湖北省潜江地区克氏原螯虾白斑综合征PCR诊断及组织病理学观察
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摘要
近年来在湖北省范围内人工养殖的克氏原螯虾暴发了严重的疾病,其中白斑综合征病毒(WSSV)已成为危害克氏原螯虾健康养殖的重要病原。2016年5月湖北省潜江市养殖区暴发了一种传染性疾病,为探究此次疾病病因和流行规律,将染病虾进行临床症状观察、对病料进行PCR检测、系统发育树分析、人工感染和组织病理学观察。结果显示,发病克氏原螯虾临床症状主要表现为摄食减少,活力下降,反应迟钝;组织病理学观察结果显示,克氏原螯虾的肝胰腺、肠、肌肉、鳃组织均出现不同程度变性和坏死以及炎性细胞浸润等典型病理学变化,与WSSV感染克氏原螯虾出现的病变相似;PCR检测患病克氏原螯虾样品,结果显示WSSV呈阳性,阳性检出率为55.56%(15/27),未检测到斑节对虾杆状病毒(MBV)和传染性皮下及造血组织坏死病毒(IHHNV);检测产物测序并进行系统发育树分析,结果显示,该基因序列与WSSV的EG3株(KR083866.1)核苷酸序列同源性为100%。将病虾的肝胰腺、肠和肌肉组织投喂健康克氏原螯虾,投喂组均表现为急性死亡(累积死亡率为100%),并出现与自然发病虾相同的症状。WSSV的巢式PCR检测结果显示,人工感染病虾为WSSV阳性。根据以上显示,本次养殖克氏原螯虾大规模死亡的病原是WSSV。
In May 2016, a severe infectious disease occurred among the farmed Procambarus clarkii in Qianjiang,Hubei Province. Histological examination, virus isolation, nested-PCR. artificial infection experiment and phylogenetic analysis were performed to explore its causes and the epidemic law. Principal symptoms included darkening of the body, reduction in vigour, low feeding. Histopathologically, hepatopancreas, intestines, muscle, gill showed varying degrees of degeneration, necrosis and inflammatory cellinvasion. Pathologic changes were the same as the white spot syndrome virus(WSSV) infected arthropod. PCR detection results also showed that tissues of dying P. clarkii were positive to WSSV, the positive rates of PCR was 55.56%(15/27), while those were negative to monodon baculovirus disease(MBV) and infectious hypodermal and hematopoietic necrosis(IHHNV).P. clarkii that died in oral infection was positive for the presence of WSSV by Nested-PCR.The sequenced amplification products shared 100% identity with white spot syndrome virus isolate EG3, complete genome(Gen Bank access number: KR083866.1). Phylogenetic analysis showed that the isolate was belonging to WSSV. The hepatopancreas, intestine and muscle tissue of the diseased P. clarkii were fed to the healthy P. clarkii. The results showed that a cumulative mortality of 100% was observed in the infected P. clarkii, while no death in control group. Therefore WSSV was determined to be the pathogen of the P. clarkii.
In May 2016, a severe infectious disease occurred among the farmed Procambarus clarkii in Qianjiang,Hubei Province. Histological examination, virus isolation, nested-PCR. artificial infection experiment and phylogenetic analysis were performed to explore its causes and the epidemic law. Principal symptoms included darkening of the body, reduction in vigour, low feeding. Histopathologically, hepatopancreas, intestines, muscle, gill showed varying degrees of degeneration, necrosis and inflammatory cellinvasion. Pathologic changes were the same as the white spot syndrome virus(WSSV) infected arthropod. PCR detection results also showed that tissues of dying P. clarkii were positive to WSSV, the positive rates of PCR was 55.56%(15/27), while those were negative to monodon baculovirus disease(MBV) and infectious hypodermal and hematopoietic necrosis(IHHNV).P. clarkii that died in oral infection was positive for the presence of WSSV by Nested-PCR.The sequenced amplification products shared 100% identity with white spot syndrome virus isolate EG3, complete genome(Gen Bank access number: KR083866.1). Phylogenetic analysis showed that the isolate was belonging to WSSV. The hepatopancreas, intestine and muscle tissue of the diseased P. clarkii were fed to the healthy P. clarkii. The results showed that a cumulative mortality of 100% was observed in the infected P. clarkii, while no death in control group. Therefore WSSV was determined to be the pathogen of the P. clarkii.
