C.A. Speer

A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM).

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in the Americas. The protozoan most commonly associated with EPM is Sarcocystis neurona. The complete life cycle of S. neurona is unknown, including its natural intermediate host that harbors its sarcocyst. Opossums (Didelphis virginiana, Didelphis albiventris) are its definitive hosts. Horses are considered its aberrant hosts because only schizonts and merozoites (no sarcocysts) are found in horses. EPM-like disease occurs in a variety of mammals including cats, mink, raccoons, skunks, Pacific harbor seals, ponies, and Southern sea otters. Cats can act as an experimental intermediate host harboring the sarcocyst stage after ingesting sporocysts. This paper reviews information on the history, structure, life cycle, biology, pathogenesis, induction of disease in animals, clinical signs, diagnosis, pathology, epidemiology, and treatment of EPM caused by S. neurona.

COMPLETION OF THE LIFE CYCLE OF SARCOCYSTIS NEURONA

Sarcocystis neurona is the most important cause of a neurologic disease in horses, equine protozoal myeloencephalitis (EPM). The complete life cycle of S. neurona, including the description of sarcocysts and intermediate hosts, has not been completed until now. Opossums (Didelphis spp.) are definitive hosts, and horses and other mammals are aberrant hosts. In the present study, laboratory-raised domestic cats (Felis domesticus) were fed sporocysts from the intestine of a naturally infected opossum (Didelphis virginiana). Microscopic sarcocysts, with a maximum size of 700 × 50 µm, developed in the muscles of the cats. The DNA of bradyzoites released from sarcocysts was confirmed as S. neurona. Laboratory-raised opossums (D. virginiana) fed cat muscles containing the sarcocysts shed sporocysts in their feces. The sporocysts were ∼10–12 × 6.5–8.0 µm in size. Gamma interferon knockout mice fed sporocysts from experimentally infected opossums developed clinical sarcocystosis, and S. neurona was identified in their tissues using S. neurona-specific polyclonal rabbit serum. Two seronegative ponies fed sporocysts from an experimentally-infected opossum developed S. neurona-specific antibodies within 14 days.

Isolation of a third species of Sarcocystis in immunodeficient mice fed feces from opossums (Didelphis virginiana) and its differentiation from Sarcocystis falcatula and Sarcocystis neurona.

Opossums (Didelphis virginiana) were found to be hosts for 3 species of Sarcocystis: Sarcocystis falcatula with an avian intermediate host, S. neurona with an undetermined intermediate host, and a third, unnamed, species. Sporocysts from the intestines of 2 opossums (nos. 26 and 47) were fed to budgerigars (Melopsittacus undulatus), nude mice, and gamma-interferon knockout (KO) mice. Sporocysts of S. falcatula were not infective to nude or KO mice. Sporocysts of S. neurona induced encephalitis in KO and nude mice; only schizonts and merozoites were found in tissues of mice, and they reacted with anti-S. neurona serum raised against the SN-2 isolate of S. neurona originally obtained from tissues of a paralyzed horse. All 3 species of Sarcocystis were present in opossum no. 47. Sarcocystis neurona was isolated in cell culture from this opossum. Sporocysts from opossum no. 47 were lethal to budgerigars, indicating S. falcatula infection. Only 1 species of Sarcocystis (the third species) was found in opossum no. 26; the sporocysts were infective to KO and nude mice. Schizonts and merozoites of this species were predominantly in the liver but were also found in other tissues; schizonts did not react with anti-S. neurona serum. Merozoites of the third species were ultrastructurally distinct from S. falcatula and S. neurona merozoites. Sarcocysts were found in leg muscles of 2 mice killed 50 and 54 days after they were fed sporocysts from opossum no. 26. These sarcocysts had steeple-shaped protrusions on the cyst wall and were distinct from sarcocysts of S. falcatula and any other species of Sarcocystis.

Ultrastructure of schizonts and merozoites of Sarcocystis neurona

The ultrastructure of Sarcocystis neurona schizonts and merozoites was studied in specimens derived from cell culture and from the brains of infected mice. Schizonts and merozoites were located in the host cell cytoplasm without a parasitophorous vacuole at any stage of development. Merozoites divided by endopolygeny. Fully formed merozoites had a pellicle, numerous polysomes and ribosomes, smooth and rough endoplasmic reticulum, 22 subpellicular microtubules, 9-16 dense granules, 25-75 micronemes, a plastid, a Golgi complex, 1-3 mitochondria, a conoid, 2 apical rings, 2 polar rings, 0-6 lipid bodies, a nucleus and nucleolus, but no rhoptries. Most micronemes were located anterior to the nucleus including 1-6 micronemes in the conoid. Merozoites were either slender (7.3 microm x 1.7 microm) or stumpy (7.7 microm x 3.1 microm). Dense granules appeared to arise from the maturation face of the Golgi complex. The ultrastructure of in vitro derived schizonts and merozoites were similar to in vivo derived organisms.

