Necrotic Enteritis Challenge Model Research Articles

Comparison of necrotic enteritis effects on growth performance and intestinal health in two different meat-type chicken strains Athens Canadian Random Bred and Cobb 500

Chickens have undergone genetic improvements in the past few decades to maximize growth efficiency. However, necrotic enteritis (NE), an enteric disease primarily caused by C. perfringens, remains a significant problem in poultry production. A study investigated the differences in intestinal health between the nonselected meat-type chicken Athens Canadian Random Bred (ACRB) and the modern meat-type Cobb 500 broilers (Cobb) when challenged with experimental NE. The study utilized a 2 × 3 factorial arrangement, consisting of two main effects of chicken strain and NE challenge model (nonchallenged control, NC; NE challenge with 2,500/12,500 Eimeria maxima oocysts + 1 × 109C. perfringens, NE2.5/NE12.5). A total of 432 fourteen-day-old male ACRB and Cobb were used until 22 d (8 d postinoculation with E. maxima on d 14, dpi), and the chickens were euthanized on 6 and 8 dpi for the analysis. All data were statistically analyzed using a two-way ANOVA, and Student's t-test or Tukey's HSD test was applied when P < 0.05. The NE12.5 group showed significant decreases in growth performance and relative growth performance from d 14 to 20, regardless of chicken strain (P < 0.01). The ACRB group exhibited significant decreases in relative body weight and relative body weight gain compared to the Cobb group from d 14 to 22 (P < 0.01). On 6 and 8 dpi, both NE challenge groups showed significant decreases in intestinal villus height to crypt depth ratio, jejunal goblet cell count, and jejunal MUC2 and LEAP2 expression (P < 0.01). Additionally, the NE12.5 group had significantly higher intestinal NE lesion score, intestinal permeability, fecal E. maxima oocyst count, intestinal C. perfringens count, and jejunal IFNγ and CCL4 expression compared to the NC group (P < 0.05). In conclusion, NE negatively impacts growth performance and intestinal health in broilers, parameters regardless of the strain.

Hyperimmune egg yolk antibodies developed against Clostridium perfringens antigens protect against necrotic enteritis

Necrotic enteritis (NE) is a widespread infectious disease caused by Clostridium perfringens that inflicts major economic losses on the global poultry industry. Due to regulations on antibiotic use in poultry production, there is an urgent need for alternative strategies to mitigate the negative effects of NE. This paper presents a passive immunization technology that utilizes hyperimmune egg yolk immunoglobulin Y (IgY) specific to the major immunodominant antigens of C. perfringens. Egg yolk IgYs were generated by immunizing hens with 4 different recombinant C. perfringens antigens, and their protective effects against NE were evaluated in commercial broilers. Six different spray-dried egg powders were produced using recombinant C. perfringens antigens: α-toxin, NE B-like toxin (NetB; EB), elongation factor-Tu (ET), pyruvate:ferredoxin oxidoreductase, a mixture of 4 antigens (EM-1), and a nonimmunized control (EC). The challenged groups were either provided with different egg powders at a 1% level or no egg powders (EN). The NE challenge model based on Eimeria maxima and C. perfringens dual infection was used. In Experiments 1 and 2, the EB and ET groups exhibited increased body weight gain (BWG; P < 0.01), decreased NE lesion scores (P < 0.001), and reduced serum NetB levels (P < 0.01) compared to the EN and EC groups. IgY against NetB significantly reduced Leghorn male hepatocellular cytotoxicity in an in vitro test (P < 0.01). In Experiment 3, the protective effect of the IgYs mixture (EM-2) against C. perfringens antigens (NetB and EFTu) and Eimeria antigens (elongation factor-1-alpha: EF1α and Eimeria profilin: 3-1E) was tested. The EM-2 group showed similar body weight, BWG, and feed intake from d 7 to 22 compared to the NC group (P < 0.05). On d 20, the EM-2 group showed comparable intestinal permeability, NE lesion scores, and jejunal NetB and collagen adhesion protein levels to the NC group (P < 0.05). In conclusion, dietary mixture containing antibodies to NetB and EFTu provides protection against experimental NE in chickens through passive immunization.

