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

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

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.     

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.     

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.     

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.     

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.     

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 of cervids (Cervus elaphus) following experimental Mycobacterium bovis infection and the implications for immunodiagnosis

Captive and free-ranging wildlife animals are implicated in the maintenance and transmission of bovine tuberculosis and therefore pose a significant obstacle to eradication of the disease from domestic livestock. The current antemortem diagnostic method, the intradermal tuberculin skin test, is impractical for routine use with many wild animals. Antibody-based assays are particularly attractive because the animals are handled only once and immediate processing of the sample is not required. This report characterizes the antibody responses of red deer-elk hybrids (Cervus elaphus) against Mycobacterium bovis and subsequently evaluates the diagnostic performance of select antigens in a rapid-test format. Sequential serum samples were collected from 10 animals experimentally infected with M. bovis and 5 noninfected animals over a 7-month period postinfection (p.i.). Samples were evaluated by enzyme-linked immunosorbent assays, immunoblot analyses, and multiantigen print immunoassays for seroreactivity to mycobacterial antigens. Although all infected animals produced antibodies to M. bovis protein antigens, there was significant animal-to-animal variation in the kinetics and magnitudes of responses and the antigens recognized. The most frequently recognized antigens included MPB83, ESAT-6, CFP10, and MPB70. Responses to some antigens, such as MPB83, were consistently detected as early as 4 weeks after inoculation, whereas other antigens were detected only much later (>140 days p.i.). Antibody responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Comparison of single-antigen (fluorescence polarization assay) with multiantigen (CervidTB STAT-PAK) rapid tests demonstrated that a highly sensitive and specific serodiagnostic test for tuberculosis in cervids will require multiple and carefully selected seroreactive antigens covering a broad spectrum of antibody specificities.     

Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine

A major drawback of current whole-cell vaccines for Mycobacterium avium subsp. paratuberculosis is the interference with diagnostic tests for bovine tuberculosis (TB) and paratuberculosis. The current study was designed to explore the effects of immunization with a heat-killed whole-cell vaccine (Mycopar) on diagnostic test performance and to characterize host immune responses to vaccination over a 12-month period. Neonatal dairy calves were assigned to treatment groups consisting of (i) controls, not vaccinated (n = 5), and (ii) vaccinates, vaccinated with Mycopar vaccine (n = 5). The results from this study demonstrated a rapid initiation of M. avium subsp. paratuberculosis-specific gamma interferon (IFN-γ) in vaccinated calves by 7 days, with robust responses throughout the study. Vaccinated calves also had responses to M. bovis purified protein derivative tuberculin (BoPPD) but minimal reactivity to ESAT-6/CFP-10, an M. bovis recombinant fusion protein. The levels of antigen-specific interleukin-4 (IL-4) and IL-10 were markedly decreased in vaccinated calves between days 7 and 90 of the study but thereafter were similar to the levels in controls. Vaccinated calves began to seroconvert at 4 months, with 4/5 calves having detectable M. avium subsp. paratuberculosis antibody by 6 months. The responses in test platforms for bovine TB were negligible in the vaccinate group, as only one calf had a response, which was in the suspect range of the comparative cervical skin test. Serum antibody responses to M. bovis antigens ESAT-6, CFP-10, and MPB83 were negative on the Vet TB STAT-PAK, DPP VetTB, and DPP BovidTB tests. These results suggest that the Mycopar vaccine will interfere with diagnostic tools for paratuberculosis but result in low interference with the comparative cervical skin test and emerging serologic tests for M. bovis.     

Protein G-based enzyme-linked immunosorbent assay for anti-MPB70 antibodies in bovine tuberculosis.

MPB70 is a highly species specific protein which is secreted from Mycobacterium bovis during culture. To investigate whether antibodies against MPB70 can be used as an indicator of infection with M. bovis, an enzyme-linked immunosorbent assay was developed, based on the use of biotinylated protein G, to provide a common indicator for antibody formation in different species. During experimental infection with M. bovis in cattle, a characteristic pattern of anti-MPB70 antibody production was observed with an initial flat plateau followed by a marked rise 18 to 20 weeks after infection. Skin testing with bovine tuberculin purified protein derivative (PPD), which was shown to contain antibody-reactive MPB70, was a potent stimulator of antibody production in infected animals. In experimentally infected cattle, we observed an inverse relationship between antibody activity and delayed-type hypersensitivity skin test reactions. In natural M. bovis infections, skin testing with PPD was also a potent stimulator of anti-MPB70 formation. Comparison between the enzyme-linked immunosorbent assay for antibodies to MPB70 and that for antibodies to the widely cross-reacting M. bovis BCG antigen 85B in animals with M. bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Corynebacterium pseudotuberculosis infections showed that formation of antibody to MPB70 was highly specific for infection with M. bovis. The use of an MPB70-containing PPD preparation for skin testing followed by this anti-MPB70 assay is a highly specific indicator of M. bovis infection. Adjustment of the test conditions is expected to provide an increased sensitivity of the procedure for the diagnosis of natural M. bovis infections.     

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.     

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.     

Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment

Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.     

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.     

Use of synthetic peptides derived from the antigens ESAT-6 and CFP-10 for differential diagnosis of bovine tuberculosis in cattle

In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best long-term prospect for tuberculosis control in British herds. A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that testing and slaughter-based control strategies can continue alongside vaccination. To date bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only available vaccine for the prevention of tuberculosis. However, tests based on tuberculin purified protein derivative cannot distinguish between M. bovis infection and BCG vaccination. Therefore, specific antigens expressed by M. bovis but absent from BCG constitute prime candidates for differential diagnostic reagents. Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection of M. bovis infection in cattle. Here we report the identification of promiscuous peptides of CFP-10 that were recognized by M. bovis-infected cattle. Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6. Using this peptide cocktail in T-cell assays, M. bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond. The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals. This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals. In summary, our results demonstrate that this peptide cocktail can discriminate between M. bovis infection and BCG vaccination with a high degree of sensitivity and specificity.     

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.     

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.     

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     

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.