辽宁一例凡纳滨对虾大规模死亡的病原研究
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摘要
为对辽宁省盘锦地区2014年出现的大规模死亡凡纳滨对虾Litopenaeus vannamei展开病原研究,对死虾进行了白斑综合征病毒(WSSV)、桃拉病毒(TSV)、传染性皮下及造血组织坏死病病毒(IHHNV)和嗜水气单胞菌Aeromonas hydrophila的分析鉴定,提取病虾鳃组织DNA/RNA后,以特异性引物进行WSSV、TSV和IHHNV病毒检测,通过16S r DNA测序及嗜水气单胞菌特异性引物和气溶素基因的双重PCR对分离菌株QD1进行分类鉴定,并对病虾的鳃、胃、肝胰腺和附肢进行了组织病理切片观察。结果表明:患病对虾WSSV和TSV检测呈阳性;菌株QD1为嗜水气单胞菌,其人工回归感染试验结果显示,分离菌株能使健康凡纳滨对虾致病死亡;组织病理学观察显示,病虾鳃及胃组织出现大量WSSV特征性的病变核,肝胰腺上皮细胞出现严重萎缩,肝胰腺管腔变大,部分肝细胞坏死,肝胰腺管之间结缔组织中血细胞明显增多。研究表明,此次凡纳滨对虾大量死亡为WSSV和TSV两种病毒与嗜水气单胞菌混合感染所致。
In this paper,white spot syndrome virus( WSSV),Taurus virus( TSV),Infectious hypodermal,hematopoietic necrosis virus( IHHNV) and pathogenic bacterium Aeromonas hydrophila were identified in diseased and mass death of Pacific white leg shrimp Litopenaeus vannamei collected from Panjin region in Liaoning Province with specific primer after extracting DNA / RNA from gill tissue,16 S r DNA sequencing and double PCR identification in specific primer and aerolysin gene of the pathogenic bacterium was identified to classify strain QD1. The tissue pathological observation was carried out on gills,stomach,hepatopancreas and appendages of the diseased shrimps. The results showed that WSSV and TSV were positive to the disease,and that the isolated strain was proved to be classified as Aeromonas hydrophila which led to the shrimp death by artificial challenged. Histopathological observation revealed that there was a large number of nuclei of WSSV in gill and stomach of the diseased shrimp. The severe atrophical epithelial cells,larger lumen,part of the hepatic cell necrosis and blood cells were observed in the hepatopancreas of the diseased shrimp. Comprehensive analysis confirmed that it is the mixed infection of 2 species of virus and Aeromonas hydrophila in a large number of Pacific white leg shrimp death.
In this paper,white spot syndrome virus( WSSV),Taurus virus( TSV),Infectious hypodermal,hematopoietic necrosis virus( IHHNV) and pathogenic bacterium Aeromonas hydrophila were identified in diseased and mass death of Pacific white leg shrimp Litopenaeus vannamei collected from Panjin region in Liaoning Province with specific primer after extracting DNA / RNA from gill tissue,16 S r DNA sequencing and double PCR identification in specific primer and aerolysin gene of the pathogenic bacterium was identified to classify strain QD1. The tissue pathological observation was carried out on gills,stomach,hepatopancreas and appendages of the diseased shrimps. The results showed that WSSV and TSV were positive to the disease,and that the isolated strain was proved to be classified as Aeromonas hydrophila which led to the shrimp death by artificial challenged. Histopathological observation revealed that there was a large number of nuclei of WSSV in gill and stomach of the diseased shrimp. The severe atrophical epithelial cells,larger lumen,part of the hepatic cell necrosis and blood cells were observed in the hepatopancreas of the diseased shrimp. Comprehensive analysis confirmed that it is the mixed infection of 2 species of virus and Aeromonas hydrophila in a large number of Pacific white leg shrimp death.
引文
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[17]霍国才.红螯螯虾消化系统细菌病病原的研究[J].内陆水产,1997(12):21-24.
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[19]胡萌.江苏地区气单胞菌分离鉴定及强毒株生物学特性分析[D].南京:南京农业大学,2012:1-93.
[2]Tang K F J,Lightner D V.Homologues of insecticidal toxin complex genes within a genomic island in the marine bacterium Vibrio parahaemolyticus[J].FEMS Microbiology Letters,2014,361(1):34-42.
[3]Han J E,Tang K F J,Tran L H,et al.Photorhabdus insect-related(Pir)toxin-like genes in a plasmid of Vibrio parahaemolyticus,the causative agent of acute hepatopancreatic necrosis disease(AHPND)of shrimp[J].Diseases of Aquatic Organisms,2015,113(1):33-40.
[4]赵永锋,宋迁红.南美白对虾养殖概况及病害防控措施[J].科学养鱼,2014(7):13-17.
[5]Stentiford G D,Neil D M,Peeler E J,et al.Disease will limit future food supply from the global crustacean fishery and aquaculture sectors[J].Journal of Invertebrate Pathology,2012,110(2):141-157.
[6]王奕玲,李卓佳,张家松,等.高位池养殖过程凡纳滨对虾携带WSSV情况的动态变化[J].中国水产科学,2012,19(2):301-309.
[7]乔毅,沈辉,万夕和,等.1株黑鲷致病性假交替单胞菌的鉴定及毒力基因分析[J].江苏农业科学,2015,43(4):229-233.
[8]王友娟,李荣峰,李华,等.辽宁地区养殖淡水鱼感染嗜水气单胞菌的流行病学调查[J].中国农业科技导报,2012,14(4):128-134.
[9]刁菁,杨秀生,李天保,等.病原微生物药敏检测方法的研究进展[J].中国农学通报,2013,29(8):1-5.
[10]陈颜锋.如何减轻南美白对虾桃拉综合征的危害[J].科学种养,2012(7):50.
[11]陈健舜,朱凝瑜,孔蕾,等.凡纳滨对虾细菌性红体病病原的分子特征与耐药性[J].水产学报,2012,36(12):1891-1900.
[12]刘飞.中国大陆几株WSSV毒株毒力测定及分析[D].青岛:中国海洋大学,2013:1-57.
[13]Tsai Y L,Wang H C,Lo C F,et al.Validation of a commercial insulated isothermal PCR-based POCKIT test for rapid and easy detection of white spot syndrome virus infection in Litopenaeus vannamei[J].PLo S One,2014,9(3):e90545.
[14]王立强.白斑病毒(WSSV)和桃拉病毒(TSV)对凡纳滨对虾(Litopenaeus vannamei)的混合感染[D].青岛:中国海洋大学,2005.
[15]冯守明.嗜水气单胞菌对水生动物的致病作用及防治[J].天津水产,2002(1):19-23.
[16]樊海平,孟庆显,俞开康.中国对虾败血病病原菌(气单胞菌)的致病性与生物学性状[J].水产学报,1994,18(1):32-38.
[17]霍国才.红螯螯虾消化系统细菌病病原的研究[J].内陆水产,1997(12):21-24.
[18]倪军.对虾“早期死亡综合征”研究现状[J].海洋与渔业,2014(12):74-75.
[19]胡萌.江苏地区气单胞菌分离鉴定及强毒株生物学特性分析[D].南京:南京农业大学,2012:1-93.