| The infectious diseases of livestock and birds are always the chief problem to jeopardize the stockbreeding, especially the virosis there are not effective therapies up to now. Vaccination is the important measure to prevent the infectious diseases and immune adjuvant can enhance the immune effect of vaccine. However, chemical adjuvants, such as oil and alum commonly used in domestic and abroad, have some side effects of strong local stimulation and carcinogenesis and teratogenesis. Therefore, it is urgent to develop high-effect and low-toxicity newtype of immunopotentiator. Propolis contained flavones, aromatic acids, and phenol is a natural activity substance with activities of immunoenhancement, antiviral and antioxidant. Sulfated polysaccharide is a class of polysaccharides containing sulfate ions in hydroxy after sulfated modification, and has more or stronger bioactivities in comparison with non-sulfated. In this research, propolis and sulfated polysaccharide were selected as object and their immune-enhancing and antiviral activities respectively for porcine parvovirus vaccine and ND vaccine were studied. The details were divided into six parts as follows:Test 1. Adjuvantivity of propolis on inactive PPV vaccine In order to investigate the adjuvantivity of propolis to inactive porcine parvovirus (PPV) vaccine. Firstly the propolis-, aluminum-and oilemulsion-adjuvant (PA, AA and OA) PPV vaccine were prepared and detected the safety. Then the immune effects of three adjuvant vaccines were compared in efficacy-detecting animals, guinea pigs and rabbits. In test of immune response,68 guinea pigs and 40 rabbits were randomly divided into 4 groups respectively, the guinea pigs and rabbits in three adjuvant groups were injected respectively with PA, AA and OA vaccine. Within 8 weeks after vaccination, blood samples were collected for determination of serum hemagglutination inhibition (hemagglutination inhibition, HI) antibody titers, lymphocyte proliferation of peripheral blood (rabbit) and spleen (guinea pigs) and content of IL-2 and IL-4 secreted by the lymphocyte, weekly. In test of immune protection,50 guinea pigs were randomly divided into 5 groups, the guinea pigs in three adjuvant groups were injected respectively with PA, AA and OA vaccine, in challenge and blank control (CC and BC) group, physiological saline. On day 42 after vaccination, the guinea pigs of 1-4 groups were challenged with PPV. At different time points after challenge, blood samples were collected for determination of serum HI antibody, peripheral lymphocyte proliferation and content of IL-2 and IL-4 secreted by the lymphocyte. On day 28 after challenge, the heart, liver, spleen, lung, kidney and gonad of the guinea pigs were were collected for determination of the content of PPV by quantitative real-time polymerase chain reaction (QR-PCR). The results showed that safety of PA was higher than those of OA and AA. Three adjuvants could enhance the immune effect of PPV vaccine in guinea pigs and rabbits. Propolis was superior to AA and inferior to OA in enhancing serum antibody, and superior to OA and AA in promoting lymphocyte proliferation and secretion of IL-2 and IL-4. After challenge, Propolis was superior to AA in promoting antibody production, and presented most significant action in promoting lymphocyte proliferation and cytokine secretion and lowering PPV content of the organs. The results indicated that propolis possessed stronger adjuvantivity for PPV vaccine and was a safe and effective adjuvant.Test 2. Immune effects of propolis on inactive PPV vaccine in pig In order to investigate the adjuvantivity of propolis, the effect of propolis on immune effect for inactivated PPV vaccine were determined. Twenty 25-30-days-old pigs were randomly divided into 4 groups, the pigs in three adjuvant groups were intramuscular injected respectively with 2.0 mL PA-, AA-and OA-vaccine, in blank control (BC) group, with 2.0 mL of physiological saline. On days 7,14,21,28,35 and 49 after vaccination, blood samples were collected for determination of serum antibody titer, on days 7,14,21, and 35, serum IgM, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, on days 7,14 and 35, lymphocyte proliferation and the content of IL-2, IL-4, IL-6 and IFN-γ secreted by the lymphocyte. The results showed that on day 7 and 14, the antibody titer of PA group was higher than those of AA group (P<0.05) and OA group (P>0.05), and on other time point, numberly or significantly higher than that of AA group. On day 7 to 35 after vaccination, IgM, IgG2, IgG3, IgG4, IgA1 and IgA2 levels of PA group were numberly or significantly higher than those of OA and AA group, and IgGl levels of PA group were higher than those of AA group (P<0.05). IL-2, IL-4, IL-6 and IFN-y level of PA groups were mostly significantly higher than those of AA and OA group (P<0.05). These results indicated that propolis could significantly enhance the immune effect of PPV vaccine in pig especially in improving cellular immunity superior to AA and OA adjuvants.Test 3. Study on anti-PPV antivity of propolis in vitro In order to investigate the anti-PPV action, the effects