The presence of differentiated C2C12 muscle cells enhances toxin production and growth by Clostridium perfringens type A strain ATCC3624.

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

Broiler chicken distal jejunum microbial communities are more responsive to coccidiosis or necrotic enteritis challenge than dietary anti-interleukin-10 in a model using Salmonella Typhimurium- Eimeria maxima- Clostridium perfringens coinfection.

Dietary anti-interleukin (IL)-10 antibodies may protect broiler performance during coccidiosis by inhibiting Eimeria host-evasion pathways; however, anti-IL-10's effects on microbial communities during coccidiosis and secondary Clostridium perfringens (necrotic enteritis) challenge is unknown. The study objectives were to assess the jejunal microbiota of broilers fed anti-IL-10 during E. maxima ± C. perfringens challenge. Two replicate studies using Ross 308 chicks placed in wire-floor cages (32 cages/ replicate study; 20 chicks/ cage) were conducted, with chicks assigned to diets ± 0.03% anti-IL-10 for 25 d. In both replicate studies, challenge-designated chicks were inoculated with 1 × 108Salmonella Typhimurium colony forming units (CFU) at placement. On d14, S. Typhimurium-inoculated chicks were gavaged with 15,000 sporulated Eimeria maxima M6 oocysts and half the E. maxima-challenged chicks received 1×108C. perfringens CFUs on d 18 and 19. Six chicks/ treatment were euthanized for distal jejunum content collection at baseline (d 14), 7 d post-inoculation (pi) with E. maxima/ 3 dpi with C. perfringens (peak) or 11 dpi with E. maxima/ 7 dpi with C. perfringens (post-peak) for 16S rRNA gene amplicon sequencing. Sequences were quality screened (Mothur V.1.43.0) and clustered into de novo operation taxonomical units (OTU; 99% similarity) using the SILVA reference database (v138). Alpha diversity and log-transformed relative abundance data were analyzed in SAS 9.4 with replicate study, diet, challenge, and timepoint main effects plus associated interactions (P ≤ 0.05). Few baseline changes were observed, but E. maxima ± C. perfringens challenge reduced Romboutsia and Staphylococcus relative abundance 4- to 800-fold in both replicate studies (P ≤ 0.008). At peak challenge with secondary C. perfringens, feeding anti-IL-10 instead of the control diet reduced Clostridium sensu stricto 1 relative abundance 13- and 1,848-fold in both replicate studies (P < 0.0001); however, OTUs identified as C. perfringens were not affected by dietary anti-IL-10. These results indicate that anti-IL-10 does not affect the jejunal microbiota of unchallenged broilers, while coccidiosis or necrotic enteritis challenge generally contributed to greater microbiota alterations than diet.

Sequencing approaches to identify distal jejunum microbial community composition and function in broiler chickens fed anti-interleukin-10 during coccidiosis and necrotic enteritis challenge.

Strategies to counteract interleukin (IL)-10-mediated immune evasion by Eimeria spp. during coccidiosis- like anti-IL-10 antibodies- may protect broiler chicken health and reduce incidence of secondary necrotic enteritis (Clostridium perfringens) via undetermined mechanisms. Objectives were to use sequencing techniques to evaluate jejunal microbial community composition and function in anti-IL-10-fed broilers during coccidiosis and necrotic enteritis. On d0, Ross 308 chicks were placed in 32 cages (15 chicks/ cage) for a 25-d study and randomly assigned to diets ± 0.03% anti-IL-10. Six chicks/ diet were euthanized for distal jejunum content and tissue collection on d 14 (baseline) before inoculating the remainder with saline or 15,000 E. maxima oocysts (M6 strain). Half the chicks challenged with E. maxima were challenged with C. perfringens (1×108 colony forming units) on d 18 and 19. Follow-up samples (6 chicks/treatment) were collected at 7 and 11 d postinoculation (pi) for the E. maxima-only group, or 3 and 7 dpi for the E. maxima + C. perfringens group with 3/7 dpi being designated as peak and 7/11dpi as postpeak challenge. DNA was extracted from digesta for microbiota composition analysis (16S rRNA gene sequencing) while RNA was extracted from tissue to evaluate the metatranscriptome (RNA sequencing). Alpha diversity and genus relative abundances were analyzed using the diet or challenge main effects with associated interactions (SAS 9.4; P ≤ 0.05). No baseline microbial changes were associated with dietary anti-IL-10. At peak challenge, a diet main effect reduced observed species 36.7% in chicks fed anti-IL-10 vs. control; however, the challenge effect reduced observed species and Shannon diversity 51.2-58.3% and 33.0 to 35.5%, respectively, in chicks challenged with E. maxima ± C. perfringens compared to their unchallenged counterparts (P ≤ 0.05). Low sequencing depth limited metatranscriptomic analysis of jejunal microbial function via RNA sequencing. This study demonstrates that challenge impacted the broiler distal jejunum microbiota more than anti-IL-10 while future research to characterize the microbial metatranscriptome may benefit from investigating other intestinal compartments.

