吉林某牛场牛病毒性腹泻流行病学调查及分析
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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.
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.
引文
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[13]李健友,刘泽余,刘占悝,等.牛病毒性腹泻病毒Nano-PCR检测方法的建立及初步应用[J].中国兽医科学,2018,48(5):580-585.LI J Y,LIU Z Y,LIU Z K,et al.Establishment and preliminary application of Nano-PCR for the detection of bovine viral diarrhea virus[J].Chinese Veterinary Science,2018,48(5):580-585.(in Chinese)
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[15]陶洁,廖金虎,张倩,等.猪感染牛病毒性腹泻病毒研究进展[J].中国预防兽医学报,2014,36(5):410-413.TAO J,LIAO J H,ZHANG Q,et al.Advances in research on swine infection with bovine viral diarrhea virus[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(5):410-413.(in Chinese)
[16]BEZEK D M,GROHN Y T,DUBOVI E J.Effect of acute infection with noncytopathic or cytopathic bovine viral diarrhea virus isolates on bovine platelets[J].American Journal of Veterinary Research,1994,55(8):1115-1119.
[17]唐新仁,陈亮,傅小平.牛病毒性腹泻病对牧场生产的直接影响[J].中国乳业,2010,5:50-52.TANG X R,CHEN L,FU X P.Direct impact of bovine viral diarrhea on pasture production[J].Chinese Dairy Industry,2010,5:50-52.(in Chinese)
[18]王炜.牛主要呼吸道病毒病血清学调查、牛病毒性腹泻病毒分离株鉴定及疫苗研究[D].北京:中国农业科学院,2014.WANG W.Serological investigation of major respiratory virus in cattle,identification of bovine viral diarrhea virus isolates and vaccine research[D].Beijing:Chinese Academy of Agricultural Sciences,2014.(in Chinese)
[19]魏伟,李岩,刘长军,等.黑龙江省部分奶牛场牛病毒性腹泻病毒感染的血清学调查[J].中国兽医科学,2009,39(6):561-563.WEI W,LI Y,LIU C J,et al.Serologic investigation of bovine viral diarrhea virus infection on some dairy cow farms in Heilongjiang province[J].Chinese Veterinary Science,2009,39(6):561-563.(in Chinese)
[20]童钦,吕晓妍,王炜,等.内蒙古牛病毒性腹泻病毒血清学调查[J].中国农学通报,2013,29(5):26-29.TONG Q,LYU X Y,WANG W,et al.Seroepidemiological survey on bovine viral diarrhea virus infection in Inner Mongolia[J].Chinese Agricultural Science Bulletin,2013,29(5):26-29.(in Chinese)
[21]姚伟.辽宁地区规模化奶牛场牛病毒性腹泻-黏膜病血清学调查[J].现代畜牧兽医,2015,4:36-40.YAO W.Serologic study on bovine viral diarrhea in dairy cattle from scale dairy farms in Liaoning[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2015,4:36-40.(in Chinese)
[22]倪惠军,王英,许利琴,等.牛病毒性腹泻的流行病学调查[J].上海畜牧兽医通讯,2018,3:34-35.NI H J,WANG Y,XU L Q,et al.Epidemiological investigation of bovine viral diarrhea[J].Shanghai Livestock and Veterinary Communication,2018,3:34-35.(in Chinese)
[23]KENNY V.Strategies for the control and prevention of bovine viral diarrhea virus[J].Veterinary Clinics Food Animal Practice,2004,20(1):171-180.
[24]WALZ P H,GROOMS D L,PASSLER T,et al.Control of bovine viral diarrhea virus in ruminants[J].Journal of Veterinary Internal Medicine,2010,24(3):476-486.
[2]KHODAKARAM-TAFTI A,FARJANIKISH G H.Persistent bovine viral diarrhea virus(BVDV)infection in cattle herds[J].Iranian Journal of Veterinary Research,2017,18(3):154-163.
[3]BIELANSKI A,ALGIRE J,LALONDE A,et al.Transmission of bovine viral diarrhea virus(BVDV)via in vitro-fertilized embryos to recipients,but not to their offspring[J].Theriogenology,2009,71(3):499-508.
[4]BROCK K V.The many faces of bovine viral diarrhoea virus[J].The Veterinary Clinics North America Food Animal Practice,2004,20(1):1-3.
[5]CAMPBELL J R.Effect of bovine viral diarrhea virus in the feedlot[J].The Veterinary Clinics North America Food Animal Practice,2004,20(1):39-50.
[6]穆昱,操时伟,周玉龙,等.Bac-to-Bac系统表达鹿源BVDV E2蛋白及其免疫原性[J].中国兽医学报,2014,34(12):1899-1905.MU Y,CAO S W,ZHOU Y L,et al.Eukaryotic expression with the bac to bac system and immunogenicity of deer BVDV E2protein[J].Chinese Journal of Veterinary Science,2014,34(12):1899-1905.(in Chinese)
[7]张淑琴,谭斌,王凤雪,等.牛病毒性腹泻病毒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.ZHANG S Q,TAN B,WANG F X,et al.Determination and analysis of whole gene sequence of bovine viral diarrhea virus BVDV-JL isolate[J].China Animal Husbandry&Veterinary Medicine,2014,41(10):23-27.(in Chinese)
[8]BHUYAN A A,MEMON A M,BHUIYAN A A,et al.The construction of recombinant Lactobacillus casei expressing BVDV E2protein and its immune response in mice[J].Journal of Biotechnology,2018,270:51-60.
