Clostridium Perfringens-induced Necrotic Enteritis Research Articles

Dietary ellagic acid ameliorated Clostridium perfringens-induced subclinical necrotic enteritis in broilers via regulating inflammation and cecal microbiota

BackgroundSubclinical necrotic enteritis (SNE), a common intestinal disease of broiler caused by Clostridium perfringens, could reduce production performance of broilers by chronic intestinal damage and poor absorption of nutrients. Ellagic acid (EA) has been reported to present antioxidant and anti-inflammatory properties on human and animals in many aspects. This study was conducted to evaluate the effect and mechanism of EA in relieving SNE in broilers induced by C. perfringens.ResultsC. perfringens challenge decreased body weight (BW), average daily gain (ADG), jejunal villi height/crypt depth (V/C) ratio, the activity of catalase (CAT) and the mRNA expression of zonula occludens 1 (ZO-1) in jejunal mucosa of broilers. While feed conversion ratios (FCR), jejunal crypt depth (CD), the activities of myeloperoxidase (MPO) and diamine oxidase (DAO), as well as the concentrations of interleukin 6 (IL-6), C-reactive protein (CRP) and procalcitonin (PCT) in serum, the activities of inducible nitric oxide synthase (iNOS) and lysozyme (LZM), the concentration of malondialdehyde (MDA), and the mRNA expressions of claudin-2, TNF-α, IL-1β, TLR-4, TLR-2, NF-κB, JAK3, STAT6 and iNOS in jejunal mucosa of broilers were increased by C. perfringens challenge. Dietary EA supplement relieved these adverse effects, and heightened jejunal villi height (VH), the concentration of D-xylose in plasma, activity of superoxide dismutase (SOD), and the mRNA expression of occludin in jejunal mucosa of broilers. The alpha diversity of cecal microbiota indicated that dietary EA supplement increased observed species and Shannon index. C. perfringens challenge increased the relative abundance of Firmicutes and decreased the relative abundance of Desulfobacterota in cecal microbiota. EA increased the relative abundance of Firmicutes in cecal microbiota. LEfSe analysis showed that C. perfringens challenge triggered the imbalance of cecal microbiota in broilers, dietary EA supplementation led to a small beneficial effect on microbiota, while the simultaneous effect of them seemed to stimulate the immune function of broilers by improving the microbiota balance.ConclusionsDietary EA ameliorated C. perfringens-induced SNE in broilers via regulating jejunal inflammation signaling pathways TLR/NF-κB and JAK3/STAT6, relieving jejunal oxidative stress and balancing cecal microbiota to inhibit intestinal barrier damage, prevent systemic inflammatory response and improve nutrient absorption capacity, finally protect and enhance growth performance of broilers.

Intestinal changes and immune responses during Clostridium perfringens-induced necrotic enteritis in broiler chickens

Clostridium perfringens-induced necrotic enteritis (NE) is an economically important disease of broiler chickens. The present study evaluated the effect of C. perfringens on the intestinal histomorphometry, enteric microbial colonization, and host immune responses using 3 experimental NE reproduction methods. The experimental groups consisted of 1) unchallenged Control diet (corn-soybean meal), 2) Control diet + Eimera inoculation at d 11 followed by C. perfringens challenge at d 15 (ECp), 3) Wheat-based diet + C. perfringens challenge (WCp), and 4) Wheat-based diet + Eimeria inoculation followed by C. perfringens challenge (WECp). The results showed that chickens receiving ECp and WECp had reduced (P < 0.05) bird performance coupled with enteric gross lesions and epithelial damage at d 17 and 24 of age compared to unchallenged control birds. These ECp and WECp administered birds also had increased (P < 0.05) ileal colonization by clostridia and E. coli at d 17 and 24, while the resident Lactobacillus counts were reduced (P < 0.05) at d 24 of age. Furthermore, at d 24, jejunal transcription of IL-6, IL-10, annexin-A1 and IL-2 genes was upregulated (P < 0.05) in the ECp group, whereas the transcription of TNF receptor associated factor (TRAF)-3 gene was increased (P < 0.05) in WECp treated birds when compared to unchallenged control group. Additionally, stimulation of chicken splenocytes and cecal tonsilocytes with virulent C. perfringens bacilli or their secretory proteins resulted in a higher (P < 0.05) frequency of T cells and their upregulation of MHC-II molecule, as determined by flow cytometry. These findings suggest that C. perfringens, while inducing epithelial damage and changes in microbiota, can also trigger host immune responses. Furthermore, NE reproduction methods using coccidia with or without the wheat-based dietary predisposition seem to facilitate an optimal NE reproduction in broiler chickens and thus, may provide better avenues for future C. perfringens research.

