Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.

Protective Effects of Lactobacillus plantarum 16 and Paenibacillus polymyxa 10 Against Clostridium perfringens Infection in Broilers.

This study aimed to investigate the protective effects of Lactobacillus plantarum 16 (Lac16) and Paenibacillus polymyxa 10 (BSC10) against Clostridium perfringens (Cp) infection in broilers. A total of 720 one-day-old chicks were randomly divided into four groups. The control and Cp group were only fed a basal diet, while the two treatment groups received basal diets supplemented with Lac16 (1 × 108 cfu·kg-1) and BSC10 (1 × 108 cfu·kg-1) for 21 days, respectively. On day 1 and days 14 to 20, birds except those in the control group were challenged with 1 × 108 cfu C. perfringens type A strain once a day. The results showed that both Lac16 and BSC10 could ameliorate intestinal structure damage caused by C. perfringens infection. C. perfringens infection induced apoptosis by increasing the expression of Bax and p53 and decreasing Bcl-2 expression and inflammation evidence by higher levels of IFN-γ, IL-6, IL-1ß, iNOS, and IL-10 in the ileum mucosa, and NO production in jejunal mucosa, which was reversed by Lac16 and BSC10 treatment except for IL-1ß (P < 0.05). Besides, the two probiotics restored the intestinal microbiota imbalance induced by C. perfringens infection, characterized by the reduced Firmicutes and Proteobacteria and the increased Bacteroidetes at the phyla level and decreased Bacteroides fragilis and Gallibacterium anatis at the genus level. The two probiotics also reversed metabolic pathways of the microbiota in C. perfringens-infected broilers, including B-vitamin biosynthesis, peptidoglycan biosynthesis, and pyruvate fermentation to acetate and lactate II pathway. In conclusion, Lac16 and BSC10 can effectively protect broilers against C. perfringens infection through improved composition and metabolic pathways of the intestinal microbiota, intestinal structure, inflammation, and anti-apoptosis.

The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation.

The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response.IMPORTANCEClostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen's growth.

Zinc source modulates intestinal inflammation and intestinal integrity of broiler chickens challenged with coccidia and Clostridium perfringens.

Two dietary sources of zinc (ZnSO4 or organic Zn) were tested in chickens challenged with coccidiosis (Co) or coccidiosis plus Clostridium perfringens (CoCPF). On day 14, the chickens were orally gavaged with ∼5,000 Eimeria maxima sporulated oocysts. On day 19, 20, and 21 chickens challenged with C. perfringens were given a broth culture containing 108 cfu of this bacterium. Productive performance parameters were determined at d 14, 21, and 28. On day 21, necrotic enteritis (NE) lesions were scored, and intestinal permeability was evaluated. Jejunum and cecal tonsils were collected for morphology and gene expression analysis. On day 21, organic Zn improved BW gain by 18.6% (P = 0.07), and FCR by 12% (P = 0.09) in CoCPF challenged chickens vs. birds fed ZnSO4. From 1 to 28, organic Zn increased BW gain (P = 0.02), and improved FCR (P = 0.03) vs. birds fed ZnSO4. At 21 d, NE lesions were only observed in CoCPF birds (P < 0.001), and mortality due to NE was only observed when CoCPF birds were fed ZnSO4 (P = 0.001). Organic Zn fed birds had increased villus height in the jejunum (P = 0.005) and decreased intestinal permeability (P = 0.01) vs. ZnSO4. In the jejunum, organic Zn fed birds showed a downregulation of expression of IL-8 (P = 0.02), and upregulation of IL-10 (P = 0.05) in CoCPF birds vs. ZnSO4- CoCPF birds. As main effect, birds supplemented with organic Zn had higher mRNA expression of TLR-2 (P = 0.02) and IgA (P = 0.01). In the cecal tonsils, organic Zn fed birds showed upregulation of iNOS (P = 0.008) in CoCPF birds vs. ZnSO4-CoCPF birds. Organic Zn supplementation reduced intestinal permeability and attenuated intestinal inflammation of broilers co-challenged with coccidia and C. perfringens.

