Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification.

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.