A triple-sugar regulated Salmonella vaccine protects against Clostridium perfringens-induced necrotic enteritis in broiler chickens

Gram-positive Clostridium perfringens type G, the causative agent of necrotic enteritis (NE), has gained more attention in the poultry industry due to governmental restrictions on the use of growth-promoting antibiotics in poultry feed. Our previous work has proved that regulated delayed lysis Salmonella vaccines delivering a plasmid encoding an operon fusion of the nontoxic C-terminal adhesive part of alpha toxin and a GST-NetB toxin fusion were able to elicit significant protective immunity in broilers against C. perfringens challenge. We recently improved our S. Typhimurium antigen delivery vaccine strain by integrating a rhamnose-regulated O-antigen synthesis gene enabling a triple-sugar regulation system to control virulence, antigen-synthesis and lysis in vivo traits. The strain also includes a ΔsifA mutation that was previously shown to increase the immunogenicity of and level of protective immunity induced by Salmonella vectored influenza and Eimeria antigens. The new antigen-delivery vaccine vector system confers on the vaccine strain a safe profile and improved protection against C. perfringens challenge. The strain with the triple-sugar regulation system delivering a regulated lysis plasmid pG8R220 encoding the PlcC and GST-NetB antigens protected chickens at a similar level observed in antibiotic-treated chickens. Feed conversion and growth performance were also similar to antibiotic-treated chickens. These studies made use of a severe C. perfringens challenge with lesion formation and mortality enhanced by pre-exposure to Eimeria maxima oocysts. The vaccine achieved effectiveness through three different immunization routes, oral, spray and in drinking water. The vaccine has a potential for application in commercial hatcher and broiler-rearing conditions.

Butyrate and Forskolin Augment Host Defense, Barrier Function, and Disease Resistance Without Eliciting Inflammation.

Host defense peptides (HDPs) are an integral part of the innate immune system with both antimicrobial and immunomodulatory activities. Induction of endogenous HDP synthesis is being actively explored as an antibiotic-alternative approach to disease control and prevention. Butyrate, a short-chain fatty acid, and forskolin, a phytochemical, have been shown separately to induce HDP gene expression in human cells. Here, we investigated the ability of butyrate and forskolin to induce the expressions of chicken HDP genes and the genes involved in barrier function such as mucin 2 and claudin 1 both in vitro and in vivo. We further evaluated their efficacy in protecting chickens from Clostridium perfringens-induced necrotic enteritis. Additionally, we profiled the transcriptome and global phosphorylation of chicken HD11 macrophage cells in response to butyrate and forskolin using RNA sequencing and a kinome peptide array, respectively. Our results showed a strong synergy between butyrate and forskolin in inducing the expressions of several, but not all, HDP genes. Importantly, dietary supplementation of butyrate and a forskolin-containing plant extract resulted in significant alleviation of intestinal lesions and the C. perfringens colonization in a synergistic manner in a chicken model of necrotic enteritis. RNA sequencing revealed a preferential increase in HDP and barrier function genes with no induction of proinflammatory cytokines in response to butyrate and forskolin. The antiinflammatory and barrier protective properties of butyrate and forskolin were further confirmed by the kinome peptide array. Moreover, we demonstrated an involvement of inducible cAMP early repressor (ICER)-mediated negative feedback in HDP induction by butyrate and forskolin. Overall, these results highlight a potential for developing butyrate and forskolin, two natural products, as novel antibiotic alternatives to enhance intestinal health and disease resistance in poultry and other animals.

