Effects of dietary sulfur amino acid levels on growth performance and intestinal immunity in broilers vaccinated and subsequently infected with coccidiosis.

Coccidia vaccination is a common practice in the poultry industry. However, research is lacking regarding the optimal nutritional support for coccidia vaccinated broilers. In this study, broilers were vaccinated with coccidia oocyst at hatch and were fed with a common starter diet from 1 to 10 d. On d 11, the broilers were randomly assigned to groups in a 4 × 2 factorial arrangement. Briefly, the broilers were fed one of four diets containing 0.6, 0.8, 0.9, and 1.0% of standardized ileal digestible methionine plus cysteine (SID M+C), respectively, from 11 to 21 d. On d 14, the broilers from each diet group were orally gavaged with either PBS (Mock challenge) or Eimeria oocysts. Compared to PBS-gavaged broilers and regardless of dietary SID M+C levels, the Eimeria-gavaged broilers had 1) decreased gain-to-feed ratio (15-21 d, P = 0.002; 11-21 d, P = 0.011); 2) increased fecal oocysts (P < 0.001); 3) increased plasma anti-Eimeria IgY (P = 0.033); and 4) increased intestinal luminal interleukin-10 (IL-10; duodenum, P = 0.039; jejunum, P = 0.018) and gamma interferon (IFN-γ; duodenum, P < 0.001; jejunum, P = 0.017). Regardless of Eimeria gavage, broilers fed 0.6% SID M+C had decreased (P<0.001) body weight gain (15-21 and 11-21 d) and gain-to-feed ratio (11-14, 15-21, and 11-21 d) when compared to those fed ≥ 0.8% SID M+C. Eimeria challenge increased (P < 0.001) duodenum lesions when the broilers were fed with 0.6, 0.8, and 1.0% SID M+C, and increased (P = 0.014) mid-intestine lesions when the broilers were fed with 0.6 and 1.0% SID M+C. An interaction between the two experimental factors was detected on plasma anti-Eimeria IgY titers (P = 0.022), as coccidiosis challenge increased plasma anti-Eimeria IgY titers only when the broilers were fed with 0.9% SID M+C. In summary, the dietary SID M+C requirement for grower (11-21 d) broilers vaccinated with coccidiosis was ranged from 0.8 to 1.0% for optimal growth performance and intestinal immunity, regardless of coccidiosis challenge.

Dietary supplementation of sulfur amino acids improves intestinal immunity to Eimeria in broilers treated with anti-interleukin-10 antibody.

Oral antibody to interleukin-10 (anti-IL-10) enhances the intestinal immune defense against Eimeria. The sulfur amino acids methionine and cysteine (M+C) play essential roles in inducing and maintaining protective immune responses during intestinal infections. Hence, increased dietary M+C may support the anti-IL-10-induced intestinal immunity to Eimeria. Broilers (n = 640) were arranged in a 2 × 2 × 2 factorial design with 2 levels of each of the 3 main factors: dietary standardized ileal digestible (SID) M+C levels (0.6% or 0.8%), dietary anti-IL-10 supplementation (with or without), and coccidiosis challenge (control or challenge). Briefly, the broilers were supplied with either 0.6% or 0.8% SID M+C, each with or without anti-IL-10 (300 µg/kg), from d 10 to 21. On d 14, broilers from each diet were gavaged with either PBS or Eimeria. The resulting Eimeria infection induced fecal oocyst shedding and intestinal lesions. Broilers fed 0.8% SID M+C (main effects, P ≤ 0.05) had decreased feed-to-gain ratio, increased duodenum and cecum luminal anti-Eimeria IgA titers, and decreased fecal oocyst counts, when compared to 0.6% SID M+C. The supplementation of anti-IL-10 (main effects, P ≤ 0.05) increased cecum luminal total IgA concentration and decreased cecum lesions. Interactions (P ≤ 0.05) were detected for growth performance and cecum luminal IFN-γ. Briefly, the highest body weight gain and feed intake were reached in PBS-gavaged broilers fed 0.8% SID M+C with no anti-IL-10 and in Eimeria-challenged broilers fed 0.8% SID M+C with anti-IL-10. In Eimeria-infected broilers, anti-IL-10 increased intestinal luminal IFN-γ and body weight gain only at 0.8% SID M+C. Collectively, anti-IL-10 increased intestinal luminal IFN-γ levels, decreased cecum lesions and restored growth only when fed with adequate amounts of sulfur amino acids. Our findings underscore the importance of providing sufficient essential nutrients to support the anti-IL-10 induced immunity against coccidiosis.

