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.

Tracking and Characterizing Spatiotemporal and Three-Dimensional Locomotive Behaviors of Individual Broilers in the Three-Point Gait-Scoring System.

Gait scoring is a useful measure for evaluating broiler production efficiency, welfare status, bone quality, and physiology. The research objective was to track and characterize spatiotemporal and three-dimensional locomotive behaviors of individual broilers with known gait scores by jointly using deep-learning algorithms, depth sensing, and image processing. Ross 708 broilers were placed on a platform specifically designed for gait scoring and manually categorized into one of three numerical scores. Normal and depth cameras were installed on the ceiling to capture top-view videos and images. Four birds from each of the three gait-score categories were randomly selected out of 70 total birds scored for video analysis. Bird moving trajectories and 16 locomotive-behavior metrics were extracted and analyzed via the developed deep-learning models. The trained model gained 100% accuracy and 3.62 ± 2.71 mm root-mean-square error for tracking and estimating a key point on the broiler back, indicating precise recognition performance. Broilers with lower gait scores (less difficulty walking) exhibited more obvious lateral body oscillation patterns, moved significantly or numerically faster, and covered more distance in each movement event than those with higher gait scores. In conclusion, the proposed method had acceptable performance for tracking broilers and was found to be a useful tool for characterizing individual broiler gait scores by differentiating between selected spatiotemporal and three-dimensional locomotive behaviors.

Undergraduate student attitudes to current poultry industry issues over four semesters: surveying an introductory poultry science course.

Individual background and demographics affect student perceptions of animal production. Understanding how science-based education alters these opinions is a critical aspect of improving university instruction as well as increasing consumer engagement in the poultry industry. The study objectives were to quantify the effects of student background, career interests, and science-based instruction on opinions regarding current issues in the poultry industry. Undergraduate students enrolled in a one semester poultry science course at Iowa State University between 2018 and 2021 were anonymously surveyed at the start and end of the semester as part of a 4-yr study. Students who opted to take the survey answered three demographic questions indicating their 1) livestock experience, 2) sex, and 3) career goals. The body of the survey consisted of 16 "poultry issue statements" where students were directed to mark a vertical dash on a 130 mm horizontal line indicating their level of agreement with each statement. Post-survey collection, the line was separated into 5 sections for discussion: responses within 0%-20% indicated strongly disagree, 21%-40% disagree, 41%-60% neutral, 61%-80% agree, and 81%-100% indicated strongly agree. Responses were analyzed using Proc Mixed in SAS Version 9.4 with a Tukey-Kramer adjustment for all pairwise comparisons using main effects including demographic categories, education (pre- or post-instruction), and year the survey was taken. Responses to various issue statements were affected by students' livestock experience (P < 0.05; 6 out of 16 statements affected), sex (P < 0.05; 5 out of 16 statements), and ultimate career goals (P < 0.05; 4 out of 16 statements). Pre- vs. post-education responses differed significantly in 6 out of 16 statements (P < 0.05), and in 2 out of 16 poultry issue statements, the year of instruction affected student response (P < 0.05). These data indicate that individual student background, sex, and differing career interests impact opinions of current topics in the broiler and layer industries. Further, science-based education as well as the year the course was taken over consecutive semesters significantly altered student opinions.

Individual experience and demographics affect perceptions of animal production. Understanding how science-based education alters these opinions is a critical aspect of improving instruction and increasing consumer engagement in the poultry industry. Undergraduate students enrolled in a poultry science course at Iowa State University between 2018 and 2021 were surveyed at the start and end of the semester as part of a 4-yr study. Students answered three demographic questions and indicated their agreeability with 16 "poultry issue statements." Responses to various issue statements were affected by students' livestock experience (6 out of 16 statements), sex (5 out of 16 statements), and ultimate career goals (4 out of 16 statements). Pre- vs. post-education responses changed in 6 out of 16 statements, and in 2 out of 16 poultry issue statements, the year of instruction affected student response. Individual student background, sex, and career interests impacted opinions on current topics in the broiler and layer industries, including laying hen housing systems, selective poultry breeding, environmental enrichment availability, culling practices, commercial stocking density, purchasing decisions, and more. Science-based instruction with hands-on farm experience as well as the year the course was taken over consecutive semesters significantly altered student opinions.

