SARCOCYSTIS INFECTIONS IN RIVER OTTER (LONTRA CANADENSIS) IN MICHIGAN
Sarcocystis infections are common in the muscles of herbivores but were, until recently, considered relatively rare in carnivores. Little is known of sarcocysts in the muscles of river otters in the United States. In a previous epidemiologic study of Toxoplasma gondii infections in North American river otters (Lontra canadensis) from Michigan in the 2018 and 2019 harvest season, Sarcocystis DNA was found in 34 (27.4%) of 124 otter muscles. Tongues from these 34 PCR-positive samples were further examined here for Sarcocystis species. An additional batch of frozen 62 samples collected at the end of the season was also tested for Sarcocystis herein. Morphologically, sarcocysts were studied in 23 otters (13 of 34 PCR-positive samples from the first batch and 10 of 62 samples of batch 2) in compression smears and paraffin-embedded histologic sections stained with hematoxylin and eosin. Morphologically, at least 2 different kinds of sarcocysts were identified, 1 with a smooth sarcocyst wall and the second with villar protrusions. By transmission electron microscopy, sarcocysts from 1 otter were similar to Sarcocystis caninum. Morphologically, sarcocysts from the river otter were different from the European otter (Lutra lutra). Sequencing amplification products from 18S rRNA, 28S rRNA, and cox1 genes, suggested S. caninum–like, Sarcocystis svanai–like, and Sarcocystis sp. We detected a third, potentially undescribed species, in 3 otters. Genetic markers for conclusive differentiation of Sarcocystis spp. from mustelids should be developed. The samples in the present study had degraded; better preserved samples are needed for further morphologic studies. This is the first report of S. caninum–like, S. svanai–like, and Sarcocystis sp. in the river otter in the United States.Abstract
Protozoa in the genus Sarcocystis are ubiquitous parasites of ectotherm and endotherm hosts (Dubey et al., 2016). These parasites have an obligatory 2-host life cycle, alternating between an herbivore and a carnivore. The sexual cycle is restricted to the intestines of carnivores; the asexual cycle occurs in extraintestinal tissues of the intermediate host after it ingests water or vegetation contaminated with sporocysts. A carnivore consuming tissues containing viable sarcocysts releases bradyzoites during digestion, which then transform into gamonts in the intestine of the carnivore. Fertilization produces oocysts in the lamina propria, where they sporulate in situ and often rupture, releasing sporocysts in the feces. Once an intermediate host ingests these sporocysts, parasites multiply in endothelium of blood vessels and finally become encysted in tissues, often in muscles. Sarcocystis species are generally host specific, especially for the intermediate host (herbivore). In some carnivore or omnivore hosts, sarcocysts occur in extraintestinal muscles; life cycles of these uncommon sarcocysts remain incomplete.
Otters of the subfamily Lutrinae are aquatic animals and feed mainly on fish and invertebrates. They consume enormous amounts of food daily compared with their body weight. They are also exposed to contaminants, including parasites in runoff from land, and thus infections in otters are an indication of environmental contamination (Miller et al., 2002; Shapiro et al., 2012). More is known of protozoan infections of sea otters (Enhydra lutris) than of the North American river otters (Lontra canadensis) in the United States (Dubey et al., 2016). Two protozoans, Toxoplasma gondii (felids as definitive hosts) and Sarcocystis neurona (the Virginia opossum, Didelphis virginiana, as a definitive host) are common causes of mortality in sea otters in the United States (Dubey et al., 2016; Dubey, 2022).
In a recent survey of river otters from the state of Michigan, Sarcocystis spp. DNA was detected in the tongues of 34 (27.4%) of 124 animals (Cotey et al., 2022). No details were provided concerning the detection of Sarcocystis DNA and morphology of the sarcocysts. Here, we report on the molecular details and morphologically characterize sarcocysts. Also, we add information on another batch of 62 river otters from the same harvest year from Michigan.
