禽霍乱巴氏杆菌的分离鉴定及基因分型

Isolation,Identification and Genotyping of Pasteurella multocida from Fowl Cholera

为确定疑似禽霍乱病例病原种类及其病原基因型,本研究采用细菌分离技术对病原菌进行实验室分离培养,应用传统方法和分子生物学方法对分离细菌进行鉴定,并应用PCR扩增和基因序列分析对分离细菌进行基因分型。结果显示,分离菌具有多杀性巴氏杆菌(Pasteurella multocida,Pm)典型培养特征,菌落形态和菌体染色特征、生理生化特性均与多杀性巴氏杆菌相符;PCR扩增到457bp的基因片段。采用5对分型引物对分离菌进行基因分型显示,仅有A型引物扩增到大小1 050bp的目的基因片段,序列分析也显示分离菌荚膜基因与A型多杀性巴氏杆菌参考菌株荚膜特异性基因同源性高达97.6%~100.0%,系统进化与A型多杀性巴氏杆菌处于同一进化分支。结果表明,疑似禽霍乱病例病原为A型多杀性巴氏杆菌,本研究结果将为禽霍乱的防控提供参考资料。

In order to determine the suspected avian cholera pathogen and geneotypes,bacteria isolation technology was used to isolate and culture pathogenic bacteria,the isolated bacteria were identified by traditional methods and molecular biological methods.Geneotyping of the isolated bacteria were determined using PCR technology and gene sequence analysis.The results demonstrated that the isolated bacteria had typical culture characteristics of Pasteurella multocida（Pm）,and the morphology of the colony,the characteristics of cell staining,the physiological and biochemical characteristics were consist with Pm;457bp fragment was amplified by PCR.Geneotyping results showed that only A primer amplified the target gene fragment of 1 050 bp,sequence analysis also showed that the isolated bacteria shared 97.6%to 100.0%sequence homology with reference strains,and phylogenetic tree analysis showed that the isolated bacteria and A type Pm were in a branch.The experimental results indicated that the isolated bacteria was identified as capsular serotype A of Pm,the results provided reference for the prevention and control of fowl cholera.