bModern pyrosequencing technology allows for a more comprehensive approach than traditional Sanger sequencing for elucidating the etiology of bovine digital dermatitis. We sought to describe the composition and diversity of treponemes in digital dermatitis lesions by using deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with species-level taxonomic identification. Treponema-specific 16S rRNA gene PCRs and pyrosequencing were performed on biopsy specimens originating from 10 different Catalan dairy herds (n ‫؍‬ 36) with digital dermatitis, and this analysis yielded 75,297 sequences. We identified 20 different taxa, including a potentially novel phylotype that displayed 95% sequence identity to members of the Treponema denticola/Treponema pedis-like cluster. Species frequencies and abundances that were determined by pyrosequencing analysis were highly correlated with the results of fluorescent in situ hybridization using phylotype-specific oligonucleotide probes. In a limited number of animals from a single geographic region, we detected most of the Treponema phylotypes that were described in previous investigations of digital dermatitis. Additionally, we identified a number of phylotypes that mapped to oral treponemes of humans and dogs that had not been reported for digital dermatitis lesions. The results presented here support previous observations of a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/ Treponema vincentii-like, and T. denticola/T. pedis-like phylotypes being highly associated with disease. Using this new approach, it has become feasible to study large herds and their surrounding environments, which might provide a basis for a better understanding of the pathogenesis of this disease. Bovine digital dermatitis (DD), which causes lameness and wasting in cattle, is the most serious foot disorder of dairy cows from economic and welfare perspectives (1). The disease, which is characterized by focal proliferative to ulcerative dermatitis that is typically located on the plantar aspect of the foot, apparently is contagious (2). Currently, a bacterial etiology of DD is well documented (3, 4). Fluorescent in situ hybridization (FISH) analyses have shown that spirochetes of the genus Treponema are found mainly in the deeper parts of DD lesions, near the interface with healthy tissue (5, 6). Additionally, cows with DD demonstrate specific humoral and cell-mediated immune responses to Treponema (7). At least 18 different treponemal species have been identified by cloning of bacterial 16S rRNA genes PCR-amplified directly from DD samples (8-11). Previous works by us and others have suggested that DD has a symbiotic treponemal pathogenesis (9-12), and Treponema phagedenis-like, Treponema denticola-like, and Treponema medium-like bacteria appear to be among the most prevalent species in the lesions. The complex interplay between infecting Treponema species, their relative contributions to pathogenesis and infection reservoirs, and their...