牛源多杀性巴氏杆菌的分离与初步鉴定

Isolation and diversity analysis of bovine Pasteurella multocida

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。

[Objective]Our research focuses on the detection and the distribution of some important virulence genes in Pasteurella multocida associated with bovine respiratory disease (BRD).[Methods]In total 237 nasal swabs were collected from August 2017 to April 2018 in 8 areas of China.The bacteria were isolated to record the colony morphology and Gram staining,and phylogenetic tree based on the 16S rRNA gene sequences of the 31 isolates was drawn.Serotype classification was performed,and total 23 virulence factors were determined by PCR. [Results]In total 31strains ofP multocida were identified from 237samples.Serum typing found all the isolates were type A strains and phylogenetic tree showed the 31 strains related to HB01 strain (China),USDA-ARS-USMARC-60712, USDA-ARS-USMARC-60214,ATCC 43137and 36950.PCR was used for detecting 23 virulence-associated genes occurred in those isolates,demonstrating that each P multocida carried 17 to 19 virulence-associated genes with high concentration ratio.[Conclusion]Type A P multocida were dominant strains in which 7 strains from different areas of China belong to one cluster,virulent genes tadD and hgbA were lost in this cluster as well as virulent hsf-1 was carried.The analysis result reminds the virulence genotyping distribution has relevant to serum typing.