Implementing real-time immunometabolic assays and immune cell profiling to evaluate systemic immune response variations to Eimeria challenge in three novel layer genetic lines.

Introduction: Evaluating differences in immune responses to Eimeria spp. between poultry genetic lines could be valuable for understanding favorable traits to address coccidiosis, a costly poultry disease. The objective was to compare peripheral blood mononuclear cell (PBMC) immunometabolism and composition during Eimeria challenge in three distinct and highly inbred genetic lines; Leghorn Ghs6, Leghorn Ghs13, and Fayoumi M5.1. Methods: At hatch, 180 chicks (60/ line) were placed in wire-floor cages (10 chicks/cage) and fed a commercial diet. Baseline PBMC were isolated on d21 (10 chicks/line) and 25 chicks/line were inoculated with 10X Merck CocciVac®-B52 (Kenilworth, NJ), creating 6 genetic line × Eimeria groups total. Chicks were euthanized on 1, 3, 7, and 10d post-inoculation (pi; 5 chicks/ line × Eimeria group) for PBMC isolation with body weight and feed intake recorded throughout. Immunometabolic assays to determine PBMC ATP production profiles and glycolytic activity were implemented along with flow cytometric immune cell profiling. Genetic line × Eimeria challenge, and line´challenge fixed effects were analyzed using the MIXED procedure (SAS 9.4; P ≤ 0.05). Results and Discussion: Before inoculation, M5.1 chicks had 14.4-25.4% greater average daily gain (ADG) with 19.0-63.6% increased monocyte/macrophage+, Bu-1+ B cell, and CD3+ T cell populations compared to both Ghs lines (P < 0.0001) but similar immunometabolic phenotype. The Eimeria main effect reduced ADG by 61.3% from 3-7dpi (P = 0.009) except in M5.1 chicks, where no ADG difference due to challenge was found. At 3dpi, Eimeria-challenged M5.1 chicks had 28.9 and 33.2% reduced PBMC CD3+ T cells and CD3+CD8α+ cytotoxic T cells than unchallenged chicks, suggesting early and preferential recruitment from systemic circulation to tissues local to Eimeria challenge (i.e., intestine; P ≤ 0.01). Both Ghs lines displayed 46.4-49.8% T cell reductions at 10dpi with 16.5-58.9% recruitment favoring underlying CD3+CD4+ helper T cells. Immunometabolic responses in Eimeria-challenged Ghs6 and Ghs13 chicks were characterized by a 24.0-31.8% greater proportion of ATP from glycolysis compared to unchallenged counterparts at 10dpi (P = 0.04). These results suggest that variable T cell subtype recruitment timelines in addition to altered systemic immunometabolic requirements may work synergistically to determine favorable immune responses to Eimeria challenge.

Dietary yeast beta 1,3/1,6 glucan supplemented to adult Labrador Retrievers alters peripheral blood immune cell responses to vaccination challenge without affecting protective immunity.

Yeast-derived 1,3/1,6 ß-glucans may alter host immunity to produce robust and quickly resolved responses that align with companion animal health goals. In adult dogs, immunomodulation by yeast 1,3/1,6 ß-glucans in extruded kibble diet have not been well documented. The study objective was to evaluate systemic immune responses in dogs fed kibble diets with two yeast 1,3/1,6 ß-glucans doses before and after vaccine challenge. Twenty-four adult Labrador Retrievers were assigned to three dietary treatments consisting of a basal diet (control) supplemented with 0.012% or 0.023% (0.5 or 1×, respectively) yeast 1,3/1,6 ß-glucan with equal sex representation within each treatment (8 dogs/diet). Animals were fed experimental diets for a 29-d acclimation period, after which baseline blood samples were collected before administration of a combination canine distemper virus, parvovirus, and adenovirus-2 vaccine. Blood samples were collected weekly for 21 d following vaccination with whole blood for CBC analysis, serum for titer and cytokine assays, and peripheral blood mononuclear cells (PBMC) isolated for flow cytometric immune cell profiling. Data were analyzed using the MIXED procedure with diet and timepoint fixed effects. Serum titer was analyzed by Kruskal-Wallis test (SAS 9.4; P ≤ 0.05). Prior to vaccination, ß-glucan diets did not affect serum cytokines, antibody titer, or immune cell populations. In the first 7 d post-vaccination (dpv), PBMC CD21low B cells increased 36.5% to 58.1% in all groups but the magnitude of change was lesser in the 0.5× ß-glucan diet resulting in 25.6% lower CD21low populations compared to control-fed dogs (P = 0.007). By 21 dpv, B-cell populations recovered to baseline levels in dogs fed 1× ß-glucan, but CD21high cells remained elevated 50.5% in dogs fed 0.5× ß-glucan diets compared with baseline (P < 0.0001). While no differences in serum titer or cytokines were observed, feeding both ß-glucan diets maintained stable blood monocytes, whereas a 53.0% decrease between baseline and 14 dpv was observed in control-fed dogs (P = 0.01). Collectively, these outcomes suggest that a 1× dose of 1,3/1,6 yeast ß-glucan in extruded kibble diets altered monocytes associated with trained immunity, did not reduce PBMC CD21low B-cell responsiveness, and simultaneously contributed to B-cell population resolution by 21 dpv in adult dogs. Additional research to assess the functionality of these changes is needed.

