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.     

Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona

The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. Eighteen horses were assigned to 1 of 3 groups: no treatment, 2.5 mg/kg ponazuril, or 5.0 mg/kg ponazuril. Horses were administered ponazuril, once per day, beginning 7 days before infection (study day 0) and continuing for 28 days postinfection. On day 0, horses were stressed by transport and challenged with 1 million S. neurona sporocysts per horse. Sequential neurologic examinations were performed, and serum and cerebrospinal fluid were collected and assayed for antibodies to S. neurona. All horses in the control group developed neurologic signs, whereas only 71 and 40% of horses in the 2.5 and 5.0 mg/kg ponazuril groups, respectively, developed neurologic abnormalities. This was significant at P = 0.034 by using Fisher exact test. In addition, seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100 vs. 40%; P = 0.028). Horses with neurologic signs were killed, and a post-mortem examination was performed. Mild-to-moderate, multifocal signs of neuroinflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses.     

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.     

The gamma interferon knockout mouse model for sarcocystis neurona: comparison of infectivity of sporocysts and merozoites and routes of inoculation.

The dose-related infectivity of Sarcocystis neurona sporocysts and merozoites of 2 recent isolates of S. neurona was compared in gamma interferon knockout (KO) mice. Tenfold dilutions of sporocysts or merozoites were bioassayed in mice, cell culture, or both. All 8 mice, fed 1,000 sporocysts, developed neurological signs with demonstrable S. neurona in their tissues. Of 24 mice fed low numbers of sporocysts (100, 10, 1), 18 became ill by 4 wk postinoculation, and S. neurona was demonstrated in their brains; antibodies (S. neurona agglutination test) to S. neurona and S. neurona parasites were not found in tissues of the 6 mice that were fed sporocysts and survived for >39 days. One thousand culture-derived merozoites of these 2 isolates were pathogenic to all 8 mice inoculated subcutaneously (s.c.). Of the 24 mice inoculated s.c. with merozoites numbering 100, 10, or 1, only 3 mice had demonstrable S. neurona infection; antibodies to S. neurona were not found in the 21 mice that had no demonstrable organisms. As few as 10 merozoites were infective for cell cultures. These results demonstrate that at least 1,000 merozoites are needed to cause disease in KO mice. Sarcocystis neurona sporocysts were infective to mice by the s.c. route.     

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.     

Experimental infection of ponies with Sarcocystis fayeri and differentiation from Sarcocystis neurona infections in horses

Sarcocystis neurona and Sarcocystis fayeri infections are common in horses in the Americas. Their antemortem diagnosis is important because the former causes a neurological disorder in horses, whereas the latter is considered nonpathogenic. There is a concern that equine antibodies to S. fayeri might react with S. neurona antigens in diagnostic tests. In this study, 4 ponies without demonstrable serum antibodies to S. neurona by Western immunoblot were used. Three ponies were fed 1 x 10(5) to 1 x 10(7) sporocysts of S. fayeri obtained from dogs that were fed naturally infected horse muscles. All ponies remained asymptomatic until the termination of the experiment, day 79 postinoculation (PI). All serum samples collected were negative for antibodies to S. neurona using the Western blot at the initial screening, just before inoculation with S. fayeri (day 2) and weekly until day 79 PI. Cerebrospinal fluid samples from each pony were negative for S. neurona antibodies. Using the S. neurona agglutination test, antibodies to S. neurona were not detected in 1:25 dilution of sera from any samples, except that from pony no. 4 on day 28; this pony had received 1 X 10(7) sporocysts. Using indirect immunofluorescence antibody tests (IFATs), 7 serum samples were found to be positive for S. neurona antibodies from 1:25 to 1:400 dilutions. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies, with heaviest infections in the tongue. All sarcocysts examined histologically appeared to contain only microcytes. Ultrastructurally, S. fayeri sarcocysts could be differentiated from S. neurona sarcocysts by the microtubules (mt) in villar protrusions on sarcocyst walls; in S. fayeri the mt extended from the villar tips to the pellicle of zoites, whereas in S. neurona the mt were restricted to the middle of the cyst wall. Results indicate that horses with S. fayeri infections may be misdiagnosed as being S. neurona infected using IFAT, and further research is needed on the serologic diagnosis of S. neurona infections.     

