牛病毒性腹泻—黏膜病病毒分离鉴定及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2015.09.027

摘要/Abstract

摘要： 为分离鉴定新疆地区牛病毒性腹泻—黏膜病病毒(BVDV)流行毒株,掌握该毒株的生物学特性,本试验在新疆北疆部分地区采集牛病毒性腹泻—黏膜病(BVD/MD)疑似病例的粪便,通过RT-PCR检测、细胞分离培养、间接免疫荧光抗体检测、免疫电镜观察及血清中和试验5种方法对毒株进行分离和鉴定。对毒株的TCID₅₀测定后,再对毒株分别进行乙醚敏感性试验、氯仿敏感性试验、胰蛋白酶敏感性试验、酸碱度敏感性试验、温度敏感性试验及核酸分型试验等理化特性检测。经RT-PCR诊断,病料在286 bp处出现了目的片段。将RT-PCR诊断为阳性的粪便,接种于密度约为80%的单层MDBK细胞出现了细胞病变,盲传5代至出现典型的细胞病变。将F5代细胞培养物采用间接免疫荧光抗体检测,结果产生了与C₂₄V标准毒株相同的特异性黄绿色荧光。免疫电镜观察到了大量呈球形的BVDV粒子,大小20~40 nm。血清中和试验中抗体阳性血清处理组细胞均未出现细胞病变,病毒完全被抗体阳性血清中和。综合以上方法确定分离株为BVDV毒株。对分离株进行毒价和理化特性测定,该毒株TCID₅₀为10^-4.5/0.1 mL,对乙醚和氯仿敏感,对胰蛋白酶敏感,耐碱不耐酸,对温度敏感,经54 ℃ 1 h完全被灭活,属于RNA病毒。本试验成功分离到一株新疆BVDV流行毒株,掌握了该毒株的生物学特性,为今后该病的诊断和防控奠定了基础。

关键词: 牛; 牛病毒性腹泻— 黏膜病病毒(BVDV); 分离鉴定; 生物学特性

Abstract: In order to isolate and identify bovine viral diarrhea-mucosal virus (BVDV) strain of Xinjiang, grasp the biological characteristics of the strain, we gathered feces which were suspected as bovine viral diarrhea-mucosal disease infected feces in the northern parts of Xinjiang.The strain was separated and identified by RT-PCR, cell isolation and culture, indirect immunofluorescence antibody test, immune electron microscopy and serum neutralization test.After determining TCID₅₀ of the strain, ether sensitivity test, chloroform sensitivity test, trypsin sensitivity test, acid and alkali sensitivity test, temperature sensitivity test and nucleic acid typing test were used to detect the physical and chemical properties of the strain.The samples appeared fragment at 286 bp by RT-PCR, and then inoculated the positive samples into a density of about 80% monolayer MDBK cells, and appeared cytopathic effect, passaged to the 5th generation caused the typical cytopathic effect.The F5 cell cultures were detected by indirect immunofluorescent antibody, the result showed that a yellow-green fluorescence appeared same as C₂₄V.A lot of spherical BVDV particles with the size of 20 to 40 nm were observed by immune electron microscopy.Serum neutralization test had no cytopathic effect, viruses were completely neutralized by positive antibody serum.The isolated strain was determined to be BVDV based on the methods above.The TCID₅₀ of the isolated strain was 10^-4.5/0.1 mL.Physicochemical assays showed that the isolated strain was sensitive to chloroform, ether, trypsin and temperature, sensitive to acid and resistant to alkali in acid and alkali sensitivity test, and was completely inactivated by 54 ℃ for 1 h.The virus genome was RNA.The study successfully isolated a BVDV strain in Xinjiang and grasped the biological characteristics of the strain, which laid a foundation for future diagnosis and prevention of the disease.

Key words: bovine; bovine viral diarrhea-mucosal disease virus (BVDV); isolation and identification; biological characteristics

中图分类号:

S858.23

包振中, 雷程红, 蔡元庆, 高窦, 卞赛赛, 葛婷. 牛病毒性腹泻—黏膜病病毒分离鉴定及生物学特性研究[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(9): 2391-2398.

BAO Zhen-zhong, LEI Cheng-hong, CAI Yuan-qing, GAO Dou, BIAN Sai-sai, GE Ting. Isolation,Identification and Biological Characteristics Analysis of Bovine Viral Diarrhoea-mucosal Disease Virus[J]. , 2015, 42(9): 2391-2398.

