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

Abstract

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.

Diagnosis of bovine tuberculosis: review of main techniques

“…The assay is based on the release of IFNγ from sensitized lymphocytes during a 16-24 hours incubation period with specific antigen and makes use of comparison of IFNγ production following stimulation with avium and bovine PPD (Alito et al, 2003). Besides high logistical demands (culture start is required within 24 h after blood sampling), and its high costs, showed the same difficulties in the standardization already discussed in relation to the TST with the tuberculin (de la Rua-Domenech et al, 2006;Schiller et al, 2010;Vordermeier et al, 2008).…”

Potential application of new diagnostic methods for controlling bovine Tuberculosis in Brazil

“…It is well established that cattle infected by M. bovis develop early immune responses, dominated by cell-mediated immunity (1,20,46,59,81). Therefore, serology is less efficient to identify cattle in the early stages of infection, when antibodies titres are low (88).…”

Only a Subset of Phosphoantigen-Responsive γ9δ2 T Cells Mediate Protective Tuberculosis Immunity

“…Consistent with our observations enhanced secondary γ 9 δ 2 T cell responses have been observed in non-human primates infected with virulent M. tuberculosis (3). In addition to activation by M. tuberculosis (4), γ 9 δ 2 T cells proliferate and develop relevant effector functions during infections with virulent M. bovis (5), Listeria monocytogenes (6), Plasmodia (7) and herpesviruses (8). Furthermore, depletion of circulating γ 9 δ 2 T cells has been associated with exacerbation of susceptibility to infectious diseases (9–11).…”