From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture.

Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.

A hybrid sequencing and assembly strategy for generating culture free Giardia genomes.

Giardia duodenalis is a pathogenic intestinal protozoan parasite of humans and many other animals. Giardia duodenalis is found throughout the world, and infection is known to have adverse health consequences for human and other mammalian hosts. Yet, many aspects of the biology of this ubiquitous parasite remain unresolved. Whole genome sequencing and comparative genomics can provide insight into the biology of G. duodenalis by helping to reveal traits that are shared by all G. duodenalis assemblages or unique to an individual assemblage or strain. However, these types of analyses are currently hindered by the lack of available G. duodenalis genomes, due, in part, to the difficulty in obtaining the genetic material needed to perform whole genome sequencing. In this study, a novel approach using a multistep cleaning procedure coupled with a hybrid sequencing and assembly strategy was assessed for use in producing high quality G. duodenalis genomes directly from cysts obtained from feces of two naturally infected hosts, a cat and dog infected with assemblage A and D, respectively. Cysts were cleaned and concentrated using cesium chloride gradient centrifugation followed by immunomagnetic separation. Whole genome sequencing was performed using both Illumina MiSeq and Oxford Nanopore MinION platforms. A hybrid assembly strategy was found to produce higher quality genomes than assemblies from either platform alone. The hybrid G. duodenalis genomes obtained from fecal isolates (cysts) in this study compare favorably for quality and completeness against reference genomes of G. duodenalis from cultured isolates. The whole genome assembly for assemblage D is the most contiguous genome available for this assemblage and is an important reference genome for future comparative studies. The data presented here support a hybrid sequencing and assembly strategy as a suitable method to produce whole genome sequences from DNA obtained from G. duodenalis cysts which can be used to produce novel reference genomes necessary to perform comparative genomics studies of this parasite.

Transmission electron microscopy on a case of Cyclospora cayetanensis infection from an immune-competent case confirms and extends prior detailed descriptions of its notably small endogenous stage.

Although infections with Cyclospora cayetanensis are prevalent worldwide, many aspects of this parasite's life cycle remain unknown. Humans are the only known hosts, existing information on its endogenous development has been derived from histological examination of only a few biopsy specimens. In histological sections, its stages are less than 10 µm, making definitive identification difficult. Here, confirmation of cyclosporiasis in a duodenal biopsy specimen from an 80-year-old man without any recognized immunodeficiency patient is reported. Asexual forms (schizonts) and sexual forms (gamonts) were located within enterocytes, including immature and mature schizonts, an immature male gamont and a female gamont. Merozoites were small (<5 µm × 1 µm) and contained two rhoptries, subterminal nucleus and numerous micronemes and amylopectin granules. These parasite stages were like those recently reported in the gallbladder of an immunocompromised patient, suggesting that the general life-cycle stages are not altered by immunosuppression.

Life Cycle and Transmission of Cyclospora cayetanensis: Knowns and Unknowns.

Although infections with Cyclospora cayetanensis are prevalent worldwide, many aspects of this parasite's life cycle and transmission remain unknown. Humans are the only known hosts of this parasite. Existing information on its endogenous development has been derived from histological examination of only a few biopsy specimens. Its asexual and sexual stages occur in biliary-intestinal epithelium. In histological sections, its stages are less than 10 µm, making definitive identification difficult. Asexual (schizonts) and sexual (gamonts) are located in epithelial cells. Male microgamonts have two flagella; female macrogametes contain wall-forming bodies. Oocysts are excreted in feces unsporulated. Sporulation occurs in the environment, but there are many unanswered questions concerning dissemination and survival of C. cayetanensis oocysts. Biologically and phylogenetically, C. cayetanensis closely resembles Eimeria spp. that parastize chickens; among them, E. acervulina most closely resembles C. cayetanensis in size. Here, we review known and unknown aspects of its life cycle and transmission and discuss the appropriateness of surrogates best capable of hastening progress in understanding its biology and developing mitigating strategies.

Blue mussel (Mytilus edulis)-A bioindicator of marine water contamination by protozoa: Laboratory and in situ approaches.

