| Animal infectious diseases,especially viral disease,were a major problem in livestoc k husbandry,which have caused huge losses and receive worldwide concern.Because of un ique biological features and pathogenesis of virus,there was no effective method for anti-vi rus therapy,so vaccination was the main preventive measure.Although with so many resear ch and application of vaccine,immunization effect was always unsatisfactory with phenom enon of immune failure.It has been demonstrated that many Chinese herbal medicines and their ingredients possess antiviral activity.Some Chinese herbal medicines and their'ingredients have been used in chickens after or before vaccination,resulting in the decrease of the incidence of infectious diseases and the increase in animal immunity.Ganoderma lucidum(G.lucidum)has been widely used as a remedy to promote health and longevity in Asia for thousands of years,especially G lucidum polysaccharides(GLP),which are water-soluble and extracted from G lucidum.Recent studies have shown that GLP has antioxidant activities,immunomodulating activities,anti-tumor activities and so on.However,there are few reports about the effects of GLP on animal infectious diseases especially the virosis,which cause great loss in the domestic animal and poultry industry.In this study,the antiviral and immune-enhancing action and mechanism of GLP were investigated through a series of experiments in vitro and vivo.The purpose of this research is to offer theoretical evidence for the development of a novel immunopotentiator in the domestic animal and poultry industry.The details are divided into eight parts as follows:Test 1 Effects of G.lucidum polysaccharide on cell infectivity of NDV and IBDVTo investigate the antiviral activity of Ganoderma lucidum polysaccharides(GLP)in vitro,GLP was added into chicken embryo fibroblasts(CEF)infected with avian infectious bursal disease virus(IBDV)and Newcastle disease virus(NDV)in three different mechanisms:pre-addition,post-addition,and mixed addition of polysaccharides with virus,respectively.The blank control group and the virus control group were included.The effects of GLP on cellular infectivity of virus were determined by MTT method.Results showed that in the pre-addition mode,the A570 values of GLP groups(9.766?625?g·mL-1)were significantly higher than that of the virus control group,in the post-addition mode,only the A570 value of GLP group at 625 ?g·mL-1 was significantly higher than that of the IBDV control group,and other GLP groups did not have significant actions;in mixed addition of polysaccharides with virus mode,the A570 values of GLP groups at 78.125?g·mL-1 to 625 ?g·mL-1 infected with IBDV were significantly higher than those of the IBDV control group,and the A570 values of all GLP groups infected with NDV were significantly higher than those of the NDV control group.The results indicated that GLP,if used properly,possesses potent antiviral effect on IBDV and NDV infection,particularly by pre-addition of polysaccharides,and it also indicated that GLP could prevent IBDV and NDV infection by blocking the virus adsorbed host cells.Test 2 Effect of G.lucidum polysaccharide on mRNA expression of IFN-P in CEFTo investigate the antiviral activity and mechanism of Ganoderma lucidum polysaccharides(GLP)in vitro,according to Primer Premier software with Oligo7,a pairs of RT-PCR primers of interferon-p were designed.Then,the effects of GLP on expression level of interferon-? mRNA was determined by semi-quantitative RT-PCR assay.The results showed that GLP could improve the expression level of interferon-? mRNA,especially at the dose of 156.25?g·mL-1,GLP significantly increased ConA-induced IFN-?mRNA expression(P<0.05).At 39.065 and 78.125 ?g·mL-1 of GLP slightly,but not significantly,increased IFN-? mRNA expression in CEF.It suggested that GLP exerts its antiviral activity at least in part via promoting IFN-? gene expression.Test 3 Effects of G.lucidum polysaccharide on proliferation of chicken peripheral and splenic lymphocytesIn order to study the action of GLP on chicken peripheral lymphocytes proliferation in vitro,the proliferation was observed in peripheral and splenic lymphocytes treated with GLP.The lymphocytes proliferation was determined by MTT assay.Results showed that GLP at 0.070?2.25 mg mL-1 alone increased the A570 values in chicken peripheral blood lymphocytes in a concentration-dependent manner,at 0.141?2.25 mg·mL-1 GLP also enhanced ConA-induced A570 values(P<0.05).Besides,GLP at 0.035?2.25 mg·mL-1 alone increased the A570 values in chicken splenic lymphocytes(P<0.05),at 0.035?2.25 mg·mL-1 GLP also enhanced ConA-induced A570 values(P<0.05).It implied that GLP not only promoted cell proliferation of the lymphocytes,but also synergized with ConA in promoting proliferation of the lymphocytes.Test 4 Effects of G.lucidum polysaccharide on mRNA expression of interleukin-6 and interferon-y in chicken peripheral lymphocyte in vitroIn order to study the action and molecular mechanism of GLP in immunoenhancement,according to Primer Premier software with Oligo7,two pairs of RT-PCR primers of Interleukin 6(IL-6)and Interferon ?