Efficacy of probiotic Bacillus licheniformis DSM 28710 on performance and the mitigation of Clostridium perfringens-induced necrotic enteritis in broiler chickens

The aim of this study was to evaluate the impact of a probiotic Bacillus licheniformis strain (DSM 28710; B-Act®) on growth performance and its capacity to mitigate necrotic enteritis (NE; induced via a Clostridium perfringens challenge) in poultry. A broiler trial was conducted, examining three treatments for 42 days under an induced NE challenge; a negative control (basal diet only); an antibiotic treated group (oxytetracycline hydrochloride (OXT), therapeutic dose of 105 mg OXT/litre in drinking water, for three days after C. perfringens challenge); and a B-Act group (500 g B-Act/tonne of feed, equalling 1.6×1012 colony forming units B. licheniformis DSM 28710/tonne of feed, supplemented from start until finish). Despite the induced NE challenge, weight gains of the B-Act and OXT groups were similar to each other but significantly higher compared to the control at the end of the study (P&lt;0.05). Weight gain of the B-Act group was already significantly higher compared to the control on day 21 (P&lt;0.05), indicating a potential benefit of the probiotic even before clinical establishment of NE. Feed conversion ratio (FCR) values followed a similar pattern throughout the study, with a significantly lower overall FCR for the B-Act and OXT groups compared to the control (P&lt;0.05; d0-42). Birds fed B-Act had significantly (P&lt;0.05) lower NE lesions compared to the control and OXT group on day 21, although OXT was not supplemented to the animals at this stage yet. Both B-Act and OXT groups had significantly (P&lt;0.05) lower NE scores than the control on day 28, demonstrating the effectiveness of the antibiotic treatment and the mitigating effect of B-Act on the effects of a Clostridium perfringens induced NE challenge.

Effect of Bacillus subtilis DSM 32315 on the intestinal structural integrity and growth performance of broiler chickens under necrotic enteritis challenge

The effect of dietary inclusion of Bacillus subtilis DSM 32315 on the intestinal health and growth performance of Cobb 500 male broilers subjected to a Clostridium perfringens-induced necrotic enteritis (NE) challenge was determined in 2 experiments. In experiment 1, chicks were randomly assigned to 4 treatments of 10 replicate/treatment. In experiment 2, chicks were randomly assigned to 4 treatments of 12 replicates/treatment. The experimental treatments were non-infected, non-supplemented control, infected, non-supplemented control (IC), infected + Bacillus subtilis DSM 32315 (B. subtilis DSM 32315), infected + bacitracin methylene disalicylate (BMD). In both experiments, NE was induced by oral inoculation of toxin producing C. perfringens on 3 consecutive days between 17 and 20 D of age, following exposure of birds to pre-disposing conditions. At day 28 (experiment 1), broilers fed diets with B. subtilis DSM 32315 exhibited a significantly higher body weight, lower mortality, and intestinal NE lesion score, compared to the IC treatment. At day 42 (experiment 2), B. subtilis DSM 32315 supplementation significantly improved BW, feed conversion ratio, production efficiency factor, NE lesion score, and mortality, compared to IC treatment. The effect of B. subtilis DSM 32315 on intestinal integrity of NE challenged chickens was evaluated with histomorphometry. A significantly shallower crypt depth and higher villus height to crypt depth ratio were observed in the mid-intestine of birds belonging to the B. subtilis DSM 32315 group, compared to the IC group. Furthermore, B. subtilis DSM 32315 supplementation significantly reduced the enteritis index associated with NE. In both experiments, the effect of B. subtilis DSM 32315 on the phenotypic measurements of NE and performance was comparable to the effect observed with BMD supplementation. In conclusion, supplementation of the direct fed microbial strain B. subtilis DSM 32315 can ameliorate the pathology and performance detriments associated with NE.

