Enterococcus faecium NCIMB 10415 administration improves the intestinal health and immunity in neonatal piglets infected by enterotoxigenic Escherichia coli K88

Background:This study aimed to investigate the effects of oral administration of Enterococcus faecium NCIMB10415(E.faecium)on intestinal development,immunological parameters and gut microbiota of neonatal piglets challenged with enterotoxigenic Escherichia coli K88(ETEC).A total of 961-day-old sow-reared piglets were randomly assigned to 2 groups,with 48 piglets in each group.The piglets were from 16 litters(6 piglets each litter),and 3 piglets each litter were allocated to the E.faecium-supplemented(PRO)group,while the other 3 piglets were allocated to the control(CON)group.After colostrum intake,piglets in the PRO group were orally administrated with 3×10~9 CFU E.faecium per day for a period of one week.On day 8,one piglet per litter from each group was challenged(CON+ETEC,PRO+ETEC)or not(CON-ETEC,PRO-ETEC)with ETEC in a 2×2 factorial arrangement of treatments.On day 10(2 days after challenge),blood and tissue samples were obtained from piglets.Results:Before ETEC challenge,there were no significant differences for the average daily gain(ADG)and fecal score between the two groups of piglets.After ETEC challenge,the challenged piglets had greater fecal score compared to the non-challenged piglets,whereas E.faecium administration was able to decrease the fecal score.Piglets challenged with ETEC had shorter villous height,deeper crypt depth,and reduced number of goblet cells in the jejunum and decreased m RNA abundance of claudin-1 in the ileum,whereas increased the percentage of lymphocytes,concentrations of IL-1βin the plasma and TNF-αin the ileal mucosa,as well as increased the m RNA abundances of innate immunity-related genes in the ileum tissue.These deleterious effects caused by ETEC were partly alleviated by feeding E.faecium.In addition,piglets in PRO-ETEC group had decreased the percentage of CD8^+T cells of the peripheral blood when compared to those in CON-ETEC group.Moreover,E.faecium administration increased Verrucomicrobia at phylum level and decreased Bilophila at genus level.Conclusions:These results suggest that oral administration of E.faecium alleviated the intestinal injury and diarrhea severity of neonatal piglets challenged by ETEC,partly through improving the intestinal microbiota and immune response.This offers a potential strategy of dietary intervention against intestinal impairment by ETEC in neonatal piglets.

Cross Sectional Study of Skin Carriage and Enterotoxigenicity of Staphylococcus aureus among Food Handlers

The prevalence of enterotoxigenic Staphylococcus aureus was investigated among 200 participants working in three different food processing plants in Egypt. Using skin swabs, 75 (38%) of the 200 tested persons were positive for the presence of S. aureus. Of the S. aureus positive persons, 28 (14%) harboured S. aureus produced staphylococcal enterotoxins. The serotypes of these enterotoxins were enterotoxin A (68%), enterotoxin B (36%), enterotoxin C (46%) and enterotoxin D (18%). Some of these isolates produced more than one type of enterotoxins namely AB, AC, BC, BD, ABC and ACD. Analysis of risk factors implicated in skin carriage of S. aureus as age, gender, marital status, education, duration in employment, frequency and method of hand wash and incidence of chronic skin infection revealed insignificant association with staphylococcal skin carriage. The obtained results put forth the risk of food contracting contamination with enterotoxigenic strains of S. aureus owing to skin colonization of S. aureus among food handlers.

Risk Factors for Prevalence of Enterotoxigenic Escherichia coli (ETEC) in Diarrheic and Non-diarrheic Neonatal and Weaner Pigs, South Africa

Enterotoxigenic Escherichio coli （ETEC） causes neonatal and post-weaning diarrhea in pigs. In order to determine the risk factors, rectal/fecal swabs and visceral organs obtained from pig farms in two regions of South Africa were analyzed microbiologically against risk variables. Seventy-two percent of young pigs were found to be positive for ETEC toxin genes; estB （38.9%）, estB/STAP （25%）, and estB/LT （13.9%） were dominant. Risk factors for ETEC-diarrhea in pigs include： leaving sick piglets in a pen with healthy piglets [odds ratio （OR） = 33.52; P 〈 0.0001]; water spillage in pen （OR = 42.87; P 〈 0.0001）; hypothermic piglets （OR = 7.29; P 〈 0.0001）; runt piglets in pen with healthy littermates （OR = 3.65; P 〈 0.0001）; and prolonged use of antibiotics （OR = 3.05; P = 0.05）.

