Citral alleviates peptidoglycan-induced inflammation and disruption of barrier functions in porcine intestinal epithelial cells.

Peptidoglycan (PGN) is a major polymer in bacterial cell walls and may constrain gut functionality and lower intestinal efficiencies in livestock. Citral has been reported to exhibit antibacterial and anti-inflammatory biological activities, improving the gastrointestinal function of swine. However, the protective effect of citral against PGN-elicited cellular responses and possible underlying mechanisms are unknown. In this study, the porcine jejunal epithelial cell line (IPEC-J2) was challenged with PGN from Staphylococcus aureus (S. aureus) or Bacillus subtilis (B. subtilis) to explore PGN-induced inflammatory responses. Our data showed that the inflammatory response stimulated by PGN from harmful bacteria (S. aureus) was more potent than that from commensal bacteria (B. subtilis) in IPEC-J2 cells. Based on the inflammatory model by PGN from S. aureus, it was demonstrated that PGN could significantly induce inflammatory cytokine production and influence nutrient absorption and barrier function in a dose-dependent manner. However, the PGN-mediated immune responses were remarkably suppressed by citral. In addition, citral significantly attenuated the effect of PGN on the intestine nutrient absorption and barrier function. The expression of TLR2 was strongly induced by PGN stimulation, which was suppressed by citral. All data nominated that citral downregulated PGN-induced inflammation via TLR2-mediated activation of the NF-κB signaling pathway in IPEC-J2 cells. Furthermore, the results also indicate that the PGN degradation through the inclusion of enzymes (e.g., muramidase) as well as the inclusion of citral for attenuating inflammation may improve pig gut health and functionality.

A Novel PilR/PilS Two-Component System Regulates Necrotic Enteritis Pilus Production in Clostridium perfringens.

Clostridium perfringens causes necrotic enteritis (NE) in poultry. A chromosomal locus (VR-10B) was previously identified in NE-causing C. perfringens strains that encodes an adhesive pilus (NE pilus), along with a two-component system (TCS) designated here as PilRS. While the NE pilus is important in pathogenesis, the role of PilRS remains to be determined. The current study investigated the function of PilRS, as well as the Agr-like quorum-sensing (QS) system and VirSR TCS in the regulation of pilin production. Isogenic pilR, agrB, and virR null mutants were generated from the parent strain CP1 by insertional inactivation using the ClosTron system, along with the respective complemented strains. Immunoblotting analyses showed no detectable pilus production in the CP1pilR mutant, while production in its complement (CP1pilR+) was greater than wild-type levels. In contrast, pilus production in the agrB and virR mutants was comparable or higher than the wild type but reduced in their respective complemented strains. When examined for collagen-binding activity, the pilR mutant showed significantly lower binding to most collagen types (types I to V) than parental CP1 (P ≤ 0.05), whereas this activity was restored in the complemented strain (P > 0.05). In contrast, binding of agrB and virR mutants to collagen showed no significant differences in collagen-binding activity compared to CP1 (P > 0.05), whereas the complemented strains exhibited significantly reduced binding (P ≤ 0.05). These data suggest the PilRS TCS positively regulates pilus production in C. perfringens, while the Agr-like QS system may serve as a negative regulator of this operon. IMPORTANCE Clostridium perfringens type G isolates cause necrotic enteritis (NE) in poultry, presenting a major challenge for poultry production in the postantibiotic era. Multiple factors in C. perfringens, including both virulent and nonvirulent, are involved in the development of the disease. We previously discovered a cluster of C. perfringens genes that encode a pilus involved in adherence and NE development, along with a predicted two-component regulatory system (TCS), designated PilRS. In the present study, we have demonstrated the role of PilRS in regulating pilus production and collagen binding of C. perfringens. In addition, the Agr-like quorum sensing signaling pathway was found to be involved in the regulation. These findings have identified additional targets for developing nonantibiotic strategies to control NE disease.

Innovative drugs, chemicals, and enzymes within the animal production chain.

