Molecular identification of two Sarcocystis species in fallow deer (Dama dama) from Lithuania

Due to the lack of molecular research conducted, little is known about Sarcocystis species diversity in the fallow deer (Dama dama). Until now, Sarcocystis jorrini and Sarcocystis morae were described to form sarcocysts in the muscles of this host. In the present study diaphragm muscle samples of free-ranging fallow deer from Lithuania were investigated for Sarcocystis species. Sarcocysts were detected in 39 out of 48 (81.3%) fallow deer examined. Under a light microscope two types of sarcocysts having hair-like and finger-like protrusions were observed. Based on DNA sequence analysis of cox1 and 18S rDNA, two species, S. morae and Sarcocystis entzerothi were identified. In prior studies, the latter species was only detected in Lithuanian roe deer (Capreolus capreolus) and in sika deer (Cervus nippon). The haplotype network of S. morae sequences specified close relationships between haplotypes found in the same country. According to current knowledge, the fallow deer is characterised by low Sarcocystis species richness as compared with other cervid species from Europe.     

Sarcocystis rommeli,n. sp. (Apicomplexa: Sarcocystidae) from Cattle (Bos taurus) and its Differentiation from Sarcocystis hominis

Cattle (Bos taurus) are intermediate hosts for three named species of Sarcocystis, S. cruzi, S. hirsuta, and S. hominis. Recently, a fourth species was identified and named S. sinensis. However, S. sinensis originally named a species of Sarcocystis in water buffalo (Bubalus bubalis) in China. Based on unverifiable evidence, it was suggested that the same parasite infects cattle. In addition, S. sinensis was recently declared as nomen nudum because its naming violated the rules of International Code of Zoological Nomenclature. Thus, the fourth species using cattle as an intermediate host does not have a valid name. Here, we propose a new name, Sarcocystis rommeli for the S. sinensis‐like parasite from cattle in Argentina, and differentiate it ultrastructurally from S. hominis sarcocysts from experimentally infected cattle. Sarcocystis rommeli sarcocysts were microscopic with a 5‐μm‐thick wall with slender villar protrusions (Vp); the Vp were up to 5 μm long, up to 0.5 μm wide, and of uneven thickness, often bent at an angle. The ground substance layer (Gs) was up to 0.8 μm thick and smooth. Vesicular structures were seen at the base of the Vp. The bradyzoites were 10–12 μm long. Sarcocystis hominis sarcocysts had Vp that were often upright, up to 7.5 μm long, and up to 1.8 μm wide; the Gs was up to 2 μm thick and without vesicles. Its sarcocyst wall was up to 5.6 μm thick, the vp were bent at an angle, up to 5.8 μm long, the Gs was up to 2 μm thick, but without vesicles seen in S. rommeli. Beef containing sarcocysts of S. rommeli was not orally infectious for two human volunteers and a red fox (Vulpes vulpes). The Sarcocystis described here is molecularly different from S. cruzi, S. hirsuta, and S. hominis based on 18S rRNA and cox1 gene sequences.     

Molecular identification targeting cox1 and 18S genes confirms the high prevalence of Sarcocystis spp. in cattle in the Netherlands

The reported prevalence of Sarcocystis infection in cattle in Europe ranges between 66 and 94%. Although in the Netherlands a prevalence of 100% was reported in 1993, this study aimed to develop a method for sensitive and specific molecular detection and species identification of Sarcocystis spp., in order to provide more recent data on the prevalence and identification of these protozoa in cattle meat intended for human consumption in the Netherlands. For this purpose, 104 cattle samples were obtained from Dutch slaughterhouses. Genomic DNA was extracted, and analysed by 18S and cox1 PCR. Magnetic capture was used to extract and amplify 18S-specific DNA. Sarcocystis DNA was detected in 82.7% of the samples. PCR amplicons of both targets were sequenced, and sequence identities of ≥97% were observed for Sarcocystis cruzi (65.4%), Sarcocystis hominis (12.5%), Sarcocystis bovifelis (8.7%), Sarcocystis hirsuta and Sarcocystis heydorni (both 1.0%). Mixed infections were observed in 17.3% of the samples. The magnetic capture was not significantly more sensitive compared with standard DNA extraction, but magnetic capture did add to the overall sensitivity. Using cox1 sequencing, all species are clearly distinguished, whereas for 18S the variation between species is limited, which particularly hampers reliable identification of thick walled Sarcocystis spp. Furthermore, the detection of 12.5% S. hominis and 1% S. heydorni points towards an established transmission route between cattle and humans in the Netherlands. The availability of four additional well-identified and well-referenced S. hominis cox1 sequences in public databases enables development of species-specific diagnostic PCRs targeting cox1, which in combination with magnetic capture could provide the means to determine the prevalence of human sarcocystosis.     

