致急性肝胰腺坏死病副溶血弧菌(Vp_(AHPND))自然感染三疣梭子蟹
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摘要
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。
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 pir Avp 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.
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 pir Avp 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.
引文
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[2]Lightner D V,Redman R M,Pantoja C R,et al.Early mortality syndrome affects shrimp in Asia[J].Global Aquaculture Advocate,2012,15(1):40.
[3]NACA-FAO.Quarterly aquatic animal disease report(Asia and Pacific region)[R].Bangkok:Network of Aquaculture Centres in Asia-Pacific,2011.
[4]Flegel T W.Historic emergence,impact and current status of shrimp pathogens in Asia[J].Journal of Invertebrate Pathology,2012,110(2):166-173.
[5]孙明玉,冯博,张昭寰,等.引起凡纳滨对虾急性肝胰腺坏死病的副溶血弧菌MLST新序列型[J].水产学报,2018,42(3):410-418.Sun M Y,Feng B,Zhang Z H,et al.New sequence type isolates of AHPND-causing Vibrio parahaemolyticus from Litopenaeus vannamei by multilocus sequence typing[J].Journal of Fisheries of China,2018,42(3):410-418(in Chinese).
[6]Huang J.Experience in EMS/AHPNS from China[C]//NACA.The Asia Pacific emergency regional consultation on the emerging shrimp disease:early mortality Syndrome(EMS)/acute hepatopancreatic necrosis syndrome(AHPNS).Bangkok,Thailand:Network of Aquaculture Centres in Asia-Pacific,2012.
[7]Thitamadee S,Prachumwat A,Srisala J,et al.Review of current disease threats for cultivated penaeid shrimp in Asia[J].Aquaculture,2016,452:69-87.
[8]Global Aquaculture Alliance.Cause of EMS shrimp disease identified:GAA news release[EB/OL].(2013-05)[2018-09-09].https://www.aquaculturealliance.org/blog/cause-of-ems-shrimp-disease-identified/.
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[10]Tran L,Nunan L,Redman R M,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp[J].Diseases of Aquatic Organisms,2013,105(1):45-55.
[11]Yang Y T,Chen I T,Lee C T,et al.Draft genome sequences of four strains of Vibrio parahaemolyticus,three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand[J].Genome Announcements,2014,2(5):e00816-14.
[12]Sirikharin R,Taengchaiyaphum S,Sanguanrut P,et al.Characterization and PCR Detection of binary,pir-like toxins from Vibrio parahaemolyticus isolates that cause acute hepatopancreatic necrosis disease(AHPND)in shrimp[J].PLoS One,2015,10(5):e0126987.
[13]Lee C T,Chen I T,Yang Y T,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803.
[14]唐小千,徐洪森,战文斌.对虾急性肝胰腺坏死综合症研究进展[J].海洋湖沼通报,2016(2):90-93.Tang X Q,Xu H S,Zhan W B.Advances in study of shrimp acute hepatopancreas necrosis syndrome[J].Transactions of Oceanology and Limnology,2016(2):90-93(in Chinese).
[15]Theethakaew C,Nakamura S,Motooka D,et al.Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp acute hepatopancreatic necrosis syndrome(AH-PNS)[J].Infection,Genetics and Evolution,2017,51:211-218.
[16]Kondo H,Van P T,Dang L T,et al.Draft genome sequence of non-Vibrio parahaemolyticus acute hepatopancreatic necrosis disease strain KC13.17.5,isolated from diseased shrimp in Vietnam[J].Genome Announcement,2015,3(5):e00978-15.
[17]Liu L Y,Xiao J Z,Xia X M,et al.Draft genome sequence of Vibrio owensii Strain SH-14,which causes shrimp acute hepatopancreatic necrosis disease[J].Genome Announcement,2015,3(6):e01395-15.
[18]Xiao J Z,Liu L Y,Ke Y Y,et al.Shrimp AHPND-causing plasmids encoding the PirAB toxins as mediated by pir AB-Tn903 are prevalent in various Vibrio species[J].Scientific Reports,2017,7:42177.
[19]Liu L Y,Xiao J Z,Zhang M M,et al.A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp,Litopenaeus vannamei[J].Journal of Invertebrate Pathology,2018,153:156-164.
[20]Ahn Y S,Piamsomboon P,Tang K F J,et al.Complete genome sequence of acute hepatopancreatic necrosis disease-causing Vibrio campbellii LA16-V1,isolated from Penaeus vannamei cultured in a Latin American country[J].Genome Announcements,2017,5(37).
[21]Dong X,Bi D X,Wang H L,et al.pirABVP-bearing Vibrio parahaemolyticus and Vibrio campbellii pathogens isolated from the same AHPND-affected pond possess highly similar pathogenic plasmids[J].Frontiers in Microbiology,2017,8:1859.
[22]Dong X,Wang H L,Zou P Z,et al.Complete genome sequence of Vibrio campbellii strain 20130629003S01 isolated from shrimp with acute hepatopancreatic necrosis disease[J].Gut Pathogens,2017,9:31.
[23]Flegel T W,Lo C F.Interim primers for specific detection of bacterial isolates that cause acute hepatopancreatic necrosis disease(AHPND)[EB/OL].(2014).https://www.was.org/Meetings/ShowAbstract.aspx?Id=32078.
[24]Dangtip S,Sirikharin R,Sanguanrut P,et al.AP4method for two-tube nested PCR detection of AHPNDisolates of Vibrio parahaemolyticus[J].Aquaculture Reports,2015,2:158-162.
[25]Arunrut N,Kampeera J,Sirithammajak S,et al.Sensitive visual detection of AHPND bacteria using loop-mediated isothermal amplification combined with DNA-functionalized gold nanoparticles as probes[J].PLoS One,2016,11(3):e0151769.
[26]Ding Z,Meng Q,Liu H,et al.First case of hepatopancreatic necrosis disease in pond-reared Chinese mitten crab,Eriocheir sinensis,associated with microsporidian[J].Journal of Fish Diseases,2016,39(9):1043-1051.
[27]杨宗英,张一柳,胡鲲,等.中华绒螯蟹肝胰腺坏死综合症病原及病理学研究[J].水生生物学报,2018,42(1):17-25.Yang Z Y,Zhang Y L,Hu K,et al.Etiological and histopathological study on hepatopancreatic necrosis syndrome in eriocheir sinensis[J].Acta Hydrobiologica Sinica,2018,42(1):17-25(in Chinese).
[28]丁立云,付辉云,侯迎梅,等.饲料磷脂对已交配和未交配雌性三疣梭子蟹卵巢发育、组织学和卵黄蛋白原表达的影响[J].水产学报,2018,42(3):366-375.Ding L Y,Fu H Y,Hou Y M,et al.Effects of dietary soy lecithin on the ovary development,histology and vitellogenin gene expression in mated and unmated female swimming crab(Portunus trituberculatus)[J].Journal of Fisheries of China,2018,42(3):366-375(in Chinese).
[29]Zhang Q L,Liu Q,Liu S,et al.A new nodavirus is associated with covert mortality disease of shrimp[J].Journal of General Virology,2014,95(12):2700-2709.
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