Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis

Abstract

Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis),responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cellmediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex andpoorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (falsepositiveresults) has been crucial to develop a more proper antigen. In the present study, we selected sixM. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by insilicoanalysis and evaluated them in experimental and natural infection; none of these antigens hadbeen previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animalswith both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected withMycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually inducedan IFN-g response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the mostvaluable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAPinfected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B amongTST and MTC specific-PCR positive animals, although this result needs to be proven in further studieswith a higher sample size. Our data confirm the feacibility to implement bioinformatic screening toolsand suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate theircontribution to bTB diagnosis.