Alternative to antimicrobial growth promoters in the diets of broilers challenged with subclinical necrotic enteritis.

Necrotic enteritis (NE) is a disease of worldwide distribution, which affects young broilers and causes economic losses on a scale of 6 billion dollars per year. For decades, NE was controlled in poultry flocks by dietary administration of low doses of antimicrobial growth promoters (AGPs). However, an increase in NE incidence was noted after the AGP ban. This study aimed to compare the effect of an antibiotic (Enramycin) diet to a combination of sodium butyrate, hydrolyzed yeast, and zinc proteinate (ViligenTM) on broiler diets regarding performance, blood parameters, intestinal permeability, morphology and lesions, and carcass yield of broilers challenged with Eimeria spp. and Clostridium perfringens to simulate subclinical necrotic enteritis. A total of 1,150 one-day-old male broiler chickens with an initial average weight of 43.9 ± 0.65 g were allocated to 50 experimental pens. Animals were divided into 5 groups: Negative control (NC) without additives; Positive control (PC) with 0.12 g/ton of Enramycin (8%); V500, V1000, and V1500 with the addition of 500, 1.000, and 1.500 g/ton of Viligen, respectively. All animals were challenged by Eimeria spp. at 7 d of age and by C. perfringens at 17, 18, and 19 d for induction of subclinical NE. The broilers fed with all concentrations of Viligen showed similar performance, blood parameters, intestinal permeability, and carcass yield compared to PC broilers. However, NC broilers showed higher FCR compared to PC broilers from 1 to 33 d (1.42 vs. 1.39) (P = 0.048) and from 1 to 42 d (1.51 vs. 1.49) (P < 0.001). V1500 broilers had fewer intestinal lesions at 28 d when compared to the PC treatment (P < 0.05) and showed that higher Viligen inclusion resulted in lower intestinal damage. At 21 d, the V500 group showed higher intestinal morphology characteristics (VH:VD 4.9 vs. 3.5) compared to the PC treatment (P < 0.001). Thus, in this study, the dietary addition of Viligen to broilers challenged by an experimental model of subclinical NE resulted in lower intestinal damage and similar performance to that obtained by the addition of Enramycin.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

In vitro and genomic mining studies of anti-Clostridium perfringens Compounds Derived from Bacillus amyloliquefaciens.

Clostridium perfringens is an important opportunistic microorganism in commercial poultry production that is implicated in necrotic enteritis (NE) outbreaks. This disease poses a severe financial burden on the global poultry industry, causing estimated annual losses of $6 billion globally. The ban on in-feed antibiotic growth promoters has spurred investigations into approaches of alternatives to antibiotics, among which Bacillus probiotics have demonstrated varying degrees of effectiveness against NE. However, the precise mechanisms underlying Bacillus-mediated beneficial effects on host responses in NE remain to be further elucidated. In this manuscript, we conducted in vitro and genomic mining analysis to investigate anti-C. perfringens activity observed in the supernatants derived from 2 Bacillus amyloliquefaciens strains (FS1092 and BaD747). Both strains demonstrated potent anti-C. perfringens activities in in vitro studies. An analysis of genomes from 15 B. amyloliquefaciens, 11 B. velezensis, and 2 B. subtilis strains has revealed an intriguing clustering pattern among strains known to possess anti-C. perfringens activities. Furthermore, our investigation has identified 7 potential antimicrobial compounds, predicted as secondary metabolites through antiSMASH genomic mining within the published genomes of B. amyloliquefaciens species. Based on in vitro analysis, BaD747 may have the potential as a probiotic in the control of NE. These findings not only enhance our understanding of B. amyloliquefaciens's action against C. perfringens but also provide a scientific rationale for the development of novel antimicrobial therapeutic agents against NE.

