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