Effects of dietary methionine plus cysteine levels on growth performance and intestinal antibody production in broilers during Eimeria challenge.

Research has shown that methionine+ cysteine (M+C) requirements may be higher when chickens are infected with Eimeria app. In a 4 × 2 factorial design, broilers (11 to 21 D) were fed one of 4 corn-soybean meal-based diets containing either 0.6, 0.8, 0.9, or 1.0% standardized ileal digestible (SID) M+C; on day 14, broilers from each diet were gavaged with either phosphate-buffered saline (PBS) or a commercial coccidiosis vaccine (at 100 × vaccine dose) which provide a mixture of live Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts. Growth performance was recorded from day 11 to 21. Plasma and intestinal luminal samples were collected on days 14 and 21. Intestine lesion scores and fecal oocyst counts were conducted on day 21. Regardless of dietary SID M+C levels, compared to PBS gavaged broilers, the Eimeria-challenged broilers had (1) decreased (P < 0.05) body weight gain (BWG), feed intake (FI), and gain-to-feed ratio (G:F); (2) increased (P < 0.05) intestinal lesion scores and fecal oocyst counts; (3) increased (P < 0.05) plasma anti-Eimeria IgG, and intestinal luminal total IgA and anti-Eimeria IgA concentrations; and (4) increased (P < 0.05) levels of duodenum luminal gamma interferon (IFN-γ) and interleukin-10 (IL-10), as well as jejunum and cecum luminal IFN-γ concentrations. Regardless of Eimeria challenge, when compared to 0.6% SID M+C, broilers fed ≥0.8% SID M+C had (1) increased (P < 0.05) BWG, FI, and G:F and (2) increased (P < 0.05) levels of jejunum luminal total IgA. After Eimeria challenge, broilers fed 0.8% SID M+C had increased (P < 0.05) levels of jejunum luminal anti-Eimeria IgA compared to broilers fed diets containing 0.6 and 1.0% SID M+C. Collectively, in 11- to 21-D broilers, the growth suppression caused by Eimeria infection could not be mitigated by further increasing dietary M+C alone ≥0.8%. Further research should investigate interactions between dietary M+C and other nutrients for support of immune function and growth in pathogen-challenged broilers.

Modular Polymer Antigens To Optimize Immunity.

Subunit vaccines can have excellent safety profiles, but their ability to give rise to robust immune responses is often compromised. For glycan-based vaccines, insufficient understanding of B and T cell epitope combinations that yield optimal immune activation hinders optimization. To determine which antigen features promote desired IgG responses, we synthesized epitope-functionalized polymers using ring-opening metathesis polymerization (ROMP) and assessed the effect of B and T cell epitope loading. The most robust responses were induced by polymers with a high valency of B and T cell epitopes. Additionally, IgG responses were greater for polymers with T cell epitopes that are readily liberated upon endosomal processing. Combining these criteria, we used ROMP to generate a nontoxic, polymeric antigen that elicited stronger antibody responses than a comparable protein conjugate. These findings highlight principles for designing synthetic antigens that elicit strong IgG responses against inherently weak immune targets such as glycans.

Effect of anti-fibroblast growth factor receptor 1 antibodies on phosphorus metabolism in laying hens and their progeny chicks.

