| Newcastle disease virus(NDV)is a highly contagious viral pathogen.It is a worldwide problem that ND has a high morbidity and mortality when poultry was infected.Up to now,the prevention and control methods of ND is still largely relied on vaccines.Many vaccines cannot reach ideal immune effect and generally require the assistance of immunologic adjuvant.To use immunopotentiator can make vaccine generate a strong immune response and provide long term protection against infection.The objective of this study is to screen out the optimal dose that KSP can improve the ND antibody levels in chicken and to probe the mechanism of action which KSP can improve the immune efficacy of Newcastledisease vaccine in chicken.In order to investigate the effect of different doses of the Kucaojuntai Soluble Powder(KSP)on immune of Newcastle disease(ND)vaccine and growth performance,two batches of experiments were performed.In the first batch of experiment,the tested broilers were divided into three groups of different immune types: live vaccine group(LVG),inactivated vaccine group(IVG),combination of inactivated vaccine and live vaccine group(CVG).Each group of immune type was divided into high dose group(500mg/L),medium dose group(100mg/L),low dose group(20mg/L),and vaccination control(VC)group.The chickens of each group received KSP via the drinking water(ad libitum),for ten successive days.The serum antibody titers were measured by hemagglutination inhibition test.In the second batch of experiment,the chickens were randomly divided into ten groups to confirm the more suitable dose.The chickens,except blank control(BC)group,vaccinated with ND vaccine at 11 days old,and repeated vaccination at 25 days old except single immune group and BC group.At days of 11,18,25,32,39 and 43,serum antibodytiters and weight were measured.To evaluate the immune effect of antibacterial peptide Soluble Powder(KSP)on chickens,the vitro experiment and vivo experiments were performed.In vitro experiment,the effects of KSP on chicken T and B peripheral lymphocyte proliferation were determined by MTT assay.In vivo experiment,450 14-day-old chickens were averagely divided into 7 groups.The chickens,except blank control(BC)group,were vaccinated with Newcastle disease(ND)vaccine at 14-day-old,and repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in three KSP groups received KSP via the drinking water(ad libitum),respectively,with the KSP at low,medium and high concentrations,in vaccination control(VC)and BC group,with water freely,the other groups were porcine skin collagen control group(PSC)and echinacea oral liquid control group(EOL),except VC group and BC group for ten successive days.On days14,21,28,35,42,the serum antibody titer was measured.On days 13,24,39,the T and B peripheral lymphocyte proliferation were measured by MTT method and T-lymphocytes subpopulation were measured by flow cytometry.On days 14,21,36 and 43,the IL-2?IL-4?IL-10 and IFN-? were determined by Elisa.On days 25 and 40,the immune organ index were measured by weight and peritoneal macrophages phagocytosis were determined by neutral red incorporation method.1?The results in first batches of pretest experiments showed that the suitable dosage range of KSP was around the low dose(20mg L-1),and the effect of combination of inactivated vaccine and live vaccine group was better.The results in second batches of pretest experiments showed that the serum antibody titers was decreased significantly by the highest doses(2500mg L-1)of the KSP and increased significantly by the low dose(50mg L-1)of the KSP(p<0.05).It is no difference that between take medicine once in test cycle and take medicine twice in test cycle.These indicated that the KSP could significantly improve the immune efficacy of Newcastledisease vaccine at the optimal dose,but it has no significant effect on the weight of chicken.2?The results in vivo experiments of official test showed that the A570 value of KSP synergistically with Con A was significantly higher than those of cell controlgroup when the KSP at the concentration of 62.5–1000?g m L-1(p<0.05)..It indicated that KSP at these concentrations could significantly stimulate chicken T lymphocytes proliferation synergistically with Con A.The A570 value of KSP synergistically with LPS was significantly higher than those of cell control group when the KSP at the concentration of 15.63–1000?gm L-1(p<0.05).It indicated that KSP at these concentrations could significantly stimulate chicken B lymphocytes proliferation synergistically with LPS.The effect of KSP at the concentration of 1000?g m L-1 was best in the all of dose.3?The index of vitro experiments were included ND serum antibody titer,T and B lymphocyte proliferation,T-lymphocytes subpopulation,the amount of cytokines,peritoneal macrophages phagocytosis and immune organ index.The ND serum antibody titer of KSPH?KSPM and KSPL groups were significantly higher than VC group except 14 days,KSPH and KSPM groups were significantly higher than PSC group(p<0.05).The A570 of every group were not statistically significant in T lymphocyte proliferation test(p>0.05).The A570 of KSPH and KSPM groups were significantly higher than VC group at 24 days and the A570 of KSPH?KSPM and KSPL groups were significantly higher than VC group at 39 days in B lymphocyte proliferation test(p<0.05).The amount of CD4+ and CD8+ and the ratio of CD4+/CD8+ in T-lymphocytes subpopulation were not significant(p>0.05).The content of IL-4 in KSPH and KSPM groups were significantly higher than VC group at 36days(p<0.05),the content of IL-2,IL-10 and IFN-? in every groups were not significant(p>0.05).The A570 of KSPH?KSPM and KSPL groups were significantly higher than VC group on 25 days and the A570 of KSPH and KSPM groups were significantly higher than VC group on 39 days in the peritoneal macrophages phagocytosis test(p<0.05).On days 25,the spleen and the bursa of fabricius index of KSPH and KSPM groups were significantly higher than VC group(p< 0.05),and the thymus index of KSPH,KSPM and KSPL groups were significantly higher than that of VC group(p< 0.05).On days 39,the spleen index of KSPH and KSPM groups were significantly higher than that in KSPL and VC groups(p< 0.05),the difference of thymus and bursa of fabricius index were not statistically significant among the testedgroups(p > 0.05).Our studies screen out that the optimal dose which KSP can improve the ND antibody levels in chicken is 50 mg L-1,and demonstrate that the KSP improve the immune efficacy of Newcastle disease vaccine by enhancing serum antibody titer,promoting lymphocyte proliferation,and improving peritoneal macrophages phagocytosis and immune organ index.So KSP would be as the candidate of a new-type immune adjuvant. |
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