Stimbiotic supplementation modulated intestinal inflammatory response and improved broilers performance in an experimentally-induced necrotic enteritis infection model

BackgroundTwo experiments were conducted to establish an optimal NE challenge model and evaluate the efficacy of stimbiotic (STB) supplementation in necrotic enteritis (NE) challenged broilers. In Exp. 1, a total of 120 Arbor Acres (AA) broilers (45.0 ± 0.21 g) were randomly assigned to 6 treatments in a 3 × 2 factorial arrangement. Vaccine treatments included non-challenge (0), × 10 the recommended dose (× 10) or × 20 the recommended dose (× 20) by the manufacturer. Clostridium perfringens (CP) treatments were non-challenge (No) or 3 mL of 2.2 × 107 CFU CP challenge (Yes). In Exp. 2, a total of 72 AA broilers (40.17 ± 0.27 g) were randomly assigned to 6 treatments in a 3 × 2 factorial arrangement. Dietary treatments included non-additive (CON), 100 mg/kg STB (STB) and 100 mg/kg STB on top of a typical commercial blend including an essential oil, probiotics, and enzyme (CB). Challenge treatments included non-NE challenge (No) and NE challenge (Yes) as established in Exp. 1.ResultsIn Exp. 1, CP and vaccine challenge decreased (P < 0.05) body weight (BW), body weight gain (BWG) and feed intake (FI), and increased (P < 0.05) the number of broilers with diarrhea and intestinal lesions. The oral administration of × 20 recommended dose of vaccines coupled with 3 mL of 2.2 × 107 CFU CP resulted in (P < 0.01) a significantly increased incidence of wet litter and intestinal lesions. Thus, this treatment was chosen as the challenge model for the successful inducement of NE in Exp. 2. In Exp. 2, the NE challenge negatively affected (P < 0.01) growth performance, ileal morphology, immunoglobulin contents in blood, caecal microbiota in the caecum, footpad dermatitis, intestinal lesion scores, tumour necrosis factor (TNF-α) and endotoxin in the serum compared with the non-NE challenged birds. The supplementation of STB and CB in diets enhanced (P < 0.05) growth performance, intestinal microbiota, and blood profiles by stimulating ileal morphology (VH and VH:CD) and propionate production in the cecum, and there were no differences in measured variables between STB and CB supplemented birds.ConclusionOverall, these results indicate that STB supplementation was able to reduce the inflammatory response and improve the performance of NE challenged birds, and the supplementation of STB alone was as effective as a typical commercial blend containing a number of other additives.

A blend of microencapsulated organic acids and botanicals reduces necrotic enteritis via specific signaling pathways in broilers

Necrotic enteritis (NE) is a devastating disease that has seen a resurgence of cases following the removal of antibiotics from feed resulting in financial loss and significant animal health concerns across the poultry industry. The objective was to evaluate the efficacy of a microencapsulated blend of organic (25% citric and 16.7% sorbic) acids and botanicals (1.7% thymol and 1% vanillin [AviPlusP]) to reduce clinical NE and determine the signaling pathways associated with any changes. Day-of-hatch by-product broiler breeder chicks were randomly assigned to a control (0) or supplemented (500 g/MT) diet (n = 23–26) and evaluated in a NE challenge model (n = 3). Birds were administered 2X cocci vaccine on d 14 and challenged with a cocktail of Clostridium perfringens strains (107) on d 17 to 19. On d 20 to 21 birds were weighed, euthanized, and scored for NE lesions. Jejunal tissue was collected for kinome analysis using an immuno-metabolism peptide array (n = 5; 15/treatment) to compare tissue from supplement-fed birds to controls. Mortality and weight were analyzed using Student's t test and lesion scores analyzed using F-test two-sample for variances (P < 0.05). The kinome data was analyzed using PIIKA2 peptide array analysis software and fold-change between control and treated groups determined. Mortality in the supplemented group was 47.4% and 70.7% in controls (P = 0.004). Lesions scores were lower (P = 0.006) in supplemented birds (2.47) compared to controls (3.3). Supplement-fed birds tended (P = 0.19) to be heavier (848.6 g) than controls (796.2 g). Kinome analysis showed T cell receptor, TNF and NF-kB signaling pathways contributed to the improvements seen in the supplement-fed birds. The following peptides were significant (P < 0.05) in all 3 pathways: CHUK, MAP3K14, MAP3K7, and NFKB1 indicating their importance. Additionally, there were changes to IL6, IL10, and IFN- γ mRNA expression in tissue between control- and supplement-fed chickens. In conclusion, the addition of a microencapsulated blend of organic acids and botanicals to a broiler diet reduced the clinical signs of NE that was mediated by specific immune-related pathways.

Supplementing Garlic Nanohydrogel Optimized Growth, Gastrointestinal Integrity and Economics and Ameliorated Necrotic Enteritis in Broiler Chickens Using a Clostridium perfringens Challenge Model.

