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

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

摘要:

【目的】美人鱼发光杆菌美人鱼亚种(Photobacterium damselaesubsp.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(extracellular products，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，5×10⁸ CFU/mL的菌液浓度24 h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死亡曲线趋势基本一致；病理切片观察发现，感染ECP后许氏平鲉组织发生明显病变，主要表现为肠黏膜层组织崩解，心脏心肌炎性细胞浸润，肝脏血细胞和炎性细胞浸润，脾脏出现血铁蛋白沉积等；2株PDD菌株的ECP也具有体外强溶血活性和强磷脂酶活性，与菌株的毒力特性基本一致；2株PDD菌株的ECP对人胚肾细胞系HEK293T和小鼠成纤维细胞系MCFS具有细胞损伤作用，均使体外培养的细胞体积萎缩，形成了圆形细胞簇等退行性变化。【结论】通过体外测定溶血性、磷脂酶活性，许氏平鲉人工感染实验和病理切片观察，以及细胞毒性研究，证实2株高致病性PDD菌株的致病性表型与其ECP致病性表型和细胞毒性高度一致，从而说明ECP是高致病性PDD菌株的重要致病机制之一。

Abstract:

[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.