Detection of anti-Mycoplasma bovis IgG in bovine nasopharyngeal swabs

Mycoplasma bovis is a significant bacterial pathogen involved in the Bovine Respiratory Disease (BRD) complex. While assessing localized mucosal immunity is vital for understanding host defenses against respiratory pathogens, there is a lack of validated diagnostic assays for detecting mucosal immunoglobulins. This study aimed to evaluate the diagnostic performance of a novel multiplex ELISA platform (Pictor PictVet ® ) in detecting anti- M. bovis IgG in bovine nasopharyngeal swab washes. Archived nasopharyngeal swab washes from crossbred feedlot steers (50 positive and 50 negative for M. bovis DNA via digital qPCR) were evaluated. The ELISA utilized three target antigens: K-0310, K-0320, and MilA. Diagnostic accuracy was assessed using both concurrent M. bovis DNA status and a Latent Class Analysis (LCA) model as ground truths. Inter-operator reproducibility and analytical sensitivity (by serial dilution) were also determined. The MilA antigen exhibited high variance and poor predictive performance (51.5% accuracy), resulting in its exclusion from the final models. When utilizing M. bovis DNA status as the ground truth, the best custom cutoff (K-0310 and K-0320 in series) yielded high specificity (88%) but low sensitivity (30%). However, employing LCA to estimate IgG status (17.1% prevalence) dramatically improved diagnostic validity. Evaluating K-0310 and K-0320 in parallel achieved an accuracy of 97.59% with 100% sensitivity and 95.18% specificity. The assay demonstrated excellent inter-operator agreement (Kappa = 0.894 to 1) and reliably detected positive samples at a 1:2 dilution. Analyzing nasopharyngeal swabs with this novel multiplex ELISA platform, specifically utilizing the K-0310 and K-0320 antigens, provides a highly accurate, reproducible, and non-invasive method for monitoring mucosal anti- M. bovis IgG. The use of LCA was paramount in establishing appropriate cutoffs, highlighting the limitations of relying solely on pathogen DNA detection to evaluate adaptive immune responses.