高致病性美人鱼发光杆菌美人鱼亚种胞外产物的毒力特性分析

【目的】美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp. damselae,PDD)是一种可导致多种海洋生物患病的重要病原菌。本研究以从我国海水养殖环境中分离的具有强磷脂酶活性、强溶血性表型且具有高致病性的2株PDD菌株为研究对象，分析PDD菌株胞外产物(extracellularproducts,ECP)的致病性及细胞毒性，及其与菌株致病性表型的相关性。【方法】利用培养基平板法测定菌株PDD1605、PDD1608的ECP体外磷脂酶活性和溶血性；通过人工感染实验测定PDD1605和PDD1608活菌株及其ECP对许氏平鲉的致病性，并进行组织病理切片观察组织的病理损伤；通过向人胚肾细胞系HEK293T和小鼠成纤维细胞系MCFS等2种培养细胞中添加菌株PDD1605和PDD1608的ECP测定其对哺乳动物的细胞毒性。【结果】人工感染PDD1605和PDD1608活菌株对许氏平鲉均表现为高致病性，5×108CFU/mL的菌液浓度24h内受试鱼全部死亡；菌株PDD1605和PDD1608的ECP对许氏平鲉同样表现为高致病性，受试鱼的死亡曲线与感染活菌株的...

Virulence of the extracellular products of highly pathogenic strains of Photobacterium damselae subsp. damselae

[Objective] Photobacterium damselae subsp. damselae (PDD) is a major pathogen which causes serious diseases in marine organisms. This study focused on two highly pathogenic PDD strains (PDD1605 and PDD1608) with strong hemolysis and phospholipase activities, which were isolated from maricultured fish in China. We analyzed the virulence and cytotoxicity of the extracellular products (ECPs) derived from the two PDD strains and their correlations with the strain pathogenicity. [Methods] The hemolysis and phospholipase activities of ECPs from PDD1605 and PDD1608 were measured on agar plates. The pathogenicity of the two live strains and their ECPs to Sebastes schlegeli were detected by artificial challenge test. Meanwhile, we prepared histopathological sections to observe the pathological damage of organs and tissues. Furthermore, we added the ECPs to the media of human embryonic kidney cell line HEK293T and mouse cardiac fibroblasts (MCFS) to study the toxicity of ECPs to mammalian cells.[Results] The live PDD1605 and PDD1608 strains showed strong pathogenicity to S. schlegeli. All the fish exposed to the strains at the concentration of 5×10⁸ CFU/mL died within 24 h. The ECPs of PDD1605 and PDD1608 also showed strong pathogenicity to S. schlegeli. The death curve trend was similar between ECPs and live strains. The ECPs caused serious tissue damage to S. schlegeli, which mainly included disintegration of intestinal mucosa, blood cell and inflammatory cell infiltration in liver, hemosiderosis in spleen, and inflammatory cell infiltration in myocardium. Additionally, the ECPs of the two PDD strains had strong hemolysis and phospholipase activities in vitro, which was basically consistent with the virulence characteristics of live strains. Moreover, they had cytotoxicity to HEK293T and MCFS cells, reducing the cell volume in vitro and causing degenerative changes such as round cell clusters.[Conclusion] TheECPs and live strains of PDD1605 and PDD1608 have highly consistent virulence and cytotoxicity. Our findings support that ECPs are the key pathogenic factor of highly pathogenic PDD strains.