| There is a complex relationship between microbiota and immune system.Gut microbiota,through pattern recognition receptors(PRRs),can modulate the expression of genes involved in inflammatory responses.Furthermore,gut microbiota exerts an influence on gut health via microbial metabolism products of proteins,carbohydrates and lipids.During the early-life period,the composition and diversity of microbiota is unstable and highly influenced by environmental conditions.In piglets,the intestine is still immature and relatively permeable to antigens.In this situation,toxins,allergenic compounds,or even bacteria may enter into bloodstream,resulting in inflammatory or immunologic responses.In-feed antibiotic use is a common practice for improving growth performance in pig farm of China,especially for piglets.Previous studies have shown that antibiotics can increase opportunities for pathogenic organisms to colonize and suppresses the host's innate immune defenses.Change in dietary protein level may affect the bacterial composition in gut.Low-protein amino acid-balanced diet(LPD)modifies bacterial communities in the gut with an increase in generally beneficial bacteria counts and a decrease in potential pathogenic Escherichia coli counts and reduces protein fermentation products,indicating that LPD may benefit gut health.Whether LPD dominates EAI in regulating gut health after antibiotic cease or not.The mammalian gastrointestinal tract harbors a complex and dense microbiota,especially in hindgut.However,little is known about effect of the changes in hindgut bacteria with antibiotic intervention on small intestinal health.This study aimed to reveal the role of intestinal microbiota on intestinal mucosal immunity by changing bacterial composition through through in feed antibiotics,feeding different protein levels diets,antibiotic infusion at the terminal ileum.1 Effects of early antibiotic intervention on gut bacteria and cecal bacterial metabolites in pigletsThe study investigated the effects of early antibiotic intervention(EAI)on gut bacteria and cecal bacterial metabolites.Eighteen litters of piglets on day(d)7 were fed either a commercial creep feed or commercial creep feed + antibiotic(Olaquindox,Oxytetracycline Calcium,Kitasamycin,50 mg/kg)until d 42.At d 42,8 litters were randomly selected in each group,and one pig of each litter was randomly slaughtered.Cecal samples were used for high-throughput sequencing and fermentation products analysis.The microbial community in the jejunum,ileum and colon was analyzed real-time quantitative analysis(RT-PCR).The results showed that EAI altered the gut bacteria by decreasing short-chain fatty acids(SCFA)producing bacteria(Clostridium cluster IV and Clostridium cluster ??a in the ileum and colon;Roseburia,Lachnospira pectinoschiza and Clostridium leptum in the cecum)and Lactobacillus(in the ileum and colon)(P<0.05).For cecal fermentation products,EAI altered bacterial fermentation patterns by increasing protein fermentation products(ammonia,putrescine,cadaverine,p-cresol and indole)and decreasing carbohydrate fermentation products(totel SCFA,acetate,propionate and butyrate).These results indicate EAI reduced generally beneficial SCFA-producing bacteria,Lactobacillus and SCFA while increasing potentially detrimental protein fermentation products,which may affect the gut health in piglets.2 Effects of early antibiotic intervention on intestinal mucosal and serum immune status in pigletsBased on the animal trial in chapter 1,jejunal,ileal and colonic mucosa and serum were collected for analyzing immune index using RT-PCR and ELISA.Results showed that EAI down-regulated(P<0.05)toll-like receptor(TLR2)mRNA expression in the ileum and colon.EAI decreased(P<0.05)intestinal mucosal cytokines mRNA and protein levels[interferon-?(IFN-?)in the jejunum;interleukin-10(IL-10)and tumor necrosis factor-?