[9]邢思毅.BVDV重组E2蛋白间接ELISA检测方法的建立及初步应用[D].大庆:黑龙江八一农垦大学,2015.XING S Y.Establishment and preliminary application of indirect ELISA for detection of BVDV recombinant E2protein[D].Daqing:Heilongjiang Bayi Agricultural University,2015.(in Chinese)
[10]GROOMS D L.Reproductive consequences of infection with bovine viral diarrheavirus[J].The Veterinary Clinics North America Food Animal Practice,2004,20(1):5-19.
[11]HOUE H,LINDBERG A,MOENNIG V.Test strategies in bovine viral diarrhea virus control and eradication campaigns in Europe[J].Journal of Veterinary Diagnostic Investigation,2006,18(5):427-436.
[12]BROCK K V.Strategies for the control and prevention of bovine viral diarrhea virus[J].The Veterinary Clinics North America Food Animal Practice,2004,20(1):171-180.
[13]李健友,刘泽余,刘占悝,等.牛病毒性腹泻病毒Nano-PCR检测方法的建立及初步应用[J].中国兽医科学,2018,48(5):580-585.LI J Y,LIU Z Y,LIU Z K,et al.Establishment and preliminary application of Nano-PCR for the detection of bovine viral diarrhea virus[J].Chinese Veterinary Science,2018,48(5):580-585.(in Chinese)
[14]HESSMAN B E,SJEKLOCHA D B,FULTON R W,et al.Acute bovine viral diarrhea associated with extensive mucosal lesions,high morbidity,and mortality in a commercial feedlot[J].Journal of Veterinary Diagnostic Investigation,2012,24(2):397-404.
[15]陶洁,廖金虎,张倩,等.猪感染牛病毒性腹泻病毒研究进展[J].中国预防兽医学报,2014,36(5):410-413.TAO J,LIAO J H,ZHANG Q,et al.Advances in research on swine infection with bovine viral diarrhea virus[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(5):410-413.(in Chinese)
[16]BEZEK D M,GROHN Y T,DUBOVI E J.Effect of acute infection with noncytopathic or cytopathic bovine viral diarrhea virus isolates on bovine platelets[J].American Journal of Veterinary Research,1994,55(8):1115-1119.
[17]唐新仁,陈亮,傅小平.牛病毒性腹泻病对牧场生产的直接影响[J].中国乳业,2010,5:50-52.TANG X R,CHEN L,FU X P.Direct impact of bovine viral diarrhea on pasture production[J].Chinese Dairy Industry,2010,5:50-52.(in Chinese)
[18]王炜.牛主要呼吸道病毒病血清学调查、牛病毒性腹泻病毒分离株鉴定及疫苗研究[D].北京:中国农业科学院,2014.WANG W.Serological investigation of major respiratory virus in cattle,identification of bovine viral diarrhea virus isolates and vaccine research[D].Beijing:Chinese Academy of Agricultural Sciences,2014.(in Chinese)
[19]魏伟,李岩,刘长军,等.黑龙江省部分奶牛场牛病毒性腹泻病毒感染的血清学调查[J].中国兽医科学,2009,39(6):561-563.WEI W,LI Y,LIU C J,et al.Serologic investigation of bovine viral diarrhea virus infection on some dairy cow farms in Heilongjiang province[J].Chinese Veterinary Science,2009,39(6):561-563.(in Chinese)
[20]童钦,吕晓妍,王炜,等.内蒙古牛病毒性腹泻病毒血清学调查[J].中国农学通报,2013,29(5):26-29.TONG Q,LYU X Y,WANG W,et al.Seroepidemiological survey on bovine viral diarrhea virus infection in Inner Mongolia[J].Chinese Agricultural Science Bulletin,2013,29(5):26-29.(in Chinese)
[21]姚伟.辽宁地区规模化奶牛场牛病毒性腹泻-黏膜病血清学调查[J].现代畜牧兽医,2015,4:36-40.YAO W.Serologic study on bovine viral diarrhea in dairy cattle from scale dairy farms in Liaoning[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2015,4:36-40.(in Chinese)
[22]倪惠军,王英,许利琴,等.牛病毒性腹泻的流行病学调查[J].上海畜牧兽医通讯,2018,3:34-35.NI H J,WANG Y,XU L Q,et al.Epidemiological investigation of bovine viral diarrhea[J].Shanghai Livestock and Veterinary Communication,2018,3:34-35.(in Chinese)
[23]KENNY V.Strategies for the control and prevention of bovine viral diarrhea virus[J].Veterinary Clinics Food Animal Practice,2004,20(1):171-180.
[24]WALZ P H,GROOMS D L,PASSLER T,et al.Control of bovine viral diarrhea virus in ruminants[J].Journal of Veterinary Internal Medicine,2010,24(3):476-486.