Targeting fibroblast growth factor 23 (FGF-23) signaling pathway is of interest in controlling body phosphate metabolism. This study investigated the effect of anti-fibroblast growth factor receptor 1 (FGFR1, major FGF-23 receptor in the kidney) antibodies on phosphate metabolism. White Leghorn laying hens (65-wk-old) were vaccinated with either a FGFR1 peptide vaccine (five 8-amino-acid peptides were selected, CrZ-1:LPEDPRWE, CrZ-2:LDKDKPNR, CrZ-3:RRPPGMEY, CrZ-4:GSPYPGVP, and CrZ-5:RMDKPSNC) or adjuvant control. At peak antibody titer, hens were artificially inseminated. Chicks from control-vaccinated hens were fed either a non-phytate phosphorus (nPP) sufficient (nPP = 0.45%, positive control) or deficient (nPP = 0.20%, negative control) diet, while chicks from each of the FGFR1 peptide vaccinated hens were fed with the above nPP-deficient diet, for 14 D. When compared to control hens, plasma phosphate in CrZ-1, CrZ-2, CrZ-3, CrZ-4, and CrZ-5 vaccinated hens were decreased by 33, 30, 24, 20, and 26%, respectively (P < 0.05); egg weight in CrZ-2 and CrZ-5 vaccinated hens were increased by 6 and 7%, respectively (P < 0.05); egg production in CrZ-3, CrZ-4, and CrZ-5 vaccinated hens tended to decrease (P = 0.085; decreased by 14, 15, and 13%, respectively). When compared to positive control, chicks from all other groups had decreased body weight gain (BWG) and feed intake (FI) during 1 to 14 D, and had decreased plasma phosphate, tibiotarsus ash, and 24-h phosphorus excretion on day 14. When compared to negative control, BWG of CrZ-1, CrZ-2, CrZ-3, and CrZ-4 antibody chicks were decreased by 23, 28, 26, and 20%, respectively (P < 0.05); FI of CrZ-1, CrZ-2, and CrZ-3 antibody chicks were decreased by 15, 15, and 18%, respectively (P < 0.05); plasma phosphate of CrZ-5 antibody chicks were decreased by 26% (P < 0.05); plasma FGF-23 levels of CrZ-4 antibody chicks were increased by 18% (P < 0.05); tibiotarsus ash content of CrZ-2, CrZ-3, and CrZ-4 antibody chicks were decreased by 20, 20, and 21%, respectively (P < 0.05). In conclusion, anti-FGFR1 peptide antibodies decreased egg production of hens and growth performance of their progeny chicks probably by activating FGF-23 signaling and stimulating FGF-23 production.

Effect of anti-fibroblast growth factor 23 antibody on phosphate and calcium metabolism in adenine gavaged laying hens.

Dietary factors such as adenine have been linked to phosphate-calcium metabolism disturbance and adverse productive outcomes. Anti-fibroblast growth factor 23 (FGF-23) antibody has been proposed to ameliorate adenine-induced abnormal FGF23/phosphate metabolism. This experiment was conducted to investigate the application of anti-FGF-23 antibody in adenine-gavaged laying hens. Single Comb White Leghorn laying hens with (n = 10) or without (control group, n = 10) systemic anti-FGF-23 antibody were orally gavaged with adenine (600 mg/hen/D) for 21 consecutive days. Adenine gavage increased (P ≤ 0.01) plasma phosphate and calcium levels and tended to increase (0.05 < P ≤ 0.1) plasma 1,25-dihydroxy-cholecalciferol [1,25(OH)2D3] level of hens without FGF-23 antibody. In hen with anti-FGF-23 antibody, adenine gavage increased (P ≤ 0.01) body weight and plasma calcium level and decreased (P ≤ 0.05) plasma FGF-23 level. Feed intake of hens in both treatments was suddenly decreased (control hens decreased from 111 to 55 g, P ≤ 0.01; anti-FGF-23 hens decreased from 96 to 46 g, P ≤ 0.01) 10 D after adenine gavage. Anti-FGF-23 antibody tended to increase (0.05 < P ≤ 0.1) plasma phosphorus level of hens before adenine gavage, interestingly, and decreased (P ≤ 0.01) plasma FGF-23 level and kidney index (% of body weight) of hens after adenine gavage. In conclusion, anti-FGF-23 antibody might be used (before or in the early stage) to delay the development of adenine-induced abnormal FGF23/phosphate metabolism. This is the first study to investigate the FGF-23 status in chickens suffering from dietary factors which may cause abnormal renal phosphate resorption.

Oral antibody to interleukin-10 receptor 2, but not interleukin-10 receptor 1, as an effective Eimeria species immunotherapy in broiler chickens.