The alarming number of recently reported human illnesses with bacterial infections resistant to multiple antibacterial agents has become a serious concern in recent years. This phenomenon is a core challenge for both the medical and animal health communities, since the use of antibiotics has formed the cornerstone of modern medicine for treating bacterial infections. The empirical benefits of using antibiotics to address animal health issues in animal agriculture (using therapeutic doses) and increasing the overall productivity of animals (using sub-therapeutic doses) are well established. The use of antibiotics to enhance profitability margins in the animal production industry is still practiced worldwide. Although many technical and economic reasons gave rise to these practices, the continued emergence of antimicrobial resistant bacteria is furthering the need to reduce the use of medically important antibiotics. This will require improving on-farm management and biosecurity practices, and the development of effective antibiotic alternatives that will reduce the dependence on antibiotics within the animal industry in the foreseeable future. A number of approaches are being closely scrutinized and optimized to achieve this goal, including the development of promising antibiotic alternatives to control bacterial virulence through quorum-sensing disruption, the use of synthetic polymers and nanoparticles, the exploitation of recombinant enzymes/proteins (such as glucose oxidases, alkaline phosphatases and proteases), and the use of phytochemicals. This review explores the most recent approaches within this context and provides a summary of practical mitigation strategies for the extensive use of antibiotics within the animal production chain in addition to several future challenges that need to be addressed.

Beneficial Impact and Molecular Mechanism of Bacillus coagulans on Piglets' Intestine.

The aim of this research was to investigate the beneficial impact and molecular mechanism of B. coagulans on piglets' intestine. Twenty-four 21 days old weaned piglets were allotted to three treatments: Control group (basal diet), B6 group (basal diet + 2 × 106 CFU/g B. coagulans), and the B7 group (basal diet + 2 × 107 CFU/g B. coagulans). The results showed that, compared with the control group, the B7 group had a reduced cholesterol content and gamma glutamyl transpeptidase (GGT) in plasma (p < 0.05); the B6 and B7 groups had a significantly decreased diarrhea rate and diamine oxidase (DAO) activity in plasma (p < 0.05), increased villus height in ileum and decreased crypt depth in the jejunum (p < 0.05); increased activities of superoxide dismutase (SOD) and catalase (CAT), and decreased the content of malondialdehyde (MDA) and H2O2 in the intestine (p < 0.05). These data suggested that supplementing B. coagulans had beneficial impacts on promoting nutrients' metabolism, maintaining intestinal integrity, and alleviating oxidative stress and diarrhea. Further research of molecular mechanisms showed changing expression levels of related proteins and genes, suggesting that these could be involved in the regulation of the impact. The community composition of the gut microbiota was also found to be altered in several operational taxonomic units within the genus, Prevotella (order Bacteroidales), and the order, Clostridiales.

Dietary Supplementation with Oleum Cinnamomi Improves Intestinal Functions in Piglets.

The present study was to determine the efficacy of dietary supplementation with oleum cinnamomi (OCM) on growth performance and intestinal functions in piglets. Sixteen piglets (24-day-old) were randomly assigned to the control or OCM groups. Piglets in the control group were fed a basal diet, whereas piglets in the OCM group were fed the basal diet supplemented with 50 mg/kg OCM. On day 20 of the trial, blood samples and intestinal tissues were obtained from piglets. Compared with the control group, dietary OCM supplementation increased (p < 0.05) average daily feed intake, plasma insulin levels, villus width and villous surface area in the duodenum and jejunum, DNA levels and RNA/DNA ratios in the ileum, the abundance of Enterococcus genus and Lactobacillus genus in caecum digesta, mRNA levels for epithelial growth factor receptor (EGFR), Ras, extracellular signal-regulated kinase 1/2 (Erk1/2), b-cell lymphoma-extra large (Bcl-xL), villin, junctional adhesion molecule A (JAM-A), myxovirus resistance (MX) 1, MX2 and regenerating islet-derived protein 3 gamma (REG3G), and protein abundances of Ras and claudin-1, but decreased (p < 0.05) diarrhoea incidence; the abundances of Enterobacteriaceae family, Enterococcus genus, Lactobacillus genus, Bifidobacterium genus, and Clostrium coccoides in the colon digesta, and AMP-activated protein kinase (AMPK) mRNA levels and caspase-3 protein abundance in the jejunal mucosa of piglets. Taken together, these data indicate that dietary OCM supplementation modulates intestinal microbiota and improves intestinal function in weanling pigs. OCM is an effective feed additive and alternative to feed antibiotics for improving intestinal health in swine.