Evaluation of long-term supplementation of a Bacillus subtilis direct-fed microbial and enzymatically hydrolyzed yeast cell culture product used alone or in combination on Clostridia, Clostridium perfringens, Escherichia coli, and Salmonella prevalence in beef steers.

The objective was to determine the influence of long-term supplementation (258 d) of a direct-fed microbial (DFM) and/or yeast cell wall (YCW) product on bacterial populations in beef steers. Single-sourced Charolais × Red Angus steers (n = 256; body weight = 246 ±â€…1.68 kg) were used in a randomized complete block design and blocked by location into one of four treatments: 1) fed no DFM and no YCW (Control); 2) fed only the DFM (DFM; Certillus CP B1801 Dry, 28 g/steer d-1 ); 3) fed only the YCW (YCW; Celmanax; 18 g/steer d-1 ); and 4) fed the DFM and the YCW (DFM+YCW). Steers were vaccinated for respiratory and clostridial diseases and treated for internal and external parasites at processing and individually weighed on days 1, 14, 42, 77, 105, 133, 161, 182, 230, and 258. To determine bacterial prevalence, fecal samples were collected on days 1, 14, 77, 133, 182, and 230 and environmental (pen area, feed, and water) samples were collected at the beginning of the week when cattle were weighed. No treatment × day interactions or treatment effects (P > 0.05) were observed between treatment groups at any sampling days for the bacterial populations. Samples on days 1, 133, and 182 had greater (P < 0.05) Clostridia levels compared to the other sampling points but were not different from each other. Clostridia levels were also greater (P < 0.05) on day 77 compared to days 14 and 230. Samples on days 77 and 230 had greater (P < 0.05) Clostridium perfringens levels compared to the other sampling points but were not different (P > 0.05) from each other. Samples on days 1 and 14 had lower (P < 0.05) total Escherichia coli levels compared to the other sampling points but were not different (P > 0.05) from each other. Escherichia coli levels on day 77 were higher (P < 0.05) compared to days 133, 182, and 230. Little Salmonella prevalence (1.5%) was observed throughout the study. This study had greater levels of Clostridia compared to small and large commercial feedlots in the Church and Dwight research database, but C. perfringens, total and pathogenic E. coli, and Salmonella prevalence were notably lower. Collectively, there were no appreciable treatment influences on bacterial populations. These data further indicate a low pathogenic bacterial challenge at the trial site, which could partially explain the lack of differences with DFM or YCW supplementation. The DFM and YCW used alone or in combination cannot be expected to show additional benefits when animals are relatively unstressed with a low pathogenic bacterial challenge.

The objective of this research was to determine the influence of long-term supplementation (258 d) of a direct-fed microbial (DFM) and/or yeast cell wall (YCW) product on bacterial populations in beef steers. Collectively, there were no appreciable treatment influences on bacterial populations. These data further indicate a low pathogenic bacterial challenge at the trial site, which could further explain the reasons for little differences. The DFM and YCW used alone or in combination cannot be expected to show productive benefits when animals are relatively unstressed with a low pathogenic bacterial challenge.

Screening and selection of eubiotic compounds possessing immunomodulatory and anti-Clostridium perfringens properties.

Eubiotics are water and/or feed additives used in poultry to promote gut health and control enteric burden of pathogens, including Clostridium perfringens. While several eubiotic compounds (ECs) are being introduced commercially, it is essential to devise an in vitro model to screen these compounds to assess their immunomodulatory and antimicrobial properties prior to their testing in vivo. A chicken macrophage cell-line (MQ-NCSU) was used to develop an in vitro model to screen the immunological and anti-C. perfringens properties of 10 ECs: monobutyrin, monolaurin, calcium butyrate, tributyrin, carvacrol, curcumin, green tea extract, rosemary extract, monomyristate, and tartaric acid. An optimal concentration for each EC was selected by measuring the effect on viability of MQ-NCSU cells. Cells were then treated with ECs for 6, 12, and 24 h. and expression of interferon-gamma (IFNγ), interleukin (IL)-1ß, IL-6, IL-10, transforming growth factor-beta (TGFß) and cluster of differentiation (CD40) genes, as well as major histocompatibility complex (MHC)-II protein were evaluated. At 6 h post-stimulation, monobutyrin, calcium butyrate, and green tea extract treatments induced a significant downregulation of IFNγ, IL-6, or IL-1ß gene transcription and MHC-II expression, while the IL-10 or TGFß gene expression in these treatments as well as those receiving rosemary extract and tartaric acid was significantly upregulated, when compared to control, suggesting immunomodulatory properties of these ECs. Finally, pretreatment of macrophages with these selected 5 ECs for 24 h followed by C. perfringens infection showed that monobutyrin, green tea extract, rosemary extract, and calcium butyrate treatments can inhibit bacterial growth significantly at 12 and/or 24 h post-infection, when compared to the control. Collectively, our findings show that ECs possessing immunomodulatory and anti-C. perfringens properties can be selected using an in vitro avian macrophage cell-based model so that such ECs can further be tested in vivo for their disease prevention efficacy.

