Subclinical Necrotic Enteritis Research Articles

241 Immunophysiology and performance of broilers supplemented with macroalgal sulfated polysaccharides during a subclinical necrotic enteritis challenge

Abstract This study evaluated the effects of a sulfate polysaccharide extracted from marine algae product on performance, intestinal lesion scores, and mRNA abundance of various tight junction proteins and immune response genes. A total of 600 one-day old male broilers (Ross 708) were allocated to one of four treatment groups (6 replicate floor pens, 25 birds/pen): negative control (NC) group fed a corn-soybean meal diet; positive control (PC) fed NC + 15 ppm Avilamycin and 125 ppm Amprolium, an additive group (AGS) fed NC + Algoguard added at 0.1% of the diet, and (AGH) fed NC + Algoguard added at 0.2% of the diet. To predispose the birds to NE, birds were orally gavaged with 2,000 Eimeria maxima sporulated oocysts on d 14 followed by a dose of approximately 1×108 CFU/mL of C. perfringens on d 19. On d 21, four birds/pen were necropsied for NE lesions and on d 14, 21, and 42 jejunal samples were collected to assess mRNA abundance. Birds and feed were weighed on d 14, 21, 28, and 42 on a per pen basis. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated during each feeding phase and adjusted for daily mortality. The data were analyzed using ANOVA (JMP, Pro 16) and significance (P ≤ 0.05) was determined by the LSD test. During d 14-21, 0-21, 0-28, and 0-42, mortality rate and ADFI were significantly less in AGS, AGH, and PC compared with NC. ADG was greater while FCR was significantly less in PC, AGS, and AGH during d 21-28, 0-28, and 0-42 compared with NC (P ≤ 0.05). Lesion severity was also significantly less in AGS, AGH, and PC compared with NC. On d 14, 21, and 42 there were no toxins present in the tested serum samples. Calprotectin concentration was greater in PC and NC on d 21 and 42 compared with the supplemented groups. C. perfringens toxin was not detected in serum samples of any of the four treatment groups. mRNA abundance of CLDN1, IL1β, IL12B, and IL10 was greater in AGS compared with NC on d 14 and 42. ZO1 was significantly greater on d 21 in PC treatment and was less than both AGS and AGH on d 42 (P ≤ 0.05). Compared with all other treatments, mRNA abundance of OCLDN was greater and those of IFN-γ and ZO2 were less in PC, while that of CLDN3 showing no significant difference among all 4 treatments. To conclude, dietary inclusion of this algal product in broiler diets could be considered as a beneficial alternative to antibiotic growth promoters in the context of this challenge model.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αβ T cells) and natural IEL (TCRαβCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαβ+CD4-CD8-, TCRαβ+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαβ+CD4-CD8- and TCRαβ+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1β and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging and/or in drinking water method twice, on d 8 and 13. Non-challenged groups included (1) without or (2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included (3) without the specific bromelain formulation; (4) gavaged with 0.4 mL/kg; (5) gavaged with 0.8 mL/kg and (6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the non-challenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Non-challenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d19, birds received bromelain in drinking water had lower faecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model

BackgroundNecrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations.MethodsThis study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches.ResultsOur findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens.ConclusionOur study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Inclusion of Quillaja Saponin Clarity Q Manages Growth Performance, Immune Response, and Nutrient Transport of Broilers during Subclinical Necrotic Enteritis.