Bacillus subtilis B21 and Bacillus licheniformis B26 improve intestinal health and performance of broiler chickens with Clostridium perfringens-induced necrotic enteritis.

This study investigated the influence of Bacillus-based probiotics on performance and intestinal health in broiler challenged with Clostridium perfringens-induced necrotic enteritis. One-day-old Arbor Acre (n=480) were randomly assigned to four treatments with 10 cages of 12 birds: (a) basal diet negative control (NC), with no probiotics nor antibiotics formulated to contain 2,930 and 3,060kcal/kg with 24.07 and 15.98% CP, for starter and finisher diet, respectively, (b) basal diet+enramycin (5mg/kg), an antibiotic growth promoter (AGP); (c) basal diet+Bacillus subtilis B21 at 2 × 109 CFU per g (BS); (d) basal diet+Bacillus licheniformis B26 at 2 × 109 CFU per g (BL); growth performance, intestinal morphology, intestinal lesion scores, short-chain fatty acids (SCFAs) and mucosal barrier tight junction's (TJ) mRNA expression were assessed. NC- and BL-fed groups showed higher (p=0.005) average daily feed intake from d1 to d21 than AGP and BS, whereas BS- and AGP-fed groups showed higher average daily weight gain from d22 to d42 and d1 to d42 of age. Higher mortality rate of (12.5%) and lower of (5.5%) were recorded in AGP and NC fed-groups respectively, lesion score was higher in BS and BL than in AGP, while no lesion was observed in NC group, results revealed higher duodenum and jejunum villus height to crypt depth (VH:CD) compared with NC and BS. Probiotics-fed groups showed higher total (SCFAs), acetic and butyric acid concentrations at d21 post-challenge (PC) than other groups. The expression of claudin-1 was upregulated in duodenum (d7) PC and in jejunum (d7) and (d21) PC in BL group, while at d21 PC, the expression of occludens was higher in jejunum and ileum by AGP and BL. The present study indicated both BS and BL have some similarity with AGP in preventing or partially preventing NE effect in broilers.

Evaluation Effect of Silver Acetate on Performance and Clostridium Perfringens-Induced Necrotic Enteritis in Broiler Chickens

Evaluation Effect of Silver Acetate on Performance and Clostridium Perfringens-Induced Necrotic Enteritis in Broiler Chickens

Optimization of solid-state fermentation conditions of Bacillus licheniformis and its effects on Clostridium perfringens-induced necrotic enteritis in broilers

ABSTRACT In the present study, we examined the growth parameters of Bacillus licheniformis in solid-state fermentation (SSF) and evaluated the effects of Bacillus licheniformis-fermented products on Clostridium perfringens-challenged broilers. During four and six days of SSF, the highest viable biomass was observed at 5% glucose, 10% soybean meal, 3% yeast, and 50% initial moisture content. The Bacillus licheniformis SSF products were heat- and acid-resistant. Furthermore, the fermented products were able to inhibit the growth of Clostridium perfringens and Staphylococcus aureus [...]

Optimization of surfactin production from Bacillus subtilis in fermentation and its effects on Clostridium perfringens-induced necrotic enteritis and growth performance in broilers.

Bacillus species are commonly used as probiotics in the poultry feed industry for preventing infectious diseases and improving productivity by altering gastrointestinal microbiota. The growth parameters of Bacillus subtilis for surfactin production in fermentation and the benefits of surfactin on broiler chickens remain unclear. In this study, we examined the growth parameters of B.subtilis in fermentation and evaluated the effects of surfactin from B.subtilis-fermented products on Clostridium perfringens-induced necrotic enteritis and growth performance in broilers. Results showed that the highest viable biomass of B.subtilis was observed at 10% molasses and 2% yeast supplementation during fermentation. The 4- and 6-day fermented B.subtilis products were heat-, acid- and bile-resistant. Furthermore, the 4-day fermented B.subtilis products with the highest surfactin concentration showed the maximal antimicrobial activity against pathogens, including Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and C.perfringens. Dietary B.subtilis-fermented product supplementation in broilers significantly improved intestinal morphology and necrotic lesions under C.perfringens challenge. Bacillus subtilis treatments could enhance broiler productivity, as well as promote bone quality and intestinal morphology. These results together indicate that B.subtilis-fermented products containing surfactin have potential for the development as feed additives and use as possible substitutes for antibiotics to treat C.perfringens in the poultry industry.