Characterization of intestinal immune response to Clostridium perfringens infection in broiler chickens.

Necrotic enteritis toxin B (NetB)-producing Clostridium perfringens (CP) type A is the etiological agent of necrotic enteritis (NE) - an economically significant disease in broiler chickens. Understanding the immune response to CP infection in broiler chickens is becoming important to develop effective vaccines against NE. An experiment was conducted to determine the expression levels of selected cytokine genes in the intestine and cecal tonsil of CP-challenged broiler chickens. In a floor-pen housing, broiler chickens were randomly assigned to the following treatment groups: 1) bacitracin methylene disalicylate (BMD)-free control diet with no CP challenge (CX), 2) BMD-supplemented diet with no CP challenge (CM), 3) BMD-free control diet with CP challenge (PCX), or 4) BMD-supplemented diet with CP challenge (PCM). The establishment of CP infection was confirmed, with the treatment groups exposed to CP having a 1.5 to 2-fold higher CP levels (P < 0.05) compared to the non-exposed groups. On day 1 and 7 post-challenge, jejunal segments and cecal tonsils were collected from experimental chickens for quantitative real-time RT-PCR analysis to determine the expression levels of interleukin (IL)-1ß, interferon-γ (IFN-γ), IL-2, IL-13, IL-17, IL-10, and transforming growth factor (TGF)-ß genes. Levels of antibodies to CP recombinant proteins were also determined in the plasma of experimental chickens. Results indicated that on day 7 post-challenge, IL-1ß (proinflammatory cytokine), IL-13 (Th2 cytokine), and IL-17 (Th17 cytokine) were upregulated (P < 0.05) in CP-challenged PCX and PCM treatments, compared to the unchallenged (control) CX and CM treatments. A reverse trend was observed for TGF-ß (anti-inflammatory cytokine), while no change was observed in IFN-γ (Th1 cytokine). Levels of plasma antibodies (IgY) to CP recombinant proteins were higher in CP-challenged treatments (PCX and PCM; P < 0.05), compared to their corresponding controls (CX and CM). It was concluded that CP infection induced inflammatory response in the intestine of broiler chickens, and the mechanisms of inflammation are probably mediated via Th2 and Th17 cells.

Zinc source influences the gene expression of zinc transporters in jejunum and cecal tonsils during broiler challenge with Eimeria maxima and Clostridium perfringens.

The objective of this experiment was to study the effects of dietary zinc (Zn) source on gene expression of Zn transporters (metallothionein [MT], ZIP 3, 5, 8, 9, 10, 11, 13, and 14, and ZnT 1, 4, 5, 6, 7, 9, and 10) in the jejunum and cecal tonsils of broilers challenged with coccidia or coccidia plus Clostridium perfringens. A 2 × 2 factorial design was used with 2 Zn sources (90 mg Zn/kg from either ZnSO4 or an organic Zn, Bioplex® Zn) and challenged with approximately 5,000 oocysts of Eimeria maxima at 14 d of age with or without C. perfringens (108 CFU/bird) at 18, 19, and 20 d of age (8 pens per treatment and 8 birds per pen) after which 1 bird/pen was sampled at 21 d of age. In the jejunum, co-infection resulted in higher ZnT 5 and 6 gene expression, while organic Zn fed birds had lower ZIP 5 and 11, and higher ZnT1. Additionally, an interaction of challenge by Zn source was noted wherein ZnT10 was unaffected by the C. perfringens in the organic Zn treatment but was 2.7-fold lower in the co-infected ZnSO4 fed birds. S100A9 gene expression, a biomarker of inflammatory response in necrotic enteritis, increased 2 and 2.8-fold in the cecal tonsils and jejunum with the co-infection, respectively. Supplementation with organic Zn lowered S100A9 by 1.9 and 4.4-fold in the cecal tonsils and jejunum, respectively, when birds were supplemented with ZnSO4. Notably, MT, ZIP 3, 8, 9, 10, 13, or 14, and ZnT 4, 7, and 9 were unaffected by Zn source and/or method of challenge. An interaction of challenge by Zn source was also noted for serum Zn concentration, which was reduced when birds were challenged with C. perfringens and fed ZnSO4 but no difference between challenge method when birds were fed organic Zn. Based on the expression of ZnT and ZIP genes, more Zn trafficking due to treatment occured in the jejunum than cecal tonsils, but further studies are needed to ascertain how Zn source regulates intracellular free Zn concentrations and whole-body Zn status during an enteric challenge.

