Ileum terminal antibiotic infusion affects jejunal and colonic specific microbial population and immune status in growing pigs

Background: Compared with oral antibiotics(primarily disrupt foregut microbiota), the present study used antibiotics with ileum terminal infusion to disrupt the hindgut microbiota, and investigated the changes in specific bacterial composition and immune indexes in the jejunum and colon, and serum of growing pigs. Twelve barrows(45 d 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 without antibiotics(Control) or with antibiotics(Antibiotic) at the terminal ileum. After 25 d experiment, all pigs were euthanized for analyzing bacterial composition and immune status.Results: Ileum terminal antibiotic infusion(ITAI) altered dominant bacteria counts, with a decrease in Bifidobacterium, Clostridium cluster IV and Clostridium cluster IV in the colon(P < 0.05), and an increase in Escherichia coli in the jejunum(P < 0.05). ITAI decreased(P < 0.05) short-chain fatty acids concentrations in the colon. ITAI decreased interleukin-8(IL-8), IL-10 and secretory immunoglobulin A(sIgA) concentrations, and down-regulated IL-10, Mucin-1(MUC1), Mucin-2(MUC2) and zonula occludens-1(ZO-1) mRNA expression in the colonic mucosa(P < 0.05). In the jejunal mucosa, ITAI decreased interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α), s IgA and IgG levels together with down-regulation of IFN-γ, TNF-α, MUC2 and ZO-1 mRNA expression(P < 0.05). Furthermore, ITAI decreased IL-10, INF-γ, TNF-α, IgA and IgG concentrations in serum(P < 0.05). Correlation analysis revealed that the change in intestinal microbiota was correlated with alterations of Ig and cytokines.Conclusions: ITAI affected jejunal and colonic specific bacteria counts, and altered some immune markers levels in the jejunal and colonic mucosa and serum. These findings implicate the potential contribution of hindgut bacteria to immune response in the intestinal mucosa and serum of growing pigs.

Diversity and community pattern of sulfate-reducing bacteria in piglet gut

Background: Among the gut microbiota,sulfate-reducing bacteria(SRB) is a kind of hydrogen-utilizing functional bacteria that plays an important role in intestinal hydrogen and sulfur metabolism.However,information is lacking regarding diversity and community structure of SRB in the gut of piglets.Middle cecum contents were collected from 6 Yorkshire and 6 Meishan piglets at postnatal days(PND) 14,28 and 49.Piglets were weaned at PND28.Real-time quantitative PCR was performed to detect the number of SRB in the cecum based on dissimilatory sulfite reductase subunit A(dsrA) gene.Prior to real-time PCR,plasmid containing the dsrA gene was constructed and used as external standard to create a standard curve,from which the gene copies of dsrA were calculated.H2S concentration in the cecal contents was measured.Illumina PE250 sequencing of dsrA gene was used to investigate SRB diversity in cecum contents.Results: The qPCR results showed that the number of SRB at PND49 was significantly higher than that at PND28 in Meishan piglets.The concentration of H2S has no significant difference between piglet breeds and between different ages.The Illumina sequencing analysis revealed that the Chao1 richness index was significantly higher at PND49 than that at PND14 and PND28 in Yorkshire piglets.Based on dsrA gene similarities,Proteobacteria,Actinobacteria,and Firmicutes were identified at the phylum level,and most sequences were classified as Proteobacteria.At the genus level,most of sequences were classified as Desulfovibrio.At the species level,Desulfovibrio intestinalis was the predominant SRB in the piglet cecum.The relative abundance and the inferred absolute abundance of Faecalibacterium prausnitzii at PND49 were significantly higher than that at PND14 in Yorkshire piglets.Pig breeds did not affect the dsrA gene copies of SRB,diversity index and community pattern of SRB.Conclusions: Sulfate-reducing bacteria are widely colonized in the cecum of piglets and D.intestinalis is the dominant SRB.The age of piglets,but not the pig breeds affects the diversity and community pattern of SRB.