of propolis on cellular infectivity of PPV in vitro was measured. First propolis was duplex diluted into ten concentrations from 2000 μg·mL-1, and added into the cultivating system of PK-15 monolayers, the maximal safe concentration of propolis was determined by MTT method. Then propolis at five concentrations within safe concentration and PPV were added into the cultivating system of PK-15 monolayers in three models (pre-, post-adding propolis and simultaneous adding propolis and PPV after mixed), the cellular infectivity of PPV (A570 value) were observed by MTT assay and the content of PPV in PK-15 was determined by quantitative real-time PCR assay. The results showed that in three different models, the A570 values of propolis at 31.2-250 μg·mL-1 groups were significantly higher and the content of PPV in the cell significantly lower than those of vrial control (P<0.05), which indicated that propolis could promote the PK-15 to resist and eliminate the PPV infection. These effects related to the dosage and pattern administration. Within safe concentration the higher concentration, the better effect, and the effect was best in pre-adding propolis pattern.Test 4 Effects of cSPSs on immune response of ND vaccine in chicken On the basis of that the sulfated polysaccharide (sPS) with the better immunoenhancement were selected out and that the action of compound sulfated polysaccharide (cSPS) was better than that of single sPS was confirmed, the effects of four cSPSs on immune response of ND vaccine were compared.27014-day-old chickens were averagely divided into 9 groups and vaccinated with ND-Ⅳ vaccine except for blank control (BC) group, repeated in two weeks later, the chickens in four cSPSs and three sPS control groups were intramuscularly injected with corresponding polysaccharide, and in non-adjuvant control (NA) and BC groups, with equal volume of physiological saline. On days 7,14 and 21 after the first vaccination, the serum antibody was determined by β-micromethod, and on day 14 and 21, peripheral lymphocyte proliferation was determined by method of MTT. On day 23 after first vaccination, the chickens except for BC group were challenged with Newcastle disease virus (NDV). On days 14 after challenged, the morbidity, mortality and immune protective rate were calculated and the serum HI antibody was measured. The results showed that in all polysaccharides groups at all time points, the antibody titers were higher and lymphocyte proliferations were significantly higher in compasison with NA group (P<0.05), the HI antibody and lymphocyte proliferation in cSPS 1 group were highest and significantly higher than those of the NA groups (P< 0.05). After challenge, the morbidity and mortality of all polysaccharide groups were lower and protective rate and antibody were higher than those of NA group. In cSPS 1 group, The morbidity was lowest and the HI antibody and protective rate were highest, with significant difference as compared with NA group (P< 0.05). These result indicated that all polysaccharides could enhance the immune response of ND vaccine and protective rate of challenge and promote antibody production after challenge, cSPS 1 possessed the best efficacy.Test 5. Effect of cSPSs on splenic lymphocyte proliferation and secretion of IL-2 and IL-6 In order to comparing the immune-enhancing activity of cSPSs, firstly the safe concentration of four cSPSs to spleen lymphocyte was determined by MTT method. Then four cSPSs and their three components (sAPS. sEPS and sLNT) at five concentrations within safe concentration and ConA were added into the cultivating system of splenic lymphocyte. The lymphocyte proliferation and the content of IL-2 and IL-6 secreted by lymphocyte were determined by MTT and ELISA method, respectively. The results showed that within 12.5-3.13 μg·mL-1, the lymphocyte proliferation of the four cSPSs, sAPS and sEPS group were significantly larger than those of ConA and cell control group. The contents of IL-2 and IL-6 in all polysaccharides groups were significantly higher than those of cell control group, and in cSPSs 1 group were highest. These result indicated that all cSPSs and their components could significantly stimulate the lymphocyte proliferation synergistically with ConA and promote lymphocyte to secrete cytokine, cSPSs 1 possessed the best action.Test 6. Effect of cSPSs on cellular infectivity of NDV in vitro In order to compared the antiviral activity of cSPSs, firstly the safe concentrations of four cSPSs to CEF were determined by MTT method. Then four cSPSs and their components at three concentrations within safe concentration were added into the cultivating system of CEF, and replaced with NDV after 2 h. After cultivation of 72 h, the cells were collected, the RNAs were extracted and the contents of NDV were assayed by QR-PCR. The results showed that the NDV contents in all polysaccharides at all concentration groups were significantly lower than that in virus control group, which indicated that four cSPSs and their components could inhibit NDV to infect CEF, and cSPS 1 possessed strongest action. |
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