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Verotoxin (VT) has been implicated in the promotion of adherence to and colonization of intestinal epithelial cells by enterohemorrhagic Escherichia coli (EHEC) 0157:H7. The present study investigated the effect of VT2 on the adherence of EHEC O157:H7 strain 86-24 to porcine jejunal (IPEC-J2), human colon (CaCo-2), and human laryngeal carcinoma (HEp-2) cell lines and on the expression in IPEC-J2 cells of synthases for β1-integrin and nucleolin, both of which are implicated in bacterial adherence. The effect on expression of globotriaosylceramide (Gb3) synthase, the receptor for VT, was also examined. Data were obtained by adherence assays and quantitative reverse transcriptase PCR, using EHEC O157 strain 86-24, a v12 deletion mutant, a v12 phage-negative strain, and complemented mutants in which the vt2 gene was restored. Compared with the adherence of the parent and complemented mutant strains, the v12-negative strains adhered significantly less to all three types of cells. Adherence of the wild-type EHEC strain to IPEC-J2 cells was accompanied by increased expression of β1-integrin, nucleolin, and Gb3 synthase. IPEC-J2 cells in association with wild-type EHEC O157:H7 or the complemented mutants expressed higher levels of 31-integrin than did cells in association with the v12-negative strains or with no bacteria. Expression of nucleolin was decreased by association with the vt2-negative mutant, but complementation failed to restore wild-type expression. The data indicate that VT2 plays a role in the adherence of EHEC 0157:H7 to intestinal epithelial cells, possibly by increasing the expression of the host receptor β1-integrin. [ABSTRACT FROM AUTHOR]

Lactobacilli are sensitive to heat, which limits their application as probiotics in livestock production. Lactobacillus rhamnosus LB1 was previously shown to reduce enterotoxigenic Escherichia coli (ETEC) and Salmonella infections in pigs. To investigate its potential in the application, the bacterium was microencapsulated and examined for its survival from feed pelleting and long-term storage as well as its function in modulating pig intestinal microbiota. The in vitro studies showed that freshly microencapsulated Lactobacillus rhamnosus LB1 had viable counts of 9.03 ± 0.049 log10 colony-forming units/g, of which only 0.06 and 0.87 Log of viable counts were reduced after storage at 4 and 22 °C for 427 d. The viable counts of encapsulated Lactobacillus rhamnosus LB1 were 1.06 and 1.54 Log higher in the pelleted and mash feed, respectively, than the non-encapsulated form stored at 22 °C for 30 d. In the in vivo studies, 80 piglets (weaned at 21 d of age) were allocated to five dietary treatments for a 10-d growth trial. The dietary treatments were the basal diet (CTL) and basal diet combined with either non-encapsulated LB1 (NEP), encapsulated LB1 (EP), bovine colostrum (BC), or a combination of encapsulated LB1 and bovine colostrum (EP-BC). The results demonstrated that weaning depressed feed intake and reduced growth rates in pigs of all the treatments during 21 to 25 d of age; however, the body weight gain was improved during 25 to 31 d of age in all groups with the numerically highest increase in the EP-BC-fed pigs during 21 to 31 d of age. Dietary treatments with EP, particularly in combination with BC, modulated pig intestinal microbiota, including an increase in Lactobacillus relative abundance. These results suggest that microencapsulation can protect Lactobacillus rhamnosus LB1 against cell damage from a high temperature during processing and storage and there are possible complementary effects between EP and BC., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food, Government of Canada, 2023.)

It is well-known that essential oil thymol exhibits antibacterial activity. The protective effects of thymol on pig intestine during inflammation is yet to be investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cells was established. Cells were pretreated with thymol for 1 h and then exposed to LPS for various assays. Interleukin 8 (IL-8) secretion, the mRNA abundance of cytokines, reactive oxygen species (ROS), nutrient transporters, and tight junction proteins was measured. The results showed that LPS stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) mRNA abundance ( P < 0.05), but the mRNA abundance of sodium-dependent glucose transporter 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1), and H + /peptide cotransporter 1 (PepT1) were decreased ( P < 0.05). Thymol blocked ROS production ( P < 0.05) and tended to decrease the production of LPS-induced IL-8 secretion ( P = 0.0766). The mRNA abundance of IL-8 and TNF-α was reduced by thymol pretreatment ( P < 0.05), but thymol did not improve the gene expression of nutrient transporters ( P > 0.05). The transepithelial electrical resistance (TEER) was reduced and cell permeability increased by LPS treatment ( P < 0.05), but these effects were attenuated by thymol ( P < 0.05). Moreover, thymol increased zonula occludens-1 (ZO-1) and actin staining in the cells. However, the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS or thymol treatments. These results indicated that thymol enhances barrier function and reduce ROS production and pro-inflammatory cytokine gene expression in the epithelial cells during inflammation. The regulation of barrier function by thymol and LPS may be at post-transcriptional or post-translational levels.

