**Background:** Emus (Dromaius novaehollandiae) are large flightless omnivorous ratites farmed for fat and meat, with emu oil used therapeutically and cosmetically. Despite their ability to digest plant fiber and their seasonal dietary intake and fat deposition patterns, little is known about their gastrointestinal microbiota, particularly in the small intestine. Most avian microbiota studies focus on the ceca due to high bacterial diversity, and few have examined regional variation along the small intestine. This study aimed to characterize bacterial communities and predict metabolic functions in the duodenum, jejunum, and ileum of emus and compare them with the ceca.
**Methods:** Gut content samples were collected from four adult emus (2 males, 2 females; 5–6 years old) at TryHarder Farm (Saskatchewan, Canada) in early November, just prior to breeding season. Birds were free-ranged but supplemented with a barley-alfalfa-canola based diet. DNA was extracted using PowerMax Soil DNA Isolation Kit. The V3-V5 region of the 16S rRNA gene was amplified with primers 341F and 926R and sequenced using the Roche 454 Junior system. Sequence processing used QIIME 1.8.0 with quality trimming (mean quality score <25, length <150 or >900 bp removed), denoising via DENOISER v. 0.9.1, and chimera removal via ChimeraSlayer. OTUs were clustered at 97% similarity using UCLUST. Taxonomy was assigned using RDP classifier 2.0.1. PICRUSt was used to predict functional genes via closed-reference OTU mapping against Greengenes, with KEGG pathway categorization. Statistical comparisons used Wilcoxon Test and Welch's t-test.
**Key Results:** After quality filtering, 165,585 sequence reads were obtained from the three SI segments (average 41,396 ± 3,266 seqs/bird). A total of 701 OTUs were identified across SI segments. Firmicutes (14–99%) and Proteobacteria (0.5–76%) were the predominant phyla in the small intestine. The duodenum yielded 52,880 sequences classified into 343 OTUs (125.0 ± 32.6 OTUs/bird), with 13 core OTUs accounting for 74.1% of reads. Turicibacter (Firmicutes) accounted for 31.7% of duodenal reads. The jejunum yielded 61,139 sequences classified into 219 OTUs (75.3 ± 21.3 OTUs/bird), with only 2 core OTUs (Escherichia and Sinobacteraceae) common to all 4 birds. The ileum yielded 51,567 sequences classified into 438 OTUs (145.3 ± 48.7 OTUs/bird), with 14 core OTUs accounting for 59.7% of reads. Only 2 OTUs (Escherichia and Sinobacteraceae) were core along the entire small intestine. Chao1 richness estimates were: duodenum 150 ± 77, jejunum 164 ± 106, ileum 91 ± 50, and ceca 624 ± 170. Cecal richness was significantly higher than all SI segments (P = 0.0011 overall; ceca vs duodenum P = 0.01429; ceca vs ileum P = 0.0286; ceca vs jejunum P < 0.01429). Shannon and Simpson indices showed no significant differences among segments. Sex differences were observed: 18 OTUs were found only in female SI contents and 59 OTUs only in male SI contents, though sample size precluded statistical comparison. PICRUSt analysis revealed that the jejunum microbiome was enriched in genes for detoxification pathways including chloroalkane/chloroalkene degradation, styrene degradation, dioxin degradation, xylene degradation, and benzoate degradation. The cecal microbiome was enriched in genes for immune-related functions including antigen processing and presentation, NOD-like receptor signaling, lipopolysaccharide biosynthesis, secondary bile acid biosynthesis, and multiple antibiotic biosynthesis pathways (streptomycin, vancomycin, butirosin/neomycin). The ceca also showed enrichment for fermentation-related functions including carbohydrate and protein digestion/absorption, energy metabolism, nitrogen metabolism, and methane metabolism.
**Clinical Implications:** This study provides the first characterization of emu small intestinal microbiota across different segments using direct gut samples. The findings suggest that the small intestine, particularly the jejunum, plays a key role in detoxification of plant materials and environmental chemicals, which is relevant for free-ranging birds exposed to diverse forage. The cecal microbiome appears specialized for immune defense and pathogen resistance, with enrichment in genes for antigen processing, antibiotic biosynthesis, and glycan degradation. The jejunum and ceca are identified as primary sites for microbial digestion and fermentation. These results establish a baseline for investigating how seasonal and physiological changes in gut microbiota influence emu nutrition and, indirectly, the fatty acid composition of emu fat used in therapeutic and cosmetic products. The high individual variation in SI microbiota (low core OTU numbers) suggests that fecal samples would not adequately represent SI communities, supporting the need for direct gut sampling in future studies.