AIMS: The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii are identified as public health priorities and are present in a wide variety of environments including the marine ecosystem. The objective of this study was to demonstrate that the marine bivalve blue mussel (Mytilus edulis) can be used as a tool to monitor the contamination of marine waters by the three protozoa over time. METHODS AND RESULTS: In order to achieve a proof of concept, mussels were exposed to three concentrations of G. duodenalis cysts and Cryptosporidium parvum/T. gondii oocysts for 21 days, followed by 21 days of depuration in clear water. Then, natural contamination by these protozoa was sought for in wild marine blue mussels along the northwest coast of France to validate their relevance as bioindicators in the field. Our results highlighted that: (a) blue mussels bioaccumulated the parasites for 21 days, according to the conditions of exposure, and parasites could still be detected during the depuration period (until 21 days); (b) the percentage of protozoa-positive M. edulis varied under the degree of protozoan contamination in water; (c) mussel samples from eight out of nine in situ sites were positive for at least one of the protozoa. CONCLUSIONS: The blue mussel M. edulis can bioaccumulate protozoan parasites over long time periods, according to the degree of contamination of waters they are inhabiting, and can highlight recent but also past contaminations (at least 21 days). SIGNIFICANCE AND IMPACT OF THE STUDY: Mytilus edulis is a relevant bioaccumulators of protozoan (oo)cysts in laboratory and field conditions, hence its potential use for monitoring parasite contamination in marine waters.

Seroepidemiology of Toxoplasma gondii in wild ruminants in Spain.

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii which infects warm-blooded species worldwide. Humans can be infected through ingestion of tissue cysts from raw or undercooked meat, including game meat. A nationwide large-scale cross-sectional study was conducted to assess exposure to T. gondii in seven wild ruminant species in Spain. A total of 2,040 serum samples from 77 sampling sites randomly distributed in the five bioregions (BRs) covering mainland Spain were tested for antibodies against T. gondii using the modified agglutination test. The overall seroprevalence was 22.0% (449/2,040). Seroprevalence by species in decreasing order was as follows: 39.6% (141/356) in roe deer (Capreolus capreolus), 37.1% (138/372) in fallow deer (Dama dama), 16.6% (92/553) in red deer (Cervus elaphus), 14.0% (26/186) in Southern chamois (Rupicapra pyrenaica), 11.5% (24/209) in mouflon (Ovis aries musimon), 7.8% (27/346) in Iberian wild goat (Capra pyrenaica) and 5.6% (1/18) in Barbary sheep (Ammotragus lervia). Seropositivity was detected in 74.0% (57/77) of the sampling sites. Results indicate widespread but not homogeneous exposure to T. gondii in wild ruminant populations in Spain during the last two decades and highlight differences related to animal species and spatial distribution of these species in this country; this implies potential consequences of T. gondii for animal health, conservation and public health.

Outbreaks of clinical toxoplasmosis in humans: five decades of personal experience, perspectives and lessons learned.

BACKGROUND: The protozoan parasite Toxoplasma gondii has a worldwide distribution and a very wide host range, infecting most warm-blooded hosts. Approximately 30% of humanity is infected with T. gondii, but clinical toxoplasmosis is relatively infrequent. Toxoplasmosis has a wide range of clinical symptoms involving almost all organ systems. In most persons that acquire infection postnatally, symptoms (when present) are mild and mimic other diseases such as flu, Lyme disease, Q fever, hematological alterations, or mumps. It is likely that clinical disease is more common than reported. The ingestion of infected meat or food and water contaminated with oocysts are the two main modes of postnatal transmission of Toxoplasma gondii. The infective dose and the incubation period of T. gondii infection are unknown because there are no human volunteer experiments. METHODS: Here, I have critically reviewed outbreaks of clinical toxoplasmosis in humans for the past 55 years, 1966-2020. Information from oocyst-acquired versus meat-acquired infections was assessed separately. RESULTS: Most outbreaks were from Brazil. There were no apparent differences in types or severity of symptoms in meat- versus oocyst-acquired infections. Fever, cervical lymphadenopathy, myalgia, and fatigue were the most important symptoms, and these symptoms were not age-dependent. The incubation period was 7-30 days. A genetic predisposition to cause eye disease is suspected in the parasites responsible for three outbreaks (in Brazil, Canada, and India). Only a few T. gondii tissue cysts might suffice to cause infection, as indicated by outbreaks affecting some (but not all) individuals sharing a meal of infected meat. CONCLUSIONS: Whether the high frequency of outbreaks of toxoplasmosis in humans in Brazil is related to environmental contamination, poor hygiene, socioeconomic conditions, or to genotypes of T. gondii needs investigation.

