浙江省患病凡纳对虾副溶血弧菌的分离、鉴定及毒力基因分析
PDF下载 (372)方 磊1,石 坚1,2,夏武强3,张 鑫1,孟庆珍1,陈铁枫1,2,徐锦余2,钱 冬1*.浙江省患病凡纳对虾副溶血弧菌的分离、鉴定及毒力基因分析[J].宁波大学学报(理工版),2017,30(1):29-34.DOI:
FANG Lei1,SHI Jian1,2,XIA Wu-qiang3,ZHANG Xin1,MENG Qing-zhen1,CHEN Tie-feng1,2,XU Jing-yu2,QIAN Dong1*.Isolation, identification and main virulence analysis of Vibrio parahaemolyticus from diseased Penaeus vannamei in Zhejiang province
[J].Journal of Ningbo University(Natural Science & Engineering Edition),2017,30(1):29-34.DOI:
| Title: | Isolation, identification and main virulence analysis of Vibrio parahaemolyticus from diseased Penaeus vannamei in Zhejiang province |
| 作者: | 方 磊1, 石 坚1, 2, 夏武强3, 张 鑫1, 孟庆珍1, 陈铁枫1, 2, 徐锦余2, 钱 冬1* |
| Author(s): | FANG Lei1, SHI Jian1, 2, XIA Wu-qiang3, ZHANG Xin1, MENG Qing-zhen1, CHEN Tie-feng1, 2, XU Jing-yu2, QIAN Dong1* |
| 关键词: | 副溶血弧菌; 分离和鉴定; 毒力基因; 凡纳对虾 |
| Keywords: | Vibrio parahaemolyticus; isolation and identification; virulence; Penaeus vannamei |
| 分类号: | P735 |
| 文献标识码: | A |
| 摘要: | 对浙江省发病凡纳对虾进行了弧菌分离、鉴定, 对分离菌株进行了API-20E生化鉴定, 不耐热毒素基因tlh、致病基因tdh、trh和毒力基因pir的PCR检测, 以及高致病性副溶血弧菌对小鼠致病性研究结果显示, 150个分离菌株中共检出tlh基因菌株61株(40.67%), ap阳性菌株21株(14.00%); 189个固定样品中检出副溶血弧菌阳性样品108个(57.14%), ap阳性样品24个(12.70%); 所有tlh阳性样品中均未检出tdh、trh毒力基因; ap阳性副溶血弧菌灌胃小鼠死亡率为20%, 且其培养上清液有弱溶血效价. 表明副溶血弧菌在浙江省凡纳对虾弧菌感染中占有重要地位, 高致病性副溶血弧菌在副溶血弧菌中占较高比例, ap阳性副溶血弧菌对小鼠具有一定毒力和溶血性. |
| Abstract: | To investigate the vibrio infection of diseased Penaeus vannamei, a certain number of diseased samples of Penaeus vannamei in Zhejiang province are collected for vibrio isolation and biochemical identification. Vibrio isolations with TCBS agar are used for biochemical identification of API-20E as well as for PCR detection of tlh, tdh, trh and pir gene. High pathogenic Vibrio parahaemolyticus are applied in order for evaluating the virulence to mice. The results show that 61 tlh isolations (accounting for 40.67%) and 21 ap positive isolations (14%) have been detected from a total of 150 isolations; 108 (57.14%) out of 189 fixed samples are tlh positive, while 24 ap positive samples are detected (12.70%). No tdh and trh positive are found. The supernatant of the bacteria manifests the weak hemolytic activity (1:2) and the bacterium can cause death of mice. The results indicate that Vibrio parahaemolyticus plays an important part in the diseased Penaeus vannamei infected by vibrio. Furthermore, high pathogenic Vibrio parahaemolyticus has been found to share a large proportion of Vibrio parahaemolyticus isolations which are separated from diseased Penaeus vannamei. High pathogenic Vibrio parahaemolyticus of ap positive suggests the hemolytic activity and virulence to some degree |
| 参考文献 /References: | [1]., 王国良. 对虾白斑综合症病毒的诊断和检测[J]. 海洋湖沼通报, 2002(3):65-70. [2].郑志肖, 林建东, 顾建明, 等. 宁波地区凡纳对虾白斑综合征病毒(WSSV)的检测与分析[J]. 南方农业学报, 2010, 41(11):1226-1228. [3].杨冰, 宋晓玲, 黄倢, 等. 对虾传染性皮下及造血组织坏死病毒IHHNV的流行病学与检测技术研究进展[J]. 中国水产科学, 2005, 12(4):520-524. [4].袁颜颜, 杨冰, 万晓媛, 等. 采用OIE标准检测养殖对虾中传染性皮下及造血组织坏死病毒(IHHNV)的PCR检出类型[J]. 渔业科学进展, 2015(1):67-72. [5].ZHANG Q, LIU Q, LIU S, et al. A new nodavirus is associated with covert mortality disease of shrimp[J]. Journal of General Virology, 2014, 95:2700-2709. [6].刘珍, 张庆利, 万晓媛, 等. 虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测[J]. 渔业科学进展, 2016, 37(2):119-126. [7].HAN J E, TANG K F TRAN L H, et al. Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepato- pancreatic necrosis disease (AHPND) of shrimp[J]. Dis Aquat Org, 2015, 113(1):33-40. [8].XU X, WU Q, ZHANG J, et al. Prevalence, pathogenicity, and serotypes of Vibrio parahaemolyticus in shrimp from Chinese retail markets[J]. Food Control, 2014, 46(46): 81-85. [9].李晶娇, 薛峰, 曾德新, 等. 副溶血弧菌毒力因子的研究进展[J]. 畜牧与兽医, 2014(12):613-619. [10].HIROTAKA H, TOSHIO K, TETSUYA I, et al. Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice[J]. Infection and Immunity, 2010, 78(4):1772-1780. [11].KOBAYASHI T, ENOMOTO S, SAKAZAKI R, et al. A new selective isolation medium for vibrio group on a modified Nakanishi’s medium (TCBS agar medium)[J]. Nihon Saikingaku Zasshi (Japanese Journal of Bacteriology), 1963, 18(10/11):387-392. [12].沈萍. 微生物学实验[M]. 北京: 高等教育出版社, 2007:120-125. [13].FLEGEL T W, LO C F. Interim primers for specific detection of bacterial isolates that cause acute hepato- pancreatic necrosis disease (AHPND)[EB/OL]. [2015-10-12]. http://www.enaca.org/modules/library/ [14].publication.php?publication_id=1128. [15].BEJ A K, PATTERSON D P, BRASHER C W, et al. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh[J]. Journal of Micro- biological Methods, 1999, 36(3):215-225. [16].钱冬, 陈月英, 沈锦玉, 等. 引起鱼类暴发性流行病的嗜水气单胞菌的血清型、毒力及溶血性[J]. 微生物学报, 1995(6):460-464. [17].翟秀梅, 王斌, 毛连菊, 等. 副溶血弧菌对凡纳对虾生理生化指标的影响[J]. 上海海洋大学学报, 2007, 16(2): 162-168. [18].宋春华, 孟庆显. 副溶血弧菌引起中国对虾红腿病的组织病理及防治[J]. 齐鲁渔业, 1998(3):24-27. [19].金培婕, 吴蓓蓓, 王淑娜, 等. 浙江沿海地区海产品及环境中副溶血弧菌的分离与主要毒力基因分析[J]. 微生物学通报, 2009, 36(7):962-967. [20].魏霜, 冼钰茵, 赵晖, 等. 多重PCR检测四种食源性病原弧菌[J]. 中国农业科学, 2013, 46(8):1682-1686. [21].林祥田. 94起副溶血弧菌食物中毒流行病学特征分析[J]. 上海预防医学, 2006, 18(3):141-143. [22].周向阳, 王淑娜, 周秀锦, 等. 沿海地区副溶血弧菌的表型及其主要毒力基因分布特征[J]. 华北农学报, 2011, 26(1):192-195. [23].THOMSON W K, THACKER C L. Effect of temperature on Vibrio parahaemolyticus in oysters at refrigerator and deep freeze temperatures[J]. Canadian Institute of Food Science and Technology Journal, 1973, 6(3):156-158. Isolation, identification and main virulence analysis of Vibrio parahaemolyticus from diseased Penaeus vannamei in Zhejiang province [24].FANG Lei1, SHI Jian1,2, XIA Wu-qiang3, ZHANG Xin1, MENG Qing-zhen1, CHEN Tie-feng1,2, XU Jing-yu2, QIAN Dong1* [25].( 1.School of Marine Sciences, Ningbo University, Ningbo 315211, China; 2.Hangzhou Sanhe Innovation Technology Co. Ltd, Hangzhou 310020, China; 3.Xiangshan Marine and Fishery Quality Surveillance Center, Xiangshan 315700, China ) [26].Abstract: To investigate the vibrio infection of diseased Penaeus vannamei, a certain number of diseased samples of Penaeus vannamei in Zhejiang province are collected for vibrio isolation and biochemical identification. Vibrio isolations with TCBS agar are used for biochemical identification of API-20E as well as for PCR detection of tlh, tdh, trh and pir gene. High pathogenic Vibrio parahaemolyticus are applied in order for evaluating the virulence to mice. The results show that 61 tlh isolations (accounting for 40.67%) and 21 ap positive isolations (14%) have been detected from a total of 150 isolations; 108 (57.14%) out of 189 fixed samples are tlh positive, while 24 ap positive samples are detected (12.70%). No tdh and trh positive are found. The supernatant of the bacteria manifests the weak hemolytic activity (1:2) and the bacterium can cause death of mice. The results indicate that Vibrio parahaemolyticus plays an important part in the diseased Penaeus vannamei infected by vibrio. Furthermore, high pathogenic Vibrio parahaemolyticus has been found to share a large proportion of Vibrio parahaemolyticus isolations which are separated from diseased Penaeus vannamei. High pathogenic Vibrio parahaemolyticus of ap positive suggests the hemolytic activity and virulence to some degree. |
| 备注/Memo: | 收稿日期: 2016?04?08. 宁波大学学报(理工版)网址: http://journallg.nbu.edu.cn/ 基金项目: 浙江省公益技术研究农业项目(2013C32SA1G0002); 宁波市科技富民强县项目(2016C10049). 第一作者: 方磊(1991-), 男, 浙江浦江人, 在读硕士研究生, 主要研究方向: 水生动物病害. E-mail: 420219351@qq.com *通信作者: 钱冬(1963-), 男, 浙江嵊州人, 研究员, 主要研究方向: 水生动物病害. E-mail: qiandong@nbu.edu.cn 宁波大学学报(理工版)网址:http://journallg.nbu.edu.cn/ |