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凡纳滨对虾急性肝胰腺坏死病(AHPND)病原分离鉴定及其致病性分析
贾 丹1,2, 史成银1,3, 黄 倢1,3, 张庆利1,3, 万晓媛1,3, 许 华1,3, 刘冉阳1,2, 王海波1,2, 郭程程1, 谢国驷1,3
1.农业部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 青岛市海水养殖流行病学与生物安保重点实验室 青岛 266071;2.上海海洋大学水产与生命学院 上海 201306;3.青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 青岛 266071
摘要:
本研究从患急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease, AHPND)的凡纳滨对虾(Litopenaeus vannamei)肝胰腺中分离到一株优势菌,编号为20160303005-1,通过16S rRNA和分子伴侣蛋白groEL基因序列分析,并结合生理生化特征,将该细菌鉴定为副溶血弧菌(Vibrio parahaemolyticus),其血清型为O1:KUT(K untypeable)。基因分析结果显示,该菌株携带可引起对虾AHPND的相关毒力蛋白基因pirAVP和pirBVP,但不携带副溶血弧菌临床菌株毒力基因:耐热直接溶血毒素(Thenmostable direct hemolysin, tdh)和相对耐热直接溶血毒素(TDH-related hemolysin, trh)基因。菌株对凡纳滨对虾具有较强的致病性,浸泡感染的半数致死剂量(LD50)为7.96×103 CFU/ml。对虾急性感染后,6 h肝胰腺颜色变浅,肠胃变空;9 h肝胰腺呈浅白色,萎缩变小。9 h死亡数过半,24 h全部死亡。组织病理学分析显示,感染后对虾肝胰腺小管崩塌,上皮细胞严重脱落,呈现出典型的AHPND病理症状。药敏实验结果显示,该菌对庆大霉素、环丙沙星和头孢他啶等16种药物敏感,对阿莫西林、替卡西林和头孢噻吩等5种药物表现为耐药。上述研究可为该病原的流行病学及药物防控研究提供基本数据。
关键词:  凡纳滨对虾  急性肝胰腺坏死病  副溶血弧菌  细菌鉴定
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Identification and Pathogenicity Analysis of Bacterial Pathogen Associated with Acute Hepatopancreatic Necrosis Disease (AHPND) in the Pacific Shrimp Litopenaeus vannamei
JIA Dan1,2, HI Chengyin1,3, HUANG Jie1,3, ZHANG Qingli1,3, WAN Xiaoyuan1,3, XU Hua1,3, LIU Ranyang1,2, WANG Haibo1,2, GUO Chengcheng1, XIE Guosi1,3
1.Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao 266071;2.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;3.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071
Abstract:
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome (EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16S rRNA and groEL gene sequence. The serotype of the bacterium is O1:KUT (K untypeable). It revealed positive amplification of the genes pirAVP and pirBVP which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose (LD50) value of 7.96×103 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100% died at 24 h. Subsequent histological analyses showed that the hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells, which was the typical pathological characteristics of AHPND. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.
Key words:  Litopenaeus vannamei  AHPND  Vibrio parahaemolyticus  Bacterial identification