Enhanced Immune Responses And Mechanisms To Live Vaccines In Chickens By Ginseng Stem-leaf Saponins Combined With Selenium

Infectious diseases are common diseases of poultry,which have caused significant economic losses to the poultry industry.Immunization with vaccines could induce immune responses against pathogenic microorganisms,which is an important measure to prevent infectious diseases in poultry.After the chicks hatched,they contain high level of maternal antibodies which have immune protective effects on chicks.However,maternal antibodies can also interfere the immune function of vaccine and reduce immune effects.In order to avoid the interference from maternal antibodies,early vaccine immunization is often performed after maternal antibodies falling to a certain level.Therefore,there is a time gap after the maternal antibody decreases to the protective level and before the vaccine-induced antibody reaches to the protective level,which we call the "dangerous window".Chickens in the "dangerous window"are susceptible to pathogenic microorganisms because their maternal and vaccine-induced antibody levels are below protective levels.Therefore,research on reducing the "dangerous window" has practical significance in poultry production.Making vaccine-induced antibodies early or increasing antibody levels can help reduce the "dangerous window" and reduce the chance of chickens infected by pathogens.Previous studies have proven that ginseng stem and leaf saponins(GSLS)and selenium(Se)(GSLS-Se)can enhance the immune function of animals and increase the level of vaccine-induced antibodies.This article studied the immune-enhancing effect of the solution containing GSLS-Se on chicken attenuated vaccines,and then observed the effect on reducing the "dangerous window" during early immunization of chickens.As the vaccines of chicken attenuated vaccine are usually immunized through intraocular-and-intranasal route,the vaccines are affected through the Harderian gland(HG),the immune organ.This paper also studied the mechanism of GSLS-Se in chicken attenuated vaccine on HG tissue and cells to enhance the immunity of the attenuated vaccine.1.Formulation of GSLS and Se as diluents in chicken attenuated vaccinesObjective:To investigate the effect of saline containing different concentrations of GSLS and Se(in the form of Sodium selenite,based on Se content)as a diluent of live attenuated chicken vaccine on immune response.Methods:Chickens at 12 days old were immunized with live bivalent vaccines of NDV and IBV using different concentrations of GSLS and Se as vaccine diluents(GSLS-Se).Blood samples were collected at 10,12,14,18 days to detect serum NDV HI titer and chicken IBV specific antibody titer.Results:It was found that 5 ?g/50 ?L GSLS and 25 ?g/50 ?L Se can significantly increase chicken NDV HI titer and chicken IBV specific antibody titer.Especially when 5 ?g/50 ?L GSLS and 25 ?g/50?L Se used together,the antibody titer reached to the highest.Therefore,saline containing(5 ?g GSLS+25 Lg Se)/50?L was selected as the vaccine dilution for further investigation.2.Effect of GSLS-Se on immunity of chicken attenuated vaccineObjective:To observe the immune-enhancing effect of GSLS-Se on chicken attenuated vaccine.Methods:Experiment 1 Effect of GSLS-Se on "dangerous window" of NDV and IBV.GSLS-Se and saline were used as vaccine diluents to resuspend live bivalent vaccines of NDV and IBV of chicken.Chickens were immunized in intraocular-and-intranasal route in 12 days old.Non-immune chickens were served as control.Blood samples were collected every three days post immunization till 27 days post immunization to detect chicken NDV HI titer and IBV specific antibody titer.Experiment 2 Effect of GSLS-Se on "dangerous window" of IBDV.GSLS-Se and saline were used as vaccine diluents to resuspend live bivalent vaccines of IBDV of chicken.Chickens were immunized in intraocular-and-intranasal route in 12 days old.Non-immune chickens were served as control.Blood samples were collected every four days post immunization till 36 days post immunization to detect chicken IBDV specific antibody titer.Experiment 3 The effect of GSLS-Se on humoral and cellular immunity in live bivalent