Morphological and molecular characterization of Sarcocystis taeniata and Sarcocystis pilosa n. sp. from the sika deer (Cervus nippon) in Lithuania.

The diaphragm muscles of eight sika deer (Cervus nippon) bred in Lithuania were examined for Sarcocystis cysts. Two Sarcocystis species, Sarcocystis taeniata, which were previously reported in Canadian moose (Alces alces) and Argentinean red deer (Cervus elaphus), and Sarcocystis pilosa n. sp. were described using light microscopy (LM), transmission electron microscopy (TEM), 18S ribosomal DNA (rDNA), and subunit I of cytochrome c oxidase (cox1) sequences analysis. By LM, cysts of S. taeniata were 424.8 × 57.9 (200-837 × 30-100) µm in size and had a thin (up to 1 µm) and smooth cyst wall, while short ribbon-like protrusions arising from broadened cone-shaped bases were seen under TEM. Cysts of S. pilosa (by LM) were ribbon-shaped, measured 848.5 × 63.8 (350-1700 × 30-125) µm and had thin 7-8-µm long hair-like protrusions. By TEM, cyst wall was type 7a-like; protrusions arose from 0.3 µm wide dome-shaped base with minute indentations of the parasitophorous vacuolar membrane near it, the surface of protrusions seemed to be smooth, and the ground substance layer was thin (0.18-0.22 µm). The 18S rDNA, in contrast to the cox1, lacked variability to discriminate S. pilosa from closely related Sarcocystis hjorti from the red deer and moose. S. taeniata, but not S. pilosa, showed a considerable intraspecific variation in both genes analyzed. The phylogenetic analyses based on 18S rDNA and cox1 sequences suggest that canids are definitive hosts of both S. taeniata and S. pilosa. This paper represents the first identification of Sarcocystis species in the sika deer by morphological and molecular methods.

Molecular identification of Sarcocystis rileyi sporocysts in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in Lithuania.

Despite the fact that Sarcocystis rileyi is one of the earliest described species of the genus Sarcocystis forming macrocysts in ducks, the life cycle of this species is still unknown in Europe. Sarcocystis spp. oocysts/sporocysts were observed in faeces of four of 23 (17.4 %) and in small intestine mucosal scrapings of four of 20 (20.0 %) red foxes (Vulpes vulpes) and in small intestine mucosal scrapings of seven of 13 (53.8 %) raccoon dogs (Nyctereutes procyonoides) hunted in Lithuania. A very small number of Sarcocystis sporocysts measuring 11.9 × 8.3 µm (n = 5) was found in faecal samples, whereas considerably more sporulated Sarcocystis oocysts and free sporocysts were detected in the small intestines of red foxes and raccoon dogs. These sporocysts measured 12.9 × 8.1 µm (n = 16) and 12.1 × 8.1 µm (n = 54) in red foxes and raccoon dogs, respectively. Using species-specific PCR and subsequent sequencing, internal transcribed spacer 1 (ITS-1) region partial sequences of oocysts/sporocysts from small intestine mucosal scrapings of six raccoon dogs and three red foxes were identified as belonging to S. rileyi. The present study provides strong evidence showing that the red fox and the raccoon dog can serve as final hosts of S. rileyi in Europe; however, transmission experiments are needed for the ultimate approval.

Description of Sarcocystis lari sp. n. (Apicomplexa: Sarcocystidae) from the great black-backed gull, Larus marinus (Charadriiformes: Laridae), on the basis of cyst morphology and molecular data.

