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.

Antimicrobial peptides as an alternative to relieve antimicrobial growth promoters in poultry.

1. This review describes different classes of antimicrobial peptides (AMP) found in the gastrointestinal (GI) tract of avian species, and their antimicrobial and immunomodulatory activities. The potential benefits of synthetic AMP in poultry production are examined, in the context of the use of AMP as alternatives to antimicrobial growth promoters (AGP).2. Since the mid-1950s, antibiotic growth promoters (AGP) have been used in feed at low prophylactic doses to modulate the homoeostasis of intestinal microbiota, decreasing the risk of intestinal dysbacteriosis and the growth of pathogens within the avian gut. Over the last three decades, AGP have faced major regulatory restrictions due to concerns of generating antimicrobial resistance (AMR). It is now well documented that the rate of infectious disease outbreaks is higher in flocks that are not fed prophylactic antibiotics, resulting in a compensatory increase in antimicrobial use for therapeutic purposes.3. Endogenous natural AMP production is associated with the presence of microbiota and their interaction with the intestinal epithelial and lamina propria lymphoid cells. Their antimicrobial activity shapes the beneficial microbiota population and controls intestinal pathogens such Clostridium and Salmonella spp., and stimulates the development and maturation of the local immune system.4. Similar to AGP, AMP can establish a well-balanced gut beneficial microbiota for adequate immune-competence, animal health and high growth performance parameters such as feed intake, daily weight, feed conversion and accumulated mortality.5. Antimicrobial proteins and peptides constitute an essential part of the innate immune system of all organisms and protect the host from invading pathogenic bacteria, viruses, fungi, and parasites by interacting with the negatively charged pathogen membranes.

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.

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.

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.

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.

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.

Innate immune response of pullets fed diets supplemented with prebiotics and synbiotics.

Prebiotics and synbiotics are considered to be among the most promising replacements for in-feed antibiotic growth promoters (AGP) in poultry feed. The current study was designed to study the effect of Bacitracin methylene disalicylate (BMD) (Control), yeast-derived carbohydrates (YDC), and a blend of YDC and probiotics [Lactobacillus acidophilus, Lactobacillus casei, Streptococcus faecium, Bacillus subtilis, and YDC] (SNB) in the performance and innate immune response of pullets. Feed intake and BW were measured on a weekly basis. At the end of the study (d 21), 10 birds/treatment were sacrificed by cervical dislocation and ileum, cecal tonsil, and spleen samples were collected for gene expression analysis. No significant difference (P > 0.05) in feed intake and G:F was observed among treatments. In the second and third wk age, higher BW gain was observed in SNB treatment compared to control and both control and YDC treatments, respectively. Expression of TLR2b was upregulated in YDC and SNB in the ileum, and in SNB in the spleen (P < 0.05). Expression of TLR4 was downregulated in SNB in the cecal tonsil. Expression of TLR21 was downregulated in YDC in the ileum, while it was upregulated in SNB in the spleen (P < 0.05). In the ileum, YDC resulted in downregulated IL-12p35, CxCLi2, and IL-13, and SNB resulted in upregulated IL-6, interferon (IFN)-γ, and IL-4 (P < 0.05). In the cecal tonsil, YDC resulted in upregulated IL-12p35, IL-2, IL-13 and IL-10, and SNB resulted in downregulated IL-2 and upregulated IL-13 and IL-10 (P < 0.05). In the spleen, YDC resulted in dowregulated IL-2 and CxCLi2, and SNB resulted in upregulated IL-6, IFN-γ, IL-4, and IL-10 (P < 0.05). In conclusion, no change in performance was observed. Innate immune response analysis showed SNB with a more potent effect compared to YDC where the former showed a balanced T-helper (Th)-1/Th-2 response locally and a more Th-2-dependent response systemically; SNB might provide a more beneficial immune modulation with maintaining immune homeostasis, which was observed in a strong IL-10 response.