The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

Issues deserve attention in encapsulating probiotics: Critical review of existing literature.

Probiotic bacteria are being increasingly added to food for developing products with health-promoting properties. However, the efficacy of probiotics in commercial products is often questioned due to the loss of their viability during shelf storage and in human gastrointestinal tracts. Encapsulation of probiotics has been expected to provide protection to probiotics, but not many commercial products contain encapsulated and viable probiotic cells owing to various reasons. To promote the development and application of encapsulation technologies, this paper has critically reviewed previous publications with a focus on the areas where studies have fallen short, including insufficient consideration of structural effects of encapsulating material, general defects in encapsulating methods and issues in evaluation methodologies and risk assessments for application. Corresponding key issues that require further studies are highlighted. Some emerging trends in the field, such as current treads in encapsulating material and recently advanced encapsulation techniques, have also been discussed.

The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review.

This review summarizes advances in understanding the pathogenesis of necrotic enteritis of chickens caused by netB-positive Clostridium perfringens. The discovery of NetB as the essential toxin trigger for the disease was followed by recognition that it forms part of a large plasmid-encoded 42 kb pathogenicity locus (NELoc-1). While the locus is critical for toxin production, it likely has additional functions related to colonization and degradation of the mucus barrier, which are essential both to multiplication and to bringing NetB close to the intestinal epithelium. Two "chitinases" (glycoside hydrolases (GHs)) present on NELoc-1 are predicted to be involved in mucin degradation, as is the large carbohydrate-binding metalloprotease, shown to be involved in mucinase activity in other clostridia. A second pathogenicity locus found in netB-positive C. perfringens, NELoc-2, also encodes a GH likely involved in mucin degradation. Upon reaching a sufficient cell density on the intestinal mucosa, the Agr-like quorum-sensing system is triggered, which in turn up-regulates the VirR/VirS regulon. This regulon includes NetB. Where NetB initiates damage is unresolved, but it may be deep in the intestinal mucosa, rather than superficially. As the disease progresses, C. perfringens line what remains of the intestinal epithelium in large numbers. This likely involves a number of different bacterial adhesins, including additional NELoc-1-encoded bacterial surface proteins, some of which may adhere to epithelial cell ligands exposed by bacterial sialidases. Further studies of the pathogenesis of necrotic enteritis should lead to development of novel ways to control the infection.

Effect of encapsulated carvacrol on the incidence of necrotic enteritis in broiler chickens.

There is an urgent need to control necrotic enteritis (NE) caused by Clostridium perfringens in chickens when antibiotics are withdrawn from feed. Carvacrol has strong antimicrobial activity and its delivery to the animal intestine can be significantly enhanced after encapsulation. The present study has investigated the potential of encapsulated carvacrol in controlling NE. In general, micro-encapsulation of carvacrol in an alginate-whey protein matrix showed no adverse effect on its antimicrobial activity towards C. perfringens in either Brain Heart Infusion (BHI) broth or a simulated gastrointestinal model. The minimum inhibitory concentrations of both encapsulated and un-encapsulated carvacrol were approximately 200 µl/l against C. perfringens in BHI. In a broiler infection model with C. perfringens, the diets supplemented with encapsulated carvacrol at the dose of either 250 or 650 µg/g significantly reduced NE in the chicken intestine, which was close to the degree of lesions observed in bacitracin/salinomycin treated birds. Supplementation with either bacitracin/salinomycin or encapsulated carvacrol showed no significant impact on intestinal burden of Lactobacillus. However, the treatment with bacitracin/salinomycin or the low dose of encapsulated carvacrol reduced the level of C. perfringens in the ileum of birds at 35 days of age. These results suggest that our encapsulated carvacrol can be used to combat NE disease in chickens.

