Identification of Novel Antigens Recognized by Serum Antibodies in Bovine Tuberculosis

Abstract

Bovine tuberculosis (TB), caused by Mycobacterium bovis, remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (ϳ95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.KEYWORDS antigen, antibody, Mycobacterium bovis, serodiagnosis, tuberculosis B ovine tuberculosis (TB) is an important zoonotic disease caused by Mycobacterium bovis, a member of the M. tuberculosis complex (1). Although M. bovis is most often isolated from tuberculous cattle, it has a broad range of susceptible host species, including humans (2-4). The control of TB in cattle is therefore difficult due to the existence of wildlife reservoirs of M. bovis, such as white-tailed deer in the United States, Eurasian badgers in Great Britain, wild boars in Spain, and brushtail possums in New Zealand (5-8).The antemortem diagnostic methods currently approved for use in cattle have limitations. The intradermal tuberculin test has suboptimal sensitivity and inconsistent performance (1, 9, 10), while the available blood-based assays, such as the Thermo Fisher Scientific Bovigam TB kit or the Idexx M. bovis antibody (Ab) test, lack the required accuracy and show a significant variability when used in different geographic areas (11-13). Given the ease of sample collection and the test procedure, antibody detection assays may be useful to identify M. bovis-infected cattle (1,14,15), but the existing methods require improvement.The primary goal of the present study was to identify novel seroreactive antigens of M. bovis. Using a multiantigen print immunoassay (MAPIA) and well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection, we screened a panel of 101 recombinant proteins of M. tuberculosis, including 10 polyepitope fusions. The performance of MAPIA-selected candidates was also evaluated in pilot experiments using the dual-path platform (DPP) technology to

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA

“…ELISA using three recombinant protein antigens, MPB70, MPB83, and ESAT6, showed 69.5% sensitivity and 96% specificity in a previous study [ 20 ]. The ELISA sensitivities of PC (MPB83, MPB70, Rv2650c, Mb1498, SerS), DID38 (MPB70, MPB83), and DID65 (MPB70, PstS1, CFP10) were 90.5%, 85.1%, and 81.1%, respectively, and the specificities were 98.1%, 99.1%, and 98.2%, respectively, in a previous study [ 23 ]. In this study, for ELISA using the antigen combination 22-kDa MPB70 and 60-kDa SahH, the sensitivity was 94.4% and the specificity was 97.3% based on the S/N ratio criteria ( Table 2 ).…”

Fatal Tuberculosis in a Free-Ranging African Elephant and One Health Implications of Human Pathogens in Wildlife

“…High intensity of the test line in the Chembio DPP VetTB assay suggested the presence of IgG antibodies to MTBC-specific fusion antigen ESAT-6/CFP10 (Figure 4). In addition, the presence of high intensity lines for the MTBC-specific antigens including CFP10 protein, ESAT-6/CFP10 fusion protein, and DID65 fusion (MPT70/PstS1/CFP10) protein (17), using the multi-antigen print immunoassay (MAPIA), supported a presumptive diagnosis of TB (Figure 5) (18, 22).…”

“…Whole blood was collected from the heart into serum separator tubes, which formed a clot, and then serum was harvested by centrifuged at 3,000 x g for 10 min. Serology to detect the presence of antibodies to Mtb complex (MTBC)-specific antigens was performed using the Chembio DPP VetTB assay (Chembio Diagnostic Systems, Inc., Medford, NY) and the multi-antigen print immunoassay (MAPIA) (17, 18).…”

Biomarkers for Detecting Resilience against Mycobacterial Disease in Animals

“…The isotype of antibody detected, as well as the antigenic target, can impact serological test efficacy. A range of antigens have been tested in an attempt to improve early disease detection in both paratuberculosis and bTB (36)(37)(38). Immunoglobulin (Ig)G antibodies are the most common isotype used for mycobacterial antibody ELISAs, however targeting different isotypes may be more informative.…”