Simple SummaryNecrotic enteritis (NE) is one of the most serious diseases in terms of economic losses. Aspects related to application of nanotechnology to control outbreaks of NE due to Clostridium perfringens (C. perfringens) are not completely understood. Hence, the purpose of this study was to evaluate the beneficial effects of garlic nano-hydrogel (G-NHG) on the performance, intestinal integrity, economic returns and alleviation of the intestinal C. perfringens levels using an NE challenge model in broiler chickens. Dietary inclusion of 400 mg/kg of G-NHG improved body weight gain (BWG) and feed conversion ratio (FCR). The digestive enzymes and tight junction and gut barrier-related genes expression were negatively impacted post C. perfringens challenge, resulting in a decrease in BWG with an increase in FCR. Meanwhile, G-NHG supplementation decreased C. perfringens levels, mortality rates and intestinal lesion score, and thereby improved intestinal permeability measurements, which consequently resulted in improved growth performance parameters. In conclusion, G-NHG markedly ameliorated the negative effects of C. perfringens challenge, which positively reflected on the growth performance of challenged birds, suggesting its beneficial effects in controlling C. perfringens infection in broiler chickens.Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds’ intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

Effect of Herbanoplex CP on broiler chicken's performance following a nondefined challenge or intestinal lesion score using a necrotic enteritis challenge model

Two independent trials were conducted to assess the effect of Herbanoplex CP (HB) on broiler chicken's performance using a 1) a nondefined challenge or 2) necrotic enteritis (NE) challenge model with intestinal lesion scoring. Herbanoplex CP is a unique combination of phytocompounds (ground hops, wheat germ, and chicory). In Exp 1, 400-day-old male chicks (Cobb 500) were individually tagged, weighed, and randomly assigned to one of 20 pens (n = 20 birds/pen). Pens were randomly assigned per treatment group: 4 treatments with 5 replicates each (n = 100 birds/treatment). T1: no modification; T2: antibiotic growth promoter bacitracin methylene disalicylate (BMD) 11% at 0.5 kg/metric ton); T3: HB 1 kg/metric ton; and T4: HB at 0.5 kg/metric ton. On day 7, 14, 15 of life, all birds in all treatment groups were challenged by drinking water with a liter filtrate as a nondefined challenge model. Their performance was evaluated at day 35. T3 had a significant (P-value < 0.05) increase in body weight gain (BWG) and a significant reduction in FCR compared with the rest of the experimental groups. Interestingly, chickens in T2, showed similar BWG and FCR when compared with chickens in T4. In Exp 2, 100-day-old male broiler chicks were divided into 5 groups and allocated in isolation cages. T1: birds without Clostridium perfringens (CP) challenge; T2: birds with CP challenge; T3: birds with CP challenge plus HB at 1 kg/metric ton; T4: birds with CP challenge plus HB at 0.75 kg/metric ton and T5: birds with CP challenge plus HB at 0.5 kg/metric ton. At day 13, all chickens were vaccinated with 10x coccidia oocysts. At day 14, all chickens were treated with a 10x dose of infectious bursal disease vaccine. From day 15 to day 19, chickens in challenge groups (T2, T3, T4, and T5) were orally gavaged with 108 cfu of C. perfringens twice a day. At day 21, all chickens were euthanized, and NE intestinal lesion scores were recorded. Chickens in T3 and T4 exposed to a NE challenge model showed significantly reduced lesion scores compared with the rest of the groups. Furthermore, in vitro minimal inhibitory concentration (MIC) assays revealed that HB had the lowest MIC at both 24 h (390 μg/mL) and 48 h (625 μg/mL) against C. perfringens. Herbanoplex CP showed similar response to BMD in a nonspecific challenge model in promoting growth performance (Exp 1). Moreover, HB reduced the severity of intestinal lesion score using a NE challenge model (Exp 2), suggesting that the antibacterial properties against C. perfringens by HB can promote growth during microbial challenges.

Differential expression of intestinal genes in necrotic enteritis challenged broiler chickens with 2 different Clostridium perfringens strains

The primary cause of necrotic enteritis (NE) disease in chickens is the NetB-positive Clostridium perfringens bacterium. Many factors are known to affect the severity of NE in the challenge models of broiler chickens, and one of these factors is the virulence of C. perfringens strain. This study was conducted to evaluate the effect of 2 pathogenic C. perfringens strains in a NE challenge model on gut health and mRNA expression of genes encoding apoptosis, tight junction, immunity, and nutrient transporters in broilers. Day-old Ross-308 male broilers (n = 468) were allocated in a 2 × 3 factorial arrangement of treatments with in-feed antibiotics (no or yes) and challenge (Non, C. perfringens strain NE18, and C. perfringens strain NE36) as the factors. The birds in the challenged groups were inoculated with Eimeria species on day 9 and with a fresh suspension of C. perfringens NE18 or NE36 on day 14 and 15. Sample collection was performed on 2 birds of each pen on day 16. Necrotic enteritis challenge, impaired feed conversion ratio during day 0 to 16 compared with the control group where the effect of the NE36 challenge was more severe than that with NE18 (P < 0.001). The mRNA expression of mucin-2, immunoglobulin-G, occludin (P < 0.001), and tight junction protein-1 (P < 0.05) genes were downregulated in both challenged groups compared with the nonchallenged counterparts. Antibiotic supplementation, on the other hand, increased weight gain, and feed intake in all challenged birds (P < 0.01), but upregulated mucin-5ac and alanine, serine, cysteine, and threonine transporter-1 (P < 0.05) only in the NE18 challenged birds. The challenge with NE36 significantly upregulated caspase-8 and claudin-1 (P < 0.001), but downregulated glucose transporter-2 (P < 0.001) compared with the NE18 challenge. These results suggest that NE challenge is detrimental to the performance of broilers through compromised intestinal health, and different C. perfringens strains can affect the severity of the disease through modulating the expression of intestinal genes encoding proteins responsible for apoptosis, gut integrity, immunity, mucus production, and nutrient transporters.