Necrotic enteritis (NE) is an intestinal disease that results in poor performance, inefficient nutrient absorption, and has a devastating economic impact on poultry production. This study evaluated the effects of a saponin-based product (Clarity Q, CQ) during an NE challenge. A total of 1200 male chicks were randomly assigned to four dietary treatments (10 pens/treatment; 30 birds/pen): treatment 1 (NC), a non-medicated corn-soybean basal diet; treatment 2 (PC), NC + 50 g/metric ton (MT) of bacitracin methylene disalicylate (BMD); and treatments 3 (CQ15) and 4 (CQ30), NC + 15 and 30 g/MT, respectively. On the day (d) of placement, birds were challenged by a coccidia vaccine to induce NE. On d 8, 14, 28, and 42, performance parameters were measured. On d 8, three birds/pen were necropsied for NE lesions. On d 8 and d 14, jejunum samples from one bird/pen were collected for mRNA abundance of tight junction proteins and nutrient transporter genes. Data were analyzed in JMP (JMP Pro, 16), and significance (p ≤ 0.05) between treatments was identified by Fisher's least significant difference (LSD) test. Compared to PC and NC, CQ15 had higher average daily gain (ADG), while CQ30 had lower average daily feed intake (ADFI) and feed conversion ratio (FCR). NE lesions in the duodenum were lower in CQ15 compared to all other treatments. On d 8, mRNA abundance of CLDN1, CLDN5, AMPK, PepT2, GLUT2, and EAAT3 were significantly greater in CQ30 (p < 0.05) compared to both PC and NC. On d 14, mRNA abundance of ZO2 and PepT2 was significantly lower in PC when compared to all treatments, while that of ANXA1, JAM3, and GLUT5 was comparable to CQ15. In summary, adding Clarity Q to broiler diets has the potential to alleviate adverse effects caused by this enteric disease by improving performance, reducing intestinal lesions, and positively modulating the mRNA abundance of various tight junction proteins and key nutrient transporters during peak NE infection.

Subclinical doses of dietary fumonisins and deoxynivalenol cause cecal microbiota dysbiosis in broiler chickens challenged with Clostridium perfringens.

Fusarium toxins are one of the most common contaminants in poultry diets. The co-occurrence of fumonisins (FUM) and deoxynivalenol (DON), even at a subclinical dose, negatively affects the growth performance, intestinal integrity and induce subclinical necrotic enteritis in broiler chickens. Loss of gut integrity can be expected to alter the intestinal microbiota's composition. The objective of this study was to identify the effects of combined FUM and DON on the cecal microbiome profile and predicted metabolic functions and a short chain fatty acid profile in broilers challenged with Clostridium perfringens. A total of 240 1 day-old chicks were randomly assigned to two treatments: a control diet and the control diet with 3 mg/kg FUM + 4 mg/kg DON each with eight replications. All the birds were received cocci vaccine at d0. All birds in both treatment groups were challenged with C. perfringens 1 × 108 CFU via feed on d 19 and 20 to achieve 5% mortality. On d 35, the FUM and DON contaminated diet numerically (P = 0.06) decreased the body weight gain (BWG) by 84 g compared to the control group. The bacterial compositions of the cecal contents were analyzed by sequencing the V3-V4 region of the 16S rRNA gene. Overall, microbial richness and diversity increased (P < 0.02) during the studied period (d 21-35). Cecal contents of birds in the FUM + DON group had greater (P < 0.05) microbial evenness and diversity (Shannon index) compared to the control group. FUM + DON exposure decreased (P = 0.001) the relative abundance of Proteobacteria in the cecal content, compared to the control group. The combined FUM + DON significantly increased the relative abundance of the Defluviitaleaceae and Lachnospiraceae families (P < 0.05) but decreased the abundances of the Moraxellaceae and Streptococcaceae (P < 0.05) compared to the control group birds. At the genus level, FUM + DON exposure decreased (P < 0.05) Acinetobacter and Pseudomonas abundance and had a tendency (P = 0.08) to decrease Thermincola abundance compared to the control group. In the ileum, no NE-specific microscopic abnormalities were found; however, the tip of the ileal villi were compromised. The present findings showed that dietary FUM and DON contamination, even at subclinical levels, altered cecal microbial composition, dysregulated intestinal functions, and impaired the gut immune response, potentially predisposing the birds to necrotic enteritis.

Intracloacal Inoculation of Broiler Chickens with Clostridium perfringens Strains: Evaluation of Necrotic Enteritis Disease Development and Lymphoid Immune Responses.

Necrotic enteritis (NE) is an economically important disease of chickens. We have recently shown that inflammatory responses in chickens inoculated orally with virulent Clostridium perfringens were spatially regulated. Here, we used previously virulence-characterized netB+C. perfringens strains, avirulent CP5 and virulent CP18 and CP26, to assess the severity of NE and immune responses in broiler chickens when inoculated intracloacally. The results showed that CP18- and CP26-infected birds had a reduced weight gain and developed milder/less severe NE lesions, as determined by the gross lesions scores, suggesting a subclinical-grade infection. Gene expression analysis in infected birds revealed three statistically significant observations compared to uninfected-control: (1) Increased expression of anti-inflammatory/immunoregulatory interleukin (IL)-10/transforming growth factor (TGF)β in cecal tonsil (CT) and bursa of Fabricius in the CP18/CP26-infected groups. (2) Increased CT transcription of pro-inflammatory IL-1β, IL-6 and interferon (IFN)γ and decreased Harderian gland (HG) expression of IFNγ in the CP18/CP26-infected birds. (3) Increased HG or bursal expression of IL-4 and IL-13 in CP5-infected birds. Collectively, intracloacal C. perfringens inoculation seems to induce a highly regulated inflammatory response in the CT and other mucosal lymphoid organs and an intracloacal infection model may be useful in evaluating immune responses in chickens with subclinical NE.