Coccidiosis is a major gastrointestinal disease caused by several Eimeria species in floor raised chickens. Feeding an antibody to interleukin 10 (aIL-10) ameliorates the negative symptoms of coccidiosis in broilers, i.e., lack of weight gain, decreased feed conversion, and mortality. IL-10 signals by forming a ligand-receptor complex with IL-10 Receptor 1 (IL-10 R1) and IL-10 Receptor 2 (IL-10 R2). In this study, we hypothesize oral antibodies to the IL-10 receptors will neutralize the IL-10 signaling pathway equal to or better than aIL-10 to act as an oral anti-coccidiosis immunotherapy. A total of 5 sequential feed trials, set up as a 4 (diet antibody) × 2 (Eimeria challenge) factorial design, tested oral egg yolk antibodies to a total of 6 IL-10 R1 epitopes and 3 IL-10 R2 epitopes compared to a control antibody diet. A total of 10 pens of 5 chicks/pen/diet antibody/Eimeria challenge were housed for 21 d. On day 3 of age, chicks were either infected or not infected with a 10× dose of an Eimeria vaccine containing Eimeria acervulina, Eimeria tenella, and Eimeria maxima. Pen feed consumption and mean body weights were assessed weekly (d1, d7, d14, and d21); fecal oocyst shedding was assessed on day 10. Data were analyzed using a 2-way ANOVA. No significant interaction on chick weight was observed in chicks fed IL-10 R1 antibodies compared to chicks fed the control antibody was observed. In studies evaluating aIL-10 R2 oral antibodies, infected chicks fed aIL-10 R2: epitope 1 overcame the negative effects of Eimeria infection and had similar 21-d body weight to uninfected chicks (P4 = 0.07). We hypothesized that feeding oral antibodies to the IL-10 receptors would result in equivalent anti-coccidial benefits to aIL-10. However, none of the 6 antibodies to IL-10 R1 epitopes yielded any benefits during Eimeria infection compared to controls. A total of 2 oral antibodies to IL-10 R2 showed promising results equivalent to the aIL-10 immunotherapeutic. Immunofluorescence staining shows that the IL-10R2 significantly increases in abundance in response to Eimeria infection, whereas IL-10R1 does not.

Conjugated Linoleic Acid Isomers Trans-10, Cis-12 and Cis-9, Trans-11 Prevent Collagen-Induced Arthritis in a Direct Comparison.

Mixed-isomer conjugated linoleic acid (CLA) and the individual isomers, trans-10, cis-12 (CLAt10c12) and cis-9, trans-11 (CLAc9t11), decrease severity of collagen-induced arthritis (CA) when consumed after disease onset. Few studies have been conducted exploring the role of CLA in the prevention of autoimmune diseases. These studies suggest that isomer-specific effects may be occurring; however, a direct comparison of CLAt10c12 and CLAc9t11 has yet to be conducted. A study to compare the ability of CLAt10c12 and CLAc9t11 to prevent CA and assess their effects on early inflammation was performed. DBA/1 mice were fed a semipurified diet containing 6% corn oil (CO), 5.5% CO and 0.5% CLAt10c12, or 5.5% CO and 0.5% CLAc9t11 (n = 27 per diet) starting three weeks before CA primary immunization. Effects on disease incidence and severity, anticollagen antibodies, plasma and paw cytokines, and hepatic fatty acids were measured. Arthritis incidence was reduced by a minimum of 34% in mice fed either CLA isomer compared to those fed CO diet (p = 0.06). In mice that did develop arthritis (n = 9-12 mice per treatment), CLAt10c12 reduced arthritic severity to a greater extent than CLAc9t11 and CO (p = 0.03). CLA isomer treatment attenuated the increased hepatic arachidonic acid (ARA; 20:4n-6) observed with arthritis at one-week postonset (p = 0.03), while no differences in anticollagen antibodies or cytokines were observed between dietary treatments. These results suggest that CLA isomers may be effective at preventing specific immune-mediated inflammatory diseases, in part, through modulation of the ARA cascade.

Trans10,cis12 conjugated linoleic acid inhibits proliferation and migration of ovarian cancer cells by inducing ER stress, autophagy, and modulation of Src.

