| The Newcastle disease(ND),commonly known as "chicken fever",is a highly contagious disease caused by the ND virus(NDV)of the family Paramyxoviridae,and is classified by the OIE as a Class A disease worldwide.Currently,there is no typical outbreak of Newcastle disease in general because the poultry industry pays more attention to vaccination against Newcastle disease.However,due to changes in the breeding environment,the genotype of NDV is evolving and the immunized flocks are affected by strong virulent attacks.In recent years,Newcastle disease has occurred in immunized flocks,often showing subclinical or atypical symptoms,with a consistent incidence of 15%-50%.Chickens of different ages with atypical Newcastle disease are susceptible to endogenous infections caused by other pathogenic bacteria such as Escherichia coli and other microorganisms,resulting in a decrease in chick performance and an increase in mortality of breeding chickens,causing huge economic losses to the poultry industry in China.Therefore,it is still important to study effective methods to improve the immunization effect of vaccines.Chinese herbal medicines,natural products and their active ingredients are natural and mild,safe and efficient,and inexpensive.Meanwhile,with the emphasis on food safety and green health,the application of natural products such as Chinese herbs and their active ingredients to enhance the immunity of animal organisms has become a hot research topic in recent years.Previous studies in our laboratory have shown that yeast cell wall polysaccharide can enhance the immune function of chicks by increasing the HI potency,intestinal specific s Ig A,Ig A+ cell count and relative expression of immune-related genes in spleen and jejunum after chickens are immunized against Newcastle disease vaccine.Yeast cell wall polysaccharides contain β-glucan and mannan-oligosaccharide.So,the present study was proposed to further investigate the immune enhancing effect of yeast cell wall polysaccharide(G70),of which β-glucan is the main component.The effect of yeast β-glucan(G70)on immunization of chickens against Newcastle disease vaccine was investigated by humoral immunity,cellular immunity and intestinal immunity,while the possible mechanism of action of G70 was explored by RNA-seq and RT-q PCR assays,aiming to provide reference for the application of yeast β-glucan(G70)as an immune booster to clinical practice and the theoretical study of the mechanism of action of yeast β-glucan.This study includes the following pilot studies:1.The effect of yeast β-glucan(G70)on humoral and cellular immunity in chickens.Forty-eight 1-day-old male commercial black-boned chickens(body weight 45±5 g)were selected for the experiment after 5 d of adaptive feeding and were randomly divided into three groups,group H,group C and group S,with 16 chickens in each group according to their largely uniform body condition.The physiological saline group(group S)and the vaccine group(group C)were fed the basal diet,while the G70 group(group H)was fed the basal diet with 1 g/kg of yeast β-glucan(G70).The ND vaccine was administered by eye and nose drops at 14 and 28 days of age for the first immunization and booster immunization,respectively,while the group S was immunized with the same dose of saline..The effects of G70 on humoral and cellular immunity in chickens is investigated by measuring immune organ indices,detecting serum NDV-specific antibody levels with HA-HI method,detecting peripheral blood lymphocyte proliferation capacity with MTT method,detecting CD4+ T cell and CD8+ T cell numbers in peripheral blood by flow cytometry,and detecting immune-related genes in the spleen by RT-q PCR.The results showed that yeast β-glucan significantly reduced the organ index of chicken bursa(P < 0.05)and increased organ index of thymus and spleen compared with immune control and saline control,but the differences were not significant.For specific antibody levels,serum NDV-specific antibody levels were higher in the G70 group than in the immunized control group at 7 d and 14 d after the first immunization(P < 0.05)and at 7 d and14 d after the second immunization.At 7 d after secondary immunization,the peripheral blood lymphocyte stimulation index was significantly higher in the G70 group compared with the immunized control group(P < 0.05).Meanwhile,the peripheral blood CD4+/CD8+ T-cell ratio was significantly increased in the G70 group(P < 0.05).In addition,the m RNA expression levels of IL-6(P < 