Bacillus subtilis DSM29784 attenuates Clostridium perfringens-induced intestinal damage of broilers by modulating intestinal microbiota and the metabolome.

Necrotic enteritis (NE), especially subclinical NE (SNE), without clinical symptoms, in chicks has become one of the most threatening problems to the poultry industry. Therefore, increasing attention has been focused on the research and application of effective probiotic strains as an alternative to antibiotics to prevent SNE in broilers. In the present study, we evaluated the effects of Bacillus subtilis DSM29784 (BS) on the prevention of subclinical necrotic enteritis (SNE) in broilers. A total of 480 1-day-old broiler chickens were randomly assigned to four dietary treatments, each with six replicates pens of twenty birds for 63 d. The negative (Ctr group) and positive (SNE group) groups were only fed a basal diet, while the two treatment groups received basal diets supplemented with BS (1 × 109 colony-forming units BS/kg) (BS group) and 10mg/kg enramycin (ER group), respectively. On days 15, birds except those in the Ctr group were challenged with 20-fold dose coccidiosis vaccine, and then with 1 ml of C. perfringens (2 × 108) at days 18 to 21 for SNE induction. BS, similar to ER, effectively attenuated CP-induced poor growth performance. Moreover, BS pretreatment increased villi height, claudin-1 expression, maltase activity, and immunoglobulin abundance, while decreasing lesional scores, as well as mucosal IFN-γ and TNF-α concentrations. In addition, BS pretreatment increased the relative abundance of beneficial bacteria and decreased that of pathogenic species; many lipid metabolites were enriched in the cecum of treated chickens. These results suggest that BS potentially provides active ingredients that may serve as an antibiotic substitute, effectively preventing SNE-induced growth decline by enhancing intestinal health in broilers.

Complex of Lauric Acid Monoglyceride and Cinnamaldehyde Ameliorated Subclinical Necrotic Enteritis in Yellow-Feathered Broilers by Regulating Gut Morphology, Barrier, Inflammation and Serum Biochemistry.

This experiment investigated the benefits of plant essential oil (EO) composed with lauric acid monoglyceride and cinnamaldehyde on necrotic enteritis-challenged broilers. A total of 180 1-day-old healthy yellow-feathered broilers were randomly divided into 4 groups with 3 replicates of 15 chicks each. The experimental groups were as follows: the control group (CON) was fed with the basal diet and was not challenged by Eimeria acervulina (EA) and Clostridium perfringens (CP); CPEA group was also fed with a basal diet, but infected with CP and EA; CPEA_EO350 group and CPEA_EO500 group were fed with a basal diet supplemented with 350 and 500 mg/kg EO, respectively, and all infected with CP and EA. On the 7th day, each bird in the CPEA group, CPEA_EO350 group and CPEA_EO500 group was orally administrated with 1 mL Eimeria acervulina containing 5000 oocytes/mL, and the birds of the CON group were orally administrated with 1 mL normal saline. From the 15th day, 1 mL of CP type A CVCC-2030 strain (about 5 × 108 cfu/mL) was orally inoculated into each bird of the CPEA, CPEA_EO350 and CPEA_EO500 groups for three consecutive days. Similarly, the CON group was orally given 1 mL of normal saline. The CPEA stimulation reduced the average daily gain (ADG) of broilers, increased the feed-to-gain ratio (F:G), and increased the intestinal lesions of the broilers (p < 0.01), indicating that CPEA stimulation was clinically successful. Compared with the CPEA group, the ADG of CPEA_EO350 and CPEA_EO500 increased, the F:G decreased (p < 0.01), and the intestinal score of CPEA_EO500 decreased (p < 0.01). The expression of the tight junction protein of the jejunum and ileum on 21d was upregulated (p < 0.01), and the expression of jejunum inflammation factors TNF-α on 21d and jejunum and ileum inflammatory factor IL-6 on 28d were also downregulated. The CPEA_EO350 and CPEA_EO500 increased antioxidant capacity. To sum up, 350 and 500 mg/kg of lauric acid monoglyceride and cinnamaldehyde complex plant essential oils can improve ADG and F:G, improve intestinal morphology and the body's antioxidant capacity, and downregulate the expression of inflammatory factors. The concentration of 500 mg/kg performed even better.

