Study On Immune Effects Of Porcine Parvovirus Inactivated Vaccines With Different Adjuvants

Porcine Parvovirus (PPV) is one of the major pathogens responsible for reproductive failure of pregnant sows. After infecting primiparous pregnant sows and then invaded embryonic or fetal placenta, causing sow abortion, fetal death, fetal malformation and the mummy,resulting in infertility or repeated estrus for sows, piglets can also cause dermatitis and diarrhea. PPV widespread and prevalent among swine throughout the world, seriously affecting and constraining the development of the pig farming industry. Since PPV was isolated by Carwnght in 1967,the virus and it’s antibody were proved to be positive in many countries,and the antibody positive rate of PPV reaches to 50%～80%. In china,the antibody positive rate of PPV was up to 80%. In recent years, PPV infection and the harm caused by the upward trend to the pig farming industry and caused great economic losses. The best method to control this kind of disease is prevention.Among the methods,developing vaccine is a key step.In present study, the biological characteristics of PPV HN-4 strain and immune effects of porcine parvovirus inactivated vaccines with different adjuvants were studied, which provided theoretic and technological base of prevention and treatment in the disease.The research contents are as follows.1. A comparative study on proliferation characteristics of PPV HN-1, HN-2, HN-3 and HN-4 isolated strains in PK-15 cells. The results showed that, the TCID50 and hemagglutination titer of PPV HN-4 strain is higher than the other three strains, and the cytopathic was easy to observed. The biological characteristics of HN-4 strain was studied, the results showed that HN-4 strain on chloroform, trypsin, pH3.0 acid, and 56℃heat treatment are not sensitive. HN-4 strain can result in stability cytopathic effect in PK-15 cells,the electron microscopy show that the virus particles are round 23nm and 26nm in diameter. To extracting DNA as a template successfully amplified NS1 gene fragment, Compared with other reference strains from Genbank, genome of HN-4 isolate shared about 98.6% homology with other strains.2. Purification PPV HN-4 strain was inactivated with 0.2% formaldehyde under 37℃, then was passaged on PK-15 cell three times and the inactivation effect was estimated. The results showed that 0.2% formaldehyde inactivated at 37℃for 20 h had a good effect. Selected the PPV HN-4 strain, adding an appropriate amount of oil emulsion, aluminum hydroxide and nano-aluminum adjuvant vaccine to inactivated PPV, then PPV oil emulsion vaccine, PPV aluminum hydroxide adjuvant vaccine and PPV nano-aluminum adjuvant vaccine were prepared. After testing, physical properties and asepsis of the three types of inactivated vaccine are qualified. In order to examine the safety, three inactivated PPV vaccines were injected to mice and piglets, the results showed that immunized animals had no deaths and any other unhealthy performance, indicated the vaccines was safe and had no side effect.3. Immunized twice to mice with the three inactivated vaccines, the interval was two weeks. Collected peripheral blood every week after first immunization. Examined humoral and cellular immunity in mice through ELISA and flow cytometry. The results showed that, three inactivated vaccines all could induce humoral and cellular immunologic response in mice. The results of the humoral immunity showed that the antibody of the PPV oil emulsion vaccine increased rapidly, and had a good effect. And the result of the cellular immunity showed that PPV nano-aluminum adjuvant vaccine significant higher than the other inactivated vaccines.4. Immunized twice to pigs with the three inactivated vaccine, the interval was two weeks. Collected peripheral blood every week after first immunization. Examined the titer of PPV antibody through HI and ELISA, and the cellular immunity by flow cytometry. The results showed that nano-aluminum adjuvant vaccine can enhance the speed of the antibody response significantly than oil emulsion adjuvant vaccine and conventional aluminum adjuvant vaccine, but the duration of antibody inferior to oil emulsion vaccine. The results of cellular immunity showed that, the level of response by all vaccines were higher than the control group, but existed no significant difference among each group.