MATERIALS AND METHODS
River otter samples
Samples came from otter heads that were collected at the time of registration of the fur and then frozen and submitted to the Michigan Department of Natural Resources (Lansing, Michigan) for the present investigation. The heads were thawed for sampling, and the collected tissues subsequently refrozen. Two batches of muscle samples from tongues or masseter muscles or both of frozen river otter heads were received (Table I). Batch 1 consisted of 124 samples; these were used by Cotey et al. (2022). A second batch of 62 samples collected in the 2018–2019 harvest season was also tested; these samples were not included in Cotey et al. (2022).
Testing of samples at the College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma
For the initial molecular testing performed on 124 samples from batch 1, the following method was used. For detection of Sarcocystis DNA, a PCR was performed to amplify a portion of the 18S rRNA gene, using the primers SarcoF (5′-CGCAAATTACCCAATCCTGA) and SarcoR (5′-ATTTCTCATAAGGTGCAGGAG) that amplify a 700-base-pair (bp) DNA fragment, as previously described by Yang et al. (2002). Briefly, a reaction mixture containing 5 μl (1×) buffer, 1.5 μl of magnesium, 1 μl of deoxynucleoside triphosphate, 0.5 μl each of 10 pmol forward and reverse primers, 0.25 μl of AmpliTaq Gold™ DNA Polymerase, and 2 to 5 μl of DNA containing approximately 10–20 ng of DNA template with water adjusted to a total reaction volume of 25 μl (Applied Biosystems™, Waltham, Massachusetts). PCR was performed in a thermocycler (Applied Biosystems™) with an initial denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 59 C for 30 sec, extension at 72 C for 60 sec, and a final extension at 72 C for 10 min. These details for PCR performed on 124 samples were not given by Cotey et al. (2022).
Many of the samples from batch 1 were dehydrated; therefore, for the present investigation, we concentrated on testing 34 of the 124 samples that were positive for Sarcocystis DNA (Cotey et al., 2022).
Testing samples at the Animal Parasitic Diseases Laboratory (APDL), Beltsville, Maryland
Cytologic and histologic examination:
At APDL, samples of muscles were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin (HE), and examined microscopically for parasites. Portions of muscle were stored at −20 C for DNA extraction and further microscopic testing. Around 5 g of muscle was thawed, and compression preparations were examined microscopically. For this, muscle snips were pressed between coverslips and glass slides (around 30 preparations per 1 g of tissue) and examined microscopically. From 2 otters (nos. 2 and 7; Table I), muscle samples were ground in aqueous 0.85% NaCl solution (saline), and homogenized preparations were examined microscopically (Fig. 1C, D). Portions of muscles containing sarcocysts were also saved in 70% ethanol before DNA extraction. Parasites detected were photographed using an Olympus AX-70 microscope with DP-73 digital camera, and measurements were made digitally using Olympus Imaging Software cellSens Standard 1.18 (Olympus Optical Ltd., Tokyo, Japan).
Citation: The Journal of Parasitology 111, 4; 10.1645/25-25
Electron microscopic examination:
Muscle snips (n = 8) containing sarcocysts were fixed with McDowell Trump’s fixative (10% formalin 37%, 1% glutaraldehyde in 1.16% sodium phosphate monobasic, and 0.27% NaOH with deionized water), placed in plastic sealed bags, and transported by air to Complutense University of Madrid, Spain, for transmission electron microscopy (TEM) examination. Samples were postfixed, sectioned, and finally contrasted with 4% uranyl acetate and 3% lead citrate, as reported by Dubey et al. (2024b). Ultrathin sections were examined at the Spanish National Centre for Electron Microscopy (Madrid, Spain) using a JEOL JEM 1400 Plus device (JEOL, Tokyo, Japan) at 80 kW.
DNA isolation and amplification:
Genomic DNA was extracted from individual sarcocysts excised from 4 positive river otters (nos. 7, 19, 20, and 23; Table I). The extraction was carried out using the DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. DNA was then stored at −20 C. The purity and concentration of the extracted DNA specimens were measured using a Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, Massachusetts).