Systemic Toxoplasmosis in a Horse.

An adult American Quarter Horse gelding with a history of weight loss presented with an acute onset of colic, fever, soft faeces and elevated liver enzymes. At necropsy, there were gastric mucosal masses and evidence of caecal necrosis. Histologically, the masses were lymph nodes with granulomatous inflammation and areas of liquefactive necrosis. Within and surrounding necrotic areas were free and intrahistiocytic clusters of protozoal tachyzoites. Similar but milder inflammation was evident in the spleen, lungs and liver. Necrotizing typhlitis was also evident. Immunolabelling for Toxoplasma gondii was positive and the ultrastructural morphology of the protozoa was compatible with T. gondii. Although studies have shown seropositivity to T. gondii in horses throughout the world, this is the first report of clinical toxoplasmosis in this species.

Dynamics of Toxoplasma gondii Oocyst Phagocytosis by Macrophages.

Oocysts are the environmentally resistant stage of the protozoan parasite Toxoplasma gondii. They are responsible for foodborne infections in humans and animals worldwide. Infectious oocysts contain sporozoites that have to exit the sporocyst and oocyst walls to initiate replication of the parasite within the host tissues. Given their robustness and resistance to chemical degradation, it is still unclear how the oocyst and sporocyst walls release the sporozoites. This process called excystation is thought to occur in the small intestine as a result of the combined action of digestive agents, yet to be identified. By using an oocyst-macrophage co-culture platform, we previously demonstrated in vitro that the excystation of sporozoites and their differentiation into replicative tachyzoites could occur in absence of digestive factors, following phagocytosis by macrophages. Here, we further characterize the dynamics of the oocyst phagocytosis at the single-cell level by using optical tweezers and micropipette aspiration techniques. Our results show that the oocyst internalization kinetics can vary among a given population of macrophages, but similar processes and dynamics could be observed. Most of the cells manipulate oocysts for ~15 min before internalizing them in typically 30 min. This process mainly involves the actin cytoskeleton of the macrophages. Liberated sporozoites within macrophages then differentiate into tachyzoites within 4-6 h following oocyst-macrophage contact. Tachyzoites appear to develop better in macrophages challenged with free sporocysts or sporozoites than with whole oocysts, suggesting that opening of the oocyst wall is one of the most limiting steps for sporozoite excystation completion.

Distribution of Toxoplasma gondii Tissue Cysts in Shoulder Muscles of Naturally Infected Goats and Lambs.

ABSTRACT: Toxoplasmosis has been recognized as a major public health problem worldwide. The consumption of uncooked or undercooked meat infected with Toxoplasma gondii tissue cysts is one of the main means of transmission of this parasite. Although sheep, goats, and pigs are commonly infected with T. gondii, little information is available on the distribution of T. gondii tissue cysts in naturally infected meat. In this study, we investigated the distribution of viable T. gondii tissue cysts in shoulder muscles of naturally infected lambs and goats. Hearts and shoulders of 46 lambs and 39 goats from a local grocery store were tested for T. gondii infection. Animals were evaluated for the presence of anti-T. gondii antibodies in heart blood and clots by the modified agglutination test. Fourteen of the 85 animals (seven lambs and seven goats) were seropositive. Six to 12 samples weighing 5, 10, and 50 g were obtained from shoulder muscles of each seropositive animal and used for bioassay in mice. The distribution of viable T. gondii differed according to the size of the sample analyzed, but in general larger sample sizes resulted in higher isolation rates (P < 0.05). Results of the study revealed an uneven distribution of T. gondii in muscle samples of lambs and goats and that T. gondii can be transmitted by consumption of very small servings (5 and 10 g) of meat when it is consumed raw or is undercooked.