STRUCTURE OF SARCOCYSTIS NEURONA SARCOCYSTS

The ultrastructure of Sarcocystis neurona sarcocysts was studied from muscle of an experimentally infected cat. The cat was killed 144 days after being fed sporocysts from a naturally infected opossum. Sarcocysts were microscopic, up to 700 μm long, and up to 50 μm wide. By light microscopy, the sarcocyst wall was 1–2 μm thick. Ultrastructurally, the sarcocyst wall consisted of numerous villar protrusions. The villar protrusions were up to 2.8 μm long and 0.4 μm wide, with a tapered end. Microtubules extended from the tip of the villus to the base and occasionally extended deep into the granular layer. The granular layer was ∼0.5 μm thick. Longitudinally cut bradyzoites were 5.2 by 1.2 (4.8–6.5 by 1.0–1.3) μm in size. Micronemes in bradyzoites were numerous and located in the anterior ⅓ of the conoidal end.

Sarcocystis arieticanis and other Sarcocystis species in sheep in the United States.

Histological sections of tongues, esophagi, and diaphragms from 512 adult ewes from the northwest United States and Texas were examined for Sarcocystis spp. Sarcocysts were found in sections of 82.1% of 504 tongues, 44.4% of 478 esophagi, and 51.7% of 89 diaphragms. Sarcocystis tenella was the predominant species and was found in 430 (84.0%) sheep; S. arieticanis was found in 18 (3.5%) sheep. The mean number of S. tenella sarcocysts in tissue sections was approximately 10 times higher than that of S. arieticanis. The identification of S. arieticanis was confirmed by ultrastructural studies and by transmission to dogs. Macroscopic sarcocysts of S. gigantea were also found but were not quantitated in all sheep; sarcocysts of S. medusiformis were not observed.

Ultrastructural differentiation between sarcocysts of Sarcocystis hirsuta and Sarcocystis hominis

The ultrastructure of macroscopic (2-7 mm) Sarcocystis hirsuta sarcocysts from naturally infected cattle from New Zealand was compared with the ultrastructure of 222-day-old S. hominis in experimentally infected cattle in the United States. The villar protrusions of S. hirsuta were approximately 8 microns long, constricted at the base, expanded laterally in the mid-region and tapered distally. Some of the villar tips were folded to form two to four conical projections. The distal portion of the villar protrusions was bent at an angle of 45-90 degrees to the sarcocyst surface. The villar core contained numerous microfilaments and rows of electron-dense granules. The villar protrusions of S. hominis were cylindrical, oriented nearly perpendicularly to the sarcocyst surface, not constricted at their base and contained relatively few electron-dense granules. Although the sarcocysts of S. hirsuta were indistinguishable from those of S. hominis by light microscopy, they were distinguishable ultrastructurally.

Ultrastructure of sarcocysts from water buffalo in India

The ultrastructure of sarcocysts of macro- and microscopic species of Sarcocystis was compared from naturally infected water buffalo from India. Grossly visible sarcocysts had walls consisting of cauliflower-like villar protrusions, typical of S. fusiformis. The sarcocyst wall of the microscopic species of Sarcocystis was 6.4 microns thick and consisted of tightly packed conical villar protrusions that were 9.6 microns long and 3.7 microns wide at the base. At approximately 3 microns above the base, the distal two-thirds of the villar protrusion became conical shaped and was bent laterally at an angle of 45 degrees to the sarcocyst surface. The granular layer beneath the villar protrusions was 0.9 microns thick. In S. levinei the granular layer was 1.9 microns thick, the villar protrusions were narrow and it had a highly undulating primary cyst wall. Whether the microscopic S. levinei-like sarcocysts of Indian and Malaysian water buffalo are distinct species of Sarcocystis will require further investigation.

Ovine sporozoan encephalomyelitis linked to Sarcocystis infection

The unidentified sporozoan causing encephalomyelitis in sheep described by Hartley and Blakemore (1974) was restudied, and the parasite was identified as a Sarcocystis species based on its location and structure. The parasite was located free in the host-cell cytoplasm, divided by endopolygeny and mature merozoites lacked rhoptries.

Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture

An isolate of Sarcocystis neurona (SN7) was obtained from the spinal cord of a horse with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The organism divided by endopolygeny and completed at least one asexual cycle in cell cultures in 3 days. The parasite was maintained by subpassages in bovine monocytes for 10 months when it was found to be non-pathogenic to gamma interferon knockout (KO) mice. Revival of a low passage (10th passage) of the initial isolate stored in liquid nitrogen for 18 months retained its pathogenicity for KO mice. Merozoites (10(6)) of the late passage (22nd passage) were infective to only one of four KO mice inoculated. Similar results were obtained with SN6 isolate of S. neurona. No differences were found in Western blot patterns using antigens from the low and high passage merozoites of the SN7 and SN6 isolates. These results suggest that prolonged passage in cell culture may affect the pathogenicity of some isolates of S. neurona.