Effects of Pediococcus acidilactici and Saccharomyces cerevisiae on broiler chickens challenged with Clostridium perfringens induced subclinical necrotic enteritis

Effects of Pediococcus acidilactici and Saccharomyces cerevisiae on broiler chickens challenged with Clostridium perfringens induced subclinical necrotic enteritis

Therapeutic effect of heat-killed Lactobacillus plantarum L-137 on the gut health and growth of broilers

Although some studies on the effects of para-probiotics on the immune system and intestinal health have been conducted independently of research on antibiotics ass growth promoters. This study investigated the effects of heat-killed Lactobacillus plantarumL-137 (L-137) and antibiotics as preventive and/or therapeutic substances for broilers against subclinical necrotic enteritis caused by Clostridium perfringens (CP). In total, 300 1-day-old broilers (46.13 ± 1.38 g) were randomly stocked at 10 birds pen−1 in five replicates and divided into six groups, namely T1 and T2, positive and negative control of CP challenge; T3 and T4, prevention with basal diet plus 10 and 50 mg/kg L-137; T5 and T6, prevention and treatment with basal diet plus 50 mg/kg of L-137 and bacitracin at 50 ppm, respectively. Broilers administered L-137 in T4, T5 and bacitracin in T6 showed an improved (p < 0.05) villus height/crypt depth ratio than control groups, suggesting that it might significantly boost growth performance. In contrast to bacitracin, a high dosage of L-137 significantly increased (p < 0.05) the spleen index value and the cytokine levels, as well as the expression of intestinal β-defensin genes on day 28. During the 42-day production period, broilers in T4 and T5 showed a significantly enhanced (p < 0.05) expression of cytokines, AvBD-1 and AvBD-7 on day 42 compared to the control and bacitracin groups. In particular, broilers given the L-137 diets demonstrated no cumulative mortality following CP exposure, compared to a 2% mortality in T6. Our findings provide insight into eco-friendly alternatives to antibiotics for maximizing growth performance, feed efficiency and long-term disease protection in chickens; however, this has to be proven in larger-scale commercial experiments.

Ileal profile of non-starch polysaccharides and oligosaccharides in response to exogenous enzymes in broiler chickens offered wheat- or maize-based diets under subclinical necrotic enteritis challenge.

The present study evaluated the impacts of fibre-degrading enzymes on the profiles of non-starch polysaccharides (NSP) and oligosaccharides (OS) in the ileum of broiler chickens offered wheat- or maize-based diets under subclinical necrotic enteritis (NE) challenge. A 2 × 2 × 4 factorial arrangement of treatments was used. Factors were the following: NE challenge, no or yes; diet type, wheat- or maize-based; and supplemental enzymes, control (no enzyme), family 10 xylanase (XYN10), family 11 xylanase (XYN11) or β-mannanase (MAN). Birds in the challenged group were inoculated with Eimeria on d 9 and Clostridium perfringens on d 14 and 15. A 3-way interaction (P = 0.047) occurred on overall (d 0 to 16) weight gain. When NE was present, all the supplemental enzymes increased weight gain in birds fed the wheat-based diet; whereas in those fed the maize-based diet supplemental XYN10 and XYN11 decreased weight gain. When NE was absent, birds fed the wheat-based diet supplemented with XYN10 or MAN presented increased weight gain compared to non-supplemented birds, but no improvements with enzyme addition were observed in birds fed the maize-based diet. A 3-way interaction (P = 0.002) was observed on insoluble NSP level in the ileum. When NE was absent, all the supplemental enzymes reduced the ileal level of insoluble NSP, regardless of diet type. In the challenged birds, supplementing XYN10 and MAN reduced insoluble NSP level in the ileum, but only in birds fed the wheat-based diet. Ileal soluble NSP level was reduced by supplemental XYN11 and MAN, but only in birds fed the wheat-based diet, resulting in a 2-way diet type × enzyme interaction (P < 0.001). Ileal OS arabinose (P = 0.030) level was highest in birds offered the wheat-based diet supplemented with XYN11. Collectively, supplementation of NSP-degrading enzymes to the wheat-based diet enhanced bird performance regardless of NE challenge, with XYN11 significantly increasing oligosaccharide release. However, enzyme addition did not improve growth performance in birds fed maize-based diet, with supplemental XYN10 and XYN11 impeding weight gain when NE was present.

