摘要
为全面了解吉林省某规模化牛场牛病毒性腹泻(BVD)的流行情况,试验采集临床血清样品157份,粪便样品18份,肝脏、精液等组织样品17份,应用BVDV抗体检测试剂盒进行血清抗体检测,利用BVDV1型引物,采用纳米PCR方法对血清及临床样品进行BVDV抗原检测,对抗原阳性样品进行测序分析;将抗原阳性样品经研磨、稀释后用0.22μm滤膜过滤除菌,接入牛肾细胞(MDBK)进行病毒分离培养,盲传3代后再次进行抗原检测;采用免疫荧光技术检测病毒对MDBK细胞的侵染作用;利用Mega软件绘制系统进化树并进行同源性比对分析。结果显示,该牛场临床血清BVDV抗体阳性率为77.1%,血清抗原阳性率为12.1%,临床粪便等样品抗原阳性率为74.3%;病料接入细胞未观察到细胞病变,分离毒株PCR产物测序分析结果与样品抗原检测结果一致;免疫荧光检测结果显示,分离株毒液正常吸附于MDBK细胞中,有明显荧光反应;抗原测序分析显示,该牛场BVD主要流行毒株与BVDV JL-1株同源性高达99.0%,且均为BVDV 1型毒株。本研究对该牛场BVDV流行情况进行了全面调查,为开展净化工作奠定了基础。
In order to totally understand the prevalence of bovine viral diarrhea(BVD)in a largescale cattle farm in Jilin province,a total of 157 clinical serum samples,18 stool samples,17 liver and semen tissue samples were collected.Serum antibody detection was performed using BVDV antibody detection kit.BVDV antigen detection was performed on serum and clinical samples by Nano-PCR method and BVDV type 1specific primers,the antigen positive results were sequenced and analyzed;The antigen positive samples were ground and diluted,filtered and sterilized by0.22μm filter membrane,and connected to MDBK cells for virus isolation and culture.The antigen detection was carried out again after three generations;The infection of MDBK cells was detected by immunofluorescence technique;Phylogenetic tree and homology analysis were drawn byMega software.The results showed that the positive rate of clinical serum BVDV antibody in the cattle farm was 77.1%,the positive rate of serum antigen was 12.1%,and the positive rate of sample antigen in clinical feces was 74.3%.After the diseased material was connected to the cells,the cytopathic effect could not be observed.PCR results of the obtained strains were consistent with the results of sample antigen detection.The immunofluorescence assay results showed that BVDV venom was normally adsorbed in MDBK cells,and there was obvious fluorescence reaction.The main strain of BVD and BVDV JL-1strain were analyzed by antigen sequencing,the homology was as high as 99.0%,and both were BVDV type 1strains.This study had conducted a comprehensive survey of the prevalence of BVDV in the cattle farm,laying the foundation for the purification work.
作者
刘泽余
李健友
刘占悝
李智杰
王超
郭利
LIU Zeyu;LI Jianyou;LIU Zhankui;LI Zhijie;WANG Chao;GUO Li(JilinAgricultural University,Changchun 130118,China;Key Laboratory of Special Animal Epidemic Disease,Ministry of Agriculture,Institute of Special Economic Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China)
出处
《中国畜牧兽医》
CAS
北大核心
2019年第1期247-255,共9页
China Animal Husbandry & Veterinary Medicine
基金
吉林省科技厅产业技术创新战略联盟项目(20170309002NY)
国家重点研发计划:畜禽重大疫病防控于高效安全养殖综合技术研发(2016YFD0500907)
作者简介
同等贡献,并列为第一作者:刘泽余(1994-),女,吉林长春人,硕士生,研究方向:临床兽医学,E-mail:Liuzy941206@163.com;同等贡献,并列为第一作者:李健友(1991-),男,山东临沂人,硕士生,研究方向:预防兽医学,E-mail:cctcs-liy@163.com;通信作者:郭利(1977-),女,吉林长春人,副研究员,研究方向:动物病毒和生物制品,E-mail:piaogl110@163.com.