Extract of Scutellaria baicalensis and Lonicerae flos improves growth performance, antioxidant capacity, and intestinal barrier of yellow-feather broiler chickens against Clostridium perfringens.

In this study, we aimed to investigate the effect of Scutellaria baicalensis and Lonicerae Flos (SL) extract on the growth performance and intestinal health of yellow-feather broilers following a Clostridium perfringens challenge. In total, 600 one-day-old yellow-feather broilers were divided into five treatments (6 replicate pens of 20 birds per treatment), including a control (Con) group fed a basal diet and the infected group (iCon) fed a basal diet and infected with Clostridium perfringens, the other 3 groups receiving different doses of SL (150, 300, and 450 mg/kg) and infected with Clostridium perfringens. The total experimental period was 80 d. When the birds were 24-days-old, a subclinical necrotizing enteritis model was induced by orally inoculating the birds with 11,000 oocysts of mixed Eimeria species on d 24, followed by C. perfringens (108 CFU/mL) from d 28 to 30. The birds were evaluated for parameters such as average weight gain (AWG), average daily feed intake (ADFI), mortality, feed conversion ration (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. Results indicated that C. perfringens infection led to reduced AWG and the levels of tight junction proteins, increased the FCR, ileum E. coli load, and intestinal permeability, causing damage to the intestinal mucosal barrier (P < 0.05). Compared with the infected group, supplementing 300 mg/kg of SL significantly increased AWG at 43 to 80 d, the ratio of villus height to crypt depth in the jejunum and ileum at 35 d, and the activity of superoxide dismutase (SOD) in serum. It also significantly reduced the FCR at 22 to 42 d, intestinal lesion score, and the amount of C. perfringens in the ileum (P < 0.05). Additionally, compared with the infected group, the addition of 300 mg/kg SL significantly increased mRNA levels of claudin-2, claudin-3, mucin-2, and toll-like receptor 2 (TLR-2) in the ileum of infected birds at 35 d of age. In conclusion, supplementation with SL extract could effectively mitigate the negative effects of C. perfringens challenge by improving intestinal barrier function and histomorphology, positively influencing the growth performance of challenged birds.

Effect of arginine supplementation on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

The objective of this study was to evaluate the effect of 25% arginine supplementation as a functional amino acid in partially alleviating the detrimental effects of necrotic enteritis (NE) on the growth performance, serum biochemistry, gut integrity, and the relative gene expression of tight junction proteins and inflammatory cytokines in broilers during NE. Three hundred and sixty 1-day-old chicks were randomly allocated to 4 treatments in a 2 × 2 factorial arrangement -basal diet and 125% arginine diet, with or without NE challenge. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21. The NE challenge had a significant effect on the BWG (p < 0.05), FCR (p < 0.05), serum AST (p < 0.05), GLU (p < 0.05), and K+ (p < 0.05) levels, and intestinal permeability (p < 0.05) and jejunal lesion score (p < 0.05). A significant challenge × diet interaction effect was observed in the cecal tonsil CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 28 (p < 0.05) and spleen CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 35 (p < 0.05). Arginine supplementation significantly increased the CD8+: CD4+ T-cell ratio in uninfected birds but decreased the CD8+: CD4+ T-cell ratio in infected birds. On d 21, a significant interaction effect was observed on the relative expression of the iNOS gene (p < 0.05). Arginine supplementation significantly downregulated the expression of the iNOS gene in infected birds. A significant effect of the challenge (p < 0.05) was observed on the relative gene expression of the ZO-1 gene in the jejunum. NE challenge significantly downregulated the expression of the ZO-1 gene on d 21. In conclusion, arginine supplementation did not alleviate the depression in growth performance and disease severity during the NE challenge. However, arginine downregulated the expression of inflammatory cytokines and enzymes, preventing inflammatory injury to the tissues during NE. Hence, arginine might be supplemented with other alternatives to downregulate inflammatory response during NE in poultry.

