Dietary Inclusion of Scent Leaf Meal (Ocimum Gratissimum) Affects Immune Genes Expression in Chicken Spleen at 28 and 56 Days

This study was conducted to examine the effects of scent leaf meal (Ocimum gratissimum) on expression of inflammatory cytokines in the spleen of two chicken strains. A total of 150birds (75 of each strain) were randomly allotted into five dietary treatments at fifteen birds per treatment. Birds were fed diet containing varying levels of Ocimum gratissimum leaf meal. Treatment one (T1) had 0% OG, while treatment two (T2), treatment three (T3), treatment four (T4) and treatment five (T5) had 0.5% OG, 1.00% OG, 1.5% OG and 2% OG respectively. Feed and water was provided adlibitum throughout the feeding trial. Three birds were slaughtered from each treatment at day 28 and day 56, spleen samples were collected and stored using RNALater in a -20oc freezer prior to RNA extraction. Real-time qPCR was performed in 40cycles using the PowerUp SYBR Green reagent and analyzed with the 2-∆∆Ct method. Gene expression data were subjected to two-way analysis of variance. Strain effect was significantly different (P<0.05) at both time points. All the genes studied significantly differed (P<0.05) in their expression patterns at 28 and 56days of age. Increased inclusion rate of the test ingredients significantly (P<0.05) reduced IL1β and NF-KB1, while increasing IL10 and NF-KB2. Ocimim gratissimum leaf meal shows promise in the regulation of inflammation in chickens and can be used to efficiently replace antibiotics in broiler production.

The Effect of Chicken Spleen Transfer Factor on Intestinal Mucosa Immunity Barrier of Laying Hens

Chicken spleen Transfer Factor (TF) is a low-molecular-weight lymphocyte extract composed of polypeptide and nucleotide. However, its role in regulating intestinal structure and function in laying hens has remained largely unknown. 100 one-day-old laying hens were randomly divided into five groups and administered with different doses of TF (0.00 [control], 0.05mL, 0.10mL, 0.25mL and 1.00mL). The results showed that the high dose of TF (1.00mL) improved the intestinal mucosa morphology and strengthened the digestive and absorption function. Furthermore, the histology analysis revealed an increase in the number of intraepithelial lymphocytes and goblet cells. Similarly, the results from ELISA demonstrated an increase in the content of IL-10 in the intestinal tract, while the content of TNF-a showed a decrease in this regard. The RT-PCR assay also demonstrated the upregulation of the relative mRNA expressions of Muc2, TLR-2, and TLR-4 genes. The intestinal antioxidant function was significantly enhanced. In conclusion, high-dose of TF can improve the intestinal mucosa morphology and structure, enhance digestion and absorption functions, enhance the intestinal mucosal barrier immune function and antioxidant function, and up-regulate Muc2, TLR-2 and TLR-4 gene relative expression.

The Effect of Chicken Spleen Transfer Factor on Intestinal Mucosa Immunity Barrier of Laying Hens

Background Chicken spleen transfer factor (TF) is a low-molecular-weight lymphocyte extract composed of polypeptide and nucleotide. This factor is obtained by repeated freeze-thaw dialysis of the specific-pathogen-free chicken spleen and can transfer cell-mediated immunity from immunocompetent donors to recipients. However, its role in regulating intestinal digestion and absorption and mucosal immunity in laying hens has remained largely unknown. Results To explore the effect of TF on intestinal mucosa barrier function, 100 one-day-old White Leghorns laying hens were randomly divided into five groups and administered with different doses of TF (0.00 [control], 0.05 mL, 0.10 mL, 0.25 mL, and 1.00 mL). The chickens received orally TF from days 5 to 12, and they were then raised a week without it. Tissue sampling was performed on the 12th and 19th days. The results showed that the high dose of TF (1.00 mL) improved the intestinal mucosa morphology and strengthened the digestive and absorption function. Furthermore, the histology analysis revealed an increase in the number of intraepithelial lymphocytes and goblet cells. Similarly, the results from ELISA demonstrated an increase in the content of IL-10 in the intestinal tract, while the content of TNF-α showed a decrease in this regard. The RT-PCR assay also demonstrated the upregulation of the relative mRNA expressions of Muc2, TLR-2, and TLR-4 genes in the small intestine and rectum. The intestinal antioxidant function was significantly enhanced. After stopping the TF administration for a week, its effect on the intestinal mucosa barrier was still detectable; however, it was followed by a weakened effect. Conclusions In conclusion, high-dose (TF-1.00mL) of TF can improve the intestinal mucosa morphology and structure, enhance digestion and absorption functions, enhance the intestinal mucosal barrier immune function and antioxidant function, and up-regulate Muc2, TLR-2 and TLR-4 gene relative expression.

