文章摘要
韩书煜,梁静真,覃志彪,黄艳华,韦慕兰,蒙兰丽,黄维,胡大胜,黄钧.山瑞鳖细菌性败血症病原菌的分离鉴定及其毒力基因检测[J].水产学报,2017,41(9):1443~1454
山瑞鳖细菌性败血症病原菌的分离鉴定及其毒力基因检测
Isolation, identification and virulence genes detection of pathogenic bacteria from Palea steindachneri with bacterial septicemia
投稿时间:2016-09-25  修订日期:2017-04-11
DOI:10.11964/jfc.20160910554
中文关键词: 山瑞鳖  细菌性败血症  嗜水气单胞菌  毒力基因
英文关键词: Palea steindachneri  bacterial septicemia  Aeromonas hydrophila  virulence gene
基金项目:广西水产畜牧兽医局专项(桂渔牧财[2013]35号,桂渔牧财[2014]52号,桂渔牧财[2015]97号);南宁市科技局科技计划项目(南科发〔2012〕51号-20122096;南科发〔2013〕46号-20132124)
作者单位E-mail
韩书煜 广西水生动物病害诊断实验室, 广西 南宁 530005
广西水产技术推广总站, 广西 南宁 530022 
 
梁静真 广西水生动物病害诊断实验室, 广西 南宁 530005
广西大学动物科学技术学院, 广西 南宁 530005 
 
覃志彪 广西水生动物病害诊断实验室, 广西 南宁 530005
广西大学动物科学技术学院, 广西 南宁 530005 
 
黄艳华 钦州市水产技术推广站, 广西 钦州 535000  
韦慕兰 都安瑶族自治县水产技术推广站, 广西 都安 530700  
蒙兰丽 田东县水产技术推广站, 广西 田东 531500  
黄维 田东县水产技术推广站, 广西 田东 531500  
胡大胜 广西水生动物病害诊断实验室, 广西 南宁 530005
广西水产技术推广总站, 广西 南宁 530022 
 
黄钧 广西水生动物病害诊断实验室, 广西 南宁 530005
广西大学动物科学技术学院, 广西 南宁 530005 
hj1351@163.com 
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中文摘要:
      查明广西南宁、贵港和桂平养殖山瑞鳖细菌性败血症的病原菌及其6种毒力基因的携带情况,为有效防控山瑞鳖细菌性败血症提供参考。本研究以常规方法从患病山瑞鳖的心脏和肝脏取样、分离细菌,人工感染方法确定分离菌株的致病性,细菌鉴定采用API 20NE生化鉴定和16S rRNA分子鉴定相结合的方法进行,PCR扩增法对菌株的溶血素基因(hemolysin gene,hly)、气溶素基因(aerolysin gene,Aer)、细胞兴奋性肠毒素基因(cytotonic enterotoxin gene,Alt)、细胞毒性肠毒素基因(cytotoxic enterotoxin gene,Act)、黏附素基因(major adhesin gene,ahal)和丝氨酸蛋白酶基因(serine protease gene,ahp)6种毒力基因进行检测。结果显示,从患病山瑞鳖心脏和肝脏中共分离到4株优势菌SRB125、SRB142、SRB143和SRB345,对健康山瑞鳖的平均致死率为97.50%~100.00%,是引起山瑞鳖细菌性败血症的病原菌;生化和分子鉴定结果显示,4株分离菌均为嗜水气单胞菌(Aeromonas hydrophila),与A.hydrophila L3-5(KP716701)菌株的亲缘关系最近,同源相似性均达到99.9%;6种毒力基因共包含2种毒力基因型,在4株菌株中的分布为hly+Aer+Alt+Act+ahal+ahp+hly+Aer+Alt+Act+ahal+ahp各2株,来源于南宁的SRB143和桂平的SRB345菌株均缺失ahp基因。
英文摘要:
      In order to provide reference for effective prevention and control of the bacterial septicemia of Palea steindachneri, the present experiment was conducted to investigate the pathogen of P. steindachneri with bacterial septicemia and the presence of six virulence genes. The pathogen was isolated from the heart and liver tissues of P. steindachneri by using conventional methods, and the pathogenicity of the strains was confirmed by artificial infection. Then the pathogen was identified based on API 20NE bacteria identification system and PCR amplification of 16S rRNA gene. Presence of virulence genes was determined using the polymerase chain reaction. Results showed that four dominant strains named SRB125, SRB142, SRB143 and SRB345 were isolated from the heart and liver tissues of diseased P. steindachneri. The mean fatality rate of healthy P. steindachneri was 97.50%–100.00% and the pathogenic bacteria of bacterial septicemia of P. steindachneri were the four isolated strains. According to the biochemical and molecular identification results, the four isolated strains were all Aeromonas hydrophila, and they had the closest genetic relationship with A. hydrophila L3-5 (KP716701), sharing homology of 99.9%. The four strains contained two virulence genotypes, hly+Aer+Alt+Act+ahal+ahp+ and hly+Aer+Alt+Act+ahal+ahp-, each accounting for two strains. Strain SRB143 from Nanning City and SRB345 from Guiping City all lacked gene ahp.
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