Comparative evaluation of loop-mediated isothermal amplification (LAMP) vs qPCR for detection of Toxoplasma gondii oocysts DNA in mussels.

The apicomplexan parasite Toxoplasma gondii can infect humans and cause toxoplasmosis. T. gondii has been highly prioritized among the foodborne parasites regarding its global impact on public health. Human infection can occur through multiple routes, including the ingestion of raw or undercooked food contaminated with T. gondii oocysts, such as fresh produce and bivalves. As filter-feeders, bivalves can accumulate and concentrate contaminants, including protozoan (oo)cysts. Although detection of T. gondii in different bivalves by molecular techniques (PCR and qPCR) has been achieved, routine application is currently limited by lack of sensitivity or equipment costs. Here, we describe the assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect T. gondii oocysts in spiked mussels. Detection limit was down to 5 oocysts/g in tissue and 5 oocyst/ml in hemolymph, and, under the experimental conditions tested, LAMP was found to provide a promising alternative to qPCR.

Lamb as a potential source of Toxoplasma gondii infection for Australians.

OBJECTIVE: Toxoplasmosis may follow consumption of undercooked meat containing Toxoplasma gondii cysts. Lamb is considered to pose the highest risk for contamination across meats. Red meat is often served undercooked, yet there are no current data on T. gondii contamination of Australian sourced and retailed lamb. We sought to address this gap in public health knowledge. METHODS: Lamb mincemeat was purchased at the supermarket counter three times weekly for six months. T. gondii was detected by real-time polymerase chain reaction (PCR) of DNA extracted from the meat following homogenisation. Purchases were also tested for common foodborne bacterial pathogens. RESULTS: Conservative interpretation of PCR testing (i.e. parasite DNA detected in three of four tests) gave a probability of 43% (95% confidence interval, 32%-54%) that lamb mincemeat was contaminated with T. gondii. None of the purchases were contaminated with Campylobacter jejuni, Salmonella species or S. enterica serovar Typhimurium, indicating sanitary meat processing. CONCLUSIONS: Australian lamb is commonly contaminated with T. gondii. Future studies should be directed at testing a range of red meats and meat cuts. Implications for public health: Consuming undercooked Australian lamb has potential to result in toxoplasmosis. There may be value in health education around this risk.

Use of the bivalve Dreissena polymorpha as a biomonitoring tool to reflect the protozoan load in freshwater bodies.

Cryptosporidium parvum, Toxoplasma gondii and Giardia duodenalis are worldwide pathogenic protozoa recognized as major causal agents of waterborne disease outbreaks. To overcome the normative process (ISO 15553/2006) limitations of protozoa detection in aquatic systems, we propose to use the zebra mussel (Dreissena polymorpha), a freshwater bivalve mollusc, as a tool for biomonitoring protozoan contamination. Mussels were exposed to three concentrations of C. parvum oocysts, G. duodenalis cysts or T. gondii oocysts for 21 days followed by 21 days of depuration in clear water. D. polymorpha accumulated protozoa in its tissues and haemolymph. Concerning T. gondii and G. duodenalis, the percentage of protozoa positive mussels reflected the contamination level in water bodies. As for C. parvum detection, oocysts did accumulate in mussel tissues and haemolymph, but in small quantities, and the limit of detection was high (between 50 and 100 oocysts). Low levels of T. gondii (1-5 oocysts/mussel) and G. duodenalis (less than 1 cyst/mussel) were quantified in D. polymorpha tissues. The ability of zebra mussels to reflect contamination by the three protozoa for weeks after the contamination event makes them a good integrative matrix for the biomonitoring of aquatic ecosystems.

Global selective sweep of a highly inbred genome of the cattle parasite Neospora caninum.