Modulations of genes related to gut integrity, apoptosis, and immunity underlie the beneficial effects of Bacillus amyloliquefaciens CECT 5940 in\xa0broilers fed diets with different protein levels in a necrotic enteritis challenge model

BackgroundThe ban of in-feed antimicrobial additives has negatively affected the poultry industry by causing necrotic enteritis (NE) to emerge in the flocks. Alternatives such as Bacillus probiotics have shown to be effective on eliminating the negative effects of this disease. Two experiments were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940 (BA) in broiler chickens under NE challenge and/or fed diets with different protein levels.MethodsIn both experiments, 480 day-old mix-sexed Ross-308 broilers were arranged in a 2 × 2 factorial arrangement of treatments. In experiment 1, the factors were NE challenge (yes or no) and probiotic (yes or no). In experiment 2, the factors were dietary crude protein levels (standard or reduced) and probiotic (yes or no) and were used under NE challenge condition. Oral administration of Eimeria oocysts (day 9) followed by inoculation with Clostridium perfringens (day 14 and 15) was used to induce NE challenge. On day 16, two birds from each treatment were gavaged with fluorescein isothiocyanate-dextran (FITC-d) and blood samples were collected for gut integrity evaluation, and jejunal samples were collected for gene expression assay.ResultsIn experiment 1, BA supplementation decreased caspase-3 (CASP3) (P < 0.001) and caspase-8 (CASP8) (P < 0.05) and increased occludin (OCLD) (P < 0.05) expression regardless of the challenge. Additionally, BA supplementation downregulated interfron-γ (IFN-γ) expression (P < 0.01) and upregulated immunoglobulin-G (IgG) (P < 0.01) and immunoglobulin-M (IgM) (P < 0.05) only in challenged birds. In experiment 2, the expression of genes encoding mucin-2 (MUC2) (P < 0.001), tight junction protein-1 (TJP1) (P < 0.05) and OCLD (P < 0.05) were upregulated by the addition of BA in the diet, regardless of the crude protein level. Further, BA supplementation downregulated INF-γ (P < 0.01) and upregulated immunoglobulin-A (IgA) (P < 0.05), IgM (P < 0.05) and IgG (P < 0.01) regardless of the crude protein level.ConclusionThese findings suggest that supplementation of BA in broiler diets can improve gut health by modulation of genes related to the mucosal barrier, tight junction, and immunity in broilers challenged by unfavourable conditions such as NE challenge.

Effects of Eimeria maxima and Clostridium perfringens infections on cecal microbial composition and the possible correlation with body weight gain in broiler chickens

With the voluntary and regulatory withdrawal of antibiotic growth promoters from animal feed, coccidiosis and necrotic enteritis (NE) emerge as the top two enteric poultry infectious diseases responsible for major economic loss worldwide. The objective of this study was to investigate the correlation between the cecal microbiota compositions with the growth trait after coccidiosis and NE. In this study, the effects of Eimeria maxima and/or Clostridium perfringens infections on the microbial composition and potential correlation with the body weight gain were investigated in broiler chickens using 16S rRNA gene sequencing. E. maxima and C. perfringens coinfection successfully induced NE with its typical gut lesions and significant reductions in the percentage of relative body weight gain (RBWG%). The NE challenge model did not affect cecal microbial diversity, but influenced the cecal microbial composition. KEGG enzymes in microbiota were significantly altered in abundance following dual infections. Furthermore, significant correlations between cecal microbiota modules and RBWG% were identified in the sham control, E. maxima or C. perfringens infected groups. Understanding of host-microbiota interaction in NE would enhance the development of antibiotics-independent strategies to reduce the harmful effect of NE on the gut microbiota structure, and improve the gut health and poultry production.

