Morphological and molecular characteristics of seven species from sika deer () in Japan, including three new species.

Samples of diaphragm were collected from 53 sika deer from Gifu Prefecture, Japan; 220 sarcocysts were isolated, examined in wet mounts and classified according to their cyst wall protrusions. The sarcocysts were then examined molecularly in order to assign them to different species. All but 11 of the 220 sarcocysts were initially identified by means of a multiplex PCR assay targeting of five species, whereas the remaining 11 sarcocysts were identified by standard PCR and sequencing. DNA from selected sarcocysts was used for PCR amplification and sequencing of (59 sequences) and 18S rDNA (23 sequences). The 220 sarcocysts comprised seven major sequence types or species. Types 4 and 7 were assigned to the known species and , whereas types 1, 3 and 5 were considered to represent three new species, for which the names and have been proposed. Types 2 and 6 were most similar to and , respectively, but could not be unequivocally assigned to these species. Sarcocysts belonging to were macroscopic with fairly thick finger-like protrusions, whereas most sarcocysts of the six other species were microscopic. Sarcocysts of cf. and were spindle-shaped and possessed thin finger-like cyst-wall protrusions. Sarcocysts of and had similar hair-like protrusions, whereas those of cf. had a smooth surface. Sarcocysts of , , cf. , , , cf. and were found in 50 (94.3%), 29 (54.7%), 22 (41.5%), 10 (18.9%), 8 (15.1%), 6 (11.3%) and 1 (1.9%) of the 53 sika deer examined, respectively. An improved multiplex PCR assay targeting was developed, through which the seven spp. found in the present study could be identified.