Morphological and molecular characteristics of seven Sarcocystis species from sika deer (Cervus nippon centralis) in Japan, including three new species

Niichiro Abe; Kayoko Matsuo; Junji Moribe; Yasuhiro Takashima; Takao Irie; Takashi Baba; Bjørn Gjerde

doi:10.1016/j.ijppaw.2019.10.002

Morphological and molecular characteristics of seven Sarcocystis species from sika deer (Cervus nippon centralis) in Japan, including three new species

Int J Parasitol Parasites Wildl. 2019 Oct 9:10:252-262. doi: 10.1016/j.ijppaw.2019.10.002. eCollection 2019 Dec.

Authors

Niichiro Abe¹, Kayoko Matsuo^{2

3}, Junji Moribe⁴, Yasuhiro Takashima^{3

5}, Takao Irie⁶, Takashi Baba¹, Bjørn Gjerde⁷

Affiliations

¹ Division of Microbiology, Osaka Institute of Public Health, 8-34 Tojo-cho, Tennoji-ku, Osaka, 543-0026, Japan.
² Hida Regional Livestock Hygiene Service Center, 7-468 Kamiokamoto-machi, Takayama, Gifu, 506-8688, Japan.
³ Department of Veterinary Parasitological Diseases, Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
⁴ Research Center for Wildlife Management, Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
⁵ Center for Highly Advanced Integration of Nano and Life Science, Gifu University (G-CHAIN), 1-1 Yanagido, Gifu, 501-1193, Japan.
⁶ Department of Infectious Diseases, Hokkaido Institute of Public Health, North 19, West 12, Kita-ku, Sapporo, Hokkaido, 060-0819, Japan.
⁷ Faculty of Veterinary Medicine, Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, P.O. Box 369 Sentrum, 0102, Oslo, Norway.

Abstract

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 cox1 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 cox1 (59 sequences) and 18S rDNA (23 sequences). The 220 sarcocysts comprised seven major cox1 sequence types or species. Types 4 and 7 were assigned to the known species Sarcocystis pilosa and Sarcocystis ovalis, whereas types 1, 3 and 5 were considered to represent three new species, for which the names Sarcocystis japonica, Sarcocystis matsuoae and Sarcocystis gjerdei have been proposed. Types 2 and 6 were most similar to Sarcocystis tarandi and Sarcocystis taeniata, respectively, but could not be unequivocally assigned to these species. Sarcocysts belonging to S. japonica were macroscopic with fairly thick finger-like protrusions, whereas most sarcocysts of the six other species were microscopic. Sarcocysts of S. cf. tarandi and S. matsuoae were spindle-shaped and possessed thin finger-like cyst-wall protrusions. Sarcocysts of S. pilosa and S. gjerdei had similar hair-like protrusions, whereas those of S. cf. taeniata had a smooth surface. Sarcocysts of S. japonica, S. pilosa, S. cf. tarandi, S. gjerdei, S. matsuoae, S. cf. taeniata and S. ovalis 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 cox1 was developed, through which the seven Sarcocystis spp. found in the present study could be identified.

Keywords: 18S ribosomal RNA gene; Cervus nippon centralis; Cytochrome c oxidase subunit I gene; Japan; Sarcocystis gjerdei; Sarcocystis japonica; Sarcocystis matsuoae.