Evaluation of encapsulated sodium butyrate with varying releasing times on growth performance and necrotic enteritis mitigation in broilers

This study was conducted to evaluate the effect of an encapsulated sodium butyrate (Na-B) with targeted releasing times on growth performance and mitigating the impact of necrotic enteritis in broilers. Two Na-B (30% of Na-B content) products, CMA (2 h releasing time) and CMP (3 to 4 h releasing time), were evaluated in a necrotic enteritis challenge model. The experiment consisted of 4 Na-B treatments (500 and 1,000 ppm of each product) plus 2 control (non-challenged and challenged). A total of 336 Cobb-Cobb male broilers were placed 8 birds per pen into 7 replicate battery cages. On day 14, birds from challenged treatments were orally gavaged with ∼5,000 oocysts of Eimeria maxima. On day 19, 20, and 21, the challenged birds received 1 mL of 108 cfu/mL Clostridium perfringens. Total pen body weight (BW) and feed weight were assessed on day 14, 21, and 28 for BW gain and mortality-adjusted feed conversion ratio (FCR). On day 21, 3 birds were randomly selected per pen and scored for intestinal lesions. The duodenum and jejunum tissue were collected on day 21 and 28 for quantifying intestinal histology. Results revealed that adding CMA at 500 ppm showed significantly higher cumulative BW gain (P < 0.05) compared to the challenged control from day 1 to 21. Adding CMA at 500 ppm also showed the equivalent cumulative FCR that was comparable to the non-challenged control on day 21. The CMA treatments and CMP at 1,000 ppm treatment showed equivalent BW gain compared to the non-challenge control after an additional 7 d post-challenge on day 28. Both products at 500 or 1,000 ppm had the significantly (P < 0.05) lower intestinal lesion scores compared to the challenged control. However, there was no difference in lesion scores among the Na-B treatments. The Na-B product targeted to release in the anterior intestinal tract shows the beneficial effects on BW gain and feed utilization efficiency in current challenge model. Adding encapsulated Na-B product has the potential to mitigate the impact of necrotic enteritis in broilers.

Effect of a Competitive Exclusion Culture in a Necrotic Enteritis Challenge Model in Broilers

SUMMARY Restrictions on the use of antibiotics in broiler chicken production have led to a resurgence in necrotic enteritis (NE) and heightened interest in non-antibiotic alternatives to control the problem. Probiotics, which are beneficial bacteria that improve the enteric microbial balance, and which range from defined single-organism products to complex mixtures of undefined normal gut flora (the latter known as competitive exclusion cultures) have been investigated as alternative control measures for NE, with varying results. In this study, a complex undefined competitive exclusion culture (Aviguard) was compared to bacitracin methylene disalicylate (BMD) in a NE challenge model. Male broiler chickens housed in floor pens were either treated with Aviguard on the day of hatch, fed rations containing 55 ppm of BMD, or untreated, then challenged with coccidiosis and Clostridium perfringens in a NE challenge model. One group was untreated and unchallenged. The challenge successfully induced NE as evidenced by challenge control having NE mortality (12%) and lesion scores (0.367). The Aviguard group had numerically lower lesion scores (0.267) and the Aviguard and BMD groups had significant lower mortality (8%; 9%, respectively). There were no significant treatment effects on adjusted feed conversion or body weight by 42 d. Poultry producers do not adjust feed conversion for mortality, therefore using this standard, the Aviguard treatment was the only treatment for which the feed conversion that was not adjusted for mortality was statistically the same as the unchallenged control group (1.653; 1.636, respectively).

Characterization of Virulent netB+/tpeL+Clostridium perfringens Strains from Necrotic Enteritis-Affected Broiler Chicken Farms.

Clostridium perfringens (CP) type A and newly created type G strains are the key etiological factors in the induction of necrotic enteritis (NE), an important enteric disease that is responsible for the annual loss of $6 billion in the worldwide poultry industry. Several CP toxin genes were found to be critical in NE pathogenesis in chickens, but limited information is available on the CP lethal toxin tpeL gene. In this study, 19 CP strains isolated from NE-affected chicken farms were characterized microbiologically and molecularly and evaluated for their pathogenicity in commercial broiler chickens. Toxin typing by PCR revealed that all strains tested were positive for the netB toxin gene, but only five strains were positive for the tpeL toxin gene (LLY-TpeL 13, -TpeL 15, -TpeL 17, -TpeL 18, and -TpeL 19, simplified as TpeL 13, TpeL 15, TpeL 17, TpeL 18, and TpeL 19). High levels of TpeL proteins were detected in the concentrated culture supernatant from strains TpeL 13, 15, 17, and 19 by western blotting. Quantitative PCR showed that strains TpeL 13, 15, 17, 18, and 19 harbored a high number of copies of tpeL genes, while TpeL 18 had the highest number of copies of the tpeL gene among all CP strains tested when normalized with copy numbers of 16S rRNA gene as a housekeeping gene marker. The in vivo NE challenge test using multiple oral CP inoculations combined with a high-protein diet showed that TpeL 17 was the most virulent in inducing typical NE lesions, followed by TpeL 19 as the next most virulent, when tested in commercial broiler chickens. Infection with TpeL 17 reduced the growth rate significantly, as shown by reduced relative body weight gain percentage at day 5 postinfection. Availability of the virulent netB+tpeL+ CP strains is essential for the development of a CP-alone NE challenge model that could provide significant tools for understanding CP pathogenesis and for development of alternative to antibiotics.