Functional assessment of encapsulated citral for controlling necrotic enteritis in broiler chickens.

Development of viable alternatives to antibiotics to control necrotic enteritis (NE) caused by Clostridium perfringensis becoming urgent for chicken production due to pessures on poultry producers to limit or stop the use of antibiotics in feed. We have previously identified citral as a potential alternative to antibiotics. Citral has strong antimicrobial activity and can be encasupsulated in a powder form for protection from loss during feed processing, storage, and intestinal delivery. In the present study, encapsulated citral was evaluated both in vitro and in vivo for its antimicrobial activity against C. perfringens Encapsulation did not adversely affect the antimicrobial activity of citral. In addition, encapsulated citral was superior to the unencapsulated form in retaining its antimicrobial activity after treatment with simulated gastrointestinal fluids and in the presence of chicken intestinal digesta. In addition, the higher antimicrobial activity of encapsulated citral was confirmed in digesta samples from broilers that had been gavaged with encapsulated or unencapsulated citral. In broilers infected with C. perfringens, the diets supplemented with encapsualted citral at both 250 and 650 µg/g significantly reduced intestinal NE lesions, which was comparable to the effect of bacitracin- and salinomycin-containing diets. However, supplementation with the encapsulated citral appeared to have no significant impact on the intestinal burden of Lactobacillus These data indicate that citral can be used to control NE in chickens after proper protection by encapsulation.

Evaluation of alginate-whey protein microcapsules for intestinal delivery of lipophilic compounds in pigs.

BACKGROUND: In animal care and management, there is an increasing demand for convenient methods of oral delivery of bioactive compounds to specific segments of an animal's gastrointestinal tract. The objective of this study was to test the suitability of microcapsules made with alginate and whey proteins of two different sizes (250 and 800 µm; containing 72 and 76 g kg(-1) of carvacrol respectively) for intestinal delivery of carvacrol in pigs. RESULTS: Encapsulated carvacrol was completely released from the microcapsules after 5 h incubation in simulated intestinal fluids or 6 h in (ex vivo) ileal digesta, whereas release in simulated gastric fluid was minimal. Tests with growing pigs showed over 95% of unencapsulated carvacrol was absorbed or metabolized in the stomach and the duodenum. Encapsulation effectively minimized carvacrol absorption in the stomach (P < 0.05), and increased carvacrol recovery in the small intestine (P < 0.05). Encapsulated carvacrol was completely released from both small and large size capsules within the gastrointestinal tract of pigs. Larger size microcapsules showed a slower in vitro release and greater in vivo recovery of carvacrol in the small intestine (P < 0.05) than the smaller ones. CONCLUSION: This study indicates alginate-whey protein microencapsulation is a feasible approach for targeted oral delivery of hydrophobic compounds to pig intestines; increasing capsule size increased delivery of carvacrol to the end of the small intestine. © 2015 Her Majesty the Queen in Right of Canada Journal of the Science of Food and Agriculture © 2015 Society of Chemical Industry.

Modulation of cytokine gene expression by selected Lactobacillus isolates in the ileum, caecal tonsils and spleen of Salmonella-challenged broilers.