vaccines of NDV and IBV.GSLS-Se and saline were used as vaccine diluents to resuspend live bivalent vaccines of NDV and IBV and immunized in intraocular-and-intranasal route in 12 days old chickens.Non-immune chickens were served as control.Blood samples were collected 48 hours post immunization to measure the levels of IFN-? and IL-4 in serum.Anticoagulated and non-anticoagulated blood samples were collected aseptically at 14 days after immunization.anticoagulated blood was used for peripheral blood lymphocyte proliferation test while non-anticoagulated blood was used for virus neutralizing antibody detection.Experiment 4 Effect of GSLS-Se on spray immunization of chicken attenuated vaccine.GSLS-Se and saline were used as diluents in live vaccine of IBDV.The 12 days old chickens were immunized in spray immunization with 15 in each group.Non-immune chickens were served as control.Blood was collected 1-5 weeks post immunization;GSLS-Se and saline were used as diluents in live vaccine of IBDV.Experiment 5 Effect of GSLS-Se on intraocular-and-intranasal immunization of chicken attenuated vaccine in large-scale commercial chicken farms.According to the immunization program in the farm,chickens at 8 days old received live bivalent vaccines of NDV and IBV with or without GSLS-Se in intraocular-and-intranasal route with 500 chickens in each group.Blood samples were collected 2,3 weeks post immunization to detect chicken NDV HI titer and IBV specific antibody titer.Experiment 6 Effect of GSLS-Se on drinking immunization of chicken attenuated vaccine in large-scale commercial chicken farms.The 12 days old chickens were immunized in drinking immunization with 5000 in each group.Blood samples were collected 1-week post booster immunization according to the immunization program in the farm to detect IBDV-specific antibody titers.Results:In terms of reducing "dangerous window",GSLS-Se can reduce chickens "dangerous window" for NDV,IBV and IBDV.promote early production and prolong duration of antibody responses;In terms of enhancing humoral and cellular immunity of live bivalent vaccine of NDV and IBV;GSLS-Se can increase the peripheral blood lymphocyte stimulation index,IFN-y and IL-4 levels,and specific neutralizing antibody titers.In terms of immunization methods,GSLS-Se can be used in intraocular-and-intranasal immunization and drinking immunization in commercial farms.,but GSLS-Se in spray immunization showed no significant enhancement effect.Results showed that GSLS-Se can shorten the "dangerous window" of chickens,improve humoral and cellular immunity,and be suitable for intraocular-and-intranasal immunization and drinking immunization.3.Effect of storage conditions of GSLS-Se in drug content and immune effectObjective:To evaluate whether GSLS-Se could be a stable vaccine diluent under different storage conditions.Try to find suitable storage conditions to provide references for subsequent research and applications.Methods:GSLS-Se in different concentrations,storage methods,and storage time was used as a vaccine diluent to dilute chicken live bivalent vaccine of NDV and IBV.The chickens were immunized in intraocular-and-intranasal route.Blood samples were collected after 14 days.The serum was tested for chicken NDV HI titer and chicken IBV-specific antibody titer,compared with the antibody titer of chickens immunized with GSLS-Se diluent newly prepared;GSLS and Se content in GSLS-Se in different concentrations,storage methods,and storage times were measured according to China Veterinary Pharmacopoeia;GSLS-Se stored for 12 months was plate streaking.Results:The GSLS-Se diluent of working solution and 20-fold concentrated solution can be stored under normal storage conditions such as room temperature,room temperature away from light,and 4? for 3,6,9,and 12 months with adjuvant effect,drug content and no bacterial growth.4.Study on the immune effect of GSLS-Se on chicken's Harderian gland(HG)Objective:To investigate the immunoregulatory mechanism of GSLS-Se diluent on HG in intraocular-and-intranasal route,which is one of the main sites of NDV infection.Methods:GSLS-Se was used as a vaccine diluent to dilute live vaccine of NDV.Chickens were immunized in