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.

iNKT cells coordinate immune pathways to enable engraftment in nonconditioned hosts.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that interact with key antigen-presenting cells to modulate adaptive T-cell responses in ways that can either promote protective immunity, or limit pathological immune activation. Understanding the immunological networks engaged by iNKT cells to mediate these opposing functions is a key pre-requisite to effectively using iNKT cells for therapeutic applications. Using a human umbilical cord blood xenotransplantation model, we show here that co-transplanted allogeneic CD4+ iNKT cells interact with monocytes and T cells in the graft to coordinate pro-hematopoietic and immunoregulatory pathways. The nexus of iNKT cells, monocytes, and cord blood T cells led to the release of cytokines (IL-3, GM-CSF) that enhance hematopoietic stem and progenitor cell activity, and concurrently induced PGE2-mediated suppression of T-cell inflammatory responses that limit hematopoietic stem and progenitor cell engraftment. This resulted in successful long-term hematopoietic engraftment without pretransplant conditioning, including multi-lineage human chimerism and colonization of the spleen by antibody-producing human B cells. These results highlight the potential for using iNKT cellular immunotherapy to improve rates of hematopoietic engraftment independently of pretransplant conditioning.

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.

Mitochondrial and Glycolytic Capacity of Peripheral Blood Mononuclear Cells Isolated From Diverse Poultry Genetic Lines: Optimization and Assessment.

Cellular metabolic preference is a culmination of environment, nutrition, genetics, and individual variation in poultry. The Seahorse XFe24 analyzer was used to generate foundational immune cellular metabolic data in layer, broiler, and legacy genetic strains using fresh chicken peripheral blood mononuclear cells (PBMCs). Baseline mitochondrial respiration [oxygen consumption rate (OCR)] and glycolytic activity [extracellular acidification rate (ECAR)] were determined in modern commercial laying hen (Bovans White) and broiler (Ross 308) lines, as well as the highly inbred lines of Iowa State University (L8, Fayoumi M-15.2, Spanish, Ghs-6), partially inbred broiler line, and advanced intercrosses of broiler by Fayoumi M-15.2 and broiler by Leghorn lines. Commercial broiler vs. Bovans layer and unvaccinated vs. vaccinated Bovans layer immune cell metabolic potential were compared following an in-assay pathway inhibitor challenge. Titrations consistently showed that optimal PBMC density in laying hens and broilers was 3 million cells per well monolayer. Assay media substrate titrations identified 25 mM glucose, 1 mM glutamine, and 1 mM sodium pyruvate as the optimal concentration for layer PBMCs. Pathway inhibitor injection titrations in Bovans layers and broilers showed that 0.5 µM carbonyl cyanide-4 phenylhydrazone (FCCP) and 1 µM oligomycin were optimal. Baseline OCR and ECAR were significantly affected by genetic line of bird (p < 0.05), with the dual-purpose, L8 inbred line showing the highest OCR (mean 680 pmol/min) and the partially inbred broiler line showing the greatest ECAR (mean 74 mpH/min). ECAR metabolic potential tended to be greater in modern layers than broilers (p < 0.10), indicating increased ability to utilize the glycolytic pathway to produce energy. OCR was significantly higher in vaccinated than unvaccinated hens (p < 0.05), while baseline ECAR values were significantly lower in vaccinated Bovans laying hens, showing increased oxidative capacity in activated immune cells. These baseline data indicate that different genetic strains of birds utilized the mitochondrial respiration pathway differently and that modern commercial lines may have reduced immune cell metabolic capacity compared with legacy lines due to intense selection for production traits. Furthermore, the Seahorse assay demonstrated the ability to detect differences in cellular metabolism between genetic lines and immune status of chickens.

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.

Eggshell and environmental bacteria contribute to the intestinal microbiota of growing chickens.

BACKGROUND: The initial intestinal microbiota acquired from different sources has profound impacts on animal health and productivity. In modern poultry production practices, the source(s) of the establishing microbes and their overall contribution during development of gastrointestinal tract communities are still unclear. Using fertilized eggs from two independent sources, we assessed the impact of eggshell- and environmental-associated microbial communities on the successional processes and bacterial community structure throughout the intestinal tract of chickens for up to 6 weeks post-hatch. RESULTS: Culturing and sequencing techniques identified a viable, highly diverse population of anaerobic bacteria on the eggshell. The jejunal, ileal, and cecal microbial communities for the egg-only, environment-only, and conventionally raised birds generally displayed similar successional patterns characterized by increasing community richness and evenness over time, with strains of Enterococcus, Romboutsia, and unclassified Lachnospiraceae abundant for all three input groups in both trials. Bacterial community structures differed significantly based on trial and microbiota input with the exception of the egg-exposed and conventional birds in the jejunum at week 1 and the ileum at week 6. Cecal community structures were different based on trial and microbiota input source, and cecal short-chain fatty acid profiles at week 6 highlighted functional differences as well. CONCLUSION: We identified distinct intestinal microbial communities and differing cecal short-chain fatty acid profiles between birds exposed to the microbiota associated with either the eggshell or environment, and those of conventionally hatched birds. Our data suggest the eggshell plays an appreciable role in the development of the chicken intestinal microbiota, especially in the jejunum and ileum where the community structure of the eggshell-only birds was similar to the structure of conventionally hatched birds. Our data identify a complex interplay between the eggshell and environmental microbiota during establishment and succession within the chicken gut. Further studies should explore the ability of eggshell- and environment-derived microbes to shape the dynamics of succession and how these communities can be targeted through interventions to promote gut health and mitigate food-borne pathogen colonization in poultry.

