Folic acid enhances proinflammatory and antiviral molecular pathways in chicken B-lymphocytes infected with a mild infectious bursal disease virus.

1. This study evaluated the effect of folic acid (FA) supplementation on the proinflammatory and antiviral molecular pathways of B-lymphocytes infected with a modified live IBDV (ST-12) mild vaccine strain during a timed post-infection analysis.2. A chicken B-lymphocytes (DT-40) cell line was cultured in triplicate at a concentration of 5 × 105 cells per well in 24-well plates; and was divided into three groups: 1: No virus, FA; 2: Virus, no FA; 3: Virus + FA at a concentration of 3.96 mM. The experiment was repeated three times.3. Cells in groups 2 and 3 were infected with a modified live IBDV (ST-12) mild vaccine strain at one multiplicity of infection (MOI: 1). After 1 hour of virus adsorption, samples were collected at 0, 3, 6, 12, 24 and 36 hours post-infection (hpi).4. The modified live IBDV (ST-12) mild vaccine strain triggered a B-lymphocyte specific immune response associated with the upregulation of genes involved in virus recognition (Igß), virus sensing (TLR-2, TLR-3, TLR-4 and MDA5), signal transduction and regulation (TRIF, MyD88 and IRF7), and the antiviral effector molecules (IFN-α, OAS, PKR, and viperin).5. FA supplementation modulated IBDV replication and regulated the proinflammatory and antiviral downstream molecular pathways.6. In conclusion, the low virulent pathotype serotype I modified live IBDV (ST-12) mild vaccine strain was able to trigger and mount an immune response in chicken B-lymphocytes without affecting B-cell viability. FA supplementation modulated B lymphocytes response and improved their innate immune proinflammatory and antiviral response molecular pathways.

Yeast cell wall polysaccharides enhanced expression of T helper type 1 and 2 cytokines profile in chicken B lymphocytes exposed to LPS challenge and enzyme treatment.

1. The objective of this study was to investigate the potential immunomodulatory effects of yeast (Saccharomyces cerevisiae) and yeast-derived products treated with a cell wall lytic enzyme mixture on the gene expression of toll-like receptors and cytokines of chicken B cell line (DT 40) stimulated with lipopolysaccharide. 2. The effect of brewer's yeast (Y), yeast cell wall (YCW), distilled dried grains with solubles (DDGS) and a processed yeast + nucleotide-rich product (PY/N) treated with a yeast cell wall lytic enzyme (E) was assessed using a chicken B cells and LPS challenge model. 3. Relative gene expression of toll-like receptors (TLRs) and cytokines was investigated. Treatment of cells with Y, YCW, YCW + E and PY/N upregulated the expression of TLR2b following LPS challenge. Gene expression of TLR4 was downregulated in E, YCW and YCW + E treatments compared to control, while adding DDGS and PY/N upregulated the expression of TLR4 either before or after enzyme treatment. Following LPS challenge, expression of IL-4 and IL-10 was upregulated in cells treated with YCW and PY/N, both pre and post enzyme incubation. Adding YCW and PY/N to the cells challenged with LPS upregulated the expression of IFN-γ and IL-12 before and after enzyme treatment. Treatment of cells with YCW, DDGS and PY/N increased the expression of IL-6 prior to LPS challenge. 4. In conclusion, the results suggested that yeast-derived products affected immunomodulatory activities by changing the expression of cytokines involved in the innate immune response.

Differential immunomodulatory effect of vitamin D (1,25 (OH)2 D3) on the innate immune response in different types of cells infected in vitro with infectious bursal disease virus.

It has been demonstrated that vitamin D (Vit D) included in diets offers a beneficial effect by improving innate immune responses in chickens. However, its mechanisms of action and the effect on immunosuppressive pathogens, such as infectious bursal disease virus, are not yet known. In the present study, we have studied the immunomodulatory effect of Vit D on the innate immune response in 3 cell lines: fibroblast cells (DF-1), macrophages (HD11), and B cells (DT-40) infected with IBDV (intermediate vaccine) at 2 multiplicity of infections (MOI) (1 and 0.1). Genes associated with innate immune responses (TLR-3, TLR-21, MDA-5, MyD88, TRIF, IRF-7, INF-α, INF-ß, PKR, OAS, viperin, IL-1ß, IL-6, and IL-12) were evaluated at different time points (3, 6, 12, 24, and 36 h after infection, h.p.i). Virus production reached a maximum at 24 h.p.i., which was significantly (P < 0.05) higher in DF-1 cells, followed by HD-11 and DT-40 cells. Mainly in HD-11 cells, there was a significant (P < 0.05) effect of Vit D supplementation on receptors TLR-3, TLR-21, and MDA-5 after 12 h.p.i, independent of MOI. DT-40 cells showed the highest antiviral activity, with a significant (P < 0.05) effect on IRF-7, IFN-ß, OAS, and PKR gene expression, where expression of IRF-7 and IFN-ß correlated positively with Vit D supplementation, while OAS and PKR were independent of Vit D. Proinflammatory cytokines were significantly (P < 0.05) upregulated and found to be Vit D and MOI dependent. In conclusion, this study demonstrated the capacity of IBDV to trigger a strong innate immune response in chicken cells and contributes to the understanding of the activation pathways of innate immunity induced by IBDV and further shows the benefitial effect of Vit D supplementation as an immunomodulator.