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.

Trans-anethole Ameliorates Intestinal Injury Through Activation of Nrf2 Signaling Pathway in Subclinical Necrotic Enteritis-Induced Broilers.

This study was conducted to investigate the alleviative effects of trans-anethole (TA) on intestinal oxidative stress by enhancing the activities of intestinal antioxidant enzymes and activating the Nrf2 signaling pathway in subclinical necrotic enteritis (NE) infected broilers. A total of 192 1-day-old male Arbor Acres broilers were randomly allocated into three treatment groups: (1) control (CON); (2) subclinical NE challenge (NE); (3) NE challenge + 600 mg/kg TA (NE+TA600). Subclinical NE was induced by oral administration of live coccidiosis vaccine containing 2 × 104 oocysts at 10 days of age and 2 ml of Clostridium perfringens type A solution (3 × 108 CFU/ml) daily from days 14 to 19. The results showed that NE infection led to a severe decline (p < 0.05) in the final body weight (BW) and average daily gain (ADG), but an increase (p < 0.05) in feed/gain (F/G) of broilers at day 10–21 and day 1–21 compared with the control group. TA administration improved (p < 0.05) the growth performance of NE birds. The intestinal villus height (VH) and villus height/crypt depth (VH/CD) were reduced (p < 0.05) by NE challenge as compared with those of the control group, which was elevated by TA administration. Subclinical NE infection decreased (p < 0.05) serum activities of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and jejunal and ileal glutathione peroxidase (GSH-PX), and T-SOD activity as well as T-AOC in the jejunum, while TA interventions positively elevated that (p < 0.05). Administration of TA protected the intestine against oxidative stress through up-regulation of intestinal nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway as compared with the NE group (p < 0.05). In addition, dietary inclusion of TA elevated (p < 0.05) mRNA abundance of c-mesenchymal-epithelial transition factor (c-Met), jejunal epidermal growth factor receptor (EGFR), and transforming growth factor-beta 1 (TGF-β1) in the jejunum and ileum of birds after subclinical NE challenge. In conclusion, 600 mg/kg of TA may be a promising tool to prevent and control subclinical NE by increasing intestinal antioxidant status in broilers.

Dietary Probiotic Supplementation Suppresses Subclinical Necrotic Enteritis in Broiler Chickens in a Microbiota-Dependent Manner.

BackgroundChicken meat is one of the most consumed meats worldwide and poultry production is increasing at an exponential rate. Reducing antibiotic usage has resulted in the recurrence of subclinical necrotic enteritis again and influenced global poultry production. Probiotics are potential antibiotic substitutes that can be used to prevent subclinical necrotic enteriti. However, the precise mechanism of action of probiotics and information on which gut microbes confer this efficacy remain elusive.Methods and resultsThe subclinical necrotic enteritis animal model was used to reveal the mechanism underlying the effect of probiotics on intestinal health through RNA sequencing and 16S rDNA amplicon sequencing. Bacillus licheniformis H2 feeding significantly reduced the relative abundance of Clostridium perfringens in the ileum and markedly ameliorated the pathological damage in the ileum and liver. In addition, oral administration of B. licheniformis H2 contributed to the enhancement of the intestinal barrier function and epithelial renewal, reducing energy consumption, and improving enteral nutrition absorption. Probiotic B. licheniformis H2 also ameliorated the inflammatory response and increased the immunity of subclinical necrotic enteritis infected broilers. Finally, B. licheniformis H2 feeding regulated liver gene expression to suppress immune response and promoted growth and metabolism depending on the gut microbiota.ConclusionsThese results indicated the mechanism of probiotic action of B. licheniformis H2 in maintaining intestinal health and thus promoting growth and B. licheniformis H2 may serve as an antibiotic substitute to prevent subclinical necrotic enteritis in poultry farming.

