Introduction

The Sarcocystis genus has a two-host life cycle, with a sexual stage in the small intestine of the definitive host and an asexual stage in the extra-intestinal tissues, especially in the striated muscle of the intermediate host. Definitive hosts are carnivorous that become infected through the ingestion of tissues containing sarcocysts (Dubey et al. 2016). Definitive hosts can also serve as intermediate hosts for other Sarcocystis spp. harboring sarcocysts in their musculature (Kubo et al. 2009; Dubey et al. 2016).

Until now, sarcocysts of several species of Sarcocystis have been described from terrestrial carnivores (reviewed by Dubey et al. 2016), but none from the wolf. Here, we describe the ultrastructure and molecular characteristics of sarcocysts found in the Alaskan wolf (Canis lupus) for the first time.

Material and methods

In November 2015, serum samples and pieces of the tongue, leg muscle, heart, and encephalon were collected from a trapped adult male wolf (C. lupus) in interior Alaska (Game Management Unit 20B location), near Fairbanks (65.12-148.0). Animal was legally trapped for fur and portions donated for scientific sampling by the Alaska Department of Fish and Game. Samples were kept chilled until arriving to the Animal Parasitic Diseases Laboratory, USDA-ARS, in Beltsville, Maryland. Study animal was part of a programmed survey on Toxoplasma gondii in the wildlife from the USA.

Fresh tissues were examined for the presence of Sarcocystis cysts by squeezing small pieces between the glass slide and coverslip. A piece of each tissue was fixed in 10 % buffered formalin and processed for histopathology by hematoxylin and eosin (H&E) stain of 5-μm-thick sections. After collecting samples for histology, tissues were homogenized in blender and incubated in acid pepsin solution at 37 °C for 1 h to release bradyzoites from sarcocysts, similar to the procedure for T. gondii tissue cysts (Dubey 2010). After centrifugation, the sediment was re-suspended in ∼5 ml saline (0.85 % NaCl) and ∼25 μl of each digest were screened for the presence of bradyzoites at ×40 magnification using a light microscope.

Unlike in the tongue where five cysts were found, no cysts were detected in other tissues by fresh examination. Sarcocysts were mechanically removed from the tongue, of which three (cyst #2, #3, #4) were directly preserved in 20 μl saline solution for molecular analyses. Two sarcocysts (cyst #5 and half of cyst #1) were fixed in 2.5 % glutaraldehyde and processed for transmission electron microscopy (TEM) as described previously by Trupkiewicz et al. (2016).

Four excised sarcocysts (cyst #2, #3, #4, and half of cyst #1) were subjected to DNA extraction using DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. DNA quantification and quality were determined by Thermo Scientific NanoDrop Lite Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

The DNA was characterized by PCR amplification and sequencing of three regions of the nuclear ribosomal DNA unit, 18S rRNA, 28S rRNA, internal transcribed spacer-1 (ITS-1), and a mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. The almost complete regions of 18S rRNA and 28S rRNA were amplified using overlapping fragments and primer pairs ERIB1/S2r, S5f/S4r, S3f/Primer Bsarc, and KL1/LS2R, LS1F/KL3, respectively, as described previously by Gjerde and Josefsen (2015). Similarly, fragments of the ITS-1 and the cox1 loci were also amplified using primer pairs SU1F/5.8SR2, and SF1/SR5, respectively (Gjerde 2013, 2014; Gjerde and Josefsen 2015). The amplified PCR products were resolved on 2.5 % (w/v) agarose gel.

The single PCR amplicons of 18S rRNA, 28S rRNA, ITS-1, and cox1 were excised from the gel and purified using QIAquick Gel Extraction (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s recommendations. The purified PCR products were sent to Macrogen Corporation (Rockville, MD, USA) for direct sequencing using the same primer set used in PCR amplification to obtain both strand reads.