Long-term effects of early antibiotic intervention on blood parameters,apparent nutrient digestibility, and fecal microbial fermentation profile in pigs with different dietary protein levels

Backgroud： This study aimed to determine the effects of early antibiotic intervention（EAI） on subsequent blood parameters, apparent nutrient digestibility, and fecal fermentation profile in pigs with different dietary crude protein（CP） levels. Eighteen litters of piglets（total 212） were randomly allocated to 2 groups and were fed a creep feed diet with or without in-feed antibiotics（olaquindox, oxytetracycline calcium and kitasamycin） from postnatal d 7 to d 42. On d 42, the piglets within the control or antibiotic group were mixed, respectively, and then further randomly assigned to a normal-（20%, 18%, and 14% CP from d 42 to d 77, d 77 to d 120, and d 120 to d 185,respectively） or a low-CP diet（16%, 14%, and 10% CP from d 42 to d 77, d 77 to d 120, and d 120 to d 185,respectively）, generating 4 groups. On d 77（short-term） and d 185（long-term）, serum and fecal samples were obtained for blood parameters, microbial composition and microbial metabolism analysis.Results： EAI increased（P 〈 0.05） albumin and glucose concentrations in low-CP diet on d 77, and increased（P 〈 0.05） urea concentration in normal-CP diet. On d 185, EAI increased（P 〈 0.05） globulin concentration in normal-CP diets, but decreased glucose concentration. For nutrient digestibility, EAI increased（P 〈 0.05）digestibility of CP on d 77. For fecal microbiota, the EAI as well as low-CP diet decreased（P 〈 0.05） E. coli count on d 77. For fecal metabolites, on d 77, EAI decreased（P 〈 0.05） total amines concentration but increased skatole concentration in low-CP diet. On d 185, the EAI increased（P 〈 0.05） putrescine and total amines concentrations in low-CP diets but reduced（P 〈 0.05） in the normal-CP diets. The low-CP diet decreased the concentrations of these compounds.Conclusions： Collectively, these results indicate that EAI has short-term effects on the blood parameters and fecal microbial fermentation profile. The effects of EAI varied between CP levels, which was characterized by the significant alteration of glucose and putrescine concentration.

The effect of increased atmospheric temperature and CO_2 concentration during crop growth on the chemical composition and in vitro rumen fermentation characteristics of wheat straw

This experiment was conducted to investigate the effects of increased atmospheric temperature and CO2 concentration during crop growth on the chemical composition and in vitro rumen fermentation characteristics of wheat straw. The field experiment was carried out from November 2012 to June 2013 at Changshu （31°32′93″N, 120°41′88″E） agro-ecological experimental station. A total of three treatments were set. The concentration of CO2 was increased to 500 pmol/mol in the first treatment （CO2 group）. The temperature was increased by 2℃ in the second treatment （TEM group） and the concentration of CO2 and temperature were both increased in the third treatment （CO2 ＋ TEM group）. The mean temperature and concentration of CO2 in control group were 10.5 ℃ and 413μmol/mol. At harvesting, the wheat straws were collected and analyzed for chemical composition and in vitro digestibility. Results showed that dry matter was significantly increased in all three treatments. Ether extracts and neutral detergent fiber were significantly increased in TEM and CO2 ＋ TEM groups. Crude protein was significantly decreased in CO2＋TEM group. In vitro digestibility analysis of wheat straw revealed that gas production was significantly decreased in CO2 and CO2 ＋ TEM groups. Methane production was significantly decreased in TEM and CO2 ＋ TEM groups. Ammonia nitrogen and microbial crude protein were significantly decreased in all three treatments. Total volatile fatty acids were significantly decreased in CO2 and CO2 ＋ TEM groups. In conclusion, the chemical composition of the wheat straw was affected by temperature and CO2 and the in vitro digestibility of wheat straw was reduced, especially in the combined treatment of temperature and CO2.

