鲫CyHV-2病毒病的诊断及组织病理损伤研究
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摘要
【目的】为了确定一例鲫(Carassius auratus)鳃出血症的病原。【方法】无菌操作条件下从患病鱼的肝脏、脾脏等病原灶处分离病原菌;利用压片法取鳃丝、体表粘液进行观察,检查是否有寄生虫感染;取鳃、肝脏、脾脏、肾脏用聚合酶链式反应(PCR)技术检测鲤疱疹病毒Ⅱ型病毒(Cyprinid herpesvirus 2,CyHV-2),并构建系统发育树,结合组织病理学技术以分析鳃出血症的病因。【结果】结果发现,病鱼尾鳍末端发白,鳃丝出血、末端轻微溃烂,有少量红色腹水;寄生虫学检测未发现鳃丝和体表黏液有寄生虫寄生;通过微生物学诊断,未见病原菌生长;PCR检测结果显示患病鲫体内CyHV-2呈阳性;组织病理学检测表明,患病鲫的主要损伤靶器官为脾脏、鳃、肾脏、肠道、心脏和肝脏,主要引起重度鳃出血和坏死性鳃炎,重度坏死性脾炎、中度至重度肠炎、中度至重度坏死性肾炎、中度坏死性心肌炎和中度心内膜炎和轻度坏死性肝炎。【结论】推断本次鲫鳃出血症为CyHV-2感染导致。
【Objective】The aim of this study was to determine the pathogen of gills hemorrhagic disease of crucian carp.【Method】The pathogenic bacteria were isolated from the liver,spleen and other pathogenic sites of the diseased fish under the condition of sterile operation. The gill filaments and mucus on the body surface were taken by means of pressure film method for observation to check whether there was parasitic infection. Gill,liver,spleen,kidney with polymerase chain reaction(PCR) technology to detect Cyprinid herpesvirus 2(CyHV-2),and construct phylogenetic tree analysis,combined with histopathological techniques to the analysis of the gill disease causes.【Result】The results showed that the diseased fish tail fin turns white. The gill filament was bleeding and ulcerated,there was red ascites. Parasitological examination showed no parasitism in branchial filaments and mucus on the body surface. The growth of pathogenic bacteria was not detected by microbiological diagnosis. PCR results showed that CyHV-2 was positive in diseased fish. The results of the phylogenetic tree showed that the virus isolated in this experiment and CyHV-2 was the same. Histopathological results showed that the main target organs of crucian carp were gills,spleen,kidney,intestinal tract,heart and liver,resulting in different degrees of damage. 【Conclusion】It could be inferred that the gills hemorrhage of crucian carp is a viral disease,which caused by CyHV-2.
【Objective】The aim of this study was to determine the pathogen of gills hemorrhagic disease of crucian carp.【Method】The pathogenic bacteria were isolated from the liver,spleen and other pathogenic sites of the diseased fish under the condition of sterile operation. The gill filaments and mucus on the body surface were taken by means of pressure film method for observation to check whether there was parasitic infection. Gill,liver,spleen,kidney with polymerase chain reaction(PCR) technology to detect Cyprinid herpesvirus 2(CyHV-2),and construct phylogenetic tree analysis,combined with histopathological techniques to the analysis of the gill disease causes.【Result】The results showed that the diseased fish tail fin turns white. The gill filament was bleeding and ulcerated,there was red ascites. Parasitological examination showed no parasitism in branchial filaments and mucus on the body surface. The growth of pathogenic bacteria was not detected by microbiological diagnosis. PCR results showed that CyHV-2 was positive in diseased fish. The results of the phylogenetic tree showed that the virus isolated in this experiment and CyHV-2 was the same. Histopathological results showed that the main target organs of crucian carp were gills,spleen,kidney,intestinal tract,heart and liver,resulting in different degrees of damage. 【Conclusion】It could be inferred that the gills hemorrhage of crucian carp is a viral disease,which caused by CyHV-2.
引文
[1]李莉娟,罗杨志,刘学芹,等.金鱼鲤疱疹病毒Ⅱ型的分子诊断[J].华中农业大学学报,2013,32(1):92-96.
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[3]STEPHENS F J,RAIDAL S R,JONES B.Haematopoietic necrosis in a goldfish(Carassius auratus)associated with an agent mor-phologically similar to herpesvirus[J].Australian Veterinary Journal,2010,82(3):167-169.
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[10]李宝山,冷向军,李小勤,等.投饲蚕豆对异育银鲫生长、肉质及肠道蛋白酶活力的影响[J].动物营养学报,2007,19(5):631-635.
[11]LIU W Z,ZENG L B,ZHANG H,et al.Studies on the change of blood biochemical indices in gibel carp,Carassius auratus,infected with Cyprinid herpesvirusⅡ[J].Journal of Shanghai Ocean University,2013,22(6):928-935.
[12]WALTZEK T B,KUROBE T,GOODWIN A E,et al.Development of a polymerase chain reaction assay to detect Cyprinid Herpesvirus 2 in goldfish[J].Journal of Aquatic Animal Health,2009,21(1):60-67.
[13]林秀秀,叶元土,吴萍,等.鲤疱疹Ⅱ型病毒(CyHV-2)感染对异育银鲫(Carassius auratus gibelio)组织器官的损伤作用[J].基因组学与应用生物学,2016(3):587-594.
[14]方进,邓院生,王俊,等.急性病毒性鲫鳃出血病的病理变化[J].中国水产科学,2016,23(2):336-343.
[15]周勇,曾令兵,张辉,等.鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用[J].水产学报,2013,37(4):607-613.
[16]WALTZEK T B,KELLEY G O,STONE D M,et al.Koi herpesvirus represents a third cyprinid herpesvirus(CyHV-3)in the family Herpesviridae[J].Journal of General Virology,2005,86(6):1659.
