Immune Effect Of Two Adjuvants On Turbot (Scophthalmus Maximus) Vaccine Against Vibrio Anguillarum Delivered By Immersion

The bacteriosis are the most serious diseases in aquaculture at present. More than20kinds of pathogens that could cause huge economic losses to the fish aquaculture wereidentfied in the world. Thus, prophylaxis and control of fish bacterial disease byvaccnine has practical significance. In the past decade, fish disease control were larglydepend on using chemical drugs and antibiotics. But today, the side effect of usingantbiotics is becoming serious. More pathogenic bacteria obtained drug resistance,immunity of fish against disease were lower than that in wild, drug residues in aquaticproducts and water could cause environment pollution. Food safety and human healthare seriously affected by the using of antibiotics. Application of vaccine in theprevention of fish bacterial diseases had achieved good results. Among three modes offish vaccinations, Immersion is the preferred method of immunization for its simpleoperation and safest. But vaccination of fish by direct immersion cannot enhance theuptake of antigen through the epithelia of gills and skin. Thus, the research and developof high efficiency and low toxicity adjuvant to increase the effectiveness of immersionvaccine have become a main task at home and abroad. In this study, two kinds ofadjuvant for fish vaccine were screened, the toxicity of saponins to Scophthalmusmaximus was evluated, the optimal dose of adjuvant and immersing time wasdetermined. To clarify the mechanism of action of adjuvant, titers of antibodyproduction in serum were studied by indirect ELISA, and IgM gene expressions inS.maximus immersed with inactivated V. anguillarum were monitored by real-timequantitative PCR.In this study, four parts of research contents were included.Part Ⅰ: Hemolytic assay of tubot red blood cell in different saponins concentrations were carried on in vitro by microscopic observation method. When theconcentration of saponins was under50mg/L with the response time in10min, thehemolytic rate was the lowest than that with the concentration above50mg/L. Differentconcentrations of saponins were used to soak turbot for the analyzing of hemolyticactivity. The results showed that when the concentration of saponins was under45mg/L,the hemolytic activity was the lowest in average. This result provided scientificevidence for seasonable using saponins.PartⅡ: In this study S.maximus was employed for the assessing of the effect ofadjuvant by the index of cumulative mortality and relative survival percent afterimmunization. The order of adding saponins and vaccine in immersion was compared.Also the effect of challenge method was anlysised. The result showed that adjuvant usedbefore vaccine has a better immune effect, and immersion challenge is better than IP.The results also provided an useful information for further experiments.PartⅢ: The main objective of this work was to study the effect of differentconcentrations dithiothreitol with formalin inactivated V. anguillarum vaccine byimmersion vaccination on the RPS of turbot challenge by V. anguillarum. The resultsshowed that when the concentration of dithiothreitol was at5mg/L, RPS wassignificantly improved. The highest of RPS was71.4%on15days after immersionimmunization. This study is the first report of dithiothreitol used as an adjuvant inaquaculture. The results will provide a reference for further applying of dithiothreitol inaquaculture.partⅣ: S. maximus received different concentrations of Saponins mixed withformalin inactivated V. anguillarum vaccine by immersion inoculation. Relative survivalpercent (RPS) was monitored in7、15、30days post immersion inoculation. In challengeexperiments, S. maximus were immersed in15-fold LD50of V. anguillarum suspension.Titers of antibody production in serum were studied by indirect ELISA. And IgM geneexpressions in S.maximus immersed with inactivated V. anguillarum were monitored byreal-time quantitative PCR. The results indicated that when the concentration ofsaponins combined with inactivated vaccine was45mg/L, RPS was significantlyimproved; peak of antibody titer appeared on the14d in serum, with the highest of210;raise of IgM mRNA transcription appeared to be primarily in the skin and gill (4d) andsecondarily in the spleen (11d) and head kidney (14d). The gene expression levels of IgM were higher in the gill, head kidney and spleen (peak value reached33.2,7.25and6.99, respectively) than that in skin (tiptop reached1.03). Taken together, these resultssuggest that immersion vaccination may induce systematic and local immune responses.Judging from time, mucosa-associated tissues made immune responses in advance,followed by lymphoid tissues. This results support that mucosa-associated tissues playimportant roles in the early phase of antigen invasion.