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.
Highlights
Bovine tuberculosis caused by Mycobacterium bovis is an animal health problem throughout the world and constitutes a major threat to public health
Culture supernatants, harvested at three different culture times (24, 38 and 73 days), and cell extracts were fractionated by FPLC anion-exchange chromatography with a linear gradient of 0-0.42 M NaCl
When culture supernatants and cell extract preparations were separated by anionexchange chromatography, four (24-day culture supernatant) to three (73-day culture supernatant and cell extract) major peaks were observed in the gradient zone
Summary
Bovine tuberculosis caused by Mycobacterium bovis is an animal health problem throughout the world and constitutes a major threat to public health. There are 50,600,000 Argentine heads of cattle, with 5% of the animals and 35% of the farms being infected with bovine tuberculosis [1] Eradication of this zoonotic disease remains an important goal in several countries. Some bovine tuberculosis eradication programs have incorporated variants of this test but suboptimal sensitivity and specificity have frequently been reported [2,3]. This is considered to be due, in part, to the nature of the poorly characterized antigens used, which are mycobacterial extracts containing components that are not species specific [3]. More sensitive and specific tests, probably incorporating better defined antigens, are required for efficient detection of this disease
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