| 养殖三疣梭子蟹十足目虹彩病毒1的检出及组织病理学分析 |
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| 引用本文： | 赵丹阳,施慧,许文军,何杰,王庚申.养殖三疣梭子蟹十足目虹彩病毒1的检出及组织病理学分析[J].中国水产科学,2023,30(8):1042-1053. |
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| 作者姓名： | 赵丹阳 施慧 许文军 何杰 王庚申 |
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| 作者单位： | 浙江海洋大学水产学院, 浙江 舟山 316022;浙江省海洋水产研究所, 浙江省海水增养殖重点实验室, 浙江 舟山 316021 |
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| 基金项目： | 浙江省农业重大技术协同推广项目(2020XTTGSC03, 2022XTTGSC04)； 象山县科技计划项目(2022C1001) |
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| 摘 要： | 为探究浙江省舟山市某养殖池塘中三疣梭子蟹(Portunus trituberculatus)暴发疾病的病因, 应用组织病理学、 分子生物学和荧光原位杂交等技术手段, 对患病三疣梭子蟹组织进行检验。研究发现, 患病三疣梭子蟹的临床症状主要表现为食欲下降, 行动迟缓, 鳃水肿; 光学显微镜下观察鳃及血淋巴液未发现寄生虫, 肝胰腺等组织中也未分离到致病菌; 采用 PCR 方法对病蟹进行血卵涡鞭虫(hematodinium)、白斑综合征病毒(white spot syndrome virus, WSSV)、青蟹双顺反子病毒(mud crab discistrovirus-1, MCDV-1)以及青蟹呼肠孤病毒(Scylla serrata reovirus, SSRV) 蟹类常见病原 PCR 检测, 结果均为阴性; 病蟹的肝胰腺、心脏、鳃等组织的病理切片中可观察到明显的细胞病变和嗜酸性包涵体; 超薄切片电镜观察显示: 病蟹的肝胰腺、心脏和鳃组织中均存在六边形病毒颗粒, 粒子直径 150 nm 左右, 与已报道的十足目虹彩病毒 1 (decapoda iridescent virus 1, DIV1)形态特征相似。采用特异性套式 PCR 检测方法对患病蟹组织样品进行 DIV1 病原检测, 所有样本均扩增出 457 bp 和 129 bp 大小的目的片段。进一步根据 GenBank 中 DIV1 的主要衣壳蛋白(major capsid protein, MCP)表达基因和三磷酸腺苷酶(ATPase)表达基因序列设计特异性引物, 均能从病蟹样品中扩增出预期大小的 MCP 和 ATPase 基因开放阅读框(open reading frame, ORF)区全长。将扩增获得的 MCP 和 ATPase 基因 ORF 区全长进行测序和同源序列比对分析, 进化树分析结果表明其与十足目虹彩病毒属(Decapodiridovirus)病毒的 MCP 和 ATPase 基因序列自然聚为一支, 判定导致此次三疣梭子蟹发病病原为 DIV1。根据原位杂交探针设计原则, 以 DIV1 的 MCP 和 ATPase 基因的保守区域为靶位点分别设计探针, 通过荧光原位杂交获得了病毒粒子在病蟹肝胰腺、心脏、肌肉和鳃组织的分布情况, 与电镜切片观察和套式 PCR 检测结果相符。研究结果可为海水养殖三疣梭子蟹十足目虹彩病毒 1 病诊断与防控提供参考。
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| 关 键 词： | 三疣梭子蟹 十足目虹彩病毒 1 组织病理 原位杂交 |
| 收稿时间： | 2023/6/13 0:00:00 |
| 修稿时间： | 2023/7/21 0:00:00 |
Detection and histopathological analysis of Decapoda iridescent virus 1 in cultured Portunus trituberculatus |
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| ZHAO Danyang,SHI Hui,XU Wenjun,HE Jie,WANG Gengshen.Detection and histopathological analysis of Decapoda iridescent virus 1 in cultured Portunus trituberculatus[J].Journal of Fishery Sciences of China,2023,30(8):1042-1053. |
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| Authors: | ZHAO Danyang SHI Hui XU Wenjun HE Jie WANG Gengshen |
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| Abstract: | In October 2022, an infectious disease outbreak was observed in farmed Portunus trituberculatus in Zhoushan, China. Samples of diseased crabs showing unspecific signs, such as anorexia, slow activity, and gill edema, were analyzed using parasitology, microbiology, histopathology, electron microscopy, and molecular identification. Parasitological and microbiological assays indicated that the diseased crabs were not infected with parasites or bacteria. The crab samples were tested by PCR methods recommended by World Organization for Animal Health and demonstrated to be free of Hematodinium, white spot syndrome virus (WSSV), mud crab discistrovirus-1 (MCDV-1), and Scylla serrata reovirus (SSRV). Histopathological examination revealed eosinophilic inclusions in hematopoietic tissue and hemocytes in gills, hepatopancreas, and the myocardial tissue of diseased crabs. Meanwhile, transmission electron microscopy showed that the virus in diseased crab tissues exhibited a typical icosahedral structure with a mean diameter of 150 nm, which was similar to the morphological characteristics of Decapoda iridescent virus 1 (DIV1). Nested PCR detection of DIV1 result showed that the first step of the PCR produced a 457 bp amplicon and the second step of the PCR produced a 129 bp amplicon. Phylogenetic analyses using gene sequences of major capsid protein (MCP) and ATPase revealed amplified sequences, DS.MCP201010 and DS.ATP202210 sequences, had the highest homology with the MCP gene and ATPase gene of DIV1, 100%. Therefore, it was determined that the pathogen of the disease was DIV1. In addition, specific probes directed to the MCP and ATPase gene of DIV1 were designed to obtain the distribution of virus particles in the hepatopancreas, heart, muscles, and gills of diseased crabs by fluorescence in situ hybridization, which was consistent with the results of electron microscopy and nested PCR. This study presents the first report of DIV1 infection in cultured Portunus trituberculatus, which will provide a strong reference for the prevention and control of the disease. |
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