PCR amplifications were performed using previously published primers specific to Sarcocystis (Table II). Each PCR mixture (14.5 μl total volume) contained 2 μl of DNA template, 6.25 μl of Platinum Hot Start PCR Master Mix (Invitrogen, Waltham, Massachusetts), 1 μl of 10 pmol/μl of each primer (Integrated DNA Technologies, Coralville, Iowa), and 4.25 μl of molecular-grade water. The thermal cycling conditions included an initial denaturation step at 94 C for 3 min, followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and extension at 68 C for 2 min, with a final elongation at 68 C for 5 min.
PCR products were visualized on a 2% agarose gel, and fragment size was determined by comparison to the 100-bp Plus DNA Ladder (Promega Corporation, Madison, Wisconsin). PCR products were purified using the ExoSAP method (Bell, 2008), followed by bidirectional Sanger sequencing performed at Psomagen, Inc. (Rockville, Maryland) on an ABI 3500xl Genetic Analyzer (Applied Biosystems™). Sequence data were processed, assembled, and edited using Geneious 11.1.5 (Biomatters Limited), and the final consensus sequences were submitted to GenBank (Table II).
Phylogenetic analysis
Sequences were compared with the NCBI database using Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al., 1990). All sequences were trimmed at each end before phylogenetic reconstruction. Multilocus genotyping using 18S rRNA, 28S rRNA, and cox1 genes was performed, and the results are provided in Table II.
The web server GUIDANCE2 (Sela et al., 2015) was used to align and remove ambiguously aligned positions. Specifically, the sequences were aligned with the MAFFT algorithm under the following options: maxiterate 1,000 and pairwise alignment method –localpair. Positions with a score below 0.93 were removed. Phylogenetic relationships were reconstructed under the maximum-likelihood (ML) criterion. ML analyses were performed with the program IQ-TREE version 1.6.12 (Nguyen et al., 2015). The analyses were run with the options –m MFP –b 1,000. The model selected based on the BIC criterion was T92+G+I, K2+G, and T92 for 18S rRNA, 28S rRNA, and cox1, respectively.
RESULTS
Cytologic and morphologic findings
Sarcocysts were found in compression smears of 23 otters (13 from the first batch, otter nos. 1–13, and in 10 of 62 samples from batch 2, otter nos. 14–23; Table I). Tongue and masseter were difficult to squash because of partial dehydration. Therefore, details of the sarcocyst wall could be photographed from only 11 of 23 positive samples. In unstained preparations, sarcocysts had smooth sarcocyst walls without any protrusions (Fig. 1A, B). In 2 otters (nos. 2 and 7), sarcocysts could be freed from the host tissue; these sarcocysts had visible serrations to conical villar protrusions (Fig. 1C, D).
Muscles from these 23 samples positive by the compression method were studied histologically in paraffin-embedded sections, and sarcocysts were found in sections of 15 samples. From 2 otters (nos. 7 and 13), several additional muscle samples were studied histologically. Sarcocysts were sparse. In total, 41 sarcocysts were examined at ×1,000 magnification of HE-stained sections. All sarcocysts were mature.
At least 2 morphologic types of sarcocysts were recognized, based on the protrusions of the sarcocyst wall (Fig. 2). Some sarcocysts had conical to slender protrusions (Fig. 1A–D; Table I), but interpretation could be clouded by the angle of the sarcocyst cut.
Citation: The Journal of Parasitology 111, 4; 10.1645/25-25
Before TEM examination, samples underwent several rounds of freezing and thawing, somewhat compromising the morphologic integrity. Nevertheless, ultrastructural morphology could be appreciated in 1 specimen (otter no. 2; Figs. 3–5). In sarcocysts examined by TEM, bradyzoites had degenerated, but amylopectin granules were visible, and the structure of the sarcocyst wall was fairly preserved (Figs. 3–5). The sarcocyst wall consisted of a parasitophorous vacuolar membrane that was wavy and lined with a remarkably thick (94 nm) electron-dense layer (Fig. 5). The sarcocyst wall had pleomorphic villar protrusions, with an average size of 1.78 × 0.59 μm (n = 15), with a smooth 0.77-μm-thick ground substance layer. By TEM, sarcocysts closely resemble the type 9c grouping by Dubey et al. (2016).
