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.

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.

Early Salmonella Typhimurium inoculation may obscure anti-interleukin-10 protective effects on broiler performance during coccidiosis and necrotic enteritis challenge.

Anti-interleukin (IL)-10 may preserve broiler performance during coccidiosis by diminishing Eimeria spp. host-evasion but has not been evaluated during secondary Clostridium perfringens challenge (necrotic enteritis). Early Salmonella Typhimurium inoculation is implemented in some models to improve repeatability-a potential confounder due to Salmonella using similar IL-10 host evasion pathways. The objective was to evaluate performance and disease outcomes in broilers fed anti-IL-10 during necrotic enteritis challenge ± S. Typhimurium. Three 42 d replicate studies in wire-floor cages (32 cages/replicate) were conducted with Ross 308 chicks assigned to diets ± 0.03% anti-IL-10 for 25 d before moving to floor pens for the study remainder. In replicates 1 and 2, 640 chicks were placed at hatch (20/cage) and inoculated with sterile saline ± 1 × 108 colony forming units (CFU) S. Typhimurium. Replicate 3 placed 480 chicks (15/cage) at hatch. On d 14, S. Typhimurium-inoculated chicks (replicates 1 and 2) or those designated for challenge (replicate 3) were inoculated with 15,000 sporulated Eimeria maxima M6 oocysts. On d 18 and 19, half the E. maxima-challenged chicks were gavaged with 1 × 108 CFU C. perfringens. Body weight (BW) and feed intake were measured throughout, while 6 chicks/ treatment were scored for jejunal lesions at 7 and 3 d postinoculation (pi) with E. maxima and C. perfringens, respectively. Oocyst shedding was measured at 8 and 4 dpi with E. maxima and C. perfringens, respectively. Performance and oocyst shedding were analyzed with diet and challenge fixed effects (SAS 9.4), whereas lesion scores and mortalities were analyzed by ordinal logistic regression (R 4.2.2; P ≤ 0.05). In replicate 3, no wk 3 feed conversion ratio (FCR) differences were observed between chicks fed anti-IL-10 challenged with E. maxima ± C. perfringens, whereas control-fed chicks had a 50 point less efficient FCR during E. maxima + C. perfringens challenge vs. E. maxima only (P = 0.04). Outcomes suggest anti-IL-10 may preserve bird feed efficiency during necrotic enteritis challenge in models without S. Typhimurium.

Dietary peptide-specific antibodies against interleukin-4 differentially alter systemic immune cell responses during Eimeria challenge with minimal impacts on the cecal microbiota.

Eimeria spp. induce host interleukin (IL)-4 production, a potent immune regulator, during coccidiosis to evade immune responses. Dietary anti-IL-4 may preserve bird performance during challenge; however, specific mechanisms have not been investigated. Study objectives were to develop peptide-specific anti-IL-4 antibodies and evaluate immune cell profiles and the cecal microbiota during Eimeria challenge. Four candidate IL-4 peptides were selected based on antigenicity and location. Hens were injected with conjugated peptide or carrier-only control (3/injection), eggs were collected post-vaccination and yolks were pooled by peptide before freeze-drying. On d 0, 300 Ross 708 broilers were placed in floor pens (10/pen) and assigned to 5 diets consisting of basal diet + 2% egg yolk powder containing antibodies against 1 of 4 target peptides or carrier-only control for 14-d starter and grower periods (28 d total). Baseline blood and cecal contents were collected on d 14 (6 birds/diet) before half the remainder were inoculated with 10X Coccivac-B52 (Merck Animal Health, Kenilworth, NJ). Body weight (BW) and feed intake (FI) were recorded weekly and blood and cecal samples were collected at 3, 7, and 14 d post-inoculation (pi; 3/treatment). Immune cell profiles in peripheral blood mononuclear cells (PBMC) were evaluated flow cytometrically and cecal microbial communities determined by 16S/18S rRNA gene amplicon sequencing. Data were log-transformed when necessary and analyzed with diet, Eimeria, and timepoint fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Anti-IL-4 did not alter baseline performance but generally increased PBMC Bu-1+ B cells 38.0 to 55.4% (P < 0.0001). Eimeria challenge reduced FI and BWG 16.1 and 30.3%, respectively, regardless of diet (P < 0.0001) with only birds fed peptide 4 antibodies not recovering feed conversion by d 28. Minimal diet-associated cecal microbiota changes were observed, indicating that anti-IL-4 effects were likely host-specific. Eimeria-challenged birds fed peptide 3 antibodies displayed minimal immune cell fluctuations compared to unchallenged counterparts, suggesting these antibodies potentially modulated intestinal immune responses to minimize systemic requirements, making them good candidates for further research.

