牛源溶血性曼氏杆菌和多杀性巴氏杆菌的分离与鉴定

Isolation and identification of bovine-derived Mannheimia haemolytica and Pasteurella multocida

摘要:

【背景】 牛呼吸疾病综合征(bovine respiratory disease complex, BRDC)是由多病原和多因素互作引起的以支气管肺炎为主的牛呼吸系统疾病。引起BRDC的细菌性病原主要有溶血性曼氏杆菌(Mannheimia haemolytica, Mh)、多杀性巴氏杆菌(Pasteurella multocida, Pm)、睡觉嗜组织菌(Histophilus somni, Hs)等。【目的】 了解我国引起BRDC相关的Mh、Pm分布和血清型种类。【方法】 自2022年7月到2024年1月间，通过PCR技术对来自河南、湖南、湖北、浙江、安徽、山东、宁夏、甘肃、贵州、黑龙江、内蒙古等16个地区的54个牧场共698头牛的698份样本(鼻拭子359份、气管拭子321份，肺脏组织18份)进行溶血性曼氏杆菌及多杀性巴氏杆菌的检测，对阳性样品进行细菌分离和血清型鉴定。【结果】 溶血性曼氏杆菌和多杀性巴氏杆菌检测为阳性的样本量分别为15/698和72/698，阳性检出率分别为2.15% (95% CI: 1.2%−3.5%)及10.32% (95% CI: 8.2%−12.8%)，二者混合感染有5/698份，阳性率为0.72% (95% CI: 0.2%−1.7%)。PCR检测后进一步对阳性样品进行细菌分离，共获得8株溶血性曼氏杆菌，其中A1型1株，A2型3株，A6型4株，多位点序列分型(multi locus sequence typing, MLST)结果为ST1型5株(5/8)，均为血清型A1和A6型；ST2型3株(3/8)，为血清型A2型；21株多杀性巴氏杆菌，其中A:L3型17株，A:L6型4株，MLST分型结果为ST1型和ST7型，ST1型17株(17/21)，均为A:L3型；ST7型4株(4/21)为A:L6型。【结论】 通过对部分地区具有呼吸道症状的病牛进行细菌分离鉴定，对牛呼吸疾病综合征主要致病的细菌性病原进行分型以及生物学特性分析，确定了病原流行的优势血清型、毒力基因和耐药基因等，为相关疫苗的制备提供了基础，为防控该病提供了理论依据。

关键词:牛呼吸疾病综合征;溶血性曼氏杆菌;多杀性巴氏杆菌;细菌分离;PCR鉴定

Abstract:

[Background] Bovine respiratory disease complex (BRDC), a bovine respiratory disease characterized by bronchopneumonia, is caused by interactions of multiple pathogens and factors. The main bacterial pathogens causing BRDC include Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), and Histophilus somni (Hs). [Objective] To understand the distribution and serotypes of Mh and Pm associated with BRDC in China. [Methods] We employed PCR to detect Mh and Pm in 698 samples (359 nasal swabs, 321 tracheal swabs, and 18 lung tissue samples) collected from 698 cattle across 54 farms in 16 provincial-level administrative regions including Henan, Hunan, Hubei, Zhejiang, Anhui, Shandong, Ningxia, Gansu, Guizhou, Heilongjiang, and Inner Mongolia from July 2022 to January 2024. Bacterial isolation and serotyping were performed on the positive samples. [Results] The positive detection rates of Mh and Pm in the samples were 2.15% (15/698, 95% CI: 1.2%–3.5%) and 10.32% (72/698, 95% CI: 8.2%–12.8%), respectively. Co-infection was detected in 5 (0.72%, 95% CI: 0.2%–1.7%) samples. From the positive samples, 8 strains (1 of serotype A1, 3 of serotype A2, and 4 of serotype A6) of Mh and 21 strains (17 of serotype A:L3 and 4 of serotype A:L6) of Pm were isolated. The multi-locus sequence typing (MLST) results for Mh showed that 5 out of 8 strains were of sequence type (ST) 1 (serotypes A1 and A6), and 3 strains were of ST2 (serotype A2). The 21 strains of Pm included 17 strains of ST1 (serotype A:L3) and 4 strains of ST7 (serotype A:L6). [Conclusion] This study isolated and identified the main bacterial pathogens causing BRDC in cattle with respiratory symptoms in specific regions and revealed the prevalent serotypes, virulence genes, and resistance genes. These findings lay a foundation for the development of related vaccines and offer theoretical support for the prevention and control of BRDC.

Key words:bovine respiratory disease complex;Mannheimia haemolytica;Pasteurella multocida;bacterial isolation;PCR identification