Effects And Mechanism Of Bacteroides And Its Metabolites On The Intestinal Immune Performance Of Broilers

The interaction between nutrition and immunity plays a fundamental role in nutrients digestion,absorption,and metabolism in poultry production.Current studies showed that the gut microbiota contributed to the development of intestinal mucosal immunity.The regulatory mechanisms of gut microbiota on the animal immune system have been extensively studied,however,the activation mechanism of intestinal microbes on the intestinal immune system of poultry is still unclear.Free-range Chinese native chickens have higher disease resistance than commercial broiler chickens,but there is a lack of research and mechanism analysis of this phenomenon from the perspective of gut microbiota.We hypothesized that the specific gut microbiota in Chinese native chickens might be a potential factor to enhances mucosal immunity,and the key gut microbes would enhance mucosal immune response.Here,we compared the intestinal mucosal immunity and gut microbiota between Arbor Acre broilers(AA)with lower gut mucosal immunity and Chinese native Wuliang Mountain Black-bone chickens(WLMB)with higher gut mucosal immunity to screen the gut microbes associated with intestinal mucosal immunity development and dissect the underlying mechanisms.Therefore,the gut immunophenotype was compared between AA and WLMB chickens,and the influence of gut microbiota on the production and secretion of IgA was analyzed.16 S r DNA sequencing and transcriptomics were used to identified key microbe related to the intestinal IgA response.Microbial transplantation was used to verify the impact of key differential microbes on gut mucosal immune responses and to elucidate the mechanism.Part Ⅰ Study on the correlation between chicken gut microbes and immune performance.In order to study the effect of gut microbiota on the intestinal immune performance of poultry.We selected commercialized AA and WLMB with strong intestinal immune performance as the research objects,to study its intestinal immune performance,intestinal transcriptomics,gut microbiota,serum immune level.Correlations between gut microbes and immune performance were analyzed.And screen the key microbiota that may promote the development of the intestinal immune system of commercial chickens.Compared with AA,WLMB had a higher concentration of immunoglobulin A(IgA)and higher proportion of IgA-coated microbiota in the intestinal contents.With IgA sequencing,it was found that WLMB intestinal IgA has higher diversity,bacterial affinity,and specificity.Transcriptome analysis revealed that AA and WLMB had significant differences in gut immune-related pathways.Through validation,compared with AA we found that the expression levels of Toll-like receptors,BAFF,TGF-β,and AID were higher in WLMB,and the phagocytic ability of antigen-presenting cells in the gut of WLMB was stronger.It was found by 16 S r DNA sequencing that the gut microbial diversity of WLMB was higher.Bacteroides were the key differential species between AA(0.03%)and WLMB(17.93%)and were highly associated with the IgA production pathway.Part Ⅱ Effects of Bacteroides on the immune performance of broilers.Bacteroides caecicola,Bacteroides uniformis and Bacteroides salanitronis were orally introduced into neonatal AA broilers for 21 days to evaluate the effect of Bacteroides on the intestinal and serum immune performance.The results showed that Bacteroides can increase the concentration of IgA in the intestinal contents by increasing the expression levels of IgA and poly-Ig receptor(PIg R),as well as up-regulates the expression levels of TNF family B cell activating factor(BAFF),interleukin(IL)-2 and IL-4 activation-induced cytidine deaminase(AID)in the duodenum.Bacteroides can significantly increase the levels of serum Ig G、IgA and Ig M.Modulate immune performance by increasing serum IL-2,IL-4 and IL-10 levels.Bacteroides can significantly improve serum total antioxidant capacity(Total antioxidant capacity,T-AOC).Among them,BC significantly increased the level of GSH-Px in chicken serum;BC and BS significantly increased the level of SOD in serum;BS significantly increased the level of CAT in serum.By treating macrophages with the equivalent SCFAs of Bacteroides fermentation medium,it was found that SCFAs significantly decreased the expression levels of pro-inflammatory factors IL-6 and significantly increased the expression levels of anti-inflammatory factors IL-10 and TGF-β.By treated IECs,it was found that SCFAs significantly inhibited the expression levels of pro-inflammatory factors IL-1β,IL-6 and Toll-like receptor TLR4,and increased the expression levels of IL-10,BAFF and My D88.By detecting the concentration of SCFAs in the intestinal contents,it was found that Bacteroides can significantly increase the concentration of isovaleric acid(IVA)in the small intestine.Part Ⅲ Effects of isovaleric acid on immune performance of broilersTo investigate the effect of IVA on AA broilers production,we treated AA broilers with IVA,used VNAM to establish a model lacking intestinal bacteria,and supplemented IVA to explore the effect of IVA on the development of the intestinal immune system.The results showed that IVA treatment did not increase the intestinal s IgA concentration,antibiotic treatment significantly decreased the intestinal s IgA concentration,while the addition of IVA could significantly increase the intestinal s IgA concentration and IgA expression level of VNAM-treated chickens.IVA significantly increased the diversity of gut microbes in broilers,increased the relative abundance of Clostridia vadin BB60 group,Defluviitaleaceae UCG-011,Lactobacillus,Ruminococcus,Bacillus,Anaeroplasma,Hydrogenoanaerobacterium,Parasutterella and decreased the relative abundance of Enterococcus and Streptococcus.Through the verification of key pathways,it was shown that IVA can significantly reduce the expression level of intestinal BAFF,and significantly inhibit intestinal inflammation;VNAM significantly reduced the expression levels of BAFF,IL-2,IL-4 and AID in the duodenum,adding IVA up-regulated the expression levels of the above genes.IVA significantly decreased the levels of IL-1 and IL-6 in serum,and increased the levels of IL-2,IL-4,IL-10 and TGF-β in serum.IVA,VNAM and VNAM+IVA all significantly increased ADG and decreased FCR.Part Ⅳ Effects of isovaleric acid on the immune properties of macrophages and IECsIVA significantly increased the expression levels of the alternative activated macrophage(M2)markers IL-10,Arg-2 and TGF-β,decreased the expression level of classical activated macrophage(M1)the marker i NOS,and significantly increased the expression levels of cytokine IL-4 and TGF-β and TLR signaling molecule My D88,enhanced phagocytic activity.Meanwhile.IVA significantly increased the expression levels of BAFF,TGF-β and My D88 in IECs.Cytokines secreted by macrophages and IECs stimulated by IVA activated gene expression of AID in lymphocyte and promoted IgA+ B cell generation.Mechanistically,IVA,the metabolite of Bacteroides,promoted the M2 polarization of macrophages through the IVA-mTOR-M2 polarization axis and elevated the expression level of several cytokines in the intestine,including IL-10,IL-4,TGF-β which induced the expression of AID to promote the production of IgA B cells.In conclusion: In this study,by analyzing the gut immunophenotype and gut microbiota of WLMB and AA,a key differential species-Bacteroides which related to the IgA production pathway was identified.Through microbial transplantation,the key role of Bacteroides on intestinal and serum immune levels,serum antioxidant levels were verified,and a differential metabolite-isovalerate in the small intestine was found.By animal experiment,it was found that IVA can significantly improve the intestinal and serum immune levels of chickens,improve serum antioxidant capacity,and improve production performance;at the same time,IVA regulates macrophages M2 polarization through the mTOR/PPAR-γ/STAT3 signaling pathway.