Epigenetic effect of folic acid (FA) on the gene proximal promoter area and mRNA expression of chicken B cell as antigen presenting cells.

1. This study evaluated and characterised the effect of folic acid (FA) on chromosomal DNA methylation and the epigenetic result on gene expression control mechanisms in chicken B cells as a model of antigen presenting cells. 2. After FA supplementation, the methylation pattern on the proximal promoter area and mRNA expression of toll-like receptor (TLR) 2b, TLR4, B cell receptor (BCR) immunoglobulin (Ig) ß and major histocompatibility complex (MHC) II ß chain genes in chicken B cells was observed 3. Chicken B cell line (DT40) cultures were incubated with 0, 1.72 or 3.96 mM of FA for 4 and 8 h and samples were taken at specific time points. After 4 h of incubation, cells were challenged with 0, 1 or 10 µg/ml of lipopolysaccharide (LPS) and samples were collected 4 h post-challenge. 4. FA supplementation modified the methylation patterns of the proximal promoter regions of TLR4, Igß, and MHCII ß chain at 4 and 8 hours of incubation; however, the single CpG dinucleotide of TLR2b remained methylated regardless of the treatment. 5. A positive association was found between FA concentration and percentage DNA methylation on the promoter area of Igß and TLR2b. However, there was a negative association between FA and MHCII ß chain. 6. There were downregulatory effects in TLR4, Igß and MHCII ß chain gene expression after 8 h of incubation, nut not at 4 h. Although incubation time did not affect TLR2b gene expression, FA concentration did, whereby it increased TLR2b expression at 1.72 mM FA (P < 0.05). 7. LPS significant downregulated TLR2b expression, while an interaction between FA and LPS concentration affected TLR4 and Igß gene expression. 8. In conclusion, the results showed that FA can have an immunomodulatory effect on chicken B cells, possibly affecting their ability to both recognise antigens through the TLR and BCR pathways, and to present it via the MHCII presentation pathway.

Short communication: Extended-spectrum cephalosporin-resistant Escherichia coli in colostrum from New Brunswick, Canada, dairy cows harbor blaCMY-2 and blaTEM resistance genes.

Dairy calves are colonized shortly after birth by multidrug resistant (MDR) bacteria, including Escherichia coli. The role of dairy colostrum fed to calves as a potential source of MDR bacteria resistance genes has not been investigated. This study determined the recovery rate of extended-spectrum cephalosporin-resistant (ESC-R) E. coli in colostrum from cows. The ESC-R E. coli isolates were further investigated to determine their phenotypic antimicrobial resistance pattern and the genes conferring ESC-R. Fresh colostrum was collected from 452 cows from 8 dairy herds in New Brunswick, Canada. The ESC-R E. coli was isolated from the colostrum by using the VACC agar, a selective media for extended-spectrum ß-lactamase producing Enterobacteriaceae. Minimum inhibitory concentration was determined for all the suspected ESC-R E. coli isolates using a commercial gram-negative broth microdilution method. Two multiplex PCR were conducted on all the suspected ESC-R E. coli isolates to determine the presence of the blaCTX-M (groups 1, 2, 9, and 8/25) blaCMY-2, blaSHV, and blaTEM resistance genes. The ESC-R E. coli were detected in 20 (4.43%) of the colostrum samples. At least 1 ESC-R E. coli isolate was detected in 6 (75%) of the dairy herds. All ESC-R E. coli had MDR profiles based on minimum inhibitory concentration testing. No blaCTX-M groups genes were detected; however, the blaCMY-2 gene was detected in 9 or 20 (45%) and blaTEM was detected in 7 of 20 (35%) of the ESC-R E. coli. No ESC-R E. coli had both blaCMY-2 and blaTEM resistance genes. This is the first report of blaCMY-2 and blaTEM genes found in E. coli isolates cultured from dairy colostrum to our knowledge.

