Towards diagnostic differentiation between MTBC and NTM: ELISA and Western blot antibody detection

Abstract

In South Africa, bovine tuberculosis (BTB) occurs sporadically in cattle and is endemic in African buffaloes. Non-tuberculous mycobacteria (NTM) may induce cross-reactive immune responses which can interfere with the diagnosis of tuberculosis. This cross-reactive immune response has not yet clearly been established for the humoral branch of the immune system, but test performance has been suboptimal. Therefore, the aim of the study was to develop an NTM specific ELISA to better classify positive reactions to Mycobacterium bovis (M. bovis). Purified protein derivatives (PPDs) for the ELISA were made of the following NTM species: M. fortuitum, M. malmesburiense and M. nonchromogenicum. The results for the checkerboard titrations for the ELISA were not what was expected, due to a lot of background and no plateau height maximum. The plateau would have made it possible to identify a region of antigen and antibody excess so that the next step for the CBT could be performed. The semidry Western blot did not demonstrate presence of antibodies to the various PPDs. The absence of antibodies could be relating to experimental (Western blot optimisation, freeze-thaw cycles of sera, PPD production) or biological factors. The question arises whether the humoral immune system is triggered enough to induce a detectable response when an animal is infected with NTM. In line with what is known about (B)TB immunity, the cell-mediated immune response may primarily handle the bacterial load. More research is needed for more accurate (B)TB tests to be developed. For now, the best approach would be to join forces and test an animal with a cell-mediated immune response-based test supported by a serological assay.