The goal of this study was to investigate the anti-cancer effects of Trans10,cis12 conjugated linoleic acid (t10,c12 CLA). MTT assays and QCM™ chemotaxis 96-wells were used to test the effect of t10,c12 CLA on the proliferation and migration and invasion of cancer cells. qPCR and Western Blotting were used to determine the expression of specific factors. RNA sequencing was conducted using the Illumina platform and apoptosis was measured using a flow cytometry assay. t10,c12 CLA (IC50, 7 µM) inhibited proliferation of ovarian cancer cell lines SKOV-3 and A2780. c9,t11 CLA did not attenuate the proliferation of these cells. Transcription of 165 genes was significantly repressed and 28 genes were elevated. Genes related to ER stress, ATF4, CHOP, and GADD34 were overexpressed whereas EDEM2 and Hsp90, genes required for proteasomal degradation of misfolded proteins, were downregulated upon treatment. While apoptosis was not detected, t10,c12 CLA treatment led to 9-fold increase in autophagolysosomes and higher levels of LC3-II. G1 cell cycle arrest in treated cells was correlated with phosphorylation of GSK3ß and loss of ß-catenin. microRNA miR184 and miR215 were upregulated. miR184 likely contributed to G1 arrest by downregulating E2F1. miR215 upregulation was correlated with increased expression of p27/Kip-1. t10,c12 CLA-mediated inhibition of invasion and migration correlated with decreased expression of PTP1b and decreased Src activation by inhibiting phosphorylation at Tyr416. Due to its ability to inhibit proliferation and migration, t10,c12 CLA should be considered for treatment of ovarian cancer.

Dietary Conjugated Linoleic Acid-c9t11 Prevents Collagen-Induced Arthritis, Whereas Conjugated Linoleic Acid-t10c12 Increases Arthritic Severity.

Two conjugated linoleic acid (CLA) isomers, cis-9, trans-11 (CLAc9t11) and trans-10, cis-12 (CLAt10c12), reduce inflammation in a number of animal models, including collagen-induced arthritis (CA). However, little is known about the ability of individual CLA isomers to prevent autoimmune disease onset. Evidence that mixed isomer CLA drives T helper cell (Th) 1 responses suggests that CLA, or a specific isomer, exacerbates onset of Th1 autoimmune diseases. In two experiments, we examined if prior dietary exposure to CLAt10c12 (experiment 1) or CLAc9t11 (experiment 2) affected the incidence or severity of CA. DBA/1 mice were fed a semi purified diet with either 6% corn oil (CO, w/w), 5.75% CO plus 0.25% CLAt10c12, or 5.5% CO plus 0.5% CLAc9t11 prior to arthritis development. Arthritis incidence and severity, anti-collagen antibodies, paw cytokines, and hepatic fatty acids were measured. CLAt10c12 had no effect on arthritis incidence but increased arthritic severity (42%, P = 0.02); however, CLAc9t11 decreased arthritis incidence 39% compared to CO fed mice (P = 0.01), but had no effect on disease severity. CLAt10c12-induced increase in anti-collagen type II IgG antibodies may be a mechanism by which this isomer increased arthritic severity, and CLAc9t11-induced increase in Th2 paw cytokines (IL-4 and IL-10, P ≤ 0.04) may explain how CLAc9t11 reduced the arthritis incidence. While both isomers are well known to reduce inflammation in arthritic mice, these new data suggest isomer differences when fed prior to autoimmune disease.

A Comparison of the Anti-Inflammatory Effects of Cis-9, Trans-11 Conjugated Linoleic Acid to Celecoxib in the Collagen-Induced Arthritis Model.

Cyclooxygenase (COX)-2 inhibitors, such as celecoxib, for chronic inflammatory disease are associated with adverse health events, while cis-9, trans-11 (c9t11) conjugated linoleic acid (CLA) is anti-inflammatory without adverse events attributed to pure intake. Mechanistically, celecoxib and c9t11 disrupt the arachidonic acid cascade; however, the equivalency of anti-inflammatory effects between these compounds is unknown. Therefore, to test the hypothesis that 0.5% dietary c9t11 reduces inflammation equivalently to a celecoxib dose intended to treat rheumatoid arthritis (RA; 5 mg/kg bw), arthritic mice received diets containing one of the following supplements: 1% corn oil (CO, w/w), 0.5% c9t11 (>91% purity) +0.5% CO, or 1% CO + 0.5, 5, or 50 mg/kg bw celecoxib, and were assessed for changes in arthritic severity over 6 weeks. Overall, arthritic severity in mice fed c9t11 was reduced (34%, P < 0.01) while celecoxib doses (0.5, 5, 50 mg/kg) reduced arthritic severity (16, 56, 48%, respectively) compared to CO-fed arthritic mice. Linear regression of the celecoxib dose-response showed 0.5% c9t11 (570 mg/kg bw) reduced arthritic severity equivalently to 1.5 mg/kg celecoxib. Interleukin-6 (IL-6) was increased in paws of arthritic mice fed CO compared to shams, but was decreased in arthritic groups fed 0.5% c9t11 and 5 mg/kg celecoxib, compared to arthritic mice fed CO (Ps ≤ 0.05). Additionally, paw and plasma IL-10 levels in arthritic mice were decreased by 5 mg/kg celecoxib, but were unaffected by c9t11 compared to CO. Results suggest dietary c9t11 may be an effective adjunct to COX-2 inhibition for treating chronic inflammation.

