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

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.

Graphical abstract

Fig. 1

Phylogenetic tree based on 322 partial sequences of cox1 of 61 taxa, including the seven Sarcocystis species (types 1–7) from this study and inferred using the neighbour-joining method and with evolutionary distances computed using the p-distance method. Bootstrap support (1000 replicates) is shown at each node. Subtrees formed by two or more haplotypes of the same species have been collapsed. The number of haplotypes included is given in parentheses. The number of sequences of each Sarcocystis species used in this analysis and their GenBank accession numbers are shown in Table S3.

Fig. 2

Light microscopic appearance of sarcocysts isolated from sika deer from Gifu Prefecture, Central Japan. a-d Thumb-like (a, b) and elongated finger-like (c, d) protrusions (P) of S. japonica. e, f Finger-like protrusions in S. cf. tarandi (e) and S. matsuoae (f). g, h Hair-like protrusions in S. pilosa (g) and S. gjerdei (h). i Indistinct protrusions on cyst S. cf. taeniata.j, k Oval sarcocyst of S. ovalis (j); cyst surrounded by fibrous layer (FL), making the slanting tongue-like protrusions (arrow) nearly invisible (k).