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.

Impact of an oligosaccharide-based polymer on the metabolic profiles and microbial ecology of weanling pigs experimentally infected with a pathogenic E.coli

Background Our previous study has reported that supplementation of oligosaccharide-based polymer enhances gut health and disease resistance of pigs infected with enterotoxigenic E.coli(ETEC)F18 in a manner similar to carbadox.The objective of this study was to investigate the impacts of oligosaccharide-based polymer or antibiotic on the host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18.Results Multivariate analysis highlighted the differences in the metabolic profiles of serum and colon digesta which were predominantly found between pigs supplemented with oligosaccharide-based polymer and antibiotic.The relative abundance of metabolic markers of immune responses and nutrient metabolisms,such as amino acids and carbohydrates,were significantly differentiated between the oligosaccharide-based polymer and antibiotic groups(q<0.2 and fold change>2.0).In addition,pigs in antibiotic had a reduced(P<0.05)relative abundance of Lachnospiraceae and Lactobacillaceae,whereas had greater(P<0.05)Clostridiaceae and Streptococcaceae in the colon digesta on d 11 post-inoculation(PI)compared with d 5 PI.Conclusions The impact of oligosaccharide-based polymer on the metabolic and microbial profiles of pigs is not fully understood,and further exploration is needed.However,current research suggest that various mechanisms are involved in the enhanced disease resistance and performance in ETEC-challenged pigs by supplementing this polymer.

Macleaya cordata extract alleviated oxidative stress and altered innate immune response in mice challenged with enterotoxigenic Escherichia coli

This study examines the effects of dietary Macleaya cordata extract(MCE) on bacterial burden and resistance to enterotoxigenic Escherichia coli(ETEC) in ICR mice. ICR mice were randomly distributed into one of the following groups:(i) basal diet;(ii)basal diet with 200 mg kg–1 MCE;(iii) basal diet challenged with ETEC;and(iv) basal diet with 200 mg kg–1 MCE and challenged with ETEC. Following a 7-day period of pre-treatment, CTRL-ETEC and MCE-ETEC mice were subjected to oral infection using 5×108 E. coli SEC 470. The results showed dietary 200 mg kg–1 MCE markedly reduced intestinal ETEC burden(P<0.05) and the disease-associated mortality was significantly alleviated in the MCE treated group(P<0.05). In addition,dietary MCE markedly alleviated ETEC-induced oxidative stress, evidenced by the lowered methane dicarboxylic aldehyde(MDA) abundance and enhanced activities of catalase and glutathione peroxidase(P<0.05). Furthermore, MCE mice exhibited higher immune activity, which might have further mediated ETEC infection. These results indicate MCE plays a preventative role with respect to ETEC infection. Future research should aim to develop MCE as a therapeutic approach to the promotion of intestinal health and a safeguard against ETEC infection.

Micro-encapsulated essential oils and organic acids combination improves intestinal barrier function,inflammatory responses and microbiota of weaned piglets challenged with enterotoxigenic Escherichia coli F4(K88^+)

This study evaluated the effects of micro-encapsulated(protected)organic acids(OA)and essential oils(EO)combination,P(OA+EO),and effects of a regular blend of free acids(FA)on the growth,immune responses,intestinal barrier and microbiota of weaned piglets challenged with enterotoxigenic Escherichia coli(ETEC)F4(K88^+).A total of 30 crossbred(Duroc×Landrace×Large White)weaned barrows(7.41±0.06 kg,28 d old)were assigned randomly to 5 treatments:1)non-challenged positive control(PC),2)ETEC F4(K88^+)-challenged negative control(NC),3)NC+kitasamycin at 50 mg/kg+olaquindox at 100 mg/kg+free acidifier(FA)at 5 g/kg,4)NC+kitasamycin at 50 mg/kg+olaquindox at 100 mg/kg+P(OA+EO)at 1 g/kg(P1),5)NC+kitasamycin at 50 mg/kg+olaquindox at 100 mg/kg+P(OA+EO)at 2 g/kg(P2).Each dietary treatment had 6 replicates of one piglet each and the study lasted for 3 wk.On d 7,pigs in NC,FA,P1 and P2 were orally dosed with 10 mL of ETEC F4(K88^+)culture(1×10^9 CFU/mL).From d 7 to 14 after the ETEC F4(K88^+)challenge,P1 increased gain-to-feed ratio(G:F)significantly(P<0.05)compared with NC and FA groups.From d 14 to 21,P2 increased the average daily gain of pigs(P<0.05)compared with NC and FA groups.Compared with NC,P2 reduced tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-10 concentrations(P<0.05)in sera collected at 4 h later after ETEC F4(K88^+)challenge.On d 21,P1 increased occludin and zonula occludens-1 protein expression in ileum compared with NC(P<0.05).After this 3-wk experiment,alpha diversity of gut microbiota was decreased by P2 compared with PC,and P1 increased the relative abundance of Lactobacillus in ileum,cecum and colon(P<0.05).In co nclusio n,dietary P(OA+EO)additive at 2 g/kg combined with antibiotics could improve piglet performance and attenuate inflammation,and P(OA+EO)additive at 1 g/kg combined with antibiotics improved intestinal barrier and increased beneficial microbiota composition after an F4(K88^+)challenge.

