bDigital dermatitis (DD) is an important cause of lameness in dairy cattle worldwide. It has now been reported in beef cattle and also sheep (contagious ovine digital dermatitis [CODD]). Three Treponema phylogroups are consistently isolated from lesions, Treponema medium-like, Treponema phagedenis-like, and Treponema pedis. The gastrointestinal (GI) tract and feces are suggested sites of treponemal infection in dairy cattle; however, isolation of DD-associated treponemes from these areas has previously failed. This study surveyed gingival tissues, rectal tissues, and feces of beef cattle and sheep for the molecular presence (PCR) and isolation of the three cultivable DD-treponeme phylogroups. Of the sheep gingival (n ‫؍‬ 40) and rectal (n ‫؍‬ 40) tissues, 1/40 gingival tissues was positive for DD-associated treponemes (T. pedis), as were 3/40 rectal tissues (one containing T. medium-like and two containing T. pedis). No DD-associated treponeme DNA was amplified from beef cattle rectal tissues (n ‫؍‬ 40); however, 4/40 beef gingival tissues were positive for DD-associated treponemes (all containing T. phagedenis-like). A T. phagedenis-like DD-associated treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n ‫؍‬ 41) and sheep (n ‫؍‬ 79) feces failed to amplify DD-associated Treponema DNA. Twenty-two treponemes were isolated from sheep feces; however, upon phylogenetic analysis, these clustered with the considered nonpathogenic treponemes. This study detected DD-associated treponemes in the GI tract tissues of sheep and beef cattle and successfully isolated a DD-associated treponeme from ruminant rectal tissue. This gives evidence that the GI tract is an important infection reservoir of DD-associated treponemes in multiple DD-infected species. Digital dermatitis (DD) is an ulcerative lesion of the digital skin (1), causing severe lameness in dairy cattle worldwide. The disease was first reported in dairy cattle in 1974, where it is known as bovine digital dermatitis (BDD) (1, 2). The disease more recently has been reported in sheep (3, 4), where it is known as contagious ovine digital dermatitis (CODD), and now has been confirmed in beef cattle (5, 6).Over the last few decades, DD has been recognized as an important cause of bovine lameness (7) with serious animal welfare and economic implications, including reduced fertility and milk yield and an increase in the risk of culling (8-10). DD is now a worldwide problem, and controlling the disease on dairy operations has proven difficult due to there being no single effective treatment available. Furthermore, there are no data on treatment of DD in beef cattle, and treatment of CODD is difficult and rarely effective.The primary causative agents of DD are considered to be the spirochetal bacteria, treponemes, with a polytreponemal etiology (11-13). Cloning bacterial 16S rRNA genes indicated five phylogroups of treponemes in BDD lesions from Germany (14). Three Treponema phylogroups have been consistently isolated from dairy cattle les...