运输后犊牛感染多杀性巴氏杆菌及溶血性曼氏杆菌的多样性分析

Diversity of Pasteurella multocida and Mannheimia haemolytica in transported calves

摘要:

多杀性巴氏杆菌(Pasteurella multocida, Pm)和溶血性曼氏杆菌(Mannheimia haemolytica, Mh)是导致牛呼吸道疾病的主要细菌性病原。目前，对这2种病原在运输后犊牛群中存在的多样性情况仍缺乏了解，严重影响了其感染的有效防控。目的 调查一批从内蒙古某犊牛交易市场购买并运送到重庆合川某养殖场的育肥犊牛群中存在的Pm和Mh多样性情况。方法 犊牛运送到牛场后分4个时间点采集有呼吸道症状的犊牛鼻拭子进行细菌分离培养。根据菌落特点、溶血特性及瑞氏染色特性挑取Pm和Mh疑似菌落，通过特异PCR鉴定及16S rRNA基因序列分析进行确定，进而测定各分离株的血清型、生化及耐药特性，以及毒力基因和耐药基因的存在情况。结果 在4个时间点所采集的68个有呼吸道症状的鼻拭子中共分离到23株A型Pm、10株A6型Mh和1株A2型Mh，总分离率达50%。仅在1个样本中同时分离到了Pm和Mh，部分菌株的生化特性及耐药特性呈现多样性，且与样本采集时间点无显著相关性。耐药性测定发现Pm及Mh分离株对多数青霉素类、氨基糖苷类及林可胺类抗生素具有耐药性，对头孢菌素类及喹诺酮类抗生素较为敏感。耐药基因检测显示，Pm及Mh分离株中β-内酰胺类基因(bla_TEM)检出率分别为73.91%、90.91%，磺胺类基因(sul2)检出率分别为69.57%、18.18%，仅1株Mh检出氨基糖苷类基因(aadA25、aadB)，部分分离株的耐药表型与所选择测定的耐药基因间存在差异。所有Pm及Mh分离株均具有致病性，毒力基因检出发现，Pm分离株均能检出tonB、hsf-1、nanB、oma87、tbpA等毒力相关基因，Mh分离株中毒力基因gapA、dnaN检出率为100%，lktA、plpB、tbpB基因检出率均为82%，并未检出ptfA。结论 长途运输到新养殖场的犊牛群所携带的Pm和Mh在生化特性及耐药性等方面呈现出多样性，这给饲养期Pm和Mh感染的防控带来了较大挑战。该研究为运输后犊牛Pm和Mh感染的防控策略制定提供了参考依据。

Abstract:

Pasteurella multocida (Pm) and Mannheimia haemolytica (Mh) are major bacterial pathogens responsible for bovine respiratory diseases. However, the diversity of these two pathogens in transported calf populations remains poorly understood, which severely hinders the effective prevention and control of their infections.Objective To investigate the diversity of Pm and Mh in a group of fattening calves purchased from a calf trading market in Inner Mongolia and transported to a breeding farm in Hechuan, Chongqing.Methods After arrival, nasal swabs were collected from calves showing respiratory symptoms at four time points for bacterial isolation and culture. Suspected Pm and Mh colonies were selected based on colony morphology, hemolytic characteristics, and Wright-Giemsa staining results, followed by PCR identification and 16S rRNA gene sequencing for confirmation. Furthermore, the serotypes, biochemical and antibiotic resistance profiles, virulence genes, and resistance genes of the isolates were analyzed.Results A total of 23 strains of Pm serotype A, 10 strains of Mh serotype A6, and 1 strain of Mh serotype A2 were isolated from 68 nasal swabs collected at 4 different time points, and only 1 nasal swab harbored both Pm and Mh. Some isolates exhibited diversity in biochemical and antibiotic resistance profiles, which had no significant correlation with sampling time points. Antimicrobial susceptibility testing revealed that Pm and Mh isolates were resistant to most penicillins, aminoglycosides, and lincosamides but remained sensitive to cephalosporins and quinolones. Resistance gene detection showed that β-lactamase resistance (bla_TEM) genes were detected in 73.91% of Pm isolates and 90.91% of Mh isolates, while sulfonamide resistance (sul2) genes were found in 69.57% of Pm isolates and 18.18% of Mh isolates. Only one Mh isolate carried aminoglycoside resistance genes (aadA25 and aadB). Discrepancies were observed between resistance phenotype and the presence of selected resistance genes. All Pm and Mh isolates were pathogenic. Virulence gene analysis confirmed that Pm isolates consistently carried tonB, hsf-1, nanB, oma87, and tbpA, while Mh isolates showed the detection rates of 100% for gapA and dnaN, 82% for lktA, plpB, and tbpB, and 0 for ptfA.Conclusion These findings suggest that calves purchased from trading markets and transported over long distances to new farms harbor Pm and Mh strains exhibiting diversity in biochemical characteristics and drug resistance, which pose challenges for effective infection control. This study provides critical insights for developing prevention and control strategies against Pm and Mh infections in transported calves.