A novel environmental enrichment device increased physical activity and walking distance in broilers.

Modern broilers are selected for fast growth and a large proportion of breast tissue, contributing to a top-heavy phenotype, leg disorders, and inactivity as birds reach market weight. Therefore, the objective was to motivate broilers to move through environmental enrichment. A total of 1,200 Ross 308 broilers were housed in pens of 30 for 6 wk: 600 birds were exposed to a novel laser enrichment device (LASER) and 600 were control. Each device projected 2 randomly moving red laser dots onto the floor 4 times/day for 4-min "laser periods." Seven LASER and 7 control pens, with 5 focal birds/pen (n = 70), were randomly selected to be video-recorded day 0 to 8 and once weekly for the remainder of the trial. Videos were analyzed to measure broiler time-budget and behaviors such as latency to feed and distance walked during laser periods. Focal birds were gait scored weekly on-farm. A test of the human-approach paradigm was carried out on weeks 1 and 6 on all pens. LASER birds were more active on days 0, 1, 3, 4, 5, 7, and 8, moving 254% more on day 7 (P ≤ 0.05). Time spent active was increased in LASER treatment by 114% on week 2; 157% on week 3; 90% on week 4; and 82% on week 5. LASER birds spent more time at the feeder on days 0, 1, 2, 5, 8, and on weeks 1 and 5, with 84% more time at feeder than control on day 5 (P ≤ 0.05). LASER birds walked further during laser periods on day 0 to 8, reaching 646.5 cm greater (day 1), and on weeks 2, 3, 4, and 5, with an increase of 367.5 cm on week 2 (P ≤ 0.05). Over week 1 to 6, 60.54 ± 7.4% of focal birds in the laser treatment were at the feeder during or within 5 min following laser periods. The laser enrichment device was successful in stimulating broiler physical activity and feeding, and did not negatively impact walking ability.

Development and Validation of Broiler Welfare Assessment Methods for Research and On-farm Audits.

Required auditing of on-farm broiler welfare in the United States has increased; however, a lack of validated tools exists for assessment of enrichment. National Chicken Council (NCC) guidelines were used on a subset of 300 Ross 308 broilers out of 1200 to validate and adapt welfare measures. Half of the broilers were exposed to environmental enrichment, hence these measures were used to evaluate the enrichment within the context of behavior and welfare, although the nature of the enrichment is not described in detail here as the aim is to serve solely as a description and validation of methods using a subset of example data. Birds were recorded in repeated 4-min periods to quantify behavior and walking distance. Outcomes were categorized to improve auditing and make recommendations to producers and researchers. Bone mineral density, content, and breaking strength were successful in determining numerical differences. Quantifying lameness using an enclosed walkway and measuring footpad dermatitis weekly are recommended on-farm. We recommend including additional measures not required by the NCC: monitoring breast condition in the flock and including a behavior component with a scoring ethogram.

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.

Oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary 1α-hydroxycholecalciferol.

While it is well established that active vitamin D treatment increases dietary phytate phosphate utilization, the mechanism by which intestinal alkaline phosphatase (IAP) participates in phytate phosphate use is less clear. The ability of human IAP (hIAP) oral antibodies to prevent dietary phytate phosphate utilization in the presence of 1α-hydroxycholecalciferol (1α-(OH) D3) in a chick model was investigated. hIAP specific chicken immunoglobulin Y (IgY) antibodies were generated by inoculating laying hens with 17 synthetic peptides derived from the human IAP amino acid sequence and harvesting egg yolk. Western blot analysis showed all antibodies recognized hIAP and 6 of the 8 antibodies selected showed modest inhibition of hIAP activity in vitro (6 to 33% inhibition). In chicks where dietary phosphate was primarily in the form of phytate, 4 selected hIAP antibodies inhibited 1α-(OH) D3-induced increases in blood phosphate, one of which, generated against selected peptide (MFPMGTPD), was as effective as sevelamer hydrochloride in preventing the 1α-(OH) D3-induced increase in blood phosphate, but ineffective in preventing an increase in body weight gain and bone ash induced by 1α-(OH) D3. These studies demonstrated that orally-delivered antibodies to IAP limit dietary phytate-phosphate utilization in chicks treated with 1α-(OH) D3, and implicate IAP as an important host enzyme in increasing phytate phosphate bioavailability in 1α-(OH) D3 fed chicks.

Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

Hyperimmunized hens are an effective means of generating large quantities of antigen specific egg antibodies that have use as oral supplements. In this study, we attempted to create a peptide specific antibody that produced outcomes similar to those of the human pharmaceutical, sevelamer HCl, used in the treatment of hyperphosphatemia (a sequela of chronic renal disease). Egg antibodies were generated against 8 different human intestinal sodium-dependent phosphate cotransporter 2b (NaPi2b) peptides, and hNaPi2b peptide egg antibodies were screened for their ability to inhibit phosphate transport in human intestinal Caco-2 cell line. Antibody produced against human peptide sequence TSPSLCWT (anti-h16) was specific for its peptide sequence, and significantly reduced phosphate transport in human Caco-2 cells to 25.3±11.5% of control nonspecific antibody, when compared to nicotinamide, a known inhibitor of phosphate transport (P≤0.05). Antibody was then produced against the mouse-specific peptide h16 counterpart (mouse sequence TSPSYCWT, anti-m16) for further analysis in a murine model. When anti-m16 was fed to mice (1% of diet as dried egg yolk powder), egg yolk immunoglobulin (IgY) was detected using immunohistochemical staining in mouse ileum, and egg anti-m16 IgY colocalized with a commercial goat anti-NaPi2b antibody. The effectiveness of anti-m16 egg antibody in reducing serum phosphate, when compared to sevelamer HCl, was determined in a mouse feeding study. Serum phosphate was reduced 18% (P<0.02) in mice fed anti-m16 (1% as dried egg yolk powder) and 30% (P<0.0001) in mice fed sevelamer HCl (1% of diet) when compared to mice fed nonspecific egg immunoglobulin. The methods described and the findings reported show that oral egg antibodies are useful and easy to prepare reagents for the study and possible treatment of select diseases.

Sevelamer hydrochloride binds phosphate released from phytate in chicks fed 1α-hydroxy cholecalciferol.

OBJECTIVE: Hyperphosphatemia in animal models of human renal disease has been linked to increased risk of death. Phosphate binders (e.g., sevelamer hydrochloride) and plant-based, low phosphate diets are used to reduce dietary phosphate load; however, animal models show that treatment with active forms of vitamin D(3) (e.g., calcitriol, a renal disease therapy) renders plant phytate phosphate available for absorption. Using an established chick model, the effectiveness of sevelamer in preventing the apparent absorption of liberated phytate phosphate during active vitamin D use was investigated in two separate experiments. DESIGN: One-day-old chicks were fed ad libitum a basal diet containing deficient levels of inorganic phosphate (0.13%), but adequate in total phosphate (0.40%, 0.23% as phytate phosphate), with or without the inclusion of sevelamer hydrochloride (a phosphate binder), available inorganic phosphate, or active vitamin D as 1α-(OH) D(3). MAIN OUTCOME MEASURES: Plasma phosphate (mg/dL), total bone ash (%), and weight gain (g). RESULTS: Adding inorganic phosphate (0.36%) or 1α-(OH) D(3) increased plasma phosphate 49% and 48%, respectively (P < .0001), and bone ash 23% and 19%, respectively (P < .001). The addition of 1% sevelamer to the basal diet with added inorganic phosphate or 1α-(OH) D(3) significantly decreased plasma phosphate by 28% and 20%, respectively (P < .01). CONCLUSION: Active vitamin D increased the availability of phytate phosphate for intestinal absorption in an animal model; however, sevelamer effectively reduced the availability of phosphate liberated from phytate. These data imply that sevelamer has phytate phosphate binding efficacy.

Maternally-derived antibody to fibroblast growth factor-23 reduced dietary phosphate requirements in growing chicks.

Phosphate in manure of monogastric animals pollutes the environment if improperly applied to soil. Strategies that reduce phosphate inputs into animal production systems reduce environmental pollution. Using a novel vaccine to fibroblast growth factor-23 (FGF-23), we induced neutralizing antibodies that reduced the phosphate requirement of growing chickens. Breeding hens were injected with a FGF-23 peptide (AFLPGMNP) conjugate. Antibody was passively transferred from hen to chick and chick response to deficient dietary phosphate intake was determined. Chicks without passive anti-FGF-23 antibody had a 43% and 21% reduction in blood phosphate and bone ash, respectively, when fed a phosphate deficient diet and compared to chicks fed a phosphate replete diet (P<0.05). Chicks with circulating anti-FGF-23 antibodies fed the phosphate deficient diet had plasma phosphate and bone ash that did not differ from chicks fed the phosphate replete diet (P>0.05). Neutralization of FGF-23 offers a new approach to reduce phosphate requirements of farmed animals and may provide a new means to reduce phosphate pollution related to animal farming.