Enterotoxigenic Escherichia coli strains induce human and animal intestinal dysfunction and injury, and cause diarrhea in weanling pigs. A recombinant E.coli strain LMG194-STa which expressed single toxin STa of ETEC was constructed by directly inserting the STa gene of ETEC into the expression vector pBAD202 and then transferring the recombinant plasmid pBAD-STa into the E.coli LMG19 host strain. Diarrhea and intestinal injury in piglets were induced by oral administration of recombinant strain LMG194-Sta. In vitro , the recombinant strain LMG194-Sta had the same toxicity to the IPEC cell as wild type strain K88, and higher toxicity than the host strain LMG194. In vivo , LMG194-STa caused severe diarrhea in piglets as K88, the diarrhea rate of LMG194-STa and K88 groups was higher than that in the LMG194 and control groups. Both K88 and LMG194-STa induced intestinal inflammation, injury, and atrophy, while adversely affecting the expression of genes for cytokines, transporters and ion channels, and nutrient metabolism. Thus, we established a porcine model of recombinant E. coli strain LMG194-STa-induced diarrhea for future nutritional and mechanistic studies of intestinal dysfunction.

Cinnamon is a traditional herb used for treatment of many human diseases. The most important chemical compounds of the essential oil are cinnamaldehyde and eugenol. Oleum cinnamomi (OCM, cinnamon oil) is increasingly used as a feed additive to animal diets. Beneficial effects of OCM in protecting tissues from inflammation and injury by endogenous and exogenous agents (such as hydrogen peroxide and lipopolysaccharide (LPS)) may result, in part, from its action on regulating amino acid metabolism in cells to favor the synthesis of glutathione (a major low-molecular-weight antioxidant) from cysteine, glycine and glutamate. In support of this notion, results of recent studies indicate that supplementing OCM (50 mg/kg diet) to a corn- and soybean meal-based diet for piglets weaned at 21 days of age enhances intestinal anti-oxidative capacity and reduces the incidence of diarrhea. Additionally, dietary supplementation with OCM ameliorates LPS-induced mucosal barrier dysfunction and mucosal damage in the small intestine. OCM holds great promise for protecting the gut from injury under conditions of inflammation, infections, and oxidative stress.

Salmonella Typhimurium is a foodborne pathogen and commonly present on pig farms. Probiotics have shown potential as a means of reducing Salmonella shedding in pigs. Three experimental challenge trials were conducted to investigate the potential application of newly isolated Lactobacillus isolates for controlling Salmonella infection in pigs. In each trial, 16 Yorkshire piglets (28-d old) were randomly allocated to 1 of 4 dietary treatments: (1) basal diet (BD), (2) naturally fermented (NF) feed, (3) Lactobacillus zeae-fermented (LZ-F) feed, and 4) Lactobacillus casei-fermented (LC-F) feed. All pigs consumed their assigned diets for 3 d prior to the challenge of Salmonella Typhimurium DT104 (approximately 6 log colony-forming units/pig) through gavage. Pediococcus pentosaceus, L. zeae, and L. casei were most abundant in NF, LZ-F, and LC-F feed, respectively. After the challenge, pigs on fermented feed had lower rectal temperature, diarrhea scores, serum haptoglobin concentrations, and intestinal Salmonella counts than the control group (BD) (p ≤ 0.01). Salmonella spp. were detected in both ileocecal lymph nodes (ICLN) and spleens from all pigs on BD, NF, and LC-F, but only 50% of spleens from pigs on LZ-F. Pigs had a dynamic spatial and temporal immune response to Salmonella infection and dietary treatments, as indicated by up- and downregulation in gene expression of inflammatory cytokines (interleukin (IL)-1β, IL-6, IL-10, interferon-γ, and tumor necrosis factor) in the ileum, ICLN, and spleen. The alternation in cytokine expression by fermented feed, particularly LZ-F, appeared to benefit pigs in combating Salmonella infection.

