Evidence for Sarcocystis as the etiologic agent of equine protozoal myeloencephalitis

Equine protozoal myeloencephalitis (EPM) was diagnosed in 10 horses. By electron microscopy, schizonts were found in intact host cells of the spinal cords or, more frequently, free in the extracellular spaces. Developmental stages of schizonts differed morphologically, and the late stage of schizogony was characterized by endopolygeny. These findings permitted tentative identification of the protozoon as a Sarcocystis sp. Free merozoites were present in the extracellular spaces or in cells of the spinal cord. Pericytes of capillaries were most frequently parasitized by merozoites were present in the extracellular spaces or in cells of te spinal cord. Pericytes of capillaries were most frequently parasitized by merozoites, but the cytoplasm of neurons, macrophages, intravascular and tissue neutrophils, and axons of myelinated nerve fibers also contained these organisms. The presence of parasites in the cytoplasm of tissue and circulating neutrophils suggest that this putative Sarcocystis sp. may have a hematogenous phase of infection.     

Sarcocystis canis n. sp. (Apicomplexa: Sarcocystidae), the etiologic agent of generalized coccidiosis in dogs

Sarcocystis canis n. sp. is proposed for the protozoon associated with encephalitis, hepatitis, and generalized coccidiosis in dogs. Only asexual stages are known in macrophages, neurons, dermal, and other cells of the body. The parasite is located free in the host cell cytoplasm without a parasitophorous vacuole; schizonts divide by endopolygeny. Schizonts are 5-25 x 4-20 microns and contain 6-40 merozoites. Merozoites are approximately 5-7 microns x 1 micron and do not contain rhoptries. The parasite is PAS-negative and reacts with Sarcocystis cruzi antiserum but not with Toxoplasma gondii, Neospora caninum, or Caryospora bigenetica antisera in an immunohistochemical test.     

Isolation and characterization of microsatellite markers from Sarcocystis neurona,a causative agent of equine protozoal myeloencephalitis

The population genetics and systematics of coccidian parasites of the genus Sarcocystis remain poorly defined, notwithstanding their relevency to veterinary and human health. Despite opportunities for sexual recombination, nonrecombinant parasite clones characterized by distinct transmission and pathogenesis traits persist in related parasites (i.e. Toxoplasma gondii). In order to determine whether this may be generally true for parasitic coccidia, and to address evolutionary and taxonomic problems within the genus Sarcocystis, we isolated 12 polymorphic microsatellite markers (four to 14 alleles) for Sarcocystis neurona, the major causative agent of equine protozoal myeloencephalitis (EPM).     

An equine protozoal myeloencephalitis challenge model testing a second transport after inoculation with Sarcocystis neurona sporocysts

Previous challenge studies performed at Ohio State University involved a transport-stress model where the study animals were dosed with Sarcocystis neurona sporocysts on the day of arrival. This study was to test a second transportation of horses after oral inoculation with S. neurona sporocysts. Horses were assigned randomly to groups: group 1, transported 4 days after inoculation (DAI); group 2, at 11 DAI; group 3, at 18 DAI; and group 4, horses were not transported a second time (controls). An overall neurologic score was determined on the basis of a standard numbering system used by veterinarians. All scores are out of 5, which is the most severely affected animal. The mean score for the group 1 horses was 2.42; group 2 horses was 2.5; group 3 horses was 2.75; and group 4 horses was 3.25. Because the group 4 horses did not have a second transport, they were compared with all other groups. Statistically different scores were present between group 4 and groups 1 and 2. There was no difference in the time of seroconversion between groups. There was a difference between the time of onset of first clinical signs between groups 1 and 4. This difference was likely because of the different examination days. Differences in housing and handling were likely the reason for the differences in severity of clinical signs. This model results in consistent, significant clinical signs in all horses at approximately the same time period after inoculation but was most severe in horses that did not experience a second transport.     

