Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, AB T1J 4B1, Canada.
University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.
J Microbiol Methods. 2024 Jun;221:106943. doi: 10.1016/j.mimet.2024.106943. Epub 2024 May 3.
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.
牛呼吸道疾病（BRD）是全球养牛业的一个重要的健康和经济负担。三种与 BRD 密切相关的细菌病原体（溶血曼海姆菌、多杀巴斯德菌和产单核细胞增生李斯特菌）可以拥有整合和共轭元件（ICEs），这是一组多样化的移动遗传元件，可获得抗生素耐药性（AMR）基因（ARGs）并降低抗生素药物的治疗效果。我们开发了一种双管重组聚合酶扩增（RPA）检测方法，用于检测这些巴斯德氏菌中多达两种变体的 ICEs。携带 ICEs 的溶血曼海姆菌、多杀巴斯德菌和产单核细胞增生李斯特菌分离株的全基因组序列分析显示，tet(H) 旁边存在 tnpA 或 ebrB，这是一个经常在与 BRD 相关细菌的 ICEs 中检测到的抗生素耐药性基因（ARG）。这种实时多重 RPA 检测方法同时针对两种 ICE 变体，分别表示为 tetH_tnpA 和 tetH_ebrB，检测限（LOD）分别为 29（95%CI[23, 46]）和 38 个基因组拷贝（95%CI[30, 59]）。从到达的牛群的 100 个深鼻咽拭子中提取 DNA。使用实时多重 RPA 检测方法检测 ICEs，并使用培养方法和 RPA 检测溶血曼海姆菌、多杀巴斯德菌、产单核细胞增生李斯特菌和牛支原体。该检测方法能够在提取的 DNA 中灵敏、准确地识别 ICEs，为及时检测与牛群抗生素耐药性 BRD 发展相关的潜在风险因素提供了有用的分子工具。