Histological parameters to evaluate intestinal health on broilers challenged with Eimeria and Clostridium perfringens with or without enramycin as growth promoter.

The maintenance of integrity of the gastrointestinal tract is an important aspect for animal productivity, since it is able to absorb nutrients more efficiently and serves as a barrier against microorganisms. To control agents detrimental to intestinal integrity, growth-promoting antibiotics (AGP) are used, which reduce the number of toxin-producing microorganisms in the intestinal lumen, acting as anti-inflammatory agents. There is a demand for restriction of use of AGP in animal feed, but there are few studies showing what parameters we should observe to search for alternative additives. The aim of this study was to establish histological parameters that explain the effect of enramycin as growth promoter on intestinal health in broilers challenged with Eimeria and Clostridium perfringens. The zootechnical performance and the histology by I See Inside (ISI) methodology were evaluated on liver and ileum samples. Chickens challenged without AGP have the worst BWG, FCR, and histological ISI score (ISI score 9) in the ileum compared to non-challenged (ISI score 5). The use of enramycin on challenged group significantly minimized the ISI score in the ileum at 21 and 28 d (ISI score 7.4 and 8.0, respectively) compared with the challenged group not fed with enramycin (ISI score 9.2 and 9.9, respectively), associated with reduced lamina propria thickness and inflammatory cell infiltration. We suggest these 2 histological parameters as a standard to compare products for gut health.

Allium hookeri supplementation improves intestinal immune response against necrotic enteritis in young broiler chickens.

Three hundred birds (1 day old) were randomly assigned to 6 groups (n = 50 birds/treatment) and fed a basal diet (control) or basal diet supplemented with Allium hookeri (AH) root (1 or 3%). At day 14, half of the birds in each group were orally challenged with E. maxima 41A (1 × 104 cells/chicken), followed by C. perfringens infection (1 × 109 cfu/chicken) on day 18. Necrotic enteritis (NE)-associated infections and intestinal immune response were assessed by average body weight gain, lesion score, and oocyst shedding. The effect of dietary supplementation, AH, on transcript levels of pro-inflammatory cytokines, and tight junction proteins and mucin protein in the jejunum, were quantified by quantitative real-time (qRT)-PCR. At day 20, birds fed with diet supplementation (3% of AH) significantly weighted more than the control group. Although the NE-challenged had significantly reduced average body weight gain, there was no significance in the effect between diet × NE-challenge interactions on the average body weight gain. Among the NE-challenged groups, gut lesion score and oocyst shedding were significantly decreased in birds given AH (1 or 3%) compared to the control group. There was a correlation between diet and NE infection with regards to interleukin (IL)-17A, and inducible nitric oxide synthase (iNOS). The up-regulated transcript levels of cytokines IL-8, IL-17A, iNOS, and LITAF by NE challenged groups were significantly reduced by AH (1 or 3%) supplementation. Down-regulated expression levels of tight junction (TJ) proteins: junctional adhesion molecule 2 (JAM2), occluding, and intestinal mucin 2 (MUC2) by NE challenge, was up-regulated by the addition of AH (1 or 3%) supplementation. All TJ proteins (JAM2, ZO1, Ocluddin and MUC2) in the jejunum had a significant diet × NE-challenge interaction. These findings demonstrate that dietary supplementation of AH in chicken feed could be beneficially used to improve chicken health against NE.

