Proteome microarray-guided identification of mycobacterial antigens and ELISA-based peptide mapping for improved serological detection of <i>Mycobacterium bovis</i> infection in European badgers

Bovine tuberculosis, a zoonotic disease caused primarily by <i>Mycobacterium bovis</i>, poses a significant threat to cattle health and farming livelihoods within the United Kingdom (UK). Disease control in cattle is complicated by the persistence of <i>M. bovis</i> in European badgers, the UK's principal wildlife reservoir. Accurate diagnostic tools for both species are essential for effective surveillance and disease control. Many existing badger serodiagnostic tests, which include MPB70, MPB83, and ESAT6-CFP10 antigens, have relatively modest sensitivities (~50%-60%), limiting their utility in surveillance. To address this issue, we used an unbiased and comprehensive antigen discovery approach to identify new diagnostic targets. This strategy identified Rv3616c as a novel antigen with promising diagnostic test potential for <i>M. bovis</i> infection in badgers. Overlapping peptides spanning the full Rv3616c amino acid sequence were screened to identify the most diagnostically informative epitopes. A pool of four Rv3616c peptides, used in an indirect enzyme-linked immunosorbent assay (ELISA), had a sensitivity of 85.71% (95% CI: 77.19-91.96), a specificity of 94.80% (95% CI: 90.35-97.59), and a diagnostic accuracy of 91.51% (95% CI: 87.54-94.54). The existing validated Badger <i>M. bovis</i> Ab Test, when used alone, had a sensitivity of 73.47% (95% CI: 63.59-81.88); however, parallel interpretation with the Rv3616c ELISA could increase overall sensitivity to 91.84% (95% CI: 84.55-96.41), with minimal loss of specificity. These findings support the use of Rv3616c-derived peptides in serodiagnostic tests to improve the detection of <i>M. bovis</i> infection in badgers and enhance tuberculosis surveillance in this wildlife reservoir.IMPORTANCEAccurate diagnosis of <i>Mycobacterium bovis</i> infection in wildlife reservoirs is essential for controlling bovine tuberculosis (bTB), a zoonotic disease that threatens human health, animal welfare, and farming livelihoods. In the United Kingdom, European badgers are the principal wildlife reservoir, complicating efforts to eradicate bTB in cattle. Existing serodiagnostic tests for badgers have moderate sensitivity, limiting effectiveness in surveillance. To address this, this study used an unbiased, comprehensive antigen discovery approach and identified several new diagnostic targets, including the Rv3616c protein. A test based on specific Rv3616c-derived peptides had a high diagnostic accuracy (91.51%) and, when used in parallel with a validated test, improved test sensitivity while maintaining specificity. These synthetic peptides are scalable, cost-effective, and adaptable to different diagnostic platforms. The findings reveal an antigen with diagnostic potential that could inform the development of new tests for bTB surveillance in wildlife, supporting One Health principles and global tuberculosis elimination strategies.