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.     

Sarcocystis neurona retinochoroiditis in a sea otter (Enhydra lutris kenyoni)

Sarcocystis neurona is an important cause of fatal disease in sea otters in the USA. Encephalitis is the predominant lesion and parasites are confined to the central nervous system and muscles. Here we report retinochoroiditis in a sea otter (Enhydra lutris kenyoni) found dead on Copalis Beach, WA, USA. Salient lesions were confined to the brain and eye. Multifocal nonsuppurative meningoencephalitis was present in the cerebrum and cerebellum associated with S. neurona schizonts. The retina of one eye had a focus of inflammation that contained numerous S. neurona schizonts and merozoites. The focus extended from the retinal pigment epithelium inward through all layers of the retina, but inflammation was most concentrated at the inner surface of the tapetum and the outer retina. The inner and outer nuclear layers of the retina were disorganized and irregular at the site of inflammation. There was severe congestion and mild hemorrhage in the choroid, and mild hemorrhage into the vitreous body. Immunohistochemistry with S. neurona-specific polyclonal rabbit antibodies stained schizonts and merozoites. To our knowledge this is the first report of S. neurona-associated retinochoroiditis in any naturally infected animal.     

Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum infections in dogs

Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum are related apicomplexans that can cause systemic illness in many species of animals, including dogs. We investigated one breeder's 25 Basset Hounds for these infections. In addition, tissues from dogs and other non-canine hosts previously reported as S. canis infections were studied retrospectively. Schizonts resembling those of S. neurona, and recognized by polyclonal rabbit anti-S. neurona antibodies, were found in six of eight retrospective cases, as well as in two additional dogs (one Basset Hound, one Springer Spaniel) not previously reported. S. neurona schizonts were found in several tissues including the central nervous system, lungs, and kidneys. Fatal toxoplasmosis was diagnosed in an adult dog, and neosporosis was diagnosed in an adult and a pup related to the one diagnosed with S. neurona. No serological reactivity to S. neurona antibodies occurred when S. canis-like liver schizonts were retrospectively assayed from two dogs, a dolphin, a sea lion, a horse, a chinchilla, a black or either of two polar bears. Sequencing conserved (18S) and variable (ITS-1) portions of nuclear ribosomal DNA isolated from the schizont-laden liver of a polar bear distinguished it from all previously characterized species of Sarcocystis. We take this genetic signature as provisionally representative of S. canis, an assumption that should be tested with future sequencing of similar liver infections in other mammalian hosts. These findings further extend the uncharacteristically broad intermediate host range for S. neurona, which also causes a neurologic disease in cats, mink, raccoons, skunks, Pacific harbor seals, ponies, zebras, lynxes, and sea otters. Further work is necessary to delineate the causative agent(s) of other cases of canine sarcocystosis, and in particular to specify the attributes of S. canis, which corresponds morphologically to infections reported from wide range of terrestrial and marine mammals.     

Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites

An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.     

Evidence to support horses as natural intermediate hosts for Sarcocystis neurona

Opossums (Didelphis spp.) are the definitive host for the protozoan parasite Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). Opossums shed sporocysts in feces that can be ingested by true intermediate hosts (cats, raccoons, skunks, armadillos and sea otters). Horses acquire the parasite by ingestion of feed or water contaminated by opossum feces. However, horses have been classified as aberrant intermediate hosts because the terminal asexual sarcocyst stage that is required for transmission to the definitive host has not been found in their tissues despite extensive efforts to search for them [Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001b. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol. 95, 89-131]. In a 4-month-old filly with neurological disease consistent with EPM, we demonstrate schizonts in the brain and spinal cord and mature sarcocysts in the tongue and skeletal muscle, both with genetic and morphological characteristics of S. neurona. The histological and electron microscopic morphology of the schizonts and sarcocysts were identical to published features of S. neurona [Stanek, J.F., Dubey, J.P., Oglesbee, M.J., Reed, S.M., Lindsay, D.S., Capitini, L.A., Njoku, C.J., Vittitow, K.L., Saville, W.J., 2002. Life cycle of Sarcocystis neurona in its natural intermediate host, the raccoon, Procyon lotor. J. Parasitol. 88, 1151-1158]. DNA from schizonts and sarcocysts from this horse produced Sarcocystis specific 334bp PCR products [Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Greiner, E.C., MacKay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J. Parasitol. 85, 221-228]. Restriction fragment length polymorphism (RFLP) analysis of these PCR products showed banding patterns characteristic of S. neurona. Sequencing, alignment and comparison of both schizont and sarcocyst DNA amplicons showed 100% identity. Although Koch's postulates have not been demonstrated in this case study, the finding of mature, intact S. neurona schizonts and sarcocysts in the tissues of this single horse strongly suggests that horses have the potential to act as intermediate hosts. Further studies are needed to demonstrate Koch's postulates with repeated transfer of S. neurona between opossums and horses.     

Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum

Migration and development of Sarcocystis neurona was studied in 50 gamma interferon knockout mice fed graded doses of S. neurona sporocysts from the intestine of a naturally infected opossum. Mice were examined at necropsy 1-62 days after feeding sporocysts (DAFS). All tissue sections were reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical (IHC) test. Between 1 and 3 DAFS, organisms were seen mainly in intestines. Between 4 and 11 DAFS, organisms were seen in several visceral tissues. Beginning with 13 DAFS, schizonts and merozoites were present in sections of brains of all infected mice. All regions of the brain were parasitized but the hind brain was most severely affected. S. neurona was found in the spinal cord of all 10 mice examined 22-30 DAFS. Of the 28 infected mice examined 20-62 DAFS, S. neurona was found in the brains of all 28, lungs of 14, hearts of 8 and eyes of 3. More organisms were seen in IHC-stained sections than in sections stained with hematoxylin and eosin. Treatment of tissues with glutaraldehyde, Karnovsky fixative, and ethylene diamino tetra acetic acid (EDTA, used for decalcification) did not affect staining of organisms by IHC.     

Clinical Sarcocystis neurona encephalomyelitis in a domestic cat following routine surgery

Sarcocystis neurona is an important cause of equine protozoal myeloencephalitis (EPM) in horses in the Americas. An EPM-like neurological disease also has been reported from other mammals but it is difficult to induce this disease in the laboratory. A 4-month-old male domestic cat developed neurological signs 3 days following castration. The cat was euthanized 12 days later because of paralysis. Encephalomyelitis was the only lesion and was associated with numerous Sarcocystis schizonts and merozoites in the brain and spinal cord. The protozoa reacted positively with S. neurona-specific polyclonal rabbit antibody. Two unidentified sarcocysts were present in the cerebellum. It may be possible that stress of surgery triggered relapse of S. neurona infection in this cat.     

Naturally occurring Sarcocystis neurona-like infection in a dog with myositis

Tissue stages similar to those of Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis, were identified in skeletal muscles of a dog. The dog, a 6-year-old Labrador retriever, was seropositive for Toxoplasma gondii infection and euthanized due to a history of polymyositis and progressive muscular atrophy. Histologically, 30, variably sized, microscopic, intracellular sarcocysts were observed in 60 sections of skeletal muscles taken from the neck, fore limbs and hind limbs. The cysts were only observed in inflamed skeletal muscles, but were mostly in myocytes at the periphery of areas infiltrated with leukocytes. Ultrastructurally, the cyst wall had villar protrusions consistent with sarcocysts. Immunohistochemistry with monoclonal S. neurona antibodies demonstrated positive labeling of zoites in merozoites or schizonts in the skeletal muscle interstitium, but no labeling of the sarcocysts. Initial PCR analysis with primers amplifying a genetic sequence encoding Apicomplexan 18s rRNA, and subsequent PCR analysis with differentiating primers indicated that the genetic sequences had 100% identity with sequences reported for S. neurona.     

Experimental inoculation of domestic cats (Felis domesticus) with Sarcocystis neurona or S. neurona-like merozoites

Sarcocystis neurona is the parasite most commonly associated with equine protozoal myeloencephalitis (EPM). Recently, cats (Felis domesticus) have been demonstrated to be an experimental intermediate host in the life cycle of S. neurona. This study was performed to determine if cats experimentally inoculated with culture-derived S. neurona merozoites develop tissue sarcocysts infectious to opossums (Didelphis virginiana), the definitive host of S. neurona. Four cats were inoculated with S. neurona or S. neurona-like merozoites and all developed antibodies reacting to S. neurona merozoite antigens, but tissue sarcocysts were detected in only two cats. Muscle tissues from the experimentally inoculated cats with and without detectable sarcocysts were fed to laboratory-reared opossums. Sporocysts were detected in gastrointestinal (GI) scrapings of one opossum fed experimentally infected feline tissues. The study results suggest that cats can develop tissue cysts following inoculation with culture-derived Sarcocystis sp. merozoites in which the particular isolate was originally derived from a naturally infected cat with tissue sarcocysts. This is in contrast to cats which did not develop tissue cysts when inoculated with S. neurona merozoites originally derived from a horse with EPM. These results indicate present biological differences between the culture-derived merozoites of two Sarcocystis isolates, Sn-UCD 1 and Sn-Mucat 2.     

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.     

Brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona and act as intermediate hosts

We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.     

Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases

Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or stayed in the US exhibited titers over 50, a higher figure than in the group which did not travel out of France or stayed in an other European country. No correlation was found between anti-S. neurona and anti-Neospora sp. antibody titers. Data prompt further study of significance of anti-S. neurona antibodies in clinically healthy or diseased European horses, and identification of putative immunizing parasite(s) and their host(s).     

Comparison of Sarcocystis neurona isolates derived from horse neural tissue

Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan.Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed that Sarcocystis spp. from the eight horses appeared the same, but different from the grackle isolate. One Michigan horse isolate (MIH6) had two bands at 72 and 25kDa that were more prominent than the UCD1 isolate and other Michigan horse isolates. Western blot analysis showed that merozoites of eight of eight equine-derived isolates, and the UCD1 S. neurona isolate had similar bands when developed with serum or CSF of an infected horse. Major bands were seen at 60, 44, 30, and 16kDa. In the grackle (Cornell) isolate, bands were seen at 60, 44, 29, and 16kDa. DNA from merozoites of each of the eight equine-derived isolates and the grackle-derived isolate produced a 334bp PCR product (Tanhauser et al., 1999). Restriction fragment length polymorphism (RFLP) analysis of these horse isolates showed banding patterns characteristic for S. neurona. The grackle (Cornell) isolate had an RFLP banding pattern characteristic of other S. falcatula species. Finally, electron microscopy examining multiple merozoites of each of these eight horse isolates showed similar morphology, which differed from the grackle (Cornell) isolate. We conclude that the eight Michigan horse isolates are S. neurona species and the grackle isolate is an S. falcatula species.     

Penetration of equine leukocytes by merozoites of Sarcocystis neurona

Horses are considered accidental hosts for Sarcocystis neurona and they often develop severe neurological disease when infected with this parasite. Schizont stages develop in the central nervous system (CNS) and cause the neurological lesions associated with equine protozoal myeloencephalitis. The present study was done to examine the ability of S. neurona merozoites to penetrate and develop in equine peripheral blood leukocytes. These infected host cells might serve as a possible transport mechanism into the CNS. S. neurona merozoites penetrated equine leukocytes within 5 min of co-culture. Infected leukocytes were usually monocytes. Infected leukocytes were present up to the final day of examination at 3 days. Up to three merozoites were present in an infected monocyte. No development to schizont stages was observed. All stages observed were in the host cell cytoplasm. We postulate that S. neurona merozoites may cross the blood brain barrier hidden inside leukocytes. Once inside the CNS these merozoites can egress and invade additional cells and cause encephalitis.     

Serologic responses of cats against experimental Sarcocystis neurona infections

Sarcocystis neurona is the most important cause of a neurologic disease of horses, equine protozoal myeloencephalitis (EPM). Cats and other carnivores can act as its intermediate hosts and horses are aberrant hosts. Little is known of the sero-epidemiology of S. neurona infections in cats. In the present study, antibodies to S. neurona were evaluated by the S. neurona agglutination test (SAT). Cats fed sporocysts from the feces of naturally infected opossums or inoculated intramuscularly with S. neurona merozoites developed high levels (> or =1:4000) of SAT antibodies. Antibodies to S. neurona were not found in a cat inoculated with merozoites of the closely related parasite, Sarcocystis falcatula. These results should be useful in studying sero-epidemiology of S. neurona infections in cats.     

Sarcocystis neurona infections in raccoons (Procyon lotor): evidence for natural infection with sarcocysts, transmission of infection to opossums (Didelphis virginiana), and experimental induction of neurologic disease in raccoons

Equine protozoal myeloencephalitis (EPM) is a serious neurologic disease of horses in the Americas and Sarcocystis neurona is the most common etiologic agent. The distribution of S. neurona infections follows the geographical distributions of its definitive hosts, opossums (Didelphis virginiana, Didelphis albiventris). Recently, cats and skunks were reported as experimental and armadillos as natural intermediate hosts of S. neurona. In the present report, raccoons (Procyon lotor) were identified as a natural intermediate host of S. neurona. Two laboratory-raised opossums were found to shed S. neurona-like sporocysts after ingesting tongues of naturally-infected raccoons. Interferon-gamma gene knockout (KO) mice fed raccoon-opossum-derived sporocysts developed neurologic signs. S. neurona was identified immunohistochemically in tissues of KO mice fed sporocysts and the parasite was isolated in cell cultures inoculated with infected KO mouse tissues. The DNA obtained from the tongue of a naturally-infected raccoon, brains of KO mice that had neurological signs, and from the organisms recovered in cell cultures inoculated with brains of neurologic KO mice, corresponded to that of S. neurona. Two raccoons fed mature S. neurona sarcocysts did not shed sporocysts in their feces, indicating raccoons are not likely to be its definitive host. Two raccoons fed sporocysts from opossum feces developed clinical illness and S. neurona-associated encephalomyelitis was found in raccoons killed 14 and 22 days after feeding sporocysts; schizonts and merozoites were seen in encephalitic lesions.     