Bacillus licheniformis normalize the ileum microbiota of chickens infected with necrotic enteritis

Necrotic enteritis (NE) is a severe intestinal disease, which can change gut microbiota and result in a high cost for the poultry industry worldwide. However, little is known regarding how the gut microbiota of NE chicken ileum are changed by Bacillus licheniformis. This study was conducted to investigate how ileum microbiota structure was changed by B. licheniformis in broiler chickens challenged with Clostridium perfringens-induced NE through Illumina MiSeq sequencing. The broilers were randomly separated into four groups: the negative control group (NC), the positive control group (PC), the fishmeal and coccidia group (FC), and the PC group supplied with feed containing B. licheniformis (BL). Compared to the PC and FC, alpha diversity, beta diversity, and the bacterial taxa of the ileum microbiota were more similar in BL and NC. Some genera, which were related to the NE control, became insignificant in BL with NC, such as Lactobacillus, Lactococcus, Bacteroides, Ruminococcus and Helicobacter. The PICRUSt analysis revealed that a tumour suppressor gene, p53, which was negatively correlated with Helicobacter, was enriched in the BL group. Our findings showed that the ileum microbiota disorder caused by NE in chickens was normalized by dietary B. licheniformis supplementation.

Effects of Bacillus coagulans supplementation on the growth performance and gut health of broiler chickens with Clostridium perfringens-induced necrotic enteritis

BackgroundThe poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) due to Clostridium perfringens.MethodsThis study was conducted to investigate the effects of feeding Bacillus coagulans on the growth performance and gut health of broiler chickens with C. perfringens-induced NE. Two hundred and forty 1-day-old broiler chicks were randomly assigned to a 2 × 2 factorial arrangement with two dietary B. coagulans levels (0 or 4 × 109 CFU/kg of diet) and two disease challenge statuses (control or NE challenged).ResultsNE-induced reduction in body weight gain was relieved by the addition of B. coagulans into broiler diets compared with the NE-infected birds. NE infection damaged intestinal morphological structure, promoted intestinal C. perfringens growth and liver invasion, and enhanced anti-C. perfringens specific sIgA concentrations in the gut and specific IgG levels in serum compared with the uninfected birds. NE infection significantly (P < 0.05) decreased mucin-2 (at 14 d post-infection (DPI), toll -like receptor 2 (TLR2, at 7 and 14 DPI), TLR4 (at 7 and 14 DPI), tumor necrosis factor super family 15 (TNFSF15, at 7 and 14 DPI), lysozyme (LYZ, at 14 DPI) and fowlicidin-2 (at 7 and 14 DPI) mRNA levels, whereas it dramatically (P = 0.001) increased IFN-γ mRNA levels at 7 DPI. However, challenged birds fed diets supplemented with B. coagulans showed a significant (P < 0.01) decrease in gut lesion scores, decreased C. perfringens numbers in the cecum and liver, and an increase in fowlicidin-2 mRNA levels in compared with the uninfected birds. In addition, compared with the non-supplemented group, dietary inclusion of B. coagulans improved intestinal barrier structure, further increased specific sIgA levels and alkaline phosphatase (IAP) activity in the jejunum, enhanced the expression of jejunum lysozyme mRNA, and inhibited the growth, colonization, and invasion of C. perfringens; in contrast, it reduced serum-specific IgG concentrations and jejunum IFN-γ mRNA levels.ConclusionThese results indicated that dietary B. coagulans supplementation appeared to be effective in preventing the occurrence and reducing the severity of C. perfringens-induced NE in broiler chickens.

