安格斯牛源多杀性巴氏杆菌的分离及其种特异性基因的克隆与序列分析

Isolation of Pasteurella multocida in Angus stud bull and its species-specific gene cloning and sequence analysis

为了研究引起青海省海西州漠河骆驼场安格斯种牛致死病因及其病原特征,试验以C47-8为质控菌株,采用常规细菌分离鉴定技术对无菌采集的病死安格斯种牛病料进行了多杀性巴氏杆菌的分离与纯化、生化鉴定、致病性试验、药敏试验及其种特异性基因Kmt-1的检测。结果表明:临床分离株为革兰氏阴性的短杆菌,病料触片经美蓝染色镜检可见其呈两极着色特性;与C47-8间存在乳糖、蔗糖、木糖、水杨苷及尿素酶等5种生化鉴定差异;对小鼠具有致死性效应;对红霉素等22种抗生素耐药,平均耐药率为62.85%(22/34),而质控菌株则无耐药性。同时对提取的临床分离株DNA进行Kmt-1基因的PCR扩增,分别获得了大小为460 bp和253 bp的Kmt-1基因扩增片段;经序列分析,临床分离株Kmt-1基因与Gen Bank公布的序列之间的基因编码区核苷酸序列同源性为97%~100%;检测的最低DNA浓度为790 ng/m L;所应用的2对Kmt-1基因检测引物对伤寒沙门氏菌、大肠埃希氏菌和克雷伯氏菌的扩增均为阴性。说明此次安格斯种公牛发病为多杀性巴氏杆菌引起,建议该养殖场在做好饲养管理的同时,应加强针对多杀性巴氏杆菌的免疫预防和消毒处理措施,以减少或杜绝本病的再次发生。

In order to research the cause and pathogen charactristics which led to the death of Angus stud bulls which lived in Mohe camel farm in Haixi prefecture of Qinghai province, C47 -8 strain was used as reference strain in this experiment. Routine bacteria isolation and identifica- tion methods were employed to isolate and purify PasteureUa multocida from sterilly collected samples of the dead Angus stud bull. Biochemical identification, pathogenicity experiment and antimicrobial susceptibility test for the bacteria were conducted, also including the determination of its species - specific gene Kmt - 1. The results showed that the clinical isolate was short gram - negative bacillus, and presented dipolar colour- ing feature by methylene blue staining. Five aspects of biochemical identification differences obtained between this bacteria and C47 - 8 strain, containing lactose, sucrose, xylose, salicin and urease tests. It had lethal effect on mice, and was resistant to 22 kinds of antibiotics with an aver- age resistance rate of 62.85 % （22/34）while reference strain had no resistance to the drugs. The isolated strain＇ s DNA was extracted and PCR was used for amplifying Kmt - 1 gene. The fragments of 460 bp and 253 bp respectively were obtained. Sequence analysis showed that nucleo- tide sequence of Kmt - l coding region of the isolate had 97% ~ 100% homology with those published in GenBank database. The minimum DNA concentration for detection was 790 ng./mL;and PCR tests for Salmonella typhi, Esctierichia coli and KlebsieUa were negative when using the applied 2 pairs of Kmt - 1 specific primers. The results indicated that Pasteurella multocida was the cause which led to the death of Angus stud bulls,and a proposal was concluded that except making better feeding management, the farm should strengthen the immunization against Pasteurella multocida and disinfection measure to reduce or completely eradicate the disease.