Dietary supplementation with a nucleotide-rich yeast extract modulates gut immune response and microflora in weaned pigs in response to a sanitary challenge.

An experiment was carried out to evaluate the short-term effect of supplementing a nucleotide-rich yeast extract (NRYE) on growth performance, gut structure, immunity and microflora of piglets raised under sanitary and unsanitary conditions. A total of 84, 21-day old piglets were used in this study; 42 piglets were raised in a room designated as the clean room that was washed once per week, whereas the other 42 piglets were raised in a room designated as the unclean room in which 7 kg of manure from the sow herd was spread on each pen floor on day 1 and 7 and the room was not washed throughout the experiment. The pigs were fed a corn-soybean meal-based diet without or with 0.1% NRYE. Each treatment had 7 replicate pens in each room, and each pen housed 3 pigs. Feed disappearance and BW were recorded on day 1 and 14. On day 14, one pig per pen was euthanized to collect ileum, mesenteric lymph nodes and spleen tissues, and cecum and colon digesta. Overall, NRYE supplementation did not affect growth performance in both clean and unclean conditions, improved kidney weight in both clean (P=0.0002) and unclean room (P<0.0001) and tended to improve the villus height/crypt depth ratio in the clean room (P=0.073). Supplementing NRYE was associated with upregulation of Ileal programmed cell death gene-1 (P=0.0003), interleukin (IL)-1ß (P<0.0001), IL-6 (P=0.0003), IL-10 (P<0.0001) and tumor necrosis factor-α (TNF-α) (P<0.0001) in pigs raised in the unclean room. Supplementing the NRYE in pigs raised in the clean room suppressed growth of cecal Enterobacteriacea (P<0.0001) members and colonic Enterococcus spp. (P<0.019), improved proliferation of cecal Lactobacillus spp. (P<0.002) and colonic Clostridium cluster IV (P<0.011) and XVIa members (P<0.0002). Supplementing the NRYE in the unclean room improved proliferation of cecal Clostridium cluster IV (P<0.026) and suppressed proliferation of colonic Enterococcus spp. (P<0.037). In conclusion, supplementing the NRYE to piglets under unsanitary conditions improved ileal immune response by upregulating inflammatory cytokines, and positively modulated proliferation of beneficial gut bacteria and suppression of harmful ones in both clean and unclean rooms.

The immunomodulatory effect of vitamin D in chickens is dose-dependent and influenced by calcium and phosphorus levels.

Vitamin D requirement is estimated to be higher than recommended values for the first two weeks of a broiler chicken's life, and is heavily dependent on the concentrations of Ca and P in the diet. There are data indicating the beneficial effect of higher vitamin D levels on performance and overall health of the chickens. However, data on the role of higher vitamin D levels on the innate immune response of chickens are limited. Therefore, in the current study, we examined the effect of higher doses of vitamin D supplementation on the innate immune response in broiler chickens receiving optimal or calcium (Ca) and phosphorus (P) deficient diets. Three hundred Ross-308 male broiler chicks were randomly allocated into 60 cages with 5 birds per cage in a 3 × 2 factorial design with three levels of vitamin D and two levels of Ca/P with each experimental diet fed to 10 cages (10 replicates). Quantitative reverse transcription PCR (n = 5) was used to assess Toll-like receptor (TLR2b and 4), cytokine/chemokine (IL-12, IFN-γ, IL-10, IL-4, IL-13, IL-18, CxCLi2) and cathelicidin (CATH1, CATHB1, CATH3) transcription levels in peripheral blood mononuclear cells (PBMCs), spleen, and bursa of Fabricius. Vitamin D supplementation of the Ca and P deficient diet considerably augmented transcription of TLR2b, TLR4, CATH1, and CATHB1 and predominantly Th2 cytokines in spleen. Supplementation of the control diet with vitamin D downregulated TLR4 transcription, and dose-dependently increased CATH1, CATHB1, Th1, and Th2 cytokine transcription (Th2>Th1). All diets downregulated CATH3 transcription. In conclusion, vitamin D or its derivative 25-OH-D3 both have a robust immunomodulatory property with a more favorable Th2 response, while at the same time enhancing observed Th2 cytokine responses under both optimal and lower Ca and P inclusion levels in the diets of broiler chickens.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance and local innate immune response of broiler chickens challenged with Clostridium perfringens.

