Study On The Probiotic Mechanism Of Lactobacillus Acidophilus On Broilers

In poultry,pathogens infection leads to a severe immunity issue and a considerable burden to farmers worldwide,in terms of decreased animal welfare and production economy.Infected chickens and contaminated poultry meat or egg are known to be the most important carriers of pathogens threatening human health.In order to keep the health intestinal status and improve the growth performance of animals without using antibiotics,there is an increasing demand to seek safety,effective and economic alternatives to replace feed antibiotics to reduce intestinal damage caused by pathogens infection and oxidative stress.Lactobacillus is an important part of broiler intestinal immune system,which can improve the intestinal barrier function and immunity of broilers by promoting intestinal flora balance.Lactobacillus showed significant advantages in the regulation of animal intestinal health in the previous research.In this study,severn experiments were conducted to explore the probiotic effect of Lactobacillus acidophilus(L.acidophilus)and its mechanism to alleviate the intestinal inflammatory response in broiler chickens in vivo and in vitro levels.In Experiment 1,we evaluated the antimicrobial properties of ten selected probiotics against the pathogens Escherichia coli O157 and Salmonella pullorum in vitro.L.acidophilus E were selected with better antibacterial effect and then explored its antibacterial substances and effect on broiler feeding.The production of lactic acid from Lactobacillus acidophilus E metabolites increased linearly with incubation time and its concentration was linearly correlated with the ability of L.acidophilus E to inhibit E.coli(R~2=0.9780)and Salmonella(R~2=0.9714).This experiment evidenced that L.acidophilus has no hemolysis and were sensitive to most antibiotics.When fed to broilers,L.acidophilus increased the average daily gain(P<0.05),and tend to increase the body weight(P=0.09)of broilers during d1?14.The serum results showed that L.acidophilus E reduced glucose,total cholesterol,urea nitrogen(P>0.05)and triglyceride(P<0.05)levels at d21.In experiment 2,we evaluated the effect of solid fermented feed of L.acidophilus E on growth performance and intestinal development of broilers according to a 2×4 factorial arrangement with fermented feed(pelletize or unpelletize)and additional levels(0,5%,10%,15%)and its interaction effects.Results showed that broiler fed diet with 10%fermented feed increased the ADG and ADFI during d 1-42(P<0.05).Fermented feed increased the total length and weight of the small intestine of broiler on d 42.The jejunal villi height and villi height/crypt depth(V/C)ratio were increased in fermented feed groups,which also showed improved nutrients digestibility.In addition,fermented improved the intestinal barrier and immune function with downregulated inflammatory factor of IL-6,IL-8 and i NOS and upregulated jejunal barrier function proteins Occludin and Claduin-1 m RNA expression,improved broilers intestinal immune function and barrier function.In experiment 3,we evaluated the effects of feeding L.acidophilus on the growth performance and immune response of broilers challenged with E.Coli O157 according to a 3×2 factorial arrangement with three dietary L.acidophilus levels(0,5×10~8CFU/kg,and 10×10~8CFU/kg of diet)and two disease challenge statuses(control or E.Coli challenged).Results showed that E.Coli challenge decreased the ADG,ADFI,and BW of broilers during d 15-21,and decreased CD3+T cell counts and serum Ig A,Ig M,and Ig G level on d 21.Dietary L.acidophilus supplementation showed higher ADG and ADFI during d 1-14 and 15-21,and showed a tendency to reduce the mortality and increased the CD3+T cell counts in E.Coli infection birds.In addition,both 5×10~8CFU/kg and 10×10~8CFU/kg levels of L.acidophilus E significantly inhibited the upregulation of jejunal NF-?B,TNF-?and IL-8 m RNA expression in E.coli infected broilers at 14 days of age(P<0.05),and upregulated the expression of the inflammatory factor IL-10;L.acidophilus E downregulated the expression of spleen i NOS and jejunum IL-8 m RNA expression in E.coli infected broilers at d 21.Dietary L.acidophilus supplementation improved broiler performance and immune performance and alleviated the inflammatory response caused by E.coli infection.In experiment 4,we evaluated the regulating