| Lateolabrax maculatus plays an important role in marine in China,and it is also the second most mariculture fish.However,sea bass diseases occur frequently and become more and more serious,causing great economic losses to sea bass breeding industry and seriously restricting the development of sea bass breeding industry.According to the epidemiological investigation of our group,Vibrio harveyi is one of the most important pathogens in sea bass culture industry.In order to control V.harveyi disease of sea bass,our group decided to carry out the laboratory development of formalin-killed vaccine of V.harveyi in Lateolabrax maculatus.1.In this experiment,21 strains of V.harveyi with different molecular types were selected from the laboratory bacterial library,and 21 strains of V.harveyi were initially screened,then rescreened from the screened strains,and finally vaccine strains and standby strains were screened.Finally,the cross protection effect of vaccine strains was tested.The results showed that two virulent strains 5#and 16#were selected as vaccine strains from 21 strains of V.harveyi,and one sub virulent strain 3#was selected as standby strain;the cross protection effect between vaccine strains was poor,so bivalent vaccine should be prepared.2.The biological and growth characteristics of the strain were studied.The growth curve of the strain was plotted;the temperature,salinity,pH and culture medium were screened by single factor experiment method,and the effects of temperature,salinity and pH on the growth of V.harveyi were compared by orthogonal experiment method;finally,the half lethal concentration of the strain was determined.The results showed that there was a good linear relationship between the absorbance and the concentration of V.harveyi;when the medium was nutrient broth medium,pH 7,salinity 3%and the temprature 28℃,the growth of V.harveyi was the best;among the three factors,salinity had the greatest effect on growth,followed by temperature anf pH;in the medium screening experiment,2216E showed the best growth effect;the LD50 of 3#,5#and 16#were 3.09×105 CFU/g,4.04×105 CFU/g and 4.02×105 CFU/g respectively.3.The safety and immune efficacy of the vaccine were studied,and its optimal inactivation conditions were determined.the safety of the vaccine in fry was tesed;the effects of the vaccine on the growth of sea basss was studieds;the minimum immune dose was determined;the immune protection period and relative protection rate of the vaccine and the serum antibody titer of the immunized fish was tested.The results showed that the best inactivation conditions were formaldehyde concentration 0.4%,inactivation time 24h,temperature 28℃,shaking speed 200rmp.The vaccine wass safe to Lateolabrax maculatus,and did not affect the growth of them.When the concentration of the vaccine was 9.6×107 CFU/g,the relative protection rate was the highest.When the final dose of the vaccine was 4.78×107 CFU/g,the relative protection rate was only 21.4%in the first week,and increased to 43.7%in the second week.From the seventh week,the relative protection rate was more than 50%and could last until the 13th week,which meant in the whole experimental period after the second week,the vaccine could provide better relative protection for sea bass,that is,the protection period was 11 weeks.The serum antibody titer of the immunized group was the lowest in the first week,ranging from 1:32 to 1:64,but there was still a significant difference between the immunized group and the blank group and the control group;it increased rapidly in the second week,and then tended to be flat,but still at a high level.In this process,the highest serum antibody titer was 1:2048,and the serum antibody titer of the blank group and the control group was stable,ranging from1:8 to 1:32.In conclusion,this study has provided a basis for the follow-up study of formalin-killed vaccine of V.harveyi in sea bass. |
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