Companion animal food trends reflect the growing demand for healthy, functional foods. Yeast-derived ß-glucans have been shown to train the human immune system to respond and resolve inflammation quickly, which aligns with companion animal health goals of providing disease protection without excessive inflammation. In this work, 24 healthy adult Labrador Retrievers were fed diets without or with yeast ß-glucans at two doses (0.5 and 1×). After feeding experimental diets for 4 wk, blood samples were collected to establish baseline immunity before dogs were challenged with a commercially available vaccine. Blood samples were collected weekly for 3 wk post-vaccination to determine changes, if any, to serum antibody, cytokine production, and blood counts. All dogs achieved protective antibody titers within 1 wk post-vaccination. Dogs fed the 1× ß-glucans inclusion showed potentially reduced reliance on cells associated with early immune responses without prolonging responses by antibody-producing cells. These outcomes suggest beneficial responses to dietary yeast ß-glucans in vaccinated adult Labrador Retrievers, but further research and refinement of immunological assessment in companion animals are needed.

Age and Staphylococcus aureus Inoculation Route Differentially Alter Metabolic Potential and Immune Cell Populations in Laying Hens.

In 2018 and 2019, Staphylococcus aureus was isolated from multiple post-molt commercial laying hens with unusually high mortality. A challenge study was conducted to elucidate the role of S. aureus in this disease outbreak and the work herein represents the assessment of immunological responses in laying hens experimentally infected with S. aureus isolates from these cases. A total of 200 laying hens at 22 or 96 weeks of age (100/ age group) were assigned to 1 of 4 experimental inoculation groups (negative control, oral gavage, subcutaneous injection, or intravenous injection) after a 72 h acclimation period. Blood samples were taken prior to inoculation (baseline), 6 h post-inoculation (pi), 24 hpi, 3 dpi, and 7 dpi. Additional spleen samples to further assess systemic immunity were taken at baseline, 3 and 8 dpi. Metabolic phenotypes of peripheral blood mononuclear cells (PBMC) were isolated and assessed by Seahorse metabolic assay. Immune cell profiles in the spleen and PBMC were assessed by multicolor flow cytometry. At baseline, 96-week-old laying hens had 26.7% fewer PBMC-derived T cells compared to 22-week-old birds. Older hens had 28.9% increased helper T cell (TH) populations and 60.5% reduced γδ T cells (P = 0.03 and < 0.0001) which may contribute to variable clinical responses between age groups; however, no age-related differences in metabolic potential were observed. Metabolic outcomes showed that birds remained stressed from transport and re-housing past a 72 h acclimation period and through 24 h- 3 days post-inoculation. Inoculation with S. aureus generally reduced oxidative and glycolytic potentials compared to the control, with the greatest reductions observed in birds inoculated by intravenous injection (P < 0.05). Overall CD3+ T cell populations showed significant reductions in the intravenous group compared to other inoculation routes from 24 hpi to 7 dpi (23.6-39.0%; P ≤ 0.0001). These results suggest that age-related baseline differences in T cell populations and changes to T cell subpopulations and other immune cells due to inoculation route may have an additive effect on S. aureus- induced reductions in metabolic potential; however, further research linking metabolic potential and immune cell profiles is needed.

Lipid Source and Peroxidation Status Alter Immune Cell Recruitment in Broiler Chicken Ileum.

