Morphological and molecular characterization and phylogenetic placement of Sarcocystis capreolicanis and Sarcocystis silva n. sp from roe deer (Capreolus capreolus) in Norway

Sarcocysts were isolated from the muscle tissue of three roe deer from southeastern Norway and examined by light microscopy, scanning electron microscopy and/or sequencing of the small subunit ribosomal RNA (ssu rRNA) gene. By light microscopy, four sarcocyst types were found, including those of Sarcocystis gracilis and Sarcocystis oviformis, which had been characterized previously. The third cyst type had about 10 mu m long, flexible, hair-like surface protrusions, consistent with those of Sarcocystis capreolicanis, and differed genetically from other known species. The name S. capreolicanis was therefore assigned to this sequence type. The fourth cyst type had densely packed, upright, finger-like surface protrusions, about 8 mu m long, and was morphologically similar to an unnamed Sarcocystis sp. reported from roe deer in other countries, and identical at the ssu rRNA gene to Sarcocystis sp. Type D previously reported from moose. This species was assigned the new name Sarcocystis silva. Both S. capreolicanis and S. silva displayed considerable intraspecific variation at the ssu rRNA gene. In phylogenetic analyses based on ssu rRNA gene sequences, S. capreolicanis grouped together with other canine-transmitted Sarcocystis species, whereas S. silva was most closely related to Sarcocystis rangiferi and Sarcocystis tarandi of reindeer. Roe deer muscles containing numerous cysts of S. gracilis were fed to a silver fox (Vulpes vulpes) and a blue fox (Vulpes lagopus), both of which started shedding Sarcocystis sporocysts in their faeces 9 days later, and harboured numerous oocysts, measuring about 20 x 15 mu m, in their intestinal mucosa upon euthanasia 14 days post-inoculation. DNA derived from these oocysts was amplified and sequenced at the ssu rRNA gene and belonged to S. gracilis, confirming for the first time by molecular methods that foxes are definitive hosts for this species.