Probiotics have been used to control Salmonella colonization in the chicken intestine. Recently, we demonstrated that certain selected Lactobacillus isolates were able to reduce Salmonella infection in the chicken spleen and liver as well as down-regulated Salmonella pathogenicity island 1 virulence gene expression in the chicken caecum. To further understand the mechanisms through which Lactobacillus protected chickens from Salmonella infection, the present study has investigated the Lactobacillus isolate(s)-induced host immune response of chickens to Salmonella enterica serovar Typhimurium infection. A thorough examination of cytokine gene expression in the ileum, caecal tonsils, and spleen on days 1 and 3 post-Salmonella infection showed a dynamic spatial and temporal response to Salmonella infection and Lactobacillus treatments. In most instances, it was evident that treatment of chickens with Lactobacillus isolates could significantly attenuate Salmonella-induced changes in the gene expression profile. These included the genes encoding pro-inflammatory cytokines [lipopolysaccharide-induced TNF factor, interleukin (IL)-6, and IL-8], T helper 1 cytokines [IL-12 and interferon (IFN)-γ], and T helper 2 cytokines (IL-4 and IL-10). Another important observation from the present investigation was that the response induced by a combination of Lactobacillus isolates was generally more effective than that induced by a single Lactobacillus isolate. Our results show that administration of certain selected Lactobacillus isolates can effectively modulate Salmonella-induced cytokine gene expression, and thus help reduce Salmonella infection in chickens.

Inactivation and potential reactivation of pathogenic Escherichia coli O157:H7 in bovine milk exposed to three monochromatic ultraviolet UVC lights.

The ultraviolet (UVC) light irradiation has been recently studied as a novel non-thermal treatment method for milk. However, the potential reactivation of microorganisms following exposure to UVC light in milk medium was not studied yet. Therefore, the present study was conducted to determine the inactivation and reactivation of pathogenic Escherichia coli O157:H7 following exposure to UV light at three monochromatic wavelengths (222, 254 and 282 nm) in bovine milk. The results showed that inactivation of E. coli O157:H7 following exposure to the UV light at 254 nm was higher (P < 0.05) than that following exposure at 222 and 282 nm at the same UV fluence of 5, 10 and 20 mJ/cm(2). The reactivation of E. coli O157:H7 was increased as the incubation time and temperature increased regardless of the UV light sources under dark incubation phases. The evaluated reactivation ratios of E. coli O157:H7 following exposure to the UV light at 254 nm in milk were lower (P < 0.05) than that following exposure at 222 nm after 1 to 6, 2 to 5 and 5-6 h incubation at 4, 20 and 37 °C, respectively. Furthermore, at most incubation time points, the reactivation ratio of E. coli O157:H7 following exposure to these three UV light sources were lower (P < 0.05) than that of non-UV treated cells regardless of the incubation temperature. The lowest reactivation ratios of E. coli O157:H7 were observed after milk exposure to the UV light at 254 nm at 4 °C incubation when compared to that following exposure to the UV light at 222 and 282 nm.

Inactivation and potential reactivation of pathogenic Escherichia coli O157:H7 in apple juice following ultraviolet light exposure at three monochromatic wavelengths.

Ultraviolet (UV) light irradiation at 254 nm is considered as a novel non-thermal method for decontamination of foodborne pathogenic bacteria. However, lower penetration depth of UV light at 254 nm in apple juice resulted in higher UV dose consumption during apple juice decontamination. In addition, no studies are available on the reactivation of pathogens following exposure to UV light in drinks and beverages. Two novel monochromatic UV light sources (λ = 222 and 282 nm) have been developed for bacterial disinfection. However, the inactivation of pathogenic Escherichia coli O157:H7 following exposure to these UV wavelengths is still unclear. Therefore, the present study was conducted to determine the inactivation and reactivation potential of pathogenic E. coli O157:H7 in apple juice following exposure to UV light at three monochromatic wavelengths: Far UV (λ = 222 nm), Far UV+ (λ = 282 nm) and UVC light (λ = 254 nm). The results showed that E. coli O157:H7 is acid-resistant, and up to 99.50% of cells survived in apple juice when incubated at 20 °C for 24 h. Inactivation of E. coli O157:H7 following exposure to Far UV light (2.81 Log reduction) was higher (P < 0.05) than the inactivation caused by UVC light (1.95 Log reduction) and Far UV+ light (1.83 Log reduction) at the similar levels of UV fluence of 75 mJ/cm(2). No any reactivation potential was observed for E. coli O157:H7 in dark incubation phases after exposure to UV light as determined by the regular plating method. In addition, the exposure to Far UV light at 222 nm followed by incubating at 37 °C significantly decreased (P < 0.05) the survival of E. coli O157:H7 during dark incubation phase compared to that of UVC and Far UV+ light.