This study evaluated the effect of yeast-derived products on growth performance, gut lesion score, intestinal population of Clostridium perfringens, and local innate immunity of broiler chickens challenged with C. perfringens. One-day-old broiler chickens were randomly assigned to eight dietary treatments providing six replicate pens of 55 birds each per treatment. Dietary treatments consisted of Control diets without and with C. perfringens challenge, and diets containing bacitracin methylene disalicylate (BMD, 55 g/tonne), nucleotides (150 g/tonne), yeast cell wall (YCW, 300 g/tonne), and a commercial product Maxi-Gen Plus (1 kg/tonne) fed to chickens challenged with C. perfringens. Diets containing 10% distillers dried grains with solubles without and with C. perfringens challenge were also used. Birds were orally challenged with C. perfringens (10(8) colony-forming units (cfu)/bird) on day 14. On day 21, intestinal samples were collected for gene expression analysis. Pathogen challenge significantly (P < 0.05) impaired feed intake, body weight gain, and feed conversion ratio (FCR) shortly after the challenge (14-21 days). Increased C. perfringens counts and intestinal lesion scores were observed for challenged birds except the BMD-containing diet. Over the entire trial (1-35 days), no difference in growth performance was observed except the BMD diet which improved FCR over the Control, challenged group. Birds receiving nucleotides showed increased expression of toll-like receptors and cytokines interleukin (IL)-4 and IL-18 compared to the Control, challenged group. Expression of macrophage mannose receptor and IL-18 was upregulated in birds receiving YCW. Increased expression of cytokines and receptors involved in innate immunity in broilers receiving nucleotides and YCW suggests the immunomodulatory properties of these products under pathogen challenge conditions.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance, gut morphology, and gene expression of pattern recognition receptors and cytokines in broiler chickens.

An experiment was carried out to investigate the effect of yeast-derived products and distillers' dried grains with solubles (DDGS) on growth performance, small intestinal morphology, and innate immune response in broiler chickens from 1 to 21 d of age. Nine replicates of 5 birds each were assigned to dietary treatments consisting of a control diet without antibiotic (C), and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus, 0.025% of nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On d 21, 5 birds per treatment were euthanized and approximately 5-cm long duodenum, jejunum, and ileum segments were collected for intestinal morphology measurements. Cecal tonsils and spleen were collected to measure the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, macrophage mannose receptor (MMR), and cytokines IFN-γ, IL-12, IL-10, and IL-4. No significant difference was observed for growth performance parameters. However, diets containing 0.05% of nucleotides and YCW significantly increased (P < 0.05) villus height in the jejunum. Furthermore, the number of the goblet cells per unit area in the ileum was increased (P < 0.05) in diets supplemented with yeast-derived products. The expression of TLR2b in the spleen was down-regulated for diets supplemented with nucleotides and antibiotic. In addition, lower expression of TLR21 and MMR was observed in the spleen of birds receiving yeast-derived products and antibiotic. However, expression of TLR4 in the spleen was up-regulated in diets supplemented with YCW and nucleotides. The expression of IFN-γ and IL-12 was down-regulated in the spleen of birds fed diets supplemented with yeast-derived products. In addition, inclusion of YCW, Maxi-Gen Plus, or 0.05% of nucleotides down-regulated the expression of IL-10 and IL-4 in the cecal tonsils. In conclusion, down-regulation of receptors and cytokines in spleen and cecal tonsils of birds fed diets supplemented with yeast-derived products may suggest that yeast products do not exert immune stimulating effect under normal health conditions.

Organic trace mineral supplementation enhances local and systemic innate immune responses and modulates oxidative stress in broiler chickens.

The effect of organic trace mineral supplementation on performance, intestinal morphology, immune organ weights (bursa of Fabricius and spleen), expression of innate immune response related genes, blood heterophils/lymphocytes ratio, chemical metabolic panel, natural antibodies (IgG), and oxidative stress of broiler chickens was studied. A total of 1,080 day-old male broilers were assigned to 1 of 3 dietary treatments, which included basal diet with Monensin (control), control diet supplemented with bacitracin methylene disalicylate (BMD), and BMD diet supplemented with organic trace minerals (OTM). No difference in feed conversion ratio was observed among treatments; ileum histomorphological analysis showed a lower crypt depth, higher villi height/crypt depth ratio, and lower villi width in the OTM treatment compared to control. Furthermore, OTM treatment resulted in higher uric acid and lower plasma malondehaldehyde (MDA), indicating lower oxidative stress. Gene expression analysis showed that OTM treatment resulted in up-regulations of TLR2 bin the ileum, and TLR2b, TLR4, and IL-12p35 in the bursa of Fabricius, and down-regulation of TLR2b and TLR4 in the cecal tonsils. In the spleen, OTM treatment resulted in up-regulation of IL-10. In conclusion, OTM supplementation to broiler diets may have beneficial effects on intestinal development, immune system status, and survival by improving ileum histomorphological parameters, modulation of Toll-like receptors and anti-inflammatory cytokines, and decreasing level of MDA, which in conjunction could enhance health status.

Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell-mediated immune response indicating the immunomodulatory activities of these products following immunization with non-inflammatory antigens.

Performance, serum biochemical responses, and gene expression of intestinal folate transporters of young and older laying hens in response to dietary folic acid supplementation and challenge with Escherichia coli lipopolysaccharide.

The present study was conducted to investigate the effects of dietary folic acid (FA) supplementation on performance, serum biochemical indices, and mRNA abundance of intestinal folate transporters in young and older laying hens after acute lipopolysaccharide (LPS) challenge. Two experiments were conducted separately involving 48 Shaver White young laying hens (24 wk of age) in experiment 1 and 48 Shaver White older laying hens (58 wk of age) in experiment 2. Birds were fed 2 diets in a complete randomized design. The diets were wheat-soybean meal based, with or without supplemental 4 mg of FA/kg of diet. Birds were fed for 8 wk, during which time feed consumption and egg production were monitored. At the end of each feeding experiment, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either Escherichia coli LPS or sterile saline. Four hours after injection, blood and intestinal samples were collected for further analysis. Compared with the control, dietary FA supplementation increased egg weight and egg mass and decreased serum glucose levels in the young laying hens, and reduced serum uric acid in the older laying hens (P < 0.05). Relative to saline injection, plasma homocysteine, serum calcium, and phosphorus levels were found to be lower in both young and older laying hens after LPS challenge (P < 0.05). Other serum biochemical variables and the mRNA expression of 2 folate transport genes in the small and large intestine were differentially affected by LPS challenge, and some of those responses varied with the age of the birds. Additionally, interactions between diet and LPS challenge were specifically found in the older laying hens. In summary, in addition to improving production performance, there were effects of dietary FA supplementation and its interaction with LPS challenge on biochemical constituents, and age played a role in the development of responses to diet and bacterial LPS infections.

Performance, histomorphology, and toll-like receptor, chemokine, and cytokine profile locally and systemically in broiler chickens fed diets supplemented with yeast-derived macromolecules.

The turnover of intestinal epithelial cells is a dynamic process that includes adequate cell proliferation and maturation in the presence of microbiota and migration and seeding of immune cells in early gut development in chickens. We studied the effect of yeast-derived macromolecules (YDM) on performance, gut health, and immune system gene expression in the intestine of broiler chickens. One thousand eighty 1-d-old birds, with 60 birds per pen and 6 pens per treatment, were randomly assigned to 3 treatment diets; a diet containing monensin (control), control diet supplemented with bacitracin methylene disalycylate (BMD), and BMD diet supplemented with YDM. Feed intake, BW, mortality, ileum histomorphology, and gene expression of Toll-like receptors (TLR2b, TLR4, and TLR21), cytokines [interferon (IFN)-γ, IFN-ß, IL-12p35, IL-1ß, IL-6, IL-10, IL-8, IL-2, IL-4, and transforming growth factor (TGF)-ß4], and cluster of differentiation (CD)40 in the ileum, cecal tonsil, bursa of Fabricius, and spleen were assessed. No significant overall difference in performance in terms of feed intake, BW gain, and G:F was observed among treatments (P > 0.05). The YDM diet resulted in significantly higher villi height and villi height:crypt depth ratio compared with BMD and control diets (P < 0.05). A significantly lower mortality was observed in the YDM treatment compared with both control and BMD treatments. Compared with the control, gene expression analysis in YDM treatment showed no major change in response in the ileum, whereas higher CD40, IFN-ß, IL-ß, IL-6, TGF-ß4, IL-2, and IL-4 in the cecal tonsil; TLR2b, TLR4, TLR21, and TGF-ß4 in the bursa of Fabricius; and TLR4, IL-12p35, IFN-γ, TGF-ß4, and IL-4 in the spleen was observed (P < 0.05). In conclusion, supplementation of YDM supports pro- and anti-inflammatory cytokine production via T helper type 1 and 2 (Th1 and Th2) cell-associated pathways both locally and systemically with a stronger additive effect in the cecal tonsil in the presence of BMD in the diet of chickens.

Response of older laying hens to an Escherichia coli lipopolysaccharide challenge when fed diets with or without supplemental folic acid.

