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The initial isolation of Helcococcus ovis from a valvular thrombus prompted us to investigate the prevalence of this bacterium in bovine valvular endocarditis. Specimens from 55 affected hearts were examined by culture using Columbia blood agar and cross streaking the inoculated plate with a Staphylococcus aureus strain. As confirmed by 16S rRNA gene sequencing, H. ovis was isolated with an unexpectedly high frequency of 33%, predominantly as heavy growth and pure culture. The majority of H. ovis isolates showed distinct satellitism around S. aureus and pyridoxal dependency, resembling "nutritionally variant streptococci" (now assigned to the genera Abiotrophia and Granulicatella). Using the API rapid ID 32 Strep, API ZYM, and Rosco Diatabs systems, incongruent results were obtained for alkaline phosphatase, beta-galactosidase, beta-glucuronidase, and leucine aminopeptidase activities. Based on the satellitism/pyridoxal dependency; hemolysis on blood agar; the API rapid ID 32 Strep results for arginine dihydrolase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, and pyroglutamic acid arylamidase activities; hippurate hydrolysis; and acidification of sucrose, a scheme for the identification of H. ovis and its differentiation from other members of the Helcococcus genus and the pyridoxal-dependent species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans is proposed. By establishing specific fluorescence in situ hybridization, large H. ovis aggregates were specifically detected within the fibrinous exudate of the valvular thrombi. Our results demonstrate for the first time that H. ovis represents an emerging pathogen in bovine valvular endocarditis that is frequently isolated if appropriate culture conditions are used.