Dietary alfalfa hay or lipid-soluble alfalfa extract may improve broiler growth, but fiber presence may be detrimental during Eimeria vaccine challenge.

Lipid-soluble components in late-cutting alfalfa are linked to beneficial immune and microbiota responses in mouse challenge models; therefore, responses in a comparative poultry Eimeria challenge model were investigated. The study objective was to evaluate performance, immunity, and the cecal microbiota in broilers fed ground hay or lipid-soluble extract from late (fifth) cutting alfalfa during Eimeria challenge. At hatch, 432 Ross 708 broilers were placed in 24 floor pens (18 birds/pen) and assigned to 3 isocaloric/isonitrogenous dietary treatments consisting of control, 5% ground hay, or 0.25% lipid-soluble extract for a 42-d trial divided into 14 d starter, grower, and finisher periods. On d 14, 4 birds/treatment were euthanized to collect blood and cecal contents before half the remainder were inoculated with 10X Merck Coccivac-B52 (Kenilworth, NJ). Tissue samples were collected at 3, 7, 14, and 28 d postinoculation (pi; 4 birds/diet × Eimeria group) with body weight (BW) and feed intake (FI) recorded weekly. Immune populations within peripheral blood mononuclear cells were characterized by flow cytometry while cecal microbial communities were profiled by 16S rRNA gene amplicon sequencing. Data were normalized when appropriate and analyzed to evaluate the effects of diet, Eimeria challenge, and timepoint (SAS 9.4; P ≤ 0.05). Before challenge, dietary alfalfa hay or extract increased FI 6.9 to 8.0% and increased CD3+ T cells 19.3 to 24.9% compared to control-fed birds (P ≤ 0.007). Alfalfa did not significantly affect post-Eimeria performance, but Eimeria-challenged birds fed hay showed the greatest numeric reduction in final BW compared to their unchallenged counterparts (0.17 kg) vs. control and extract-fed birds (0.02-0.04 kg). Immune cell changes did not indicate recruitment from peripheral blood to local infection sites; however, alfalfa hay may have accelerated Bu-1+ B cell development by 7 d in unchallenged birds (P < 0.0001). During Eimeria-challenge, dietary alfalfa extract preserved alpha diversity measures related to species richness (P ≤ 0.007). Collectively, these results indicate potential benefits of feeding lipid-soluble extract from late cutting alfalfa to broilers during Eimeria challenge.

Evaluating a Salmonella Typhimurium, Eimeria maxima, and Clostridium perfringens coinfection necrotic enteritis model in broiler chickens: repeatability, dosing, and immune outcomes.

Coccidiosis and necrotic enteritis negatively impact poultry production, making challenge model repeatability important for evaluating mitigation strategies. Study objectives were: 1) evaluate Salmonella Typhimurium-Eimeria maxima-Clostridium perfringens necrotic enteritis coinfection model repeatability and 2) investigate E. maxima dose and early S. Typhimurium challenge on immune responses. Three trials using Ross 308 chicks assigned to 12 cages/trial (7 chicks/cage) in wire-floor brooders were completed. Trials 1 and 2 determined E. maxima dose for necrotic enteritis challenge in trial 3. Chicks in trials 1 and 2 were inoculated with 0 (control), 5, 15, or 25,000 sporulated E. maxima M6 oocysts on d 14 and jejunal lesion scores evaluated on d 20. In trial 3, chicks were assigned to control or necrotic enteritis challenge (42 chicks/group). Necrotic enteritis challenge chicks were inoculated with 1 × 105 colony forming units (CFU) S. Typhimurium on d 1, 15,000 E. maxima oocysts on d 14, and 1 × 108 CFU C. perfringens on d 19 and 20 with lesion scoring on d 22. Bird and feeder weights were recorded throughout each trial. Peripheral blood mononuclear cells (PBMC) were isolated from 1 chick/cage at baseline (all trials), 4 chicks/dose (trials 1 and 2) or 8 chicks/challenge (trial 3) 24 h post-inoculation (pi) with E. maxima for immunometabolic assays and immune cell profiling. Data were analyzed by mixed procedure (SAS 9.4) with challenge and timepoint fixed effects (P ≤ 0.05, trends 0.05 ≤ P ≤ 0.01). Inoculating chicks with 15,000 E. maxima oocysts increased d 14 to 20 FCR 79 points (trials 1 and 2; P = 0.009) vs. unchallenged chicks and achieved a target lesion score of 2. While C. perfringens challenge reduced trial 3 performance, average lesion scores were <1. Salmonella inoculation on d 1 tended to increase PBMC ATP production 41.6% 24 hpi with E. maxima vs. chicks challenged with E. maxima only (P = 0.06). These results provide insight for future model optimization and considerations regarding S. Typhimurium's effect on E. maxima immune response timelines.