引文
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[5]Wongteerasupaya C,Vickers J E,Sriurairatana S,et al.A non-occluded,systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon[J].Diseases of Aquatic Organisms,1995,21(1):69-77.
[6]兰江风,代云佳,林蠡.养殖克氏原螯虾体内白斑综合征病毒的绝对定量分析[J].水产学报,2016,40(3):318-325.Lan J F,Dai Y J,Lin L.Quantitative analysis of white spot syndrome virus in the tissues of cultured red swamp crayfish(Procambarus clarkii)[J].Journal of Fisheries of China,2016,40(3):318-325(in Chinese).
[7]潘子豪,杨政霖,陆承平.安徽地区克氏原螯虾白斑综合征的诊断及朔源[J].微生物学报,2013,53(5):492-497.Pan Z H,Yang Z L,Lu C P.Diagnosis of white spot syndrome virus in farm crawfish in Anhui province and its epidemiological source[J].Acta microbiologica Sinica,2013,53(5):492-497(in Chinese).
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[9]杨冰,宋晓玲,黄倢,等.对虾传染性皮下及造血组织坏死病毒(IHHNV)PCR检测方法的建立[J].海洋水产研究,2005,26(2):1-5.Yang B,Song X L,Huang J,et al.Development of a polymerase chain reaction(PCR)for the detection of infectious hypodermal and hematopoietic necrosis virus[J].Marine Fisheries Research,2005,26(2):1-5(in Chinese).
[10]徐丽美,杨丰.利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究[J].高技术通讯,2005,15(5):101-104.Xu L M,Yang F.Simultaneous detection of white spot syndrome virus(WSSV)and monodon baculovirus(MBV)by multiplex polymerase chain reaction(PCR)in Penaeus monodon[J].Chinese High Technology Letters,2005,15(5):101-104(in Chinese).
[11]吴昊.对虾传染性皮下及造血组织坏死病毒(中国福建株)的基因组克隆及其重组衣壳蛋白自组装成病毒样颗粒的初步研究[D].厦门:国家海洋局第三海洋研究所,2007.Wu H.Genome cloning of the infectious hypodermal and hematopoietic necrosis virus(IHHNV)isolated from Fujian in China and assembly of vires-like particles of recombinant IHHNV capsid protein[D].Xiamen:Third Institute Of Oceanography,State Oceanic Administration,2007(in Chinese).
[12]Rajendran K V,Vijayan K K,Santiago T C,et al.White spot syndrome virus(WSSV)infection in tiger shrimp Penaeus monodon:a non-lethal histopathological rapid diagnostic method using paraffin and frozen sections[J].Aquaculture International,2005,13(4):341-349.
[13]Penaeus Monodon-type baculovirus disease,MBVD[J].Penaeus Monodon-type baculovirus disease,MBVD[J].China Fisheries,2011(11):56-57.
[14]朱斐,吕梦园,许梓荣.白斑综合征病毒感染克氏原螯虾后的PCR检测及组织病理学研究[J].淡水渔业,2012,42(6):43-48.Zhu F,Lv M Y,Xu Z R.PCR detection of white spotsyndrome virus infected Procambarus clarkii and histo-pathological study[J].Freshwater Fisheries,2012,42(6):43-48(in Chinese).
[15]Withyachumnarnkul B,Chayaburakul K,Laoaroon S,et al.Low impact of infectious hypodermal and hematopoietic necrosis virus(IHHNV)on growth and reproductive performance of Penaeus monodon[J].Diseases of Aquatic Organisms,2006,69(2-3):129.
[16]闫冬春,董双林,黄倢.PCR检测对虾白斑综合征病毒(WSSV)中假阴性的探讨[J].海洋与湖沼,2007,38(2):146-150.Yan D C,Dong S L,Huang J.PCR false negative result in white spot syndrome virus(WSSV)detection[J].Oceanologia et Limnologia Sinica,2007,38(2):146-150(in Chinese).
[17]周宸,曾志南.对虾杆状病毒病组织病理研究[C]//第二届全国人工养殖对虾疾病综合防治和环境管理学术研讨会.青岛:中国海洋潮沼学会,2007.Zhou C,Zeng Z N.Preliminary studies on baculoviral disease in Chinese penaeid shrimps[C]//The 2nd National Cultured Prawn Disease Integrated Control and Environmental Management Seminar,2007(in Chinese).