Citation: The Journal of Parasitology 111, 4; 10.1645/25-25
Citation: The Journal of Parasitology 111, 4; 10.1645/25-25
Citation: The Journal of Parasitology 111, 4; 10.1645/25-25
Molecular and phylogenetic analysis
The molecular analyses based on the conserved regions of the 18S rRNA, 28S rRNA, and cox1 genes confirm the close genetic relationship of the Sarcocystis isolates from river otters to sequences of Sarcocystis caninum and Sarcocystis svanai derived from domestic dogs with identities of >99%. The 18S rRNA gene also showed absolute identity to Sarcocystis sp. previously described from fisher (Martes pennanti) in Pennsylvania (Larkin et al., 2011). The cox1 gene, a more variable marker, provided high-resolution insights into interspecies differences and affirmed the identity of these Sarcocystis species. The sequence types obtained during the study appeared closely related (but not identical) to each other: 98.8% (for 18S rRNA); 98.4% (for 28S rRNA); and 98.8% (for cox1).
Based on the 18S rRNA alignment using undescribed species from M. pennanti (GenBank accession nos. HQ709138 and HQ709139) and some sequences of S. caninum (GenBank accession nos. MH469238 KM362427), Sarcocystis arctica (GenBank accession nos. MF596237, MF596227, KX022100, and KX022101), and S. svanai (GenBank accession nos. OR921255, KM362428, and OR921256), 8 single nucleotide polymorphisms (SNPs) were found comparing HQ709138 and HQ709139 (100% identity to 18S rRNA sequences isolated from otter nos. 7, 20, and 23 and S. svanai). Only 1 fixed SNP comparing HQ709138, HQ709139 with S. caninum or S. arctica (Y = C or T) was also found. Such a small difference does not rule out the possibility that it is an intraspecific variation.
The phylogenetic analysis of 18S rRNA included 18 taxa and 1,678 positions, 13 taxa and 1,085 positions for cox1, and 15 taxa and 1,509 positions for 28S rRNA. Isolates of Sarcocystis myodes were used as an out-group (Fig. 6).
Citation: The Journal of Parasitology 111, 4; 10.1645/25-25
The 18S rRNA gene
The 18S rRNA sequences isolated from otters (nos. 7, 20, and 23) were identical to previously reported isolates of an undescribed species from M. pennanti (GenBank accession nos. HQ709138, HQ709139, and HQ709143). These formed a group, supported in 67 of 100 bootstrap replicates, distinct from sequences attributed previously to S. caninum and S. arctica (GenBank accession nos. KM362427, KX022102, and MH469238). Strong bootstrap support (97/100) distinguishes the 2 groups discussed previously as a monophyletic group to the exclusion of all others, including the 18S rRNA sequence amplified from river otter no. 20 that resembles, but appears slightly distinct from, prior exemplars of S. svanai (GenBank accession nos. OR921254 and KM362428). Excluded from this analysis are 3 partial 18S rRNA sequences obtained from parasites infecting river otters nos. 7, 19, and 23. Over their length, these 3 sequences are identical to the sequence obtained from river otter no. 20 whose phylogenetic position appears in Fig. 6A.
The cox1 gene
Sarcocystis svanai sequences (GenBank accession nos. PQ490734 and PP819578) grouped with S. svanai sequences isolated during the study from river otters (nos. 7, 20, 23), but this assemblage does not form a strictly monophyletic clade due to the presence of other Sarcocystis species within the grouping. The high bootstrap support value (93) for the S. svanai cluster suggests strong confidence in the relationships within the cox1 dataset. Sarcocystis caninum and S. arctica formed a distinct grouping, separate from other species, consistent with their taxonomic identity. Also, Sarcocystis halieti, Sarcocystis corvusi, and Sarcocystis columbae clustered together, separating them from S. arctica and S. caninum, further supporting their evolutionary divergence (Fig. 6B).