intraocular-and-intranasal route.HGs were collected 14 days later,the sIgA content in the tissue supernatant,IgG+,IgA+,IgM+cell in the tissue sections were measured;GSLS-Se diluent was administered to chickens in intraocular-and-intranasal route,and the mRNA relative expression levels of IFN-?,IL-6 and IgA in HGs were measured at 24,48,and 72 hours after administration to determine the transcriptome sequencing sampling time;the samples and the non-administration control samples were sequenced for RNA-seq and bioinformatics analysis such as DEGs,GO function,and KEGG pathway enrichment analysis.Results:GSLS-Se was found to significantly increase the sIgA content,IgG+,IgA+,and IgM+cell index in chicken's HGs;The mRNA relative expression of IFN-y,IL-6,and IgA in HGs 48 hours after GSLS-Se administration reached to the highest;RNA-seq results showed that DEGs,immune-related GO terms,and KEGG pathways were significantly enriched in the GSLS-Se group.TLR signaling pathway and MAPK signaling pathway may be the main signals involved in immune regulation.Therefore,GSLS-Se may exert immunoregulatory effect on immune-related gene profiles in the HG immune cells to make chickens immunized with live vaccine of NDV produce antibodies in advance and increase immune response.5.Study on the effect of GSLS-Se on chicken HG's single cellsObjective:To investigate the immunoregulatory effect of GSLS-Se on HG's single cells in vitro.Methods:HGs in chickens were aseptically removed to make single-cell suspensions.After plating,GSLS-Se/GSLS/Se were added in different concentrations,The cell viability was detected using the cck8 method;Fluorescence microplate reader was used to detect cell apoptosis;JC-1 was used to detect mitochondrial membrane potential ratio;Flu-3/AM probe was used to detect Ca2+ channel concentratioL;ELISA was used to measure cytokines(IFN-?and IL-4)content in cell culture supernatant;RT-qPCR was used to detect intracellular cytokines(IFN-y,IL-1?,IL-4,IL-6,IL-17),IgA and the HG tissue transcriptome results verification in the cell level.Results:Adding a small amount of GSLS-Se(concentration range between 5ng Se-1?g GSLS?1ng Se-0.2?g GSLS)to HGs'single cells cultured in vitro can increase cell viability and reduce early cell apoptosis,increase the mitochondrial membrane potential JC-1 ratio.reduce the concentration of Ca2+ channels;increase the level of cell supernatant cytokines(IFN-?,IL-4),intracellular cytokines(IFN-y,IL-1?,IL-4,IL-6,IL-17)and IgA mRNA relative expression levels,and the trend of HGs tissue RNA-seq results in vitro had also been verified.In conclusion,live vaccine diluted in saline containing 5 ?g GSLS+25 ?g Se could significantly increase chicken NDV.IBV and IBDV specific antibody titers,promote early antibody production and prolong the duration antibodies,reduce chicken infection dangerous window.The effect had been confirmed in large farm clinical tests;GSLS-Se could increase the content of neutralizing antibodies of NVD and IBV,lymphocyte proliferation index,and the levels of serum IFN-y and IL-4.GSLS-Se could be used in intraocular-and-intranasal immunization and drinking immunization,could be stored for one year under normal storage conditions such as room temperature,room temperature away from light and 4? underworking solution concentration and 20-fold concentration,which could maintain a good adjuvant effect and drug content.GSLS-Se might be a suitable diluent for poultry vaccines worthy for promotion.In the study of the immune effect on the HG,it was found that GSLS-Se could significantly increase the sIgA content,IgG+,IgA+,and IgM+cell index in the chicken's HGs and significantly enriched immune-related signaling pathways in the HG;GSLS-Se added to HG single cells cultured in vitro could increase cell viability,reduce early cell apoptosis,increase mitochondrial Gembrane potential JC-1 ratio,and reduce Ca2+ channel concentration,improve cell culture supernatant cytokine(IFN-y,IL-4)levels,mRNA relative expression of intracellular cytokines(IFN-?,IL-1?,IL-4,IL-6,IL-17)and IgA.It is speculated that GSLS-Se may enhance the immune response by affecting the anti-oxidation and regulating the activity of immune-related enzymes in chickens' HGs.