Effect of 125% and 135% arginine on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

The objective of this study was to evaluate the effects of 25% and 35% arginine supplementation in partially alleviating the effects of necrotic enteritis (NE) challenge on the production performance, intestinal integrity, and relative gene expression of tight junction proteins and inflammatory cytokines in broilers. Four hundred and eighty 1-day-old chicks were randomly allocated to the 4 treatments- Uninfected + Basal, NE + Basal, NE + Arg 125%, and NE + Arg 135%. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21 of age by oral gavage. The NE challenge significantly decreased body weight gain (BWG) (p < 0.05) and increased the feed conversion ratio (FCR) (p < 0.05). On d 21, the NE challenge also increased the jejunal lesion score (p < 0.05) and relative gene expression of IL-10 and decreased the expression of the tight junction proteins occludin (p < 0.05) and claudin-4 (p < 0.05). The 125% arginine diet significantly increased intestinal permeability (p < 0.05) and the relative gene expression of iNOS (p < 0.05) and IFN-γ (p < 0.05) on d 21 and the bile anti-C. perfringens IgA concentration by 39.74% (p < 0.05) on d 28. The 135% arginine diet significantly increased the feed intake during d 0 - 28 (p < 0.05) and 0 to 35 (p < 0.05) and increased the FCR on d 0 to 35 (p < 0.05). The 135% and 125% arginine diet increased the spleen CD8+: CD4+ T-cell ratio on d 28 (p < 0.05) and 35 (p < 0.05), respectively. The 135% arginine diet increased the CT CD8+:CD4+ T-cell ratio on d 35 (p < 0.05). In conclusion, the 125% and 135% arginine diets did not reverse the effect of the NE challenge on the growth performance. However, the 125% arginine diet significantly increased the cellular and humoral immune response to the challenge. Hence, the 125% arginine diet could be used with other feed additives to improve the immune response of the broilers during the NE challenge.

Research Note: Bovine lactoferrin in chickens: an investigation into its viability as an antibiotic alternative.

Finding effective antibiotic alternatives is crucial to managing the re-emerging health risk of Clostridium perfringens (CP) type A/G-induced avian necrotic enteritis (NE), a disease that has regained prominence in the wake of governmental restrictions on antibiotic use in poultry. Known for its antimicrobial and immunomodulatory effects, the use of bovine lactoferrin (bLF) in chickens is yet to be fully explored. In this study, we hypothesized that bLF can accumulate in the small intestines of healthy chickens through gavage and intramuscular supplementation and serves as a potential antibiotic alternative. Immunohistochemistry located bLF in various layers of the small intestines and ELISA testing confirmed its accumulation. Surprisingly, sham-treated chickens also showed the presence of bLF, prompting a western blotting analysis that dismissed the notion of cross-reactivity between bLF and the avian protein ovotransferrin. Although the significance of the route of administration remains inconclusive, this study supports the hypothesis that bLF is a promising and safe antibiotic alternative with demonstrated resistance to the degradative environment of the chicken intestines. Further studies are needed to determine its beneficial pharmacological effects in CP-infected chickens.

Mechanisms of intestinal epithelial cell damage by Clostridiumperfringens.

Clostridium perfringens, a Gram-positive bacterium, causes intestinal diseases in humans and livestock through its toxins, related to alpha toxin (CPA), beta toxin (CPB), C. perfringens enterotoxin (CPE), epsilon toxin (ETX), Iota toxin (ITX), and necrotic enteritis B-like toxin (NetB). These toxins disrupt intestinal barrier, leading to various cell death mechanisms such as necrosis, apoptosis, and necroptosis. Additionally, non-toxin factors like adhesins and degradative enzymes contribute to virulence by enhancing colonization and survival of C. perfringens. A vicious cycle of intestinal barrier breach, misregulated cell death, and subsequent inflammation is at the heart of chronic inflammatory and infectious gastrointestinal diseases. Understanding these mechanisms is essential for developing targeted therapies against C. perfringens-associated intestinal diseases.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology.