Maternally derived anti-fibroblast growth factor 23 antibody as new tool to reduce phosphorus requirement of chicks.

Novel means to reduce phosphate input into poultry feeds and increase its retention would preserve world phosphate reserves and reduce environmental impact of poultry production. Here we show that a maternally derived antibody to a fibroblast growth factor-23 (FGF-23) peptide (GMNPPPYS) alleviated phosphorus deficiency in chicks fed low non-phytate phosphorus (nPP) diets. White Leghorn laying hens were vaccinated with either an adjuvant control or the synthetic FGF-23 peptide, and chicks with control or anti-FGF-23 maternal antibodies were fed a diet containing either 0.13 or 0.45% nPP (experiment 1), and 0.20 or 0.45% nPP (experiment 2) for 14 d. In both experiments, decreasing nPP from 0.45 to 0.13 or 0.20% decreased BW gain, G:F, excreta phosphorus, plasma phosphate, and plasma FGF-23 at all time periods examined (nPP main effect, P < 0.05). In experiment 1, chicks with maternal anti-FGF-23 antibody had increased tibiotarsi ash (d 14), and decreased excreta phosphate (d 7, 14) and plasma intact parathyroid hormone (d 7) when compared to chicks with control antibody (antibody main effect, P < 0.05). Mortality (d 7 to 14, 1 to 14), posture scores (d 7, 14) and bone lesion scores (d 14) decreased and plasma phosphate (d 14) increased in anti-FGF-23 chicks fed 0.13% nPP, compared to those with control antibody on the same diet (P < 0.05). In experiment 2, chicks with maternal anti-FGF-23 antibody had increased tibiotarsi ash (d 14), and plasma phosphate (d 14) and 1,25(OH)2D3 (d 14) levels, compared to chicks with control antibody (antibody main effect, P < 0.05). BW gain and G:F were increased in chicks with anti-FGF-23 antibody fed 0.20% nPP, compared to control antibody chicks on the same diet, at all time periods examined (P < 0.05). In conclusion, maternally-derived anti-FGF-23 antibody increased phosphorus retention in chicks fed diets containing either 0.13 or 0.20% nPP and thereby, reduced signs of phosphorus deficiency.

Antibody to fibroblast growth factor 23-peptide reduces excreta phosphorus of laying hens.

Novel strategies to minimize the excretion of phosphorus in swine and poultry are critical in minimizing environmental degradation. We have developed a synthetic peptide vaccine to produce autoantibodies to fibroblast growth factor 23 (FGF-23), a bone-derived hormone that blocks kidney phosphate resorption and indirectly reduces intestinal phosphate absorption. Single Comb White Leghorn laying hens, fed a standard diet (inorganic phosphorus, Pi = 0.4%), were immunized over the course of 4 weeks with either a FGF-23 peptide vaccine or adjuvant control (without FGF-23 peptide). At peak antibody titer to the peptide (week 5), 24-h excreta were collected and hens were blood sampled (represents 0.4% Pi treatment). Hens were then fed a 0.8% Pi diet and blood was sampled at 24 and 72 h and 24-h excreta were collected at 12 to 36 and 60 to 84 h (represents 0.8% Pi treatment). Increasing Pi from 0.4 to 0.8% increased (P < 0.05) percent excreta phosphorus, total 24-h phosphorus excretion, and plasma levels of FGF-23 and phosphate in either control or FGF-23 peptide vaccinated hens as early as the first sampling period. FGF-23 peptide vaccinated hens fed 0.4% Pi had reduced (P < 0.05) percent excreta phosphorus, total 24 h phosphorus excretion, and plasma levels of FGF-23 and iPTH, and increased (P < 0.05) plasma levels of phosphate and 1,25(OH)2D3 when compared to control vaccinated hens fed 0.4% Pi. In the first collection period post 0.8% Pi feeding, FGF-23 peptide vaccinated hens had reduced (P < 0.05) plasma levels of FGF-23 and iPTH, and increased (P < 0.05) plasma levels of phosphate and 1,25(OH)2D3, and tended to have reduced percent excreta phosphorus (P = 0.085) and total 24 h phosphorus excretion (P = 0.078) when compared to control vaccinated hens. Results during the second collection period post 0.8% Pi feeding were similar to that at the first collection period. These results are the first to show that the inhibition of FGF-23 action by a peptide vaccine (via neutralizing antibody) reduced phosphorus excretion. The approach presented provides new information on phosphorus metabolism in the laying hen.