Polysaccharide from the seeds of Plantago asiatica L. was given via oral administration to mice (0.4 g/kg body weight, 30 days) to observe its effects on mouse nutrient metabolism and colon microbiota. It was found the polysaccharide intake could lower the apparent absorption of lipid. Total triglyceride, cholesterol, and atherogenic index in blood serum with total lipid and cholesterol levels in liver of polysaccharide group mice were all significantly lower than those of the control group (p < 0.05). Furthermore, the effect of the polysaccharide intake on mouse colon bacterial communities was investigated. Mice from the polysaccharide group showed a higher colon bacterial diversity than the control group. Bacteroides sp., Eubacterium sp., butyrate-producing bacteria Butyrivibrio sp., and probiotics Bifidobacterium bifidum , Lactobacillus fermentum , and Lactobacillus reuteri in mouse colon were all increased after polysaccharide intake. These indicated that the intake of polysaccharide from P. asiatica L. could be beneficial for lipid metabolism and colon microbiota.

The objectives of this study were to determine the effect of bacterial culture conditions on adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 in vivo to pig enterocytes and to compare the results with adherence in vitro to cultured HEp-2 and IPEC-J2 cells. Growth of O157:H7 in MacConkey broth (MB) resulted in almost no adherence to both HEp-2 and IPEC-J2 cells; prior exposure of the bacteria to pH 2.5 reduced adherence. There was greater adherence by bacteria from static cultures than by those from shaken cultures and by bacteria cultured in brain-heart infusion (BHI) plus NaHCO₃ (BHIN) than by bacteria cultured in BHI. In contrast, in pig ileal loops, bacteria cultured in MB adhered well to enterocytes, and prior exposure to pH 2.5 had no effect on adherence. Among several media tested for their effect on bacterial adherence in the pig intestine, MB and BHIN proved to be the best. Bacterial adherence was dose-dependent and was more extensive in the ileum than in the colon. This study demonstrated that there are remarkable differences between culture conditions that promote adherence of an EHEC O157:H7 strain in vitro and in vivo, that culture conditions profoundly affect adherence to epithelial cells in vitro and in vivo, and that pig ileal loops are better suited to adherence studies than are colon loops.

Verotoxin (VT) has been implicated in the promotion of adherence to and colonization of intestinal epithelial cells by enterohemorrhagic Escherichia coli (EHEC) O157:H7. The present study investigated the effect of VT2 on the adherence of EHEC O157:H7 strain 86-24 to porcine jejunal (IPEC-J2), human colon (CaCo-2), and human laryngeal carcinoma (HEp-2) cell lines and on the expression in IPEC-J2 cells of synthases for beta1-integrin and nucleolin, both of which are implicated in bacterial adherence. The effect on expression of globotriaosylceramide (Gb3) synthase, the receptor for VT, was also examined. Data were obtained by adherence assays and quantitative reverse transcriptase PCR, using EHEC O157 strain 86-24, a vt2 deletion mutant, a vt2 phage-negative strain, and complemented mutants in which the vt2 gene was restored. Compared with the adherence of the parent and complemented mutant strains, the vt2-negative strains adhered significantly less to all three types of cells. Adherence of the wild-type EHEC strain to IPEC-J2 cells was accompanied by increased expression of beta1-integrin, nucleolin, and Gb3 synthase. IPEC-J2 cells in association with wild-type EHEC O157:H7 or the complemented mutants expressed higher levels of beta1-integrin than did cells in association with the vt2-negative strains or with no bacteria. Expression of nucleolin was decreased by association with the vt2-negative mutant, but complementation failed to restore wild-type expression. The data indicate that VT2 plays a role in the adherence of EHEC O157:H7 to intestinal epithelial cells, possibly by increasing the expression of the host receptor beta1-integrin.