[18]吴友吕,王方国,洪健.长毛对虾杆状病毒病研究[J].电子显微学报,1994(5):356.Wu Y L,Wang F G,Hong J.Preliminary studies on penaeus penicillatus baculovirus[J].Journal of Chinese Elec-tron Microscopy Society,1994(5):356(in Chinese).
[19]杨冰.对虾传染性皮下及造血组织坏死病毒(IHHNV)的检测及其流行情况初步调查[D].青岛:中国海洋大学,2004.Yang B.Detection and prevalence of infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp[D].Qingdao:Ocean University of China,2004(in Chinese).
[20]雷质文,黄倢,莫照兰.人工感染白斑综合征病毒的克氏原螯虾的病理学试验[J].中国兽医科技,2002,32(10):23-25.Lei Z W,Huang J,Mo Z L.A pathological experiment of Procambarus clarkii artificially-infected with white spot syndrome virus[J].Chinese Journal of Veterinary Science and Technology,2002,32(10):23-25(in Chinese).
[21]魏克强.白斑综合征病毒浙江分离株在动物模型克氏原螯虾组织中的分布[J].细胞生物学杂志,2006,28(6):897-901.Wei K Q.The distribution of white spot syndrome virus Zhejiang strain in tissue cells of Procambarus clarkii[J].Chinese Journal of Cell Biology,2006,28(6):897-901(in Chinese).
[22]Manjusha M.White spot syndrone virus in penaeids:histopathology,development of polyclonal antisera and a cocktail vaccine[D].Cochin:Cochin University of Science and Technology.
[2]陈昌福,杨军,刘远高,等.克氏原螯虾暴发性疾病病原及其传播途径的初步研究[J].华中农业大学学报,2008,27(6):763-767.Chen C F,Yang J,Liu Y G,et al.Pathogenic organisms for explosive epidemic disease of red swamp crayfish,Procambarus clarkii[J].Journal of Huazhong Agricultural University,2008,27(6):763-767(in Chinese).
[3]闫冬春.白斑综合征病毒(WSSV)的PCR检测条件优化及浮游动物中间宿主的调查研究[D].青岛:中国海洋大学,2004.Yan D C.White spot syndrome virus(WSSV)PCR detection optimization and zooplankton mid-host investigation[D].Qingdao:Ocean University of China,2004(in Chinese).
[4]黄倢,宋晓玲,于佳,等.杆状病毒性的皮下及造血组织坏死:对虾暴发性流行病的病原和病理学[J].海洋水产研究,1995,16(1):1-10.Huang J,Song X L,Yu J,et al.Baculoviral hypodermal and hematopoietic necrosis-study on the pathogen and pathology of the explosive epidemic disease of shrimp[J].Marine Fisheries Research,1995,16(1):1-10(in Chinese).
[5]Wongteerasupaya C,Vickers J E,Sriurairatana S,et al.A non-occluded,systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon[J].Diseases of Aquatic Organisms,1995,21(1):69-77.
[6]兰江风,代云佳,林蠡.养殖克氏原螯虾体内白斑综合征病毒的绝对定量分析[J].水产学报,2016,40(3):318-325.Lan J F,Dai Y J,Lin L.Quantitative analysis of white spot syndrome virus in the tissues of cultured red swamp crayfish(Procambarus clarkii)[J].Journal of Fisheries of China,2016,40(3):318-325(in Chinese).
[7]潘子豪,杨政霖,陆承平.安徽地区克氏原螯虾白斑综合征的诊断及朔源[J].微生物学报,2013,53(5):492-497.Pan Z H,Yang Z L,Lu C P.Diagnosis of white spot syndrome virus in farm crawfish in Anhui province and its epidemiological source[J].Acta microbiologica Sinica,2013,53(5):492-497(in Chinese).
[8]世界动物卫生组织.水生动物疾病诊断手册[M].北京:中国农业出版社,2001.World Organization for Animal Health.Diagnostic Manual for Aquatic Animal Diseases[M].Beijing:China Agriculture Press,2001(in Chinese).
[9]杨冰,宋晓玲,黄倢,等.对虾传染性皮下及造血组织坏死病毒(IHHNV)PCR检测方法的建立[J].海洋水产研究,2005,26(2):1-5.Yang B,Song X L,Huang J,et al.Development of a polymerase chain reaction(PCR)for the detection of infectious hypodermal and hematopoietic necrosis virus[J].Marine Fisheries Research,2005,26(2):1-5(in Chinese).
