| Furunculosis is an emerging problem in farming of rainbow trout(Oncorhynchus mykiss)and Salmo salar in China and affects different species of marine and freshwater fish,being salmonid species highly susceptible to the infection.The bacteriosis of Aeromonas salmonicida are the most serious diseases in aquaculture at present.Nowadays,furunculosis control were largly depend on using chemical drugs and antibiotics.The side effect of using antbiotics is becoming serious.More pathogenic bacteria obtained drug resistance,immunity of fish against disease were lower than that in wild,drug residues in aquatic products and water could cause environment pollution.Food safety and human health are seriously affected by the using of antibiotics.Therefore,in order to prevent outbreaks of A.salmonicida infection,immunization of fish by vaccination appears to be the most efficient measure of control.Vaccination is an effective method for controlling diseases of fish.Different vaccines against A.salmonicida have been developed,especially for Atlantic salmon(Salmo salar)and rainbow trout(Oncorhynchus mykiss),resulting in variable levels of efficacy.For purpose of studying the influence of immune adjuvant on expression of immune related genes,rainbow trout were intraperitoneal injected with inactivated A.salmonicida vaccine in this paper.Firstly,50 strains were selected from As clinical isolates which were identified from farming Atlantic salmon and rainbow trout in the lab.Combining the gram staining,physiological and biochemical identification,and molecular characteristics,these strains were identified and the results showed that 7 strains were all gram-negative ferment glucose and produce acid and gasand oxidizing enzyme positive,and so on.All of the biochemical and physiological characteristics were consistent with the national standard of As.Blast analysis indicated that 16 S r RNA andfst A sequences of the strains were highly identical to A.salmonicida subsp.salmonicida in Gen Bank database with 99% identity.According to the results above,the selected 7strains of bacteria were all identified to be A.salmonicida subsp.salmonicida.The results of serum typing showed that all of the 7 strains were the same serotype.The main objective of this work was to study the fatality rate of the 7 strains by the RPS of rainbow trout challenge by A.salmonicida.The results showed that only the NO.7strain of the 7 has the highest of RPS.This starin was as the vaccine.Secondly,Based on the ferric siderophore receptor(fst A)gene sequence of Aeromonas salmonicida,a pair of primers was designed and used in a real time quantitive polymerase chain reaction(q PCR)technique.The specificity,sensitivity and repeatability of the system were also evaluated.A.salmonicida and its subspecies can be clearly discriminated from the other 10 bacteria species by SYBR Green I real-time q PCR,which indicated that the primer pair has good inter-species specificity.The standard curve established by recombinant plasmid showed a fine linear relationship between initial templates and threshold cycle,which can be described as y=-4.2552x+39.644(R2=0.988).The sensitivity analysis showed that the detection limit was 35 copies/?L,which suggested that the sensitivity of real-time q PCR was about 100 times higher than that of the conventional PCR assay.The established method was applied to detect the samples in rainbow trout after artificial infection.Results showed that 15 of those samples were positive,which had good agreement(100%)with bacteriological analysis by isolation and culture.In conclusion,the developed real-time PCR assay for A.salmonicida is fast,highly specific,sensitive.This method had a broad application for basic and clinical investigations.Thirdly,healthy rainbow trout were selected and randomly divided into 8 groups.Definition of the treatment groups are following.The groups were: A)Un-vaccinated control(Un Vac),i.p.injection of 0.1 ml PBS(7.3),B)i.p.injection of 0.1 ml experimental vaccine + propolis adjuvant(Exp Vac Pro)(1*108CFU/fish),C)0.1 ml propolis adjuvant(Pro)(no antigen),D)i.p.injection of 0.1ml experimental vaccine(Exp Vac)(1*108CFU/fish),E)i.p.injection of 0.1 ml experimental vaccine +Freund's incomplete adjuvant(Exp Vac FCA)(1*108 CFU/fish),F)i.p.injection of 0.1 ml Freund's complete adjuvant(FCA)+ PBS(FCA)(no antigen).Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvantas the challenge produced 100% mortality in the F group,and only 10 and 15%mortalities in the A and the C groups,respectively.The results showed that,the expression of all the tested genes varied significantly.In the four organs,the expression of IL-8?IL-1??MHC??CD8 and Ig T 5 reached the peak between 1-3d but IFN-? and Ig M reached the peak between 3-7d in gills.The gene maximum expression was 2-70 times higher than that of control group.Our findings indicated that the inactivated vaccine against A.salmonicola could significantly induce the rapid changes of immune-related genes in the four organs of rainbow trout.In addition,the propolis could enhance the non-specific immunity of turbot.Data are referring for the evaluation of vaccine. |
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