Evaluation of the Epithelial Barrier Function and Ileal Microbiome in an Established Necrotic Enteritis Challenge Model in Broiler Chickens.

Necrotic enteritis (NE) is a recognized multifactorial disease that cost annually to the poultry industry around $2 billion. However, diverse aspects related to its presentation are not completely understood, requiring further studies using known induction experimental models. Therefore, the purpose of this study was to measure the changes occurring in performance, intestinal integrity and ileal microbiome using a previously established NE-challenge model. Chickens were assigned to a negative control group (NC) or a positive control group (PC). In the PC, broilers were orally gavaged with Salmonella Typhimurium (ST) (1 × 107 cfu/chick) at day 1, Eimeria maxima (EM) (2.5 × 104 oocyst/chick) at day 18 and Clostridium perfringens (CP) (1 × 108 cfu/chick/day) at 23–24 days of age. Weekly, body weight (BW), body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) were evaluated. Morbidity and mortality were determined throughout the study, and NE lesion scores were recorded at day 25. Additionally, blood and liver samples were collected to measure gut permeability as determined by levels of serum fluorescein isothiocyanate-dextran (FITC-d) and bacterial translocation (BT). Ileal contents were processed for 16S rRNA gene-based microbiome analysis. Performance parameters and intestinal permeability measurements were negatively impacted in the PC resulting in elevated serum FITC-d and BT with a −6.4% difference in BWG. The NE lesion score in PC (1.97 vs. 0.00) was significantly higher in comparison to NC, although there was no difference in mortality. The microbiome analysis showed a dramatic shift of ileal microbiomes in PC groups as compared to NC (ANOSIM: R = 0.76, P = 0.001). The shift was characterized by reduced abundance of the phylum Actinobacteria (P < 0.01), and increased abundance of the genera Butyrivibrio, Lactobacillus, Prevotella and Ruminococcus in PC compared to NC (P < 0.05). Expectedly, Clostridium was found higher in PC (2.98 ± 0.71%) as compared to NC (1.84 ± 0.36%), yet the difference was not significant. In conclusion, results of the present study showed the different intestinal epithelial and microbiological alterations occurring in an established NE-challenge model that considers paratyphoid Salmonella infections in young chicks as an important predisposing factor for presentation of NE.

Valeric acid glyceride esters in feed promote broiler performance and reduce the incidence of necrotic enteritis

Valeric acid is a C5 fatty acid, naturally produced in low concentrations by specific members of the microbiota of the lower intestinal tract. Effects of valeric acid on intestinal health have been poorly investigated. Valeric acid derivatives can be produced as glyceride esters and added to broiler feed. In the current study, experiments were carried out to evaluate the effect of valeric acid glycerides (GVA) on growth performance, on the morphology of the small intestinal mucosa and on protection against necrotic enteritis. In a first feeding trial, Ross-308 chicks were randomly divided into 2 dietary treatment groups and fed either a non-supplemented diet or a diet supplemented with GVA (1.5g/kg). In the GVA supplemented group, the feed conversion ratio was significantly decreased during the entire trial period (D1-37). In a second trial, gut wall morphology was evaluated. In broilers fed a GVA-containing diet at 5g/kg, the villus height/crypt depth ratio in the jejunum was significantly increased (P ≤ 0.05), and the crypt depth was significantly decreased at 28 d. In a third trial, immunohistochemistry showed that the density of glucagon-like peptide-2 immunoreactive cells in jejunal and ileal villi from broilers supplemented with GVA (5g/kg) was significantly increased (P ≤ 0.05) on d 10. In a necrotic enteritis challenge model, a significant reduction of the number of birds with necrotic lesions was found at d 21, using in-feed supplementation of low and high regimen of GVA. These data show that GVA supplementation to broiler feed can decrease the feed conversion, positively affect the morphology of the small intestinal mucosa, increase the density of glucagon-like peptide-2 producing enteroendocrine cells, and reduce the incidence of necrotic enteritis, making GVA a valuable candidate feed additive for broilers.

