone, phenotype CD4+, raised from an animal experimentally infected with Mycobacterium bovis was shown to proliferate in response to a panel of sonicates derived from different mycobacterial species indicating recognition of an antigen with broad specificity. This antigen was subsequently shown to be MPB59. Recognition of MPB59 at the epitope level was determined in experimental and field cases of bovine tuberculosis using a panel of synthetic peptides (20-mers with 10-residue overlaps) incorporating the signal sequence and mature protein. The results showed that in vitro interferon-gamma was predominantly produced in response to adjacent peptides numbers 10 and 11, suggesting that the dominant epitope was contained in the overlap, correlating to residues 101-110 (YYQSGLSIVM). This epitope was recognized by 54% of tuberculous cattle of mixed breeds, which suggests that it may be genetically permissive in terms of major histocompatibility complex presentation. Sequence analysis confirmed that there were only minor differences in the amino acid composition within this region for various mycobacterial species, which could explain the common T-cell recognition described in this study. Common recognition of this epitope indicates that it would have limited potential for use as a diagnostic reagent per se but may have potential for inclusion in a subunit vaccine.     

Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG.

Substrains of Mycobacterium bovis BCG have been divided in two major groups, high producers and low producers of the secreted proteins MPB64 and MPB70. Of these, Mycobacterium tuberculosis secretes only the analog MPT64 during growth on Sauton medium. It has been confirmed that high-producer and low-producer substrains of BCG as well as M. tuberculosis contain the gene for the MPB/MPT70 protein. By contrast, polymerase chain reaction and hybridization experiments are reported here which indicate that the MPB64 gene is absent in the BCG substrains Copenhagen, Pasteur, Glaxo, and Tice, in which previous methods did not permit distinction between secretion of small amounts or absence of the protein in culture fluids.     

Cloning and characterization of the gene for immunogenic protein MPB64 of Mycobacterium bovis BCG.

The gene for immunogenic protein MPB64 found in culture filtrates of only Mycobacterium tuberculosis and some strains of Mycobacterium bovis BCG was cloned by using a single-probe method and was sequenced. The gene analysis revealed that the structural gene for MPB64 consisted of 618 base pairs, and its deduced molecular weight was 22,400. Twenty-two amino acids for a putative signal peptide and 205 amino acids for the MPB64 protein were observed. In the coding region, the third letter of the codon showed a biased codon and a high G+C content (78.5%). The gene was expressed in Escherichia coli by using an E. coli expression vector. The product showed migration similar to that of the authentic MPB64 protein by electrophoresis and reacted with the polyclonal and the monoclonal antibodies raised against the MPB64 protein. The strict specificity of MPB64 could be applied to immunodiagnosis of tuberculosis.     

T-Cell Stimulation with Purified Mycobacterial Antigens in Patients and Healthy Subjects infected with Mycobacterium leprae: Secreted Antigen 85 is Another Immunodominant Antigen

Peripheral blood leucocytes from 9 paucibacillary and 12 multibacillary leprosy patients, from 18 healthy controls and from 34 healthy leprosy contacts were stimulated with three mycobacterial heat shock proteins with respective molecular weights of 70,65 and 18 kDa and with the secreted 30–32 kDa protein, also called antigen 85. Antigen 85 was found to be the most powerful T-cell antigen (as measured by lymphoproliferation and IFN-γ secretion), eliciting a positive response in all (100%) paucibacillary patients and in all lepromin-positive controls and contacts. The three heat shock proteins (hsp) were less active T-cell stimuli. Reactivity to the 70 kDa hsp was found in only 44% of the paucibacillary patients, in 80% of the lepromin-positive controls and in 60% of the lepromin-positive leprosy contacts. The 65 kDa hsp stimulated T cells in 89% of the paucibacillary patients and in 80% of the lepromin-positive controls and contacts. Responsiveness to the 18 kDa hsp, finally, was clearly more frequent in tuberculoid leprosy patients (78%) than in lepromin-positive controls (40%) or lepromin-positive leprosy contacts (4%). T-cell reactivity of 8 lepromin-negative controls, of 9 lepromin-negative contacts and of 12 multibacillary leprosy patients was low to all the antigens tested. Although proliferative and IFN-γ responses were generally closely related, some subjects demonstrated a dissociation of these two immune parameters. Our data confirm previous findings on the powerful T-cell stimulatory properties of antigen 85 during M. leprae infection and suggest that this antigen is indeed a potentially protective T-cell immunogen.     