Effect of Lactobacillus plantarum NCU116 on loperamide-induced constipation in mice.

Lactobacillus plantarum, as a probiotic, has many functional properties in human intestinal tract. This study examined the effects of L. plantarum NCU116 on loperamide-induced constipation in a mouse model. Loperamide (5 mg kg(-1)) was injected subcutaneously to induce constipation. Animals were divided to five groups: normal group, constipation group, constipation plus three doses of L. plantarum NCU116 groups (NCU116-L, 10(7) CFU/mL; NCU116-M, 10(8) CFU/mL; NCU116-H, 10(9) CFU/mL; respectively). Mice were treated with the probiotic for 15 d to assess the anti-constipation effects. Fecal parameters, intestinal transit ratio and the production of fecal short chain fatty acids, histological of colon and immunohistochemical in colonic interstitial cells of Cajal (ICC) by c-kit were all improved in L. plantarum NCU116-treated mice as compared to the constipation group. These results demonstrate that L. plantarum NCU116 enhanced gastrointestinal transit and alleviated in mice with loperamide-induced constipation.

Effects of Tributyrin on Intestinal Energy Status, Antioxidative Capacity and Immune Response to Lipopolysaccharide Challenge in Broilers.

This study was carried out to investigate the effects of tributyrin (TB) on the growth performance, pro-inflammatory cytokines, intestinal morphology, energy status, disaccharidase activity, and antioxidative capacity of broilers challenged with lipopolysaccharide (LPS). A total of 160 one-day-old Cobb broilers were allocated to 1 of 4 treatments, with 4 replicated pens per treatment and 10 birds per pen. The experiment consisted of a 2×2 factorial arrangements of treatments with TB supplementation (0 or 500 mg/kg) and LPS challenge (0 or 500 µg/kg body weight [BW]). On days 22, 24, and 26 of the trial, broilers received an intraperitoneal administration of 500 µg/kg BW LPS or saline. Dietary TB showed no effect on growth performance. However, LPS challenge decreased the average daily gain of broilers from day 22 to day 26 of the trial. Dietary TB supplementation inhibited the increase of interleukin-1ß (in the jejunum and ileum), interleukin-6 (in the duodenum and jejunum), and prostaglandin E2 (in the duodenum) of LPS-challenged broilers. Similar inhibitory effects of TB in the activities of total nitric oxide synthase (in the ileum) and inducible nitric oxide synthase (in the jejunum) were also observed in birds challenged with LPS. Additionally, TB supplementation mitigated the decrease of ileal adenosine triphosphate, adenosine diphosphate and total adenine nucleotide and the reduction of jejunal catalase activity induced by LPS. Taken together, these results suggest that the TB supplementation was able to reduce the release of pro-inflammatory cytokines and improve the energy status and anti-oxidative capacity in the small intestine of LPS-challenged broilers.

Dietary supplementation with tributyrin alleviates intestinal injury in piglets challenged with intrarectal administration of acetic acid.