Recolonizing gray wolves increase parasite infection risk in their prey

The recent recolonization of Central Europe by the European gray wolf (Canis lupus) provides an opportunity to study the dynamics of parasite transmission for cases when a definitive host returns after a phase of local extinction. We investigated whether a newly established wolf population increased the prevalence of those parasites in ungulate intermediate hosts representing wolf prey, whether some parasite species are particularly well adapted to wolves, and the potential basis for such adaptations. We recorded Sarcocystis species richness in wolves and Sarcocystis prevalence in ungulates harvested in study sites with and without permanent wolf presence in Germany using microscopy and DNA metabarcoding. Sarcocystis prevalence in red deer (Cervus elaphus) was significantly higher in wolf areas (79.7%) than in control areas (26.3%) but not in roe deer (Capreolus capreolus) (97.2% vs. 90.4%) or wild boar (Sus scrofa) (82.8% vs. 64.9%). Of 11 Sarcocystis species, Sarcocystis taeniata and Sarcocystis grueneri occurred more often in wolves than expected from the Sarcocystis infection patterns of ungulate prey. Both Sarcocystis species showed a higher increase in prevalence in ungulates in wolf areas than other Sarcocystis species, suggesting that they are particularly well adapted to wolves, and are examples of “wolf specialists”. Sarcocystis species richness in wolves was significantly higher in pups than in adults. “Wolf specialists” persisted during wolf maturation. The results of this study demonstrate that (1) predator–prey interactions influence parasite prevalence, if both predator and prey are part of the parasite life cycle, (2) mesopredators do not necessarily replace the apex predator in parasite transmission dynamics for particular parasites of which the apex predator is the definitive host, even if meso‐ and apex predators were from the same taxonomic family (here: Canidae, e.g., red foxes Vulpes vulpes), and (3) age‐dependent immune maturation contributes to the control of protozoan infection in wolves.     

Genetic characterisation by isoenzyme markers of North American and Australasian isolates of species of Sarcocystis (Protozoa: Apicomplexa) from mice,sheep, goats and cattle

The technique of isoenzyme (enzyme isotype) electrophoresis was used to compare genetic profiles of extracts of zoites of sarcocysts from North America and Australasia. The species examined were Sarcocystis muris (Railliet, 1886) from mice, S. gigantea (Railliet, 1886) (syn. S. ovifelis Heydorn et al., 1975) from sheep, S. capracanis Fischer, 1979 from goats and S. cruzi (Hasselmann, 1923) (syn. S. bovicanis Heydorn et al., 1975) from cattle. Sarcocysts from the four host animals had different alleles at almost all loci studied. This was not affected by having a common definitive host. Extracts of two cat-borne Sarcocystis species shared alleles at only 3 out of 16 loci, while two dog-borne Sarcocystis species had different alleles at 8 out of 16 loci. The extent of genetic divergence among sarcocysts confirmed the existance of distinct species in each host sampled. By contrast, the isolates from the United States of America and Australasia for any particular host were essentially identical, sharing at least one allele at every locus tested. ac]19860908     

Bovine sarcocystosis: Sarcocystis species,diagnosis, prevalence,economic and public health considerations,and association of Sarcocystis species with eosinophilic myositis in cattle

