| Gl(?)sser’s disease(Gl(?)sser’s disease)is an important bacterial infectious dise ase of swine with high morbidity and mortality caused by Haemophilus parasu is(HPS)and characterized by fibrinous polyserositis,pericarditis,meningitis an d arthritis,to the global pig industry has brought huge economic losses.Under the general trend of reducing resistance,limiting resistance and gradually mov ing towards anti-resistance,vaccination has become the main measure to preven t and control the disease.However,there are many serotypes of HPS with obv ious local specificity,and there is no effective cross-protection among different serotypes.Therefore,the development of cross-protective HPS vaccine is still an inevitable requirement for the prevention and control of Gl(?)sser’s disease.T he inactivated vaccine is safe,reliable,stable,short development period and ea sy to prepare multivalent vaccine,so it can produce immune protection against different serotype strains.In the early stage,the laboratory carried out an epid emiological investigation of porcine bacterial diseases on family farms of a gro up company in Anhui,Jiangsu,Shandong,Hebei and Guangxi provinces,as we ll as pig farms(households)of different sizes in Anhui area,to confirm the n ormal prevalence of HPS serotypes 4,5,7 and 13.In this study,4 strains of HPS vaccine-making bacteria were selected through pathogenicity test and imm une protection test in guinea pigs.The HPS tetravalent(serotypes 4,5,7 and13)inactivated vaccine was prepared,the aim of this study is to provide a tec hnical reserve for the development of a new type of HPS tetravalent inactivate d vaccine and to lay a foundation for the effective prevention and control of Gl(?)sser’s disease.Four selected HPS strains(serotypes 4,5,7 and 13,numbered AT-2,SX-1P,HF-1P and HQ-2 in turn)for vaccine preparation were used to carry out t he following experiments:(1)Inactivation of HPS strains at final concentration of 0.3%formaldehyde for 24 hours at 37°C,emulsifying with ISA 201 VG d ual-phase oil emulsion adjuvant;Three kinds of HPS tetravalent inactivated vac cine with 1.0×10~9CFU/m L,2.0×10~9CFU/m L and 4.0×10~9CFU/m L were prepared and named V-1,V-2 and V-3 respectively.(2)The immune efficacy of V-1,V-2 and V-3 and commercial vaccine(pre-inactivated MD0332 serotype 4 and SH0165 serotype 5,4.0×10~9CFU each)were evaluated by immunizing group I,group II,group III and group IV respectively:The level of Ig G antibody in s erum was determined by Indirect ELISA,the activity of Phagocytosis was dete rmined by SBT,the level of spleen lymphocyte was determined by MTT,and the level of Cytokines(IL-4,IL-10,IFN-γ,TNF-β,MCP-1)was determined by double antibody Sandwich ELISA.The immune protection rate was measured by intraperitoneal challenge,the number of colonized strains was measured by tissue charge count,and the pathological changes were observed by pathologica l sections of lung,liver,spleen and kidney.The results showed that:(1)All the four-valent inactivated HPS vaccines with three concentrations of live bacteria were oil-in-water(W/O/W),which met the qualification requirements of viscosity test,stability test,sterility test a nd safety test.(2)During the whole immune cycle,there were no significant c hanges in the detection indexes in the blank control group.The titer of Ig G a ntibody in immune group I～IV was significantly increased(1:12800),and t he level of Ig G antibody in immune group I～IV was continuously increased(P>0.05),it has strong bactericidal activity of antibody.The levels of serum-associated cytokines(IL-4,IL-10,IFN-γ,TNF-β,MCP-1)in the immune group I～IV were increased continuously without significant difference(P>0.05),the overall protective rates of group I～IV were 85%,100%,100%and 60%in turn,and the bacterial colonization of organs(lung,liver,kidney and sp leen)in group I～III was significantly lower than that in group IV(P<0.05),the pathological changes of organs in immune group I～III guinea pigs we re slight.The results showed that:(1)Four-valent inactivated HPS vaccines(1.0×10~9CFU/m L,2.0×10~9CFU/m L and 4.0×10~9CFU/m L)with stable properties,sterility t est and safety test were successfully prepared by emulsifying with strain AT-2,SX-1P,HF-1P and HQ-2,formaldehyde as inactivating agent and ISA 201VG dual-phase oil emulsion as adjuvant,V-1,V-2 and V-3,respectively.(2)Good humoral and cellular immunity were observed in Guinea pigs immunized with V-1,V-2 and V-3.However,V-2 and V-3 were 100%resistant to lethal dose s of HPS serotypes 4,5,7 and 13,and could be used as candidates for inacti vated HPS tetravalent(serotypes 4,5,7 and 13)vaccines,to achieve the curre nt commercialization of vaccines can not meet the actual production needs of t he improvement and supplement. |
PDF Full Text Request