| Aeromonas salmonicida is a common pathogen of cold water farming fish,but in recent years,it has also been found in a variety of warm water framing fish,resulting in more and more frequent diseases,which lead to huge economic losses and seriously hinder the development of aquaculture industry in China.Traditional chemical treatments lead to the overuse of antibiotics and increase drug resistance of bacterial,which is not conducive to the green and sustainable development of aquaculture,and vaccine is an effective means to prevent and control infectious diseases.The effect of Aeromonas salmonicida vaccine has been fully verified in the Norwegian Atlantic salmon industry,but there is no commercial vaccine against Aeromonas salmonicida in China at present.In this study,the pathogens of Thamnaconus septentrionalis and Sebastes schlegeli with red mouth disease were isolated,identified and verified,and many kinds of Aeromonas salmonicida microparticle vaccines with different wall materials were prepared.The stability and protective efficiency of the microsphere vaccine were preliminarily evaluated.The results provide important information for the prevention and control of Aeromonas salmonicida disease and vaccine research in China.1.Isolation and identification of the pathogen of the greenfin horse-faced filefish（Thamnaconus septentrionalis）and rockfish（Sebastes schlegeli）with red mouth disease.In Nov.2018,T.septentrionalis disease outbroke was observed in a farm located in Penglai city,Shandong province,and S.schlegeli disease outbroke was also occurred in the same farm in Apr.2019.The main symptoms of diseased fish were ulcers,redness,swelling,and bleeding in the mouth.From the liver,spleen,and kidney of all the diseased fish,a large number of homogeneous colonies were observed.All strains had the same shape,color,and size,T.septentrionalis（infected by 2018TS-1）and S.schlegeli（infected by 2019SS-1）.Bacteria identification was carried out by 16S r RNA genes analysis and Biolog Gen III characterization and the vap A gene typing was also used in this research to identify the subspecies of strains isolated from T.septentrionalis and S.schlegeli.The results of all the strains were the same,high identity with Aeromonas salmonicida subsp.masoucida.The dead fish of experimental infection showed ulcers and red mouth,the same symptoms as the naturally infected fish.The virulence of the isolates was tested experimental infection via injection with T.septentrionalis（infected by 2018TS-1）and S.schlegeli（infected by2019SS-1）in the lab to calculate the lethal dose 50%(LD50).Results showed that the LD50 of2018TS-1 to T.septentrionalis was 1.78×105 CFU/fish,2019SS-1 to S.schlegeli was 0.89×105 CFU/fish.The above results show that Aeromonas salmonicida is the pathogen of greenfin horse-faced filefish and rockfish red mouth disease.In this study,it is the first time to find the cases of Aeromonas salmonicida infection in domestic factory cultured greenfin horse-faced filefish and rockfish,and it is also the first disease report of artificially cultured greenfin horse-faced filefish.2.Preparation of microparticle vaccines.The formaldehyde inactivated whole cells of Aeromonas.salmonicida subsp.masoucida RZ6S-1 were used as antigen.Nine kinds of microparticle vaccines,Gelatin-sodium alginate,Gelatin-Arabic,Gelatin-cellulose,Sodium alginate,Cellulose,Polylactic acid,Polycaprolactone,and Polylactic acid glycolic acid copolymer microparticle were prepared by different methods.Then the morphology,particle size and entrapment efficiency of various microparticle were characterized.Two kinds of microparticle have excellent performance and the potential as oral vaccine.The sodium alginate microparticle prepared by exogenous emulsification method have high yield,small particle size and uniform distribution,with an average particle size of 10.85±2.77μm.The encapsulation efficiency was 93.10%;The average particle size distribution of gelatin-sodium alginate microspheres prepared by complex coacervation method was 24.12±2.45μm,and the encapsulation efficiency of microparticle was 58.21%;Both microparticle have the advantages of simple preparation,high encapsulation efficiency and uniform particle size,which are suitable for large-scale production and have the potential to be used as oral vaccines.Gastric juice tolerance and intestinal release of gelatin sodium alginate and exogenous sodium alginate microparticle were simulated.The two microparticle can be stable in artificial gastric juice（p H=1.5）.In artificial intestinal fluid（p H=8.0）,both microparticle can disintegrate rapidly and release antigens.Safety evaluation showed that the microparticle vaccine did not cause fish death after oral administration and injection into the fish.3.Evaluation of the immune effect of microparticle vaccines.Polylactic acid,cellulose and sodium alginate were used as wall materials to encapsulate inactivated Aeromonas.salmonicida,and prepare microparticle vaccines for injection immunization of turbot（Scophthalmus maximus）.The results showed that at 30 days after the turbot immunized by intraperitoneal injection,the serum antibody titers of PLA microparticle group,cellulose microparticle group and sodium alginate microparticle group were 1:320,1:133.33 and1:426.67,respectively,which were significantly higher than those of the control group（p<0.05）,indicating that the microparticle vaccine could induce the immune response of fish.The challenge results showed that the death of three kinds of microparticle group fish was delayed compared with control group.But there was no significant difference in survival rate between the three kinds of microparticle and the control group.Using Aeromonas.salmonicida isolated from Brown trout as antigen,sodium alginate microsphere vaccine against Aeromonas.salmonicida was prepared by exogenous emulsification,gelatin-sodium alginate microsphere vaccine was prepared by complex coacervation method,and oral immunization of Yadong salmon（Salmo trutta fario）was carried out on a field scale.After immunization,the antibody titer of each group was determined.The serum antibody titer of the control group was 1:80,which was not significantly different from that of the gelatin-sodium alginate oral immunization group.The antibody titer of the sodium alginate oral vaccine group was 1:160,which was significantly higher than that of the control group and gelatin-sodium alginate group（p<0.05）.The serum antibody titer of fish injected with oil adjuvant was 1:2560 at 30 days post vaccination.In the process of breeding,there was no large-scale disease in brown trout immunized with oral vaccine and injection vaccine,and the survival rate was more than 95%.The mortality decreased by more than 40%compared with the same period last year.Exogenous emulsified sodium alginate Aeromonas.salmonicida microparticle were also used as oral vaccine in blue gourami（Trichogaster trichopterus）.At 30 days post vaccination,challenge experimen showed that all the fish in the control group died on the 8th day.The cumulative mortality rate of the immunization group was 81.82%,and the relative percent survival rate was 18.18%.The delay of death was also observed in vaccinated group.The above results show that oral immunization can induce fish to produce a certain level of specific antibodies,which is lower than that of oil emulsion injection vaccine,but it can also exhibit protective effect in the production process.It is of great significance for the prevention and control of diseases for fish that are not suitable for injection or for juvenile fish. |