参考文献

[1] 张淑琴,谭斌,王风雪,等.牛病毒性腹泻BVDV-JL分离株全基因序列测定与分析[J].中国畜牧兽医,2014,41(10):23-27.

[2] 李文文,刘情,刘亚刚.牛病毒性腹泻病毒间接ELISA方法的建立[J].中国畜牧兽医,2015,42(3):558-562.

[3] 陆承平.兽医微生物学[M].第4版.北京:中国农业出版社,2007.

[4] Abu E E,Alkhalyifa M.Studies on BVD involving establishment of sentinel calves and assessment of herd immunity in a large dairy farm in Saudi Arabia[J].Trop Anim Health Prod,2012,44(3):413-417.

[5] 王春庆.牛病毒性腹泻病毒一步法RT-PCR检测方法的建立和应用[J].中国畜牧兽医,2013,40(1):66-68.

[6] Bachofen C,Bollinge R B,Peterhans E,et al.Diagnostic gap in bovine viral diarrhea virus serology during the periparturient period in cattle[J].J Vet Diagn Invest,2013,25(5):655-661.

[7] Karakayae E,Alpay G,Yilmazbas M G,et al.Perosomuselumbis in a Holstein calf infected with bovine viral diarrhea virus[J].Tierarztl Prax Ausg Grosstiere Nutztiere,2013,41(6):387-391.

[8] Olafson P,MacCallum A D,Fox F H.An apparently new transmissible disease of cattle[J].Cornell Vet,1946,36:205-213.

[9] 李佑民,刘振润,武银莲.牛病毒性腹泻—粘膜病病毒株(长春184)的分离和鉴定[J].兽医大学学报,1983,3(2):113-120.

[10] 孔德繁,陆承平.牛病毒性腹泻—黏膜病的最新研究进展[J].福建畜牧兽医,2005,27(3):9-13.

[11] 赵霞,岳海宁,李福红,等.牛病毒性腹泻病毒抗原抗体检测方法研究进展[J].安徽农业科学,2014,42(18):5876-5878.

[12] 王治才,刘崇向,赵泽森,等.牛腹泻病病毒感染羔羊的调查[J].中国畜禽传染病,1992,3:1-43.

[13] 郭燕.新疆北疆部分集约化奶牛场牛病毒性腹泻病毒分子流行病学调查[D].石河子:石河子大学,2007.

[14] 李娜,韩猛立,黄新,等.新疆石河子地区牛病毒性腹泻病毒的分子流行病学调查[J].石河子大学学报(自然科学版),2009,27(6):706-710.

[15] Zhong F,Li N,Huang X,et al.Genetic typing and epidemiologic observation of bovine viral diarrhea virus in Western China[J].Virus Genes,2011,42(2):204-207.

[16] 郭芙莉.牛病毒性腹泻/黏膜病的血清学调查及病毒分离[D].乌鲁木齐:新疆农业大学,2013.

[17] 马秋明,王学艳,王晶钰,等.牛病毒性腹泻病毒陕西株的分离与NS5b基因序列测定[J].西北农林科技大学学报(自然科学版),2008,36(12):1-6.

[18] 季新成,牛国辉,员丽娟,等.不同方法对牛病毒性腹泻检测比较[J].中国动物检疫,2009,26(7):54-56.

[19] 侯佩莉,杨宏军,宋玲玲,等.牛病毒性腹泻病毒山东株的分离鉴定[J].山东农业科学,2013,45(2):41-44.

[20] Zhu L Q,Lin Y Q,Ding X Y,et al.Genomic sequencing and characterization of a Chinese isolate of bovine viral diarrhea virus 2[J].Acta Virologica,2009,53(2):197-202.

[21] 杨小燕,魏春华,刘建奎,等.猪源牛病毒性腹泻病毒的分离鉴定[J].中国兽医科学,2011,4(11):9-13.

[22] 赵月兰,左玉柱,范京惠,等.牛病毒性腹泻/黏膜病病毒河北分离株的生物学特性[J].中国农学通报,2006,23(11):1-4.

[23] 邓宇,孙春清,张宏彪,等.一株猪源牛病毒性腹泻病毒的分离与鉴定[J].畜牧兽医学报,2012,43(3):416-423.

[24] 郑全.梅花鹿粘膜病病毒的分离鉴定[D].长春:吉林农业大学,2004.

[25] 杜锐,王新平,王树志,等.鹿粘膜病病毒的分离鉴定[J].中国预防兽医学报,2004,26(5):377-379.