Use of recombinant proteins MPB70 or MPB83 as capture antigens in ELISAs to confirm bovine tuberculosis infections in Brazil.

The objective was to evaluate the use of two indirect IgG-ELISA tests (with recombinant proteins MPB70 or MPB83, respectively, as capture antigens) as confirmatory tests for diagnosis of bovine tuberculosis in a herd of naturally infected dairy cows. Results for ELISA-MPB70 and ELISA-MPB83 were similar (kappa statistic=0.92) on Days 0 (day of intradermal injection with purified protein derivatives, PPD), 7, and 21. The kappa statistic between ELISA and the Comparative Intradermal Tuberculin Test, as well as ELISA sensitivity and specificity (relative to culture or PCR as standards) were: 0.7, 34.4% and 75% on Day 0; 0.25, 53.8% and 66.6% on Day 7; and 0.01, 1.8% and 77.7% on Day 21, respectively. In conclusion, although ELISAs using MPB70 or MPB83 as antigens were not reliable indicators of infection status, especially on Days 7 and 21, they were of potential value as complementary tools to intradermal PPD testing.

Detection of bovine tuberculosis in herds with different disease prevalence and influence of paratuberculosis infection on PPDB and ESAT-6/CFP10 specificity.

Bovine tuberculosis (BTB) is a major animal health problem with zoonotic implications. Current control programs are based on test and slaughter strategies utilizing skin tests with tuberculins as antigens. The low specificity and associated operative difficulties of these tests have driven the search for new antigens and diagnostic assays. In this multicenter study, using herds from Argentina, Mexico and Northern Ireland, we selected skin test positive and negative animals from herds with different prevalence's of BTB and compared tuberculin (PPDB) and ESAT-6+CFP10 as antigens ex vivo. In low prevalence herds, crossreactivity of PPDB was apparent since up to 60% of the PPDB skin test and ex vivo positive animals did not responded to ESAT-6+CFP10 ex vivo. The superior specificity of ESAT-6+CFP10 was confirmed in a Mycobacterium avium sp. paratuberculosis infected herd where several of the animals had strong crossreactivity to PPDB and PPDA but not to ESAT-6+CFP10. In high prevalence herds 85% of the skin test-positive animals, were confirmed ex vivo using either PPDB or ESAT-6+CFP10 as antigen. However, within this group 60% of the skin test negative animals were PPDB and ESAT-6+CFP10 positive ex vivo indicating that the skin test can in some herds yield a significant number of false negative results. In conclusion, the ex vivo test is recommended as an ancillary test to accelerate BTB eradication. In high prevalence herds, PPDB or ESAT-6+CFP10 can be used as antigen whereas in low and medium prevalence herds ESAT-6+CFP10 is the preferred choice.

Optimizing antigen cocktails for detection of Mycobacterium bovis in herds with different prevalences of bovine tuberculosis: ESAT6-CFP10 mixture shows optimal sensitivity and specificity.

Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain.

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.