Infections by Sarcocystis in cattle are ubiquitous worldwide. There is considerable debate concerning the identity of Sarcocystis spp. in cattle. Proper diagnosis of Sarcocystis spp. is important to assess their economic and public health importance. Currently there are seven named species: Sarcocystis hirsuta, Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis bovifelis, arcocystis heydorni, Sarcocystis bovini and Sarcocystis rommeli. Additionally, there are unnamed Sarcocystis spp. Two species, S. hominis and S. heydorni, are zoonotic. One out of seven species (S. hirsuta, contracted from cats) forms macroscopic cysts which can be visible during carcass inspection. Current molecular characterization is based on DNA extracted from sarcocysts from naturally infected cattle because DNA was not characterized from tissues of experimentally infected cattle or feces of experimentally infected definitive hosts. Sarcocystis cruzi (transmitted via canids) is recognized as the most pathogenic species and it causes abortion, low milk yield, poor body growth, and outbreaks of clinical sarcocystosis and death. Additionally, Sarcocystis infections have been linked to an inflammatory condition of striated muscles termed bovine eosinophilic myositis (BEM). Cattle affected by BEM appear clinically normal. Diagnosis of BEM at slaughter occurs when inspecting the carcass surface, or once the carcass has been divided into prime cuts or quarters. Sex and breed have no apparent influence on prevalence of BEM. The condition evidently occurs with equal frequency in steers, cows, and heifers. Virtually all striated muscles can be affected including skeletal muscles, the muscles of the eye, larynx, and the heart. In the USA, regulations require condemnation of BEM-affected parts, or (in severe cases) the entire carcass. These aesthetic considerations result in economic losses. Cattle experimentally infected with Sarcocystis did not have BEM at slaughter. Here, we review the status of Sarcocystis spp. and BEM in cattle including prevalence, lesions, epidemiology, and association of BEM with different species of Sarcocystis.     

Infection survey and morphological characteristics of Sarcocystis spp. in naturally infected Tibetan sheep from Qinghai in northwestern China

Sarcocystosis is a parasitic disease caused by intracellular coccidian protozoans that belong to the genus Sarcocystis. These parasites can cause diseases of the nervous system, abortion and economically significant losses in host animals. Previous studies have reported that Sarcocystis is found in mammals, birds and reptiles, while molecular and morphological studies of infected Tibetan sheep have not been performed in the Qinghai region. The aim of this study was to determine the prevalence of Sarcocystis spp. in Tibetan sheep in Qinghai, northwestern China. The results showed that in 1155 samples, sarcocysts from unspecified species were found in 50% (577/1155) of the sheep tissues by microscopy detection. The positive rates of sarcocysts in the diaphragmatic, esophageal and cardiac muscles were 78.4% (175/223), 29.1% (207/711), and 88.2% (195/221), respectively. Ultrastructural features were exclusively observed in Sarcocystis gigantea in the esophageal tissues. The specific architecture was characterized as a space between the two layers of the original capsule wall, which was filled with fiber bundles and tissue fluid. Cauliflower-like protrusions of the original capsule wall were observed toward the outer surface of the capsule. Prominent protrusions contained fibers and matrix. In addition, the Sarcocystis 18S rRNA genes from 6 esophageal tissue samples were cloned, sequenced, and aligned to related sequences from GenBank. All 5 S. gigantea sequences examined in this study were grouped into the same cluster and belonged to the same genotype. The other 5 Sarcocystis tenella sequences were obtained from cardiac muscle and diaphragm muscle and belonged to the same clade. Overall, this study revealed a high infection rate of Sarcocystis in Tibetan sheep in the region. The results of this study may provide a reference for further research investigating the sarcocystosis epidemic in Qinghai, China.     

<Emphasis Type="Italic">Sarcocystis menglaensis</Emphasis> n. sp. (Apicomplexa: Sarcocystidae) from Williamson’s mouse deer <Emphasis Type="Italic">Tuagulus williamsoni</Emphasis> (Kloss) (Artiodactyla: Tragulidae)

Williamson’s mouse deer, Tuagulus williamsoni (Kloss), is one of the smallest ungulates among tragulid species found in northern Thailand, and Yunnan Province, China. Here we describe Sarcocystis menglaensis n. sp., infecting two of 14 (14.3%) Williamson’s mouse deer from south-western China. By light microscopy, sarcocysts of S. menglaensis are microscopic, up to 2,170 μm in length, and have a striated sarcocyst wall with 1.5–3.6 μm long palisade-like protrusions. Transmission electron microscopy observations revealed that sarcocyst wall is of “type 10f”, and has numerous villar protrusions folded over the cyst wall. The villar protrusions contained microtubules dispersed throughout the protrusions. Phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences indicated that S. menglaensis shared a close affinity with species of Sarcocystis Lankester, 1982 from ruminants, which utilise felids as definitive hosts.     