0.05),TGF-β(P < 0.01)and TLR5(P < 0.05)were significantly up-regulated in spleen tissues of the G70 group compared with the vaccine control group,while the changes in the m RNA expression levels of IFN-γ,TLR4,TLR3,and CD40,CD80 and CD86 were not significantly different(P > 0.05).2.Effect of yeast β-glucan(G70)on intestinal immunity in chickens In this chapter,experiments were conducted to further investigate the effect of yeast β-glucan on intestinal immunity in chickens using HE staining microscopy to observe intestinal villi structure,MTT method to detect intestinal lymphocyte proliferation ability,flow cytometry to detect intestinal CD4+ T cell and CD8+ T cell numbers,and RT-q PCR to detect immune-related genes in jejunum.The results showed that yeast β-glucan significantly increased the crypt depth(P < 0.05).At 1 week after secondary immunization,the intestinal lymphocyte stimulation index was significantly higher in the G70 group compared with the immunized control group(P < 0.05).Also,the intestinal CD4+/CD8+ T-cell ratio was significantly increased in the G70 group(P < 0.05).In addition,the m RNA expression levels of GATA-3,MHC-I,MHC-II,and CCR7 were significantly upregulated in the intestinal tissues of the G70 group compared with the vaccine group(P < 0.05),while the m RNA expression levels of IL-6,TGF-β,NF-κB,TLR3,TLR4,TLR5,IFN-γ,CCR7,CCR9,J-chain,and CD40,CD80,CD86 were not significantly different in m RNA expression levels(P >0.05).3.Effect of yeast β-glucan(G70)on gene expression in chicken spleen.Transcriptome sequencing technology was used to analyze the differences in expression profiles of relevant genes and pathways in the spleen to elucidate the mechanism of immune enhancing effects.The results showed that 198 differentially expressed genes were identified between group H and group C,of which 47 were up-regulated and 151 were down-regulated.13 GO terms were identified by GO significant enrichment analysis,and the enriched GO terms included These included humoral immune response,chemokine response,antimicrobial humoral response,defense response against bacteria,immune response against antimicrobial humoral response mediated by antimicrobial peptides,defense response against Gram-negative bacteria,defense response against Gram-positive bacteria,mitochondrial respiratory chain complex I,NADH dehydrogenase complex,respiratory chain,CCR chemokine receptor binding,chemokine receptor binding and lipopolysaccharide binding.A total of seven significantly enriched metabolic pathways were identified in the KEGG enrichment analysis,namely neuroactive ligand-receptor interaction,gap junctions,MAPK signaling pathway,ABC transporter,amino acid biosynthesis,drug metabolism,and protein export.In addition,transcriptome sequencing results showed that 13 DEGs were significantly enriched in the MAPK signaling pathway.This suggests that yeast β-glucan may regulate the immune response in chicken spleen through MAPK signaling pathway.Therefore,to further investigate the mechanism of immune enhancing effect of yeast β-glucan,RT-q PCR was used to detect MAPK signaling pathway related genes.The results showed that the m RNA expression levels of FAS and CACNA2 were significantly reduced,while the expression levels of other related genes of MAPK signaling pathway were not significantly changed.In summary,the addition of 0.1% yeast β-glucan to the basal diet significantly increased the serum-specific antibody HI titers,the ratio of peripheral blood CD4+/CD8+ T lymphocytes and the proportion of CD4+ CD8+ T cells in the intestine,enhanced promotion of peripheral blood and intestinal lymphocyte proliferation,and regulated the m RNA levels of genes related to immunity in the chicken after Newcastle disease vaccination.The m RNA levels of immune-related genes in the intestine and spleen were regulated.In addition,the spleen transcriptome was screened for 198 differentially expressed genes,and the differential genes were enriched to a total of 13 GO terms and 7 KEGG metabolic pathways.Suggestively,the immune enhancing effect of yeast β-glucan(G70)may be associated with upregulation of G-protein-coupled 5-HT receptor and MHC class I peptide-related m RNA expression in spleen,inhibition of MAPK signaling pathway and immune cell apoptosis.Additionally,it is interesting to note that yeast β-glucan significantly down-regulates the expression of m RNAs related to antimicrobial peptides and avian β-defensins. |
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