摘要
为探究河南省某大型养猪场育肥猪发生疫情的原因,本研究从该猪场采集的病料样品中进行细菌分离,并对分离菌经革兰氏染色及电镜观察其形态、16S rRNA PCR鉴定;采用多重PCR鉴定其血清型并将扩增的funAB基因与GenBank中的其它副猪嗜血杆菌(HPS)参考株的相应基因进行同源性分析并构建该基因的进化树;采用纸片扩散法鉴定分离菌株的药敏特性并利用小鼠进行了其致病性试验。结果显示:经形态观察、培养特性鉴定以及16S rRNA的PCR鉴定,该分离菌为HPS;经多重PCR并经测序鉴定分离菌为14型HPS,将其命名为JZ-2019;funAB基因的同源性分析及进化树结果显示,JZ-2019株与14型HPS IA-84-22113株(KC795520.1)的同源性为100%。药敏试验结果显示,分离菌对β-内酰胺类、大环内酯类以及氨基糖苷类药物敏感,其中对大环内酯类药物最为敏感;小鼠致病性试验结果显示,小鼠均出现临床症状,甚至死亡,且小鼠各脏器组织均出现不同程度的出血等剖检症状;经PCR扩增从死亡小鼠的脑、腹水中扩增出与分离菌株一致的细菌。表明,该分离菌对小鼠有较强的致病性,且能通过小鼠血脑屏障。本研究证明了河南省猪群中存在14型HPS的感染,为中原地区防控HPS病提供了参考依据。
In order to explore the causes of the epidemic situation of fattening pigs in a large pig farm in Henan province, this study isolated bacteria from the biopsy collected from the farm. The morphology of the isolated bacteria was observed by gram-staining and electron microscopy, and the species was identified by 16 S rRNA PCR and sequencing. Multiplex PCR was used to identify the serotype and homology analysis was conducted between the amplified funAB gene and the corresponding genes of other HPS reference strains in GenBank, and the evolutionary tree of the genes was constructed. The drug sensitivity of the isolates was identified by disk diffusion method and its pathogenicity was tested in mice. The results showed that the isolates was Haemophilus parasuis(HPS) by morphological observation, culture characteristics identification and PCR identification of 16 S r RNA. The isolates was identified as type 14 HPS by multiplex PCR and sequencing, and was named JZ-2019. The homology analysis of funAB gene and the results of evolutionary tree showed that the homology of JZ-2019 strain and type 14 HPS strain IA-84-22113(KC795520.1) was 100%. The results showed that the isolates were sensitive to β-lactams, macrolides and aminoglycosides, among which macrolides were the most sensitive. The pathogenicity test results of mice showed that the mice all had clinical symptoms, even death, and various organs of the mice had different degrees of bleeding and other autopsy symptoms. PCR amplification from the brain and ascites of the dead mice identified the same bacteria as the isolate. These data indicate that the isolates is highly pathogenic to mice, and can pass the blood-brain barrier of mice. This study demonstrates that there are 14 types of HPS infection in pigs of Henan, which provides a reference for the prevention and control of HPS disease in Central Plains.
作者
王玲彩
王青
王晓斐
董萌萌
孙亚伟
杭柏林
胡建和
徐彦召
WANG Ling-cai;WANG Qing;WANG Xiao-fei;DONG Meng-meng;SUN Ya-wei;HANG Bo-lin;HU Jian-he;XU Yan-zhao(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2020年第8期766-771,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发计划项目(2016YFD0500708-4)
国家自然科学基金(31402208)
河南省教育厅项目(19A230001)。
关键词
14型副猪嗜血杆菌
猪
分离
血清型分型
药敏试验
Haemophilus parasuis type 14
swine
isolation
serotyping
drug sensitivity test
