Evaluation of recombinase polymerase amplification assays for targeted detection of bovine respiratory disease bacterial pathogens and antimicrobial-resistance genes in feedlot calves

Antimicrobial resistance (AMR) challenges the effective treatment of bovine respiratory disease (BRD). We evaluated the performance of a recombinase polymerase amplification (RPA) assay, a rapid, isothermal nucleic-acid amplification method, compared with bacterial culture (BC), antimicrobial susceptibility testing (AST), and real-time PCR (rtPCR) testing. We cultured deep nasopharyngeal swabs collected from 800 beef calves within 36 d on feed and at first treatment for BRD for <i>Mannheimia haemolytica, Pasteurella multocida</i>, and <i>Histophilus somni</i>, and screened for these species and <i>Mycoplasmopsis bovis</i> using RPA (<i>M. haemolytica</i> serotypes 1 and 6 only) and rtPCR (<i>M. bovis</i> only). We then tested samples that were RPA-positive for <i>Pasteurellaceae</i> for integrative and conjugative element (ICE) variants containing <i>tetH</i> (ICE<i>tnpA</i>, ICE<i>ebrB</i>) and macrolide antimicrobial-resistance genes (ARGs; <i>msrE-mphE</i>, <i>erm42</i>). Bayesian latent class models estimated the clinical sensitivity of BC to be higher than RPA for <i>Pasteurellaceae</i> detection. Both methods were highly specific. RPA sensitivity for <i>M. bovis</i> detection was comparable to rtPCR, but RPA specificity was higher. RPA specificity for detection of macrolide resistance was lower (93.5%) than BC-AST (99.9%), reflecting the identification of ARGs by RPA in non-target bacteria. However, the sensitivity of both tests was low (BC-AST: 20.5%; RPA: 13.3%). Limited RPA sensitivity for <i>Pasteurellaceae</i> identification constrained its downstream performance for detecting ARGs. With our large-scale study, we demonstrated that RPA could detect key BRD-associated pathogens and AMR determinants directly from respiratory samples. Although our RPA results were not sufficient to inform AMU treatment strategies, RPA testing could prove valuable for addressing focused investigations with rapid turnaround.