新疆石河子规模化牛场多杀性巴氏杆菌的分离鉴定及药敏试验

Isolation,Identification and Drug Sensitivity Test of Pasteurella multocida from A Large-scale Cattle Farm in Shihezi Xinjiang

为了确定石河子地区某规模化牛场出现呼吸困难、咳嗽、病牛消瘦甚至死亡的病因,本研究以病牛病变组织为研究对象,采用常规细菌分离鉴定和细菌16SrRNA序列分析来鉴定菌种,以及多杀性巴氏杆菌种特异性基因Kmt-1和各个荚膜血清型特异性基因(hyaD-hyaC、bcbD、dcbF、ecbJ、fcbD)PCR扩增来确定细菌的血清型,同时应用纸片扩散法对分离细菌进行药物敏感性试验和小鼠感染试验。结果表明,从病变的肺组织中分离到1株菌落为灰白色、露珠状、不溶血,染色为革兰阴性球杆状细菌,生化鉴定结果符合巴氏杆菌特征,同时16SrRNA序列分析与NCBI上已公布的多杀性巴氏杆菌16SrRNA序列同源性在99%以上;对多杀性巴氏杆菌特异性基因Kmt-1以及各血清型特异性基因PCR扩增只扩增到Kmt-1和hyaD-hyaC特异性基因片段;分离菌株对链霉素、庆大霉素、卡那霉素耐药,对其他30种药物敏感,同时感染小鼠全部死亡。结果显示从病牛体内分离到1株毒力较强的血清A型多杀性巴氏杆菌。

To identify the pathogeny of causing dyspnea,cough,emaciation and death of cattle in an intensive cattle farm in Shihezi region,we isolated bacteria from tissue lesions and used routine isolation and identification methods and 16 SrRNA sequence analysis to identify its species.With the use of primers fromPasteurella multocida species-specific gene Kmt-1and serotype-specific genes（hyaD-hyaC,bcbD,dcbF,ecbJ,fcbD）,we employed PCR to amplify relevant genes for the identification.K-B disk diffusion method and a murine infection model were also used.Results showed that we isolated a strain with a hoar,dewdrop-like,none hemolysis clone which is a type of Gram-negative rhabditiform bacteria.Biochemical identification showed that it is consistent with Pasteurella.And alignments to published sequences on NCBI showed a99%identity with P.multocida;only Kmt-1and hyaD-hyaC genes were amplified with PCR;The isolate was resistant to streptomycin,gentamicin,kanamycin and susceptible to 30 other antimicrobials,and all the infected mice died after challenge.Results showed we isolated a strain of serotype A P.multocida with strong virulence from a sick cattle.