Tributyrin (TBU) is a good dietary source of butyrate and has beneficial effects on the maintenance of normal intestinal morphology. The present study tested the hypothesis that dietary TBU supplementation could alleviate intestinal injury in the acetic acid (ACA)-induced porcine model of colitis. A total of eighteen piglets (25 d old) were randomly allocated to one of three treatment groups (control, ACA and TBU). The control and ACA groups were fed a basal diet and the TBU group was fed the basal diet supplemented with 0·1 % TBU. On day 15 of the trial, under anaesthesia, a soft catheter was inserted into the rectum of piglets (20-25 cm from the anus), followed by administration of either saline (control group) or ACA (10 ml of 10 % ACA solution for ACA and TBU groups). On day 22 of the trial, after venous blood samples were collected, piglets were killed to obtain mid-ileum and mid-colon mucosae. Compared with the control group, the ACA group exhibited an increase (P< 0·05) in lymphocyte counts, creatinine, PGE2, and malondialdehyde concentrations and diamine oxidase and inducible NO synthase activities in the plasma and lymphocyte density in the colon and a decrease in insulin concentrations and glutathione peroxidase activity, ileal villus height:crypt depth ratios and goblet cell numbers in the colon. These adverse effects of ACA were attenuated by TBU supplementation. Moreover, TBU prevented the ACA-induced increase in caspase-3 levels while enhancing claudin-1 protein and epidermal growth factor receptor (EGFR) mRNA expression in the colonic mucosa. Collectively, these results indicate that dietary supplementation with 0·1 % TBU alleviates ACA-induced intestinal injury possibly by inhibiting apoptosis, promoting tight-junction formation and activating EGFR signalling.

Reduction of Salmonella enterica serovar typhimurium DT104 infection in experimentally challenged weaned pigs fed a lactobacillus-fermented feed.

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)-1ß, 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.

Determination of the virulence status of Clostridium perfringens strains using a chicken intestinal ligated loop model is important for understanding the pathogenesis of necrotic.

Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.

Functional evaluation of Bacillus licheniformis PF9 for its potential in controlling enterotoxigenic Escherichia coli in weaned piglets.

During the bacterial selection, isolate PF9 demonstrated tolerance to low pH and high bile salt and an ability to extend the lifespan of Caenorhabditis elegans infected with enterotoxigenic Escherichia coli (ETEC; P < 0.05). Thirty-two weaned piglets susceptible to ETEC F4 were randomly allocated to four treatments as follows: 1) non-challenged negative control group (NNC; basal diet and piglets gavaged with phosphate-buffered saline), 2) negative control group (NC; basal diet and piglets challenged with ETEC F4, 3 × 107 CFU per pig), 3) positive control (PC; basal diet + 80 mg·kg-1 of avilamycin and piglets challenged with ETEC F4), and 4) probiotic candidate (PF9; control basal diet + 2.5 × 109 CFU·kg-1 diet of B. licheniformis PF9 and piglets challenged with ETEC F4). The infection of ETEC F4 decreased average daily gain and gain:feed in the NC group when compared to the NNC group (P < 0.05). The inoculation of ETEC F4 induced severe diarrhea at 3 h postinoculum (hpi), 36, 40 hpi in the NC group when compared to the NNC group (P < 0.05). The supplementation of B. licheniformis PF9 significantly relieved diarrhea severity at 3 hpi when compared to the NC group (P < 0.05). The inoculation of ETEC F4 reduced duodenal, jejunal, and ileal villus height (VH) in the NC group when compared to the NNC group. A significant (P < 0.05) decrease was detected in the duodenal VH in the PC and NNC groups. Moreover, the NNC group had a reduced relative mRNA level of Na+-glucose cotransporter 1 (SGLT1) when compared to the NC group (P < 0.05). Compared to the NC and NNC groups, the supplementation of B. licheniformis PF9 increased the relative mRNA levels of aminopeptidase N, occludin, zonula occludens-1, and SGLT1 (P < 0.05). The supplementation of B. licheniformis PF9 also significantly increased the relative mRNA level of excitatory amino acid transporter 1 when compared to the NC group (P < 0.05). Piglets supplemented with B. licheniformis PF9 showed lower relative abundance of Bacteroidetes in the colon than piglets from the NNC group (P < 0.05). The NNC group had a higher relative abundance of Firmicutes in the ileum than all the challenged piglets (P < 0.05); however, a lower relative abundance of Proteobacteria in the ileum and colon was observed in the NC group (P < 0.05). This study provides evidence that B. licheniformis PF9 has the potential to improve the gut health of piglets under challenging conditions.