Folic acid plays a key role in nucleic acids and protein synthesis, and has been associated with anti-inflammatory effects in LPS-induced infections. To this end, a study was conducted to investigate the effects of dietary folic acid (FA) supplementation in older laying hens (58 to 66 wk of age) challenged with Escherichia coli lipopolysaccharide (LPS). A total of 24 Shaver White laying hens at 58 wk were fed 2 diets. The diets were wheat-soybean-based, with either 0 or 4 mg of supplemental FA per kg of diet. After 8 wk of feeding and at 66 wk, the hens were injected intravenously with 8 mg of LPS or saline per kg of BW. Four hours after injection, blood was collected and hens were euthanized to obtain spleen and cecal tonsils. The T cell subsets in the blood and the spleen (CD4+ and CD8+), total IgG, and biochemical constituents (total protein, albumin, globulin, and fibrinogen) were not influenced (P > 0.05) by dietary FA supplementation. However, LPS injection decreased (P < 0.05) biochemical constituents, CD4+, and CD8+ cells in the blood, whereas CD4+:CD8+ ratio and total IgG increased (P < 0.05), and fibrinogen was not influenced. Gene expression in the spleen and cecal tonsils was not influenced by dietary FA supplementation except a diet × challenge interaction for interleukin (IL)-8 in the spleen; IL-8 decreased in FA-fed hens that were treated with LPS. Also, FA supplementation decreased the expression of IL-8 in cecal tonsils. Relative to saline-injected hens, expression of IL-1ß, interferon-γ, and IL-10 increased in the LPS-injected hens in the spleen and cecal tonsils, IL-8 increased in LPS-injected hens only in the cecal tonsils, whereas Toll-like receptor 4, IL-4, IL-17, and IL-18 increased in the LPS-injected hens only in the spleen; however, LPS decreased expression of IL-13 in the cecal tonsils. In conclusion, FA did not affect inflammatory responses in older laying hens; more studies are required to investigate possible protective effects of FA in laying hens.

Immunomodulation in young laying hens by dietary folic acid and acute immune responses after challenge with Escherichia coli lipopolysaccharide.

We investigated the effects of dietary folic acid (FA) supplementation on immunological parameters in young laying hens under acute conditions of lipopolysaccharide (LPS) challenge. Twenty-four Shaver White laying hens at 24 wk were fed 2 diets in a completely randomized design. The diets were wheat-soybean based, with either 0 or 4 mg of supplemental FA per kilogram of diet. At 32 wk of age, 6 hens from each dietary treatment were injected intravenously with 8 mg/kg of BW of either LPS or saline. Four hours after injection, blood was collected and the hens were euthanized to obtain spleen and cecal tonsils. Heterophil:lymphocyte ratio, CD3+, CD4+, CD8+ T cells, and CD4+:CD8+ cells in the blood and spleen were not affected by dietary FA. Relative to saline-injected hens, LPS-injected hens had fewer (P < 0.05) CD3+, CD4+, CD8+, and CD4+:CD8+ cells in the blood, and no difference was found in the spleen. Total protein, albumin, and globulin were found to be higher (P < 0.05) in FA-supplemented hens compared with the control. However, total protein, albumin, and globulins decreased (P < 0.05) in the LPS-injected hens compared with the saline control. Expression of interleukin (IL)-1ß in cecal tonsils decreased (P < 0.05) in FA-supplemented hens, but no dietary influence was found on the expression of other genes in both the spleen and cecal tonsils. Lipopolysaccharide upregulated (P < 0.05) expression of IL-10 and interferon (IFN)-γ in the spleen, and IL-1ß, IL-10, and IFN-γ in the cecal tonsils, whereas the expression of Toll-like receptor (TLR)-4 and IL-8 was not influenced by LPS in the spleen and cecal tonsils. There was a diet × challenge interaction for total IgG, and cytokines IL-1ß and IL-18 in the spleen as well as IL-18 in the cecal tonsils. In conclusion, there were few interactions of dietary FA and LPS; however, FA increased biochemical constituents, enhanced generation of total IgG, as well as exhibiting pleoitropic effects in inflammatory responses.

Local and systemic innate immunity in broiler chickens supplemented with yeast-derived carbohydrates.