Selection for pro-inflammatory mediators produces chickens more resistant to Campylobacter jejuni

Campylobacter spp. are the second leading cause of bacterial-induced foodborne illnesses with an estimated economic burden of nearly $2B USD per year. Most human illness associated with campylobacteriosis is due to infection by C. jejuni and chickens are recognized as a reservoir that could lead to foodborne illness in humans resulting from handling or consuming raw or undercooked chicken. We recently developed a novel breeding strategy based on identification and selection of chickens with an inherently high and low phenotype of pro-inflammatory mediators including IL-6, CXCLi2, and CCLi2, hereafter referred to as the high and low lines, respectively. We have shown the high line chickens are more resistant to the foodborne and poultry pathogens Salmonella enterica serovar Enteritidis, Eimeria tenella, and Clostridium perfringens-induced necrotic enteritis compared to the low line. The objective of this study was to determine whether the same trend of enhanced resistance in the high line birds was observed for C. jejuni. Birds were challenged at 2 d of age by oral gavage (0.5 mL) with 5 × 106 colony forming units (cfu) of C. jejuni/mL, necropsied 4 d post challenge, and cecal content collected to determine if there was a difference in C. jejuni resistance between the high and low line chickens. There were fewer (P = 0.01) chickens from the high line (28/40 = 71.8%) that were colonized by C. jejuni compared to the low line (37/39 = 94.9%). The amount of C. jejuni recovered from the ceca of infected birds was quantified; however, no differences were observed (P = 0.10). Since the high line birds were also more resistant to C. jejuni, it provides additional validation of selection based on pro-inflammatory mediators producing a line of chickens with increased natural resistance against diverse foodborne and poultry pathogens. The poultry industry is moving towards reduced therapeutics and, as such, our breeding strategy would be a viable method to incorporate into traditional poultry breeding programs.

The Effects of Berberine on Clostridium Perfringens Induced Necrotic Enteritis in Broiler Chickens

Background: Necrotic Enteritis, caused by C. perfringens is a major bacterial disease in chickens that results in substantial economic losses to the poultry industry. Drug resistance and increased pressure to reduce the use of antimicrobial growth promoters has stimulated the need to search for alternatives. This two-part study investigated the use of the natural herbal compound Berberine in broiler chickens for the control of Necrotic Enteritis. Methods and findings: Phase 1 evaluated Berberine in-water at 0.1 g/L and 1.0 g/L in vivo against C. perfringens induced disease in broiler chickens. Results demonstrated efficacy towards the disease based on significantly decreased mortality and lesion scores at 1.0 ml/L Berberine treatment. Despite this, bodyweight, and feed and water consumption were greatly decreased in treated groups. Bursa of fabricus to bodyweight ratio results indicate there was no distinct damage to the immune system, suggesting palatability of Berberine in-water may have been the principal cause. The follow-up Phase 2 trial investigated the in vivo palatability of Berberine in-feed at 2.0 g/kg in non-challenged broiler chickens. Bodyweight, feed consumption and feed conversion ratio were found to not be affected compared to controls. However, water consumption was significantly increased in treated groups. Conclusions: Therefore from the present study, it can be concluded that Berberine has the potential to contribute to the control of Necrotic Enteritis, and that Berberine in-feed treatment alleviates the bird productivity concerns present when Berberine is administered via water.

In Vitro Selective Growth-Inhibitory Effect of 8-Hydroxyquinoline on Clostridium perfringens versus Bifidobacteria in a Medium Containing Chicken Ileal Digesta.