The role of supplemental glycine in establishing a subclinical necrotic enteritis challenge model in broiler chickens

Subclinical necrotic enteritis (NE) is an economically important disease in the broiler industry. With the move towards removal of antibiotics from feeds, solutions to control subclinical NE are desperately being sought. Dietary glycine has been shown to promote proliferation of Clostridium perfringens (Cp) and may thus be useful to include in a NE challenge model. A study was conducted to evaluate the effect of increased dietary glycine levels on subclinical NE. A 2 × 2 × 2 factorial arrangement of treatments was carried out using day-old male Ross 308 chicks (n = 624) allocated to 48 floor pens with 8 treatments of 6 replicates with 11 birds per treatment. Factors were: Cp challenge (C− or C+), Eimeria spp. challenge (E− or E+), and dietary glycine in the grower diet (0 or 10 g/kg). Birds had higher FCR when challenged with Eimeria (P < 0.01) or Cp (P < 0.05) on d 24 or Cp on d 35 but FCR was lower when fed glycine on d 24 (P < 0.01). Supplementation of glycine reduced feed intake on d 24 and increased weight gain on d 35 (P < 0.05). A Cp × Eimeria × glycine interaction (P < 0.05) showed a higher jejunal lesion scores in birds challenged with a combination of Cp and glycine compared with those with Eimeria and glycine or the unchallenged birds. Lesion score interactions between Cp and glycine (P < 0.05) in the ileum and Cp and Eimeria in the duodenum (P < 0.05) and ileum (P < 0.05) illustrated higher lesion scores in birds challenged with Cp without Eimeria or glycine compared to those not challenged with Cp. This study suggests that using glycine can partially replace Eimeria in a subclinical NE challenge model in promoting the intestinal lesions but not impairing chicken performance.

An early feeding regime and a high-density amino acid diet on growth performance of broilers under subclinical necrotic enteritis challenge.

Broilers that have early access to feed have been shown to have enhanced immune system and gut development and heightened resilience against necrotic enteritis (NE). This study examined the effect of early feeding a high amino acid density diet on performance of broilers under a sub-clinical NE challenge model. Ross 308 broilers (n = 576) were assigned to a 2 × 2 × 2 factorial design with 2 feeding regimes (feed access either within 6 h post-hatch or after 48 h post-hatch), 2 diets (control diet or the control diet with an additional 10% digestible amino acids [HAA]) and either presence or absence of NE challenge. Oral administrations of Eimeria species (d 9) and a field strain of Clostridium perfringens (d 14) were used to induce NE. Broiler performance was analysed at d 13, 23, 30 and 35. Intestinal lesion score and bacterial count were analysed on d 16. The NE challenge reduced overall bird performance and induced severe intestinal lesions, without causing notable mortality. At d 23 bird weight was significantly lower (P < 0.001) in the challenged birds compared with the unchallenged birds, but by d 30 the challenged birds had recovered and challenge no longer had an impact on bird performance. Birds fed the HAA diet had greater body weight by d 35 and heightened Lactobacillus content in the ileum at d 16 (P < 0.05). Birds that were fed the HAA diet after a period of fasting performed better in terms of feed conversion ratio (FCR) under challenge. The findings from this study suggest there are beneficial effects of feeding high amino acid diets to birds in response to external stresses, such as post-hatch fasting and subclinical NE.

Use of yeast cell wall extract as a tool to reduce the impact of necrotic enteritis in broilers

The use of a yeast cell wall extract derived from Saccharomyces cerevisiae (Actigen®) has been proposed as an alternative to in-feed antibiotics. This experiment was conducted to investigate the efficacy of yeast cell extract as an alternative to zinc bacitracin or salinomycin using a necrotic enteritis challenge model. A feeding study was conducted using 480-day-old male Ross 308 chicks assigned to 48 floor pens. A 2 × 4 factorial arrangement of treatments was employed. The factors were: challenge (− or +) and feed additive (control, zinc bacitracin at 100/50 mg/kg, yeast cell wall extract at 400/800/200 mg/kg, or salinomycin at 60 mg/kg in starter, grower, and finisher, respectively). Diets based on wheat, sorghum, soybean meal, meat and bone meal, and canola meal were formulated according to the Ross 308 nutrient specifications. Birds were challenged using a previously established protocol (attenuated Eimeria spp oocysts) on d 9 and 108 to 109Clostridium perfringens (type A strain EHE-NE18) on d 14 and 15). Challenged and unchallenged birds were partitioned to avoid cross contamination. Challenged birds had lower weight gain, feed intake and livability compared to unchallenged birds on d 24 and d 35 (P < 0.05). Birds given zinc bacitracin, yeast cell wall extract, or salinomycin had improved weight gain and livability when compared to control birds given no additives. Challenge × additive interactions were observed for feed intake and weight gain on d 24 and d 35 (P < 0.01). The additives all had a greater positive impact on feed intake, weight gain, and livability in challenged than unchallenged birds. All challenged birds showed higher necrotic enteritis lesion scores in the small intestine sections when compared to unchallenged birds (P < 0.01). Birds fed yeast cell wall extract exhibited increased villus height, decreased crypt depth, and increased villus:crypt ratio when challenged. Yeast cell wall extract, zinc bacitracin, and salinomycin were effective in preventing performance decline from necrotic enteritis in the current study. This study indicates that yeast cell wall extract has promise as a tool for controlling necrotic enteritis.

