| Salmonellosis is a common zoonotic disease caused by Salmonella.In production,salmonellosis damaged the intestinal mucosal barrier of chickens,caused systemic inflammation and immune system disorders,led to slow growth and even death,thus seriously endangered the healthy production of chickens.Under the circumstance of"antibiotic-free",it has become a safer and more effective way to protect chickens against Salmonella infection through nutritional regulation.Anthocyanin(AC)is a flavonoid plant extract with antioxidant and anti-inflammatory activities.This study was conducted to investigate the effects of AC on the growth performance,immune function and intestinal health of yellow-feathered chickens challanged with Salmonella,thus to reveal the molecular mechanism of AC regulating the intestinal immune function to resist Salmonella infection to yellow-feathered chickens.In this experiment,300 1-day-old male yellow-feathered chickens were randomly allocated into 5 treatment groups with 6 replicates in each group and 10 chickens per replicate.The control group(CON)and the Salmonella infection group(ST)were fed basal diet,the antibiotic group(ST+ER)was supplemented with 200 mg/kg enramycin in the basal diet,and the low-or high-dose anthocyanin group(ST+ACL or ST+ACH)were supplemented with 100 mg/kg or 400 mg/kg AC in the basal diet,respectively.On the 14th and 16th day of trial,the birds in the ST,ST+ER,ST+ACL and ST+ACH were challenged with Salmonella typhimurium(10~9 CFU per chicken),and the CON was given intragastric administration with the same volume of PBS solution.The results were shown as follows:(1)Growth performance:Salmonella infection significantly reduced the body weight at 16,18,21,28(P<0.01)and 60(P<0.05)days,and significantly decreased ADG from 1 to 18,1 to28 and 1 to 60 days of age(P<0.01);Salmonella infection significantly increased F/G from 1 to 18,1 to 28(P<0.01)days of age and 1 to 60(P<0.05)days of age.Compared with the ST,supplementation with AC significantly increased the body weight of chickens challanged with Salmonella at 16(P<0.05),18(P<0.01),21(P<0.05)and 28(P<0.05)days of age,significantly increased ADG from1 to 18(P<0.01)and 1 to 28(P<0.05)days of age,significantly decreased F/G from1 to 18,1 to 28,29 to 60 and 1 to 60 days of age(P<0.05).Compared with the ST+ER,the body weight of chickens in the AC group increased significantly at 16 and 18 days of age(P<0.05).(2)Bacterial load and immune organ index:Salmonella infection significantly increased the number of Salmonella in the liver and spleen of chickens(P<0.01),significantly increased liver index(P<0.01)and spleen index(P<0.05),and significantly decreased the bursal of Fabricius index(P<0.05).Compared with the ST,supplementation with AC significantly reduced the number of Salmonella in the liver and spleen of chickens challenged with Salmonella(P<0.01),significantly decreased the liver index and spleen index(P<0.05),and significantly increased the bursal of Fabricius index(P<0.05).(3)Plasmal biochemical variables:Salmonella infection significantly increased the plasmal diamine oxidase(DAO)activity and nitric oxide(NO)content of chickens(P<0.01),significantly increased the plasmal Ig G,IL-8,IFN-βcontent(P<0.01)and Ig A,IL-1β,TNF-α,IFN-γcontent(P<0.05).Compared with the ST,supplementation with AC significantly decreased the plasmal DAO activity and NO content in chickens challenged with Salmonella(P<0.05),significantly decreased plasmal IFN-βcontent(P<0.01)and IL-1β,IL-8 content(P<0.05).The plasmal Ig G,Ig A and IFN-γcontents were significantly decreased by dietary addition of 400 mg/kg AC(P<0.05).(4)Intestinal mucosal barrier function:Salmonella infection led to rupture and shedding of villi in the jejunum and ileum of chickens,which significantly decreased the villus height and the ratio of villus height to crypt depth(V/C,Villus height/Crypt depth)(P<0.05),significantly increased the depth of ileal crypts(P<0.01),significantly decreased the number of jejunal goblet cells(P>0.05);Salmonella infection significantly decreased the expression of Occludin(P<0.05)and Claudin-1(P<0.01)in the jejunal mucosa,significantly decreased the expression of ZO-1,Occludin(P<0.05)and Claudin-1(P<0.01)in the ileal mucosa.Compared with the ST,supplementation with AC significantly increased the height of the jejunum and ileum villi and the number of goblet cells in the jejunum of chickens challanged with Salmonella(P<0.01),the jejunal V/C and the expression levels of Claudin-1 and Muc2 were significantly increased by dietary addition of 100 mg/kg AC(P<0.05),the crypt depth of the ileum(P<0.05),the V/C of the jejunum and ileum(P<0.01),and the expression levels of ZO-1,Occludin were significantly increased(P<0.05)by dietary addition of 400 mg/kg AC.