Long‑chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis

Meijerink, Nathalie; De Oliveira, Jean E.; Van Haarlem, Daphne A.; Lamot, David M.; Velkers, Francisca C.; Smidt, Hauke; Stegeman, J. Arjan; Rutten, Victor P.M.G.; Jansen, Christine A.

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Long‑chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis

Meijerink, Nathalie; De Oliveira, Jean E.; Van Haarlem, Daphne A.; Lamot, David M.; Velkers, Francisca C.; Smidt, Hauke; Stegeman, J. Arjan; Rutten, Victor P.M.G.; Jansen, Christine A.

URI: http://hdl.handle.net/2263/90140

Date: 2022-02-04

Abstract:

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

Description:

ADDITIONAL FILE 1. The gating strategies used to analyze numbers and function of NK cells, γδ T cells and cytotoxic CD8+ T cells in the ileum. Gating included consecutive selection for lymphocytes (FSC-A vs SSC-A), viable cells (Live/Dead marker-negative) followed by selection of the specific cellular subsets and the expression of activation markers by NK and T cells according to the staining panels (Table 1). NK cell subsets were gated on CD3− cells expressing either IL-2Rα or 20E5 and NK cell activation was gated on CD3− CD41/61− cells expressing CD107 or on CD3− cells expressing IFNγ. T cell subsets were gated on CD3+ CD4− cells positive for TCRγδ (γδ) or negative ( CD8+ αβ) with both γδ and cytotoxic αβ T cells expressing either CD8αα or CD8αβ. T cell activation was gated on CD3+ CD41/61−CD8α+ cells expressing CD107 or on CD3+ TCRγδ+CD8α+ and CD3+ TCRγδ−CD8α+ cells expressing IFNγ, and only in spleen also CD3+ TCRγδ−CD8α− ( CD4+) cells expressing IFNγ. The marker CD41/61 is included in the CD107 assay to exclude thrombocytes from analysis, since activated thrombocytes have been reported to express CD107 [69]. In the CD107 assay NK cells are gated by excluding T cells and thrombocytes since a pan NK marker is missing while for phenotyping the NK cells are gated based on expression of the NK markers IL-2Rα and 20E5, which are known to be expressed on cells with NK function [48].

ADDITIONAL FILE 2. Effect of GM on numbers of intraepithelial and splenic γδ T cells and cytotoxic T cells expressing either CD8αα+ and CD8αβ+ before and during SE infection in broiler chickens. A Numbers (cells/mg) of intraepithelial CD8αα+ γδ T cells, B CD8αβ+ γδ T cells, C cytotoxic CD8αα+ T cells and D CD8αβ+ T cells in chickens either fed standard (control) or long-chain glucomannan supplemented (GM) diet in course of time before and during SE infection. E Numbers (cells/mg) of splenic CD8αα+ γδ T cells, F CD8αβ+ γδ T cells, G cytotoxic CD8αα+ T cells and H CD8αβ+ T cells in chickens either fed standard or GM diet before and during SE infection. Mean + SEM per diet group and time point are shown (n = 6), if n = 5; one chicken was excluded due to numbers of events acquired in the gate of interest were < 100. Statistical significance between diet groups is indicated as ***(p < 0.001).

ADDITIONAL FILE 3. Effect of GM on T cell activation in IELs and spleen before and during SE infection in broiler chickens. A Percentages of intraepithelial CD8+ T cells expressing CD107 (including both γδ and αβ T cells), B CD8+ γδ T cells expressing IFNγ and C CD8+ αβ T cells expressing IFNγ in chickens either fed standard (control) or long-chain glucomannan supplemented (GM) diet in course of time before and during SE infection. D Percentages of splenic CD8+ T cells expressing CD107 (including both γδ and αβ T cells), E CD8+ γδ T cells expressing IFNγ, F CD4+ αβ T cells expressing IFNγ and G CD8+ αβ T cells expressing IFNγ in chickens either fed standard or GM diet before and during SE infection. Mean + SEM per diet group and time point are shown (n = 6), for IFNγ expression of CD8+ γδ T cells in the IEL population at 0 dpi percentages were not determined (nd) due to numbers of events acquired in the gate of interest were < 100.

ADDITIONAL FILE 4. Correlation between serum antibody titers and SE-CFUs in broiler chickens. Correlation between SE-specific antibody titers and splenic SE-CFUs of chickens either fed standard (control) or long-chain glucomannan (GM) diet using the Spearman rank correlation test. Statistical significance is indicated as p = 0.01.

ADDITIONAL FILE 5. Intestinal microbial taxa significantly increased with diet at 0, 3 and 7 dpi of SE in broiler chickens. Standardized relative fluorescence intensities of the microbial taxa as measured by the microarray in the ileum and caeca (Table 3) that were significantly increased either with standard (control) or long-chain glucomannan supplemented (GM) diet at 0, 3 and 7 dpi of SE in broiler chickens. LS mean per microbial taxa and diet group are shown (n = 6) with statistical significance of FDR adjusted p values set at < 0.05.

ADDITIONAL FILE 6. Correlation between microbial taxa and intraepithelial and splenic immune parameters at 0, 3 and 7 dpi of SE in broiler chickens. A Correlation values between intestinal microbial taxa in the ileum or B caeca significantly increased with diet and percentages of NK cell activation (CD107 or IFNγ expression) or numbers of NK and T cell subsets of the ileum (IEL) and spleen (Spln) per diet (control, GM) at 0, 3 and 7 dpi of SE in broiler chickens. Pearson’s correlation (r) values are depicted in a heatmap as positive (yellow) or negative (dark blue) correlations.

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