青海省刚察县某牧场牦牛源坏死杆菌的分子鉴定与分析

Molecular Identification and Analysis of Fusobacterium necrophorum from Yak in a Pasture Farm in Gangcha of Qinghai Province

为确定青海省刚察县某牧场发生耗牛脾脓肿的病原菌,以便于及时防治,本研究于患病解剖牛的脾脓肿部位进行无菌采样,并进行染色形态观察、16S rRNA基因和lktA基因扩增与序列分析等。结果显示:脾脏脓汁涂片瑞氏染色后在显微镜下呈现紫色、丝状、长短不一的杆菌。基于细菌16S rRNA基因通用引物和lktA基因特异性引物扩增,均得到预期的基因目的片段。经测序后分析,两基因分别与坏死杆菌两种基因参考序列具有较高的同源性,都高达99%以上,此病原在分子生物学上鉴定为坏死杆菌,命名为Fusobacterium necrophorum QHGC;基于16S rRNA基因和lktA基因构建的进化树显示:本研究鉴定的克隆序列(16S rRNA和lktA基因)均与其他F.Tiecrop加亚肌分离株聚在一个分支上,在进化角度鉴定了此菌确实为坏死杆菌。本研究表明引起耗牛脾脓肿的病原菌是坏死杆菌,这为本地区耗牛脾脓肿的病原学研究和流行病学调查提供了初步数据,为耗牛脾脓肿的防治提供了参考依据。

In order to determine the pathogens of yak spleen abscess in a pasture farm in Gangcha County of Qinghai Province for timely prevention and control.In this study,the spleen abscess was collected from the died yak,the staining morphology observation,16S rRNA gene and lktA gene amplification and sequence analysis were performed.The results showed that the bacteria was purple,filamentous and different length bacilli in the spleen pus smear after Wright staining under the microscope.Based on the bacterial 16S rRNA gene universal primers and lktA gene specific primers amplification,both expected target fragments were obtained.After sequencing analysis,the two genes fragments were both with high homology to the reference genes sequences of Fusobacterium necrophorum reaching up to 99%,so the pathogen was identified as F.necrophorum and named F.necrophorum QHGC.The phylogenetic trees constructed based on the 16S rRNA gene and lktA gene showed that the identified and cloned sequences(16S rRNA and lktA genes)in this study were clustered with other F.necrophorum isolates in the same branch,so the pathogen was indeed identified as F.necrophorum on evolutionary perspective.This study indicated that the pathogen causing yak spleen abscess was F.necrophorum,which provided preliminary data for the etiological and epidemiological investigation of yak spleen abscess,and provided reference for the prevention and treatment of yak spleen abscess in this area.