| Vibrio anguillarum is an important bacterial fish pathogen that can causes vibriosis extensively in economic fish including Scophthalmus maximus,Paralichthys olivaceus,Salmo salar,Anguilla anguilla and Lateolabrax japonicus in China.Vaccination is proven to be a safety and efficient way to prevent and control vibriosis.However,no commercial V.anguillarum vaccine is available in China.In our preliminary studies,we have carried out the epidemiological work of V.anguillarum and demonstrated that V.anguillarum O1,O2 and O3 serotype strains are prevalent in the marine fish farm.Therefore,in this study,we prepared the V.anguillarum inactivated bivalent-vaccine consisted of O1 and O2 serotype antigens,carried out the vaccination tests to evaluate the antibody production,persistence and immune protective duration of the bivalent-vaccine in turbot(Scophthalmus maximus).The relationship among the vaccination doses,antibody titers and immune protection were analysed.In addition,we studied the immunogenicity and protective efficacy of three V.anguillarum O3 strains.These results obtained will provide more experimental data to enhance the commercialization of V.anguillarum vaccines in China.The experimental contents and results in this study are demonstrated as follows:Ⅰ.Antibody production,persistence,and immune protective duration of inactivated bivalent-vaccine in turbot administrated by injecton vaccination.The formalin-inactivated bivalent-vaccine was prepared with O1 serotype strain VAM003 and O2 serotype strain VAM007.This bivalent-vaccine was administrated intraperitoneally into turbot with once vaccination and twice vaccination at different dosages.The serum antibody production,persistence,and immune protective duration were evaluated during a period of 150 days.1.Primary vaccination.Groups of 2500 turbot were vaccinated by intraperitoneal injection with dosages of 107 cells/ind,108 cells/ind and 109 cells/ind,respectively.The control group fish were injected intraperitoneally with sterilized PBS.The serum antibody titers and relative percent survival rate(RPS)of each group were determined at 3 d,7 d,14 d,21 d,30 d,45 d,60 d,90 d,120 d,150 d post vaccination.The result showed that at 7 d post-vaccination,the serum antibody titers were detected at a range of 1:20-1:100.8 in all vaccinated groups of fish.During the experimental period,the titers exhibited a tendency of increase gradually and then decrease gradually.In detail,the highest titers were detected at 21 d-30 d post-vaccination with anti-VAM003 titers at a value of 1:320 in three vaccinated groups,and anti-VAM007 titers at 1:1015.9 in 107 cells/ind,1:1280 in 108 cells/ind and 1:320 in 109 cells/ind.The higher the vaccination dosage,the longer the higher serum antibody titers were detected in the vaccinated fish.Taking titers value over 1:20 as a line of effective antibody titer,the effective antibody duration of anti-VAM003 was detected at 7 d-90 d in 107 cells/ind,7 d-120 d in 108 cells/ind,and 7 d-150 d in 109 cells/ind,and the effective antibody duration of anti-VAM007 was 7 d-150 d in all vaccination dosages.Taken together,the results indicated the V.anguillarum inactivated bivalent-vaccine could produce effective antibody titers in turbot over 120 d when administrated with a single dosage of 108 cells/ind or 109 cells/ind.The RPS results showed that at 3 d post-vaccination,the RPS against VAM007 was detected at 26.7%-30% in three vaccination dosages;at 7 d post-vaccination,the RPS against VAM003 was 30%-60%,while the RPS against VAM007 was 5.9%-47.1%.These data suggested the bivalent-vaccine could provide immune protection at 3 d-7 d post-vaccination in turbot administrated with a single vaccination.At 14 d-90 d post-vaccination,the PRS at 78.9%-100% was detected in three vaccination dosages.The higher the vaccination dosage,the longer the immune protection duration in each vaccinated group of fish.Taking RPS over 70% as an effective immune protection,the effective immune protection duration against VAM003 were 14 d-120 d in 107 cells/ind,14 d-120 d in 108 cells/ind and 14 d-150 d in 109 cells/ind,while the effective immune protection duration against VAM007 were 14 d-90 d in 107 cells/ind,14 d-120 d in 108 cells/ind,and 14 d-150 d in 109 