Non-starch polysaccharide-degrading enzymes may improve performance when included in wheat- but not maize-based diets fed to broiler chickens under subclinical necrotic enteritis challenge.

The present study investigated whether supplementing fibre-degrading enzymes can ameliorate the severity of subclinical necrotic enteritis (NE) in broiler chickens offered wheat- or maize-based diets. A total of 1,544 mixed-sex broiler chickens were assigned to 16 experimental treatments as a 2 × 2 × 4 factorial arrangement of treatments. The factors were the following: NE challenge, yes or no; diet type, wheat- or maize-based; and enzyme supplementation, control (no enzyme), family 10 xylanase (XYN10), family 11 xylanase (XYN11) or β-mannanase (MAN). Each treatment was replicated 6 times, with 16 birds per replicate pen. A three-way challenge × diet type × enzyme interaction occurred for body weight at 21 d of age (P = 0.025) and overall feed conversion ratio (P = 0.001). In the non-challenged birds fed the wheat-based diet, supplementing MAN increased d 21 body weight compared to the control. In challenged birds fed the maize-based diet, supplemental XYN11 impeded body weight and overall FCR compared to the control. Birds offered the maize-based diet presented heavier relative gizzard weights at both 16 and 21 d of age (P < 0.001) and reduced liveability (P = 0.046) compared to those fed the wheat-based diet. Enzyme supplementation reduced ileal and jejunal digesta viscosity at 16 d of age only in birds fed the wheat-based diet (P < 0.001). XYN11 increased ileal digesta viscosity in birds fed the maize-based diet, and MAN reduced it in birds fed the wheat-based diet at 21 d of age (P = 0.030). Supplementing XYN11 improved ileal soluble non-starch polysaccharides (NSP) digestibility in birds fed the wheat-based diet compared to non-supplemented birds (P < 0.001). Birds fed the wheat-based diet displayed a higher abundance of Bifidobacterium, Lactobacillus and Enterobacteriaceae and butyric acid in the caeca at 16 d of age compared to birds fed the maize-based diet (P < 0.05). In conclusion, supplemental XYN11 exacerbated the negative impact of NE on growth performance in birds fed the maize-based diet. Supplementing wheat-based diets with fibre-degrading enzymes ameliorates production losses induced by NE.

Supplementation of reduced protein diets with

Context Improving immune status through nutritional adjustments may be part of an effective strategy to reduce reliance on antibiotic growth promoters for controlling necrotic enteritis (NE) in broiler chickens. Aims This study examined the effect of dietary protein level and the replacement of crystalline l-arginine (Arg) with l-citrulline (Cit) in the reduced-protein diet on the performance of broilers challenged with subclinical NE. Methods Ross 308 cockerels (n = 720) were randomly allocated to six dietary treatments, with eight replicates of 15 birds per pen, during a 35-day feeding experiment. The treatments were as follows: standard protein without NE challenge (SP−); standard protein with NE challenge (SP+); reduced protein (two percentage points lower crude protein) without NE challenge (RP−); reduced protein with NE challenge (RP+); RP+ plus added Arg (103% of RP, RPA+) and RPC+ where supplemental Arg in RPA+ was replaced with Cit. The first four treatments were considered as a 2 × 2 factorial arrangement, with factors being NE (− or +) and protein level (SP or RP). Treatments SP+, RP+, RPA+, and RPC+ were analysed by one-way ANOVA. Key results Subclinical NE challenge reduced feed intake (FI), reduced body weight gain (BWG) and increased feed to gain ratio (FCR) from Day 0 to Day 35, increased intestinal lesion scores on Day 16, and reduced relative breast yield on Day 35 (P &lt; 0.05). Feeding RP diets increased FI (P &lt; 0.001), increased BWG (P &lt; 0.01) and reduced FCR (P &lt; 0.01) during the grower phase compared with SP diets when birds were challenged with NE. Birds in the RPC+ treatment had a lower overall FCR than did those in the SP+ treatment (P &lt; 0.001). Birds in the RPA+ treatment had similar FI, BWG and FCR to those in the RP+ treatment (P &gt; 0.05). Conclusions Collectively, the results showed protective effects of replacing the supplemental Arg with Cit against NE in RP diets, as indicated by higher performance during and after the challenge. Implications Feeding the RP diets supplemented with Cit may be part of an effective strategy to reduce reliance on antibiotic growth promoters for controlling NE in broiler chickens.

