致急性肝胰腺坏死病副溶血弧菌(VpAHPND)自然感染三疣梭子蟹

近年来包括急性肝胰腺坏死病(AHPND)在内的多种新发疫病的流行,使我国甲壳类养殖业遭受了严重的经济损失。为了筛查导致山东潍坊某养殖场中一虾蟹混养池塘内患病三疣梭子蟹感染的可能病原,本研究采用分子生物学检测方法,对三疣梭子蟹样品进行了白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、虾血细胞虹彩病毒(SHIV)、致急性肝胰腺坏死病副溶血孤菌(Vp_(AHPND))、虾肝肠胞虫(EHP)、偷死野田村病毒(CMNV)、黄头病毒(YHV)和肝胰腺细小病毒(HPV)等8种病原的检测,并对样品进行了组织病理和原位杂交分析。分子生物学检测结果显示,患病三疣梭子蟹样品呈Vp_(AHPND)阳性,而呈现WSSV、IHHNV、SHIV、EHP、CMNV、YHV和HPV阴性。对样品进行Vp_(AHPND)套式PCR第二轮扩增产物的序列测定、比对和进化树分析,结果显示,扩增产物序列与致病副溶血弧菌质粒上pirA~(vp)毒力基因片段具有99%的同源性,该序列与已报道的多个致病副溶血弧菌PirA聚在进化树的同一主分支上。组织病理学分析显示,患病三疣梭子蟹的肝胰腺小管上皮细胞坏死,心肌纤维呈溶解样病变,鳃丝上皮柱突细胞明显坏死,胸神经节的神经细胞损伤严重,并且这些组织中还可见大量的细胞核固缩现象;原位杂交结果显示,肝胰腺、心肌、鳃组织及胸神经节中的病变部位均存在Vp_(AHPND)探针的蓝紫色杂交信号。以上表明,虾蟹混养池塘中三疣梭子蟹在自然状态下感染了Vp_(AHPND),并导致肝胰腺、心肌、鳃和胸神经节发生了严重病理损伤。本研究首次在养殖三疣梭子蟹中检测到Vp_(AHPND)感染并揭示了感染所致的病理变化,相关结果为揭示Vp_(AHPND)自然宿主种类和养殖三疣梭子蟹病害防控提供了基础信息。

Natural infection of Portunus trituberculatus with acute hepatopancreas necrosis disease causing by Vibrio parahaemolyticus (VpAHPND)

The prevalence of multiple emerging diseases including acute hepatopancreatic necrosis disease (AHPND) has caused serious economic losses of crustacean aquaculture industry in China in last several years. In order to analyzing the possible pathogenic agents related to the slow growth of Portunus trituberculatus from Weifang City, Shandong Province, the presence and infection of nine suspected pathogens in the disease P. trituberculatus individuals were investigated by using the molecular detection methods in present study. The nine pathogens included white spot syndrome virus (WSSV), Infectious Hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), AHPND-causing Vibrio parahaemolyticus (Vp_AHPND), enterocytozoon hepatopenaei (EHP), covert mortality nodavirus (CMNV), yellow head virus (YHV) and hepatopancreatic parvovirus (HPV). Meanwhile, histopathological analysis and in situ hybridization (ISH) were performed on the clinical samples. Results of molecular analysis showed that the P. trituberculatus samples were determined to be positive of Vp_AHPND and negative of other seven pathogens. Multiple sequence alignment based on the amplicons of Vp_AHPND nested-PCR of the samples revealed that sequences of the amplicons from P. trituberculatus samples shared 99% similarity to the pirA^vp virulence gene from the pathogenic V. parahaemolyticus. Phylogenetic analysis indicated that the sequence from P. trituberculatus samples was clustered into the same branch with the reported PirA of pathogenic V. parahaemolyticus. Histological examination revealed necrosis of epithelial cells in hepatopancreas tubules, myonecrosis of cardiac muscle in heart, necrosis of epithelial columnar cells of gills, severe damage of nerve cell in the thoracic ganglion, as well as nuclear pyknosis in these tissues. Micrographs of ISH showed that blue-violet hybridization signals of the Vp_AHPND probes were present in the lesions of the hepatopancreas, myocardium, gills, and thoracic ganglia. The results indicated that the P. trituberculatus samples from the shrimp and crab polyculture ponds were naturally infected by Vp_AHPND, which caused serious pathological damages of the hepatopancreas, myocardium, gills, and thoracic ganglia. The present study revealed, for the first time, that Vp_AHPND infection in P. trituberculatus and the pathological changes caused by the infection. The results provided basic information for revealing the natural host species of Vp_AHPND, and for the prevention and control of AHPND of P. trituberculatus.