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-ß) and IL-1ß post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Bacillus subtilis HW2 enhances growth performance and alleviates gut injury via attenuation of endoplasmic reticulum stress and regulation of gut microbiota in broilers under necrotic enteritis challenge.

This study investigated the effects of Bacillus subtilis HW2 on the growth performance, immune response, endoplasmic reticulum (ER) stress, and intestinal health in broilers with necrotic enteritis. Three hundred 1-day-old male Cobb 500 broilers (33.88 ± 2.34 g) were randomly allocated to 5 groups including non-infected control (NC group), basal diet + necrotic enteritis challenge (NE group), basal diet + 1 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (L-Pro group), basal diet + 5 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (M-Pro group), and basal diet + 1 × 107 CFU/g B. subtilis HW2 + necrotic enteritis challenge (H-Pro group), with 6 replicates per group. All broilers except NC group were orally given with sporulated coccidian oocysts at day 14 and Clostridium perfringens from days 19 to 21. Results showed that L-Pro and M-Pro groups improved growth performance and intestinal morphology in necrotic enteritis-challenged broilers, and L-Pro, M-Pro, and H-Pro groups improved intestinal barrier function and immune response and decreased ER stress in necrotic enteritis-challenged broilers. Analysis of the gut microbiota revealed that L-Pro group increased the abundances of Alistipes, Coprobacter, Barnesiella, and Limosilactobacillus, decreased Erysipelatoclostridium abundance on day 42 in necrotic enteritis-challenged broilers. M-Pro group increased Turicibacter abundance on day 28 and the abundances of Alistipes, Barnesiella, and Limosilactobacillus on day 42 in necrotic enteritis-challenged broilers. H-Pro group decreased Romboutsia abundance on day 28 and unidentified_Clostridia abundance on day 42 in necrotic enteritis-challenged broilers. Analysis of short-chain fatty acids (SCFAs) revealed higher isobutyric acid and isovaleric acid levels in L-Pro and M-Pro groups than NE group. Correlation analysis revealed the correlations between the biochemical parameters and gut microbiota as well as SCFAs, especially Romboutsia, Barnesiella, Coprobacter, isobutyric acid, and isovaleric acid. Overall, our results indicated that B. subtilis HW2 supplementation could ameliorate necrotic enteritis infection-induced gut injury. The optimal dietary supplementation dosage of Bacillus subtilis HW2 was 5 × 106 CFU/g.

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.

Validating the use of a newly developed cinnamaldehyde product in commercial broiler production.

Essential oils (EOs) have been considered as an alternative to antibiotics for animal production. In the current study, 4 trials were conducted on a commercial broiler farm to investigate the effects of dietary supplementation of an encapsulated cinnamon EO product (NE-OFF) on the bird growth performance, gut health, and gene expression in the ileum, spleen, and liver relating to the host response to heat and other stresses, including potential NE challenge. In each trial, approximately 30,000 Cobb or Ross broilers were randomly allocated to 4 treatments: a raised without antibiotics (RWA) commercial diet as positive control, an adjusted RWA commercial diet as negative control, and the negative control diet supplemented with 2 different dosages of NE-OFF, which was added during feed pelleting. Although the final average body weight did not differ significantly among treatment groups, birds fed NE-OFF had an increased ratio of villus height and crypt depth in the jejunum, and reduced fecal oocyst counts. Trial 2 was conducted in the summer and had a necrotic enteritis (NE) outbreak. The supplementation of NE-OFF reduced the NE incidence and bird mortality. The samples from Trial 2 were hence selected for the analyses of Clostridium perfringens and NetB toxin gene abundance in the ileum, and host responses. The C. perfringens population appeared to be positively correlated with the NetB gene abundance. The gene expression analysis suggested that NE-OFF supplementation improved nutrient absorption and transportation as well as antioxidant activities to help the birds against stress. These on-farm trial results support the hypothesis that the use of NE-OFF as a feed additive can improve bird gut health and performance in commercial broiler production, especially for preventing NE outbreaks when birds are under stress.

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 CD8αß T cells) and natural IEL (TCRαßCD8αα, 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 TCRαß+CD4-CD8-, TCRαß+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαß+CD4-CD8- and TCRαß+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1ß 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.