[17]GOODWIN A E,KHOO L,LAPATRA S E,et al.Goldfish hematopoietic necrosis Herpesvirus(Cyprinid Herpesvirus 2)in the USA:Molecular confirmation of isolates from diseased fish[J].Journal of Aquatic Animal Health,2006,18(1):11-18.
[18]HE J,SHI X,YU L,et al.Development and evaluation of a loopmediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2[J].Journal of Virological Methods,2013,194(1):206-210.
[19]徐进,曾令兵,杨德国,等.鲤疱疹病毒2型武汉株的分离与鉴定[J].中国水产科学,2013,20(6):1303-1309.
[20]GOODWIN A E,SADLER J,MERRY G E,et al.Herpesviral haematopoietic necrosis virus(CyHV-2)infection:case studies from commercial goldfish farms[J].Journal of Fish Diseases,2009,32(3):271-278.
[21]张琳琳.鲤疱疹病毒Ⅱ型灭活疫苗免疫应答机理与保护效果研究[D].武汉:华中农业大学,2016.
[2]JUNG S J,MIYAZAKI T.Herpesviral haematopoietic necrosis of goldfish,Carassius auratus(L)[J].Journal of Fish Diseases,2010,18(3):211-220.
[3]STEPHENS F J,RAIDAL S R,JONES B.Haematopoietic necrosis in a goldfish(Carassius auratus)associated with an agent mor-phologically similar to herpesvirus[J].Australian Veterinary Journal,2010,82(3):167-169.
[4]GOODWIN A E,KHOO L,LAPATRA S E,et al.Goldfish hematopoietic necrosis herpesvirus(cyprinid herpesvirus 2)in the USA:molecular confirmation of isolates from diseased fish[J].Journal of Aquatic Animal Health,2006,18(1):11-18.
[5]CHANG P H,HWAE L S,CHOUNG C H,et al.Epizootic of herpes-like virus infection in goldfish,Carassius auratus in Taiwan[J].Fish Pathology,1999,34(4):209-210.
[6]JEFFERY K R,BATEMAN K,BAYLEY A,et al.Isolation of a cyprinid herpesvirus 2 from goldfish,Carassius auratus(L),in the UK[J].Journal of Fish Diseases,2007,30(11):649.
[7]DOSZPOLY A,BENKO M,CSABA G,et al.Introduction of the family Alloherpesviridae:the first molecular detection of herpesviruses of cyprinid fish in Hungary[J].Magyar AllatorvosokLapja,2011,133:174-181.
[8]DAN魫K T,KALOUS L,VESEL T,et al.Massive mortality of Prussian carp Carassius gibelio in the upper Elbe basin associated with herpesviral hematopoietic necrosis(CyHV-2)[J].Diseases of Aquatic Organisms,2012,102(2):87-95.
[9]WANG L,HE J G,LIANG L,et al.Mass mortality caused by Cyprinid Herpesvirus 2(CyHV-2)in Prussian carp(Carassius gibelio)in Chin[aJ].Bulletin-European Association of Fish Pathologists,2012,32(5):164-173.
[10]李宝山,冷向军,李小勤,等.投饲蚕豆对异育银鲫生长、肉质及肠道蛋白酶活力的影响[J].动物营养学报,2007,19(5):631-635.
[11]LIU W Z,ZENG L B,ZHANG H,et al.Studies on the change of blood biochemical indices in gibel carp,Carassius auratus,infected with Cyprinid herpesvirusⅡ[J].Journal of Shanghai Ocean University,2013,22(6):928-935.
[12]WALTZEK T B,KUROBE T,GOODWIN A E,et al.Development of a polymerase chain reaction assay to detect Cyprinid Herpesvirus 2 in goldfish[J].Journal of Aquatic Animal Health,2009,21(1):60-67.
[13]林秀秀,叶元土,吴萍,等.鲤疱疹Ⅱ型病毒(CyHV-2)感染对异育银鲫(Carassius auratus gibelio)组织器官的损伤作用[J].基因组学与应用生物学,2016(3):587-594.
[14]方进,邓院生,王俊,等.急性病毒性鲫鳃出血病的病理变化[J].中国水产科学,2016,23(2):336-343.
[15]周勇,曾令兵,张辉,等.鲤疱疹病毒Ⅱ型TaqMan real-time PCR检测方法的建立及应用[J].水产学报,2013,37(4):607-613.
[16]WALTZEK T B,KELLEY G O,STONE D M,et al.Koi herpesvirus represents a third cyprinid herpesvirus(CyHV-3)in the family Herpesviridae[J].Journal of General Virology,2005,86(6):1659.
[17]GOODWIN A E,KHOO L,LAPATRA S E,et al.Goldfish hematopoietic necrosis Herpesvirus(Cyprinid Herpesvirus 2)in the USA:Molecular confirmation of isolates from diseased fish[J].Journal of Aquatic Animal Health,2006,18(1):11-18.
[18]HE J,SHI X,YU L,et al.Development and evaluation of a loopmediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2[J].Journal of Virological Methods,2013,194(1):206-210.
[19]徐进,曾令兵,杨德国,等.鲤疱疹病毒2型武汉株的分离与鉴定[J].中国水产科学,2013,20(6):1303-1309.
[20]GOODWIN A E,SADLER J,MERRY G E,et al.Herpesviral haematopoietic necrosis virus(CyHV-2)infection:case studies from commercial goldfish farms[J].Journal of Fish Diseases,2009,32(3):271-278.
[21]张琳琳.鲤疱疹病毒Ⅱ型灭活疫苗免疫应答机理与保护效果研究[D].武汉:华中农业大学,2016.