A study was conducted to assess the effect of yeast-derived carbohydrates (YDC) on performance and innate immune responses of broiler chickens. In total, 1,080 one-day-old birds were randomly assigned to one of 3 dietary treatments (n = 360): a standard broiler diet containing monensin (control), control + bacitracin methylene disalycylate (BMD), and YDC treatment (control + YDC at 0.02%, 0.01%, and 0.005% for starter, grower, and finisher, respectively). Weekly BW, feed intake (FI), and feed conversion ratio (FCR) were recorded. Immune organ weights, gut morphology, gene expression, heterophil:lymphocyte (H:L), and serum IgG were determined at d 42. No significant difference in FCR, FI, and mortality was observed among treatments. However, BW gain in starter phase was higher in control and YDC treatments compared with BMD treatment. Ileal villi height, crypt depth, and their ratio were not significantly different among treatments, whereas villi width was lower in control and YDC treatments compared with BMD treatment. The number of goblet cells per unit area in the ileum was lower in BMD treatment compared with control and YDC treatments. Expression of TLR2b and IL-6 in the ileum and cecal tonsils was not significantly different among treatments (P > 0.05). Expression of TLR4 was downregulated in YDC treatment compared with control in the ileum. Expression of IL-12p35 and IFN-γ were downregulated in the YDC treatment only in the cecal tonsils. Compared with the control, the expression of IL-10 in both the ileum and the cecal tonsils was downregulated in YDC treatment. Serum IgG and H:L ratio were lower and higher, respectively, in the YDC treatment compared with control and BMD treatments. In conclusion, dietary inclusion of YDC affected intestinal cytokines anti-inflammatory profile on a gut location associated immune pathways manner, suggesting different immune pathways that require further studies in this field.

The effect of microbial-nutrient interaction on the immune system of young chicks after early probiotic and organic acid administration.

The combined effects of probiotics (Lactobacillus acidophilus, Lactobacillus casei, Streptococcus faecium, and Saccharomyces cerevisiae) and organic acids (sorbic and citric acid) on intestinal morphology and expression of immune-related genes were investigated. One-day-old chicks were randomly assigned to 1 of 3 groups: birds not receiving probiotic or organic acids (control; T1), or birds receiving an oral combination (1 g/L in water) of 10(8) CFU/g of each of the aforementioned probiotics and organic acids (1% sorbic acid and 0.2% citric acid) for 7 (T2) or 14 d (T3). Each group was divided into 5 replicate pens of 20 birds each, and 5 birds from each group (1 from each pen) were killed on d 11 and 22. Intestinal sections were collected for histological assessment, and reverse transcriptase-PCR analysis was used to assess defensin and cathelicidins expression. Quantitative real-time PCR was used to assess toll-like receptors (TLR) and cytokine expression. Duodenal villus height was greater in T2 and T3 at d 11 (P ≤ 0.036) and 22 (P ≤ 0.015) compared with T1. At d 11, duodenal goblet cell/unit area was less in T3, whereas it was greater in T2 compared with T1 in the jejunum (P = 0.009). Ileal goblet cell/unit area was greater in T3 at d 22 compared with T1 (P < 0.001). Avian beta-defensin-3 was expressed in all tissues except the bursa of T3 birds at d 11, and TLR-2 was down regulated in the cecal tonsil of birds in T2 and T3 at d 11 compared with T1 (P = 0.020 and 0.003, respectively). Expression of IL-12p35 in the ileum at d 11 was down regulated in T2 and T3 compared with T1 (P = 0.030 and 0.012, respectively). Reduced expression of INF-γ was observed in the ileum in T3 compared with T1 at d 11 (P = 0.047). Ileal IL-6 and IL-10 and cecal tonsil interferon-gamma (INF-γ) expressions were greater T2 at d 22 (P ≤ 0.047) than T1. In conclusion, supplementation of combined probiotics and organic acids resulted in inconsistent gut morphology associated responses, and avian beta-defensins and cathelicidins expression were not associated with combined probiotics and organic acids supplementation. Birds supplemented with combined probiotics and organic acids for 7 d showing similar responses in TLR-2, IL-12p35, and IFN-γ compared with those supplemented for 14 d indicates that shorter periods of supplementation might be enough to elicit beneficial responses.

The adaptive transport of folic acid in the intestine of laying hens with increased supplementation of dietary folic acid.

Different aspects of folic acid (FA) transport in the intestine of the laying hen have been characterized. Less is known about the adaptive response of this process to a dietary challenge. To this end, a study was conducted to evaluate the effect of increased dietary FA supplementation on the rate of intestinal FA transport and the expression of the intestinal folate transporter genes, the proton-coupled folate transporters (PCFT) and the reduced folate carrier (RFC), in the laying hen. Twenty-four Shaver White hens at 34 wk of age were randomly assigned to receive 1 of 3 dietary treatments: 1) basal diet with no supplemental folate (n = 8), 2) basal diet + 10 mg/kg of crystalline FA (n = 8), and 3) basal diet + 100 mg/kg of crystalline FA (n = 8). A completely randomized design with 3 dietary treatments was used. Production performance was not affected by FA supplementation; however, egg and plasma folate concentrations increased (P < 0.001), whereas plasma homocysteine concentrations decreased (P < 0.011) in birds fed 10 or 100 mg of FA/kg of diet, relative to controls. Mucosal to serosal uptake of FA in the duodenum was decreased (P < 0.002), but the mRNA levels of the duodenal PCFT and RFC genes were not affected when birds were fed 10 or 100 mg of FA/kg of diet. In the jejunum, the mucosal to serosal uptake of FA, as well as the mRNA levels of the PCFT and RFC genes, were not influenced by increased FA supplementation. Overall, increased dietary levels of FA resulted in decreased transport of FA in the duodenum but not in the jejunum of laying hens. This decrease was not associated with decreased mRNA expression of the duodenal PCFT and RFC genes. Therefore, a posttranscriptional or translational adaptation of the intestinal folate transporters may be involved in the much lower transport of FA in the duodenum of laying hens during increased dietary supplementation of FA.