Neospora caninum, a cyst-forming apicomplexan parasite, is a leading cause of neuromuscular diseases in dogs as well as fetal abortion in cattle worldwide. The importance of the domestic and sylvatic life cycles of Neospora, and the role of vertical transmission in the expansion and transmission of infection in cattle, is not sufficiently understood. To elucidate the population genomics of Neospora, we genotyped 50 isolates collected worldwide from a wide range of hosts using 19 linked and unlinked genetic markers. Phylogenetic analysis and genetic distance indices resolved a single genotype of N. caninum Whole-genome sequencing of 7 isolates from 2 different continents identified high linkage disequilibrium, significant structural variation, but only limited polymorphism genome-wide, with only 5,766 biallelic single nucleotide polymorphisms (SNPs) total. Greater than half of these SNPs (∼3,000) clustered into 6 distinct haploblocks and each block possessed limited allelic diversity (with only 4 to 6 haplotypes resolved at each cluster). Importantly, the alleles at each haploblock had independently segregated across the strains sequenced, supporting a unisexual expansion model that is mosaic at 6 genomic blocks. Integrating seroprevalence data from African cattle, our data support a global selective sweep of a highly inbred livestock pathogen that originated within European dairy stock and expanded transcontinentally via unisexual mating and vertical transmission very recently, likely the result of human activities, including recurrent migration, domestication, and breed development of bovid and canid hosts within similar proximities.

Coccidiosis in dogs-100 years of progress.

Until 1970, coccidian parasites of dogs were considered to have a direct fecal-oral life cycle like Eimeria in poultry. They were thought to be non-host specific and infect both dogs and cats. Studies conducted in the 1970s revealed that dog coccidia were host-specific and had transport or paratenic hosts that were infected with an encysted stage containing a single organism, the monozoic tissue cyst. There are still considerable confusion and uncertainties concerning the life cycles and pathogenicity of coccidian parasites of dogs. The present paper reviews the history, taxonomy, life cycles, pathogenicity, epidemiology, diagnosis, and treatment of conventional coccidian parasites previously called Isospora spp., currently designated Cystoisospora spp. that infect canines.

Structure, composition, and roles of the Toxoplasma gondii oocyst and sporocyst walls.

Toxoplasma gondii is a coccidian parasite with the cat as its definitive host but any warm-blooded animal, including humans, may act as intermediate hosts. It has a worldwide distribution where it may cause acute and chronic toxoplasmosis. Infection can result from ingestion either of tissue cysts in infected meat of intermediate hosts or oocysts found in cat faeces via contaminated water or food. In this review, we highlight how the oocyst and sporocyst walls sustain the persistence and transmission of infective T. gondii parasites from terrestrial and aquatic environments to the host. We further discuss why targeting the oocyst wall structure and molecules may reduce the burden of foodborne and waterborne T. gondii infections.

EXPOSURE OF ALASKA BROWN BEARS (URSUS ARCTOS) TO BACTERIAL, VIRAL, AND PARASITIC AGENTS VARIES SPATIOTEMPORALLY AND MAY BE INFLUENCED BY AGE.

We collected blood and serum from 155 brown bears (Ursus arctos) inhabiting five locations in Alaska, US during 2013-16 and tested samples for evidence of prior exposure to a suite of bacterial, viral, and parasitic agents. Antibody seroprevalence among Alaska brown bears was estimated to be 15% for Brucella spp., 10% for Francisella tularensis, 7% for Leptospira spp., 18% for canine adenovirus type 1 (CAV-1), 5% for canine distemper virus (CDV), 5% for canine parvovirus, 5% for influenza A virus (IAV), and 44% for Toxoplasma gondii. No samples were seropositive for antibodies to Trichinella spp. Point estimates of prior exposure to pathogens among brown bears at previously unsampled locations generally fell within the range of estimates for previously or contemporaneously sampled bears in Alaska. Statistical support was found for variation in antibody seroprevalence among bears by location or age cohort for CAV-1, CDV, IAV, and T. gondii. There was limited concordance in comparisons between our results and previous serosurveys regarding spatial and age-related trends in antibody seroprevalence among Alaska brown bears suggestive of temporal variation. However, we found evidence that the seroprevalence of CAV-1 antibodies is consistently high in bears inhabiting southwest Alaska and the cumulative probability of exposure may increase with age. We found evidence for seroconversion or seroreversion to six different infectious agents in one or more bears. Results of this study increase our collective understanding of disease risk to both Alaska brown bear populations and humans that utilize this resource.

