河北省奶牛场牛呼吸道疾病综合征病原分离鉴定  

Pathogen isolation and identification of bovine respiratory disease complex in dairy farm of Hebei Province

作　　者：高亚桃 王紫燕 马亚宾[2] 蒋桂娥[2] 倪俊卿[2] 张明勇[3] 李妍[1] 武二斌 秦建华[1] 赵月兰[1] GAO Yatao;WANG Ziyan;MA Yabin;JIANG Guie;NI Junqing;ZHANG Mingyong;LI Yan;WU Erbin;QIN Jianhua;ZHAO Yuelan(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 071001,China;Animal Husbandry and Breeding Station of Hebei,Shijiazhuang 050061,China;Animal Husbandry and Aquatic Products Bureau of Huailai 9Zhangjiakou,Hebei 075400,China;Animal Husbandry Technology Promotion Station of Zhangjiakou,Zhangjiakou,Hebei 075000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省畜牧良种工作站,河北石家庄050061 [3]怀来县畜牧水产局,河北张家口075400 [4]张家口市畜牧技术推广站,河北张家口075000

出　　处：《中国兽医学报》2021年第7期1247-1258,共12页Chinese Journal of Veterinary Science

基　　金：河北省现代农业产业技术体系奶牛创新团队建设资助项目(HBCT2018120205);河北省农业厅科技计划资助项目(冀农科17016)。

摘　　要：为了分离与鉴定河北省奶牛场牛呼吸道疾病综合征(bovine respiratory disease complex,BRDC)病原,自河北省张家口、秦皇岛、保定、衡水、沧州、石家庄、邯郸等地区规模化奶牛场采集奶牛呼吸道疾病临床病例鼻拭子样品及死亡奶牛的肺脏组织样品,用牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)、牛副流感病毒3型(bovine parainfluenza virus stype 3,BPIV3)、牛冠状病毒(bovine coronavirus,BCoV)、牛病毒性腹泻病毒(bovine viral diarrhoea virus,BVDV)基因特异性引物进行PCR扩增,初筛阳性的样品进行病毒分离鉴定。用多杀性巴氏杆菌(Pasteurellamultocida,Pm)、肺炎克雷伯杆菌(Klebsiellapneumoniae,Kp)、牛支原体(Mycoplasma bovis,MYPB)和溶血性曼氏杆菌(Mannheimiahaemolytica,Mh)基因特异性引物对所采集样品进行PCR扩增,将初筛阳性样品进行细菌分离鉴定、致病性及药物敏感试验。结果显示,分离出BVDV和IBRV,分别命名为HB-BDZ和HB-XTZ。BVDV分离毒HB-BDZ株E0基因序列与GenBank中发表的其他毒株的同源性为80.5%~99.2%,IBRV分离株HB-XTZ与GenBank其他IBRV参考株gB基因核苷酸同源性为93.8%~100%,同源性分析结果进一步表明,分离毒为BVDV和IBRV。系统发育分析结果显示,分离毒株HB-BDZ与HB-DCZ株(JX046799.1)、JL-1株(KF501393.1)、国外毒株Powder株(JN380089.1)的遗传距离较近,与国内毒株HB-DCZ株同属于Ib亚型。分离株HB-XTZ株与国内XT-IBRV(MF287966)亲缘关系最近,与BHV1.1USA株(KJ652515)和BH80株(AY745877)遗传距离相对较近,而与其他毒株遗传距离较远。利用绵羊血琼脂平板培养基和麦康凯培养基分离出Pm、Kp等2种致病菌,通过生化鉴定和PCR鉴定进一步确定分离菌为Pm、Kp。序列测定及分析结果显示,Pm分离株kmt基因与GenBank其他参考菌株核苷酸序列的同源性为96.4%~100%,与MH06873.1的亲缘关系最近。Kp分�In order to isolate and identify the pathogen of bovine respiratory disease complex(BRDC)in dairy farms in Hebei Province,Nasal swab samples from clinical cases of dairy cows with respiratory diseases and lung tissue samples of dead cows were collected from dairy farms in Zhangjiakou,Qinhuangdao,Baoding,Hengshui,Cangzhou,Shijiazhuang,Handan,etc.To isolate and identify the virus,gene specific primers of infectious bovine rhinotracheitis virus(IBRV),bovine respiratory syncytial virus(BRSV),bovine parainfluenza virus type 3(BPIV3),bovine coronavirus(BCoV),bovine viral diarrhea virus(BVDV)were used for PCR amplification,virus isolation and identification were performed on samples which were positive in preliminary screening.Gene specific primers of Pasteurellamultocida(Pm),Klebsiellapneumoniae(Kp),Mycoplasma bovis(MYPB)and Mannheimiahaemolytica(Mh)were used to carry out PCR amplification on the collected samples,and the positive samples in preliminary screening were subjected to bacterial isolation,identification,pathogenicity and drug sensitivity tests.The results showed that BVDV and IBRV were isolated,they were named as HB-BDZ and HB-XTZ,respectively.The homology of the E0 gene sequence of the BVDV isolated strain HB-BDZ with other strains published in GenBank was 80.5%-99.2%;the nucleotide homology of the gB gene of the IBRV isolated strain HB-XTZ with other IBRV reference strains in GenBank was 93.8%-100%,the result of homology analysis further showed that the isolated viruses were BVDV and IBRV.The results of phylogenetic analysis showed that the genetic distance between the isolated strain HB-BDZ and strain HB-DCZ(JX046799.1),strain JL-1(KF501393.1),and the foreign strain Powder(JN380089.1)was relatively close,and the isolated strain HB-BDZ belonged to the subtypeⅠb,as same as domestic strain HB-DCZ.The isolated strain HB-XTZ strain had the closest genetic relationship with domestic strain XT-IBRV(MF287966)and had relatively close genetic distance with strain BHV1.1 USA(KJ652515)and strain BH80(AY745877),it

关 键 词：牛呼吸道疾病综合征 牛病毒性腹泻病毒 牛传染性鼻气管炎病毒 多杀性巴氏杆菌 肺炎克雷伯杆菌 分离鉴定 

分 类 号：S852.65[农业科学—基础兽医学]