cells/ind.These results indicated that the V.anguillarum inactivaged bivalent-vaccine could provide the effective immune protection duration respectively over 90 d,120 d,150 d in turbot when administrated at single dosage of 107 cells/ind,108 cells/ind and 109 cells/ind.The analyzing results showed that a certern of positive correlation existed between serum antibody titers and RPS post primary vaccination.The effective protection RPS were to achieve over 70%,serum titers should be in a range of 1:40-1:320 in a dosage of 107 cells/ind,1:20-1:320 in 108 cells/ind,1:80-1:201.6 in 109 cells/ind against V.anguillarum VAM003,and be 1:201.6-1:1015.9 in 107 cells/ind,100.8-1280 in 108 cells/ind,1:63.5-1:320 in 109 cells/ind against V.anguillarum VAM007.2.Secondary vaccination.After 60 d of primary vaccination,a secondary vaccination was carried out in 107 cells/ind and 108 cells/ind groups of fish with the same dosages as the primary vaccination.The serum titers and the RPS values of the boosted fish were determined and compared with those of the primary vaccinated fish.The results showed that the serum titers in the boosted fish were higher than those primary vaccinated fish.The higher the vaccination dosage,the higher the serum antibody titers in each group of boosted fish.In detail,the anti-VAM003 titers of secondary fish were 1:20-1:40 in 107 cells/ind,and 1:20-1:80 in 108 cells/ind,higher than those in the primary vaccinated fish,i.e.,0-1:40 in 107 cells/ind and 1:7.4-1:40 in 108 cells/ind.The anti-VAM007 titers of secondary fish were 1:100.8-1:320 in 107 cells/ind and 1:160-1:640 in 108 cells/ind,higher than those in the primary vaccinated fish,i.e.,1:63.5-1:253.9 in 107 cells/ind and 1:40-1:320 in 108 cells/ind.The higher the vaccination dosage,the longer the effective protective duration was detected in the boosted vaccination fish.The effective immune protective duration against VAM003 was over 120 d in 107 cells/ind and over 150 d in 108 cells/ind respectively,and effective immune protective duration against VAM007 were over 150 d in both 107 cells/ind and 108 cells/ind.In conclusion,when administrated with a single dosage,the V.anguillarum inactivated bivalent-vaccine could provide the effective antibody persistence and the effective immune protective duration both over 90 d in dosage of 107 cells/ind,both over 120 d in 108 cells/ind and both over 150 d in 109 cells/ind.When administrated with a boosted dosage,the V.anguillarum inactivated bivalent-vaccine was able to provide the effective antibody persistence over 150 d,the effective immune protective duration over 120 d in dosage of 107 cells/ind andover 150 d in 108 cells/ind.A similar positive correlation discovered between serum titers and RPS post booster vaccination.The effective protection RPS were to obtain above 70%,anti-VAM003 serum titers should be in a range of 1:20-1:40 in 107 cells/ind and 1:20-1:80 in 108 cells/ind;anti-VAM007 serum titers should be in a range of 1:100.8-1:253.9 in 107 cells/ind and 1:160-1:640 in 108 cells/ind.Ⅱ.Evaluation of the immungenecity and immune protection of V.anguillarum serotype O3 strains.Three V.anguillarum serotype O3 strains,SMP1,SMP3,SMP4,were used in this study.Their 50% percent lethal dose(LD50)was 105.1 CFU/m L,104.7 CFU/mL and 105.4 CFU/m L after rejuvenation against flounder fish.Afterwards,the inactivated vaccine preparations of three strains were produced by inactivated with 0.2% formaldehyde for 36 h at 28 ℃ and 140 rpm.The obtained vaccine preparations were used to vaccinate flounder P.olivaceus via intraperitoneal injection with a dosage of 107 CFU/ind.The specific serum antibody titers against V.anguillarum cells were detected evidently(P < 0.05)at 7 days post vaccination,and the highest titers of 1:1280(SMP1),1:640(SMP3)and 1:905(SMP4)were detected at 4 weeks post vaccination(P < 0.05).Meanwhile,the RPS was 94.4%(SMP1),100%(SMP3)and 73.7%(SMP4)respectively.These results indicated that three V.anguillarum serotype O3 strains were able to induce good immune response in fish and provide good protective efficacy against V.anguillarum infection.V.anguillarum strain SMP3 was suggested to be a candidate for vaccine development and application. |
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