Cecal microbiome profile of Hawaiian feral chickens and pasture-raised broiler (commercial) chickens determined using 16S rRNA amplicon sequencing

Abstract

This study investigated the taxonomic profile and abundance distribution of the bacterial community in the ceca of feral and pasture-raised broiler (commercial) chickens. Cecal content from feral and commercial chickens (n = 7 each) was collected, and total DNA was isolated. Next-Generation Sequencing (Illumina MiSeq) was performed to characterize the cecal microbiota. Specific bacteria explored were: Bacteroides, Bifidobacterium, Lactobacillus, Enterococcus, Escherichia, and Clostridium. At the phylum level, 92% of the bacteria belonged to Firmicutes, Bacteroidetes, and Proteobacteria for both feral and commercial chickens. The proportional abundance of Firmicutes was 55.3% and 63.3%, Bacteroidetes was 32.5% and 24.4%, and Proteobacteria was 7.0% and 5.9% in the feral and commercial chickens, respectively. The alpha-diversity Shannon index (P = 0.017) and Simpson index (P = 0.038) were significantly higher for commercial than for feral chickens. Predictive functional profiling by PICRUSt showed enriched microbial metabolic pathways for L-proline biosynthesis in the feral group (P < 0.01). There were a greater percentage of specific bacteria in the feral than commercial chickens, albeit with lower diversity but a more functional microbiota. In conclusion, feral birds have distinguished microbial communities, and further microbiome analysis is mandated to know the specific functional role of individual microbiota. The difference in microbiota level between feral and commercial birds could be accounted to the scavenging nature, diverse feed ingredients, and distinct rearing localities.

Keywords: Next-Generation Sequencing; broiler chickens; cecal microbiome; feral chicken; pasture-raised chicken; qPCR.

Figure 1

Alpha diversity: Shannon Index and Simpson Index for commercial and feral chicken samples. It illustrates the differences in species diversity within feral and commercial samples.

Figure 2

Beta diversity: Bray-Curtis distance based between community diversity analysis for commercial and feral chicken samples.

Figure 3

Microbial relative abundance at the genus level for both commercial and feral chicken samples identified from amplicon high-throughput sequencing results.

Figure 4

Microbial relative abundance at the family level for both commercial and feral chicken samples identified from amplicon high-throughput sequencing results.

Figure 5

Relative abundance of bacteria in commercial chickens at the phylum level analyzed from the amplicon high-throughput sequencing results and GreenGenes database. It shows that the most abundant bacteria at the phylum level in both groups are Firmicutes, Bacteroidetes, and Proteobacteria.

Figure 6

Left panel, the heatmap showing significant differences between bacteria in commercial and feral chicken groups at the genus level. Right panel, box plot quantified the significant difference genera between the commercial and feral chicken groups. “*” represent the significance at P < 0.05; “***” represent the significance at P < 0.001.

Figure 7

Left panel, the heatmap showing significant differences between bacteria in commercial and feral chicken groups at the phylum level. Right panel, box plot quantified the significant difference phylum between the commercial and feral chicken groups. “*” represent the significance at P < 0.05; “***” represent the significance at P < 0.001.

Figure 8

Histogram of the LDA scores computed for genera differentially abundant between commercial and feral chickens. Genera enriched in feral chickens are indicated with a positive LDA score, and genera enriched in commercial chickens have a negative score. The LDA score (>2 considered) indicates the effect size and ranking of each differentially abundant taxon.

Figure 9

Significant enriched microbial metabolic pathways between the feral chicken and the commercial chicken groups.

Figure 10

Relative abundance of selected bacteria for feral and commercial chickens by normalized qPCR results. The asterisk * signifies areas of statistical significance (P < 0.01).