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.This article is published as Fries-Craft, K., and E. A. Bobeck. "Coccidiosis and necrotic enteritis model may have a greater impact than dietary anti-interleukin-10 on broiler chicken systemic immunometabolic responses." Poultry Science 103 (2024): 103551. doi:10.1016/j.psj.2024.103551. © 2024 The Authors.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 × 108 Salmonella 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 1×108 C. 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.This article is published as Fries-Craft, K., S. Schmitz-Esser, and E. A. Bobeck. "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." Poultry Science 103 (2024): 104000. doi:10.1016/j.psj.2024.104000. © 2024 The Authors. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/)

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.This article is published as Fries-Craft, K., C. Anderson, S. Schmitz-Esser, and E. A. Bobeck. "Sequencing approaches to identify distal jejunum microbial community composition and function in broiler chickens fed anti-interleukin-10 during coccidiosis and necrotic enteritis challenge." Poultry Science 103 (2024): 104001. doi:10.1016/j.psj.2024.104001. © 2024 The Authors. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/)

The role of oscillatory brain activity in object processing and figure-ground segmentation in human vision

The perception of an object as a single entity within a visual scene requires that its features are bound together and segregated from the background and/or other objects. Here, we used magnetoencephalography (MEG) to assess the hypothesis that coherent percepts may arise from the synchronized high frequency (gamma) activity between neurons that code features of the same object. We also assessed the role of low frequency (alpha, beta) activity in object processing. The target stimulus (i.e. object) was a small patch of a concentric grating of 3. c/°, viewed eccentrically. The background stimulus was either a blank field or a concentric grating of 3. c/° periodicity, viewed centrally. With patterned backgrounds, the target stimulus emerged - through rotation about its own centre - as a circular subsection of the background. Data were acquired using a 275-channel whole-head MEG system and analyzed using Synthetic Aperture Magnetometry (SAM), which allows one to generate images of task-related cortical oscillatory power changes within specific frequency bands. Significant oscillatory activity across a broad range of frequencies was evident at the V1/V2 border, and subsequent analyses were based on a virtual electrode at this location. When the target was presented in isolation, we observed that: (i) contralateral stimulation yielded a sustained power increase in gamma activity; and (ii) both contra- and ipsilateral stimulation yielded near identical transient power changes in alpha (and beta) activity. When the target was presented against a patterned background, we observed that: (i) contralateral stimulation yielded an increase in high-gamma (> 55. Hz) power together with a decrease in low-gamma (40-55. Hz) power; and (ii) both contra- and ipsilateral stimulation yielded a transient decrease in alpha (and beta) activity, though the reduction tended to be greatest for contralateral stimulation. The opposing power changes across different regions of the gamma spectrum with 'figure/ground' stimulation suggest a possible dual role for gamma rhythms in visual object coding, and provide general support of the binding-by-synchronization hypothesis. As the power changes in alpha and beta activity were largely independent of the spatial location of the target, however, we conclude that their role in object processing may relate principally to changes in visual attention. © 2010 Elsevier B.V

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.This article is published as Fries-Craft, K., and E. A. Bobeck. "Early Salmonella Typhimurium inoculation may obscure anti-interleukin-10 protective effects on broiler performance during coccidiosis and necrotic enteritis challenge." Poultry Science 103 (2023): 103187. doi:10.1016/j.psj.2023.103187. Posted with permission.© 2023 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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 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 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 This article is published as Fries-Craft, K., M. M. Meyer, and E. A. Bobeck. "Algae-based feed ingredient protects intestinal health during Eimeria challenge and alters systemic immune responses with differential outcomes observed during acute feed restriction." Poultry Science 100, no. 9 (2021): 101369. doi:10.1016/j.psj.2021.101369.</p

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 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)