A morphological type of Sarcocystis cysts found in one of two examined great black-backed gull, Larus marinus (Linnaeus) (Laridae), is considered to represent a new species for which the name Sarcocystis lari sp. n. is proposed and its description is provided. The cysts are ribbon-shaped, very long (the largest fragment found was 6 mm long) and relatively narrow (up to 75 microm). Under a light microscope the cyst wall reaches up to 1 microm and seems to be smooth. Using a computerized image analysis system, knolls, which resemble protrusions on the wall surface, are visible. Lancet-shaped cystozoites measure in average 6.9 x 1.4 microm (range 6.3-7.9 microm x 1.2-1.5 microm) in length. Observed using Transmission electron microscopy (TEM), the cyst wall is wavy and measures up to 1.2 microm in thickness. The parasitophorous vacuolar membrane has regularly arranged small invaginations. Cyst content is divided into large chambers by septa. Sarcocystis lari sp. n. has type-1 tissue cyst wall and is morphologically indistinguishable from other bird Sarcocystis species characterized by the same type of the wall. On the basis of 18S rRNA gene, 28S rRNA gene and ITS-1 region sequences, S. lari is a genetically distinct species, being most closely related to avian Sarcocystis species whose definitive hosts are predatory birds.

Molecular and morphological investigations of Sarcocystis corvusi sp. nov. from the jackdaw (Corvus monedula).

One type of sarcocyst was found in two of eight investigated jackdaws (Corvus monedula) and proposed as Sarcocystis corvusi sp. nov. By light microscope, cysts resembled a thick thread and were very long (the largest fragment found amounted to 6 mm) and relatively thin (up to 60 µm). The cyst wall measured <1 µm and seemed smooth. Using a computerized image analysis system, knolls, which resembled protrusions, were visible on the wall surface. Ultrastructurally, the cyst wall was wavy and reached up to 1.1 µm. The waves were of different heights and resembled low protrusions. The parasitophorous vacuolar membrane had many invaginations. Lancet- or orange segment-shaped cystozoites were 5.9-7.3 µm long. These sarcocysts had type-1 tissue cyst wall. According to 18S rRNA, 28S rRNA genes and ITS-1 region sequences, it was shown that S. corvusi is a genetically separate species. On the basis of these genetic markers, S. corvusi was most closely related to S. columbae, S. calchasi and S. wobeseri which parasitize birds and are characterized by the same type of sarcocyst wall.

Description of Sarcocystis anasi sp. nov. and Sarcocystis albifronsi sp. nov. in birds of the order Anseriformes.

On the basis of the already published morphological, 18S rDNA, 28S rDNA data (Kutkiene et al., Parasitol Res 99:562-565, 2006; Parasitol Res 102:691-696, 2008; Parasitol Res 104:329-336, 2009), and ITS-1 region investigation results of sarcocysts presented in this paper, Sarcocystis albifronsi sp. nov. from the white-fronted goose (Anser albifrons) and Sarcocystis anasi sp. nov. from the mallard duck (Anas platyrhynchos) are described.

Sarcocystis sp. from the herring gull (Larus argentatus) identity to Sarcocystis wobeseri based on cyst morphology and DNA results.

Having studied 11 herring gulls (Larus argentatus) Sarcocystis cysts were found in neck and leg muscles of 4 birds. One type of sarcocysts (cyst type I) that have a thin (∼1.0 µm), smooth, or slightly wavy cyst wall without clearly visible protrusions and small (6.0-8.0 µm) lancet- or banana-shaped cystozoites was identified by the light microscopy. Sarcocysts extracted from one herring gull were used for electron microscopy and DNA analysis. Ultrastructurally, Sarcocystis sp. from the herring gull had the same tissue cyst wall type-1 as S. calchasi, S. columbae, and S. wobeseri parasitizing in birds. According to first internal transcribed spacer (ITS-1) region, 18S rRNA and 28S rRNA gene sequences, Sarcocystis sp. from the herring gull belongs to S. wobeseri. Nevertheless, without evidences of cross-transmission experiment sarcocysts extracted from herring gull at present time are named as S. wobeseri-like.

Identification of Sarcocystis rileyi from the mallard duck (Anas platyrhynchos) in Europe: cyst morphology and results of DNA analysis.

Macroscopic cysts of Sarcocystis in ducks were recorded in Europe, but they were not investigated in more detail. Results of light and electron microscopy as well as 18S rDNA, 28S rDNA and ITS-1 region sequences of Sarcocystis macrocysts isolated from naturally infected mallard duck (Anas platyrhynchos) from Lithuania are presented in this paper. According to ultrastructure results, macrocysts examined corresponds to S. rileyi. Phylogenetic investigation showed S. rileyi to be the most closely related to two unnamed Sarcocystis species from anseriforms and to the S. mucosa. This is the first well-documented case of S. rileyi in Europe.

