牛源荚膜血清A型多杀性巴氏杆菌的分离鉴定

Isolation and identification of bovine capsular serotype A Pasteurella mutocida

从采集于黑龙江、天津的病牛肺组织中分离到2株病原菌,经菌落形态学观察、培养特性、生化反应和小鼠毒力试验,初步鉴定为多杀性巴氏杆菌,分别命名为Pm-HLJ和Pm-TJ。参考多杀性巴氏杆菌种特异性基因kmt1和荚膜血清型特异性基因hyaD-hyaC、bcbD、dcbF、ecbJ和fcbD,合成引物,通过多重PCR扩增2株菌的种特异性基因和荚膜血清型特异性基因。选取Pm-HLJ的目标PCR产物进行克隆、序列测定、Blast搜索同源序列并且比较分析。结果显示,Pm-HLJ的kmt1基因片段全长460bp,与GenBank中各血清型kmt1基因同源性均在96.6%以上;荚膜血清型A菌株特异性基因同源性为99.9%;而与其他荚膜血清型B、D、E、F的型特异性基因的同源性均低于50%。由此确认,分离的2株多杀性巴氏杆菌均为荚膜血清A型,这是我国A型多杀性巴氏杆菌引发牛出血性败血症的首例报道。

Two Pasteurella mutocida isolates, designated as Pm-HLJ and Pm-TJ, were isolated from affected bovine lung tissues collected from Heilongjiang and Tianjin, and identified by colonial morphology, culture characteristic, biochemistry reaction and mouse pathogenicity test. Subsequently, the genes of the isolates were amplified by multiple PCR, and PCR products of the Pm-HLJ were sequenced for Blast search of species-specific kmt1 gene and serogroup-specific hyaD-hyaC, bcbD, dcbF, ecbJ and fcbD genes. Nucleotide sequence of Pm-HLJ showed more than 96.6 % identity to the kmt1 gene and 99.9 % homologous with the A serogroup-specific gene of other Pasteurella mutocida. Results indicated that the two bacterial isolates belong to capsular serotype A of Pasteurella mutocida, which was emerged in some areas of China.