Regulation of serotonin production by specific microbes from piglet gut

Background Serotonin is an important signaling molecule that regulates secretory and sensory functions in the gut.Gut microbiota has been demonstrated to affect serotonin synthesis in rodent models.However,how gut microbes regulate intestinal serotonin production in piglets remains vague.To investigate the relationship between microbiota and serotonin specifically in the colon,microbial composition and serotonin concentration were analyzed in ileum-cannulated piglets subjected to antibiotic infusion from the ileum when comparing with saline infusion.Microbes that correlated positively with serotonin production were isolated from piglet colon and were further used to investi-gate the regulation mechanisms on serotonin production in IPEC-J2 and a putative enterochromaffin cell line RIN-14B cells.Results Antibiotic infusion increased quantities of Lactobacillus amylovorus(LA)that positively correlated with increased serotonin concentrations in the colon,while no effects observed for Limosilactobacillus reuteri(LR).To understand how microbes regulate serotonin,representative strains of LA,LR,and Streptococcus alactolyticus(SA,enriched in feces from prior observation)were selected for cell culture studies.Compared to the control group,LA,LR and SA supernatants significantly up-regulated tryptophan hydroxylase 1(TPH1)expression and promoted serotonin production in IPEC-J2 cells,while in RIN-14B cells only LA exerted similar action.To investigate potential mechanisms mediated by microbe-derived molecules,microbial metabolites including lactate,acetate,glutamine,andγ-aminobutyric acid were selected for cell treatment based on computational and metabolite profiling in bacte-rial supernatant.Among these metabolites,acetate upregulated the expression of free fatty acid receptor 3 and TPH1 while downregulated indoleamine 2,3-dioxygenase 1.Similar effects were also recapitulated when treating the cells with AR420626,an agonist targeting free fatty acid receptor 3.Conclusions Overall,these results suggest that Lactobacillus amylovorus showed a positive correlation with sero-tonin production in the pig gut and exhibited a remarkable ability to regulate serotonin production in cell cultures.These findings provide evidence that microbial metabolites mediate the dialogue between microbes and host,which reveals a potential approach using microbial manipulation to regulate intestinal serotonin biosynthesis.

微生物-肠-脑轴的研究进展

肠道微生物群能够调节宿主肠道稳态,同时参与调节宿主神经系统功能和行为。肠道菌群失调可能导致宿主神经系统功能障碍,从而引发神经退行性疾病。因此,研究微生物在肠-脑轴中发挥的作用及其机制,靶向调控肠道微生物菌群结构和功能,将为神经系统疾病的诊断与治疗提供新的手段。近年来,有关肠道微生物与机体神经系统间的互作研究受到了广泛关注,然而其具体的调控机制还未明晰。因此,本文综述了肠道微生物对宿主神经健康的调节作用,以及肠道微生物与宿主间的互作在调节神经功能、行为的潜力等研究进展,为更好地了解肠道微生物在调控宿主神经系统功能和行为的作用机制提供参考。

肠道微生物与线粒体之间的互作

肠道微生物与肠道细胞线粒体功能之间的关系十分密切。一方面,肠道微生物可直接或通过短链脂肪酸、硫化氢和一氧化氮等代谢产物间接影响与线粒体相关的能量代谢过程,调节线粒体活性氧的产生,调控线粒体甚至整个机体的免疫反应。另一方面,肠道细胞线粒体功能紊乱和基因组的遗传变异也会影响肠道微生物的组成和功能。本文主要介绍了肠道微生物和线粒体之间的互作关系的最新研究进展,为靶向作用于肠道菌群和线粒体以调节肠道健康提供理论依据。

一株猪肠道Lactobacillus animalis LGM对结肠炎小鼠Th细胞分化转录因子基因表达的影响

【目的】从体外和体内研究Lactobacillus animalis LGM对Th细胞分化转录因子T-bet、GATA-3、ROR-γt和Foxp3的调节作用,以及探究L.animalis LGM对小鼠结肠炎的影响。【方法】本试验采用改良型的Hungate滚管技术从猪结肠内容物中分离一株L.animalis LGM,根据其16S rRNA序列进行鉴定。收集L.animalis LGM培养液上清,与细菌脂多糖(LPS,2μg/mL)同时孵育Caco-2细胞24 h,体外研究L.animalis LGM对Caco-2细胞内Th细胞分化转录因子(T-bet,GATA3,ROR-γt和Foxp3)mRNA表达的影响;配制L.animalis LGM细菌悬液,研究L.animalis LGM灌胃对DSS诱导结肠炎小鼠症状及结肠Th细胞分化转录因子和细胞因子(IFN-γ,IL-4,IL-17和IL-10)mRNA表达的影响,表达结果采用荧光定量PCR法检测。【结果】与对照组相比,L.animalis LGM培养液上清显著上调Caco-2细胞内ROR-γt与Foxp3 mRNA表达(P<0.05),显著下调GATA3、IL-4、IL-17和TGF-βmRNA表达(P<0.05)。L.animalis LGM灌胃显著上调小鼠结肠内ROR-γt和Foxp3的表达(P<0.05),显著降低了促炎因子IL-4和IL-17的表达(P<0.05),阻止了小鼠结肠长度缩短(P<0.05)。【结论】猪肠道分离L.animalis LGM表现出对Th细胞分化转录因子的选择性调节,显著上调Caco-2细胞及结肠炎小鼠ROR-γt与Foxp3 mRNA表达。降低DSS诱导结肠炎小鼠炎症水平,对DSS诱导结肠炎起保护作用,有助于维护肠道环境稳态。

