Pathogenicity and Innate Immune Response to Vibrio Parahaemolyticus Infection in the Zebrafish

The aquaculture industry has grown tremendously during last few years, however it is threatened by the occurrence of diseases caused by bacteria and viruses. Vibrio parahaemolyticus, Vp has been recognized as one of the most significant pathogens in marine aquaculture. Since the disease outbreaks are a significant constraint to the development of the aquaculture, causing severe economical losses, the vaccination appear to broad prospects in fish disease prevention. Therefore, the fish innate immunity mechanism draw much more attention, but the mechanism and the immune response for the fish challenged by Vp need to be fully understood. The zebrafish (Danio rerid) has been extensively used to study vertebrate development, hematopoiesis and infectious disease in recent years because they have complete (innate and adaptive) immune systems, small size, relatively rapid life cycle and ease of breeding. As another valuable vertebrate model organism after the mice, the zebrafish is the most appropriate model animal for basic researches such as the pathogenesis of fish infectious diseases and the immune response.In this study, Vibrio parahaemolyticus fish pathogenic strain (Vp1.2164) and human pathogenic strain (Vp17) were used to challenge zebrafish and the pathogenicity and innate immune response were characterized. First of all, we systematically analyzed the effect of variety preservation methods for the virulence and biological property of Vp, and it was the necessary prerequisite to the follow-up research. Second, we tried to find out an effective way of zebrafish infection and pathogenicity. At the last, mRNA expression levels of inflammatory cytokines, such as interleukin-1β (IL-1β)、interferon (IFN) and tumor necrosis factor (TNF) were assessed by quantitative real-time PCR in infected zebrafish exposured to immersion following dermal abrasion and intraperitoneal (ip) injection.We got following conclusions:1、This study found the virulence of Vibrio parahaemolyticus weakened in different preservation method. Slope preservation at room temperature was better than slope preservation at4℃, and it was fit for short-term preservation. Freeze-drying preservation and glycerin preservation at-80℃could be lasted more than24months, and they both were fit for long-term preservation. Skim milk as protecting agent was better than glycerine at-20℃, and glycerin as protecting agent was good at-80℃. It showed that using glycerin as protecting agent was more suitable for ultra low temperature to preserve Vp.2、This study found that the pathogenicity difference between fish pathogenic strain (Vp1.2164) and human pathogenic strain (Vp17) to zebrafish was not remarkable; meanwhile, typical symptoms of septicemia occur when the zebrafish was infected with Vp which can be found in the body of the dead fish, and the tissue section reveals exfoliation and inflammatory edemma of the intestinal epithelial cells, and hyperemia and edema of the spleen. Zebrafish was susceptible to infected by intramuscular injection and ipinjection method but not static immersion unless the epithelial layer was perturbed by scraping prior to exposure. Ip injection was comparatively easy to operate and it was a dominate infection route.3n We used fish pathogenic strain (Vp1.2164) and human pathogenic strain (Vp17) to challenge zebrafish with scraped and then immersion and ipinjection method, and mRNA expression levels of inflammatory cytokines, such as interleukin-1β (IL-1β)、 interferon (IFN) and tumor necrosis factor (TNF) were assessed by quantitative real-time PCR. The results indicated that three genes transcription level follows a high-low pattern in zebrafish under the condition of different infection comparing with their controls. Gene expression of IL-1β in zebrafish scraped and then immersed in Vp1.2164and Vp17peaked at8h, showing125.7-fold and100.9-fold increase when compared with control samples, respectively. Gene expression of TNF in zebrafish scraped and then immersed in Vp1.2164and Vp17also peaked at8h, showing a249-fold and55-fold increase when compared with control samples, respectively. Two strains challenge zebrafish with scraped and then immersion showed a different result on the time of the expression peak of IFN. Gene expression of IFN in zebrafish scraped and then immersed in Vp1.2164reached the peak at8h, and showing an approximate11.6-fold increase over the control. Gene expression of IFN in zebrafish scraped and then immersed in Vp17reached the peak at4h, and showing an approximate10.4-fold increase over the control. Gene expression of IL-1β in zebrafish injected ip with Vp 1.2164and Vp17peaked at4h, showing a56.5-fold and48.8-fold increase when compared with control samples, respectively. Gene expression of IFN in zebrafish injected ip with Vp1.2164and Vp17also peaked at4h, showing11-fold and2-fold increase when compared with control samples, respectively. Two strains challenge zebrafish with ip injection showed a different result on the time of the expression peak of TNF. Gene expression of TNF in zebrafish injected ip with Vp1.2164reached the peak at4h, and showing an approximate143-fold increase over the control. Gene expression of TNF in zebrafish injected ip with Vp17reached the peak at2h, and showing an approximate46.2-fold increase over the control. The experimental results showed that Vp infection can be capable to induce typical acute inflammatory response, the zebrafish that were scraped and then immersed in Vp showed a slower response to infection than that in ip injected zebrafish. But at8h, a very strong inflammatory response was induced and with much higher levels of three inflammatory cytokine transcripts than that in ip injected zebrafish.Overall, when the virulence stableness of Vibrio parahaemolyticus was maintained using reasonable preservation method, the mutual action between Vp and zebrafish in the infection and anti-infection was studied with effective infection methods by testing the expression changes of the inflammatory cytokines, which provided research foundation for the anti-bacteria innate immune response mechanism study of the fish. In one word, this study provided good theoretical basis on the innate immune response mechanism and the mutual action between the host and Vp’s infection in the future.