Supplementation of reduced protein diets with

Context The impact of necrotic enteritis (NE) on acute-phase proteins, interleukins, blood mineral profiles, and gene expression have not been well documented. Aims This study aimed to determine the effects of l-arginine (Arg) or l-citrulline (Cit) supplementation on serum immunological parameters, serum mineral composition and gene expression in broilers fed reduced-protein diets (RP) during subclinical NE challenge. Methods Ross 308 cockerels (n = 720) were randomly assigned to six experimental treatments, with eight replicates of 15 birds per pen. The treatments were standard protein without and with NE challenge (SP−, SP+); reduced protein (2% points lower crude protein) without and with NE challenge (RP−, RP+), RP plus added Arg (103% of Ross 308 requirement) with NE challenge (RPA+) and RPC+ where supplemental Arg in RPA+ was replaced with Cit. A 2 × 2 factorial arrangement was employed for the first four treatments. Additionally, treatments SP+, RP+, RPA+, and RPC+ were analysed by one-way ANOVA. Key results The NE × protein interactions indicated that serum calcium concentration decreased in birds fed the RP diets only when challenged with NE (P &lt; 0.05). The NE × protein interactions showed that the NE challenge downregulated the mRNA expression of jejunal y+ L amino acid transporter-2, and mucin 2 only in birds fed the RP diets (P &lt; 0.05). Feeding the RP decreased expression of catenin alpha 1, but increased expression of claudin 5 and tight junction protein genes compared with the SP (P &lt; 0.05). Birds in the RPC+ treatment had increased gene expression of tight junction protein and claudin 5 compared with the SP+ treatment (P &lt; 0.05). Conclusions Dietary protein level and infection with NE both have an impact on immune response and expression of genes involved in immunity and nutrient digestibility. In part replacement of Arg with Cit in the RPC diet may have beneficial effects on gene expression in NE-challenged birds. Implications Feeding RP diets may alleviate a decline in growth during subclinical NE by increasing gene expression of tight junction proteins compared with the SP diets.

Supplementation of reduced protein diets with

Context Necrotic enteritis (NE) has been considered a major threat to broiler gut health and growth performance. Aims This study aimed at investigating the effects of l-arginine (Arg) or l-citrulline (Cit) supplementation on intestinal morphology, short-chain fatty acid (SCFA), microbiota count, gut permeability, and pH in broilers fed reduced-protein diets during subclinical NE challenge. Methods Ross 308 cockerels (n = 720) were randomly assigned to six experimental treatments with eight replicates of 15 birds per pen. The treatments were standard protein without NE challenge (SP−), or with NE challenge (SP+); reduced protein (two percentage points lower crude protein) without NE challenge (RP−), or with NE challenge (RP+); RP+ plus added Arg (103% of Ross 308 requirement, RPA+) and RPC+ where supplemental Arg in RPA+ was replaced with Cit. A 2 × 2 factorial arrangement was employed for the first four treatments. Factors were NE (− or +) and protein concentration (SP or RP). Treatments SP+, RP+, RPA+, and RPC+ were analysed by one-way ANOVA. Key results Necrotic enteritis × protein interactions were detected for serum fluorescein isothiocyanate dextran (FITC-d) level, C. perfringens (P &lt; 0.05) count in the caeca (P &lt; 0.01), and acetic acid (P &lt; 0.01) and total SCFA concentrations in the ileum on Day 16 (P &lt; 0.001). Feeding the RP diet reduced serum FITC-d concentration, number of C. perfringens in the caeca, and increased acetic acid and total SCFA concentrations in the ileum compared with the SP group only in birds challenged with NE. Birds in the RPC+ treatment had greater jejunal villus height (P &lt; 0.001), and lower caecal C. perfringens and Enterobacteriaceae count than did those in the SP+ treatment (P ≤ 0.001). Conclusions The results indicated a benefit to gut health of broilers during NE challenge when replacing crystalline Arg with Cit in RP diets. Implications In part, replacement of Arg by Cit in the RP diets is of great potential to increase gut health, reduce growth loss, thus, minimising negative effects of NE in broilers.

Comparison of the Effects of Antibiotic Alternative Feed Additives on Prevention of Subclinical Necrotic Enteritis in Broilers

Comparison of the Effects of Antibiotic Alternative Feed Additives on Prevention of Subclinical Necrotic Enteritis in Broilers