Screening And Evaluation Of The Protective Additives For Oral Immunization Of CSF Vaccine

Classical swine fever(CSF) is a highly contacting infectious disease caused by classical swine fever virus(CSFV) with the clinical symptoms of hemorrhage, fever, abortion, fetus distortion, chronic nutritional comsuption, lymphocytes and thrombocytopenia, etc. CSF was reported in many countries and regions which caused great damage to pig industry. Vaccination is the main way for prevention of CSFV infection. Several CSF vaccines in in our country market, including hog cholera lapinized virus(HCLV), rabbit splenic tissue virus, suckling pig kidney cells virus, are all inoculated via injection. Reports showed that oral inoculation of CSF vaccines produced well immune response.And it can induce less stress and save time and labour. Based on this, the study aims to screen out a perfect protective agent for oral vaccination of CSF vaccine.Two kinds of oral antigens were prepared in this study: recombinant protein CSFV E2 and LTRG were microcapsuled using chitosan-sodium alginate to make a microcapsuled antigen. Another, CSFV and LTRG protein, was freeze-dried using freeze-drying protective additive to make a freeze-dried antigen. The antigens were orally immunized the mice for immune response evaluation in order to screen out ideal protective additive for CSFV antigen. 1 Immunity evaluation of the microcapsuled E2 protein antigenAfter expression and purification, the CSFV E2 protein and LTRG were microcapsuled by chitosan-alginate and then oral immuned the mice. Results showed that chitosan-alginate microspheres with E2 and LTRG proteins induced higher Ig G and Ig A antibodies than the other groups. It’s not only indicated that LTRG as a mucosal adjuvant enhanced the immune response in mice, but also confirmed the carrier function of chitosan-alginate microspheres which presented the antigen efficiently. 2 Screening and evaluation of the freeze-drying protective additives for oral immunizationTo screen out a freeze-drying protective additive with good heat-resisting protective value for CSF vaccine, different ingredients including gelatin, glycine and lactose, were used to prepare CSF freeze-dried vaccines, together with mucosal adjuvant LTRG protein. After optimizing the vacuum freeze-drying conditions, we obtained four CSF vaccines and then oral immuned the mice. The immune responses were evaluated by detecting serum Ig G and mucosal Ig A antibody. Results showed that the appearance of the second formula consisting of gelatin and glycine, and the forth formula consisting of gelatin had good appearance and water solubility. Serum Ig G and mucosal Ig A antibodies induced by CSF vaccines prepared with the two formulas were higher than the other groups. And the m RNA levels of IFN-γ and IL-4 cykotines induced by CSF vaccine prepared with the second formula were significantly higher than other groups. It confirmed that gelatin and glycine could effectively protect CSFV virulence during the freeze-drying process and improve the immune effect through oral route.Recombinant protein antigen microcapsuled by chitosan-alginate can induce the body to produce good humoral and cellular immune response which confirms microcapsuled protein antigen has good immune effect. The freeze-dried CSF vaccine with protective additives can effectively improve antibody levels, which confirms freeze-drying protective additives have good protection of CSFV. In conclusion, this study preliminary screened out protective additives for oral vaccine which lay a foundation for further development of CSF oral vaccine.