Development An Attenuated Aeromonas Hydrophila Vaccine Candidate And The Immune Effects Of The Vaccine

Aeromonas hydrophila,as a strong Gram-negative bacterium,can infect a wide range of freshwater fish,including common carp Cyprinus carpio,and cause the huge economic loss.To create the effective vaccine is the best way to control the outbreak of the disease caused by A.hydrophila.Fish vaccine development has become a hot topic of fish immunology.However,most vaccines indicated some defects in producing,vaccine applying or immune effect.The live attenuated vaccine is an effective agent but incurred in virulence recovery.Therefore,to screen an effective and stable live attenuated vaccine is very important for applying in aquaculture.In this study,we create a live attenuated vaccine to control the outbreak of the disease caused by A.hydrophila.Meanwhile we provides the suggestion to establish the system of fish vaccine immune effect.1.A strain Aeromonas hydrophila of live attenuated screened by rifampicinIn this study,a live attenuated A.hydrophila strain,XX1 LA,was screened from the pathogenic A.hydrophila strain XX1 cultured on medium containing the antibiotic rifampicin,which was used as a live attenuated vaccine candidate.Virulence results showed that the LD50 of XX1 LA was 4.5×109 CFU/ml,which was higher than that of parent strain XX1 in 200-fold,therefore,the virulence of XX1 LA was reduced significantly and,generally,could not result in fish disease.To examine whether XX1 LA could revert to the wild type in vitro and in vivo without selective pressure,the strain was sub-cultured ten times in LB medium and passed ten rounds in common carp(Cyprinus carpio L)respectively.The results showed that the sub-cultures and XX1 LA isolated from infected fish were indistinguishable from XX1 in LD50.In summary,the live attenuated bacterial vaccine XX1 LA,screened in this study,indicates the better protect effect on common carp against A.hydrophila,which can be applied in aquaculture as the candidate vaccine.2.Immune effects of the vaccine of live attenuated Aeromonas hydrophilaTo compare the immune protection between XX1 LA and formalin-killed A.hydrophila(FKA)in common carp,four immunity indexes were evaluated,such as the relative percent survival(RPS),the specific IgM antibody titers,serum lysozyme activity and the expression profiles of multiple immune-related genes at the different time points following immunization.The results showed that the variable up-regulations of the immune-related genes,such as the pro-inflammatory cytokine IL-1?,the chemokine IL-10 and IgM,were observed in spleen and liver of common carp injected in the vaccines with the formalin-killed A.hydrophila(FKA)and the live attenuated XX1 LA.Specific antibody to A.hydrophila was found to gradually increase during 28 days post-vaccination(dpv),and the RPS(83.7%)in fish vaccinated with XX1 LA,was significant higher than that(37.2%)in fish vaccinated with FKA(P<0.05)on Day 28 after challenged by pathogen.It was demonstrated that the remarkable immune protection presented in the group vaccinated with XX1 LA.During the late stage of 4-week immunization phase,compared with FKA and the control,specific IgM antibody titers significantly increased(P< 0.05)in the XX1 LA group.The activity of the lysozyme in serum indicated no significant change among three groups.In summary,the live attenuated bacterial vaccine XX1 LA,screened in this study,indicates the better protect effect on common carp against A.hydrophila,which can be applied in aquaculture of common carp to prevent from the disease outbreak in the future.3.Immune effects of diffenrent immunity routes to vaccinate common carp with XX1LAIn our study,immersion and intraperitoneal injection methods have applied to vaccinate common carp with XX1 LA.The immune protection of the two vaccinate methods of XX1 LA was evaluated by the relative percent survival(RPS),the specific IgM antibody agglutinating titer and serum lysozyme activity.The results showed that the specific antibody to A.hydrophila was found to gradually increase during 28 days post-vaccination(dpv),and the RPS reached the value of 78.85% and 80.77%,on Day 14 after challenged by pathogen,in fish vaccinated by immersion and intrapertoneal,respectively.It was demonstrated that the remarkable immune protection presented in the groups vaccinated by immersion and intraperitoneal.During the late stage of 4-week immunization phase,compared with control group,specific IgM antibody agglutinating titers significantly increased(P< 0.05)in two immunity groups.The activity of the lysozyme in serum indicated significant change between immunity groups and control group at 7 and 14 dpv.But there is no significant change in the three immunity indexes,that has been found in immersion group and intraperitoneal group.In conclusion,the immune effect in common carp of two vaccinate methods was basically the same,immersion vaccination indicates the protect effect on common carp against A.hydrophila,which can be applied in aquaculture of common carp to prevent from the disease outbreak in the future.