【作者】 马秋明；

【导师】 何维明； 王晶钰；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2008， 硕士

【摘要】 牛病毒性腹泻-黏膜病(Bovine viral diarrhea-mucosal disease ,BVD-MD)是由牛病毒性腹泻-黏膜病病毒(Bovine viral diarrhea virus ,BVDV)引起的一种以发热、肠粘膜糜烂溃疡、白细胞减少、腹泻、咳嗽、持续性感染与免疫耐受及怀孕母牛流产或产出畸形胎儿为主要特征的,复杂的,呈多种临床类型的传染病,对养牛业危害非常大,是世界各国重点防制和检疫的对象。BVD-MD主要感染牛、羊、鹿、猪等。此病的分布几乎遍布全世界,我国自1980年首次报道该病以来,已有20多个省份相继报道有本病的发生。1994年陈茂盛等对陕西省13个县的肉牛、奶牛BVDV的感染情况做了血清学调查,结果发现阳性率为12.7%。近年来该病在陕西牛群中的流行有扩大的趋势,但至今未见BVDV陕西分离株的相关研究报道。2007年陕西某规模化奶牛场多头犊牛发生顽固性腹泻,疑为BVDV或轮状病毒(Rotavirus,RV)感染所致,笔者随即采集病死犊牛的肠系淋巴结,对病原进行了分离鉴定。参考NS5B基因保守序列设计1对引物::P1:5′-TGG AGA TCT TTC ACA CAA TAG C -3′;P2:5′-GCT GTT TCA CCC AGT TAT ACA T -3′,用于BVDV NS5b基因的扩增;另外参照RV的VP1基因保守序列设计1对引物,P3:5′-AGA ATT TCC GTT TGG CTA-3′;P4:5′-CGG TCA CAT CAT ACA ACT CT-3′,用于RV VP1基因扩增。取发病犊牛的肠系淋巴结,进行研磨处理后,取上清液,按Invitrogen公司的TRIZOL LS? Reagent试剂盒操作说明书提取病毒基因组RNA,进行RT-PCR扩增。结果扩增到约360 bp的BVDV NS5B基因,未扩增到RV VP1基因,初步鉴定为牛病毒性腹泻-黏膜病。取病料上清液接种于MDBK细胞,盲传4代后出现典型、规律的细胞病变。采用病毒蚀斑技术对病毒进行克隆纯化,提取其基因组RNA,再次进行RT-PCR鉴定。用Gel Extraction Mini Kit W5212纯化回收RT-PCR产物,将扩增的NS5B基因片段与pMD18-T载体连接构建重组质粒,将重组质粒转化DH5α感受态细胞,采用碱裂解法小量提取质粒,并对质粒进行酶切鉴定(EcoRⅠ和SalⅠ双酶切及SalⅠ单酶切)和PCR鉴定。将阳性克隆送上海生工生物工程技术服务有限公司和上海捷瑞生物工程有限公司分别测序后进行序列分析。结果显示,分离毒株为致细胞病变型(CP型)BVDV,与匈牙利VEDEVAC株的核苷酸同源性最高按Reed-Muench法计算病毒的TCID50为99%。取病毒克隆株的细胞培养物上清作10倍系列稀释,接种96孔培养板,逐日观察并记录细胞病变孔数,按Reed-Muench法计算病毒的TCID50,结果显示BVDV陕西分离株的TCID50为10-3.62/0.1mL。更多还原

【Abstract】 Bovine viral diarrhea-mucosal disease (BVD-MD) caused by the Bovine viral diarrhea virus (BVDV) is a serious disease for the cattle , this disease showed many clinical type, infects a large proportion of cattle worldwide and causes a number of clinical forms of the disease ranging from transiently detectable mild clinical symptoms to a fatal diarrheic condition known as mucosal disease.BVD-MD mainly infects cattle, caprine, deer, swine et. The common symptoms shows fever, diarrhea, mucosal anabrosis, aleucocytosis, milk-reduction, abortion or featus anomalies.BVDVcauses persistence infection, immune tolerance,breeding disable, plate-reduction. Since BVD-MD was firstly reported in 1980 in China, it was reported in many other province in the later years. In 1994, Chen Mao-sheng detected this disease by serology in 13 counties of Shannxi province, the results showed that the positive proportion is 12.7%. Recently years, this disease is spreading in Shannxi, but there is not any BVDV strain separated in Shannxi. In 2007, the cattles of one of the scale cattle nursery showed refractoriness diarrhea, and these disease was suspected to be caused by BVDV or RV. Therefore, the cattle intestine absorbent gland was separated and two pairs of primers were designed. The first pair of primers , P1:5′-TGG AGA TCT TTC ACA CAA TAG C- 3′, P2:5′-GCT GTT TCA CCC AGT TAT ACA T- 3′,are used for the amplification of NS5b gene of BVDV, and the other pair of primers P3:5′-AGA ATT TCC GTT TGG CTA-3′;P4:5′-CGG TCA CAT CAT ACA ACT CT-3′was designed according to conventional sequence of VP1 gene of RV.The total RNA was isolated from the intestine absorbent gland according to the procotals of TRIZOL LS? Reagent(Invitrogen), and the results of the RT-PCR show that the 360bp fragments of NS5 gene of BVDVwas succssfully amplyfied,VP1 gene of RV was not amplified.The above results suggest that this disease is Bovine viral diarrhea-mucosal disease. The supernatant of the intestine absorbent gland was innoculated to MDBK cell line, afer 4 passages, the cells showed classical and regular cytopathic effect. The virus was purified by the plaque technic, and the RNA was isolated and detected by RT-PCR, the fragments of NS5b gene were purified by Gel Extraction Mini Kit W5212 and successfully cloned into the pMD18-T vector, the recommbanant plasmid was identified by incision enzyme digestion and sequenced by Sangon CO.Ltd. The determined sequences share great homolgy(99%) with VEDEVAC strain. The supernatant of cell culture infected by the isolated BVDV was serially diluted and innoculated to 96 well plate, the results of the detection by Reed-Muench of TCID50 ranged is 10-3.62/0.1mL更多还原