(IFN-?)were designed.Then,the effects of GLP on expression level of IL-6 and IFN-? mRNA were determined by semi-quantitative RT-PCR assay.The results showed that GLP at 562.5?g·mL-1?281.25 ?g·mL-1 significantly enhanced ConA-induced the IL-6 mRNA expression(P<0.05),and at 140.63 ?g·mL-1 significantly enhanced ConA-induced the IFN-y mRNA expression(P<0.05).This might be one of molecular mechanisms of GLP in immunoenhancement.Test 5 Effects of G lucidum polysaccharide on mucosal immunity in vaccinated chickensTo investigate the action and mechanism of GLP in mucosal immunity,225 14-day-old chickens were randomly assigned into equal five groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in vaccination control(VC)and BC group with physiological saline,once a day for three successive days.On days 14 after the second vaccination,five chickens were sampled randomly from each group for determination of secretory immunoglobulin A(SIgA)that is released into intestinal mucosa and cecal tonsil by immunohistochemistry method.The results showed that GLP at 50?25?12.5 mg·kg-1 could raise the content of SIgA and increase the number of SIgA positive cells,especially at 25 mg kg-1,the level of SIgA positive cells was the highest.It demonstrated that GLP could enhance the number of SIgA positive cells as so to enhance the immune function of mucosa,and improve the ability of anti-infection.Test 6 Effects of G.lucidum polysaccharide on mRNA expression of interleukin-6?interferon-y in vaccinated chickens peripheral lymphocyteTo evaluate the adjuvanticity and molecular mechanism of GLP in immunoenhancement in vaccinated chickens in vivo,225 14-day-old chickens were randomly assigned into equal five groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in vaccination control(VC)and BC group with physiological saline,once a day for three successive days.On days 28 after the first vaccination,five chickens were sampled randomly from each group for determination of the expression level of IL-6?IFN-? mRNA by semi-quantitative RT-PCR assay.The results showed that GLP at 50 mg·kg-1?25 mg·kg-1 significantly enhanced the IL-6?IFN-? mRNA expression(P<0.05),This might be one of molecular mechanisms of GLP in immunoenhancement.Test 7 Effects of G lucidum polysaccharide on immune response of Newcastle disease vaccine for chickensTo evaluate the adjuvanticity of GLP on immune response of Newcastle disease vaccine for chickens in vivo,300 14-day-old chickens were randomly assigned into equal six groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in drug control chickens were orally administrated respectively with Astragalus polysaccharide(APS),in vaccination control(VC)and BC group with physiological saline,once a day for three successive days.On Day 7,14,21 and 28 after the first vaccination,the peripheral lymphocytes proliferation,the serum ND antibody titer and the immune organ index were determined.The results showed that GLP at 50?25 mg·kg-1 could significantly promote lymphocyte proliferation,raise serum antibody titer,improve immune organ index of spleen,bursa and thymus.The results indicated that GLP may be a novel immunomodulator.Test 8 Effects of G lucidum polysaccharide on immune protective effects of Newcastle disease vaccine for chickensTo further study the antiviral activity of GLP on immune response of Newcastle disease vaccine for chickens in vivo,280 14-day-old chickens were randomly assigned into equal 7 groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in drug control chickens were orally administrated respectively with Astragalus polysaccharide(APS),in vaccination control(VC)?virus control group and BC group with physiological saline,once a day for three successive days.On days 28 after the first vaccination,the chickens were attacked with New castle disease virus(NDV).The symptom and the death time were recorded,and the morbidity,mortality and protection rate were calculated.The results showed that the morbidities and mortalities of GLP at high and middle dose were significantly lower than that of the virus control group(P<0.05),slightly,but not significantly lower than that of the APS control group and the vaccination group.The results indicated that GLP could increase the immune protective effects of Newcastle disease vaccine for chickens,and increase the antiviral action of the body. |
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