Mycobacterium tuberculosis complex-specific antigens for use in serodiagnosis of bovine tuberculosis

Abstract

Bovine tuberculosis (BTB) is a zoonotic disease that affects domestic and wild animals, and humans. It is caused by Mycobacterium bovis (M. bovis) and has a wide host range. The effective control of BTB is of paramount importance and this can be achieved through the use of accurate and comprehensive diagnostic tests. The most widely used methods to detect BTB are the skin test and in vitro gamma interferon assay which do not detect anergic animals, but serological tests such as ELISA and fluorescence polarization assay (FPA) have been found promising in ancilliary tuberculosis diagnosis. The overall aim was to study M. tuberculosis complex (MTBC) protein, mycobacterial protein bovis 70 (MPB70) as a target for serological assays in the detection of antibodies to bovine tuberculosis. The MPB70 protein was expressed, purified and labeled with fluorescein (FITC). The mpb70 gene was fragmented into three regions without disrupting predicted epitopes. The resulting protein Fragments were expressed as fusion proteins with the monster green fluorescent protein (MGFP). The recombinant MPB70 (rMPB70) and the expressed gene fragments 2&3 were tested in immunoblots and ELISAs. The rMPB70 and fragment 2-MGFP reacted with chicken antibodies raised against rMPB70 and immune sera from BTB infected buffaloes. MPB70 peptides were synthesized as an approach to identify even smaller antigenic regions. The peptides BT1G (residues 31-45) and BT51L (residues 81-95) were recognised by anti-MPB70 chicken antibodies in the ELISA and fall within fragment 1 and 2, respectively. The tracers (rMPB70-FITC, fragment 2-MGFP fusion and peptides BT1G&BT51L) were tested in the FPA, but the results failed to distinguish between immune sera from chickens immunized with rMPB70 and negative control sera. Even though the FPA was not successful, the MPB70 fragment 2-MGFP fusion protein, which was recognized by sera from BTB infected buffaloes, was tested in an ELISA using panels of sera from uninfected and naturally M. bovis infected buffaloes and cattle. The diagnostic performance of the ELISA was, however, overall unsatisfactory and hence of very limited use as a serological test to detect antibody responses to BTB as a stand-alone assay. Sera from some of the animals gave false positive reactions indicating that MPB70 was not sufficiently specific for serodiagnosis of M. tuberculosis complex infections.