| Vibrio anguillarum is the causative agent of vibriosis as haemorrhagic septicaemic in many kinds of marine fish and fresh water/ brackish water fish,also bivalve and curstaceans,causing great economic losses worldwide in aquaculture.I n V.anguillarum,there are 23 serotypes(O1~O23),among those,O1,O2 and part of O3 are virulence strains,the others are avirulence strains.Epidemiological investigations show that O1,O2 and O3 seroptype strains are prevalent in mariculturing environment of China,including of turbot,Japanese flounder,rainbow trout and salmon.With the development of fish immunology and vaccination technology,fish vaccines have been taking place of antibiotic and became the main method of aquatic animal diseases control.1.In this experiment,the growth conditions of the V.anguillarum VAM003 in laboratory were optimized.Single factor experiments were used to investigate the effect of Na Cl,nitrogen source and carbon source in V.anguillarum culture.Results showed that the concentration of Na Cl in the medium should be more than 1%,and typtone was the best nitrogen source.When the concentration of typtone was 2%,the production of V.anguillarum reached maximum.Sucrose was the best carbon source and the concentration was 1.5%.In addition,carbon/nitrogen rate is also an important factor of fermentation.2.In this experiment,on the basis of a 5 L fermenter,TSB medium containing 2.5% sodium chloride was used as the fermentation medium.The preliminary fermentation process of V.anguillarum VAM003 strain was established: at first,the seed liquid was inoculated with the TSB medium with a concentration of 2.5% Na Cl,incubated for 12~14 h under 28?,180 r/min,then the production was used as secondary seed fluid and inoculate the secondary seed liquid to the 5 L fermentation tank by 10% inoculation,the fermentation liquor was aerobicculture under 28?.After 4 h fermentation,the glucose was added at a constant speed,and after 6 h,the 10 times TSB was added at a constant rate.After 10~12 h of fermentation culture,the production was harvested.Subsequently,the amplification culture was carried out in 500 L fermentation tanks,and the OD600 value of the production was raised to a maximum value at 10 h,and the number of alive bacteria was 12.0×109 CFU/m L.3.In this subject,we evaluated the turbot V.anguillarum inactivated vaccine(strain VAM003),including the effect,procedure,biosecurity,minimum vaccination dose,fish size,retention period and cross protection to non-O1 serotype strains.Result shows the vaccine is of good stability and biosecurity,the minimum immune dose was 0.025 m L/ind,the RPS were over 90% in 50 g turbot,vaccine could be stored at least 18 months under 4?.4.In this subject,the inactivated V.anguillarum trivalence vaccine(O1/O2/O3)which carried out on the basis of inactivated V.anguillarum bivalence vaccine(O1/O2)was made of V.anguillarum strains VAM003(O1),VAM007(O2),M2016261(O3),with two different dose,108cells/m L and 109cells/m L.107cells/ind group and 108cells/ind group were vaccinated with 0.1 m L 108 cells/m L and 109 cells/m L vaccine respectively,control group mock-vaccinated with sterile PBS.A booster vaccination was given to 108 cells/ind group at 60 day post-vaccination.All groups of turbot were monitored over a period of 150 day.At certainly time post-vaccination,fish was collected for sampled blood samplings and infected with VAM003,VAM007,M2016261 recpectively.Results shows that in immune group(107 cells/ind group and 108 cells/ind group),serum antibody titer anti-VAM003,anti-VAM007,anti-M2016261 serum antibody titer could be detected at 3 day post-vaccination(dpv),and increased fast at 14 dpv.At 28 dpv serum antibody titer reached the maximum(1:248.89~1:320)and significantly higher than control group(P<0.05).At 28~60dpv,serum antibody titer of 107 cells/ind group was stay around 1:151.11~1:320,significantly higher than control group(P<0.05).28~150dpv serum antibody titer of 107 cells/ind group was stay around 1:106.67~1:320,significant higher than control group(P<0.05).At 3 day post booster vaccination(dpbv),anti-VAM003,anti-VAM007,anti-M2016261 serum antibody titer of booster vaccination group was higher than primary vaccination group,14 dpbv serum antibody titer of booster vaccination group also significant higher than primary vaccination group(P<0.05).Then serum antibody titer of booster vaccination group began descended,60~90dpbv there was no significant difference between booster vaccination group and primary vaccination group.At 7dpv,RPS of immune group was around 5.56~43.75%;at 14 dpv,RPS increased rapidly;at 28 dpv,RPS reached the maximum(100%);at 60 dpv,RPS of VAM003 in 107 cells/ind group was descended to 71.43%,while the others still more than 70%.At 150 dpv,RPS of VAM003 and VAM007 in 108 cells/ind group was descended to 35 and 65% respectively,RPS of M2016261 was still 100%.At 30~60 dpbv(day post booster vaccination),RPS of VAM003,VAM007,M2016261 in booster vaccination group was more than 80%,higher than primary vaccination group at same period.At 90 dpbv,RPS of VAM003 in boosted fish descended to 75%,the others still more than 80%.According to there results,serum antibody titer of trivalent vaccine was product at 14 dpv and could keep at least for 150 day,with RPS more than 70%.The protection period were 120 day for primary vaccination group and 150 day for booster vaccination group. |
PDF Full Text Request