effect of L.acidophilus on the intestinal development and intestinal barrier function of broilers challenged with E.Coli O157 based on the results of experiment3.Results showed that E.Coli challenge significantly increased the serum concentration of CRP,DAO and LPS(P<0.05)over the whole infection period(d 14 and d 21,P<0.05),and decreased the total length of small intestine on d 21(P<0.10).E.Coli challenge downregulated the expression of jeunum Occludin and Claudin-1 m RNA expression at d 14.Dietary supplement with 5×10~8CFU/g L.acidophilus reduced the serum CRP,DAO and LPS concentration in E.coli infected broilers.Also,the m RNA expression of Occludin and Claudin-1 in jejunum at d 14 and 21 and Occludin,ZO-1 and Claudin-1 in ileum at d 21 was upregulated compared with E.coli infected birds.L.acidophilus supplement alleviates the inflammatory reaction and intestinal damage caused by E.coli infection.In experiment 5,we evaluated the effect of L.acidophilus E on the ileal microflora of broilers challenged with E.Coli O157.E.coli infection increased the relative abundance of pathogenic bacteria such as Proteobacteria?Streptococcus?Enterococcus?Escherichia-Shigella and Staphylococcus(P<0.05),while L.acidophilus E intervention significantly reduced the relative abundance of these pathogens in the E.coli infection broilers.Both L.acidophilus E and E.coli infections increased the microbial Ace and Chao indices of ileal chyme in broilers at d 21 days.L.acidophilus E and E.coli infections reduced the abundance of the Firicutes and increased the abundance of the broiler ileal cyanobacteria,Bacteroides and Actinobacteria(P<0.05).L.acidophilus E improves intestinal flora disorders caused by E.coli infections.In experiment 6,the effect of L.acidophilus E on reducing intestinal damage of broilers infected with E.coli was studied based on Lab-free proteomics technique.Results showed that E.coli infection upregulates blood coagulation?hemostasis?wound healing?regulation of body fluid levels?response to wounding?defense response to bacterium?cell death and necroptosis biological processes and initiation immune response in broilers through PPAR and NOD signaling pathway.L.acidophilus E intervention upregulated the expression of Pyrin-like,lysozyme,histone and ribosomal proteins in the ileum of E.coli infected broilers.L.acidophilus E reduced the level of molecular transcription and related gene silencing regulation in the broiler intestine,blocked the signaling of pathogens in the inflammatory response.Also,dietary with L.acidophilus E down-regulated coagulation,hemostasis,wound healing,regulation of body fluid levels,response to injury,redox processes and stress responses,and other unfavorable biological processes in the ileum of broiler chickens infected with E.coli,mitigate the damage to broiler intestine caused by E.coli infection.In Experiment 7,an in vitro study was to investigated the mechanism of L.acidophilus E on the inflammatory response of E.coli induced Caco-2 cells.E.coli infection induced a significant inflammatory reaction in Caco-2 cells with multiplicity of infection is 1(MOI=1)and duration at 4 h post infection.E.coli significantly upregulated the m RNA expression of inflammatory factors IL-6,IL-8 and TNF-?in Caco-2 cells by recognizing the TLR-4/My D88 dependent pathway to activate the NF-?B signaling pathway(P<0.05).E.coli infection significantly upregulated the expression of Caspase-3,Capase8 and P53-R m RNAs,key proteins that regulate the apoptotic process(P<0.05),and increased the number of necrotic cells.Advance intervention of L.acidophilus E and its supernatant reduced the adhesion of E.coli on Caco-2 cells(P<0.05),alleviated the inflammatory response to E.coli infection,reduced the number of necrotic cells and decreased the permeability of Caco-2monolayers,which had a protective effect on the cells.In conclusion,L.acidophilus E could promote the growth performance and intestinal health of broilers.E.coli infection produces an inflammatory response through activation of the NF-?B signaling pathway via a TLR-4/My D88 dependent pathway,increases the biological process in intestinal.L.acidophilus E intervention alleviated the inflammatory response and intestinal damage caused by E.coli infection in terms of reducing adhesion,improving intestinal flora structure and mechanical barrier,immunologic barrier,and promoting intestinal development.