Basal diet composition contributes to differential performance, intestinal health, and immunological responses to a microalgae-based feed ingredient in broiler chickens.

Novel feed ingredients may improve poultry health, but functionality of these ingredients may vary across basal diet formulations. This study evaluated a proprietary algae ingredient's effects on broiler performance, intestinal health, systemic immunity, and metabolic/immune kinotypes between corn- or wheat-based diets. Ross 308 broilers were housed in 80 floor pens (14 birds/pen) and assigned to 1 of 4 corn or wheat-based diets ± 0.175% algae ingredient for 42 d. At the end of each 14 d starter, grower, and finisher period, 10 birds/treatment were euthanized for tissue collection to assess intestinal histomorphology, systemic immune cell populations by flow cytometry and kinotypes by peptide arrays. On d 28 and 29, forty-three birds/treatment underwent a 12 h feed restriction challenge followed by a fluorescein isothiocyanate-dextran intestinal permeability assay. For the entire 42 d study, wheat-based diets improved feed conversion rate (FCR) by 5 points compared to corn-based diets (P < 0.0001). Performance benefits related to algae inclusion were diet dependent, with algae inclusion improving 42 d FCR by 6 points only in corn-based diets (P = 0.006). Birds fed wheat-based diets had reduced splenic monocyte/macrophage, CD1.1+, and T cell populations in the first 14 d (P < 0.0001) and reduced serum fluorescence on d 28/29 (P = 0.0002). Algae inclusion in the corn-based diet increased villus height in the duodenum on d 28 and jejunum on d 42, while reducing splenic CD3+CD8α+ cytotoxic T cells 13.4 to 27.5% compared to the corn-based control at the same timepoints (P < 0.0001). Kinome results showed a significant innate immune toll-like receptor (TLR) response via MyD88 at d 14 in the small intestine of birds fed corn-based diets with algae that shifted to a more growth factor and adaptive immune-oriented response by d 42. Concurrent with immune changes, signaling changes indicative of lipid metabolism in the small intestine, ceca, and liver were seen in birds fed the corn-based diet with algae. The observed differential responses to basal diet composition and algae inclusion emphasize the need to comparatively evaluate feed ingredients in various diet formulations.

Algae-based feed ingredient protects intestinal health during Eimeria challenge and alters systemic immune responses with differential outcomes observed during acute feed restriction.

Compounds in microalgae-derived feed ingredients in poultry diets may improve intestinal physiology and immunity to protect against damage induced by physiological and pathogen challenges, but mechanisms are examined sparingly. The study objective was to evaluate changes to intestinal morphology, permeability, and systemic immunity in broilers fed a proprietary microalgae ingredient during 2 separate challenge studies. In study 1, two replicate 28 d battery cage trials used 200 Ross 308 broilers each (n = 400) fed a control diet ± 0.175% algae ingredient. Half of the birds were subjected to a 12 h feed restriction challenge and fluorescein isothiocyanate dextran (FITC-D) intestinal permeability assay on d 28. Study 2 used 800 broilers randomly assigned to the same dietary treatments and housed in floor pens for 42 d. At d 14, intestine and spleen samples were collected from 10 birds/ diet. Half of the remainder was orally inoculated with 10X Coccivac-B52 vaccine in a 2 × 2 factorial treatment design (diet and Eimeria inoculation). The FITC-D assay was conducted at 1, 3, 7, and 14 d post-inoculation (pi) while intestinal and spleen samples were collected at 3, 7, 14, and 28 dpi for histomorphology and flow cytometric immune cell assessment. Study 1 validated intestinal leakage via FITC-D absorbance induced by feed restriction but showed no algae-associated protective effects. In study 2, algae preserved intestinal integrity during coccidiosis (P = 0.04) and simultaneously protected jejunal villus height as early as 7dpi (P < 0.0001), whereas intestinal damage resolution in control birds did not occur until 14 dpi. Algae inclusion increased splenic T cells in unchallenged broilers at d 14 by 29.6% vs. control (P < 0.0001), specifically γδ T cell populations, without impacting performance (P < 0.03). During Eimeria challenge, splenic T cells in algae-fed birds did not show evidence of recruitment to peripheral tissues, while control birds showed a 16.7% reduction compared to their uninoculated counterparts from 3 to 7 dpi (P < 0.0001). This evidence suggests the algae ingredient altered the immune response in a manner that reduced recruitment from secondary lymphoid organs in addition to protecting intestinal physiology.