Sarcocystis neurona: molecular characterization of enolase domain I region and a comparison to other protozoa

Sarcocystis neurona causes protozoal myeloencephalitis and has the ability to infect a wide host range in contrast to other Sarcocystis species. In the current study, five S. neurona isolates from a variety of sources, three Sarcocystis falcatula, one Sarcocystis dasypi/S. neurona-like isolate, and one Besnoitia darlingi isolate were used to compare the enolase 2 gene segment containing the domain I region to previously sequenced enolase genes from Neospora caninum, Neospora hughesi, Toxoplasma gondii, Plasmodium falciparum, and Trypanosoma cruzi; enolase 2 segment containing domain I region is highly conserved amongst these parasites of veterinary and medical importance. Immunohistochemistry results indicates reactivity of T. gondii enolase 1 and 2 antibodies to S. neurona merozoites and metrocytes, but no reactivity of anti-enolase 1 to the S. neurona bradyzoite stage despite reactivity to T. gondii bradyzoites, suggesting expression differences between organisms.     

Non-invasive methods for identifying oocysts of Sarcocystis spp. from definitive hosts

Because the excreted sporocysts and/or oocysts of various species of Sarcocystis may not be discriminated morphologically, we sought to validate a diagnostic technique based on variation in the 18S rDNA sequence. Oocysts and/or sporocysts from three taxa of Sarcocystis were collected from human, feline, and canine definitive hosts that had fed upon meats infected with the muscle cysts of Sarcocystis hominis, Sarcocystis fusiformis and a species of Sarcocystis from water buffalo that could not be distinguished from Sarcocystis cruzi. Using a new collection method employing filter paper, these excreted oocysts and sporocysts were subjected to DNA extraction, as were the corresponding muscle cysts. Methods employing PCR–RFLP and DNA sequencing of a partial 18S rDNA gene (ssrRNA) sequence were then used to successfully distinguish among the three taxa. The same, unique restriction digestion pattern characterizes the tissue cysts and oocysts and/or sporocysts of each parasite taxon. The technique makes possible amplification and identification of species specific gene sequences based on DNA extracted from as few as 7 excreted sporocysts (the equivalent of 3 and 1/2 oocysts) from freshly prepared material, or as few as 50 sporocysts from feces samples that had been stored in potassium dichromate (K₂Cr₂O₇) for as long as 6 years. This represents the first report using molecular diagnostic procedures to diagnose oocysts of Sarcocystis in faecal samples, describing a valuable new tool for studying the epidemiology of various Sarcocystis species.     

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

Sarcocystosis is a parasitic disease caused by an intracellular protozoan parasite Sarcocystis belonging to the phylum Apicomplexa. These parasites have a requisite two-host life cycle. Recently, there are many Sarcocystis species that identified morphologically. In the present study, diaphragmatic muscle samples from the domestic horse (Equus caballus) were examined for Sarcocystis infection. The natural infection with sarcocysts was recorded to be 62·5% for only microcysts in the infected muscles. Molecular analysis using the 18S rRNA gene was conducted to swiftly and accurately identify the recovered species. Studies on the expression of the 18S rRNA gene have confirmed that the present parasite isolates belong to the Sarcocystis genus. The sequence data showed significant identities (>80%) with archived gene sequences from species within the Sarcocystidae family, and a dendrogram showing the phylogenetic relationship was constructed. The most closely related species were the previously described Sarcocystis fayeri and Sarcocystis bertrami. The current data showed that the present species was identified as S. fayeri and deposited in GenBank (accession number MF614956.1). This study highlights the importance of the genetic data in the exact taxonomy within sarcocystid species.     

Staphylorchis cymatodes (Gorgoderidae: Anaporrhutinae) from carcharhiniform, orectolobiform and myliobatiform elasmobranchs of Australasia: Low host specificity, wide distribution and morphological plasticity

Anaporrhutine gorgoderids (Digenea: Gorgoderidae: Anaporrhutinae) found in the body cavity of six species of elasmobranchs from the orders Carcharhiniformes, Myliobatiformes and Orectolobiformes from Australian waters were found to belong to the genus Staphylorchis. Although these specimens were morphologically variable, sequences of ITS2 and 28S ribosomal DNA from specimens from three host families and two host orders were identical. Based on morphological and molecular data these specimens were identified as the type-species of the genus, Staphylorchis cymatodes. New measurements are provided for S. cymatodes, and for the first time genetic data are presented for this species. In addition to providing new morphological and molecular data for S. cymatodes, the previously described species S. gigas, S. parisi and S. scoliodonii, are here synonymised with S. cymatodes. This implies that S. cymatodes, as conceived here, has remarkably low host-specificity, being recorded from eight elasmobranch species from four families and three orders, has a wide geographical distribution in the Indo-west Pacific from off India, in the Bay of Bengal, to Moreton Bay in the Coral Sea, and is morphologically plastic, with body size, size of specific organs and body shape differing dramatically between specimens from different host species. The genus Staphylorchis now contains only two valid species, S. cymatodes and S. pacifica.     