Intestinal challenge with enterotoxigenic Escherichia coli in pigs,and nutritional intervention to prevent postweaning diarrhea

Gut health of nursery pigs immediately after weaning is tightly associated with their growth performance and economic values. Postweaning diarrhea(PWD) is one of the major concerns related to gut health of nursery pigs which often is caused by infections of enterotoxigenic Escherichia coli(ETEC),mainly including F4(K88)^+ and F18^+ E. coli. The main virulence factors of ETEC are adhesins(fimbriae or pili) and enterotoxins. The common types of fimbriae on ETEC from PWD pigs are F18^+ and F4^+. Typically, PWD in pigs is associated with both F18^+ and F4^+ ETEC infections whereas pre-weaning diarrhea in pigs is associated with F4+ ETEC infection. Enterotoxins including heat-labile enterotoxins(LT) and heatstable peptide toxins(ST) are associated with causing diarrhea in pigs. At least 109 to 1010 ETEC are required to induce diarrhea in nursery pigs typically lasting 1 to 5 days after ETEC infection. Antibiotics used to be the most effective way to prevent PWD, however, with the increased bacterial resistance to antibiotics, alternatives to the use of antibiotics are urgently needed to prevent PWD. Immunopropylaxis and nutritional intervention of antimicrobial minerals(such as zinc oxide and copper sulfate), organic acids, functional feedstuffs(such as blood plasma and egg yolk antibodies), direct fed microbials, phytobiotics, and bacteriophage can potentially prevent PWD associated with ETEC. Some other feed additives such as nucleotides, feed enzymes, prebiotic oligosaccharides, and clay minerals can enhance intestinal health and thus indirectly help with preventing PWD. Numerous papers show that nutritional intervention using selected feed additives can effectively prevent PWD.

Combined effects of chitosan and microencapsulated Enterococcus faecalis CG1.0007 probiotic supplementation on performance and diarrhea incidences in enterotoxigenic Escherichia coli K88^+ challenged piglets

The aim of this study was to investigate the combined effects of chitosan oligosaccharide(COS) and a microencapsulated Enterococcus faecalis CG1.0007 probiotic(PRO) on growth performance and diarrhea incidences in enterotoxigenic Escherichia coli(ETEC) K88^+ challenged piglets in a 14-d study. Thirty piglets,7.19 ± 0.52 kg initial BW weaned at 21 ± 1 d.were allotted to 5 treatment groups(n = 6)consisting of a corn-soybean meal diet with no additive(negative control, NC), NC + 0.25% chlortetracycline(positive control, PC), NC + 400 mg/kg COS(COS), NC + 100 mg/kg PRO(PRO) and NC + a combination of COS and PRO(CPRO). Pigs were individually housed in cages, acclimated to treatments for a 7-d period and had ad libitum access to feed and water throughout the study, On d 8, pigs were weighed, blood samples were collected, and then orally challenged with 6 mL(1 ×10^(11) cfu/mL) of freshly grown ETEC inoculum. During post-challenge period, blood was sampled at 24 and 48 h to determine plasma urea nitrogen(PUN), and diarrhea incidences and fecal consistency scores were recorded from d 9 to 12. On d 14, all pigs were weighed and then euthanized to obtain intestinal tissue samples for histomorphometric measurements. Growth performance responses were similar among treatments during the pre-and post-challenge periods. There were no significant differences in PUN content, incidences of diarrhea, and fecal consistency scores among treatments. The intestinal histomorphology results did not differ significantly among treatments except for PC with increased(P = 0.0001) villus:crypt ratio compared with the NC. Under the conditions of the present study, it can be concluded that supplementation of piglet diets with 400 mg/kg COS, 100 mg/kg microencapsulated PRO or their combination did not significantly improve piglet growth performance both during the pre-and post-ETEC K88+ oral inoculation. Also, there were no significant reduction of incidences and severity of diarrhea after challenge compared with the control group.