Clostridium perfringens-induced necrotic enteritis is generally controlled by antibiotics. However, because of increasing antibiotic resistance, other antibacterial agents are required, preferably ones that do not affect the beneficial intestinal microbiota of the host. This study evaluated the in vitro selective growth-inhibitory effect of 8-hydroxyquinoline (8HQ) on C. perfringens vs. bifidobacteria in a medium containing chicken ileal digesta. Prior to the experiments, the minimum inhibitory concentrations of 8HQ and penicillin G were determined by broth microdilution assay. The minimum inhibitory concentration values of 8HQ for C. perfringens were 16–32 times lower than the values for bifidobacteria. Treatment of autoclaved and non-autoclaved chicken ileal digesta with 8HQ showed a selective anticlostridial effect. After incubation of C. perfringens with autoclaved ileal digesta for 3 h, all 8HQ concentrations tested (32–2048 μg/mL) significantly reduced C. perfringens bacterial count. In contrast, the same treatment had no or only a slight effect on bifidobacteria counts. Unlike 8HQ, penicillin G did not exhibit any selectivity. Similar results were obtained after incubation for 24 h. In non-autoclaved ileal digesta, all 8HQ concentrations tested significantly reduced C. perfringens bacterial counts after incubation for 30 min and 3 h, while no effect was observed on bifidobacteria. These results suggest that 8HQ may serve as a prospective veterinary compound for use against necrotic enteritis in poultry.

Effects of Bacillus licheniformis on the growth performance and expression of lipid metabolism-related genes in broiler chickens challenged with Clostridium perfringens-induced necrotic enteritis.

BackgroundNecrotic enteritis (NE), caused by Clostridium perfringens, has cost the poultry industry $2 billion in losses. This study aimed to investigate the effect of Bacillus licheniformis as dietary supplement on the growth, serum antioxidant status, and expression of lipid-metabolism genes of broiler chickens with C. perfringens-induced NE.MethodsA total of 240 one-day-old broilers were randomly grouped into four: a negative control, an NE experimental model (PC), chickens fed a diet supplemented with 30 % of fishmeal from day 14 onwards and challenged with coccidiosis vaccine (FC), and NE group supplied with feed containing 1.0 × 106 CFU/g B. licheniformis (BL).ResultsBody weight gain, feed conversion ratio, serum antioxidant status, and lipid-metabolism-gene expression were analyzed. In the PC group, FCR increased significantly whereas serum catalase and glutathione peroxidase activity decreased compared with NC group. Dietary B. licheniformis supplementation improved FCR and oxidative stress in experimental avian NE. Using Bacillus licheniformis as a direct-fed microbial (DFM) could also significantly upregulate catabolism-related genes, namely, peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase-1, in livers and changed the expression of lipid-anabolism genes.ConclusionThese results suggested that dietary B. licheniformis supplementation can enhance growth and antioxidant ability, as well as change the expression of genes related to fatty-acid synthesis and oxidation in the livers of NE-infected broilers.

Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens-induced necrotic enteritis

We developed a novel selection method based on an inherently high and low phenotype of pro-inflammatory mediators and produced "high" and "low" line chickens. We have shown high line birds are more resistant to Salmonella enterica serovar Enteritidis and Eimeria tenella compared to the low line. Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illness, and is also an economically important poultry pathogen and known etiologic agent of necrotic enteritis (NE). The objective of this study was to determine if high line birds were also more resistant to NE than low line birds using an established model. Birds were reared in floor pens and challenges were conducted twice (high line = 25/trial, 50 birds total; low line = 26/trial, 52 birds total). Day-old chicks were provided a 55% wheat-corn-based un-medicated starter diet. A bursal disease vaccine was administered at 10× the recommended dose via the ocular route at 14-d-of-age. Birds were challenged daily for 3 d beginning at 16-d-of-age by oral gavage (3 mL) with 10(7) colony forming units (cfu) of C. perfringens/mL then necropsied at 21-d-of-age. All birds had sections of the intestine examined and scored for lesions while the first 10 necropsied also had gut content collected for C. perfringens enumeration. Chickens from the high line were more resistant to C. perfringens-induced NE pathology compared to the low line, as indicated by reduced lesion scores. Ninety percent of the high line birds had lesions of zero or one compared to 67% of the low line birds. Wilcoxon rank sum test showed significantly higher lesion scores in the low line birds compared to the high line (P < 0.0001). There were no differences in the C. perfringens recovered (P = 0.83). These data provide additional validation and support selection based on elevated levels of pro-inflammatory mediators produces chickens with increased resistance against foodborne and poultry pathogens.

The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens.

Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens.