Faecalibacterium prausnitzii and a Prebiotic Protect Intestinal Health in a Mouse Model of Antibiotic and Clostridium difficile Exposure.

BACKGROUND: Clostridium difficile (CD) infection (CDI) increases patient morbidity, mortality and healthcare costs. Antibiotic treatment induces gut dysbiosis and is both a major risk factor for CD colonization and treatment of CDI. Probiotics have been trialed to support commensal gut microbiota and reduce CDI. This study investigated commensal microbe Faecalibacterium prausnitzii (FP) and a prebiotic, both known to yield butyrate and be anti-inflammatory and immunomodulatory, on CD colonization and gut integrity in mice. METHODS: Mice were randomly grouped and supplemented daily with FP, prebiotic, FP + prebiotic, FP/prebiotic supernatant, or saline throughout the entire study. Following treatment with clindamycin for 3 days, mice were exposed to CD. Feces were collected at baseline, the day after antibiotic, and 1, 3, and 5 days after CD exposure and cultured for bacterial overgrowth and CD colonization. On days 1 and 5 after CD exposure, mice were randomly euthanized, and proximal colon was dissected for histological analysis and preparation of RNA for analysis of proinflammatory and anti-inflammatory cytokines. RESULTS: Although all mice exhibited bacterial overgrowth and CD colonization, bacterial burden resolved quicker in the FP + prebiotic group. This was associated with induction and resolution of innate immune responses, anion exchanger, and tight junction protein preservation in proximal colon. CD toxin virulence potential was questionable as expression of CD toxin B receptor was depleted in the FP + prebiotic group. CONCLUSION: Supplementation with anti-inflammatory butyrate-supporting commensal bacteria and prebiotic may support innate immune responses and minimize bacterial burden and negative effects during antibiotic and CD exposure.

Influence of different yeast cell wall preparations and their components on performance and immune and metabolic pathways in Clostridium perfringens-challenged broiler chicks.

A study was conducted to evaluate the influence of the purification of yeast cell wall (YCW) preparations on broiler performance and immunogenic and metabolic pathways under microbial challenge. A total of 240 (day old) chicks were distributed among two battery brooder units (48 pens; 5 birds/pen; 8 replicates/treatment). A basal starter diet was divided into 5 batches to create 6 dietary treatments; non-challenge (NCh-C) and challenge (Ch-C) controls, semi-purified YCW containing cytosol contents (SPYCW; 250 mg/kg), purified YCW (PYCW; 250 mg/kg), 50% purified beta-glucan (BG; 130 mg/kg), and 99.9% purified mannan-oligosaccharide (MOS; 53 mg/kg). All birds were immunocompromised with infectious bursal disease vaccine (10× the recommended dose) on day 10 and then all birds except NCh-C birds were challenged with Clostridium perfringens (Cp) (107 cfu/mL) via oral gavage on days 16 and 17. On day 21, tissue samples were collected from the jejunum and duodenum for analysis with chicken-specific, peptide arrays to study the influence of YCW supplementation on immune and metabolic kinase pathways. On day 16, SPYCW had significantly lower body weight (BW) and weight gain (WG) than other treatments except BG (P < 0.05). The productivity index (PI) was lower in SPYCW and BG than in NCh-C, Ch-C, and PYCW. On day 21, after the Cp challenge, NCh-C was higher than Ch-C, SPYCW, and BG in BW, WG, and PI (P = 0.03). The PI of PYCW was similar to NCh-C. The addition of purified YCW to the starter broiler diets influenced the immune and metabolic pathways in the gut. A total of 459 and 367 peptides in the duodenum and jejunum, respectively, were changed due to the Cp challenge. The YCW treatments had different degrees of influence on these peptides for both the duodenum and jejunum. These results suggest that relative purification of YCW and specific fractions of the YCW can influence broiler performance differently during microbial challenges and can alleviate the impact of these stressors.

Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

Amelioration of Clostridium difficile Infection in Mice by Dietary Supplementation With Indole-3-carbinol.