Dietary effects of chelated zinc supplementation and lysine levels in ISA Brown laying hens on early and late performance, and egg quality.

It has been hypothesized that zinc (Zn) levels beyond those that are nutritionally required may favor the utilization of dietary lysine, and consequently reduce the level of its inclusion into the diet. Therefore, the possible effects of interaction between chelated Zn and the level of lysine (Lys) on egg production and egg quality of laying hens were evaluated. In total, 720 ISA Brown layer hens aged 24 to 36 wk (early phase) and 48 to 60 wk (late phase) were allotted in a completely randomized factorial design that used 3 Zn and 5 Lys levels (6 replications, 8 birds/replication). All birds aged 37 to 47 wk (between early and late phases) were fed a standard diet and maintained under the same experimental design. The Zn levels used were 137, 309, and 655 mg/kg; and the Lys levels were 0.560, 0.612, 0.677, 0.749, and 0.851%. The optimal levels of Lys digestibility were based on laboratory analyses with regard to the weighted average relationship between 83.5% digestibility and the total Lys from principal ingredients. There was no effect of interaction found between the dietary levels of Zn and Lys for most of the variables studied; however, each had an independent effect on the variables. An increase in Zn from 137 to 655 mg/kg had no significant effect (P > 0.05) on the performance of hens in both phases; however, it showed a significant effect on egg quality (P < 0.01), principally on mineral composition. Increased Zn resulted in decreased shell weight, percentage of ash, yolk ash deposition, and total ash deposition. On the other hand, an increase in Lys from 0.560 to 0.851% significantly affected (P < 0.002) several performance parameters and the chemical composition of the eggs, including feed intake, feed conversion efficiency, BW gain, egg weight, and production. In conclusion, there was no interaction found between Zn and Lys, but higher dietary levels of chelated Zn reduced bird performance and egg quality parameters, whereas higher Lys levels could be beneficial to bird performance and egg quality.

Histomorphology and small intestinal sodium-dependent glucose transporter 1 gene expression in piglets fed phytic acid and phytase-supplemented diets.

An experiment was conducted to determine the effect of dietary phytic acid (PA) and phytase supplementation on small intestinal histomorphology and Na-dependent glucose transporter 1 (SGLT1) gene expression in piglets. Twenty-four piglets with an average initial BW of 7.60 ± 0.73 kg were randomly assigned to 3 experimental diets, to give 8 piglets per diet. The diets were a casein-cornstarch-based diet that was supplemented with 0 or 2% PA, or 2% PA (as Na phytate) plus an Escherichia coli-derived phytase at 500 phytase units/kg. The basal diet was formulated to meet the 1998 NRC energy, digestible AA, mineral, and vitamin requirements for piglets. After 10 d of feeding, the piglets were killed to determine small intestinal histomorphology and small intestinal SGLT1 gene expression. Phytic acid supplementation did not affect (P > 0.1) villus height (VH) and the VH-to-crypt depth (CD) ratio, but did decrease (P < 0.05) CD in the jejunum. Phytase supplementation did not affect (P > 0.1) VH, CD, and the VH-to-CD ratio. Phytic acid supplementation reduced SGLT1 gene expression in the duodenum, jejunum, and ileum by 1.1-, 5.4-, and 2.4-fold, respectively. Phytase supplementation increased SGLT1 gene expression in the jejunum by 2.6-fold, but reduced SGLT1 gene expression in the duodenum and ileum by 2.0- and 4.0-fold, respectively. In conclusion, PA reduced CD in the jejunum and SGLT1 gene expression in the duodenum, jejunum, and ileum, whereas phytase supplementation increased the expression of SGLT1 in the jejunum. The reduced SGLT1 gene expression by PA implies that PA reduces nutrient utilization in pigs partly through reduced expression of SGLT1, which is involved in glucose and Na absorption. The increased expression of SGLT1 in the jejunum by phytase supplementation implies that phytase alleviated the negative effects of PA partly through increased expression of SGLT1.