We previously reported that Clostridium perfringens in vivo proliferation and alpha-toxin gene expression were highly correlated, both progressing in a parabolic curve pattern during the development of necrotic enteritis (NE). The present study investigated the response of dominant ileal bacteria in abundance to C. perfringens infection using PCR-based denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (QPCR) techniques. Chickens were fed antibiotic-medicated (bacitracin, 55 mg/kg) or non-medicated diets, and were challenged with C. perfringens through the diet at 18 days of age. Ileal digesta was collected daily before and after the challenge for 5 days. Bacterial profiles of PCR-DGGE from both bacitracin-treated and untreated chickens responding to clostridial infection were analyzed by the principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Treatment-related differences in PCR-DGGE bacterial profiles on day 2 post-infection (PI) were detected. Subsequent QPCR assays identified changes in the abundance of lactobacilli, L. aviarius in particular. Changes in both populations correlated negatively with the population of C. perfringens in bacitracin-untreated group of chickens that displayed a high incidence of NE lesions. The results indicated that L. aviarius was suppressed by C. perfringens infection. This observation warrants further studies on the mechanisms underlying the ecological change and to assist in further development of novel probiotics to control NE disease.

Lactobacillus acidophilus has been shown to exert immunostimulating activities in a number of species, including the chicken. To examine the molecular mechanisms of this phenomenon, we investigated spatial and temporal expression of immune system genes in chicken cecal tonsil and spleen mononuclear cells in response to structural constituents of L. acidophilus. Using a low-density chicken immune system microarray, we found that cecal tonsil cells responded more rapidly than spleen cells to the bacterial stimuli, with the most potent stimulus for cecal tonsil cells being DNA and for splenocytes being the bacterial cell wall components. We also discovered that in both splenocytes and cecal tonsil cells, STAT2 and STAT4 genes were highly induced. Given the close interactions between cecal tonsil cells and commensal bacteria, we further examined the involvement of STAT2 and STAT4 signaling pathways in cellular responses to bacterial DNA. Our results revealed that the expression of STAT2, STAT4, IL-18, MyD88, IFN-alpha, and IFN-gamma genes were up-regulated in cecal tonsil cells after treatment with L. acidophilus DNA.

Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.

The Chinese green tea extract was found to strongly inhibit the growth of major food-borne pathogens, Escherichia coli O157:H7, Salmonella Typhimurium DT104, Listeria monocytogenes, Staphylococcus aureus, and a diarrhoea food-poisoning pathogen Bacillus cereus, by 44-100% with the highest activity found against S. aureus and lowest against E. coli O157:H7. A bioassay-guided fractionation technique was used for identifying the principal active component. A simple and efficient reversed-phase high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of four bioactive polyphenol compounds, epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), and caffeine (CN). The structures of these polyphenols were confirmed with mass spectrometry. Among the four compounds, ECG and EGCG were the most active, particularly EGCG against S. aureus. EGCG had the lowest MIC90 values against S. aureus (MSSA) (58 mg/L) and its methicilin-resistant S. aureus (MRSA) (37 mg/L). Scanning electron microscopy (SEM) studies showed that these two compounds altered bacterial cell morphology, which might have resulted from disturbed cell division. This study demonstrated a direct link between the antimicrobial activity of tea and its specific polyphenolic compositions. The activity of tea polyphenols, particularly EGCG on antibiotics-resistant strains of S. aureus, suggests that these compounds are potential natural alternatives for the control of bovine mastitis and food poisoning caused by S. aureus.

The study was to determine effects of dietary supplementation of chitosan (COS) and galacto-mannan-oligosaccharides (GMOS) on some serum biochemical indices, serum growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels, and hepatic and long gissimus muscle IGF-I mRNA expression in early-weaned piglets. Twenty six Duroc x Landrace x Yorkshire piglets at the age of 15 days were used. The piglets had access to creep feed during the suckling. Six piglets were sacrificed for sampling at the beginning of the study. The other 20 piglets were individually housed in metabolic cages and randomly allotted to four corn and soybean meal-based diets including the control group, the antibiotic group with 110 mg lincomycin/kg diet, the COS group containing 0.025% COS, and the GMOS group with 0.20% GMOS, respectively, in a 2-week feeding experiment. Blood urea nitrogen (BUN) level was reduced whereas serum total protein concentration was increased (P<0.05) in responses to the COS and GMOS supplementation. Dietary supplementation of COS and GMOS also increased (P<0.05) the serum GH and IGF-I levels along with enhanced hepatic and the muscle IGF-I mRNA abundance. Dietary supplementation of oligosaccharides such as COS and GMOS may improve growth and feed conversion efficiency by increasing plasma GH and IGF-I levels, in the early-weaned piglets.