Preparation Of Inactivated Streptococcus Suis Serotype 2 Vaccine And Its Immunological Efficacy In Mice

Swine Streptococcosis is a general term for swine infectious diseases caused by a variety of different groups of Streptococcus.Among them,Streptococcus suis(SS)is the most important pathogen causing Swine Streptococci worldwide,and it is also an important zoonotic pathogen.According to the difference of SS capsular antigen characteristics,it can be divided into 35 serotypes(types 1-34 and 1/2)of which Streptococcus suis serotype 2(SS2)has the highest clinical separation rate.The strongest pathogenicity not only causes huge economic losses to the pig industry,but also poses threats and challenges to public health.Vaccination is an effective measure to prevent and control Swine streptococcosis.However,due to the large number of SS serotypes and the poor mutual immunity between serotypes,identification and screening of dominant strains for vaccines will benefit SS in endemic areas.Based on the serotype and immunogenicity of vaccine strains,the preliminary research results show that SS2 is the dominant serotype of SS in the Jianghuai region.On this basis,through pathogenicity test,antigenicity test,and stability test,comprehensive screening of 2 vaccine strains(codenamed HF2,HF3).Based on the optimized culture conditions,this study improved and ensured the good antigenicity of the prepared SS2 inactivated vaccine by comparing the formaldehyde inactivation conditions of vaccine strains and the selection of immune adjuvants,and futher comparing the immune effects of the corresponding commercial vaccines.This provides a scientific basis for the prevention and control of Swine Streptococcosis and the study of multiple vaccines.The selected two vaccine strains(HF2,HF3)were subjected to the following tests:(1)The formaldehyde solutions with final concentration of 0.1%,0.2%,0.3%and 0.4%were added to HF2 and HF3 which were grown to stable phase,and inactivated for 5 h,9 h,10 h,11 h,12 h,13 h respectively.After 14 h,15 h,and 20 h,the completeness of HF2 and HF3 inactivation was tested by liquid medium,solid medium and susceptible animals.(2)HF2,HF3 inactivated vaccine was prepared by inactivation of 0.3%formaldehyde for 15 h with five adjuvants(aluminum adjuvant,Freund’s adjuvant,propolis adjuvant,GEL 01 polymer adjuvant,ISA 201 VG mineral oil adjuvant).The mice were immunized with physical properties test,aseptic test and safety test.The level of IgG antibody and the content of cytokines(IL-4,IL-10,IFN-y,TNF-β,MCP-1)in serum of mice were detected by indirect ELISA method.The T cell subsets of CD4+/CD3+and CD8+/CD3+were measured by flow cytometry,the immune protection was measured by challenge test,and the colonization of SS2 in mice was measured by tissue load test.The pathological sections of lung,liver,spleen and kidney of mice were collected and observed.(3)The HF2,HF3 was cultured to the stable stage and inactivated by adding 0.3%formaldehyde for 15 h.The inactivated vaccine was prepared by emulsification with ISA 201 VG mineral oil adjuvant.At the same time,the mice were immunized with SS2 commercial inactivated vaccine(HA9801 strain).The levels of IgG antibody and cytokines(IL-4,IL-10,IFN-y,TNF-β,MCP-1)in serum of mice were detected by indirect ELISA,and CD4+/CD3+,CD8+/CD3+T cell subsets were measured by flow cytometry.The immune protection was measured by challenge test,the colonization of SS2 was measured by tissue load test,and the pathological tissue sections of lung,liver,spleen and kidney of mice were collected to observe the pathological changes.The results showed that:(1)Formaldehyde with a final concentration of 0.3%and 0.4%for 15 h and 13 h respectively can completely inactivate HF2 and HF3.(2)The ISA 201 VG group induced the highest antibody titer in mice,which was significantly higher than other adjuvant groups.The ISA 201 VG group induced mice to produce higher levels of cytokines than other adjuvant groups;The ratio of CD4+/CD3+and CD8+/CD3+T cells produced by the ISA 201 VG group was higher than that of the other adjuvant groups,and most of them had significant differences(P<0.05).After challenge,compared with the other four adjuvant groups,the ISA 201 VG group had 100%protection rate,and the colonization of lung,liver,spleen and kidney was lower.Combined with pathological changes,ISA 201 VG The group showed no lesions or minor lesions.(3)The serum IgG antibody titers of the SS2 inactivated vaccine(HF2 strain,HF3 strain)and the SS2 commercial inactivated vaccine(HA9801 strain)after the second immunization of mice were 1:25600,1:12800,1:25600,respectively.SS2 inactivated vaccine(HF2 strain)induced cytokine production in mice higher than SS2 inactivated vaccine(HF3 strain)and SS2 commercial inactivated vaccine(HA9801 strain).The SS2 commercial inactivated vaccine(HA9801 strain)had the highest CD4+/CD3+and CD8+/CD3+T cell ratios,but the CD8+/CD3+T cell ratio was not significantly different from the SS2 inactivated vaccine(HF2,HF3 strain)(P>0.05).After the challenge,the SS2 inactivated vaccine(HF2,HF3 strain)and the SS2 commercial inactivated vaccine(HA9801 strain)have an immune protection rate of 100%.The pathological changes of the SS2 inactivated vaccine(HF3 strain)were more obvious than the SS2 inactivated vaccine(HF2 strain)and the SS2 commercial inactivated vaccine.The results showed that the tested vaccine strain(HF2)was inactivated(150 r/min)with a final concentration of 0.3%formaldehyde at 37℃ for 15 h after the growth of the modified BHI medium to stable phase,with ISA 201 VG mineral oil.The preparation of the inactivated vaccine has the best immune effect,and the immunization of the mouse not only produces a higher level of humoral immunity and cellular immunity,but also completely resists the attack of the virulent strain.