Preparation Of Bivalent Tetravalent Inactivated Vaccine Aginst Actinobacillus Pleuropneumoniae And Haemophilus Parasuis And Evaluation Of Its Immune Effect On Mice

Actinobacillus pleuropneumoniae(APP)and Haemophilus parasuis(HPS)are pathogenic bacteria that cause infectious pleuropneumonia in pigs and Haemophilus parasuis(HPS),and mixed or secondary infections are common,resulting in greater economic losses in the pig industry.APP and HPS have the characteristics of numerous serotypes,poor cross-immunity,rapid changes in epidemic strains,and increasing drug resistance,so the strategy of combined immunization,multi-polyvalent,and multi-prevention with one shot is the current epidemic prevention demand.In this study,the two tetravalent inactivated vaccines were developed for the dominant strains APP serum 7 and 8 and HPS serum 4 and 13,so as to lay the foundation for the development of APP-HPS multivalent vaccine.In this study,two strains of APP(serotypes 7 and 8,H170,HB1)and two strains of HPS(serotypes 4 and 13,AT-2 and HQ-2)were used to prepare three live bacterial concentrations of APP and HPS bivalent inactivated vaccine(AH-1,AH-2,AH-3),according to the Regulations for the Manufacturing and Inspection of Veterinary Biological Products Tests for vaccine traits,sterility,safety,and endotoxin are qualified.Mice were immunized with AH-1,AH-2,AH-3,APP commercial vaccine,HPS commercial vaccine,APP and HPS commercial vaccine as the immune group I～VI,and a negative control was set up,indirect ELISA was used to determine the level of Ig G antibody in serum,SBT to determine functional antibodies,MTT to determine SI value,flow cytometry to determine the percentage of CD4+ and CD8+ T peripheral blood T lymphocyte subsets,double antibody sandwich ELISA to determine cytokines(IL-2,IL-4,IL-6,IL-10,IFN-γ,TNF-β,MCP-1)content;The immunoprotective rate of intraperitoneal challenge was measured,and the colonization amount of challenger strains in each tissue was measured by tissue load number.Observation of pathological tissue sections was prepared to comprehensively evaluate the immune effect of each group of vaccines.The results show that:(1)AH-1,AH-2 and AH-3 are all W/O/W types,and meet the relevant inspection requirements.(2)During the entire immunization period,the detection indicators of the negative control group never changed significantly.The serum Ig G antibody titer of the immune group I～VI was 1:12800;The sterilization rate of functional antibodies was 61%,65%,71%,66%,63% and 65% respectively;SI values,CD4+ and CD8+ peripheral T cell subsets and related cytokine levels continued to increase.The protection rate of intraperitoneal attack against HPS4 and HPS13 in the immune group I～III was 100%,60%,70% and 80% for APP7,and 60%,60% and 70% for APP8,all of which were significantly higher than IV～VI(P<0.05).After 7 days of challenge,the bacterial colonization rate of organs in the immune group I～III was significantly reduced(P<0.05),and there was no obvious change in IV～VI;The tissue lesions of the immune group I～III were lighter than those of IV～VI.The results showed that:(1)In this study,APP7/8 and HPS4/13 two-quadrrivalent inactivated seedlings(AH-1,AH-2 and AH-3)with three viable bacterial concentrations with stable traits,sterility,safety and endotoxin tests were successfully prepared with H170,HB1,AT-2 and HQ-2 as seedling strains,formaldehyde as inactivators and ISA 201 VG mineral oil as adjuvants.(2)AH-1,AH-2 and AH-3 can produce good humoral immunity and cellular immunity after immunizing mice,effectively resisting the attack of lethal doses of APP7,APP8,HPS4 and HPS13 strains,of which AH-3 immunity has the best effect and can be used as a candidate for APP-HPS dual quadrivalent inactivated vaccine.