2株牛源多杀性巴氏杆菌的分离鉴定及生物特性

Isolation,Identification and Biological Characteristics Analysis of Two Bovine Pasteurella multocida Strains

为了诊断广东某养殖场肉牛连续出现发热、呼吸困难、病死等原因,对死亡牛进行病理剖检,采集肺、肝组织进行细菌分离。采用染色镜检和16S rRNA序列测定对分离菌进行鉴定;分型PCR对分离菌进行分型,并分析其系统进化;药敏试验分析药物敏感性;绘制生长曲线,挑取对数期细菌进行小鼠攻毒试验确定分离菌的毒性。结果显示:2株分离菌均为革兰阴性杆菌;分型及测序结果显示,2株分离菌均为荚膜A型多杀性巴氏杆菌;生长曲线显示,2株分离菌在6 h后进入平台期,12 h活菌数达到峰值;药敏试验结果显示,分离菌对克林霉素和阿米卡星耐药,对头孢哌酮和左氧氟沙星最敏感;测序发现,分离株与海南株具有较高亲源性;小鼠攻毒试验的结果显示,本菌具有强毒性,可引起小鼠出血性败血症,导致小鼠死亡。结果表明肉牛病死原因可能为多杀性巴氏杆菌感染,应选用敏感药物及时控制牛群继续发生该病。

In order to diagnose the cause of continuous fever,dyspnea,and death of cattle in Guangdong.Dead cattle were dissected,the bacteria were separated from bovine liver and lung,and examined by staining microscopy,16S rRNA sequencing,genotyping and phylogenetic analysis.The antibiotic resistance profile and pathogenicity of the isolates were determined by antibiotic sensitivity test and mouse virulence assay.The results showed that the two isolates were Gram-negative bacilli.Genotyping and sequencing analyses confirmed that they were Pasteurella multocida(P.multocida),serotype A.The growth curve showed that they entered the plateau after 6 h,and the number of viable bacteria reached the peak at 12 h.The results of drug sensitivity tests showed that they were resistant to clindamycin and amikacin,and were most sensitive to cefoperazone and levofloxacin.Sequencing analysis revealed that the isolate had a high affinity with the Hainan strain.The mice virulence assay showed the strains were highly pathogenic and could cause death in mice.Thus,P.multocida is potentially deadly to cattle and can be treated by cefoperazone and levofloxacin.