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Besnoitia besnoiti and B. caprae, which infect bovids (cattle and antelopes) and goats, respectively, are responsible for besnoitiosis, a chronic and debilitating disease. Bovine besnoitiosis is considered to be a reemerging disease in Central and Western Europe. In addition, infection by Besnoitia spp. has been reported in reindeer from Sweden and Finland. Recently, the parasite was also detected in roe deer and red deer from Spain, where an interconnection between the domestic and sylvatic cycles of B. besnoiti has been presumed. In contrast, caprine besnoitiosis seems to be enzootic to Kenya and Iran. The presence of Besnoitia spp. in small domestic ruminants has never been explored in Europe, and the role that these species might play in the epidemiology of bovine besnoitiosis, as intermediate hosts or reservoirs of B. besnoiti, remains unknown. Herein, the first serosurvey conducted in European sheep and goats from areas in Spain where bovine besnoitiosis is endemic is described. Convenience sampling was conducted of 1943 sheep and 342 goats close to cattle from the Pyrenees and Central Spain that were infected with endemic Besnoitia spp. Serum samples were first analyzed by ELISA and then by confirmatory Western blot. Specific antibodies were not found in any sampled animal. Thus, sheep are unlikely to play a role in the epidemiology of bovine besnoitiosis, at least in the sampled areas. A larger serosurvey is necessary to determine whether goats might be a putative reservoir. To confirm the results of this study, sheep and goats should be further studied in other European countries and regions where their numbers are high and where bovine besnoitiosis is spreading. [ABSTRACT FROM AUTHOR]