OBJECTIVE: To determine the therapeutic effects of dietary supplementation on Clostridium difficile infection (CDI). BACKGROUND: With limited treatment options, the rise of C. difficile-associated disease has spurred on the search for novel therapies. Recent data define a role for the aryl hydrocarbon receptor (AHR) and diet-derived AHR ligands in mucosal immunity. We investigated the efficacy of indole-3-carbinol (I3C), a dietary supplement, and AHR precursor ligand in a murine model of CDI. METHODS: C57BL/6 (B6), AHR, and AHR mice were placed on either grain-based or semipurified diets with or without I3C before and during CDI. Mice were followed clinically for a minimum of 6 days or euthanized between days 0 and 4 of inoculation for analysis of the inflammatory response and microbiota. RESULTS: B6 mice fed an AHR ligand-deficient, semipurified diet have significantly increased disease severity (P<0.001) and mortality (P < 0.001) compared with mice fed on diet containing I3C. The addition of I3C to the diet of AHR null mice had less of an impact than in AHR heterozygous littermates, although some protection was seen. Mice on semipurified I3C-diet had increased cecal Tregs, ILC3s, and γδ T cells and an increased neutrophilic response without increased inflammation or bacterial translocation compared with controls. CONCLUSIONS: I3C is a powerful treatment to reduce impact of CDI in mice. The findings indicate I3C may be acting through both AHR-dependent and -independent mechanisms in this model. Dietary supplementation with I3C is a potential new therapy for prevention and amelioration of C. difficile disease.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance and local innate immune response of broiler chickens challenged with Clostridium perfringens.

This study evaluated the effect of yeast-derived products on growth performance, gut lesion score, intestinal population of Clostridium perfringens, and local innate immunity of broiler chickens challenged with C. perfringens. One-day-old broiler chickens were randomly assigned to eight dietary treatments providing six replicate pens of 55 birds each per treatment. Dietary treatments consisted of Control diets without and with C. perfringens challenge, and diets containing bacitracin methylene disalicylate (BMD, 55 g/tonne), nucleotides (150 g/tonne), yeast cell wall (YCW, 300 g/tonne), and a commercial product Maxi-Gen Plus (1 kg/tonne) fed to chickens challenged with C. perfringens. Diets containing 10% distillers dried grains with solubles without and with C. perfringens challenge were also used. Birds were orally challenged with C. perfringens (10(8) colony-forming units (cfu)/bird) on day 14. On day 21, intestinal samples were collected for gene expression analysis. Pathogen challenge significantly (P < 0.05) impaired feed intake, body weight gain, and feed conversion ratio (FCR) shortly after the challenge (14-21 days). Increased C. perfringens counts and intestinal lesion scores were observed for challenged birds except the BMD-containing diet. Over the entire trial (1-35 days), no difference in growth performance was observed except the BMD diet which improved FCR over the Control, challenged group. Birds receiving nucleotides showed increased expression of toll-like receptors and cytokines interleukin (IL)-4 and IL-18 compared to the Control, challenged group. Expression of macrophage mannose receptor and IL-18 was upregulated in birds receiving YCW. Increased expression of cytokines and receptors involved in innate immunity in broilers receiving nucleotides and YCW suggests the immunomodulatory properties of these products under pathogen challenge conditions.

Hyperimmune bovine colostrum as a novel therapy to combat Clostridium difficile infection.

BACKGROUND: Clostridium difficile is a primary cause of antibiotic-associated diarrhea that typically develops when gut microbiota is altered. Conventional treatment for C. difficile infection (CDI) is additional antimicrobial administration, which further disrupts normal intestinal microbiota, often resulting in poor treatment outcomes. METHODS: A pregnant dairy cow was repeatedly immunized with recombinant mutants of toxins A and B produced by C. difficile, and the resultant hyperimmune bovine colostrum (HBC) was evaluated for therapeutic efficacy in gnotobiotic piglets with diarrhea due to CDI. Control piglets received nonimmune colostrum. To determine the impact of HBC on gut microbiota, 1 of 2 groups of piglets transplanted with normal human gut microbiota was treated with HBC. RESULTS: Nonimmune colostrum-treated piglets developed moderate to severe diarrhea and colitis. In contrast, HBC-treated piglets had mild or no diarrhea and mild or no colitis. Lyophilization had no detectable impact on HBC efficacy. HBC had no discernible effect on the composition of normal human gut microbiota in the porcine intestinal tract. CONCLUSIONS: HBC provides an oral, cost-effective, and safe alternative to antibiotic therapy for CDI. By preserving intestinal microbiota, HBC may be more efficacious than antibiotics. Additional studies are warranted to establish HBC as a viable immunotherapeutic agent for human use against CDI.