The 28S rRNA gene
Sarcocystis caninum sequences obtained from 4 river otters (nos. 7, 19, 20, and 23) during the study formed a closely related cluster with other isolates of S. caninum (GenBank accession nos. MH469239 and PQ482648) with strong support, demonstrating genetic consistency within the species for the 28S rRNA gene. Moreover, the sequences of S. columbae and S. halieti are grouped distinctly, reflecting species-specific differences. Also, S. svanai sequences showed clear clustering, differentiating them from other species such as Sarcocystis lutrae, S. arctica, Sarcocystis turdusi, and Sarcocystis lari (Fig. 6C).
Phylogenetic trees reconstructed also indicated a close relationship between S. caninum and S. arctica, as previously discussed by Gupta et al. (2024) and can be seen in the present study. This finding further suggests that S. arctica cannot be genetically differentiated from S. caninum. Therefore, sequencing additional loci, such as the ITS2, cox3, or cytb genes, can provide finer resolution for phylogenetic insights and confirm transmission pathways.
DISCUSSION
There is considerable uncertainty concerning the Sarcocystis species in otters and the relationship with Sarcocystis species in other mustelids. Sarcocystis infections were first recognized in 4 European otters (Lutra lutra) from Norway (Wahlström et al., 1999; Gjerde and Schulze, 2014; Table III). During an introduction project, 70 otters were imported into Sweden from Norway. One of these otters died in captivity before it could be released to the wild, and sarcocysts were found in the skeletal muscle; however, sarcocysts were not found in the remaining 69 otters (Wahlström et al., 1999). Sarcocysts were up to 2.3 mm long and 0.25 mm wide. With a light microscope, the sarcocyst wall was serrated. Ultrastructurally, the sarcocyst wall had small blebs at regular intervals, corresponding to the type 1 classification of Dubey et al. (2016). Bradyzoites were 8 × 2 μm (Wahlström et al., 1999). Wahlström et al. (1999) did not name the parasite. Gjerde and Josefsen (2014) found sarcocysts in 3 otters in Norway and named the parasite S. lutrae, primarily based on molecular characteristics. Sarcocysts were up to 970 μm long. By light microscopy, the sarcocyst wall was thin (<0.5 μm) and smooth without any serrations or projections; TEM examination was not performed. Sarcocyst DNA was characterized using 18S and 28S ribosomal and mitochondrial cox1 genes (Gjerde and Josefsen, 2014). Molecularly, S. lutrae resembled Sarcocystis kalvikus from wolverines in Canada (Gjerde and Josefsen, 2014). Since then, based on molecular characteristics, S. lutrae has been reported from other mustelids in Europe, including the American mink (Neovison vison), beech martins (Mates foina), European pole cat (Mustela putorius), and the European badger (Meles meles) (Lepore et al., 2017; Kirillova et al., 2018; Máca, 2018; Prakas et al., 2018). In conclusion, it is uncertain if there is more than 1 Sarcocystis species infecting European otters.
Máca and González-Solís (2022) found Sarcocystis sporocysts in the intestines of a white-tailed eagle (Haliaeetus albicilla) in the Czech Republic; molecularly, these (18S rRNA, 28S rRNA, ITS1, and cox1) were considered S. lutrae, thus confirming the eagle as its definitive host (Table III). Molecularly, S. lutrae has been also identified in the small intestines of the hooded crow (Corvus cornix) from Lithuania; however, only oocysts not sporocysts were seen in this omnivorous bird (Juozaitytė-Ngugu et al., 2021).
Little is known of Sarcocystis infections in the North American river otter in the United States. As stated earlier, using PCR (18S rRNA gene), Cotey et al. (2022) found Sarcocystis sp. DNA in 34 of 124 otters from Michigan; sarcocyst morphology was not described.