Differentiation between Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens

Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins of M. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected with M. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovis culture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.     

New Latex Bead Agglutination Assay for Differential Diagnosis of Cattle Infected with Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis

Extensive studies have shown that the current assays used to identify cattle infected with Mycobacterium bovis or Mycobacterium avium subsp. paratuberculosis are not sufficiently sensitive and specific to detect all infected animals, especially animals recently infected with the pathogens. In the present report we show that these limitations might be overcome with a latex bead agglutination assay (LBAA). With the specific immunodominant epitope (ESAT6-p) of M. bovis, we developed an LBAA and enzyme immunoassay (EIA) for that purpose and compared them with the “gold standard” culture method and skin test for their efficacy in detecting bovine tuberculosis. When sera from control healthy cows (n = 10), M. avium subsp. paratuberculosis-positive cattle (naturally infected, n = 16; experimentally infected, n = 8), and M. bovis-positive cattle (naturally infected, n = 49;experimentally infected, n = 20) were applied to an EIA and an LBAA developed with ESAT6-p, the two tests showed similar sensitivity (97.1% by EIA, 95.7% by LBAA), high specificity (94.2% by EIA, 100% by LBAA), and a positive correlation (kappa value, 0.85; correlation rate, 93.2%; correlation coefficient, 0.64). Receiver operating characteristic analysis of EIA results and comparison with the culture method determined a suitable cutoff value at 0.469, with an area under the curve of 0.991 (95% confidence interval, 0.977 to 1.0). As LBAA didn't show any positive reactions with sera from uninfected control cows or M. avium subsp. paratuberculosis-infected cattle, which were confirmed to be free of M. bovis by culture or PCR, LBAA using the ESAT6-p can be a rapid and useful M. bovis diagnostic assay. The data suggest that rapid, sensitive, and specific assays can be developed with peptides containing immunodominant epitopes present in proteins uniquely expressed in M. bovis or M. avium subsp. paratuberculosis for differential diagnosis of cattle infected with M. bovis or M. avium subsp. paratuberculosis.     

Relationship of secretion pattern and MPB70 homology with osteoblast-specific factor 2 to osteitis following Mycobacterium bovis BCG vaccination.

Significant homology was found between MPB70 and each of four repeat domains of osteoblast-specific factor 2 (OSF-2). Two internal homology regions within each repeat domain of OSF-2 presumed to be related to the active site(s) of this bone adhesion molecule showed the highest homology. A literature search concerning osteitis after Mycobacterium bovis BCG vaccination in neonates revealed that MPB70-high-producer substrains were associated with an increased incidence of osteitis following vaccination. These observations indicate that the function of MPB70 is related to the interaction between bacilli and the host following vaccination or infection with mycobacteria.     

Nucleotide sequence of MPB63 gene in Mycobacterium bovis BCG Tokyo

Mycobacterium bovis BCG has been used for the prevention of tuberculosis and as therapy for bladder tumor. MPB63 in M. bovis BCG is one of the immunogenic proteins and is secreted in large quantities. Therefore, it is of interest that the MPB63 gene be examined for the determination of its nucleotide sequence. A fragment of 820 base pairs (bp) including the MPB63 gene was prepared by amplification using polymerase chain reaction (PCR) employing M. bovis BCG Tokyo chromosomal DNA as a template and its nucleotide sequence was determined. The nucleotide sequence of mpb63 in M. bovis BCG was then compared with that of mpt63 in M. tuberculosis. The result indicated that the nucleotide sequences between two protein genes were quite agreeable in the genes' structural and upstream regions, except that one base change from C in mpt63 to G in mpb63 was detected in the downstream trailer sequence. This suggests that the genetic information of M. bovis BCG is not entirely identical to that of M. tuberculosis, although the characteristics of both microorganism are very similar to each other.