Sarcocystis mephitisi n. sp. (Protozoa: Sarcocystidae), Sarcocystis neurona-like and Toxoplasma-like infections in striped skunks (Mephitis mephitis)

Two structurally distinct types (A, B) of microscopic sarcocysts were found in muscles of 4 of 5 feral skunks. Type A sarcocysts had sarcocyst walls of up to 6 microm thick. The villar protrusions (Vp) on the sarcocyst wall were up to 5 microm long. The Vp were constricted at the base, expanded in the middle, and had a blunt tip. Numerous microtubules were present in the Vp and in the granular layer. Bradyzoites were up to 11 microm long and up to 3.2 microm wide. Based on the distinctiveness of the Vp, a new name, Sarcocystis mephitisi is proposed for type A sarcocysts. Type B sarcocysts had a relatively thin (approximately 1-2 microm thick) sarcocyst wall and the Vp were slender and tapered toward the tip. These sarcocysts were structurally similar to S. neurona sarcocysts. A Toxoplasma gondii-like tissue cyst was found in a section of tongue of 1 of the 4 skunks.     

Sarcocystis dubeyella n. sp. and Sarcocystis phacochoeri n. sp. (Protozoa: Sarcocystidae) from the Warthog (Phacochoerus aethiopicus) in South Africa

ABSTRACT Sarcocystis dubeyella n. sp. and S. phacochoeri n. sp. from muscle fibers of the skeletal musculature of two warthogs in South Africa are described by light and and electron microscopy. Sarcocystis dubeyella sarcocysts are macroscopic (up to 12 mm long and 1 mm wide), with a parasite-induced encapsulation of the host muscle fiber in which the plasma membrane of the latter remained unaltered. The sarcocyst wall is characterized by evenly arranged, irregularly semicircular or rectangular villar protrusions (5.0 T. 2.8-11.0 μm) with indented margins and no specific content. Sarcocystis phacochoeri formed filiform microcysts (up to 4 mm long and 0.13 mm wide). Its cyst wall is provided with tightly packed, molarlike villar protrusions (1.6-3.3 T. 1.7-3.3 μm), with smooth margins, hollow on one side, and with longitudinal condensations of the fine granular matrix at various locations in the interior.     

In vitro cultivation of Sarcocystis neurona from the spinal cord of a horse with equine protozoal myelitis

Asexual stages of Sarcocystis neurona were seen in cultured bovine monocytes (M617) inoculated with tissue homogenates from the spinal cord of a horse with naturally acquired protozoal myelitis. Organisms first were observed as intracytoplasmic schizonts and later as motile extracellular zoites capable of infecting surrounding M617 cells. Parasites most often occurred as clusters of merozoites dispersed throughout the host cell cytoplasm; however, schizonts also contained merozoites arranged in a radial fashion surrounding a prominent residual body. Schizonts divided by endopolygeny. The parasite has been maintained beyond 280 days in the laboratory by serial passage of infected M617 cells.     

Sarcocystis neurona (Protozoa: Apicomplexa): description of oocysts, sporocysts, sporozoites, excystation, and early development

Equine protozoal myeloencephalitis is a major cause of neurological disease in horses from the Americas. Horses are considered accidental intermediate hosts. The structure of sporocysts of the causative agent, Sarcocystis neurona, has never been described. Sporocysts of S. neurona were obtained from the intestines of a laboratory-raised opossum fed skeletal muscles from a raccoon that had been fed sporocysts. Sporocysts were 11.3 by 8.2 microm and contained 4 sporozoites. The appearance of the sporocyst residuum was variable. The residuum of some sporocysts was composed of many dispersed granules, whereas some had granules mixed with larger globules. Excystation was by collapse of the sporocyst along plates. The sporocysts wall was composed of 3 layers: a thin electron-dense outer layer, a thin electron-lucent middle layer, and a thick electron-dense inner layer. The sporocyst wall was thickened at the junctions of the plates. Sporozoites were weakly motile and contained a centrally or posteriorly located nucleus. No retractile or crystalloid body was present, but lipidlike globules about 1 microm in diameter were usually present in the conoidal end of sporozoites. Sporozoites contained 2-4 electron-dense rhoptries and other organelles typical of coccidian zoites. Sporozoites entered host cells in culture and underwent schizogony within 3 days.     