Melatonin shapes bacterial clearance function of porcine macrophages during enterotoxigenic Escherichia coli infection

Due to the immature gastrointestinal immune system,weaning piglets are highly susceptible to pathogens,e.g.,enterotoxigenic Escherichia coli(ETEC).Generally,pathogens activate the immune cells(e.g.,macrophages)and shape intracellular metabolism(including amino acid metabolism);nevertheless,the metabolic cues of tryptophan(especially melatonin pathway)in directing porcine macrophage function during ETEC infection remain unclear.Therefore,this study aimed to investigate the changes in the serotonin pathway of porcine macrophages during ETEC infection and the effect of melatonin on porcine macrophage functions.Porcine macrophages(3D4/21 cells)were infected with ETEC,and the change of serotonin pathway was analysed by reverse transcription PCR and metabolomic analysis.The effect of melatonin on porcine macrophage function was also studied with proteomic analysis.In order to investigate the effect of melatonin on bacterial clearance function of porcine macrophages during ETEC infection,methods such as bacterial counting,reverse transcription PCR and western blotting were used to detect the corresponding indicators.The results showed that ETEC infection blocked melatonin production in porcine macrophages(P<0.05)which is largely associated with the heat-stable enterotoxin b(STb)of ETEC(P<0.05).Interestingly,melatonin altered porcine macrophage functions,including bacteriostatic and bactericidal activities based on proteomic analysis.In addition,melatonin pretreatment significantly reduced extracellular lactate dehydrogenase(LDH)activity(P<0.05),indicating that melatonin also attenuated ETEC-triggered macrophage death.Moreover,melatonin pretreatment resulted in the decrease of viable ETEC in 3D4/21 cells(P<0.05),suggesting that melatonin enhances bacterial clearance of porcine macrophages.These results suggest that melatonin is particularly important in shaping porcine macrophage function during ETEC infection.

体外发酵条件下嗜热链球菌S131对肠道健康的调控机制

该研究以HT-29细胞与人体肠道菌群批量发酵模拟系统相结合,探究嗜热链球菌S131对肠道屏障的影响、拮抗致病菌造成的肠道损伤及对有益菌的增殖效果。结果表明,与对照组相比S131使HT-29细胞黏蛋白MUC2、紧密连接蛋白ZO-1、ZO-2、Claudin-1与Occludin mRNA相对表达分别提升至1.34、4.59、2.17、5.81和4.25,同时能够缓解产肠毒素性大肠杆菌(Enterotoxigenic E.coli,ETEC)对相应基因表达的抑制,各基因分别提升至1.42、6.28、3.27、5.03和4.79。S131对ETEC造成的HT-29细胞炎症因子分泌增加具有显著抑制作用,使IL-8和IL-1β分别由821.79 pg/mL和2.22 pg/mL降至573.92 pg/mL和0.29 pg/mL。S131改善了ETEC造成的上皮细胞损伤,将细胞增殖率提升至21.07%。同时S131缓解了ETEC造成的AQP-3 mRNA相对表达下调,使AQP-3基因表达提升至1.21。并且通过qPCR检测发现S131提升了体外发酵体系中柔嫩梭菌属(Faecalibacterium)和多形拟杆菌属(Bacteroides thetaiotaomicron)的相对丰度,改善了ETEC造成的乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)、多形拟杆菌和柔嫩梭菌属的降低。综上,嗜热链球菌S131可能通过增强肠道屏障、拮抗致病菌损伤和调节肠道菌群来改善肠道健康。

A Multiplex PCR Assay for the Detection of Pathogenic Genes of EPEC,ETEC and EIEC

