| Porcine circovirus type 2 (PCV2) is considered to be the etiologic agent of postweaning multisystemic wasting syndrome (PMWS), and is distributed worldwide, and immunization is one of measures to prevent and control PCV2 infection.Four strains of PCV2 isolates from Anhui were evaluated by indirect immunofluorescence, antigenicity and immunogenicity test, and a vaccine candidate strain was identified as PCV2-BH0801 for developing oil-adjuvant inactivated vaccine. The inactivated vaccine of PCV2 based on PCV2-BH0801 strain was developed by virus proliferation in PK-15 cells, virus titer assay, inactivation and concentration. The immune dose and immune programme of the inactivated vaccine were optimized, and the immune efficacy of the inactivated vaccine was evaluated by challenge-protection test.1. Obtained a virus PCV2-BH0801 strain as vaccine candidate with the virus titre of 10-4.59 TCID50/0.1mL from four PCV-2 Anhui strains. Animal inoculation test on the piglets with PCV-2-antibody negative showed that PCV2-BH0801 strain had strong antigenicity and immunogenicity, which provided the basis for developing inactivated vaccine of PCV2.2. A final virus titre of 10-4.55 TCID50/0.1mL was obtained by inoculating PK-15 cells with PCV2-BH0801 for proliferating virus. The virus was inactivated with 0.2% formaldehyde under 37℃for 30h, and concentrated to the virus titre of≥10-5.0 TCID50/0.1mL through the hollow fiber ultrafiltration membrane. The inactivated vaccine was prepared with concentrated virus and appropriate oil-adjuvant, and was evaluated by sterility test, physical characteristic and safety of the vaccine due to the standard of inactivated vaccine. Immune efficiency of the vaccine was evaluated by ELISA serum antibody titer on experimental animal post immunization and the optimum immune dosage of the inactivated vaccine was identified.3. The piglets of 21 days were immunized with 1mL, 2mL and 4mL of inactivated vaccine respectively and the optimum immune dosage was evaluated by the growth performance and PCV-2 antibody level post-immunization. The piglets of 14 days, 21 days and 28 days old were immunized with the optimum immune dosage of the inactivated vaccine respectively, and booster immunization were conducted at 14 days and 21 days intervals for exploring the best immune program of the vaccine with high growth performance and PCV-2 antibody level. Results showed that the piglets can obtain high growth performance and PCV-2 antibody level with the first-immune at 28 days with 2mL of inactivated vaccine and booster immunization interval at 21 days with 4mL of inactivated vaccine,4. The immune efficacies of the vaccine against PCV-2 were evaluated by the growth performance, PCV-2 antibody detection, clinical signs, viremia and viral DNA in different tissues. Results showed that the immune group showed high growth performance and PCV-2 antibody level, which is higher than the control group. The viremia was positive in control group from the 7th day to the 21st day post challenge, but was negative in test group. No clinical signs were observed in test group and control group, but the gross lesion in lung, lymph node, spleen and kidney were showed and viral DNA were detected in lung and lymph node in negative control group. The results indicated that the PCV-2 inactivated vaccine could provide protection to piglets against PCV2 infection.5. IL-2,IFN-γsecreted by Th1 cell and IL-4,IL-10 secreted by Th2 cell in piglets serum were detected. Results demonstrated that the content of cytokines secreted by Th2 cell was higher than Th1 cell in test group and the content of cytokine secreted by Th1 cell showed the rising tendency, which suggested that the immune response of the body trended to normal under the protection of the humeral immune reaction in immune group; and the content of cytokine in negative control group lower than immune group at the later stage of infection. The results showed that the PCV-2 inactivated vaccine could prevent piglets from the immunosuppression caused by PCV-2 infection at the late stage of infectionTo sum up, PCV-2 inactivated vaccine could induce the effective immune response in piglets after immunization, and could prevent piglets from PCV-2 infections. |