(TNF-?)in the ileum;IL-8,IL-10,IFN-? and TNF-? in the cecum;IL-10 and IFN-? in the colon].EAI also down-regulated(P<0.05)mucin-2 in the ileum and colon.Furthermore,EAI significantly decreased(P<0.05)intestinal mucosal immunoglobulin protein level[secretory immunoglobulin A(SIgA)in the jejunum;SIgA and IgG in the ileum;SIgA in the cecum;IgG in the colon].For serum immune index,pigs in antibiotic group decreased(P<0.05)concentrations of pro-inflammatory cytokines(IL-8 and INF-?)and immunoglobulin(IgG and IgA).Our findings suggest that EAI suppressed mucosal and serum immunity of piglets,which affected immune homeostasis.3 Effects of early antibiotic intervention on subsequent gut bacterial communities and their metabolic profiles in growing pigsThe study investigated the effects of EAI on subsequent intestinal bacterial communities and their metabolic profiles in pigs.Eighteen litters of piglets on d 7 were fed either a commercial creep feed or commercial creep feed + antibiotic(Olaquindox,Oxytetracycline Calcium and Kitasamycin)until d 42.At d 42,pigs within each group were further randomly fed a normal crude protein diet(NPD)(20%and 18%CP from d 42 to d 77 and d 77 to d 120,respectively)or a low protein amino acid-balanced diet(LPD)(16%and 14%CP from d 42 to d 77 and d 77 to d 120,respectively),generating 4 groups,Control-Low CP(Con-LP),Control-normal CP(Con-NP),Antibiotic-Low CP(Ant-LP)and Antibiotic-Normal CP(Ant-NP),respectively.At d 77 and d 120,5 pigs per group were slaughtered,and jejunal,ileal,cecal and colonic materials were collected for bacterial analysis.Results showed EAI increased potential pathogenic Escherichia coli counts in the jejunum and ileum at d 77(P<0.05),decreased generally beneficial bacteria counts(Bifidobacterium in the jejunum and ileum at d 77;Clostridium cluster ??a in the jejunum,ileum and colon at d 120)(P<0.05).LPD modified microbial communities with an increase in generally beneficial bacteria counts(Bifidobacterium,Clostridium cluster IV,Clostridium cluster ??a and Lactobacillus)and a decrease in potential pathogenic Escherichia coli counts in the gut of pigs at d 77 and d 120(P<0.05).For metabolites in the large intestine,EAA increased(P<0.05)cecal protein fermentation products(p-cresol and skatole at d 77;putrescine and spermidine at 120)under LPD and colonic aromatic amino acid fermentation(p-cresol and indole at d 77)under NPD.LPD increased SCFA(propionate and butyrate at d 77),reduced protein fermentation products(ammonia,phenol and indole at d 77;branched chain fatty acid(BCFA),ammonia,tyramine,cadaverine,phenol and indole at d 120)in the cecum and colon of pigs(P<0.05).These results indicate that EAI increased potential pathogenic Escherichia coli counts in the small intestine of growing pigs in short term,decreased generally beneficial bacteria counts.EAI increased protein fermentation in the large intestine under different CP level diets.LPD increased generally beneficial bacteria and SCFA while reducing potentially detrimental Escherichia coli and protein fermentation products,which may benefit gut health in growing pigs.4 Effects of early antibiotic intervention on subsequent intestinal mucosal and serum immune status in growing pigsAlteration of gut microbiota and its metabolite may affect intestinal musocal immune response.Based on the animal trial in chapter 3,jejunal,ileal and colonic mucosa and serum were collected for immune index using RT-PCR and ELISA.Results showed that EAI decreased(P<0.05)IL-10 mRNA and protein levels in the jejunum and ileum.EAI decreased(P<0.05)colonic mucosal pro-inflammatory cytokines(IL-8 and IFN-?)mRNA and protein levels under NPD at d 77(P<0.05).Furthermore,EAI decreased intestinal mucosal immunoglobulin protein level(SIgA and IgG in the ileum and IgG in the colon)at d 77.LPD down-regulated TLR4,myeloid differentiating factor 88(MyD88)and nuclear factor-?B p65(NF-?B p65)mRNA expressions in the colon.LPD decreased(P<0.05)intestinal mucosal pro-inflammatory cytokines mRNA and protein levels(IL-8 and TNF-a in the ileum and IFN-y in the colon at d 77;IL-8 and IFN-? in the jejunum,TNF-?in the ileum and IL-8 and TNF-a in the colon at d 120).LPD also up-regulated(P<0.05)intestinal mucosal barrier function gene(ZO-1 in the jejunum;mucin-2 and occludin in the ileum)at d 120.Furthermore,LPD significantly increased(P<0.05)intestinal mucosal immunoglobulin protein level(SIgA in the jejunum and colon at d 77;IgG in the jejunum at 120).For serum immune