湖北省潜江地区克氏原螯虾(procambarus clarkii)白斑综合征(White Spot Syndrome)PCR诊断及组织病理学观察

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。

Histopathological study and PCR detection of White Spot Syndrome Virus from infected Procambarus 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 (GenBank 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.