Coccidiosis and necrotic enteritis model may have a greater impact than dietary anti-interleukin-10 on broiler chicken systemic immunometabolic responses.

Dietary egg yolk-derived anti-interleukin (IL)-10 may preserve broiler chicken performance during coccidiosis due to Eimeria spp. infection while effects on secondary Clostridium perfringens (necrotic enteritis) are unknown. Some necrotic enteritis models implement Salmonella Typhimurium to improve repeatability; however, Salmonella upregulation of IL-10 may be a confounder when evaluating anti-IL-10. The study objective was to investigate anti-IL-10 effects on systemic cytokine concentrations and immunometabolism during E. maxima ± C. perfringens challenge in models ± S. Typhimurium. Three 25 d replicate studies using Ross 308 chicks were conducted in wire-floor cages (32 cages/ replicate) with chicks assigned to diets ± 0.03% anti-IL-10. 640 chicks (20/ cage; replicates 1 and 2) were inoculated with sterile saline ± 1×108 colony forming units (CFU) S. Typhimurium while 480 chicks (15/ cage) were placed in replicate 3. In all replicates, blood samples were collected on d 14 (6 chicks/treatment) before administering 15,000 sporulated E. maxima M6 oocysts to S. Typhimurium-inoculated (replicates 1 and 2) or challenge-designated chicks (replicate 3). Half the E. maxima-challenged chicks received 1×108 CFU C. perfringens on d 18 and 19. Blood samples were collected at 1, 3, 7, and 11 d post-inoculation (dpi) with E. maxima and 1, 3, and 7 dpi with secondary C. perfringens. Plasma cytokines were determined by ELISA while immunometabolic assays evaluated peripheral blood mononuclear cell ATP production and glycolytic rate responses. Data were analyzed with diet and challenge fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Replicates 1 and 2 showed few immunometabolic responses within 3 dpi with E. maxima, but 25 to 31% increased ATP production and 32% increased compensatory glycolysis at 1 dpi with C. perfringens in challenged vs. unchallenged chicks (P ≤ 0.04). In replicate 3, total ATP production and compensatory glycolysis were increased 25 and 40%, respectively, by the E. maxima main effect at 1dpi (P ≤ 0.05) with unobserved responsiveness to C. perfringens. These outcomes indicate that model type had greater impacts on systemic immunity than anti-IL-10.

A microbial muramidase improves growth performance and reduces inflammatory cell infiltration in the intestine of broilers chickens under Eimeria and Clostridium perfringens challenge.

The objective of the present studies was to evaluate muramidase (MUR) supplementation in broilers under Eimeria and/or Clostridium perfringens challenge. For this, 2 experiments were conducted. Experiment 1. A total of 256 one-day old male Cobb 500 chicks were placed in battery cages in a completely randomized design, with 5 treatment groups, 7 replicate cages per treatment and 8 birds per cage. The treatments were: nonchallenged control (NC), challenged control (CC), CC + MUR at 25,000 or 35,000 LSU(F)/kg, and CC + Enramycin at 10 ppm (positive control-PC). Challenge consisted of 15× the recommended dose of coccidiosis vaccine at placement, and Clostridium perfringens (108 CFU/bird) inoculation at 10, 11, and 12 d. Macro and microscopic evaluation, immunohistochemistry, and gene expression were evaluated at 7, 14, 21, and 28 d of age. Experiment 2. A total of 1,120 one-day old male Cobb 500 chicks were placed in floor pens with fresh litter in a completely randomized design, with 4 treatment groups, 8 replicate pens per treatment, and 35 birds per pen. The treatments were: Control, supplementation of MUR at 25,000 or 45,000 LSU(F)/kg, and a positive control (basal diet plus Enramycin). At 10, 11, and 12 d of the experiment all the birds were inoculated by oral gavage with a fresh broth culture of a field isolate Clostridium perfringens (0.5 mL containing 106 CFU/bird). It was observed that in Experiment 1 MUR supplementation reduced the infiltration of macrophages and CD8+ lymphocytes in the liver and ileum of infected birds, downregulated IL-8 and upregulated IL-10 expression. In Experiment 2, MUR linearly improved the growth performance of the birds, increased breast meat yield, and improved absorption capacity. MUR supplementation elicited an anti-inflammatory response in birds undergoing a NE challenge model that may explain the improved growth performance of supplemented birds.