Horses experimentally infected with Sarcocystis neurona develop altered immune responses in vitro

Equine protozoal myeloencephalitis (EPM) due to Sarcocystis neurona infection is 1 of the most common neurologic diseases in horses in the United States. The mechanisms by which most horses resist disease, as well as the possible mechanisms by which the immune system may be suppressed in horses that develop EPM, are not known. Therefore, the objectives of this study were to determine whether horses experimentally infected with S. neurona developed suppressed immune responses. Thirteen horses that were negative for S. neurona antibodies in serum and cerebrospinal fluid (CSF) were randomly assigned to control (n = 5) or infected (n = 8) treatment groups. Neurologic exams and cerebrospinal fluid analyses were performed prior to, and following, S. neurona infection. Prior to, and at multiple time points following infection, immune parameters were determined. All 8 S. neurona-infected horses developed clinical signs consistent with EPM, and had S. neurona antibodies in the serum and CSF. Both infected and control horses had increased percentages (P < 0.05) of B cells at 28 days postinfection. Infected horses had significantly decreased (P < 0.05) proliferation responses as measured by thymidine incorporation to nonspecific mitogens phorbol myristate acetate (PMA) and ionomycin (I) as soon as 2 days postinfection.     

Sheep experimentally infected with sarcocystis from dogs. I. Disease in young lambs.

Eight Polled Dorset lambs were orally inoculated with Sarcocystis ovicanis sporocysts. Two lambs that received 100,000 or 200,000 sporocysts became clinically ill, recovered, and were killed 67 and 88 days after inoculation (DAI). Numerous intramuscular cysts were found in their skeletal and cardiac muscles. Three lambs received 100,000 sporocysts, three lambs received 1 million sporocysts, and three lambs received no sporocysts. After an acute clinical illness characterized by anemia, inappetence, weight loss, fever, and reduced serum protein, all lambs that received 100,000 sporocysts died 27 to 29 DAI and all that received 1 million sporocysts died 24 or 25 DAI. Hemorrhage involving the striated muscle and visceral organs was the most apparent gross lesion. The heart appeared most severely affected. Schizonts were found in vascular endothelial cells of all six inoculated lambs. Uninoculated lambs remained healthy, and neither lesions nor parasites were found in any tissues. Dogs fed tissues containing S. ovicanis cysts produced sporocytes 11 to 37 days after feeding; cats fed similar stages produced no sporocysts. Dogs fed tissues containing schizonts produced no sporocysts.     

Simplified technique for isolation, excystation, and culture of Sarcocystis species from opossums

Sarcocystis neurona is a protozoan parasite that causes a neurological disease in horses called equine protozoal myeloencephalitis. The route of transmission is speculated to be by fecal-oral transfer of sporocysts shed from opossums. Controversy exists regarding both the natural life cycle for this parasite as well as the species identity of opossum Sarcocystis. To provide stage-specific material for species comparison, 27 opossums from southern Michigan were screened for Sarcocystis spp. sporocysts. Seven opossums were positive for Sarcocystis sporocysts by fecal flotation. A simplified, effective technique for isolation, excystation, and culture of opossum Sarcocystis sp. from mucosal scrapings was developed. All 7 Sarcocystis sp. isolates were successfully cultured to grow long term in equine dermal cells to the merozoite stage. Merozoites were observed between 5 and 15 days after inoculation. In conclusion, opossums shed Sarcocystis sp. sporocysts that may be manipulated to excyst and grow in vitro in equine dermal cell lines to the merozoite stage using the simplified technique described.     