Differential responses of cecal microbiota to fishmeal, Eimeria and Clostridium perfringens in a necrotic enteritis challenge model in chickens.

Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for progressing towards clinical symptoms of necrotic enteritis.

Natural plant extracts and prebiotic compounds as alternatives to antibiotics in broiler chicken diets in a necrotic enteritis challenge model

An experiment was conducted to determine the effects of two different water-soluble carbohydrate extracts (renga renga lily extract and Acacia extract), and two commercially available prebiotic compounds, Fibregum and Raftifeed-IPE, on the performance of broiler chickens subjected to a necrotic enteritis (NE) challenge model. These treatments were compared with negative control and a positive (Zn-bacitracin) control treatments. An overall 8.8% NE-related mortality was recorded, with mean jejunal and ileal lesion scores in dead birds ranging from 3.03 to 3.90 in all challenged groups except the positive control groups. NE-specific deaths or clinical abnormalities were not observed with unchallenged control and positive control groups. At 7 days post-challenge, the concentration of specific IgY antibodies against the α-toxin of Clostridium perfringens in the serum was lower (P &lt; 0.05) in birds fed the positive control and Fibregum-supplemented diets than in the negative control group. However, birds fed Fibregum had increased (P &lt; 0.05) IgM concentration compared with those fed Acacia extract and lily extract. The Fibregum-fed group also had higher (P &lt; 0.05) IgA concentrations in serum than did the positive-control and lily extract-supplemented groups at 14 days but this effect did not persist to 21 days. The results from the present study demonstrated that supplementation with water-soluble carbohydrates from two plant sources was not effective in controlling NE. However, the prebiotic compound Fibregum was found to be having some immunomodulatory effects. Addition of Zn-bacitracin and monensin was highly effective in counteracting the negative effects of the disease challenge.

The Effect of a&amp;lt;i&amp;gt; Lactobacillus&amp;lt;/i&amp;gt;-Based Probiotic for the Control of Necrotic Enteritis in Broilers

Necrotic Enteritis (NE) caused by Clostridium perfringens (CP) in poultry is probably the most important bacterial disease in terms of economic implications. The disease is multi-factorial and is invariably associated with predisposing factors. The present study investigated the effect of a commercially available Lactobacillus-based probiotic (FM-B11) for the control of necrotic enteritis in broiler chickens. In experiment 1, one-day-of-hatch broiler chicks were randomly allocated to the following treatment groups: 1) Non-challenged (NC); 2) Challenged (C); 3) Challenged + probiotic (C+ FM-B11). Prior to placement, chicks in groups 2 and 3 received 0.25 mL of Salmonella typhimurium (ST) containing 10 5 cfu of viable cells by oral gavage. At 14, 15 and 16 days of age, all chicks in group 3 were treated with FM-B11 in the drinking water at a concentration of 10 6 cfu/ml. At 21d of age, all chicks in groups 2 and 3, were individually challenged with 5 × 10 4 sporulated oocysts of E. maxima by oral gavage. At 26d of age, all chicks in groups 2 and 3, were individually challenged with 10 8 cfu CP; body weight (BW) was recorded prior to challenge. The experiment was terminated at 29 days of age and the following parameters were evaluated: NE-associated mortality, CP lesion scores, CP concentrations in ileum, BW, and body weight gain (BWG). Chicks treated with FM-B11 had significantly (P 0.05) difference in lesion score between C and C + FM-B11, group C + FM-B11 had significantly (P < 0.05) lower total number of cfu of CP recovered from the ileal mucosa and content samples when compared to group C. Experiment 2 was a unique and remarkable case report of a field outbreak of NE in a commercial broiler farm in Argentina. A reduction and control of the mortality associated with NE following 3 days of administration of FM-B11 was observed as compared with the control non treated house. These results imply that the commercially available Lactobacillus-based probiotic FM-B11 was able to reduce the severities of NE, as a secondary bacterial infection, in an experimental NE challenge model; as well as, in a commercial field outbreak of NE.