A review of Cystoisospora felis and C. rivolta-induced coccidiosis in cats.

Until the discovery of Toxoplasma gondii oocysts in cat feces in 1970, little was known of coccidiosis in cats. Until 1970, three coccidian parasites based on different sized oocysts were recognized, the parasite with large oocysts (∼40 µm long and called Isospora felis), medium sized oocysts (∼25 µm long, called Isospora rivolta), and small sized oocysts (14 µm or less, called Isospora bigemina) were known and they were considered not host-specific. Later, it was demonstrated that these parasites were host-specific and had also extra-intestinal stages. The Isospora bigemina turned out to be more than 25 organisms belonging to T. gondii, Hammondia spp., Sarcocystis spp., Besnoitia spp., and Neospora spp.; these subjects have been reviewed previously in detail. The present paper summarizes biology of Isospora felis, and I. rivolta (now transferred to genus Cystoisospora), including taxonomy, life cycle, diagnosis, and treatment. Re-excretion of T. gondii oocysts from chronically infected cats after superinfection with Cystoisospora felis oocysts is discussed. There are only two species of Cystoisospora species in cats, C. felis and C. rivolta; Isospora novocati and Cystoisospora frenkeli named for I. rivolta-like parasites of cats are considered synonym of C. rivolta. Clinical coccidiosis occurs more commonly in recently weaned kittens and C. felis infections are more prevalent than C. rivolta.

Histopathological, morphological, and molecular characterization of Sarcocystis species in elk (Cervus elaphus) from Pennsylvania, USA.

Sarcocystis sarcocysts are common in many species of domestic and wild animals. Here, we report sarcocystosis in muscles from 91 free range elk (Cervus elaphus) from Pennsylvania, USA, tested by histopathology, transmission electron microscopy (TEM), and DNA sequencing. Sarcocysts were detected in hematoxylin and eosin (HE)-stained sections from 83 of 91 (91.2%) elk, including 83/91 (91.2%) tongues and 15/17 (88.2%) hearts. With respect to age, sarcocysts were found in 0/5 calves, 8/9 (88.8%) yearlings, and 75/77 (97.4%) adults. Sarcocysts were identified in 62/69 (89.4%) females and 21/22 (91.2%) males. Associated lesions were mild and consisted of inflammatory foci around degenerate sarcocysts. There were two morphologically distinct sarcocysts based on wall thickness, thin (< 0.5 µm) and thick-walled (> 4.0 µm). Thin-walled sarcocysts had a TEM "type 2" and villar protrusions (vps), identical to Sarcocystis wapiti previously described from elk in western USA. This species was present both in tongue and heart samples and was detected in all infected elk. Thick-walled sarcocysts consisted of three morphologic variants, referred to herein as subkinds A, B, C. Subkind A sarcocysts were rare; only four sarcocysts were found in three elk. Histologically, they had a 5-8-µm thick wall with tufted vp. By TEM, the sarcocyst wall was "type 12" and appeared similar to Sarcocystis sybillensis, previously described from elk in USA. Subkind B, Sarcocystis sp.1 sarcocysts were also rare, found in only 1 elk. These sarcocysts had 6.7-7.3-µm-thick wall with TEM "type 15b" vp. Subkind C Sarcocystis sp.2 sarcocysts were more common (22/91). Morphologically, the sarcocyst wall was 6.1-6.8 µm thick and contained "type 10b" vp. Comparisons of ribosomal DNA loci with published sequences indicated all sarcocysts were similar to what has previously been isolated from cervid hosts across the northern hemisphere. Phylogenetic analysis placed the thin-walled S. wapiti within a strongly supported clade with S. linearis and S. taeniata, while the thick-walled cysts were very closely related to S. truncata, S. elongata, S. silva, and S. tarandi. Further sequencing is needed to produce molecular diagnostics to distinguish among these species. North American elk are hosts to multiple Sarcocystis species with diverse morphology, deriving from two separate evolutionary lineages.