The taxonomy of Sarcocystis (Protozoa, Apicomplexa) species

The taxonomy of the heteroxenous apicomplexan protozoan genus Sarcocystis was reviewed, and a list of 122 species with their synonyms and hosts given. Both definitive and intermediate hosts are known for only 56 species. The fine structure of the sarcocyst wall may change with age and is not considered necessarily satisfactory for separating species. Specificity for the intermediate host is not narrow for all species. Earlier work on transmission of the parasite from one intermediate host to another should be repeated in the light of present knowledge of the life cycle of species of Sarcocystis.     

Sarcocystis greineri n. sp. (Protozoa: Sarcocystidae) in the Virginia opossum (Didelphis virginiana).

Sarcocysts were found in the skeletal muscles of road-killed and live-trapped opossums collected in north central Florida. Sarcocysts were spindle-shaped and macroscopic and had an average measurement of 3.8 mm by 154.6 microm. Sarcocysts were only observed in skeletal muscle. Sarcocysts have invaginations throughout the sarcocyst wall, which is approximately 1 microm thick. Protrusions on the sarcocyst wall are stumpy and digitlike and contain fibrillar elements that extend from the interior portion of the cyst wall through the villi. A new name, Sarcocystis greineri, is proposed for this species.     

Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona.

Schizonts of Sarcocystis neurona were identified microscopically in hematoxylin-eosin-stained spinal cord sections from 2 native Panamanian horses that exhibited clinical signs of equine protozoal myelitis (EPM). Spinal cord homogenate from a third Panamanian horse with EPM was inoculated onto monolayers of cultured bovine monocytes (M617). Intracytoplasmic schizonts containing merozoites arranged in rosette forms surrounding a central residual body first were observed 13 wk postinoculation. Parasites divided by endopolygeny and lacked rhoptries. Schizonts from each horse reacted with Sarcocystis cruzi antiserum in an immunohistochemical test.     

Named species and hosts of Sarcocystis (Protozoa: Apicomplexa: Sarcocystidae)

Summary A list is given of the present 93 species of the apicomplexan protozoan genus Sarcocystis together with their definitive and intermediate hosts (if known), synonyms, homonyms, lapsi calami, etc. The names of many species of this genus are poorly known, in doubt or controversial due to lack of access to some of the literature and to failure to accept the International Code of Zoological Nomenclature.The following taxonomic innovations are introduced: New species—S. nontenella for S. tenella Eble, 1961 [non] S. tenella (Railliet, 1886) from the buzzard Buteo buteo; S. scotti for Sarcocystis sp. from the housemouse completing its sexual development in the tawny owl, Strix aluco (see Tadros & Laarman, 1980); New combinations—S. ctenosauris for Cryptosporidium ctenosauris Duszynski, 1969 from the lizard Ctenosaura similis; S. lampropeltis for Cryptosporodium lampropeltis Anderson, Duszynski & Marquardt, 1968 from the king snake Lampropeltis c. calligaster; S. roudabushi for Isospora roudabushi Pellérdy, 1974 from the gopher snake Pituophis s. sayi; and S. tropicalis for Isospora tropicalis Mukherjea & Krassner, 1965 from the golden jackal Canis aureus.Supported in part by National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland research grant AI15367.Supported in part by National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland research grant AI15367.     

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.     

Comparative epidemiology of coccidia: clues to the etiology of equine protozoal myeloencephalitis

The organism associated with EPM is a protozoan. It bears greatest resemblance to the genus Sarcocystis based on the mode of replication, staining characteristics, location in host cells, serology and epidemiology. However, attempts to produce the disease in equids by oral inoculation with many species of Sarcocystis have been unsuccessful. Because the distribution of EPM is limited to North America, further attempts to identify the organism and its source should focus on animal sources unique to that continent.     

Experimental induction of equine protozoan myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease

The effect of inoculation dose of Sarcocystis neurona sporocysts on the development of clinical neurologic disease in horses was investigated. Twenty-four seronegative weanling horses were subjected to the natural stress of transport and then randomly assigned to 6 treatment groups of 4 horses each. Horses were then immediately inoculated with either 10(2), 10(3), 10(4), 10(5), or 10(6) S. neurona sporocysts or placebo using nasogastric tube and housed indoors. Weekly neurologic examinations were performed by a blinded observer. Blood was collected weekly for antibody determination by Western blot analysis. Cerebrospinal fluid was collected before inoculation and before euthanasia for S. neurona antibody determination.Horses were killed and necropsied between 4 and 5 wk after inoculation. Differences were detected among dose groups based on seroconversion times, severity of clinical neurologic signs, and presence of microscopic lesions. Seroconversion of challenged horses was observed as early as 14 days postinfection in the 10(6) sporocyst dose group. Mild to moderate clinical signs of neurologic disease were produced in challenged horses from all groups, with the most consistent signs seen in the 10(6) sporocyst dose group. Histologic lesions suggestive of S. neurona infection were detected in 4 of the 20 horses fed sporocysts. Parasites were not detected in equine tissues by light microscopy, immunohistochemistry, or bioassay in gamma-interferon gene knockout mice. Control horses remained seronegative for the duration of the study and had no histologic evidence of protozoal infection.     

Selenidium fauchaldi sp. n. and S. telepsavi (Stuart) comb. nov. (Protozoa,Apicomplexa), Gregarines from Polychaetes*

Gregarine Selenidium fauchaldi sp. n. is described from the intestinal lumen of the polychaete Phragmatopoma californica (Fewkes, 1889) in the intertidal zone of the Pacific Ocean on Santa Catalina Island, California. In addition, the new combination Selenidium telepsavi (Stuart, 1871) comb. nov. is introduced for the gregarine originally described as Monocystis telepsavi from the polychaete Telepsavus castarum in the Black Sea.     

Sarcocystis lindsayi n. sp. (Protozoa: Sarcocystidae) from the South American opossum, Didelphis albiventris from Brazil

A new species, Sarcocystis lindsayi n. sp., is proposed for a parasite resembling Sarcocystis falcatula. It was obtained from the lungs and muscles of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris, from Jaboticabal, Brazil. Sarcocysts of S. lindsayi n. sp. in budgerigars are microscopic, up to 600 microm long and up to 50 microm wide. The cyst wall is up to 2 microm thick. Ultrastructurally, the sarcocyst wall consists of numerous slender villar protrusions (up to 2.0 microm long and up to 0.3 microm wide), each with a stylet at its tip. Schizonts in cell culture divide by endopolygeny leaving a residual body. Sporocysts are approximately 12 x 7 microm. The parasite is genetically distinct from other organisms that also cycle between opossums and avian species and resemble S. falcatula. Diagnostic genetic variation has been observed in the nuclear large subunit ribosomal RNA gene, the internal transcribed spacer (ITS-1), and each of two other genetic loci. Although the structure of the sarcocyst wall may not provide sufficient grounds for differential diagnosis, several other attributes including schizont morphology and genetic variation at each of these genetic loci permit identification of S. lindsayi n. sp.. Natural intermediate hosts for S. lindsayi n. sp. are not known, and fuller characterization of these and other Sarcocystis species would benefit from experimental avian hosts that are more permissive to the maturation of sarcocysts.     

Cochleomeritus emersoni sp. n. (Protozoa,Apicomplexa), a Lecudinid Gregarine from the Pacific Ocean Polychaete Diopatra ornata Moore, 1911*

The gregarine Cochleomeritus emersoni sp. n. is described from the intestine of the marine polychaete Diopatra ornata from the Pacific Ocean off Point Hueneme, California.