The mallard duck (Anas platyrhynchos) as intermediate host for Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis).

Morphometric and DNA investigation results of Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis) and Sarcocystis sp. (cyst type IV) from the mallard duck (Anas platyrhynchos) are presented. No significant morphometric differences between the investigated Sarcocystis species were found. ITS-1, 18S rRNA, and 28S rRNA gene sequences of these species showed 100% identity. The conclusion is drawn that it is one and the same Sarcocystis species in different intermediate hosts.

Sarcocystis in the birds family Corvidae with description of Sarcocystis cornixi sp. nov. from the hooded crow (Corvus cornix).

Having studied 67 birds of six species of the family Corvidae, Sarcocystis cysts were found in 16 (23.9%) individuals belonging to three species. The highest prevalence of infection (35.9%) was determined in the hooded crow (Corvus cornix). Two types of sarcocysts, which were temporarily called cysts type I and type V, were determined in the corvids examined. By light microscope, type I cyst wall seemed to be thin (< 1.0 microm) and smooth. Banana shaped cystozoites measured 6.0-8.0 microm in length. By light microscope, type V cyst wall seemed striated and reached up to 2.5 microm. Banana shaped cystozoites measured 6.1-7.9 x 1.4-1.8 microm. Ultrastructurally, the cyst wall amounted to 2.1 microm and had stump-like protrusions that differed greatly in size and shape. The parasitophorous vacuolar membrane had indentations and clearly visible (up to 0.2 microm in length) microprojections, which also differed considerably in size and shape. The ultrastructure of type V cyst wall differed from all those Sarcocystis spp. described thus far. On the basis of this-Sarcocystis cornixi sp. nov.-is proposed for this type of sarcocysts. Partial sequences of 18S rRNA and 28S rRNA genes were determined for this species and a phylogenetic analysis of the Sarcocystidae family was performed. In the phylogenetic tree, S. cornixi is grouped together with Frenkelia microti, F. glareoli, S. muris, S. neurona and the unnamed Sarcocystis species whose intermediate hosts are birds. S. cornixi is the most closely related to Sarcocystis sp. (cyst type I) from the white-fronted geese.

Sarcocystis sp. from the goldeneye (Bucephala clangula) and the mallard (Anas platyrhynchos): cyst morphology and ribosomal DNA analysis.

By light microscopy, cysts of Sarcocystis sp. (cyst type I) from the goldeneye (Bucephala clangula) seemed filamentous with a smooth and thin (<1 microm) cyst wall. Ultrastructurally, the cyst wall surface was irregular with minute undulations of the primary cyst wall. These sarcocysts had type-1 cyst wall. Cystozoites were banana-shaped and measured 7.0-8.5 microm in length. By light microscopy, cysts of Sarcocystis sp. (cyst type II) from the mallard (Anas platyrhynchos) were ribbon-shaped, very long, and thin. On the surface of the wall (up to 1.5 microm), they had palisade-like villar protrusions closely crowded together. Electron micrographs showed villar protrusions (up to 1.3 microm in length) different in size and shape. The latter had short microprojections especially obvious in the oblique sections. Cystozoites were slightly bent with blunt ends, broader at one end, and measured 13.0-16.1 x 1.8-2.5 microm. Phylogenetic analysis based on the comparison of partial 28S rRNR gene sequences of Sarcocystis sp. (cyst type II) derived from the mallard, Sarcocystis sp. (cyst type I) and Sarcocystis sp. (cyst type III) both derived from the white-fronted goose (Anser albifrons) suggested that these sequences belonged to separate Sarcocystis species.

Sarcocystis spp. in birds of the order Anseriformes.

Having studied 342 birds of 20 species of the order Anseriformes, we found Sarcocystis cysts in 100 individuals (29.2+/-2.5%) belonging to 15 species. One macrocyst and four microcysts types have been determined. By means of light microscopy, the morphology of cyst walls and merozoites have been examined and morphometric data are presented. According to morphological features, macrocysts correspond to the S. rileyi species.