致肉牛运输热溶血曼氏杆菌的分离鉴定及部分生物学特性研究

Isolation,Identification and Partial Biological Characteristics of Mannheimia haemolytica in Shipping Fever of Beef Cattle

为探明一起肉牛运输热的病原及生物学特性,本研究无菌采集病死牛心血、肺脏、肝脏和脾脏,对其进行细菌分离、生化试验和PCR鉴定,并对分离株进行毒力基因检测、致病性研究。结果显示,7株分离菌均为革兰氏阴性短杆菌,具有微弱的β-溶血,瑞氏染色可见两极浓染及明显的荚膜。生化试验结果显示,分离菌能发酵葡萄糖、麦芽糖、阿拉伯糖、甘露醇、甘露糖、木糖等碳水化合物,不发酵脲酶、MR-VP和吲哚,产生少量酸而不产气,结果符合溶血曼氏杆菌生化特性。PCR鉴定均为荚膜血清A2型,分离菌均含有四型菌毛相关基因ptfA、参与复制相关基因dnaN、白细胞介素相关基因LktC3种毒力基因。分离菌对小鼠的LD50值在107.83～108.50 CFU/mL之间,不同菌株间小鼠LD50值存在一定差异,但差异不明显。结果表明,引起该批肉牛运输热的病原为携带毒力基因的荚膜血清A2型溶血曼氏杆菌,本研究结果为进一步研究溶血曼氏杆菌的致病机制提供参考。

In order to identify the pathogens and biological characteristics of shipping fever of beef cattle.The bacteria of blood(heart),lung,liver and spleen were collected and isolated,biochemical tested,and PCR identified,the virulence gene detection and pathogenicity of the isolates were studied.The results showed that 7 isolates were Gram-negative brevibacterium with weakβ-hemolysis,Wright’s staining result showed that there was two-pole dense staining and obvious capsule.The biochemical test results showed that the isolated bacteria could ferment carbohydrates such as glucose,maltose,arabinose,mannitol,mannose,xylose etc.and do not ferment urease,MR-VP and indole.It could utilize carbohydrates such as glucose to produce a small amount of acid without gas production,and the results were consistent with the biochemical characteristics of Mannheimia haemolytica.The isolates were capsular serum A2 type by PCR,which contained three virulence genes of the typeⅣfimbriae-related gene ptfA,the copy-related gene dnaN,and the interleukin related gene LktC.The LD50 values of the bacteria in mice ranged from 107.83 to108.50 CFU/mL,there were some difference of LD50 values in different strains.The results showed that the pathogen which caused the shipping fever in the batch of beef cattle was Mannheimiahaemolytica with capsular serum A2 type,which provided a reference for further study of the pathogenic mechanism of Mannheimia haemolytica.