A multiplex polymerase chain reaction （PCR） was developed to detect three pathogenic genes of enteropathogenic, enterotocigenic and enteroinvasive Escherichia coli In this study three different sets of oligonucleotide primer were simultaneously used, and in this way, specific fragments of 880, 600, 150 bp for EPEC eaeA, EIEC ipaH and ETEC ST genes were amplified, respectively. The best condition of the multiplex PCR was： after an initial heat denaturation step at 95℃for 5 min, followed by 30 cycles of denaturation at 94 ℃ for 40 s, primer annealing at 51.3℃ for 40 s and extension at 72 ℃ for 1 min, final extension at 72 ℃ for 10 min. The detection limit of the eaeA, ipaH and ST primers was 38.7423, 3.60519, 29.9448 ng·mL^-1 （4.3×10^4, 1.5×10^3, 2.6×10^4 CFU·mL^-1）, respectively. It may be a good way for the detection and identification of Diarrhea-causing E. coli.

Trace amounts of antibiotic altered metabolomic and microbial profiles of weaned pigs infected with a pathogenic E.coli

Background:Our previous study has shown that supplementation of trace amounts of antibiotic exacerbated the detrimental effects of enterotoxigenic E.coli(ETEC)infection and delayed the recovery of pigs that may be associated with modified metabolites and metabolic pathways.Therefore,the objective of this study was to explore the impacts of trace levels of antibiotic(carbadox)on host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18.Results:The multivariate analysis highlighted a distinct metabolomic profile of serum and colon digesta between trace amounts of antibiotic(TRA;0.5 mg/kg carbadox)and label-recommended dose antibiotic(REC;50 mg/kg carbadox)on d 5 post-inoculation(PI).The relative abundance of metabolomic markers of amino acids,carbohydrates,and purine metabolism were significantly differentiated between the TRA and REC groups(q<0.2).In addition,pigs in REC group had the highest(P<0.05)relative abundance of Lactobacillaceae and tended to have increased(P<0.10)relative abundance of Lachnospiraceae in the colon digesta on d 5 PI.On d 11 PI,pigs in REC had greater(P<0.05)relative abundance of Clostridiaceae compared with other groups,whereas had reduced(P<0.05)relative abundance of Prevotellaceae than pigs in control group.Conclusions:Trace amounts of antibiotic resulted in differential metabolites and metabolic pathways that may be associated with its slow responses against ETEC F18 infection.The altered gut microbiota profiles by labelrecommended dose antibiotic may contribute to the promotion of disease resistance in weaned pigs.

Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity

Background:There is a great demand for antibiotic alternatives to maintain animal health and productivity.The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1antigen oligosaccharides-based polymer(Coligo)on growth performance,diarrhea severity,intestinal health,and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli(ETEC),when compared with antibiotics.Results:Pigs in antibiotic carbadox or Coligo treatment groups had greater(P<0.05)body weight on d 5 or d 11post-inoculation(PI)than pigs in the control group,respectively.Supplementation of antibiotics or Coligo enhanced(P<0.05)feed efficiency from d 0 to 5 PI and reduced(P<0.05)frequency of diarrhea throughout the experiment,compared with pigs in the control group.Supplementation of antibiotics reduced(P<0.05)fecalβ-hemolytic coliforms on d 2,5,and 8 PI.Pigs in antibiotics or Coligo groups had reduced(P<0.05)neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI.Pigs in Coligo had reduced(P<0.05)total coliforms in mesenteric lymph nodes on d 5 and 11 PI,whereas pigs in antibiotics or Coligo groups had reduced(P<0.05)total coliforms in spleen on d 11 PI compared with pigs in the control group.On d 5 PI,pigs in the Coligo group had greater(P<0.05)gene expression of ZO1 in jejunal mucosa,but less(P<0.05)m RNA expression of IL1B,IL6,and TNF in ileal mucosa,in comparison with pigs in the control group.Supplementation of antibiotics enhanced(P<0.05)the gene expression of OCLN in jejunal mucosa but decreased(P<0.05)IL1B and IL6 gene expression in ileal mucosa,compared with the control.On d 11 PI,supplementation of antibiotics or Coligo up-regulated(P<0.05)gene expression of CLDN1 in jejunal mucosa,but Coligo reduced(P<0.05)IL6 gene expression in ileal mucosa compared to pigs in the control group.Conclusions:Supplementation of Coligo improved growth performance,alleviated diarrhea severity,and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics.