Morphological and molecular characterizations of Sarcocystis miescheriana and Sarcocystis suihominis in domestic pigs (Sus scrofa) in China.

In this study, 36.8% (28/76) of tissue samples collected from domestic pigs (Sus scrofa) contained sarcocysts, as determined by light microscopy. The organisms were identified as Sarcocystis miescheriana and Sarcocystis suihominis based on their morphological and molecular characteristics. Four genetic markers, i.e., 18S rDNA, 28S rDNA, ITS-1 region (ITS-1), and the mitochondrial COX1 gene (COX1), of the two parasites were sequenced and analyzed, and the 28S rDNA and ITS-1 of S. suihominis obtained from pigs constituted the first records of these markers in GenBank. The sequences of the four loci (18S rDNA, 28S rDNA, ITS-1, and COX1) of S. miescheriana shared high identities with those of S. miescheriana obtained from domestic and/or wild pigs in GenBank, with similarities of 99.6%, 99.6%, 95.9%, and 95.4%, respectively. The 18S rDNA sequences of S. suihominis exhibited 99.4% identity with those of S. suihominis from domestic and wild pigs. The comparison of the newly obtained sequences of the four genetic markers between the two parasites revealed that the interspecific similarities of 18S rDNA, 28S rDNA, ITS-1, and COX1 were 97.7%, 96.6%, 80.3%, and 81.2%, respectively. Therefore, the two species could be better discriminated with ITS-1 and mitochondrial COX1 compared with 18S rDNA or 28S rDNA. The phylogenetic analysis using 28S rDNA indicated that the two Sarcocystis species in domestic pigs had a close relationship.

Morphological and molecular description of Sarcocystis ratti n. sp. from the black rat (Rattus rattus) in Latvia.

Rodents have been widely studied as intermediate hosts of Sarcocystis; however, only a few reports on these parasites in the black rat (Rattus rattus) are known. Having examined 13 black rats captured in Latvia, sarcocysts were found in skeletal muscles of two mammals and were described as Sarcocystis ratti n. sp. Under a light microscope, sarcocysts were ribbon-shaped, 0.9-1.3 × 0.09-0.14 mm in size and had a thin (0.8-1.3 µm) and smooth cyst wall. The lancet-shaped bradyzoites were 8.3 × 4.3 (7.5-9.3 × 3.9-4.8) µm. Under a transmission electron microscope, the cyst wall was up to 1.3 µm thick, wavy, the ground substance appeared smooth, type 1a-like. Morphologically, sarcocysts of S. ratti were somewhat similar to those of S. cymruensis, S. rodentifelis, and S. dispersa-like previously identified in the brown rat (Rattus norvegicus). On the basis of 18S rDNA, 28S rDNA, and cox1, significant genetic differences (at least 2.3, 4.5, and 5.8%, respectively) were observed when comparing S. ratti with other Sarcocystis species using rodents as intermediate hosts. While ITS1 sequences of S. ratti were highly distinct from other Sarcocystis species available in GenBank. Phylogenetic and ecological data suggest that predatory mammals living near households are definitive hosts of S. ratti.

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.

Molecular characterization and development of Sarcocystis speeri sarcocysts in gamma interferon gene knockout mice.

The North American opossum (Didelphis virginiana) is the definitive host for at least three named species of Sarcocystis: Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. The South American opossums (Didelphis albiventris, Didelphis marsupialis and Didelphis aurita) are definitive hosts for S. falcatula and S. lindsayi. The sporocysts of these Sarcocystis species are similar morphologically. They are also not easily distinguished genetically because of the difficulties of DNA extraction from sporocysts and availability of distinguishing genetic markers. Some of these species can be distinguished by bioassay; S. neurona and S. speeri are infective to gamma interferon gene knockout (KO) mice, but not to budgerigars (Melopsittacus undulatus); whereas S. falcatula and S. lindsayi are infective to budgerigars but not to KO mice. The natural intermediate host of S. speeri is unknown. In the present study, development of sarcocysts of S. speeri in the KO mice is described. Sarcocysts were first seen at 12 days post-inoculation (p.i.), and they became macroscopic (up to 4 mm long) by 25 days p.i. The structure of the sarcocyst wall did not change from the time bradyzoites had formed at 50-220 days p.i. Sarcocysts contained unique villar protrusions, 'type 38'. The polymerase chain reaction amplifications and sequences analysis of three nuclear loci (18S rRNA, 28S rRNA and ITS1) and two mitochondrial loci (cox1 and cytb) of S. speeri isolate from an Argentinean opossum (D. albiventris) confirmed its membership among species of Sarcocystis and indicated an especially close relationship to another parasite in this genus that employs opossums as its definitive host, S. neurona. These results should be useful in finding natural intermediate host of S. speeri.

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

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

Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona.

Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes. IMPORTANCE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii.

Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease outbreaks.

GAMOGONY OF SARCOCYSTIS STRIXI IN MAMMALIAN CELL CULTURES.

We are interested in the disease ecology of Sarcocystis species that infect birds of prey as definitive and intermediate hosts. The present study was done to test our hypothesis that a laboratory model can be developed for sarcocystis infection in mammals using gamma interferon gene knockout (KO) mice as a source of Sarcocystis strixi bradyzoites and mammalian cell cultures as a source of sporulated S. strixi oocysts. Sporocysts of S. strixi from a naturally infected barred owl (Strix varia) were fed to KO mice to produce sarcocysts, and the enclosed bradyzoites were obtained by acid-pepsin digestion of abdominal and thigh muscles. Bradyzoites, metrocytes, and an unusual spherical stage were seen in digest before the inoculation of host cells. The spherical stages stained dark with Giemsa stain, but no nucleus was observed, and they were seen free and associated with the concave portion of some bradyzoites. Examination of infected cell cultures demonstrated that macrogamonts and microgamonts were present at 24 hr post-inoculation. Since sporulated oocysts were not observed, we had to reject our current hypothesis.

Corvid birds (Corvidae) act as definitive hosts for Sarcocystis ovalis in moose (Alces alces).

Epidemiological data and a unique phylogenetic position had suggested that Sarcocystis ovalis in moose and red deer might use a definitive host other than canids, felids, or humans. Corvid birds and rats were therefore evaluated as potential definitive hosts for this species in a small pilot study. Four laboratory rats were each inoculated with 10 or 25 sarcocysts of S. ovalis isolated from moose, but no Sarcocystis oocysts were detected in their intestinal mucosa upon euthanasia 2 to 3 weeks later. At a site where large flocks of corvid birds (hooded crows, ravens and European magpies) fed on remnants of moose carcasses during the hunting period in October, fresh bird droppings were collected on the ground and examined microscopically and by molecular methods. By microscopy, a small number of typical Sarcocystis sporocysts, measuring 12.8 × 8.4 µm, were found in the faecal samples. These sporocysts were identified as belonging to S. ovalis by a polymerase chain reaction assay using specific primer pairs targeting the ssu rRNA gene, followed by sequence analysis. The intestinal contents of a crow and two magpies shot near the dumping site were also examined. Sarcocystis oocysts (16.1 × 12.4 µm) and free sporocysts (12.5 × 7.9 µm) were found in the intestinal mucosa/contents of one magpie (Pica pica). These oocysts/sporocysts were also found to belong to S. ovalis by the same molecular assay. This is the first report of corvid birds acting as definitive hosts for a species of Sarcocystis.

Sarcocystosis among wild captive and zoo animals in Malaysia.

Sarcocystis sp. infection was investigated in 20 necropsied captive wild mammals and 20 birds in 2 petting zoos in Malaysia. The gross post-mortem lesions in mammals showed marbling of the liver with uniform congestion of the intestine, and for birds, there was atrophy of the sternal muscles with hemorrhage and edema of the lungs in 2 birds. Naked eye examination was used for detection of macroscopic sarcocysts, and muscle squash for microscopic type. Only microscopically visible cysts were detected in 8 animals and species identification was not possible. Histological examination of the sections of infected skeletal muscles showed more than 5 sarcocysts in each specimen. No leukocytic infiltration was seen in affected organs. The shape of the cysts was elongated or circular, and the mean size reached 254 x 24.5 µm and the thickness of the wall up to 2.5 µm. Two stages were recognized in the cysts, the peripheral metrocytes and large numbers of crescent shaped merozoites. Out of 40 animals examined, 3 mammals and 5 birds were positive (20%). The infection rate was 15% and 25% in mammals and birds, respectively. Regarding the organs, the infection rate was 50% in the skeletal muscles followed by tongue and heart (37.5%), diaphragm (25%), and esophagus (12.5%). Further ultrastructural studies are required to identify the species of Sarcocystis that infect captive wild animals and their possible role in zoonosis.

Molecular identification of four Sarcocystis species in the herring gull, Larus argentatus, from Lithuania.

BACKGROUND: Birds of the family Laridae have not been intensively examined for infections with Sarcocystis spp. To date, sarcocysts of two species, S. lari and S. wobeseri, have been identified in the muscles of gulls. The aim of the present study was to evaluate the species richness of Sarcocystis in the herring gull, Larus argentatus, from Lithuania. METHODS: In the period between 2013 and 2019, leg muscles of 35 herring gulls were examined for sarcocysts of Sarcocystis spp. Sarcocystis spp. were characterised morphologically based on a light microscopy study. Four sarcocysts isolated from the muscles of each infected bird were subjected to further molecular examination. Sarcocystis species were identified by means of ITS1 sequence analysis. RESULTS: Sarcocysts were detected in 9/35 herring gulls (25.7%). Using light microscopy, one morphological type of sarcocysts was observed. Sarcocysts were microscopic, thread-like, had a smooth and thin (about 1 µm) cyst wall and were filled with banana-shaped bradyzoites. On the basis of ITS1 sequences, four Sarcocystis species, S. columbae, S. halieti, S. lari and S. wobeseri, were identified. Furthermore, it was demonstrated that a single infected herring gull could host two Sarcocystis species indistinguishable under light microscopy. CONCLUSIONS: Larus argentatus is the first bird species found to act as intermediate host of four Sarcocystis spp. According to current knowledge, five species, S. falcatula, S. calchasi, S. wobeseri, S. columbae and S. halieti can use birds belonging to different orders as intermediate hosts.

Sarcocystis: development in cultured avian and mammalian cells.

Sarcocystis, a protozoan that parasitizes muscle tissue of reptiles, birds, and mammals, including man, developed in avian and mammalian cell cultlures. Mctile banana-shaped organisms, released from cysts in grackle muscle, entered cells and transformed into enlarged ellipsoid or oblong stages, which developed into either multinucleate or cyst-like stages, or both.

Gametogony of Sarcocystis sp. in cell culture.

Sexual stages and cystlike bodies of Sarcocystis sp., a protozoan parasite found in muscles of reptiles, birds, and mammals, including man, developed in cell culture. Motile organisms, obtained from leg muscles of wild grackles, were inoculated into cell line cultures of embryonic bovine kidney. Mature micro-and macrogametes and the cystlike forms were found 30 and 42 hours after inoculation respectively. These observations indicate that the parasite is probably a coccidium.

Life cycle of Sarcocystis camelicanis infecting the camel (Camelus dromedarius) and the dog (Canis familiaris), light and electron microscopic study.

In the present study, the heteroxeneous life cycle of Sarcocystis sp. infecting camels were studied. A total of 180 slaughtered camels collected from different localities in Egypt were investigated for sarcocysts. Only 116 animals were found to be infected (the infection rate was 64%). Muscle samples of esophagus, diaphragm, tongue, skeletal, and heart muscles were examined. Exclusively, microscopic sarcocysts were detected in all examined organs. The infection rates of the esophagus, diaphragm, tongue, skeletal, and heart muscles were 60%, 50%, 40%, 40%, and 10%, respectively. By means of transmission electron microscopy, details of the ultrastructure of the sarcocysts were studied. The specific architecture and ornaments of the cyst wall, its protrusions, and the cyst interior were recorded. Unique features of protrusions of the primary cyst wall, the knob-like structures, arise around each protrusion. Experimental infection of carnivores by feeding heavily infected camel muscles revealed that the dog, Canis familiaris, is the only final host of the present Sarcocystis species. Gamogony, sporogonic stages, and characteristics of sporulated oocysts were also investigated.

Effects of temperature, pH and curing on the viability of Sarcocystis, a Japanese sika deer (Cervus Nippon centralis) parasite, and the inactivation of their diarrheal toxin.

Recently, the Sarcocystis parasite in horse and deer meat has been reported to be a causative agent of acute food poisoning, inducing nausea, vomiting and diarrhea. Compared with other causative agents, such as bacteria, viruses and other parasites, in deer meat, the Sarcocystis species parasite, including its stability under various conditions, is poorly understood. In this study, we assessed the viability of Sarcocystis spp. and the activity of their diarrhea toxin (a 15-kDa protein) in deer meat under conditions of freezing, cold storage, pH change and curing. In addition, the heat tolerance was assayed using purified bradyzoites. The results showed that the species lost viability by freezing at -20, -30 and -80°C for <1 hr, heating at 70°C for 1 min, alkaline treatment (pH 10.0) for 4 days and addition of salt at 2.0% for <1 day. Immunoblot assays showed that the diarrhea toxin disappeared together with the loss of viability. However, the parasite survived cooling at 0 and 4°C and acidification (pH 3.0 and 5.0) for more than 7 days with the diarrhea toxin intact. These results provide useful information for developing practical applications for the prevention of food poisoning induced by diarrheal toxin of Sarcocystis spp. in deer meat during cooking and preservation.

High-throughput screen of drug repurposing library identifies inhibitors of Sarcocystis neurona growth.

The apicomplexan parasite Sarcocystis neurona is the primary etiologic agent of equine protozoal myeloencephalitis (EPM), a serious neurologic disease of horses. Many horses in the U.S. are at risk of developing EPM; approximately 50% of all horses in the U.S. have been exposed to S. neurona and treatments for EPM are 60-70% effective. Advancement of treatment requires new technology to identify new drugs for EPM. To address this critical need, we developed, validated, and implemented a high-throughput screen to test 725 FDA-approved compounds from the NIH clinical collections library for anti-S. neurona activity. Our screen identified 18 compounds with confirmed inhibitory activity against S. neurona growth, including compounds active in the nM concentration range. Many identified inhibitory compounds have well-defined mechanisms of action, making them useful tools to study parasite biology in addition to being potential therapeutic agents. In comparing the activity of inhibitory compounds identified by our screen to that of other screens against other apicomplexan parasites, we found that most compounds (15/18; 83%) have activity against one or more related apicomplexans. Interestingly, nearly half (44%; 8/18) of the inhibitory compounds have reported activity against dopamine receptors. We also found that dantrolene, a compound already formulated for horses with a peak plasma concentration of 37.8 ±â€¯12.8 ng/ml after 500 mg dose, inhibits S. neurona parasites at low concentrations (0.065 µM [0.036-0.12; 95% CI] or 21.9 ng/ml [12.1-40.3; 95% CI]). These studies demonstrate the use of a new tool for discovering new chemotherapeutic agents for EPM and potentially providing new reagents to elucidate biologic pathways required for successful S. neurona infection.

Isolation, Culture and Cryopreservation of Sarcocystis species.

More than 200 valid Sarcocystis species have been described in the parasitological literature. The developmental life cycle in the intermediate host and definitive host has only been described for a few species. Sarcocystis parasites are common pathogens infecting a wide range of animals, including humans, and this unit reviews the methods used for isolating infective stages of the parasite from both definitive and intermediate host(s), as well as methods used to initiate cultures from sporocysts and merozoites and for cryopreservation of various Sarcocystis spp. These methods are based on published reports and our experience with Sarcocystis species in cell culture over many years. The information presented is suitable for the efficient culture of many Sarcocystis species; however, some minor modifications may be needed based on the unique developmental patterns of some species. © 2017 by John Wiley & Sons, Inc.

Isolation, molecular characterization, and in vitro schizogonic development of Sarcocystis sp. ex Accipiter cooperii from a naturally infected Cooper's hawk (Accipiter cooperii).

Raptors serve as the definitive host for several Sarcocystis species. The complete life cycles of only a few of these Sarcocystis species that use birds of prey as definitive hosts have been described. In the present study, Sarcocystis species sporocysts were obtained from the intestine of a Cooper's hawk (Accipiter cooperii) and were used to infect cell cultures of African green monkey kidney cells to isolate a continuous culture and describe asexual stages of the parasite. Two clones of the parasite were obtained by limiting dilution. Asexual stages were used to obtain DNA for molecular classification and identification. PCR amplification and sequencing were done at three nuclear ribosomal DNA loci; 18S rRNA, 28S rRNA, and ITS-1, and the mitochondrial cytochrome c oxidase subunit 1 (cox1) locus. Examination of clonal isolates of the parasite indicated a single species related to S. columbae (termed Sarcocystis sp. ex Accipiter cooperii) was present in the Cooper's hawk. Our results document for the first time Sarcocystis sp. ex A. cooperii occurs naturally in an unknown intermediate host in North America and that Cooper's hawks (A. cooperii) are a natural definitive host.

Sarcocystis strixi n. sp. from a Barred Owl ( Strix varia) Definitive Host and Interferon Gamma Gene Knockout Mice as Experimental Intermediate Host.

Here we report a new species of Sarcocystis with a barred owl ( Strix varia) as the natural definitive host and interferon gamma gene knockout (KO) mice as an experimental intermediate host. A barred owl submitted to the Carolina Raptor Center, Huntersville, North Carolina, was euthanized because of paralysis. Fully sporulated 12.5 × 9.9 µm sporocysts were found in intestinal scrapings from the owl. Sporocysts from the barred owl were orally fed to 4 laboratory-reared outbred Swiss Webster (SW) ( Mus musculus) and 8 KO mice. All mice remained asymptomatic. Microscopic sarcocysts were found in all 5 KO mice euthanized on day 32, 59, 120, 154, and 206 post-inoculation (PI), not in KO mice euthanized on day 4, 8, and 14 PI. Sarcocysts were not found in any SW mice euthanized on day 72, 120, 206, and 210 PI. Sarcocysts were microscopic, up to 70 µm wide. By light microscopy, the sarcocyst wall < 2 µm thick had undulating, flat to conical, protrusions of varying dimensions. Numerous sarcocysts were seen in the histological sections of tongue and skeletal muscles from the abdomen, limbs, and eye but not in the heart. By transmission electron microscopy, the sarcocyst wall was "type 1j." The ground substance layer (gs) was homogenous, up to 2 µm thick, with very fine granules, and a few vesicles concentrated toward the villar projections. No microtubules were seen in the gs. Longitudinally cut bradyzoites at 206 days PI were 7.8 × 2.2 µm. Based on molecular characterization using 18S rRNA, 28S rRNA, and cox1 genes and morphology of sarcocysts, the parasite in the present study was biologically and structurally different from species so far described, and we therefore propose a new species name, Sarcocystis strixi n. sp.

Morphology, Molecular Characteristics, and Demonstration of a Definitive Host for Sarcocystis rommeli from Cattle (Bos taurus) in China.

Sarcocysts of Sarcocystis rommeli were found for the first time in 6 of 34 (17.6%) cattle (Bos taurus) in China. With light microscopy, sarcocysts of S. rommeli were up to 1,130 µm long, with a striated, 4-8-µm-thick cyst wall. Using transmission electron microscopy, the villar protrusions (vp) were 4.7-5.2 × 0.2-0.3 µm, and 0.3-0.5 µm apart from each other. The vp contained microtubules extending from the top of the vp to the middle of the ground substance layer (gsl). A BLAST search of the near full-length 18S rRNA and partial mitochondrial cox1 sequences of S. rommeli revealed 98.7% identity and 99.2% identity with sequences of Sarcocystis bovini in GenBank, respectively. Two domestic cats (Felis catus) fed sarcocysts of S. rommeli shed oocysts/sporocysts in their feces with a prepatent period of 14 to 15 days; the partial mitochondrial cox1 sequences of these oocysts/sporocysts shared the high identities, that is, 99.4% and 99.5%, with cox1 sequences of S. rommeli sarcocysts and S. bovini sarcocysts, respectively. This is the first demonstration of a definitive host for S. rommeli.