Side Effects of In ovo Given Sunset Yellow FCF on the Embryonic Development of the Spleen by Means of Histological and Enzyme Histochemical Methods

Background: E110 is one of the food colorants which is widely used in dairy products, fast foods, jam and dry beverage powders, aqueous drug solutions, tablets, capsules, toothpastes mouthwashes, hair care products and cosmetics. Doubts have accumulated in recent years that food additives might cause allergic reactions in humans or increase these ailments. In this study, side effects of Sunset yellow FCF (E110) on the embryonic development of chicken spleen were evaluated by means of histological, histomorphometrical and enzyme histochemical methods. Methods: In the study, 250 fertilized broiler eggs obtained from a commercial broodstock were used. Eggs were divided into 5 groups each having 50 eggs. Control eggs were either nontreated or distilled water injected via air sac. The eggs in the experimental groups were injected with 100ng/egg, 500ng/egg and 1.000ng/egg E110 prior to incubation. Blood and spleen samples were taken from randomly selected 10 eggs of each group at 11th, 15th, 18th and 21st days of incubation. Leukocyte formula, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte percentages were determined in the blood samples and embriyonic development of the spleen was assessed in the tissue sections. Results: In the 500ng/egg and 1.000ng/egg E110 injected experimental groups, embryonic development of the spleen retarded, alpha-naphtyl acetate esterase-positive and acid phosphatase-positive lymphocyte rates were significantly depressed. Conclusions: Significant disturbances in the immune system functions of the affected animals might occure at post-natal period of their life.

Characterization of Conventional Dendritic Cells and Macrophages in the Spleen Using the CSF1R-Reporter Transgenic Chickens

The spleen is a major site for the immunological responses to blood-borne antigens that is coordinated by cells of the mononuclear phagocyte system (MPS). The chicken spleen is populated with a number of different macrophages while the presence of conventional dendritic cells (cDC) has been described. However, a detailed characterization of the phenotype and function of different macrophage subsets and cDC in the chicken spleen is limited. Using the CSF1R-reporter transgenic chickens (CSF1R-tg), in which cells of the MPS express a transgene under the control elements of the chicken CSF1R, we carried out an in-depth characterization of these cells in the spleen. Immunohistological analysis demonstrated differential expression of MRC1L-B by periarteriolar lymphoid sheaths (PALS)-associated CSF1R-tg+ cells. In the chicken's equivalent of the mammalian marginal zone, the peri-ellipsoid white-pulp (PWP), we identified high expression of putative CD11c by ellipsoid-associated cells compared to ellipsoid-associated macrophages. In addition, we identified a novel ellipsoid macrophage subset that expressed MHCII, CD11c, MRC1L-B, and CSF1R but not the CSF1R-tg. In flow cytometric analysis, diverse expression of the CSF1R-tg and MHCII was observed leading to the categorization of CSF1R-tg cells into CSF1R-tgdim MHCIIinter−hi, CSF1R-tghi MHCIIhi, and CSF1R-tghi MHCIIinter subpopulations. Low levels of CD80, CD40, MHCI, CD44, and Ch74.2 were expressed by the CSF1R-tghi MHCIIinter cells. Functionally, in vivo fluorescent bead uptake was significantly higher in the CSF1R-tghi MHCIIhi MRC1L-B+ cells compared to the CSF1R-tgdim and CSF1R-tghi MHCIIinter MRC1L-B+ subpopulations while LPS enhanced phagocytosis by the CSF1R-tghi MHCIIinter subpopulation. The analysis of bead localization in the spleen suggests the presence of ellipsoid-associated macrophage subsets. In addition, we demonstrated the functionality of ex vivo derived CSF1R-tg+ MRC1L-Bneg cDC. Finally, RNA-seq analysis of the CSF1R-tg subpopulations demonstrated that separating the CSF1R-tghi subpopulation into CD11chi and CD11cdim cells enriched for cDC and macrophage lineages, respectively, while the CSF1R-tghi MHCIIinter subpopulation was enriched for red pulp macrophages. However, our analysis could not define the cell lineage of the heterogeneous CSF1R-tgdim subpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation.