Host Ontogeny Structures Gut Microbiome Composition and Pathogen-Associated Bacterial Communities in the Endangered Ethiopian Wolf (Canis simensis)

The Ethiopian wolf ( Canis simensis ), the world's rarest canid and a highly specialized Afroalpine carnivore, faces increasing threats from habitat fragmentation, human encroachment, and infectious disease. Understanding gut microbiome variation in this endangered species is important for elucidating host–microbe interactions, pathogen ecology, and conservation risks at the wildlife–livestock–human interface. We used shotgun metagenomic sequencing to characterize the gut bacterial microbiome of 31 Ethiopian wolves from Bale Mountains National Park, Ethiopia, and evaluated age- and pack-associated variation in community composition and pathogen burden. Shannon alpha diversity did not differ significantly among juvenile, subadult, and adult wolves. In contrast, beta diversity analyses revealed significant age-associated differences in gut microbial community composition, whereas pack membership had little influence. Species- and genus-level analyses identified several nominal age-associated abundance trends, but no taxa remained significant after false discovery rate correction, indicating that age-related variation reflected diffuse community-wide restructuring rather than large shifts in individual bacterial taxa. Fourteen bacterial taxa with documented pathogenic potential, including several zoonotic species, were detected. Pathogen burden differed significantly among age classes, with adult wolves exhibiting a higher cumulative abundance of potentially pathogenic bacteria than subadults, while no significant differences were observed among packs. These findings demonstrate that host development is an important determinant of gut microbiome composition and pathogen burden in Ethiopian wolves and provide the first shotgun metagenomic baseline for this endangered species, supporting future conservation and One Health surveillance initiatives.