黄沙鳖致病性温和气单胞菌的分离鉴定及其毒力基因检测  

Isolation,identification and virulence gene detection of pathogenic Aeromonas sobria from Truogx sinensis

作　　者：农新闻 黄艳华[1,3] 龙苏[1] 梁静真[1] 牛志伟[1] 刘杰[1] 米强 黄钧[1] 

机构地区：[1]广西大学动物科学技术学院/广西水生动物病害诊断实验室,南宁530005 [2]广西水产畜牧学校,南宁530021 [3]梧州市水产技术推广站,广西梧州543002

出　　处：《南方农业学报》2015年第7期1322-1328,共7页Journal of Southern Agriculture

基　　金：广西自然科学基金项目(2011GXNSFA018065);广西水产畜牧兽医局专项项目(桂渔牧财[2014]52号;桂渔牧财[2013]35号);南宁市科技计划项目(20132123)

摘　　要：[目的]明确广西不同养殖区域患病黄沙鳖的病原菌及其毒力基因携带情况,为黄沙鳖细菌性疾病的有效防控及病原菌毒力监测研究提供科学依据。[方法]以常规方法进行病原菌分离鉴定,通过16S r RNA基因序列确定其系统发育地位,并以PCR检测病原菌的6种毒力基因。[结果]从广西6个养殖区10个黄沙鳖养殖场的病样中分离获得10株病原菌,经API 20NE半自动微生物鉴定系统和16S r RNA基因鉴定均为温和气单胞菌（Aeromonas sobria）,与Aeromonas sobria ATCC43979亲源关系最近（同源性达99.5%~99.9%）。6种毒力基因中hly、Aer和Alt基因的阳性检出率为100%,Act基因为90%,ahal和ahp基因为80%。病原菌株的毒力基因型共有4种,其中,hly^＋Aer^＋Alt^＋Act^＋ahal^＋ahp^＋是主要毒力基因型（占70%）,其余3种毒力基因型分别是hly^＋Aer^＋Alt^＋Act^-ahal^＋ahp^-、hly^＋Aer^＋Alt^＋Act^＋ahal^-ahp^＋和hly^＋Aer^＋Alt^＋Act^＋ahal^-ahp^-,各占10%。[结论]hly和Aer基因是黄沙鳖源温和气单胞菌的主要毒力基因,而Alt、Act、ahal和ahp基因对其致病力起协同作用,即在所检测的6种毒力基因中若只缺失Alt、Act、ahal和ahp基因中的1~2种对黄沙鳖源温和气单胞菌致病力影响不明显。[Objective ]The present experiment was conducted to investigate pathogen of diseased Truogx sinensis and its virulence genes in different culture areas of Guangxi, in order to provide scientific basis for effectively controling bacte- r/osis of T. sinensis and detecting virulence gene of its pathogen. [Method]The pathogen was isolated and identified by us- ing conventional method, and its phylogenesis status was confirmed based on 16S rRNA gene sequence. And six kinds of virulence genes in pathogen were detected by applying PCR technique. [Result]Ten strains of pathogens were isolated from diseased T. sinensis in ten farms of six culture areas in Guangxi. All strains were identified as Aeromonas sobffa by using API 20NE semi-automatic microbial identification system based on 16S rRNA gene sequence, and which had the closest relationship with A. sobria ATCC43979, sharing the homology of 99.5%-99.9%. The positive detecting rates of 6 virulence genes were as follows ： 100% of hly, Aer and Alt genes, 90% of Act gene, 80% of ahal and ahp genes. There were 4 kinds of virulence genotypes among ten strains of pathogens, and hly^＋Aer^＋Alt^＋Act^＋ahal^＋ahp^＋ was main genotype, accounting for 70% of all strains. Genotype hly^＋Aer^＋Alt^＋Act^-ahal^＋ahp^-、hly^＋Aer^＋Alt^＋Act^＋ahal^-ahp^＋ and hly^＋Aer^＋Alt^＋Act^＋ahal^-ahp^- each accounted for about 10% of all strains. [Conclusion]The hly and Aer genes are main virulence genes of A. sobria isolated from T. sinensis. Furthermore, the Alt, Act, ahal and ahp genes of A. sobria have significant synergistic effect on its pathogenicity to T. Sinensis, thus, only lack of any one or two of 4 genes has no apparent impact on A. sobria＇s pathogenicity.

关 键 词：黄沙鳖 温和气单胞菌 分离鉴定 毒力基因 

分 类 号：S947.12[农业科学—水产养殖]