Dietary sodium selenite affects host intestinal and systemic immune response and disease susceptibility to necrotic enteritis in commercial broilers.

1. This study was to evaluate the effects of supplementary dietary selenium (Se) given as sodium selenite on host immune response against necrotic enteritis (NE) in commercial broiler chickens. 2. Chicks were fed from hatching on a non-supplemented diet or diets supplemented with different levels of Se (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, broiler chickens were orally infected with Eimeria maxima at 14 d of age and then with Clostridium perfringens 4 d later using our previously established NE disease model. 3. NE-associated clinical signs and host protective immunity were determined by body weight changes, intestinal lesion scores, and serum antibodies against α-toxin and necrotic enteritis B (NetB) toxin. The effects of dietary Se on the gene expression of pro-inflammatory cytokines e.g., interleukin (IL)-1ß, IL-6, IL-8LITAF (lipopolysaccharide-induced TNFα-factor), tumour necrosis factor (TNF) SF15, and inducible nitric oxide synthase (iNOS), glutathione peroxidase 7 (GPx7), and avian ß-defensins (AvBD) 6, 8, and 13 (following NE infection) were analysed in the intestine and spleen. 4. The results showed that dietary supplementation of newly hatched broiler chicks with 0.25 Se mg/kg from hatch significantly reduced NE-induced gut lesions compared with infected birds given a non-supplemented diet. The levels of serum antibody against the NetB toxin in the chicks fed with 0.25 and 0.50 mg/kg Se were significantly higher than the non-supplemented control group. The transcripts for IL-1ß, IL-6, IL-8, iNOS, LITAF, and GPx7, as well as AvBD6, 8, and 13 were increased in the intestine and spleen of Se-supplemented groups, whereas transcript for TNFSF15 was decreased in the intestine. 5. It was concluded that dietary supplementation with optimum levels of Se exerted beneficial effects on host immune response to NE and reduced negative consequence of NE-induced immunopathology.

Passive and active immunization strategies against Clostridium difficile infections: state of the art.

The pathophysiology of Clostridium difficile infections (CDI) could be considered as a three-step process that takes place after disruption of the digestive microbiota by antibiotics: 1) germination of spores; 2) multiplication and persistence of C. difficile in the colonic niche thanks to colonization factors; 3) production of the two toxins TcdA and TcdB and for some strains an additional toxin, the binary toxin CDT. Different immunization strategies against C. difficile have been developed, first regarding the toxins. Immunization assays with colonization factors have followed, and allowed accumulation of new data concerning theirs functions and immunogenicity. Here, we present the toxins, the colonization factors and their use in passive and active immunizations to treat and/or to prevent C. difficile infections. The various experiments performed in animal models and the first clinical trials in humans are reported.

Fecal microbiota transplantation in the treatment of refractory Clostridium difficile infection in children: an update.

PURPOSE OF REVIEW: The use of transplanted fecal material for the treatment of diarrheal illness dates back to the fourth-century China. While fecal microbiota transplant has gained increasing popularity over the past 50 years for the treatment of refractory Clostridium difficile infections (RCDIs) in adults, it has only been recently utilized in children. The purpose of this article is to review the use of fecal microbiota transplant (FMT) in the treatment of pediatric RCDIs. RECENT FINDINGS: Minimal pediatric data, including few case reports and series, document the successful use of FMT for treatment of RCDI in the past 2 years. Patients in these reports included otherwise healthy children, those with inflammatory bowel disease as well as significantly immunocompromised children. Donor fecal infusion via nasogastric tube, gastroscope or colonoscope in children aged 16 months and older demonstrated a high rate of symptom resolution and organism eradication. No complications to date have been reported in children who have undergone FMT. SUMMARY: FMT is emerging as a well-tolerated and effective treatment for RCDI in not only adults but also children.

