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凡纳滨对虾感染致急性肝胰腺坏死病副溶血弧菌(VpAHPND)的定量分析
陈蒙蒙,董宣,邱亮,万晓媛,谢国驷,黄倢
1.青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 青岛市海水养殖流行病学与生物安保重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.上海海洋大学水产与生命学院 上海 201306
摘要:
对虾急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease, AHPND)是由致AHPND副溶血弧菌(AHPND-causing Vibrio parahaemolyticus, VpAHPND)携带的pVA1-like质粒所表达的PirAVp和PirBVp毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×105 CFU/ml VpAHPND分离株20130629002S01对凡纳滨对虾(Litopenaeus vannamei)进行浸泡感染,于感染后2~9 d采集对虾的肝胰腺、鳃、肠道、肌肉组织,采用实时荧光定量PCR方法,检测各组织中的pirAVp拷贝数。结果显示,感染后凡纳滨对虾各组织均能检测到pirAVp,其中,肝胰腺在感染后第4天达到峰值, 为8.71×104 copies/mg,而鳃、肌肉、肠道分别在第3、4、5天达到峰值,分别为9.08×103、2.59×104、5.76×104 copies/mg。早期感染鳃组织中先出现VpAHPND的富集,在高死亡发生期,VpAHPND数量在肝胰腺和肠道出现高峰,在死亡数量逐渐下降的后期,各组织的VpAHPND均快速下降,肠道、肝胰腺和肌肉中的VpAHPND水平趋于接近。对虾肝胰腺组织病理切片显示,同一时间有临床症状的病虾和濒死对虾相比,濒死对虾表现出更严重的AHPND病理特征,且二者的组织病理特征均随着感染时间的延长变得更为严重,但检测到的VpAHPND数量呈下降趋势。研究表明,在VpAHPND感染过程中,组织中的pirAVp基因数量不能代表对虾的发病程度,发病程度及组织病理严重的AHPND样品中VpAHPND的数量不一定处于高水平状态。
关键词:  致急性肝胰腺坏死病  副溶血弧菌  浸浴感染  凡纳滨对虾  qPCR  时间进程
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Quantitative analysis of acute hepatopancreatic necrosis disease causing Vibrio parahaemolyticus (VpAHPND) in infected Litopenaeus vannamei
CHEN Mengmeng1,2,3,4,5, DONG Xuan1,2,3,4, QIU Liang1,2,3,4,5, WAN Xiaoyuan1,2,3,4, XIE Guosi1,2,3,4, HUANG Jie1,2,3,4,5
1.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao);2.Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs;3.Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity;4.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;5.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306
Abstract:
Acute hepatopancreatic necrosis disease (AHPND) results from acute toxicity in the hepatopancreas of infected shrimp caused by the toxic proteins PirAVp and PirBVp, which are expressed by the pVA1 plasmid carried by AHPND-causing Vibrio parahaemolyticus (VpAHPND). In this study, Litopenaeus vannamei were exposed to 2.19×105 CFU/ml VpAHPND strain 20130629002S01 by immersion to explore the dynamic changes of VpAHPND in the tissues of shrimp. The hepatopancreas, gills, midgut, and muscle of infected shrimp were collected 2~9 days after immersion infection, and the quantity of pirAVP was measured by qPCR. The results showed that VpAHPND could be detected in all sampled tissues of infected shrimp. The amount of VpAHPND in the hepatopancreas reached a peak on day 4 post-infection at 8.71×104 copies/mg, while the gills, muscle, and midgut reached peaks on day 3, 4, and 5 post-infection at 9.08×103、2.59×104、5.76×104 copies/mg, respectively. The highest amount of VpAHPND was detected in the gills during the early stage of infection, followed by the hepatopancreas and midgut in sequence during heavy disease, with frequent deaths. Subsequently, the amount of VpAHPND declined rapidly in all tissues, with similar levels in the midgut, hepatopancreas, and muscle. Histopathology revealed that AHPND lesions were denser in hepatopancreas samples from moribund shrimp compared with those from morbid shrimp, when taken at the same time from infection. Furthermore, the histopathologic symptoms of both became more severe along the infection process, but with decreasing levels of VpAHPND. The results showed that the copy number of pirAVp in tissues of VpAHPND-infected shrimp does not represent the real-time condition of diseased shrimp and the quantity of VpAHPND may not be high in a severe AHPND sample.
Key words:  Acute hepatopancreatic necrosis disease (AHPND)  Vibrio parahaemolyticus  Immersing infection  Litopenaeus vannamei  qPCR  Time courses