Generated forward and reverse sequences were visually inspected using the Chromas Lite version 2.1 sequence analysis program (http://chromaslite.software.informer.com/2.1/). True or ambiguous polymorphic sites and double peaks were carefully recorded and annotated. Sequence similarity searches with sequences deposited in the NCBI database were conducted using the BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Only good-quality readings were used to generate consensus sequences at each specific locus. Multiple alignment analyses with appropriate reference sequences obtained from GenBank were conducted by ClustalW in MEGA version 6.0 to determine Sarcocystis species. Phylogenetic trees were constructed based on the neighbor-joining method using the same software (Tamura et al. 2013).

Results

Light microscopy examination

By muscle squash, Sarcocystis spp. cysts were only found in tongue. Sarcocysts were slender, up to 900 μm long. Sarcocystis bradyzoites were also seen in the leg muscle digest.

After examination of H&E-stained sections, three spindle-shaped sarcocysts were found in sections of tongue (two cysts) and skeletal muscle (one cyst). They measured 77 × 29, 86 × 30, and 228 × 24 μm in size. The sarcocyst wall appears thin and indistinct (Fig. 1).

Fig. 1
figure 1

Section of sarcocyst in tongue of the Alaskan wolf. Note cyst wall (cw) with indistinct protrusions (arrowheads) into the host cell (hc), and numerous bradyzoites (br). Hematoxylin and eosin stain

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Transmission electron microscopy examination

Two sarcocysts were studied by TEM and the sarcocyst wall in both appeared to be ultrastructurally similar; they closely resemble “type 9c” by Dubey et al. (2016). The parasitophorous vacuolar membrane (pvm) was folded into villar protrusions (vp) and lined by an electron dense layer, 60 nm thick. The pvm was wavy and the electron dense layer (edl) was thinned out or absent at different levels of the vp (Fig. 2). The vp were up to 1.5 μm long and 0.5 μm wide, without any microtubules, pleomorphic, and some appeared anastomosing (Fig. 2). The ground substance was smooth, without any microtubules or granules, and up to 2.0 μm thick. The total thickness of the wall including vp and the gs was 3.5 μm. Both sarcocysts were mature and contained few metrocytes (Fig. 3) and numerous bradyzoites (Fig. 4). Longitudinally cut bradyzoites were 9.5 (9.4–9.8) μm long, and 1.5 (1.3–1.6) μm wide. They contained organelles typical of Sarcocystis bradyzoites, including conoid, micronemes, rhoptries, mitochondrion, nucleus, and amylopectin granules (Fig. 4). The micronemes were relatively few in number and confined to the conoidal third of the bradyzoite. There were at least two rhoptries with a large bulbous blind end.

Fig. 2
figure 2

TEM micrographs of 2 sarcocysts from tongue of the Alaskan wolf. a Cyst #1. b Cyst #5. Note, host cell nucleus (hcn), pleomorphic villar protrusions (vp), with occasional anastomosing of vp (arrow), thick ground substance (gs), with prominent septa (se), metrocytes (me), and bradyzoites (br)

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Fig. 3
figure 3

TEM of sarcocyst #5 from the Alaskan wolf. Note wavy parasitophorous vacuolar membrane (pvm) lined with electron dense layer (edl) that is thinned out or interrupted at places (arrows) at different levels of the vp. The ground substance layer (gs) was 2.0 μm thick, without microtubules but contained finer granular material. A few electron dense granules were present in the gs (arrowheads). a Note 1 metrocyte with a micropore (mp), few amylopectin granules (am), prominent nucleus (nu), and no micronemes and rhoptries. b High magnification of the villar protrusions with interruptions in edl (arrows) and few granules in the gs (arrowheads)

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Fig. 4
figure 4

TEM of bradyzoites in sarcocyst #5 from the Alaskan wolf. a Sections of several bradyzoites. b Conoidal end of a bradyzoite. Note a conoid (co), many amylopectin granules (am), numerous micronemes (mn), an elongated nucleus (nu), and a micropore (mp). There are at least two rhoptries (rh) in each bradyzoite with a narrow neck (rhn) and large blind ends

