Study On The Infective Effects And Apoptosis Of Vibrio Harveyi Hemolysin On Cells And Tissues Of Flounder (Paralichthys Olivaceus)

Vibrio harveyi is a Gram-negative, luminous marine bacterium, which is widely distributed in marine environment. This organism is a major pathogen of cultured penaeid shrimp, and has also been associated with disease in fish. The disease caused by V.harveyi has become a serious problem in marine aquaculture.However, we know little about the pathogenicity mechanism of V. harveyi.After the flounder were infected with V. harveyi VIB645, the histopathological changes of the diseased fish were studied, and it was demonstrated that the kidney, spleen, liver, gill, brain and intestine of the infected flounder had serious histopathological changes.The localization of V. harveyi on different tissues of flounder was also studied by indirect fluorescent antibody technique (IFAT) that used rabbit anti-V.harveyi serum, and we found that positive reaction with V. harveyi were identified in kidney, spleen, liver and brain of the infected flounder. Moreover, the ultrastructure changes of flounder erythrocytes were observed with scanning electron microscopy. The results demonstrated that erythrocyte membranes formed thin tubular protrusions after infected with V. harveyi for 4 h. When the incubation time attained to 8 h, the membrane corrugations were observed. The erythrocytes were finally lysed after 12 h incubation.Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH activity-site mutant lost all the hemolytic and phospholipase activities as well as pathogenicity.In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated. Erythrocyte membranes formed thin tubular protrusions immediately after exposure to VHH,and membrane corrugations were evident after extended incubation.In contrast, the mutant VHH did not induce any gross morphological changes.With VHH-treated FG-9307 cells,a cell line derived from flounder gill, destruction of organelles and formation of features resembling apoptotic bodies were observed. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in VHH-treated FG-9307 cells using DAPI staining. Moreover, cell cycle analysis showed that VHH increased the proportion of cells in G1 phase. In addition, VHH significantly increased the percentage of apoptosis, the number of TUNEL positive apoptotic cells, and the caspase-3 activity in FG-9307 cells when compared with the untreated controls. These data suggested that VHH killed the cells through apoptosis via the caspase activation pathway. Immunogold staining showed that a large amount of VHH was deposited on the membranes and membrane debris of erythrocytes and FG-9307 cells after treatment with VHH. After flounder were intraperitoneally injected with VHH, the histopathological changes of the diseased fish were studied, and the results demonstrated that the kidney, liver and intestine of the infected flounder had serious histopathological changes. The results of immunohistochemistry illustrated that intense immunoreactive VHH was mainly localized to the intestinal and gill epithelial cells, indicating that intestine and gill may be the main target-sites for invasion by V. harveyi. This study facilitated the understanding of pathogenesis of V. harveyi and the mode of action of VHH/TLH-like hemolysin and would be helpful to the development of diseases control and prevention technique against V. harveyi.