Sarcocystis svanai and S. caninum are primarily known to infect canids and other intermediate hosts (Dubey et al., 2015; Hagner et al., 2018; Gupta et al., 2024; Table III). The presence of parasites similar to these species in river otters, which are not their expected typical hosts, broadens the understanding of their host range. This finding might indicate the spread or introduction of Sarcocystis species into aquatic ecosystems, potentially through definitive hosts (e.g., dogs or wild canids) defecating near water sources. Nevertheless, our TEM observations indicated the presence of a Sarcocystis that resembled S. caninum (Dubey et al., 2015); therefore, additional descriptive studies would be of interest. The Sarcocystis spp. with characteristics of an S. caninum–like sarcocyst is structurally different from S. lutrae named from European otters from Norway by Gjerde and Josefsen (2014) and Sarcocystis sp. reported by Wahlström et al. (1999). Here, we are postponing naming the species awaiting further studies.
The use of 18S, 28S, and cox1 genes as molecular markers provided robust genetic evidence for species identification. The cox1 gene proved valuable due to its high resolution in distinguishing closely related species (Gjerde, 2013, 2016; Dubey et al., 2024a; Gupta et al., 2024). Further genotyping studies using microsatellites or more discriminatory markers by multilocus sequencing typing assays could provide additional insight into Sarcocystis species that are closely related and are host and tissue specific.
Sarcocysts in compression smears of muscles of river otters (Lontra canadensis) from Michigan. Unstained. (A) Sarcocyst with a thin smooth wall (arrow) without any visible protrusions. Note septa of the same thickness (arrowheads) as the cyst wall. Otter no. 23. (B) Thin-walled sarcocyst (arrows). Otter no. 22. (C) Part of a sarcocyst freed from the muscle. Arrowheads point to elongated, slender villar protrusions. Otter no. 2. (D) Sarcocyst with conical villar protrusions (arrowheads). Otter no. 7. Color version available online.
Sarcocysts in histologic sections of the tongues of river otters (Lontra canadensis) from Michigan. Hematoxylin and eosin stain. Sarcocysts have degraded, but cyst wall protrusions are recognizable. (A) Indistinct protrusions (arrow) on the wall. Otter no. 6. (B) Eosinophilic sarcocyst wall (arrow) with villar protrusions (arrowhead). Note prominent septa divide bradyzoites into groups. Otter no. 13. (C) Eosinophilic sarcocyst wall (arrow) with villar protrusions (arrowhead). Otter no. 5. (D) Slender villar protrusions (arrowheads) on the sarcocyst wall (arrow). Otter no. 1. (E, F) Degenerated sarcocysts with smooth cyst walls (arrowheads). Otter no. 7. Color version available online.
Transmission electron micrograph of Sarcocystis caninum–like sarcocysts in the tongue of river otter no. 2. Note 3 sarcocysts (1, 2, and 3). Sarcocyst marked 1 is slender, and the sarcocyst wall is visible on both sides. Note homogenous ground substance layer (gs) and pleomorphic villar protrusions (arrows).
Transmission electron micrograph of 2 adjacent Sarcocystis caninum–like sarcocysts in tongue of river otter no. 2. The ground substance layer (gs) is smooth. Note pleomorphic villar protrusions. The bradyzoites are degenerated, but amylopectin is visible as dark spots.
Transmission electron micrograph of a Sarcocystis caninum–like sarcocyst in the tongue of river otter no. 2. Higher magnification of the sarcocyst wall. The parasitophorous vacuolar membrane (pvm) is wavy and lined by a thick electron-dense layer (edl). The villar protrusions are pleomorphic, and there are no microtubules. The ground substance layer (gs) is smooth without granules. The bradyzoite (br) is degenerated.
Phylogenetic relationships of the various Sarcocystis species identified from river otter (Lontra canadensis) in Michigan, inferred using multilocus genotyping: (A) 18S rRNA, (B) cox1, and (C) 28S rRNA. Branch support values are shown near the respective nodes. Species highlighted represent those identified in this study. Color version available online.
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