Host and Environmental Factors Modulate the Exposure of Free-Ranging and Farmed Red Deer (Cervus elaphus) to Coxiella burnetii

The control of multihost pathogens, such as Coxiella burnetii, should rely on accurate information about the roles played by the main hosts. We aimed to determine the involvement of the red deer (Cervus elaphus) in the ecology of C. burnetii. We predicted that red deer populations from broad geographic areas within a European context would be exposed to C. burnetii, and therefore, we hypothesized that a series of factors would modulate the exposure of red deer to C. burnetii. To test this hypothesis, we designed a retrospective survey of 47 Iberian red deer populations from which 1,751 serum samples and 489 spleen samples were collected. Sera were analyzed by enzyme-linked immunosorbent assays (ELISA) in order to estimate exposure to C. burnetii, and spleen samples were analyzed by PCR in order to estimate the prevalence of systemic infections. Thereafter, we gathered 23 variables—within environmental, host, and management factors—potentially modulating the risk of exposure of deer to C. burnetii, and we performed multivariate statistical analyses to identify the main risk factors. Twenty-three populations were seropositive (48.9%), and C. burnetii DNA in the spleen was detected in 50% of the populations analyzed. The statistical analyses reflect the complexity of C. burnetii ecology and suggest that although red deer may maintain the circulation of C. burnetii without third species, the most frequent scenario probably includes other wild and domestic host species. These findings, taken together with previous evidence of C. burnetii shedding by naturally infected red deer, point at this wild ungulate as a true reservoir for C. burnetii and an important node in the life cycle of C. burnetii, at least in the Iberian Peninsula.     

Pythons,parasites, and pests: anthropogenic impacts on Sarcocystis (Sarcocystidae) transmission in a multi‐host system

Parasites are essential components of ecosystems and can be instrumental in maintaining host diversity and populations; however, their role in trophic interactions has often been overlooked. Three apicomplexan parasite species of Sarcocystis (S. singaporensis, S. zamani, and S. villivillosi) use the reticulated python as their definitive hosts and several species within the Rattus genus as intermediate hosts, and they form a system useful for studying interactions between host–parasite and predator–prey relationships, as well as anthropogenic impacts on parasite transmission. Based on predictions from a 1998 survey, which detected an inverse relationship between urban development and Sarcocystis infection in Rattus, we tested the hypothesis that Sarcocystis transmission in Singapore will decrease over time due to anthropogenic activities. Despite a large proportion of the reticulated python diet consisting of Rattus species at all sizes of pythons, Sarcocystis infection rates decreased from 1998 to 2010. Pythons found in industrial areas had lower Sarcocystis infection rates, particularly in the western industrial area of Singapore Island. Average python size also decreased, with implications that we predict may disrupt host–parasite relationships. Anthropogenic activities such as habitat modification, fragmentation, and systematic removal and translocation of pythons have negative impacts on Sarcocystis transmission in Singapore, which in turn may augment pest rat populations. Trends observed may ultimately have negative impacts on human health and biodiversity in the region.     

Ultrastructure of the cyst wall of Sarcocystis spp. from some rodents in Malaysia.

The cyst wall of Sarcocystis cysts from the skeletal muscles and subcutaneous tissues of 4 species of rats and 1 species of bandicoot in Malaysia was examined under the electron microscope. Three types of sarcosysts with morphologically distinct cyst walls were found in these rodents. These morphologically distinct types of sarcocysts occurred as single or mixed infections in their various rodent hosts, suggesting that these rodents are important, though non-specific, intermediate hosts in the life cycle of at least 3 species of Sarcocystis. The final hosts of these Sarcocystis species are unknown.     