Evaluation of the systemic innate immune response and metabolic alterations of nonlactating cows with diet-induced subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) increases lipopolysaccharide endotoxin in the rumen, which might translocate into the systemic circulation, triggering a cascade of clinical and immunological alterations. The objective of this study was to characterize the clinical immune and metabolic responses to ruminal-derived lipopolysaccharide in nonlactating cows induced with SARA using 2 challenges, a grain-based SARA challenge (GBSC) or an alfalfa-pellet SARA challenge (APSC). Six dry, nonlactating Holstein cows were used in a 3 × 3 Latin square arrangement of treatments with 4-wk experimental cycles. All cows received the control diet containing 70% forage and 30% mixed concentrates (dry matter basis) for 3 wk. In wk 4, cows received a control diet, GBSC (38% wheat-barley pellets, 32% other mixed concentrate, and 30% forages), or APSC (45% mixed concentrate, 32% alfalfa pellets, and 23% other forages). Total plasma proteins and immunology-related proteins, acute phase proteins, blood cells, serum chemistry, mRNA gene expression of peripheral blood cell surface markers, and selected proinflammatory cytokines were evaluated. Ruminal pH was lower in both groups with induced SARA compared with a control group. Ruminal endotoxins were higher in GBSC; however, plasma endotoxin was not detected in any study group. No significant differences in feed intake, rectal temperature, white blood cell counts, or differentials were found between control and SARA challenge groups; changes in glucose, urea, Ca, and Mg were observed in SARA groups. Total plasma proteins were lower in both SARA groups, and acute phase proteins were higher in GBSC. The expression of CD14, MD2, and TLR4 mRNA in peripheral blood leukocytes was not affected by SARA induction. The induction of SARA as a result of GBSC or APSC challenge was successful; however, LPS was not detected in plasma. Changes in clinical, metabolic, and inflammatory responses were not observed in the SARA-challenged cows, suggesting that, in this study, SARA was not associated with a systemic response to inflammation.

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.

Effect of supplementing direct-fed microbials on broiler performance, nutrient digestibilities, and immune responses.

Direct-fed microbials (DFM) are used to improve livestock health and performance. The effects of 2 DFM products, a blend of 3 Bacillus strains (DFMB) and a Propionibacteriumspp. (DFMP), on broiler performance, nutrient utilization, and immune responses were investigated. Day-old (n = 120) male broilers were divided into 24 groups of 5 birds and fed 3 wheat-based diets in mash form (8 groups per diet) from d 1 to 22. The control diet was fed without or with 7.5 × 10(4) cfu/g of either DFMB or DFMP. From d 19 to 21 fecal samples were collected for determination of total tract apparent retention (TTAR) of nutrients and AMEn. On d 21, feed intake and BW were determined. On d 22, 5 birds per treatment were killed by cervical dislocation to collect jejunal and ileal contents for determination of digesta viscosity and apparent ileal digestibility (AID) of nutrients, respectively, and ileum, cecal tonsil, and spleen tissues for Toll-like receptors (TLR) and cytokine expressions. Compared with the control, DFM did not affect BW gain and feed intake but DFMP reduced G:F (P < 0.01). Compared with the control (2,875 kcal/kg), birds fed on DFMB and DFMP had higher AMEn (2,979 and 2,916 kcal/kg, respectively; P < 0.05), whereas both DFM reduced the AID of DM (P < 0.001) and CP (P < 0.01). Furthermore, DFMP reduced TTAR of NDF (29.0 vs. 18.4%; P < 0.001), whereas both DFM increased TTAR of DM and fat (P < 0.001). Supplementing DFMP downregulated ileal expression of TLR-2b, IL-2, IL-4, IL-6, IL-10, and IL-13, whereas DFMB downregulated TLR-2b, IL-2, IL-4, and IL-6 in all 3 tissues, IL-10 in the spleen, and upregulated IL-13 in the spleen. In conclusion, the DFM did not improve performance but increased the AMEn of diet by possibly increasing DM and fat retention. Overall, both DFM showed an antiinflammatory effect in the ileum, but DFMB had more effects on local and systemic immunity than DFMP.

Adenoviral-mediated gene transfer into bone marrow: an effective surgical technique in rat.

