Age-Related Differences in the Luminal and Mucosa-Associated Gut Microbiome of Broiler Chickens and Shifts Associated with Campylobacter jejuni Infection

Abstract

Despite the importance of gut microbiota for broiler performance and health little is known about the composition of this ecosystem, its development and response towards bacterial infections. Therefore, the current study was conducted to address the composition and structure of the microbial community in broiler chickens in a longitudinal study from day 1 to day 28 of age in the gut content and on the mucosa. Additionally, the consequences of a Campylobacter (C.) jejuni infection on the microbial community were assessed. The composition of the gut microbiota was analyzed with 16S rRNA gene targeted Illumina MiSeq sequencing. Sequencing of 130 samples yielded 51,825,306 quality-controlled sequences, which clustered into 8285 operational taxonomic units (OTUs; 0.03 distance level) representing 24 phyla. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Tenericutes were the main components of the gut microbiota, with Proteobacteria and Firmicutes being the most abundant phyla (between 95.0 and 99.7% of all sequences) at all gut sites. Microbial communities changed in an age-dependent manner. Whereas, young birds had more Proteobacteria, Firmicutes, and Tenericutes dominated in older birds (>14 days old). In addition, 28 day old birds had more diverse bacterial communities than young birds. Furthermore, numerous significant differences in microbial profiles between the mucosa and luminal content of the small and large intestine were detected, with some species being strongly associated with the mucosa whereas others remained within the luminal content of the gut. Following oral infection of 14 day old broiler chickens with 1 × 10⁸ CFU of C. jejuni NCTC 12744, it was found that C. jejuni heavily colonized throughout the small and large intestine. Moreover, C. jejuni colonization was associated with an alteration of the gut microbiota with infected birds having a significantly lower abundance of Escherichia (E.) coli at different gut sites. On the contrary, the level of Clostridium spp. was higher in infected birds compared with birds from the negative controls. In conclusion, the obtained results demonstrate how the bacterial microbiome composition changed within the early life of broiler chickens in the gut lumen and on the mucosal surface. Furthermore, our findings confirmed that the Campylobacter carrier state in chicken is characterized by multiple changes in the intestinal ecology within the host.

Keywords: 16S rRNA gene; Campylobacter jejuni; MiSeq sequencing; age; broiler chickens; luminal content; microbiota; mucosa.

Figure 1

Relative abundances (%) of the most abundant phyla in the infected birds compared with the controls at the two sampling points post infection of (A) jejunum and (B) cecum. Data are presented as the mean values and standard deviation (SD). JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content; control (c); infected (i).

Figure 2

Heatmap showing the relative abundances (%) of the 50 most-abundant OTUs sorted by gut sites of the infected birds compared with the controls at the two sampling points post infection. The heat map integrates relative abundance of a given phylotype. Colour scaling is ranged from 0 to ≥ 25%. n.d, not detected.

Figure 3

Relative abundances (%) of the most relevant OTUs in the infected birds compared with the controls at the two sampling points post infection (A) OTUs 1 and 12, (B) OTUs 4 and 29. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content.

Figure 4

Species richness and diversity measures of the microbial community at all gut sites in the control (A), infected birds (B), and (C) species richness and diversity estimates for bacteria from cecum content of the infected birds compared with the controls. Left Y-axis for number of observed OTUs (Sobs), Chao 1 and ACE, and Right Y-axis for Shannon and Simpson. Significant differences were calculated with Kruskal-Wallis-tests and Mann-Whitney-tests, and significance was declared at P < 0.05. Data are presented as the mean values and SD. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content.

Figure 5

Microbial community similarity between all samples calculated with Bray-Curtis similarities, which displays the similarity results between the control and infected groups according to age and gut sites. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content.

Figure 6

Principal component analysis (PCA) was analyzed for the control (A) and infected birds (B). Orange (day 1), Red (day 7), Green (day 14), Blue (day 21), and Black (day 28). Each symbol indicates an individual bird. All PCA plots include the data of all samples; symbols not belonging to the sample group indicated in the header are displayed faded. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content.

Figure 7

Venn diagrams showing the shared OTUs between the control and infected birds at different gut sites at the two sampling points post infection (A) jejunum; (B) cecum. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content; control (c); infected (i); d, day.