Serotonin Deficiency Rescues Lactation on Day 1 in Mice Fed a High Fat Diet.

Obesity is an inflammatory state associated with delayed lactogenesis stage II and altered mammary gland morphology. Serotonin mediates inflammation and mammary gland involution. The objective of this study was to determine if a genetic deficiency of tryptophan hydroxylase 1, the rate-limiting enzyme in peripheral serotonin synthesis, would result in an improved ability to lactate in dams fed a high fat diet. Twenty-six female mice were fed a high (HFD) or low fat (LFD) diet throughout pregnancy and lactation. Fourteen mice were genetically deficient for Tph1 (Tph1-/-), and twelve were wild type. Milk yield, pup mortality, and dam weights were recorded and milk samples were collected. On day 10 of lactation, dams were sacrificed and mammary glands were harvested for RT-PCR and histological evaluation. HFD dams weighed more than LFD dams at the onset of lactation. WT HFD dams were unable to lactate on day 1 of lactation and exhibited increased pup mortality relative to all other treatments, including Tph1-/- HFD dams. mRNA expression of immune markers C-X-C motif chemokine 5 and tumor necrosis factor alpha were elevated in WT HFD mammary glands. Mammary gland histology showed a reduced number of alveoli in WT compared to Tph1-/- dams, regardless of diet, and the alveoli of HFD dams were smaller than those of LFD dams. Finally, fatty acid profile in milk was dynamic in both early and peak lactation, with reduced de novo synthesis of fatty acids on day 10 of lactation in the HFD groups. Administration of a HFD to C57BL/6 dams produced an obese phenotype in the mammary gland, which was alleviated by a genetic deficiency of Tph1. Serotonin may modulate the effects of obesity on the mammary gland, potentially contributing to the delayed onset of lactogenesis seen in obese women.

Low Dietary c9t11-Conjugated Linoleic Acid Intake from Dairy Fat or Supplements Reduces Inflammation in Collagen-Induced Arthritis.

Dietary cis-9,trans-11 (c9t11) conjugated linoleic acid (CLA) fed at 0.5 % w/w was previously shown to attenuate inflammation in the murine collagen-induced (CA) arthritis model, and growing evidence implicates c9t11-CLA as a major anti-inflammatory component of dairy fat. To understand c9t11-CLA's contribution to dairy fat's anti-inflammatory action, the minimum amount of dietary c9t11-CLA needed to reduce inflammation must be determined. This study had two objectives: (1) determine the minimum dietary anti-inflammatory c9t11-CLA intake level in the CA model, and (2) compare this to anti-inflammatory effects of dairy fat (non-enriched, naturally c9t11-CLA-enriched, or c9t11-CLA-supplemented). Mice received the following dietary fat treatments (w/w) post arthritis onset: corn oil (6 % CO), 0.125, 0.25, 0.375, and 0.5 % c9t11-CLA, control butter (6 % CB), c9t11-enriched butter (6 % EB), or c9t11-CLA-supplemented butter (6 % SB, containing 0.2 % c9t11-CLA). Paw arthritic severity and pad swelling were scored and measured, respectively, over an 84-day study period. All c9t11-CLA and butter diets decreased the arthritic score (25-51 %, P < 0.01) and paw swelling (8-11 %, P < 0.01). Throughout the study, plasma tumor necrosis factor (TNFα) was elevated in CO-fed arthritic mice compared to non-arthritic (NA) mice but was reduced in 0.5 % c9t11-CLA- and EB-fed mice. Interleukin-1ß and IL-6 were increased in arthritic CO-fed mice compared to NA mice but were reduced in 0.5 % c9t11-CLA- and EB-fed mice through day 42. In conclusion, 0.125 % c9t11-CLA reduced clinical arthritis as effectively as higher doses, and decreased arthritis in CB-fed mice suggested that the minimal anti-inflammatory levels of c9t11-CLA might be below 0.125 %.

Interleukin-10 neutralizing antibody for detection of intestinal luminal levels and as a dietary additive in Eimeria challenged broiler chicks.

Interleukin-10 (IL-10) mRNA levels are increased within intestinal mucosa after Eimeria infection. IL-10 apical receptor presence on enterocytes suggests IL-10 is secreted into the intestinal lumen. Increased IL-10 has been shown to be central to the pathogenesis of numerous intracellular pathogens; we hypothesize luminal secretion of IL-10 enables Eimeria spp. infection in chickens. This study examines intestine luminal IL-10 levels and performance in broilers challenged with Eimeria when fed an anti-IL-10 antibody. Chicks were fed a diet (1 to 21 d) with control or anti-IL-10 antibody (0.34 g egg yolk antibody powder/Kg diet) with a saline or 10× dose of Advent coccidiosis vaccine on d 3. One chick per pen was euthanized on days 2, 4, 7, 10, 13, 16, and 19 post-challenge, bled, and intestines were collected for luminal fluid IL-10 concentrations. Body weight and feed intake were measured on d 21, and oocyst shedding was assessed on d 7 post-challenge. A significant Eimeria × antibody interaction on d 21 body weight (P < 0.05) showed chicks fed control antibody, but not anti-IL-10, had significant reductions in body weight when challenged with Eimeria spp. Oocyst shedding was increased with Eimeria challenge, but dietary antibody had no effect. Plasma carotenoid levels were reduced in Eimeria challenged chicks 4, 7, 10, and 16 days post-challenge compared to unchallenged chicks. Lack of an Eimeria × antibody interaction showed anti-IL-10 was not protective against Eimeria-induced decreases in plasma carotenoids. Eimeria challenge increased intestine luminal IL-10 on days 4 and 7 post-challenge in the cecum and jejunum, respectively, compared to unchallenged. Dietary anti-IL-10 decreased luminal IL-10 in the ileum on day 2 post-challenge when compared to control antibody fed chicks. No interaction between Eimeria challenge and antibody was observed on intestine luminal contents of IL-10, suggesting anti-IL-10 was ineffective at preventing increased Eimeria-induced luminal IL-10. In conclusion, Eimeria challenge increased intestinal luminal IL-10 and anti-IL-10 was effective at preventing Eimeria-induced decreased body weight, however the mechanism anti-IL-10 antibody protects body weight during Eimeria challenge remains unknown.

Oral antibody to interleukin-10 reduces growth rate depression due to Eimeria spp. infection in broiler chickens.

Eimeria spp. must be controlled in floor-reared poultry to prevent the onset of coccidiosis. Here we use an oral antibody to chicken IL-10 to prevent growth depression due to Eimeria spp. infection. Egg antibody directed against an antigenic peptide of IL-10 was produced in laying hens and measured using an ELISA. In the first experiment, egg yolk powder containing antibody to chicken IL-10 (vlpramqt conjugate) (anti-IL-10 yolk powder) was fed at 3.4 g/kg feed to determine growth response following mixed Eimeria spp. challenge. Chicks were fed either anti-IL-10 antibodies or control antibodies and challenged (d3) with either sterile saline or a 10× attenuated Eimeria spp. vaccine. Control-fed and Eimeria-challenged chicks grew 8.8% slower than those challenged with saline (P < 0.04), whereas anti-IL-10-fed Eimeria challenged chicks were not different from untreated controls. In the second trial a dose response was performed with doses of either 0 (control antibody), 0.34-, or 3.4-g anti-IL-10 yolk powder/kg feed. Control-fed, Eimeria-challenged chicks grew 10.6% slower than control saline-challenged chicks (P < 0.05); however, anti-IL-10-fed chicks fed either dose of anti-IL-10 were not different from saline-challenged chicks. Finally, the effect of anti-IL-10 on acquired immunity was investigated. Chicks were fed control or anti-IL-10 yolk powder and vaccinated with a 1× dose of Eimeria vaccine at d 3. After 14 d, antibody was removed from the diet. Chicks were either saline or 10× Eimeria challenged at d 17. We found that the anti-IL-10-fed chickens did not show a reduction in growth due to challenge; hence anti-IL-10 does not appear to affect adaptive immunity during the primary immunization. Overall, use of an antibody to IL-10 is a novel method in preventing adverse effects of Eimeria spp. infection in poultry.

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.