Oral Treatment With Ileal Spores Triggers Immunometabolic Shifts in Chicken Gut

Abstract

The animal gut is a major site affecting productivity via its role in mediating functions like food conversion and pathogen colonization. Live microorganisms like probiotics are widely used to improve poultry productivity. However, given that chicks receive their microbiota from the environment at-hatch, a bacterial treatment that can stimulate gut immune maturation in early life can benefit animal health. Thus, our lab has begun investigating alternative means to improve poultry health via single inoculation with microbial spores. In this study, we orally-inoculated day-old chicks with ileal scrapings (ISs) enriched for spores via chloroform treatment (SPORE) or non-treated (CON). At 3, 7, and 14 days post-inoculation (dpi), gut permeability was measured via FITC-dextran assay in serum. Additionally, small intestinal scrapings (SISs) were tested for in vitro Salmonella killing and total IgA. Lastly, distal ileum was either fixed or flash-frozen for microscopy or kinome peptide array, respectively. Using bacterial 16S rRNA gene sequencing, SPORE and CON inocula were highly-similar in bacterial composition. However, spores were detected in SPORE but not in CON inoculum. Segmented filamentous bacteria (SFB) filaments were observed in the distal ileum in SPORE birds as early as 3 dpi and all birds at 7 and 14 dpi. Additionally, SFB were detected via PCR in the ceca, colonizing all SPORE birds at 3 dpi. At 3 dpi, SPORE birds exhibited lower gut permeability vs. CON. In SPORE birds, SISs induced greater Salmonella growth in vitro at 3 dpi yet significantly-reduced Salmonella load at 7 and 14 dpi compared to CON in an IgA-independent manner. SPORE distal ileal tissue exhibited unique upregulation of several immunometabolic processes vs. CON birds, including innate (Toll-like receptor, JAK-STAT) and adaptive (T/B cell receptor, T_H17 differentiation) immune pathways, PI3K/Akt signaling, mTOR signaling, and insulin-related pathways. Collectively, these data suggest oral inoculation with ileal spores generally-improved gut health. Importance: We report that ileal, spore-forming commensal microbes have potent effects on ileum immunometabolism. Additionally, we identify a functional ileal phenotype in spore-treated chickens, which matched several of the observed immunometabolic changes and was associated with SFB colonization in the ileum.

Keywords: IgA; SFB; Salmonella; TH17; gut permeability; kinome; spores.

Figure 1

TEM images of bacterial spores in SPORE and CON inocula. (A,B), CON inoculum. (C–F), SPORE inoculum. Atrophied or dead microbes are indicated by faint bodies with contorted outer membranes (white arrows). Spores or viable bacteria are seen as electron-dense (i.e., dark) with discernable membranes (black arrows).

Figure 2

16S reads identified in SPORE and CON inocula. Bacterial taxa from spore-enriched (SPORE) and control (CON) inocula were identified via 16S rRNA gene sequencing and QIIME2 pipeline analysis.

Figure 3

SEM detection of SFB in the distal ileum. SEM images were taken for CON (A–C) and SPORE (D–F) birds at multiple days post-inoculation (dpi) to track SFB colonization over time. (A,D), 3 dpi. (B,E), 7 dpi. (C,F), 14 dpi. In addition, a table summarizing the total findings is provided.

Figure 4

SFB detection in ceca content via PCR. SFB-specific primers were used to detect SFB in ceca content at 3, 7, and 14 dpi time points.

Figure 5

Measures of weight gain and gut morphology. (A) Chick weight was measured at 1 and 11 days post-hatch to assess average weight gain per animal. (B) Intestinal segment lengths were measured via ruler at 14 days post-inoculation (dpi). (C) Gut permeability was measured at 3 dpi via orally-delivered FITC-dextran leakage in serum. ^*P < 0.05, ^**P < 0.01.

Figure 6

Salmonella resistance assays in vitro. Salmonella enterica resistance was measured using in vitro bactericidal assays against multiple Salmonella isolates (summarized in Table 1). Salmonella killing was performed in small intestinal scrapings taken at 3 (A), 7 (B), and 14 dpi (C) in experimental duplicate. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001.

Figure 7

Total IgA production. Endpoint titers for total IgA were measured in small intestinal scrapings from birds at 3, 7, and 14 days post-inoculation (dpi) via ELISA. Assays were performed in experimental duplicate. ^****P < 0.0001.

Figure 8

Heatmap and clustering of kinome profiles. The raw kinome signal from the peptide array was input into the custom software package PIIKA 2. Red indicates relative increased phosphorylation, whereas green indicates relative decreased phosphorylation of each peptide on the array.