美洲鳗鲡“脱黏败血综合征”病原的分离鉴定及其致病性研究

olation,identification and pathogenicity of the pathogen of"mucus sloughing and hemorrhagic septicemia disease"in American eel,Anguilla rostrata

为明确美洲鳗鲡“脱黏败血综合征”的病原,本研究采集福建省某养殖场的美洲鳗鲡病料组织样品制备病理切片,观察各组织病变,结果显示,病鳗的鳃、肝脏和肾脏呈现广泛性红细胞渗出和组织细胞变性、坏死。将病料样品接种鳗鲡肾脏细胞系(EK)进行病毒的分离,对分离病毒经电镜观察,结果显示,接种病料样品匀浆液的EK细胞出现合胞体病变,可见细胞核内有大量直径约120 nm的病毒。提取病毒基因后作为模板,采用PCR鉴定,结果证实其为鳗鲡疱疹病毒(AngHV),将其命名为AngHV-20713。进一步扩增分离病毒的ORF71基因并分析其同源性和遗传进化关系,结果显示,AngHV-20713与其它AngHV的同源性为93.6%~99.0%,且与分离自美洲鳗鲡的AngHV亲缘关系最近。将分离病毒以106 pfu/尾腹腔注射健康的美洲鳗鲡和欧洲鳗鲡,进行人工感染实验,结果显示,AngHV-20713对鳗鲡具有致病性,感染鳗鲡可见体表黏液脱落、胸鳍出血等“脱黏败血综合征”症状,并导致美洲鳗鲡60%的发病率和20%的死亡率,而感染的欧洲鳗鲡死亡率高达56.7%。利用qPCR从感染病毒的美洲鳗鲡各组织内均可检测到AngHV,从内脏可再次分离培养到AngHV-20713,表明AngHV是美洲鳗鲡“脱黏败血综合征”的病原。本研究为深入开展鳗鲡“脱黏败血综合征”的防控研究奠定了基础。

To clarify the pathogenic agent of"mucus sloughing and hemorrhagic septicemia disease"of American eel,Anguilla rostrata,samples of the diseased American eel farming in Fujian province were employed for virus isolation,identification and pathogenicity analysis.Histopathological examinations showed extensive erythrocyte bleeding,degeneration and necrosis in the gills,liver and kidney of the diseased eel.The eel kidney cell line(EK)was used for virus cultivation,and syncytial lesions were detected.In the nuclei of EK cells,a large number of viral particles with a diameter of approximate 120nm were observed under electron microscope.Then the virus was identified to be Anguillid herpesvirus(AngHV)by PCR,named it strain AngHV-20713.The ORF71 gene of the isolated virus was further amplified,and genetic evolution relationship were analyzed using its homology.The results showed that the homology of AngHV-20713 with other AngHV was 93.6%-99.0%,and had closest relationship with AngHV isolated from A.rostrata.The challenge assay was conducted with the healthy American eel and European eel by intraperitoneally injected with 106pfu of isolated AngHV-20713.The results showed that AngHV-20713 caused 60%morbidity to American eel with typical symptoms of"mucus sloughing and hemorrhagic septicemia disease"such as mucus shedding,pectoral fin bleeding,and accompanied with 20%mortality,while the mortality of the infected European eel was as high as 56.7%.And the AngHV could be detected from the organs and tissues of infected American eel by qPCR,and AngHV-20713 strain was re-isolated from challenged eel,indicated that AngHV is the pathogen of"mucus sloughing and hemorrhagic septicemia disease"of American eel.This study provides a research foundation for further research on the prevention and control of"mucus sloughing and hemorrhagic septicemia disease"of American eel.