Bacterial community analysis of treponeme-associated hoof disease in free-ranging elk ( <i>Cervus canadensis</i> ): evidence for a polybacterial etiology with geographic consistency

Treponeme-associated hoof disease (TAHD) is an emerging infectious disease in free-ranging elk (<i>Cervus canadensis</i>) characterized by ulcerative and necrosuppurative pododermatitis with spirochete bacteria that leads to lameness and apparent increased mortality. While TAHD is hypothesized to have a polybacterial etiology, the causative agents remain poorly characterized, particularly across its geographic range. In this study, we developed a histologic categorization system for hoof lesions and employed 16S rRNA gene amplicon sequencing to characterize bacterial communities in samples from 129 free-ranging elk across regions with endemic or sporadic TAHD and where TAHD remains undetected. Differential abundance analysis revealed strong associations between TAHD-positive lesions and the bacterial genera <i>Treponema</i>, unidentified <i>Spirochaetaceae</i>, <i>Mycoplasma</i>, and <i>Fusobacterium</i>, along with their respective families and amplicon sequence variants. Many of these TAHD-associated operational taxonomic units (OTUs) were also more frequently detected at increased abundance in more severe, histologic lesions of pododermatitis. Correlation analysis demonstrated a strong positive association between <i>Treponema</i> and <i>Mycoplasma</i> in TAHD lesions, suggesting a more significant role of <i>Mycoplasma</i> in TAHD's etiology than previously recognized. Additionally, we identified novel TAHD-associated OTUs, including <i>Corynebacterium freneyi-xerosis</i>, that warrant further investigation. Comparative analysis of TAHD-positive lesions from endemic and sporadic areas revealed minimal differences in the microbial community. These findings advance our understanding of the bacterial contributors to TAHD, highlighting putative pathogens for further investigation and as potential targets for diagnostic development.IMPORTANCEWhile detection of <i>Treponema</i> is a hallmark of treponeme-associated hoof disease (TAHD), a comprehensive understanding of other bacterial contributors is necessary to improve diagnostic testing and inform control measures. Our study confirmed strong associations between <i>Spirochaetaceae</i> and TAHD lesions and revealed a previously underappreciated role of <i>Mycoplasma</i> in TAHD's etiology. <i>Treponema</i> and <i>Mycoplasma</i> were significantly enriched in TAHD-positive lesions, absent from TAHD-negative tissues, and strongly and positively correlated with each other, suggesting a potential synergistic relationship. By developing and applying a histologic categorization system, we characterized shifts in bacterial communities as lesion severity progressed. Comparisons of TAHD-positive lesions from endemic and sporadic regions revealed minimal differences in the microbial composition, indicating strong geographic consistency. These findings enhance our understanding of TAHD's etiology and provide a foundation for future research, including the development of improved diagnostic tests and targeted disease management strategies.