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Molecular analyses

PCR amplification and sequencing were successful at all four markers in four cysts (#1 to #4). Consensus sequences obtained for 18S rRNA, 28S rRNA, ITS-1, and cox1 fragments of each individual cyst were deposited in NCBI GenBank under accession numbers KX022100 to KX022115. Using the BLAST tool, 18S rRNA, ITS-1, 28S rRNA, and cox1 sequences were compared with previously deposited sequences in GenBank, allowing the assignment of the organism to Sarcocystis arctica, previously described by Gjerde and Schulze (2014) in Norwegian Arctic foxes (Vulpes lagopus). In detail, 18S rRNA sequences obtained from four cysts showed 100 % identity among them and with S. arctica (KF601301) from arctic foxes, also with S. caninum (KM362427) isolated from a domestic dog. 28S rRNA sequences were identical in all four cysts but differed by a single nucleotide polymorphism (SNP) A to G in position 797 of the reference sequence for S. arctica (KF601312). A higher degree of genetic variability was observed for ITS-1 sequences, with all sarcocysts presenting SNPs at positions 451 (A to G) and 683 (G to A) compared to reference sequence KF601308. Interestingly, two additional SNPs in the form of clear C/T double peaks at the electropherogram level were also identified at positions 261 (cysts #1 and #2) and 327 (cysts # 3 and #4) of KF601308. Finally, coxI sequences showed 100 % identity in all four cysts and matched 100 % with that of S. arctica from Arctic fox (KF601319, KF601320) and 99 % identity with S. lutrae, S. turdusi, and S. calchasi with otters and birds as intermediate hosts; 98 and 97 % similarities were detected for S. speeri (KT207461) and S. neurona (KF854272), opossum-related species, respectively.

Phylogenetic study based on cox1 sequences (Fig. 5) showed the positioning of S. arctica isolates from Alaskan wolf in the same clade than S. arctica from Arctic fox, S. lutrae from Eurasian otter (Lutra lutra), S. speeri from North American opossum (Didelphis virginiana) with unknown intermediate host, and other species infecting avian hosts (S. turdusi, S. rileyi), clearly separated from other clusters of Sarcocystis spp. with ruminants as intermediate hosts.

Fig. 5
figure 5

Evolutionary relationships among Sarcocystis arctica isolates from Alaskan wolf and different Sarcocystis spp. at the cytochrome c oxidase subunit 1 (cox1) locus inferred by a neighbor-joining analysis of the nucleotide sequence covering a 1019-bp region (positions 2 to 1020 of GenBank accession number KF601319) of the gene. GenBank accession numbers are provided for each sequence used. The bootstrap consensus tree was inferred from 1000 replicates. Branches corresponding to partitions that were reproduced in less than 50 % of bootstrap replicates are collapsed. The bootstrap values are indicated at the branch points. The evolutionary distances were computed using the Kimura 2-parameter method. Eimeria tenella was used as outgroup taxa

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Discussion

The sarcocysts found in the Alaskan wolf in the present study most closely resemble sarcocysts of S. arctica from two Arctic foxes (V. lagopus) from Norway (Gjerde and Schulze 2014). The S. arctica sarcocysts were up to 12 mm long and up to 250 μm wide. By light microscopy, the sarcocyst wall was thin and had short knob-like or dome-shaped protrusions that were 1–1.5 μm long (Gjerde and Schulze 2014). In the initial description of S. arctica, sarcocysts were not examined by TEM. Ultrastructurally, the wall of sarcocysts from the wolf was up to 3.5 μm thick and slightly different than type 9c. S. arctica sarcocyst wall in the present study was thicker and the villar protrusions were more anastomosing than reported for type 9c walls (Dubey et al. 2016). Based on molecular characteristics, sarcocysts from the Arctic fox are similar to sarcocysts from the wolf; more conservated regions as 18S rRNA, 28S rRNA, and cox1 showed higher degrees of similarity (99.9–100.0 %). As expected, more genetic variability at the nucleotide level was revealed at the ITS-1 marker, as demonstrated by the presence of a number of SNPs. Two of these SNPs correspond to clear C/T double peaks that may be derived from two different copies within the same genome. Intraspecific sequence variation within the ITS-1 region has been reported in other Sarcocystis species (Gjerde and Josefsen 2015). The definitive hosts of the sarcocysts in the fox from Norway and the wolf from Alaska remain unknown. The present study indicates that S. arctica has a wider range of distribution.