Effects of in ovo injection with selenium on immune and antioxidant responses during experimental necrotic enteritis in broiler chickens.

This study was conducted to investigate the effects of in ovo injection of Se on modulating the immune system and antioxidant responses in broiler chickens with experimental necrotic enteritis. Broiler eggs were injected at 18 d of embryo age with either 100 µL of PBS alone or sodium selenite (Na2SeO3) in PBS, providing 0 (SS0), 10 (SS10), or 20 (SS20) µg of Se/egg. At 14 d posthatch, PBS-treated and uninfected chickens were kept as the control group, whereas the remaining chickens were orally infected with 1.0 × 10(4) sporulated oocysts of Eimeria maxima (SS0, SS10, SS20). At 18 d posthatch, E. maxima-infected chickens were orally infected with 1.0 × 10(9) cfu of Clostridium perfringens. Infected control SS0 group showed significantly decreased BW compared with the uninfected control. However, SS20 group showed significantly increased BW compared with the infected control SS0 group, whereas the BW were similar among uninfected control and infected SS10 and SS20 groups. The SS10 group showed significantly lower intestinal lesions compared with the SS0 group, and oocyst production was decreased in both SS10 and SS20 groups. Serum malondialdehyde level and catalase activity were also decreased in both SS10 and SS20 groups, whereas the superoxide dismutase level was significantly lower in the SS10 group compared with the SS0 group. The SS20 group showed significantly higher levels of transcripts for IL-1ß and IL-6 in intestine, and SS10 and SS20 groups had higher levels of transcripts for IL-8 and inducible nitric oxide synthase expression and decreased glutathione peroxidase 7 mRNA levels compared with the SS0 group. The SS10 and SS20 groups also showed increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with the SS0 group. These collective results suggest that the injection of Se into the amniotic cavity of developing eggs may be beneficial for enhancing immune and antioxidant responses in the hatched chickens exposed to the necrotic enteritis-causing pathogens.

Effect of 3',5'-cyclic diguanylic acid in a broiler Clostridium perfringens infection model.

In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains. Birds were treated with saline (control group) or 20 nmol of c-di-GMP by gavage or intramuscularly (IM) on d 24, all in conjunction with penicillin G in water for 5 d. Weekly samplings of ceca and ileum were performed on d 21 to 35 for C. perfringens and Lactobacillus enumeration. On d 35 of age, the IM treatment significantly (P < 0.05) reduced C. perfringens in the ceca, suggesting possible synergistic activity between penicillin G and c-di-GMP against C. perfringens in broiler ceca. Moreover, analysis of ceca DNA for the presence of a series of C. perfringens virulence genes showed a prevalence of 30% for the Clostridium perfringens alpha-toxin gene (cpa) from d 21 to 35 in the IM-treated group, whereas the occurrence of the cpa gene increased from 10 to 60% in the other 2 groups (control and gavage) from d 21 to 35. Detection of ß-lactamase genes (blaCMY-2, blaSHV, and blaTEM) indicative of gram-negative bacteria in the same samples from d 21 to 35 did not show significant treatment effects. Amplified fragment-length polymorphism showed a predominant 92% similarity between the ceca of 21-d-old control birds and the 35-d-old IM-treated c-di-GMP group. This suggests that c-di-GMP IM treatment might be effective at restoring the normal microflora of the host on d 35 after being challenged by C. perfringens. Our results suggest that c-di-GMP can reduce the colonization of C. perfringens in the gut without increasing the selection pressure for some ß-lactamase genes or altering the commensal bacterial population.