The role of wild ruminants as reservoirs of Besnoitia besnoiti infection in cattle.

Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer (Cervus elaphus) and roe deer (Capreolus capreolus)) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer (n=309), roe deer (n=417), Pyrenean chamois (Rupicapra p. pyrenaica, n=383) and Iberian wild goat (Capra pyrenaica hispanica, n=288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer (n=820), roe deer (n=37), fallow deer (Dama dama, n=166), Iberian wild goat (n=86) and European mouflon (Ovis orientalis musimon, n=102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti, although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle.