Studies On The Imumune Efficacy Of Proplis Inactivated Vaccine Against PRRS

Porcine reproductive and respiratory syndrome (PRRS) is a hyperinfection disease which causes fever, anorexy in susceptible swine, respiratory failure in preg. sows, and respiratory problems in piglets. It is often endemicity which has severely hazarded the industry of raising swine. The prevention and control of PRRS rely immunization in our country is a fact we have to face in the prevention and control of PRRS in fairly a long time for the low level of the industry of raising swine and synthetic technology of prevention and control. Propolis has the function of antibacteria , antivirus and immune enhancement et al. Its particles linking with antigens each other come into immune stimulating composites structure which has the function of"warehouse"and plays the role of immune adjuvant. In order to study the immune efficacy of the vaccine agaist PRRS and find an efficacious way to control it,we obtain the proplis inactivated vaccine against PRRS with the immunoenhancer of propolis and the know-how of Shen Zhiqiang. And then study on its imumune efficacy contrasting with oil vaccine.1.The effect of the proplis inactivated vaccine against PRRS to humoral immunity Choose 15 piglets of 50-day age and divide to 3 groups randomly. Then we inject proplis inactivated vaccine against PRRS with one multiple dose, oil miky inactivated vaccine against PRRS with one multiple dose and partes aequales sodium chloride. Blooding from jugular vein at7 d,14 d,21 d,28 d,42 d and 60 d to detect the ELISA antibody and neutralizing antibody. It shows that the two group has the similar growth and decline of ELISA antibody. But the level of ELISA antibody induced by group propolis is higher than group oil at all times. The neutralizing antibody induced by group propolis is rapider and the level is higher2.The effect of the proplis inactivated vaccine against PRRS to cellular immunityThe grouping same as above. Blooding from jugular vein at 0d,7 d,14 d,28 d and 42 d to detect the CD3+,CD4+,CD8+ on surfac of eperipheral blood lymphocyte by flow cytometry. It results that the proportions of T subset of CD3+,CD4+,CD8+ in control are almost invariably in the whole experimental stage. Compare with group oil, the proportions of CD3+ T subset in group propolis increase fast and the level is higher at crest-time. the increasing speed of the proportions of CD4+ T subset in two groups is similar. But at crest-time, the proportions of CD4+ T subset of group propolis is higher than group oil obviously. The proportions of CD8+ T subset of experimental group are all have the tendency of decrease after immunization, and increase slowly after 7 d until the end of experiment. It has higher increasing speed of the proportions of CD4+ T subset in group propolis, and the cell proportion is higher than group oil.3. Establishment of competitive RT-PCR and quantification of porcine IFN-γmRNAThe total RNA was extracted from the aseptically-isolated porcine peripheral blood mononuclear cell(PBMC),which were cultured with induction in vitro. An IFN-γgene of 435 bp was amplified by RT-PCR. The recombinant plasmid pMD-IFN435 was constructed by means of the amplified IFN-γgene being ligated into the vector pMD18-T. According to the gene deletion, The recombinant plasmid pMD-IFN367 with 367 bp of the target gene was constructed which is the internal standard DNA template.The linear regression equation of the standard competitive curve(y=0.414x-1.864) for the accurated and convenient detection of porcine IFN-γmRNA was develop by using quantitative pMD-IFN367 and double diluted pMD-IFN435 as PCR competitive templates. Get the reverse transcript by the step above with the sample waiting for test, and amplification with quantitative pMD-IFN367 by competitive RT-PCR. Recording the reinhoit zahl of pMD-IFN435 and pMD-IFN367 and converting the corresponding ratios of molecula, substituting into the standard competitive curve, we can get the contents of IFN-γmRNA in samples. The contents of IFN-γmRNA in group propolis and group oil are rather similar at 14 d after immunization , but after 14 d , the group oil increasing slowly and the group propolis increasing fast with a high speed which can reach 2.5632×108/μL at 42 d.Above all, compare with oil adjuvant vaccine, propolis adjuvant vaccine can not only cause higher level of ELISA antibody, but also induce specific neutralizing antibody more efficaciously which have the neutralization to organism. Meanwhile, propolis adjuvant can stimulate organism to generate T lymphocyte which can raise the level of cellular immunity. The increase of IFN-γobviously, has proved its immunologic enhancement to nonspecific immunity furtherly. Proplis adjuvant inactivated vaccine is a new generation vaccine deserving of popular generalization which also provide a new key to the vaccine of PRRS.