| Since July 2020,feed additives other than Chinese herbal medicine for growth promotion have been prohibited in China,marking the full opening stage of antibiotic-free breeding comes.The development of safe Chinese herbal immunoenhancer with wide functions,low toxicity,no drug resistance will have a broad application prospect.The pharmacological function of artificial Isaria Cicadas is similar with wild Isaria Cicadas,with the function of tonify,antitumor and immunomodulatory.In this study,the effects of the Isaria Cicada Polysaccharides(ICPs)extracted from Mycelium culture of Isaria cicadae(ICM)on humoral and cellular immunity of chicken were tested in vivo and in vitro,by the antibody and cytokine secretion of chicken immunized with Newcastle disease vaccine.The study aims to provide a basis for the role of ICPs as an immunostimulant in safety production of chicken.Experiment 1:Effect of ICPs on proliferation of chicken lymphocytes in vitro To investigate the effect of ICPs on proliferation of chicken T and B lymphocytes in vitro,the proliferation of chicken lymphocytes was tested using CCK-8 method in vitro.The results showed that the maximum safe concentration of ICPs was>1000 g/mL.The concentration of SI was significantly increased when the peripheral blood lymphocytes were stimulated by ICPs alone at 62.5-1000 μg/mL,and the concentration of SI was significantly increased when the spleen B lymphocytes were stimulated ICPs at 15.63μg/mL.When ICPs and ConA were used to stimulate peripheral blood lymphocytes,the concentration of SI was significantly higher than that of other concentrations.When ICPs was used to stimulate spleen lymphocytes at 1000 μg/mL,the concentration of SI was significantly higher than that of other concentrations.In conclusion,ICPs can stimulate the proliferation of T and B lymphocytes alone or in coordination with ConA and LPS.Experiment 2:Screening dosage of Isaria cicadae polysaccharides In order to determine the dose range of Isaria cicadae polysaccharides(ICPs)in the application of chicken,the influence of ICPs on the effect of broiler and breeding chickens chicken immunized with the Newcastle disease vaccine was studied.Brood stage:A total of 300 1-day-old healthy chickens were randomly divided into immune control group(Group Ⅰ),and groups Ⅱ,Ⅲ,Ⅳ of ICPs(supplemented with 100,200,400 mg/kg.BW,respectively)and astragalus polysaccharide group Ⅴ(100 mg/kg.BW).The chickens in each group were administrated by gavage for cicadae polysaccharides or soline from 7 to 28 days old.All of the chickens were immunized with Newcastle attenuated virus vaccine by nose and eye drops 14 and 28 days old respectively.Blood sample,and immune organs of spleen,bursa,and thymus were collected and immune organ indices were analyzed.Results showed that survival rate of Group Ⅱ,Ⅲ,Ⅳ were higher than that group Ⅰ and Ⅴ(from 4%to 12%).Maternal antibody level of Newcastle disease of Group Ⅱ,Ⅲ,Ⅳ was significantly higher than that of group Ⅰ,Ⅴ at 14 day.On 28 day,the bursa index and spleen index of group Ⅲ were significantly higher than those of other groups.The immune organ index was significantly higher than that of control group I on 42 day.Meanwhile,the splenic tissue developed quickly,the lymph nodes were larger,and the lymphatic sheath could be seen around the central artery in Group Ⅳ.Grower chicken:A total of 120 50-day-old healthy chickens were randomly divided into immune control group(Group Ⅰ),and groups Ⅱ,Ⅲof ICPs(supplemented with 50,100 mg/kg.BW,respectively)and astragalus polysaccharide group Ⅳ(100 mg/kg.BW).The chickens in each group were administrated by gavage for soline or cicadae polysaccharides from 60 to 73 days.Newcastle disease oil emulsion inactivated vaccine were used by Subcutaneous injection on 63 day.The titer of Newcastle disease antibody,amino acids and nutrients of pectoral muscle,physical and chemical indexes such as meat color and shear force were detected.Results showed that Maternal antibody level of ND-HI of Group Ⅱ,Ⅳwas significantly higher than that of group I at 70 day.The contents of amino acid,glutamic acid,alanine,methionine,leucine,tyrosine,lysine and crude protein in pectoral muscles of group II were significantly higher than those of group I.The results indicated that the addition of ICPs had the effect of improving humoral immunity,immune organ index and tissue development,but had no significant effect on growth performance in brood stage.The ideal dose was 100mg/kg·BW.Meanwhile,ICPs can significantly increase the antibody level of inactivated vaccine against Newcastle disease,increase the content of amino acid and protein in muscle,and have no significant influence on the physical and chemical properties of muscle in grower chicken.The best dose was 50mg/kg·BW.Experiment 3:Effect of ICPs on efficacy of ND vaccine in chickens This study was to investigate the effect of Isaria cicadae polysaccharides on chickens immunized with the Newcastle disease vaccine.A total of 225 chickens were randomly divided into three groups:control group,immune control group,and Isaria cicadae experimental group.The chickens in each group were administrated by gavage for Isaria cicadae polysaccharides or soline once a day for 14 days from 11 to 13 days old and 25 to 27 days old.All of the chickens were immunized with Newcastle attenuated virus vaccine by nose and eye drops 14 and 28 days old respectively.On 28,35,and 42 days,blood sample,and