文章摘要
半滑舌鳎溃疡病病原菌的分离与鉴定
Isolation and Identification of Pathogens from Cynoglossus semilaevis Günther
投稿时间:2019-07-11  修订日期:2019-10-07
DOI:
中文关键词: 半滑舌鳎  溃疡病  病原菌  分离鉴定  细胞凋亡  免疫基因
英文关键词: Cynoglossus semilaevis Günther  ulcer disease  pathogenic bacteria  separation and identification  cell apoptosis  immune gene
基金项目:天津市高等学校创新团队建设规划项目(TD13-5076);天津市人才发展特殊支持计划高层次创新创业团队项目(ITTFRS2017007);天津市水产产业技术体系创新团队项目(ITTFRS2017007);天津市人才发展特殊支持计划高层次创新创业团队项目(06202-52K16001)
作者单位E-mail
李文悦 天津师范大学生命科学学院 64301143@qq.com 
左志晗 天津师范大学生命科学学院 zhihanzuo@163.com 
张晶晶 天津师范大学生命科学学院  
尚碧娇 天津师范大学生命科学学院  
孙金生 天津师范大学生命科学学院  
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中文摘要:
      半滑舌鳎( Cynoglossus semilaevis Günther)为我国重要经济水产养殖品种,但养殖过程中疾病的频发严重影响其产量,其中半滑舌鳎溃疡病尤为常见。本实验首先采用2216E和TCBS培养基分别从患溃疡病半滑舌鳎的肠道和体表病灶分离获得菌株40株,经血平板检验分离所得菌株的溶血活性,体外筛选并鉴定出3株潜在致病性菌株:哈维氏弧菌(Vibrio harveyi),溶藻弧菌(Vibrio alginolyticus)以及副溶血性弧菌(Vibrio Parahaemolyticus)。其次,通过将3株潜在致病菌与健康半滑舌鳎的肠道上皮细胞共培养,检测病原菌刺激后半滑舌鳎肠道上皮细胞相关免疫基因的相对表达量及共培养后肠道上皮细胞的凋亡率,对病原菌的致病性进行细胞水平的筛选,结果表明:哈维氏弧菌刺激后肠道上皮细胞白介素10(IL-10)表达量极显著上调,说明哈维氏弧菌引起的共培养上皮细胞免疫反应最为剧烈;且哈维氏弧菌与肠道上皮细胞共培养后导致细胞的凋亡率高达48.3%,其次为溶藻弧菌(36%)和副溶血性弧菌(34.5%),推测该哈维氏弧菌为半滑舌鳎溃疡病高致病性弧菌。最后,对半滑舌鳎进行浸浴回感实验,结果表明哈维氏弧菌、溶藻弧菌、副溶血性弧菌人工回感后第6天半滑舌鳎的累计死亡率分别为100%、95%和75%,与细胞水平检测结果相吻合。上述结果说明本实验所筛选到的3株弧菌均具有较强的毒性和致病性,尤其以哈维氏弧菌致病性最强,并由此推测半滑舌鳎溃疡病可能由多种致病菌共同感染所致。
英文摘要:
      Cynoglossus semilaevis Günther is an important economic aquaculture species in China, but the frequent occurrence of diseases in the breeding process seriously affects its yield. Among them, Cynoglossus semilaevis Günther ulcer disease is especially common. In this experiment, 40 bacterial strains were isolated from the intestinal and surface lesions of the diseased Cynoglossus semilaevis Günther with 2216E and TCBS medium . The hemolytic activity of the strains was tested, and three potential pathogenic strains were screened and identified in vitro: Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus. Secondly, by co-culturing the three kinds of potential pathogenic bacteria with the intestinal epithelial cells of the healthy Cynoglossus semilaevis Günther, the relative expression of the immune genes and the apoptosis rate of the intestinal epithelial cells after co-culture were detected separately to evaluate the pathogenicity of these strains at the cellular level. The results showed that the expression of interleukin-10 (IL-10) in intestinal epithelial cells was significantly up-regulated after stimulated with Vibrio harveyi, suggesting that the Vibrio harveyi elicited the most intense immune response after co-culturing; and the apoptosis rate of the intestinal epithelial cells after co-cultured with Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus was 48.3%, 36% and 34.5% respectively. It is speculated that the Vibrio harveyi is a highly pathogenic strain of the Cynoglossus semilaevis Günther ulcer disease. Finally, the results of the infected test showed that the mortality of the Cynoglossus semilaevis Günther on the 6th day was 100%, 95% and 75% respectively after artificial infection by Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus, which is consistent with the results of the cellular level test. The above results indicated that the three Vibrio strains screened in this experiment had strong toxicity and pathogenicity, especially the pathogenicity of Vibrio harveyi was the strongest, and it is speculated that the Cynoglossus semilaevis Günther ulcer disease may be caused by co-infection of various pathogenic bacteria.
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