| Grouper is an important marine economic fish in China.Due to factors such as the excessive pursuit of aquaculture benefits and deterioration of the environment,disease problems frequently occur.For example,Vibrio harveyi is the pathogen causing vibriosis was particularly serious,leading to massive deaths in culture grouper,use of antibiotics to treat and prevent disease was no efficacy.To achieve the healthy and sustainable development of the aquaculture industry,vaccination is one of the best strategies for preventing and controlling aquatic animal diseases.In the preliminary work of our lab,an V.harveyi formalin-killed cells vaccine(FKC)has been developed to protect the grouper from vibriosis.In this study,many carbohydrate adjuvants were screening and evaluation on pearl gentian grouper by challenged of V.harveyi ZJ0603.The results provide a reference value for further improving the protective effect of the inactivated vaccine on grouper against vibriosis.In this study,chitosan oligosaccharide(COS),astragalus polysaccharides(APS)and β-glucan as FKC adjuvant to evaluate the immune responses and protective effect by injected intraperitoneally(IP.).After 5 weeks of immunization,the relative percentage of survival(RPS)of group FKC + COS,FKC + APS,FKC+β-glucan and FKC were 80%,72%,68% and 55%,respectively,significantly higher than the control group(32%),and while RPS of COS,APS and β-glucan was only 52%,47% and 42%.The results have shown that fish vaccination with FKC+COS could raise antibody titers to the highest level of 1: 512 by ELISA,while injection with FKC+APS,FKC+β-glucan and FKC was only 1: 256,significant differences between the immunized group and the control group during the immune period(P <0.05).Serum total protein was detected,the result was found that the content change trend was similar to the antibody titer.The detection and analysis of the activity of lysozyme(LZM),catalase(CAT)and superoxide dismutase(SOD).The results showed that APS,COS and β-glucan as FKC adjuvant can significantly increase the activity of LZM in grouper serum(P <0.05),and vaccination with FKC + APS could raise LZM to the highest level at two weeks,followed with FKC + β-glucan at three weeks.There was no significant difference between injection with FKC,APS,COS and β-glucan(P > 0.05).All experimental groups had a significantly higher levels of CAT activity than the control group(P < 0.05).Such as fish vaccination with FKC+APS could raise CAT activity to the highest value in two week,follow by the FKC+COS group.Vaccinated with FKC+β-glucan could SOD increase and raise to the highest level in three weeks.The above results show that FKC combined with COS although LZM and CAT activity is lower than other groups,but it can provide higher immunity protection and higher antibody titer for pearl gentian grouper.The effect of FKC combined with different concentrations of COS on the immune protection of pearl gentian grouper was evaluated.Preparation of FKC combined with COS,and concentrations of COS in vaccine was 2 mg/m L,4 mg/m L,6 mg/m L,8 mg/m L and 10 mg/m L,respectively.After 5 w post-vaccination,RPS of FKC,FKC+COS2,FKC+COS4,FKC+COS6,FKC+COS8 and FKC+COS10 groups were 66%,68%,70%,83%,100% and 100%,respectively.The results indicating that RPS depends on COS concentration.Serum antibody titers from vaccination vaccine fish were significant differences compared with the control during the immunization(P <0.05).Antibody titers levels depends on COS concentration,and it was the highest level from FKC+COS10 group at three week,follow by FKC+COS8 group,both antibody titers levels were 1: 1024.The above results show that FKC combined with high concentration of COS(8 mg/m L,10 mg/m L)could provide good immune protection and enhance the specific immune response for the pearl gentian grouper.As an immunostimulatory adjuvant,further study whether COS could provide longer-term and stable immune protection when used in combination with Oil and FKC.After 8 w post-vaccination,RPS of FKC、FKC+COS、FKC+Oil and FKC+Oil+COS groups were 62%,70%,82% and 87%,respectively.However,RPS of control groups(Na Cl,Oil)were only 33% and 22%,indicating that FKC combined with Oil+COS could enhance immune protection for the pearl gentian grouper.The highest level from FKC+Oil+COS was up-regulated to the highest level at five week,follow by FKC+Oil,both antibody titers levels were 1: 512,and while FKC group was only 1: 256.Meanwhile,the serum total protein,LZM,CAT and SOD activity were also enhanced,and they could be raised to the highest levels from vaccinated with FKC + Oil + COS vaccine compare with other groups for the pearl gentian grouper.Also,after vaccination,immune-related genes such as Ig M,IL-1β,TNF-α,My D88,CD8α and MHC-Iα in the liver,spleen,kidney and thymus tissues of groupers can be up-regulated at different times.The pearl gentian grouper was vaccinated with the FKC+Oil+COS vaccine could raise immune-related genes to the highest level,like as Ig M and IL-1β transcription levels of the liver,spleen,head kidney and thymus,TNF-α transcription levels of the liver and head kidney,CD8α transcription levels of the spleen,head kidney and thymus.While fish was vaccinated with FKC+Oil vaccine could raise immune-related genes to the highest level,such as TNF-α transcription levels of the spleen and thymus,My D88 transcription levels of the thymus,CD8α transcription levels of the liver,MHC-Iα transcription levels of the liver,spleen,head kidney and thymus.The results indicating that vaccination can make immune factors show different expression levels in immune tissues and depend on the type of vaccine.In summary,the pearl gentian grouper vaccinated with the inactivated V.harveyi vaccine can improve the body’s immunity,and when combined with an adjuvant,it can provide good immune protection.As an immunostimulatory adjuvant,COS could effectively against infection of V.harveyi and depends on its concentration.When both of COS and Oil were used as FKC vaccine adjuvants,they can stably enhance specific and non-specific immune responses for a long time,thereby improving the immune protection of grouper.Therefore,COS is a potential and high-quality aquatic vaccine immune enhancer. |
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