牛支原体新疆株的分离鉴定

Isolation and Identification of Mycoplasma bovis in Xinjiang

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。

In order to investigate and determine the pathogen from calf which showed pneumonia symptoms occurred in cattle farms in Xinjiang,one strain of Mycoplasma bovis was isolated from seven pneumonic lung tissue samples of calves using liquid and solid Mycoplasma medium.The isolated strain was characterized by the colonial morphologic observation and biochemistry test.Specific primers of Mycoplasma bovis and universal 16S rRNA primers of Mycoplasma were employed to amplify and the amplicons were sequenced as well.DNAStar software was used to compare the sequence between the isolate and the other strains deposited in GenBank.A phylogenetic tree of different strains based on 16S rRNA sequences was constructed by Mega 6.0 software with the Neighbor-Joining method.The results showed that colonies of isolated strain grew on solid medium were typical fried-eggs,the central depression of the colony was deep into the medium and thin and transparent periphery,and Dienes staining was dark blue.The isolated strain could not ferment glucose,not decompose urea,and not hydrolyze arginine.Hemadsorption test and hemolysis test were negative,while film and spot test and triphenyl tetrazolium chloride reduction reaction test were positive.PCR products of Mycoplasma bovis showed the specific band of 1 911 bp. Sequence analysis indicated that 16S rRNA of the isolated strain showed 99.8% homology with that of Mycoplasma bovis strain PG45,and 99.3% to 99.7% homology with that of local strains of Mycoplasma bovis (Mb NM2012,Mb HB0801,Mb Hubei-1,Mb Ningxia-1,Mb CQ-W70 and Mb 08M strains).The phylogenetic tree showed that the 16S rRNA gene of the isolated strain was closely relative with Mb Ningxia-1 and Mb 08M strains,which were in the same branch.The results confirmed that the pathogenic bacteria causing cattle death was Mycoplasma bovis ,which provided theoretical basis and scientific basis for the prevention and treatment of Mycoplasma bovis in Xinjiang.