immune organs of spleen,bursa,and thymus were collected and immune organ indices were analyzed.Serum was separated and the levels of maternal antibodies,interleukin IL-4 and IFN-γ were analysed.The peripheral anticoagulant was collected,lymphocytes were extracted,and flow cytometry was used to determine the influence of Isaria cicadae polysaccharides on dynamic changes in CD4+ and CD8+lymphocyte subsets.The effects of Isaria cicadae polysaccharides on the expression levels of MHC II,TLR-2,and TNF-α in spleen were analysed.Results showed that the antibody levels in chickens from the Isaria cicadae polysaccharide group were significantly higher than those from the immune control group.Furthermore,the immune organ indices and CD4+/CD8+ cell ratio in Isaria cicadae polysaccharide group was significantly higher,respectively,which the levels of IL-4,IFN-γwas higher than that in the immune control group,respectively.Therefore we conclude that Isaria cicadae polysaccharides can significantly enhance the immune efficacy of the Newcastle disease vaccine,with higher levels of immunity and lasting longer in chickens.Experiment 4:The Proliferation of chicken lymphocytes in vivo induced by ICPs and its transcriptome analysis The molecular mechanism of the effect of ICPs on chicken immune function was further analyzed by lymphocyte proliferation in vivo and transcriptome analysis.180 7-day-old healthy chickens were randomly divided into blank control group(group A),immune control group(group B),and groups C,D and E of ICPS(supplemented with 25,50,100 mg/kg.BW respectively)and bacitracin control group F(50 mg/kg.BW).All of the chickens were immunized with the attenuated Newcastle disease vaccine by nosal and eye drops on 14 and 28 days old,respectively.The chickens were oral administrated with ICPs or bacitracin from 11 to 17 days old and 25 to 31 days old.The experimental period was from 7 to 42 days old.At the end of the drug feeding,6 chickens were randomly selected from each group for lymphocyte proliferation test and transcriptome analysis.The results showed that the SI of lymphocytes in group D was significantly increased post stimulation with ConA,LPS and NDV;IFN-γ,IL-4 and T-AOC in group D were significantly higher than those in group B with ConA stimulation;IL-2,IL-4,IFN-γ,TNF-α,T-AOC,CAT,GSH-Px and SOD in group C were significantly higher than those in group F;T-AOC and IFN-γ in group C were significantly higher than those in group B post stimulation with NDV attenuated vaccine;IFN-y in group E and IL-2 in Group F was significantly increased.For the transcriptome analysis,a total of 782 differentially expressed genes were screened between B and D,of which 731 genes were up-regulated and 51 genes were down-regulated.The most significant enrichment was found in the two pathways of herpes simplex infection and IgA production.The immune function may be enhanced by biological functions such as antigen presentation,nuclear transcription,lymphocyte activation and interferon expression.It is suggested that ICPs can stimulate lymphocyte proliferation in vivo,CD40,STAT2,TAP1,IL-15,Mavs may be the key genes,and the possible mechanism is to enhance the immune function of chickens through the up regulation of genes in the intestinal immune network pathway produced by herpes simplex infection and IgA.The results showed that lymphocyte proliferation could be stimulate by the polysaccharide in vivo,and the possible mechanism is the enhance the immune function of chicks through the up regulation of genes in the intestinal immune network pathway of herpes simplex infection and IgA production.Experiment 5:The Application of ICPs and ICM in chicken breeding In order to explore the effect of ICPs on growth performance and immune function of Sanhuang chicken and compare the application effects of ICPS and ICM,1500 1-day-old Sanhuang chickens were randomly divided into control group(Group A),ICPs group(Group B)and ICM group(Group C),with 500 chickens in each group.Chickens in group A were fed with basal diet,chickens in group B were fed with basal diet added by 0.5 g/kg ICPs,and chickens in group C were fed with basal diet added by 20 g/kg ICM.The experiment lasted for 51 days.The results showed that the survival rate of group B and group C at 51 days old were increased by 3%and 2%respectively compared with group A;the antibody levels of H5-Re8 and H7N9 in group C were significantly higher than those in group A;the IL-4 in group B was significantly higher than that in group A or group C at 27 days old,and IL-4 in group B or group C was significantly higher than that in group A at 51 days old.The results suggested that the immune level and the activity of related cytokines of free-range chickens could be improved by ICPs and ICM.In Vitro and in Vivo experiments showed that polysaccharides from cordyceps cicadae could stimulate the proliferation of chicken lymphocyte,enhance the initial immune effect of Newcastle Disease Vaccine,induce the secretion of cytokines such as IFN-and TNF-,and increase the serum antioxidant index,the Mrna expression of TLR-2 and TNF-gene increased significantly.The Intestinal Immune Network pathway produced by IgA may be related to the effect of ICPs on the immune function of chickens.0.5 g/kg mixture was added continuously for 50 days and 50 mg/kg.BW for 2 consecutive times for 7 days before and after vaccination. |
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