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，经血平板检验分离菌株的溶血活性，体外筛选并鉴定出3株潜在致病性菌株，即哈维氏弧菌、溶藻弧菌以及副溶血性弧菌。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。结果显示，经哈维氏弧菌刺激后肠道上皮细胞白介素10基因(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%)，推测哈维氏弧菌为半滑舌鳎溃疡病高致病性弧菌。通过对半滑舌鳎进行浸浴回感实验，结果发现，哈维氏弧菌、溶藻弧菌和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。实验表明，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。

In this study, 40 bacterial strains were isolated from the intestinal tract and surface lesions of the diseased Cynoglossus semilaevis with 2216E and TCBS medium. The hemolytic activity of the strains was tested, and three strains i.e. Vibrio harveyi, V. alginolyticus and V. parahaemolyticus with the strongest hemolysis activity were screened and identified as the three potential pathogenic strains in vitro. By co-culturing the three kinds of potential pathogenic bacteria with the intestinal epithelial cells of the healthy C. semilaevis, 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 gene (IL-10) in intestinal epithelial cells was significantly up-regulated after being stimulated with V. harveyi, suggesting that V. harveyi elicited the most intense immune response after co-culturing, and the apoptosis rate of the intestinal epithelial cells after co-culture with V. harveyi, V. alginolyticus and V. parahaemolyticus was 48.3%, 36% and 34.5%, respectively. It is speculated that V. harveyi is a highly pathogenic strain of C. semilaevis ulcer disease. Finally, the results of the artificial infection showed that the mortality rates of V. harveyi, V. alginolyticus and V. parahaemolyticus were 100% and 95% and 75% respectively on the sixth day after artificial re-infection of C. semilaevis, which is consistent with the results of the cellular level test. The study indicated that the three Vibrio strains screened in this experiment had strong toxicity and pathogenicity, especially the pathogenicity of V. harveyi was the strongest, and it is speculated that C. semilaevis ulcer disease may be caused by co-infection of various pathogenic bacteria.

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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)；天津市高等学校创新团队建设规划项目(TD13-5076)