Molecular characterization of Sarcocystis spp. in seabirds from southern Brazil

Sarcocystis spp. are cyst forming apicomplexan parasites that infect many vertebrates including birds. Sarcocystis spp. infection was investigated in tissue samples (pectoral muscles, heart, and brain) of 47 dead seabirds collected from the coastline of Santa Catarina State SC – Brazil, between August 2019 and March 2020. A portion of each tissue was fixed in 10% buffered formalin for histopathologic analysis while DNA was extracted from another portion and screened using nested-PCR targeting ITS1. Based on molecular analysis, Sarcocystis spp. were identified in 15/47 (31.9%) seabirds of five species, kelp gull (Larus dominicanus), manx shearwater (Puffinus puffinus), neotropic cormorant (Phalacrocorax brasilianus), brown booby (Sula leucogaster) and great skua (Stercorarius skua). Microscopically visible sarcocysts were observed only in the pectoral muscle of four seabirds 8.5% (4/47), while in one brown booby, sarcocysts were seen in both pectoral and cardiac muscles. Two types of sarcocysts, thin walled (≤1 μm) and thick-walled (≥ 2 μm) were identified. Based on ITS1 sequence comparison, S. halieti, S. falcatula and three not yet described Sarcocystis spp. were detected. Phylogenetically, S. falcatula isolates were classified as two distinct clusters. This is the first confirmation of S. halieti in seabird's species in South America and S. falcatula in birds of the order Charadriiformes. Further molecular studies are needed to understand the epidemiology of the Sarcocystis spp. infection and its impact on the health of seabirds.     

Sarcocystis eothenomysi n. sp. (Apicomplexa: Sarcocystidae) from the large oriental vole Eothenomys miletus (Thomas) (Cricetidae: Microtinae) from Anning,China

Fifty-six oriental voles, Eothenomys miletus (Thomas), were collected in Anning prefecture of Yunnan Province (China) between March 2012 and December 2013 and examined for the presence of sarcocysts. Sarcosysts of a new species, Sarcocystis eothenomysi n. sp., were found in 14 out of 56 E. miletus (25%); they possessed a striated cyst wall, c.1–2 μm thick. Under transmission electron microscopy the cysts of S. eothenomysi exhibited numerous small, irregular protrusions, which may appear T-shaped in some sections. A phylogenetic analysis based on 18S rRNA gene sequences indicated that S. eothenomysi shares closest affinity with those species of Sarcocystis Lankester, 1982, which use cobra or viperid snakes as definitive hosts. We therefore, hypothesise that a venomous snake may serve as the definitive host for S. eothenomysi. This is the first species of Sarcocystis reported from Eothenomys spp.     

Sarcocystis caninum and Sarcocystis svanai n. spp. (Apicomplexa: Sarcocystidae) Associated with Severe Myositis and Hepatitis in the Domestic Dog (Canis familiaris)

There are several reports of Sarcocystis sarcocysts in muscles of dogs, but these species have not been named. Additionally, there are two reports of Sarcocystis neurona in dogs. Here, we propose two new names, Sarcocystis caninum, and Sarcocystis svanai for sarcocysts associated with clinical muscular sarcocystosis in four domestic dogs (Canis familiaris), one each from Montana and Colorado in the USA, and two from British Columbia, Canada. Only the sarcocyst stage was identified. Most of the sarcocysts identified were S. caninum. Sarcocysts were studied using light microscopy, transmission electron microscopy (TEM), and polymerase chain reaction. Based on collective results two new species, S. caninum and S. svanai were designated. Sarcocystis caninum and S. svanai were structurally distinct. Sarcocystis caninum sarcocysts were up to 1.2 mm long and up to 75 μm wide. By light microscopy, the sarcocyst wall was relatively thin and smooth. By TEM, the sarcocyst wall was “type 9”, 1–2 μm thick, and contained villar protrusions that lacked microtubules. Bradyzoites in sections were 7–9 μm long. Sarcocysts of S. svanai were few and were identified by TEM. Sarcocystis svanai sarcocysts were “type 1”, thin walled (< 0.5 μm), and the wall lacked villar protrusions but had tiny blebs that did not invaginate. DNA was extracted either from infected frozen muscle biopsies or formalin‐fixed paraffin‐embedded sections. Dogs were either singly infected with S. caninum or multiply co‐infected with S. caninum and S. svanai (the result of a mixed infection) based on multilocus DNA sequencing and morphology. BLASTn analysis established that the sarcocysts identified in these dogs were similar to, but not identical to Sarcocystis canis or Sarcocystis arctosi, parasites found to infect polar bears (Ursus maritimus) or brown bears (Ursus arctosi), respectively. However, the S. caninum sequence showed 100% identify over the 18S rRNA region sequenced to that of S. arctica, a parasite known to infect Arctic foxes (Vulpes lagopus).