Morphological and molecular characterization of four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy

Abstract

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n = 24) or to a Sarcocystis taeniata-like species (n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n = 27) and Sarcocystis capreolicanis (n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2 μm long, 0.3-0.4 μm wide and about 0.02-0.03 μm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4 μm wide and 0.7-0.9 μm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the boar...

Molecular identification of Sarcocystis halieti n. sp., Sarcocystis lari and Sarcocystis truncata in the intestine of a white-tailed sea eagle (Haliaeetus albicilla) in Norway

“…Thus, reverse primer SR8D had a complete match against cox1 of S. truncata , but a fairly poor match against sequences of the other species found to be present. However, since primer SR8D tends to perform poorly with forward primer SF1 (probably due to primer-dimer formation), forward primer SF4 was used instead ( Gjerde et al, 2017a ). Moreover, primer SF4 had a better match against sequences of S. truncata than against those of the other species present.…”

Molecular identification of sarcocysts from tissue of fallow deer (Dama dama) farmed in the open pasture system based on ssu rRNA gene

“…The definitive hosts of S. morae and S. linearis are unclear. However, phylogenetic data indicate canids as most likely definitive host of these two species [18,28]. It is possible that some Sarcocystis species in red deer may use other cervids as their principal intermediate hosts, and this may also indicate that many species of Sarcocystis are not as specific to their intermediate host as once believed.…”

“…Lately, S. morae was reported in fallow deer as a new host in Spain by De las Cuevas et al [19]. Sarcocystis gracilis is considered to be parasites of roe deer (Capreolus capreolus) [28] while S. morae occur in red deer (Cervus elaphus) [8] and fallow deer (Dama dama) [19]. The Sarcocystis isolates obtained in the present study shared 96.58-100% identity with those taken previously from different tissues of various hosts from other geographical localities.…”

“…Although approximately 200 species of Sarcocystis are recognized, the definitive and intermediate hosts are known for only 62 of them [4]. In the case of S. gracilis, the definitive hosts are known to be dogs, red foxes (Vulpes vulpes) or blue foxes (V. lagopus); the parasite must be consumed by these to complete its life cycle [4,28]. Domestic dogs, wild dogs and red foxes (Vulpes vulpes) are also very common in the region covered by the present study, and these may also serve as definitive hosts; foxes, in particular, have unlimited access to farm animals, unlike dogs.…”

Molecular phylogeny of Sarcocystis fayeri (Apicomplexa: Sarcocystidae) from the domestic horse Equus caballus based on 18S rRNA gene sequences and its prevalence

“…2015; Gjerde and Josefsen 2015; Gjerde et al . 2017; Rudaitytė‐Lukošienė et al . 2018; Huang et al .…”