Pathogenicity Of Fish Pathogen Pseudomonas Plecoglossicida And Preparation Of Its Inactivated Vaccine

With the continuous development of social and economic level,the increasing population pressures caused a growing demand for freshwater fish.As a result,the scale of aquaculture has expanded and the density of aquaculture has being continue increasing,which resulted in frequent outbreaks of fish diseases.Therefore,massive misuse of antibiotics was used to prevent the continually occurrence of fish diseases,which caused the deterioration of the aquaculture environmen and the damage of healthy aquaculture.The vast majority of the research on the pathogenic mechanism of fish pathogens has not yet to be carried out clearly.It is urgent to found a more safer,environmentally friendly and effective measure to prevent and control diseases in aquaculture.Plenty of fishes infected with Pseudomonas plecoglossicida generally suffer from“visceral white spot disease”or even die.Therefore,the research on the pathogenic mechanism and preventive measures against P.plecoglossicida has important scientific meaning and value for the sustainable development of aquaculture.In this paper,a dominant pathogen strain was screened from the intestinal tract of diseased crucian carp in Wangcheng Lake area,Changsha,and it was identified as P.plecoglossicida.The selected strain was a new strain in the pseudomonas species,and named as P.plecoglossicida LQJ06.The semi-lethal does of the pathogenic bacteria infected zebrafish was 2.98×10~6cfu/tail,showing strong toxicity.Strain LQJ06 was labeled with red fluorescent protein to track its dynamic distribution in zebrafish.The results showed that the strain LQJ06 basically was colonized the intestine and poisoned zebrafish.The pathological structure analysis of tissue sections indicated that the intestinal tract was seriously damaged,and the epithelial cells of intestinal tissue were necrotic,intestinal villi were fall off,the liver cells were vacuolated and the nucleus were pyknotic and shifted,and lymphocytes were proliferated in the spleen.Strain LQJ06 could invade and proliferate in grass carp liver cell L8824,and the number of LQJ06 strain was decreased during the invasion.It was speculated that the invasion of LQJ06 strain into grass carp liver cell L8824 was induced cell stress responses,such as apoptosis,and led to the reduction of the number of intracellular pathogens.Apoptosis of L8824 cells was induced by LQJ06 strain,which was detected by Hoechst and PI double staining method.Then the number of internalized bacteria are gradually growing.Cell morphology was changed owing to the toxicity of culture supernatant of LQJ06 strain,which was mainly manifested as aggregation between cells,the cell pyknosisd and grow slowly or even death.The inactivated vaccine against P.plecoglossicida LQJ06 prepared in this study was safe and nontoxic to grass carp liver cells.Compared with the oral administration group,most of the cellular immune factors were expressed earlier and at a higher level by injection immunization.The intestinal tract and liver from zebrafish mainly expressed IFN-γ2gene and IL-1βgene respectively after immunization.The up-regulation of these immune-related genes proved that the vaccine could strengthen the immunity of zebrafish,induce inflammation and resist pathogenic infection.When challenged with the homologous pathogen,the protective rate of the injection and oral immunization on zebrafish were 66%and46.6%,respectively.In summary,it can be seen that the best protection effect was achieved by injection.The results of this study provide a scientific basis for further research on the prevention and control of P.plecoglossicida,and an essential preliminary basis for the development of an inactivated vaccine against P.plecoglossicida.