[10]徐丽美,杨丰.利用多重PCR同时检测WSSV和MBV两种对虾病毒的研究[J].高技术通讯,2005,15(5):101-104.Xu L M,Yang F.Simultaneous detection of white spot syndrome virus(WSSV)and monodon baculovirus(MBV)by multiplex polymerase chain reaction(PCR)in Penaeus monodon[J].Chinese High Technology Letters,2005,15(5):101-104(in Chinese).
[11]吴昊.对虾传染性皮下及造血组织坏死病毒(中国福建株)的基因组克隆及其重组衣壳蛋白自组装成病毒样颗粒的初步研究[D].厦门:国家海洋局第三海洋研究所,2007.Wu H.Genome cloning of the infectious hypodermal and hematopoietic necrosis virus(IHHNV)isolated from Fujian in China and assembly of vires-like particles of recombinant IHHNV capsid protein[D].Xiamen:Third Institute Of Oceanography,State Oceanic Administration,2007(in Chinese).
[12]Rajendran K V,Vijayan K K,Santiago T C,et al.White spot syndrome virus(WSSV)infection in tiger shrimp Penaeus monodon:a non-lethal histopathological rapid diagnostic method using paraffin and frozen sections[J].Aquaculture International,2005,13(4):341-349.
[13]Penaeus Monodon-type baculovirus disease,MBVD[J].Penaeus Monodon-type baculovirus disease,MBVD[J].China Fisheries,2011(11):56-57.
[14]朱斐,吕梦园,许梓荣.白斑综合征病毒感染克氏原螯虾后的PCR检测及组织病理学研究[J].淡水渔业,2012,42(6):43-48.Zhu F,Lv M Y,Xu Z R.PCR detection of white spotsyndrome virus infected Procambarus clarkii and histo-pathological study[J].Freshwater Fisheries,2012,42(6):43-48(in Chinese).
[15]Withyachumnarnkul B,Chayaburakul K,Laoaroon S,et al.Low impact of infectious hypodermal and hematopoietic necrosis virus(IHHNV)on growth and reproductive performance of Penaeus monodon[J].Diseases of Aquatic Organisms,2006,69(2-3):129.
[16]闫冬春,董双林,黄倢.PCR检测对虾白斑综合征病毒(WSSV)中假阴性的探讨[J].海洋与湖沼,2007,38(2):146-150.Yan D C,Dong S L,Huang J.PCR false negative result in white spot syndrome virus(WSSV)detection[J].Oceanologia et Limnologia Sinica,2007,38(2):146-150(in Chinese).
[17]周宸,曾志南.对虾杆状病毒病组织病理研究[C]//第二届全国人工养殖对虾疾病综合防治和环境管理学术研讨会.青岛:中国海洋潮沼学会,2007.Zhou C,Zeng Z N.Preliminary studies on baculoviral disease in Chinese penaeid shrimps[C]//The 2nd National Cultured Prawn Disease Integrated Control and Environmental Management Seminar,2007(in Chinese).
[18]吴友吕,王方国,洪健.长毛对虾杆状病毒病研究[J].电子显微学报,1994(5):356.Wu Y L,Wang F G,Hong J.Preliminary studies on penaeus penicillatus baculovirus[J].Journal of Chinese Elec-tron Microscopy Society,1994(5):356(in Chinese).
[19]杨冰.对虾传染性皮下及造血组织坏死病毒(IHHNV)的检测及其流行情况初步调查[D].青岛:中国海洋大学,2004.Yang B.Detection and prevalence of infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp[D].Qingdao:Ocean University of China,2004(in Chinese).
[20]雷质文,黄倢,莫照兰.人工感染白斑综合征病毒的克氏原螯虾的病理学试验[J].中国兽医科技,2002,32(10):23-25.Lei Z W,Huang J,Mo Z L.A pathological experiment of Procambarus clarkii artificially-infected with white spot syndrome virus[J].Chinese Journal of Veterinary Science and Technology,2002,32(10):23-25(in Chinese).
[21]魏克强.白斑综合征病毒浙江分离株在动物模型克氏原螯虾组织中的分布[J].细胞生物学杂志,2006,28(6):897-901.Wei K Q.The distribution of white spot syndrome virus Zhejiang strain in tissue cells of Procambarus clarkii[J].Chinese Journal of Cell Biology,2006,28(6):897-901(in Chinese).
[22]Manjusha M.White spot syndrone virus in penaeids:histopathology,development of polyclonal antisera and a cocktail vaccine[D].Cochin:Cochin University of Science and Technology.