| Objective: As an important kind of vertebrates, zebrafish is widely used in the study of disease model with many advantages such as small size, short time of generations’ alternation, large reproduction and so on. After 48 h fertilization, zebrafish’s neutrophils play a role. The 72 h to 3 w of zebrafish larvae stage is a special period with only innate immunity. The development and maturation of the zebrafish immune system are very similar to human and the genome of zebrafish is highly homologous with the human genome. Considering both innate immunity and adaptive immunity, zebrafish is a very good model to study the pathogenic infection and immunity response. For studying the influence of Salmonella plasmid virulence gene spv on zebrafish infection and immunity, this study uses wild strains of Salmonella enterica serovar typhimurium(S. typhimurium, STM) STM-WT and lacking of spv gene mutations STM-Δspv to infect respectively. In the first part, this study uses zebrafish larvae after 72 h fertilization with only innate immunity model to discusse the effects of spv on the body infection and innate immune function. In the second part, with establishing 8-month-old adults with adaptive immunity zebrafish adult infection model, this study discusses the influence of the spv gene on the body infection and Th1/Th2 immune response, in order to further clarify the pathogenic mechanism of spv and the effects on the immune function to provide theoretical and experimental basis.Methods: 1. The influence of Salmonella plasmid virulence genes spv on larval infection and innate immune function(1) The dissemination of Salmonella in zebrafish larvae Infection has been established by zebrafish larval model method in our laboratory. 72 hours post-fertilization(hpf) zebrafish larvae were immersed in the water with wild strains of Salmonella typhimurium STM-WT and mutant strains STM-Δspv carrying redfluorescent protein(RFP) markers, divided into STM-WT and STM-Δspv group with 30 larvae each group. After 2, 4 and 8 h post-infection(hpi), the dissemination of Salmonella in 10 zebrafish larvae each group was observed with fluorescent microscopy.(2) The influence of Salmonella plasmid virulence genes spv on neutrophils recruitment 72 hpf zebrafish larvae were infected with STM-WT and STM-Δspv, with 30 larvae each group. Collecting 10 zebrafish larvae each group after 2, 4 and 8 hpi, the larvae were fixed and specifically stained on neutrophils with Sudan black staining method, observing the neutrophils recruitment by microscope.(3) The influence of Salmonella plasmid virulence genes spv on macrophages phagocytosis function The control, 2, 4 and 8 h after infected by STM-WT and STM-Δspv group was established with 10 larvae each group. Culturing 72 hpf zebrafish with neutral red dye solution, the degree of phagocytosed dye was used to judge the phagocytosis function after infection. At different time points, STM-WT and STM-Δspv were respectively joined into the water. After 2, 4 and 8 hpi, the dye of macrophages were observed by microscope after zebrafish anesthesia.(4) The influence of Salmonella plasmid virulence genes spv on bacteria survival after macrophage phagocytosis 72 hpf zebrafish larvae were infected with STM-WT and STM-Δspv with 40 larvae each group. Collecting 10 zebrafish larvae after 2, 4, 8 and 12 hpi, the macrophages were separated and counted the colony-forming units(CFU) in the cells. 2. The influence of the spv gene on the infection and Th1/Th2 immune response(1) The establishment of a Salmonella-zebrafish infection model 8-month-old zebrafish was infected by oral administration with 103, 104, 105, 106 and 107 CFU with 10 adults each group. The survival curve was drawn to show the life-or-death situation. The infection of adults’ viscera was observed with the naked eye after that they are dissected after 3 weeks. The transcriptional expression levels of Th1 cell specific T-box factor expressed in T-cells(TBX21) gene tbx21, Th2 cell GATA binding protein3(GATA-3) gene gata3 and Th1 cell cytokines genes Interferon-γ(IFN-γ), interleukin-12(IL-12) and tumor necrosis factor-α(TNF-α), Th2 cell cytokines genes IL-4, IL-10 and IL-13 m RNA in liver were measured by real-time quantitativePCR and the ratio of corresponding cytokines for Th2/Th1 were also calculated.(2) The influence of the spv genes on the infection of zebrafish adults Zebrafish infection model was established with the above conditions. The control, STM-WT and STM-Δspv group was established. 105 CFU STM-WT and STM-Δspv oral infected the adults after 1 d, 7 d and 14 d with 5 adults each point-in-time. The liver and intestine were separated after dissection. Subsequently, they were respectively counted by CFU, observed after hematoxylin-eosin(HE) staining and under transmission electron microscope(TEM) to observe the ultrastructural changes in the intestine.(3) The influence of the spv genes on Th1/Th2 immune response The control, STM-WT and STM-Δspv group was established. After oral infected with STM-WT and STM-Δspv 7 d and 14 d with 5 adults each point-in-time, the liver and intestine was separation to extract the total RNA. The transcriptional levels of tbx21 and gata3, Th1 cell cytokines genes IFN-γ, IL-12 and TNF-α and Th2 cell cytokines genes IL-4, IL-10 and IL-13 m RNA were measured by q PCR. To show the Th1/Th2 immune deviation, the ratios of gata3/tbx21 and IL-4/IFN-γ were calculated. At the same time, the contents of Th1 cell cytokines IFN-γ, IL-12 and TNF-α, Th2 cell cytokines IL-4, IL-10 and IL-13 were measured by enzyme linked immunosorbent assay(ELISA).Results: 1. The influence of Salmonella plasmid virulence genes spv on larval infection and innate immune function(1) The dissemination of Salmonella in zebrafish larvae According to the results of Fluorescence microscope observation, 2 hpi in the two groups, a small amount with red fluorescent bacteria disseminated in zebrafish and there were no difference between groups; 4 hpi, in the wild strains of STM-WT with red fluorescence labeling group(RFP-STM-WT) larva head and yolk sac area has the obvious red dot, and the mutant strains STM-Δspv group(RFP-STM-Δspv) only has small red dots; 8 hpi, there around the mouth gathered a large number of red bacteria in STM-WT group.(2) The influence of Salmonella plasmid virulence genes spv on neutrophils recruitment There has been seen no black particles for neutrophils recruitment before infection.As the extension of infection time, black particles increased; 4 hpi and 8 hpi black particles were more significantly in STM-Δspv group than STM-WT group(P<0.05).(3) The influence of Salmonella plasmid virulence genes spv on macrophages phagocytosis function There has been seen no red dot aggregations for macrophages phagocytosis function before infection. As the extension of infection time, red dot aggregations increased; 2, 4 and 8 hpi red dot aggregations were more significantly in STM-Δspv group than STM-WT group with 8 hpi was the most significant(P<0.05).(4) The influence of Salmonella plasmid virulence genes spv on bacteria survival after macrophage phagocytosis According to the results of Intracellular CFU counts, microbial infection extend increasing with time. 4, 8 and 12 hpi, the CFUs of STM-WT group were significantly higher than that of STM-Δspv group(P<0.05). 2. The influence of the spv gene on the infection and Th1/Th2 immune response(1) The establishment of a Salmonella-zebrafish infection model All of adults were alive within 2 weeks infected by 105 CFU infectious dose, while the death occurred on the 15 th day. The death rate was 50 % three weeks later. With serious red, swollen and erosion, the lesions of liver, spleen and intestine were significant, which were gained by means of dissecting the adults. It was discovered that an obvious Th1/Th2 immune response was associated with 105 CFU infectious dose. Under this dose, a Th2 response swift appeared evidently.(2) The influence of the spv gene on the infection of zebrafish adults According to the CFU results of organs, as infection time increased, the bacteria in liver and intestine were all increased, but the STM-WT group were significantly higher than that of STM-Δspv group(P<0.05). The HE staining of liver showed that with increased infection time, in STM-WT group there are a lot of liver steatosis, and local inflammatory cell infiltration. In STM-Δspv group 1, 7 day post-infection(dpi) there had not seen liver tissue steatosis, 14 dpi there were steatosis, but few inflammatory cells infiltration. The HE staining of intestinal tissue showed that in STM-WT group with time increasing the intestinal wall was thinning, intestinal epithelium microvilli arrangement was not the whole, and had inflammatory cells infiltration. In STM-Δspv group 1, 7 dpi intestinal wall thickness was not seen obvious, microvilli arranged neatly, with mild disorder 14 dpi.Under TEM, uninfected zebrafish intestine in the control group showed that intestinal epithelial neatly, the cell boundaries clear, the microvilli of free surface is closely and tidy. In STM-WT group 1 dpi the intestinal microvilli fracture could be seen, epithelial cells form had no obvious change. 7 dpi the boundaries of cells were blurred, and cell lumen saw bacteria distribution. In STM-Δspv group 1 dpi there had no significant difference with the control group. 7 dpi microvilli had slight fracture and cellular structure has also changed.(3) The influence of the spv gene on Th1/Th2 immune response The q PCR detection results showed that in STM-WT group 14 dpi, the relative expression ratios of gata3/tbx21 and IL-4/IFN-γ in liver and intestine were significantly increased(P<0.05). 14 dpi, in STM-WT group the expression of IL-12 and TNF-α were significantly lower than STM-Δspv group, and the expression of IL-10 and IL-13 were significantly higher than that of STM-Δspv group(P<0.05). ELISA results showed that 7 and 14 dpi, in STM-WT group the IFN-γ levels of liver and intestine were significantly lower than STM-Δspv group(P<0.05). 14 dpi, in STM-WT group the contents of TNF-α and IL-12 were significantly lower STM-Δspv group and the contents of IL-4 and IL-10 in STM-WT group were significantly higher than that of STM-Δspv group(P<0.05). The content of the IL- 13 in the liver did not see obvious difference, but in the intestine 7 and 14 dpi, in STM-WT group it was significantly higher than that of STM-Δspv group(P<0.05).Conclusions: 1. By inhibiting the neutrophils and macrophages to chemotaxis,aggregation to inflammatory site and devour kill function, Salmonella plasmid virulence genes spv benefit pathogenic bacteria to escape the body’s innate immune response, thus promoting the spread of bacteria and aggravating infection. 2. The established Salmonella-zebrafish infection model was appropriate for reflecting infection and the immune function in the zebrafish adults, which could be used to investigate Th1/Th2 immune response induced by pathogens. It was an available experimental implement to in-depth study the mechanism of infection and immune induced by Salmonella typhimurium. 3. By inhibiting Th1 cells tbx21 gene transcription and the expression of cell cytokines IFN-γ, IL-12 and TNF-α, promoting the Th2 cells gata3 gene transcriptionand the expression of cytokines IL-4, IL-10 and IL-13, Salmonella plasmid virulence genes spv result in the Th1/Th2 immune deviation, enhancing bacterial virulence to aggravate the infection. |
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