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.

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.

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.

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.

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.

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 25d study and randomly assigned to diets +/- 0.03% antiIL-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 pound 10 8 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-IL10. 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 bene fit from investigating other intestinal compartments.