Host immunity and the colon microbiota of mice infected with Citrobacter rodentium are beneficially modulated by lipid-soluble extract from late-cutting alfalfa in the early stages of infection.

Alfalfa is a forage legume commonly associated with ruminant livestock production that may be a potential source of health-promoting phytochemicals. Anecdotal evidence from producers suggests that later cuttings of alfalfa may be more beneficial to non-ruminants; however, published literature varies greatly in measured outcomes, supplement form, and cutting. The objective of this study was to measure body weight, average daily feed intake, host immunity, and the colon microbiota composition in mice fed hay, aqueous, and chloroform extracts of early (1st) and late (5th) cutting alfalfa before and after challenge with Citrobacter rodentium. Prior to inoculation, alfalfa supplementation did not have a significant impact on body weight or feed intake, but 5th cutting alfalfa was shown to improve body weight at 5- and 6-days post-infection compared to 1st cutting alfalfa (P = 0.02 and 0.01). Combined with the observation that both chloroform extracts improved mouse body weight compared to control diets in later stages of C. rodentium infection led to detailed analyses of the immune system and colon microbiota in mice fed 1st and 5th cutting chloroform extracts. Immediately following inoculation, 5th cutting chloroform extracts significantly reduced the relative abundance of C. rodentium (P = 0.02) and did not display the early lymphocyte recruitment observed in 1st cutting extract. In later timepoints, both chloroform extracts maintained lower splenic B-cell and macrophage populations while increasing the relative abundance of potentially beneficially genera such as Turicibacter (P = 0.02). At 21dpi, only 5th cutting chloroform extracts increased the relative abundance of beneficial Akkermansia compared to the control diet (P = 0.02). These results suggest that lipid soluble compounds enriched in late-cutting alfalfa modulate pathogen colonization and early immune responses to Citrobacter rodentium, contributing to protective effects on body weight.

Evaluation of a high-protein DDGS product in broiler chickens: performance, nitrogen-corrected apparent metabolisable energy, and standardised ileal amino acid digestibility.

1. New production processes and additional uses for corn co-products have increased the availability of distiller's dried grains with solubles (DDGS) with varying energy and amino acid digestibility, for use in poultry feed. The objective of this study was to determine the performance, N-corrected metabolisable energy (AMEn), and amino acid (AA) digestibility of a 34% CP (as fed) high-protein DDGS (HP-DDGS) included in poultry diets for Cobb 500 broiler chickens.2. A total of 832 Cobb 500 broilers were randomly assigned to four dietary treatments containing 5% conventional DDGS (CV-DDGS) as a control or 10%, 15% and 20% HP-DDGS and fed for 42 d. After the performance trial, 240 birds from the original 832 were selected for a concurrent AMEn and AA digestibility experiment consisting of two AMEn diets and two AA diets.3. Birds fed diets containing 15% and 20% HP DDGS gained less weight than birds fed the CV-DDGS (P < 0.05) but did not differ in feed intake (FI), and therefore had a less efficient FCR than the control (P < 0.05). The AMEn of HP-DDGS was determined to be 11.4 MJ/kg. The standardised ileal amino acid digestibility (SIAAD) of the essential amino acids Lys and Met were determined to be 80.9% and 88.6%, respectively.4. HP-DDGS can be included in broiler diets up to 10% without any negative impact on performance or requiring supplemental Lys and Arg. The results from the AA digestibility study indicated that HP-DDGS could be a good source of digestible Lys.