index,EAI decreased(P<0.05)IgA concentration at d 77.At d 120,LPD decreased IL-8 concentration(P<0.05),and increased IgA concentration(P<0.05).Furthermore,reductions of Escherichia coli under LPD were positively correlated with down-regulated gene expressions of pro-inflammatory cytokines(P<0.05).In the colon,down-regulated gene expressions of pro-inflammatory under LPD were also positively correlated with down-regulated TLR4-MyD88-NF-?B signaling pathway and reductions of ammonia(P<0.05).In conclusion,EAI affected intestinal immune function of growing pigs in the short term,not long term.LPD had beneficial effects on immune function of growing pigs,with more profound effect in the long term.5 Effects of ileum terminal antibiotic infusion on gut bacterial communities and immune status in the growing pigsThe bacteria in the large intestine,which may be involved in nutrient metabolism and the maintenance of immunological functions,is specific and complex.However,little is known about effect of the changes in hindgut bacteria with antibiotic intervention on small intestinal health.The present study aimed to investigate the effects of ileum terminal antibiotic infusion(ITAI)on bacterial composition and mucosal immune response in the jejunum and colon,and serum immune index of pigs.Twelve DurocŚLandraceŚLarge barrows(45 days of age,12.08 ± 0.28 kg)fitted with a T-cannula at the terminal ileum,were randomly assigned to two groups and infused either saline(10 mL/d)or saline wtih antibiotics(ampicillin at 150 mg/kg/day,gentamicin at 4 mg/kg/day and metronidazole at 30 mg/kg/day)at the terminal ileum.After 25 days' experiment,six pigs from each group were euthanized to collect serum and intestinal samples.Results showed ITAI altered dominant bacteria counts in jejunal and colonic contents.This alteration was characterized by a decrease in beneficial Bifidobacterium,Clostridium cluster IV and Clostridium cluster IV in colon(P<0.05),and an increase in potential pathogenic Escherichia coli in jejunum(P<0.05).ITAI decreased(P<0.05)SCFA concentration(totel SCFA,propionate,butyrate and isobutyrate)in the colon.For mucosal immunity,ITAI suppressed immune responses in the jejunum and colon.In the colon,ITAI significantly decreased(P<0.05)the concentrations of(IL-8,IL-10 and sIgA),and dwonregulated(P<0.05)the mRNA expression levels of IL-8,IL-10,mucin-1,mucin-2 and ZO-1 in the colon.In the jejunum,from antibiotic group,we observed the decreased concentrations of IFN-?,TNF-?,sIgA and IgG(P<0.05)together with down-regulation of IFN-?,TNF-?,mucin-2 and ZO-1 mRNA levels(P<0.05).ITAI suppressed systemic immune responses with a decrease in serum concentrations of pro-inflammatory cytokines(IL-10,INF-? and TNF-?)and immunoglobulin(IgA and IgG)(P<0.05).Our findings suggest that ITAI altered colonic intestinal microbiota,increased opportunities for pathogenic potential pathogenic Escherichia coli to colonize in the jejunum,and suppressed mucosal and serum immune responses in growing pigs.As stated above,(1)EAI reduced generally beneficial SCFA-producing bacteria,Lactobacillus and SCFA while increasing potentially detrimental protein fermentation products,which may affect the gut health in piglets.(2)EAI suppressed mucosal and serum immunity of piglets by decreasing cytokines and immunoglobulin,which affected immune homeostasis.(3)EAI increased potential pathogenic Escherichia coli counts in the small intestine of growing pigs in short term,decreased generally beneficial bacteria counts.EAI increased protein fermentation in the large intestine under different CP level diets.LPD increased generally beneficial bacteria and SCFA while reducing potentially detrimental Escherichia coli and protein fermentation products,which may benefit gut health in growing pigs.(4)EAI affected intestinal immune function of growing pigs in the short term,not long term.LPD had beneficial effects on immune function of growing pigs,with more profound effect in the long term.(5)ITAI altered colonic intestinal microbiota,increased potential pathogenic Escherichia coli counts in the jejunum,and affected mucosal and serum immune responses in growing pigs. |
PDF Full Text Request