(5)Intestinal mucosal immune function:Salmonella infection significantly increased the jejunal mucosal TNF-α,IFN-γand ileal mucosal Ig G,Ig M,IL-8,IFN-βcontents of chickens(P<0.05),significantly increased the content of s Ig A,IL-1β,IL-6,TNF-αand IFN-γin the ileal mucosa(P<0.01),significantly decreased the expression of IL-1β,TLR2,NF-κB and My D88 in the ileal mucosa(P<0.05),significantly decreased the expression of IL-2,TNF-α,TLR2,TLR4 in the jejunal mucosa and IL-2,TLR4 in the ileal mucosa(P<0.01),the expression of IL-6 in the jejunal mucosa was significantly increased(P<0.01).Compared with the ST,supplementation with AC significantly decreased the jejunal mucosa Ig G,Ig M,IL-1β,IL-6,IL-8,TNF-α,IFN-βcontents and ileal mucosa IL-1β,IL-6,TNF-α,IFN-γcontents(P<0.01),in the ST+ACL,the jejunal mucosa s Ig A and the ileal mucosa Ig G,Ig M,s Ig A,IL-8,IFN-βcontents were significantly decreased(P<0.01),the expression levels of IL-2,TNF-α(P<0.01)and IL-1β,TLR4(P<0.05)in the jejunum mucosa were significantly increased,the expression levels of IFN-γin the jejunum mucosa were significantly decreased(P<0.05),the expression levels of IL-2,TLR4,NF-κB,My D88 in the ileal mucosa were significantly increased(P<0.01),in the ST+ACH,the jejunal mucosa s Ig A and the ileal mucosa Ig M,s Ig A,IL-8,IFN-βcontents were significantly decreased(P<0.05),the expression levels of IL-2,TNF-α,TLR4(P<0.01)and TLR2(P<0.05)in the jejunum mucosa were significantly increased,the expression level of NF-κB in the jejunum mucosa was significantly decreased(P<0.05),the expression level of IL-2(P<0.05)and TLR4,My D88(P<0.01)in the ileal mucosa were significantly increased.(6)Intestinal flora:Salmonella infection affected the composition of the caecal flora in chickens,resulted in decline the diversity of the flora.At the phylum level,Salmonella infection significantly decreased the relative abundance of Firmicutes(P<0.01),significantly increased the relative abundance of Bacteroidetes(P<0.01)and Proteobacteria(P<0.05);at the genus level,Salmonella infection significantly increased the relative abundance of Bacteroides(P<0.01),significantly increased the relative abundance of Escherichia-Shigella and Enterococcus(P<0.05),the relative abundance of Lactobacillus and Lachnospiraceae were significantly decreased(P<0.05).Supplementation with AC improved the diversity and composition of the caecal flora.At the phylum level,supplementation with AC significantly increased the relative abundance of Firmicutes in the cecum of chickens challenged with Salmonella(P<0.01),significantly decreased the relative abundance of Bacteroidetes(P<0.01)and Proteobacteria(P<0.05);at the genus level,supplementation with AC significantly increased the relative of abundance Lactobacillus,Clostridia and Lachnospiraceae in the caecum of chickens challenged with Salmonella(P<0.05),significantly decreased the relative abundance of Enterococcus,Escherichia-Shigella(P<0.05)and Bacteroides(P<0.01).In summary,AC inhibited the colonization of Salmonella in the liver and spleen,and alleviated the decline in the growth performance of yellow-feathered chickens challenged with Salmonella;dietary AC supplementation decreased the secretion of pro-inflammatory cytokines and immunoglobulins in the plasma,jejunum and ileum mucosa of chickens challenged with Salmonella,and decrease the expression of pro-inflammatory cytokines in the jejunal and ileal mucosa,thereby inhibited the inflammatory damage in intestine of chickens.AC played a role in resisting Salmonella infection and maintaining intestinal health by increased the expression of gene related to intestinal mucosal barrier and TLR pathway,and regulating the diversity and composition of the cecal flora.Based on the results of this experiment,100 mg/kg AC(the active ingredient was 61 mg/kg)in diet was recommended to broiler. |
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