Preparation And Immunogenicity Analysis Of A Lipopolysaccharide-protein Conjugate Vaccine For Haemophilus Parasuis

Haemophilus parasuis is one of the commensal bacteria which colonize the upper respiratory tract of pigs. Under some appropriate conditions, H. parasuis can infect and cause severe systemic disease (Glasser's disease), characterized by polyarthritis, fibrinous polyserositis, meningitis and pneumonia. H. parasuis has more than 15 distinct serotypes. The antigens and virulence varied among H. parasuis strains of different serotypes, thus the vaccines for H. parasuis lack cross-protection efficiency. Therefore, development of sensitive, specific and cross-protective vaccines is critical for H. parasuis prevention. Lipopolysaccharides(LPS) are important and effective immunity units of bacteria, can protect animals by immunity responses,but the immunogenicity is not strong enough. The lipopolysaccharides-protein conjugate vaccines can well resolve this problem. Different from other reports, in this article, this study used a protein-H. parasuis PilA protein instead of BSA or tetanus toxoid at el. Because PilA protein has been confirmed strong immunogenicity against H. parasuis in.In this study, the LPS of field isolates of H. parasuis 0322 and 0165 were extracted by 68℃hot phenol methord and confirmed the reducibility by Fehling reaction and Biuret reaction. Ultraviolet Spectrum Analysis (USA) showed the characteristic of LPS at 255nm. Acetic acid was used to cut off the toxic part-lipid A in LPS. The O-specific polysaccharide (OSP) sub-units were purified and covalently bound to H. parasuis PilA protein, forming OSP-PilA conjugates:OSP0322-PilA, OSP0165-PilA and OSP0322-PilA+OSP0165-PilA. In order to evaluate the immunogenicity of OSP-PilA conjugates, BalB/C mice were first subcutaneously injected with the OSP-PilA conjugates and intraperitoneal injected 14days later. Took blood of mice after 14 days after both injections. And evaluated the results by OD630 of ELISA. Compared with adjuvant control group,the result showed that OSP0322-PilA, OSP0165-PilA and OSP0322-PilA +OSP0165-PilA conjugates could induce IgG antibodies and the levels of antibodies elevated significantly after the second injection.Then, mice were challenged with HPS0322 and HPS0165 at the dose of 2×LD50 and 5×LD50 respectively, the results demonstrated that OSP0322-PilA and OSP0165-PilA conjugates produced efficient protections against HPS0322 and HPS0165.But OSP0322-PilA+OSP0165-PilA conjugate didn't have such high protection.