Prebiotic Driven Increases in IL-17A Do Not Prevent Campylobacter jejuni Colonization of Chickens

Abstract

Worldwide Campylobacter jejuni is a leading cause of foodborne disease. Contamination of chicken meat with digesta from C. jejuni-positive birds during slaughter and processing is a key route of transmission to humans through the food chain. Colonization of chickens with C. jejuni elicits host innate immune responses that may be modulated by dietary additives to provide a reduction in the number of campylobacters colonizing the gastrointestinal tract and thereby reduce the likelihood of human exposure to an infectious dose. Here we report the effects of prebiotic galacto-oligosaccharide (GOS) on broiler chickens colonized with C. jejuni when challenged at either an early stage in development at 6 days of age or 20 days old when campylobacters are frequently detected in commercial flocks. GOS-fed birds had increased growth performance, but the levels of C. jejuni colonizing the cecal pouches were unchanged irrespective of the age of challenge. Dietary GOS modulated the immune response to C. jejuni by increasing cytokine IL-17A expression at colonization. Correspondingly, reduced diversity of the cecal microbiota was associated with Campylobacter colonization in GOS-fed birds. In birds challenged at 6 days-old the reduction in microbial diversity was accompanied by an increase in the relative abundance of Escherichia spp. Whilst immuno-modulation of the Th17 pro-inflammatory response did not prevent C. jejuni colonization of the intestinal tract of broiler chickens, the study highlights the potential for combinations of prebiotics, and specific competitors (synbiotics) to engage with the host innate immunity to reduce pathogen burdens.

Keywords: Campylobacter; Th17; broiler chicken; galacto-oligosaccharide; innate immunity; microbiota; prebiotic; pro-inflammatory response.

FIGURE 1

GOS improves growth performance of broiler chickens, but does not affect C. jejuni colonization of the ceca. (A,B) Chicken live total mass from day of hatch to 35 days for 6-dc birds (A) and 20-dc birds (B). The plots show the performances of the experimental treatments GOS + Campylobacter (GOS diet) Campylobacter (control diet) and the Ross 308 performance objective as the Target (Aviagen Performance Objectives, 2014). The dashed line indicates age at challenge. (C,D) Viable counts of Campylobacter recovered from the cecal content of 6-dc birds (C) and 20-dc birds (D). Data markers indicate Campylobacter CFU isolated from individual birds. Bars indicate mean Campylobacter CFU, excluding cohorts where Campylobacter levels were below the limit of detection. There are no significant differences in the Campylobacter counts for the GOS diet compared to control diet post colonization (p > 0.05). The dashed line indicates minimum level of detection.

FIGURE 2

Modulation of cecal and ileal innate immune responses to dietary GOS in Campylobacter-challenged broiler chickens. The figures report cytokine and chemokine normalized gene expression for 6-dc birds (A) and 20-dc birds (B) fed a control diet (Campylobacter treatment) or a GOS diet (GOS + Campylobacter treatment) recorded as log₁₀ of the ratio for gene of interest/GAPDH from qPCR data of individual birds. Corresponding probabilities (p-adj) were calculated using non-parametric Wilcoxon rank sum tests with Benjamini-Hochberg FDR correction, where differences at p-adj < 0.05 were considered significant and are summarized in the plot using asterisks (^∗p-adj ≤ 0.05; ^∗∗p-adj ≤ 0.01).

FIGURE 3

Dietary GOS promotes differential shifts in the cecal microbiota post Campylobacter challenge. (A,B) Cecal α-diversity for 6-dc birds (A) and 20-dc birds (B) described as Chao richness, Inverse Simpson diversity and Shannon diversity (as indicated by panels on right hand side of the figure). The age of the birds are in days as indicated by the numerals in the strip at the top of the figure. Significant differences between groups are indicated by asterisks (^∗p ≤ 0.05; ^∗∗p ≤ 0.01). (C,D) OTU relative abundance for 6-dc birds (C) and 20-dc birds (D) summarized at Phyla level.

FIGURE 4

Summary of age dependent differences for dietary GOS in C. jejuni colonized broiler chickens. Significant changes in the live weight, cecal C. jejuni colonization levels, ileal villus and crypt metrics are indicated for the GOS-fed birds compared to the birds on the control diet colonized at either 6 or 20 days of age (da) with C. jejuni. Relative changes in the transcription of ileal and cecal cytokines and chemokines for GOS-fed bird compared to controls are indicated for age matched birds by up arrows (↑) for increases and down arrows for decreases in cytokine/chemokine expression (↓). NS indicates no significant differences between the birds on the GOS or control diet.