Study On Preparation And Immune Efficacy Of Erysipelothrix Rhu Siopathiae Inactivated Vaccine In Mice

Swine erysipelas is an acute,hot zoonotic infectious disease caused by Erysipelothrix rhusiopathiae.In recent years,the morbidity and mortality of the disease have shown a clear upward trend,causing huge economic losses to the pig industry.In the previous stage of experiment,three strains of pathogenic strains with strong pathogenicity,good antigenicity,and stable genetic stability were screened out in 42 clinical isolates of Erysipelothrix rhusiopathiae?all serotype are 1a?in the Jianghuai region.This study intends to improve the antigenicity of the inactivated vaccine of Erysipelothrix rhusiopathiae through three aspects:selection of growth medium for producing seedlings,determination of formaldehyde inactivation concentration,inactivation time and selection of vaccine adjuvant.The efficacy of immunization against the vaccine was compared with commercial vaccine to provide experimental basis for the prevention and control of the disease and the study of combined vaccines.Three strains for producing seedlings?AEr 21,AEr31,and AEr 32?were used as test strains.Firstly,using the method of plate colony count,determination of OD₆₀₀ value and determination of total bacterial protein concentration,compare the growing state of the test strains which were put in 0.6%yeast extract of trypticase soy broth?TSB-YE?,in erysipelas vaccine medium and modified erysipelas vaccine medium.Secondly,the formaldehyde solution with a concentration of 0.1%⁰.4%was added to the test strains grown to a stable phase and inactivated for 5,9,10,11,12,13,14,15,and 20 hours respectively.The inactivation of the strains was tested with liquid medium and animal experiments.Thirdly,after completely inactivated with formaldehyde,the test strains were divided into five adjuvants according to different proportions?Freund's adjuvant,aluminum adhesive adjuvant,propolis adjuvant,mineral oil adjuvant ISA 201 VG,polymer adjuvant?and made into an inactivated vaccine against E.rhusiopathiae.After the mice were immunized with physical traits,sterility and safety tests,ELISA was used to detect the levels of antibodies and cytokines?IL-4?IL-10?MCP-1?TNF-??IFN-??in the serum of the mice.Vaccine immunoprotective of each inactivated vaccines was determined by means of oral gavage and virulent strains of intraperitoneal challenge.Fourthly,combining the above results,three strains were made into inactivated vaccine against Erysipelothrix rhusiopathiae and compared with a commercial vaccine.Last measure the antibody?IgG?titer,cytokine content,peripheral whole blood CD4⁺/CD3⁺,and CD8⁺/CD3⁺subpopulations in mice after immunization;calculate the protective rate of each immunization group after challenge,and prepare mouse organs?spleen,lung,liver,and kidneys?to make pathological sections and observed for pathological changes.The results showed that the highest bacterial contents of the three strains?AEr 21,AEr31,and AEr 32?in the three media were 3.8×10⁸⁶.1×10⁸,3×10⁹³.8×10⁹,4.75×10⁹⁵×10⁹ CFU/mL respectively and OD₆₀₀ values were between 1.01¹.39,1.11¹.52,1.39².07 and the highest protein concentrations were between 8.14¹0.31,9.46¹1.42,and 11.21¹3.41 mg/mL.By inactivating the whole-bacteria test,it was determined that 0.2%of formaldehyde is the optimum inactivation concentration and 11 h is the best inactivation time for whole bacterias.The mice immunized by both the test strain and the inactivated vaccine prepared with 5 adjuvants can produce better cellular and humoral immunity,while the mineral oil adjuvant ISA 201 VG immunized group stimulated the highest production of antibody levels and cytokine levels?IL-4?IL-10?MCP-1?TNF-??IFN-??in mice,which significantly different with Freund's adjuvant,aluminum adhesive adjuvant immune group?P<0.05?.After the infection,the mineral oil adjuvant ISA 201 VG immunization group has the highest protection rate,the rates of oral gavage and intraperitoneal challenge protection are 80%¹00%and 60%⁸0%respectively.The serum IgG antibody titers of AEr 21,AEr31,AEr 32 total bacteria inactivated vaccine group and commercialized weak virus and inactivated vaccine group were 1:3200,1:6400,1:1600,1:6400,1:3200?with whole cell ultrasonic pyrolysis as coating antigen?and1:3200,1:6400,1:3200,1:25600,1:6400?with recombinant SpaA as coating antigen?after secondary immune mice.The cytokine production content and percentage of CD4⁺/CD3⁺and CD8⁺/CD3⁺T cells in mice induced by commercialized weak virus vaccine group were the highest,with the significant difference when compared with the inactivated vaccine group?P<0.05?,and there was no significant difference between the inactivated vaccine groups?P>0.05?;the attack protection rate of oral gavage and intraperitoneal injection of AEr 21,AEr31,AEr 32 total bacterin inactivated vaccine group and commercialized weak virus and inactivated vaccine groups were 80%,100%,100%,100%,100%and 80%,100%,60%,100%,100%;the pathological change of the AEr 21 and AEr 32 total bacteria inactivated vaccine group were more obvious than that of the AEr31 total bacteria inactivated vaccine group and the commercialized weak virus and inactivated vaccine group.The results showed that the tested strain?AEr31 strain?was prepared with the modified erysipelas vaccine medium as a growth condition and inactivated?150 r/min?at 37°C with0.2%formaldehyde for 11 h,and then was prepared with the mineral oil adjuvant ISA 201VG.After that,the inactivated vaccines have the best immune effects.After immunizing mice,they not only produce high levels of cellular and humoral immunity,but also completely resist the attack of virulent strains.