Serological Detection of Antibodies Against Mycoplasma bovis Infection by Competitive Enzyme-Linked Immunosorbent Assay (cELISA).

Mycoplasma bovis (M. bovis) is a major pathogen in cattle, primarily responsible for bovine respiratory disease complex (BRDC). Characterized by high infectivity and morbidity, M. bovis spreads rapidly within herds and is challenging to control and eradicate, underscoring the need for early and accurate diagnosis. To address this, we developed a competitive enzyme-linked immunosorbent assay (cELISA) for detecting M. bovis-specific antibodies in bovine sera. The assay uses recombinant MbovP579 protein (1 μg/mL) as the coating antigen and an HRP-conjugated 1A2 monoclonal antibody against MbovP579 (mAb, 0.35 μg/mL) as the competitor. With a cut-off value of 40.69% (percentage inhibition, PI), the cELISA demonstrated apparent sensitivity (90%; 95% CI: 78.64-95.65%) and specificity (96.15%; 95% CI: 90.53-98.49%) using the known background positive and negative sera. In addition, it yielded 100% positivity (9/9) against sera collected from 21 days post-immunization. The assay showed no cross-reactivity with other Mycoplasma species or two common bovine bacterial pathogens, confirming its high specificity. The intra- and inter-assay coefficients of variation (CVs) were below 10%. In clinical evaluations, this cELISA generated a 55.57% positivity rate for field samples (n = 1069) and exhibited 90.46% overall agreement (967/1069; 95% CI: 88.54-92.15%) with the commercial indirect ELISA kit, demonstrating strong diagnostic consistency. Discrepant specimens were further analyzed by a metabolic inhibition test (MIT), which suggested improved specificity, although further validation is required of this cELISA.