Optimization Of The Production Process Of The Bivalent Inactivated Vaccines Against Vibrio Harveyi And Aeromonas Veronii In Lateolabrax Japonicus

Vibrio harveyi and Aeromonas veronii are two important pathogenic bacteria that endanger the health of aquatic animals and the aquaculture industry.They have a wide range of infections and cause a variety of animals and humans.Disease,seriously endangering the healthy and sustainable development of the breeding industry.There are more and more reports of the abuse of antibiotics for the prevention and treatment of fish diseases.The abuse of antibiotics has great harm to the water environment and human health.The prevention and treatment of diseases with aquatic vaccines as the main method is getting more and more attention.And development.In the overall experiment,by optimizing the optimal growth medium,fermentation conditions and inactivation process of the strains of V.harveyi and A.veronii,the optimal medium,fermentation and inactivation conditions were optimized,and A bivalent inactivated vaccine was further prepared for immunization and follow-up experiments on Lateolabrax japonicus.The specific research contents and results are as follows:1.In this experiment,the growth medium of V.harveyi strains V11HK1,V12YD22 and A.veronii strains 18BJ181,18QW2352 was optimized by constant temperature shaking culture in a triangular flask,and a single factor experiment was used to determine the sodium chloride for the growth of the strain.The concentration,nitrogen source,carbon source,phosphorus source types and concentrations were optimized and screened,and further verified by orthogonal experiments.The results show that the optimal salinity for growth of V.harveyi was 20‰,and that of A.veronii the growth salinity of the bacterial strain does not exceed 1%,and the optimal medium for bacterial growth is tryptone 20 g/L,glucose 5 g/L,and dipotassium hydrogen phosphate 5 g/L.At this time,the OD value of the bacteria and the viable bacteria are the highest.2.On the basis of obtaining the most suitable growth medium for the strain,further optimize its fermentation conditions,and use single factor experiment and orthogonal experiment to optimize and screen the temperature,the initial p H of the medium,the rotation speed of the shaker,and the amount of inoculation,etc.The results show that the optimal fermentation conditions are 28°C,the initial p H of the medium do not change,the rotating speed of the shaker is 210 r/min,and the 10% inoculum amount.After optimization,the OD value and the number of viable cells increased.3.Obtain the optimized growth medium and fermentation conditions of the strain,and prepare the bacterial solution on this basis.Use different final concentrations of formaldehyde solution to inactivate at different temperatures for different lengths of time.The results show that the final concentration is at 28 °C.The inactivation of 0.3% and 0.4% formaldehyde solution for 48 hours can completely inactivate the bacterial solution;at 37℃,the final concentration of 0.2%,0.3% and 0.4% formaldehyde solution can be completely inactivated for 48 hours.However,the A.veronii bacteria solution requires 0.3% and 0.4% formaldehyde solution to inactivate for 48 hours before it can be completely inactivated.4.Flagellin,Quorum Sensing and type III secretion system are important virulence factors of A.veronii.They are closely related to their pathogenicity and are regulated by varieties of environmental conditions.We took asc F,fli E and lux R virulence factors of A.veronii as the research objects through real-time fluorescent quantitative PCR method.The response of asc F,fli E and lux R genes to environmental factors such as temperature,p H,rotating speed and ions were explored at the transcription level.The results show that the three genes of A.veronii have a positive response to acidic environment(p H6.5-7.0),low and medium speed(150-210 r/min),Zn2+ and Mg2+.The response patterns of the three genes of the two strains of A.veronii with different molecular typing are different,indicating that the pathogenic mechanisms of A.veronii with different molecular typing types are significantly different.The important virulence factors of A.veronii are regulated by environmental conditions and show different regular changes.5.The prepared bivalent inactivated A.veronii vaccine was used to immunize the Lateolabrax japonicus,and the safety,efficacy,minimum immunization dose,repeated vaccination efficacy,overdose vaccination efficacy and the efficacy of different vaccination routes had been tested for the bivalent inactivated vaccine.The results show that the prepared vaccine is safe,and there is no adverse reaction or death after immunization of Lateolabrax japonicus.It is most suitable to immunize sea bass with a dose of 0.1 ml once through the abdominal cavity.The higher the vaccine concentration,The higher the RPS,the stronger the immune effect for Lateolabrax japonicus.