Determination of the activity of pyrimethamine, trimethoprim, sulfonamides, and combinations of pyrimethamine and sulfonamides against Sarcocystis neurona in cell cultures.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. The activities of pyrimethamine, trimethoprim, sulfachloropyridazine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfamethazine, and sulfathiazole were examined against developing S. neurona merozoites in bovine turbinate cell cultures. A microtiter plate host cell lesion based assay was used to determine the effects of agents on developing merozoites. A cell culture flask assay was used to determine if selective concentrations of the agents killed or only inhibited development of S. neurona. Pyrimethamine was coccidiocidal at 1.0 microg/ml and trimethoprim was coccidiocidal at 5.0 microg/ml. None of the sulfonamides had activity when used alone at 50.0 or 100.0 microg/ml. Combinations of sulfonamides (5.0 or 10.0 microg/ml) with 0.1 microg/ml pyrimethamine demonstrated improved activity.     

Detection of Sarcocystis neurona in the brain of a Grant's zebra (Equus burchelli bohmi).

An 8-yr-old intact male Grant's zebra (Equus burchelli bohmi) was referred to the Veterinary Medical Teaching Hospital of the University of California-Davis after being found in the owner's pasture obtunded and in lateral recumbency. The animal was hypothermic, weak, and unwilling to rise. There was no evidence of trauma, and the zebra had seemed normal the preceding evening. There was no extensor rigidity, and cranial nerve reflexes were normal. Flexor and extensor reflexes were weak upon initial examination. A complete blood count and serum biochemistry analysis revealed a mild leukocytosis, hyperfibrinogenemia, hypoglycemia, hyponatremia, hypochloremia, hypocalcemia, and hypoalbuminemia. Urinalysis was normal, and a urine toxicology screen for alkaloids was negative. No toxic substance was found in the hay or pasture grasses although the owner reported the presence of yellow star thistle and mushrooms in the pasture. The cerebrospinal fluid cytologic and biochemical analyses were normal, but antibodies to Sarcocystis neurona were detected. The zebra died despite aggressive supportive therapy over a 4-day period. The necropsy demonstrated severe gastrointestinal nematodiasis that could account for hypoalbuminemia and electrolyte abnormalities. Histopathologic examination of the nervous system revealed focal areas of perivascular cuffing in the brainstem that were comprised mainly of lymphocytes, monocytes, and plasma cells. Immunohistochemical staining identified the presence of S. neurona merozoites associated with the lesions. This zebra probably died from severe endoparasitism that resulted in malabsorption, weakness, and recumbency rather than from encephalitis associated with S. neurona merozoites. Equine protozoal myeloencephalitis has not been reported previously in nondomestic equids.     

Inoculation of Sarcocystis neurona merozoites into the central nervous system of horses

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. A horse model of EPM is needed to test the efficacy of chemotherapeutic agents and potential vaccines. Five horses that were negative for antibodies to S. neurona in their serum and cerebrospinal fluid (CSF) were injected in the subarachnoid space with living merozoites of the SN2 isolate of S. neurona. None of the horses developed clinical disease or died over a 132-day observation period. All five horses developed antibodies to S. neurona in their CSF and serum 3-4 weeks after injection. Two of the horses were examined at necropsy and no parasite induced lesions were observed in their tissues and no parasites were recovered from portions of their spinal cords inoculated on to cell cultures. Results of this study demonstrate that merozoites of the SN2 isolate of S. neurona will induce seroconversion but not clinical disease when inoculated directly into the CSF of nonimmune horses.     

Prevalence of antibodies to Sarcocystis neurona, Toxoplasma gondii and Neospora caninum in horses from Argentina.

Sera from 76 horses from Argentina were examined for antibodies to Sarcocystis neurona, Toxoplasma gondii and Neospora caninum. Antibodies to S. neurona were found in 27 (35.5%) of 76 horses using immunoblots with culture derived merozoites as antigen. Antibodies to T. gondii were found in 10 (13.1%) of 76 horses by using the modified agglutination test with formalin-fixed tachyzoites and mercaptoethanol; titers were 1:25 (two horses), 1:50 (six horses), 1:100 (two horses), and 1:200 (one horse). Antibodies to N. caninum were not found in any of the 76 horses by the use of N. caninum agglutination test. This is the first report of S. neurona infection in horses in Argentina.