Bacillus subtilis PB6 improves intestinal health of broiler chickens challenged with Clostridium perfringens-induced necrotic enteritis

Necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens that results in significant economic losses, averaging damage of $0.05 per bird. The present study investigated the influence of a dietary supplement, Bacillus subtilis PB6, on performance, intestinal health, and gut integrity against C. perfringens-induced NE in broiler birds. Bacillus subtilis PB6 (ATCC-PTA 6737) is a natural strain isolated from healthy chicken gut that has been shown in in vitro to produce antimicrobial substances with broad activity against various strains of Campylobacter and Clostridium species. The animal study was conducted on broiler chickens (Cobb 400) for the period of 35 d using a completely randomized design. The experimental design included 3 treatments groups. Each treatment group contained 6 replicates, 3 male and 3 female, with 12 birds in each replicate. The 3 treatment groups were an uninfected control, an infected control, and an infected group supplemented with B. subtilis PB6 at 500 g/t of feed, containing 5 × 1011 cfu/kg. Necrotic enteritis was induced in the broiler birds via oral inoculation of 30,000 oocysts of mixed strains of Eimeria species on d 14 followed by C. perfringens (108 cfu/mL) on d 19 through 21 of trial. The birds were analyzed for BW gain, mortality, feed conversion ratio (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. The infected control group showed markedly thickened mucosa, hemorrhages, intestinal lesions, and ballooning of intestine. The supplementation of B. subtilis PB6 reduced the FCR (P < 0.05) and intestinal C. perfringens counts significantly (P < 0.05) compared with the infected control group. It was also observed that B. subtilis PB6 improved villi length by 10.88 and 30.46% (P < 0.05) compared with uninfected and infected control groups, respectively. The group supplemented with B. subtilis PB6 significantly (P < 0.05) increased the villi length to crypt depth ratio by 49.11% compared with the infected group. In conclusion, the supplementation of B. subtilis PB6 not only controlled C. perfringens-induced NE, but also improved intestinal health in the broiler birds.

Reduced lesions in chickens with Clostridium perfringens-induced necrotic enteritis by Lactobacillus fermentum 1.2029 ,

Previous studies have demonstrated that Lactobacillus has anti-inflammatory properties, but the protective functions of Lactobacillus and mechanisms of inhibition of necrotic enteritis (NE) in the intestines of chickens have not been fully clarified. In the present study, we selected a probiotic strain, Lactobacillus fermentum 1.2029, which has good adhesive ability and a high survival rate in low pH and bile salts. The objective of this study was to examine the anti-inflammatory properties of L. fermentum 1.2029 against NE in chickens. Two hundred forty 1-d-old male Arbor Acres broilers were blocked into 3 experimental groups as follows: (I) nonchallenge control group, (II) Clostridium perfringens challenge group, and (III) C. perfringens challenge + L. fermentum 1.2029 group. Lactobacillus fermentum 1.2029 (1.0 mL/d, 108 cfu/mL) was orally administered daily to group III during the course of the experiment, and all uninfected control chickens were inoculated accordingly with the same volume of PBS. Clostridium perfringens (0.5 mL on d 1 and 1.0 mL on d 14 to 21, 108 cfu/mL) was administered to chickens in group II. At 28 d, scoring of gross NE lesions was performed. Ileal segments of approximately 2 cm from 24 chickens in each experimental group were collected and fixed in 4% (wt/vol) neutral-buffered formalin solution for histological scoring. Ileal mucosa samples were also collected for mRNA analysis by real-time PCR. The results showed that L. fermentum 1.2029 reduced the severity of NE lesions in chickens. Histological scores revealed that L. fermentum 1.2029 also reduced the inflammation damage of NE in chickens. Changes in cytokines and Toll-like receptors (TLR) were determined, and L. fermentum 1.2029 was found to increase interleukin-10 levels and reduce interferon-γ and TLR2 levels in NE-infected chickens. The results showed that L. fermentum 1.2029 was able to regulate the intestinal mucosal immune response and ameliorate inflammation by changing expression levels of cytokines and TLR.