An increase in corn resistant starch decreases protein fermentation and modulates gut microbiota during in vitro cultivation of pig large intestinal inocula

High-protein diet could cause an increase in protein fermentation in the large intestine, leading to an increased production of potentially detrimental metabolites. We hypothesized that an increase in corn resistant starch content may attenuate the protein fermentation. The aim of this study was to evaluate the effect of resistant starch on protein fermentation by inocula from large intestine of pigs using in vitro cultivation. Fermentation patterns were analyzed during a 24-h incubation of cecal and colonic digesta with varying corn resistant starch contents, using casein protein as sole nitrogen source. The results showed that the concentration of short-chain fatty acids(SCFA) and cumulative gas production were significantly increased(P < 0.05), while ammonia-nitrogen(NH_3-N) and branched-chain fatty acids(BCFA), which indicated protein fermentation, decreased when the corn resistant starch levels increased(P < 0.05). The copies of total bacteria, Bifidobacterium and Lactobacillus were significantly increased with the increased corn resistant starch levels after incubation(P < 0.05). The copies of the Bifidobacterium and Lactobacillus in cecum were significantly higher than those in colon(P < 0.05). We conclude that the addition of corn resistant starch weakens the protein fermentation by influencing microbial population and reducing protein fermentation in the cecum and colon in vitro.

Low crude protein diets supplemented with casein hydrolysate enhance the intestinal barrier function and decrease the pro-inflammatory cytokine expression in the small intestine of pigs

To reduce nitrogen excretion and lower feeding costs,low crude protein(CP)diets are sometimes pro-posed,however,a great reduction of dietary CP concentration(>4%reduction vs.recommended con-centration),even supplemented with essential and nonessential amino acids(AA)can detrimentally affect small intestinal barrier function and immunity,possibly due to the excessive lack of peptides.Here we hypothesize that with an extremely low CP concentration diet,protein-derived peptides,rather than AA supplementation,can improve intestinal barrier development and health.To test this hypothesis,21 growing pigs(19.90±1.00 kg body weight)were randomly assigned to 3 treatments with control diet(16%CP),or low CP diets(13%CP)supplemented with AA(LCPA)or casein hydrolysate(LCPC)for 28 days.In comparison with the control diet,the LCPA diet decreased the protein expression level of jejunal barrier factor zonula occludens-1(ZO-1)and stem cell proliferation factor leucine-rich repeat-containing G-protein-coupled receptor-5,whereas the LCPC diet enhanced intestinal barrier function by increasing the protein expression level of jejunal occludin and ZO-1 and ileal mucin-2.The LCPA diet reduced Lactobacillus counts,whereas the LCPC diet increased Lactobacillus counts and reduced Escherichia coli counts in the ileum.The LCPA diet also increased protein expression levels of pro-inflammatory cytokine interleukin-6(IL-6)and IL-22,whereas the LCPC diet decreased protein expression levels of pro-inflammatory IL-1β,IL-17A and tumor necrosis factor-αin the ileum.Collectively,the casein hydroly-sate supplementation of low CP diets showed beneficial effects on the small intestinal barrier,bacterial community,and immunity in pigs,pointing to the important role of protein-derived peptides in small intestinal health in cases of low crude protein diets.

Dietary citrus pectin drives more ileal microbial protein metabolism and stronger fecal carbohydrate fermentation over fructooligosaccharide in growing pigs