BACKGROUND: The role of transforming growth factor-beta 1 (TGF-ß1) in the onset of bone marrow fibrosis has been confirmed in some animal models. To further understand the genetic expression of some myeloproliferative disorders affecting marrow stem cells, however, it is necessary to develop a specific and reliable procedure to deliver modified adenoviral vectors into the bone marrow cavity. The aim of this paper is to report a surgical technique designed to deliver an adenoviral vector-mediated gene expressing TGF-ß1 into the bone marrow of rat femurs. METHODS: Forty-two Sprague-Dawley rats were used in the study. Rat femurs were exposed and the compact and trabecular bones at the proximal head removed. An intrabone marrow injection of a mutated TGF-ß1 adenoviral vector, a null adenoviral vector, or PBS was delivered into the bone. Three groups were accounted (n = 14 per group): fibrogenic and positive and negative controls. The quality of the surgical entrance was assessed by means of computerized tomography and histological changes were assessed by histochemistry. The concentration of TGF-ß1 in the bone marrow was determined by ELISA. RESULTS: The surgical technique was conducted under ideal timing (approx. 10 min) and no surgical or postsurgical complications were observed. Computerized tomography revealed no changes in the bone tissue and a clean entrance was delimited through the bone to the bone marrow. HE and Masson's trichrome staining indicated highly fibrotic areas in the profibrotic group and bone marrow lavage reported a significantly higher concentration of TGF-ß1 (p < 0.05) in that same group. CONCLUSIONS: The present study confirmed that the proposed surgical technique is an effective method to deliver adenoviral vectors into the femoral bone marrow to investigate the physiopathology of bone marrow fibrosis in rats.

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.

Effects of subacute ruminal acidosis challenges on fermentation and endotoxins in the rumen and hindgut of dairy cows.

The effects of a grain-based subacute ruminal acidosis (SARA) challenge (GBSC) and an alfalfa-pellet SARA challenge (APSC) on fermentation and endotoxins in the rumen and in the cecum, as well as on endotoxins in peripheral blood, were determined. Six nonlactating Holstein cows with cannulas in the rumen and cecum were used in the study. A 3×3 Latin square arrangement of treatments with 4-wk experimental periods was adopted. During the first 3 wk of each experimental period, all cows received a diet containing 70% forages [dry matter (DM) basis]. In wk 4 of each period, cows received 1 of the following 3 diets: the 70% forage diet fed during wk 1 to 3 (control), a diet in which 34% of the dietary DM was replaced with grain pellets made of 50% ground wheat and 50% ground barely (GBSC), or a diet in which 37% of dietary DM was replaced with pellets of ground alfalfa (APSC). Rumen pH was monitored continuously using indwelling pH probes, and rumen fluid, blood, cecal digesta, and fecal grab samples were collected immediately before feed delivery at 0900 h and at 6 h after feed delivery on d 3 and 5 of wk 4. The time for which rumen pH was below 5.6 was 56.4, 225.2, and 298.8 min/d for the control, APSC, and GBSC treatments, respectively. Compared with the control, SARA challenges resulted in similar reductions in cecal digesta pH, which were 7.07, 6.86, and 6.79 for the control, APSC, and GBSC treatments, respectively. Compared with the control, only GBSC increased starch content in cecal digesta, which averaged 2.8, 2.6, and 7.4% of DM for the control, APSC, and GBSC, respectively. Free lipopolysaccharide endotoxin (LPS) concentration in rumen fluid increased from 10,405 endotoxin units (EU)/mL in the control treatment to 30,715 and 168,391 EU/mL in APSC and GBSC, respectively. Additionally, GBSC increased the LPS concentration from 16,508 to 118,522 EU/g in wet cecal digesta, and from 12,832 to 93,154 EU/g in wet feces. The APSC treatment did not affect LPS concentrations in cecal digesta and feces. All concentrations of LPS in blood plasma were below the detection limit of >0.05 EU/mL of the technique used. Despite the absence of LPS in blood, only GBSC increased the concentration of LPS-binding protein in blood plasma, which averaged, 8.9, 9.5, and 12.1mg/L for the control, APSC, and GBSC treatments, respectively. This suggests that GBSC caused translocation of LPS from the digestive tract but that LPS was detoxified before entering the peripheral blood circulation. The higher LPS concentration in cecal digesta in the GBSC compared with the APSC suggests a higher risk of LPS translocation in the large intestine in GBSC than in APSC.

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.

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.