BACKGROUND: Restaurant oil in poultry diets increases energy content, reduces production costs, and promotes sustainability within the food supply chain. However, variable oil composition and heating temperatures among restaurant oil sources can impact broiler chicken health due to heat-induced lipid modifications. OBJECTIVES: A 21-d experiment was conducted to evaluate ileal morphology, liver cytokine gene expression, and ileal immune cell populations in broilers fed control or peroxidized lipids with varying chain and saturation characteristics. METHODS: Day-old broilers were housed in battery cages (5 birds per cage) and fed diets containing 5% control or peroxidized oils. Eight diets were randomly assigned in a 4 × 2 factorial arrangement consisting of oil source (palm, soybean, flaxseed, or fish) and peroxidation status (control or peroxidized). At day 21, samples were collected for ileal histomorphology [villus height (VH), crypt depth (CrD), and the VH:CrD ratio], and liver cytokine expression (qPCR). Ileum cytokine expression and T-cell markers were analyzed by RNAscope in situ hybridization (ISH). Data were analyzed as a mixed model (SAS 9.4) with fixed effects of lipid source, peroxidation, and lipid × peroxidation interaction. RESULTS: CD3+ T-cells in the ileum decreased 16.2% due to peroxidation (P = 0.001) with 30.3% reductions observed in birds fed peroxidized flaxseed oil (P = 0.01). Peroxidation increased IL6+ and IL1B+ cells by 62.0% and 40.3%, respectively (P = 0.01). Soybean oil increased IFNG+ cells by 55.1% compared with palm oil, regardless of peroxidation status (P = 0.007). Lipid source and peroxidation did not alter ileal histomorphology or liver cytokine expression. CONCLUSIONS: Lipid peroxidation increased ileal IL1B and IL6 in broiler chickens, whereas soybean oil diets increased IFNG. Generally, peroxidation decreased overall CD3+ T-cell populations, suggesting impaired T-cell presence or recruitment. These results identify potential immunomodulatory lipid profiles in restaurant oil while supporting RNAscope-ISH as a method to describe avian tissue-level immune responses.

Composition and inclusion of probiotics in broiler diets alter intestinal permeability and spleen immune cell profiles without negatively affecting performance1.

Probiotic feed additives with potential to enhance performance, health, and immunity have gained considerable popularity in commercial broiler production. The study objectives were to measure broiler performance, gut integrity, and splenic immune cell profiles in birds fed one of two probiotics at two inclusion levels. Nine hundred sixty Ross 708 broilers (12 per pen) were randomly assigned to no additive control, 0.05% or 0.10% LactoCare (Lactobacillus reuteri), or 0.05% or 0.10% LactoPlan (Lactobacillus plantarum) dietary treatments for 6 wk. On day 27, a 20-pen subset was utilized for a fluorescein isothiocyanate dextran (FITC-d) assay, where half of the pens were subject to a 12-h feed restriction (FR) pregavage. Serum collected from blood drawn 1-h postgavage was analyzed for relative fluorescence of FITC-d absorbed across the intestinal barrier as a gut leakiness indicator. On day 42, spleens from eight birds per treatment were collected for immune cell profile analysis by multicolor flow cytometry. Although performance outcomes were not affected by dietary treatment, FITC-d absorption post-FR was increased 57% in the 0.05% LactoPlan treatment, and was decreased by 12.6% in the 0.05% LactoCare diet, 12% in the 0.10% LactoCare diet, and 22% in the 0.10% LactoPlan diet compared with the control. This indicates a positive impact in barrier integrity maintenance due to 0.05% and 0.10% LactoCare and 0.10% LactoPlan diet following a challenge. Immune cell profiles varied between the two probiotic compositions, with an approximately 50% reduction in splenic innate immune cells (monocyte/macrophage+) in birds fed LactoPlan (P < 0.0001) and greater overall percentages of CD45+ leukocytes and CD3+ T cells in birds fed 0.10% LactoCare (P < 0.0001). LactoPlan diets shifted splenic T-cell populations in favor of CD8α + cytotoxic T cells (TC; P = 0.007), while higher inclusions (0.10%) of either probiotic increased the percentage of activated CD4+ helper T cells (TH; P < 0.0001). These results indicate that compositionally different probiotics had varying effects on the gut permeability and splenic immune cell profiles in broiler chickens, particularly at higher inclusion rates, but observed changes to underlying physiology did not negatively impact performance outcomes. The ability of a probiotic to alter gut permeability and immune cell profile, therefore, may depend on the compositional complexity of the product as well as inclusion rate.