Fructo-oligosaccharide(FOS)and pectin are known soluble dietary fibers and can influence gut microbiota and consequently modulate gut health.To understand the differential impact patterns of pectin vs.FOS in modulating gut microbiota in the small and large intestine,an ileal-cannulated pig model was adopted to compare the temporal and spatial effects of FOS and citrus pectin(CP)on the gut microbiota.Sixteen terminal ileal-cannulated pigs were randomly divided into 2 groups and fed with a standard diet supplemented with either 3% FOS or 3% CP for 28 d.The CP group and FOS group showed different microbial composition,especially in the feces,with time and location as major factors affecting microbiota in the CP group,and with only location contribution in the FOS group.In the feces,relative to the FOS group,the CP group showed higher abundance of Christensenellaceae R-7 group and Ruminococcaceae UCG-010 and lower abundance of Mitsuokella and Olsenella(adjusted P<0.05),a higher level of shortchain fatty acids and a lower level of lactate at both d 14 and 25(P<0.05),and more copy numbers of genes encoding key enzymes related to propionate(mmd A)and butyrate(BCo AT)production and lactate utilization(Lcd A)(P<0.05),indicating a greater degree of microbial carbohydrate fermentation.In the ileum,as compared with FOS,CP increased the bacteria with high capability of fermenting amino acids,including Escherichia-Shigella and Klebsiella(adjusted P<0.05),and the expression of enzymes responsible for amino acid fermentation(i.e.lysine decarboxylase),as well as the amino acid fermentation products(cadaverine and tyramine)(P<0.05),indicating a greater degree of amino acid fermentation.Overall,our results highlight a differential dynamic impact of dietary CP vs.FOS on microbial composition and metabolism in the gut.The dietary CP has a stronger ability to promote microbial amino acid fermentation in the ileum and carbohydrate fermentation in the feces than FOS.These findings provide a new insight into the role of different fibers in gut nutrition and guidelines for the choice of fibers in manipulating gut health.

Comparisons of blood biochemical parameters,digestive enzyme activities and volatile fatty acid profile between Meishan and Yorkshire piglets

This study was conducted to compare physiological characteristics between Meishan and Yorkshire piglets in their early lives. Six healthy purebred Meishan sows and Yorkshire sows with close farrowing dates were used in this research. The piglets sucked their respective sow’s milk for 14 days, then they were slaughtered to collect samples of blood, pancreas, contents of stomach, jejunum, cecum, colon as well as feces for analysis of blood biochemical parameters, digestive enzymes, and volatile fatty acid(VFA). The results showed that Yorkshire piglets had higher concentrations of high-density lipoprotein cholesterol(HDL-C) and total cholesterol(TC)(P < 0.05). Gastric lipase activity was higher in Meishan piglets but Yorkshire piglets had higher lactase activity(P < 0.05). The total VFA together with acetate and propionate in cecum and colon were higher in Meishan piglets than in Yorkshire piglets(P < 0.05),but acetate in jejunum and ratio of acetate to propionate in colon were lower in Meishan piglets than in Yorkshire piglets(P < 0.05). In conclusion, in early suckling period, significant differences exist in host metabolism and intestinal microbial metabolism between Meishan and Yorkshire piglets.

Lactiplantibacillus plantarum L47 and inulin alleviate enterotoxigenic Escherichia coli induced ileal inflammation in piglets by upregulating the levels of a-linolenic acid and 12,13-epoxyoctadecenoic acid

Alternatives to antibiotics for preventing bacteria-induced inflammation in early-weaned farm animals are sorely needed. Our previous study showed that Lactiplantibacillus plantarum L47 and inulin could alleviate dextran sulfate sodium(DSS)-induced colitis in mice. To explore the protective effects of L. plantarum L47 and inulin on the ileal inflammatory response in weaned piglets challenged with enterotoxigenic Escherichia coli(ETEC), 28 weaned piglets were assigned into four groups, namely, CON group—orally given 10 mL/d phosphate buffer saline(PBS), LI47 group—orally given a mixture of 10 m L/d L. plantarum L47 and inulin, ECON group—orally given 10 mL/d PBS and challenged by ETEC, and ELI47group—orally given 10 mL/d L. plantarum L47 and inulin mixture and challenged by ETEC. The results demonstrated that the combination of L. plantarum L47 and inulin reduced inflammatory responses and relieved the inflammatory damage caused by ETEC, including ileal morphological damage, reduced protein expression of ileal tight junction, decreased antioxidant capacity, and decreased antiinflammatory factors. Transcriptome analysis revealed that L. plantarum L47 and inulin up-regulated the gene expression of phospholipase A2 group IIA(PLA2G2A)(P < 0.05) as well as affected alphalinolenic acid(ALA) metabolism and linoleic acid metabolism. Moreover, L. plantarum L47 and inulin increased the levels of ALA(P < 0.05), lipoteichoic acid(LTA)(P < 0.05), and 12,13-epoxyoctadecenoic acid(12,13-EpOME)(P < 0.05) and the protein expression of Toll-like receptor 2(TLR2)(P = 0.05) in the ileal mucosa. In conclusion, L. plantarum L47 and inulin together alleviated ETEC-induced ileal inflammation in piglets by up-regulating the levels of ALA and 12,13-EpOME via the LTA/TLR2/PLA2G2A pathway.