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.     

Development of Sarcocystis fayeri in the equine

Eight ponies and a horse were inoculated orally with sporocysts of Sarcocystis fayeri from dogs. They were examined for clinical signs of infection and killed 10, 15, 20, 25, 30, 50 (horse), 77, 101, and 156 days after inoculation (DAI). Elevated temperature was observed in three ponies 20 and 26 DAI and anemia was observed in three ponies and the horse 15 to 69 DAI. Schizonts were found in or near cells lining capillaries or arteries of the heart, brain, and kidney 10, 20, and 25 DAI. Immature cysts containing only metrocytes were first found in muscles 50 DAI. Mature intramuscular cysts containing metrocytes and zoites or zoites alone were found 77, 101, and 156 DAI and produced patent infections in dogs fed infected meat.     

Assessment of Sarcocystis neurona sporocyst viability and differentiation between viable and nonviable sporocysts using propidium iodide stain

Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of dead sporocysts. After live and dead sporocysts were mixed at various ratios, the number of unstained sporocysts detected after the staining procedure correlated significantly (r2 = 0.9978) with the expected numbers of live sporocysts. Sporocyst mixtures were also assayed for in vitro excystation and development in tissue cultures. The correlation between the percentage of plaques formed in tissue cultures and the percentage of expected infectious (live) sporocysts in each mixture was r2 = 0.6712. By analysis of variance, no statistically significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts (P = 0.3902) in each mixture. In addition, there was evidence of a relation between PI impermeability of sporocysts and animal infectivity. These results suggest that the PI dye-exclusion technique can be a useful tool in identifying viability and potential infectivity of S. neurona sporocysts and in differentiating between viable and nonviable sporocysts.     

Sarcocystis neurona: parasitemia in a severe combined immunodeficient (SCID) horse fed sporocysts

Sarcocystis neurona was isolated from the blood of a 5-month-old Arabian foal with severe combined immunodeficiency. The foal had been inoculated approximately 3 weeks previously with 5 x 10(5) sporocysts that were isolated from the intestines of an opossum and identified by restriction enzyme analysis of PCR products as S. neurona. The isolate obtained from the blood of this foal was characterized by genetic, serologic, and morphologic methods and identified as S. neurona (WSU1). This represents the first time that S. neurona has been isolated from any tissue after experimental infection of a horse. This is also the first time a parasitemia has been detected during either natural or experimental infection. The severe combined immunodeficiency foal model provides a unique opportunity to study the pathogenesis of S. neurona infection in horses and to determine the role of the immune system in the control of infection with and development of neurologic disease.     

Protective Immunity to Sarcocystis capracanis-Induced Abortion in Dairy Goats1

ABSTRACT. Eleven female goats (Nos. 1 to 11) were each inoculated orally with 10⁴ sporocysts of Sarcocystis capracanis, and four female goats (Nos. 12 to 15) were not inoculated. Between 31 and 69 days after inoculation (DAI) goats were mated with a single buck; one goat (No. 5) did not breed. Eight inoculated goats were challenged with 10⁵or 10⁶ sporocysts, 135 DAI. Two of four goats challenged with 10⁶ sporocysts and one of three goats challenged with 10⁵ sporocysts aborted one month before the expected time of parturition. The three inoculated goats that were not challenged delivered healthy kids. All inoculated goats including the nonpregnant one (No. 5) were only mildly ill from the primary or challenge inoculations. Two of the four control goats challenged with 5 × 10⁴ or 10⁵ sporocysts aborted 21 days later, and both died of sarcocystosis 25 and 88 DAI. The two remaining control goats delivered normal kids. The results indicate that immunization prior to pregnancy protects some but not all goats from .Sarc0c.es/is-induced abortion.     

Parasitemia and early tissue localization of Sarcocystis neurona in interferon gamma gene knockout mice fed sporocysts.

Early localization and parasitemia of Sarcocystis neurona were studied in gamma interferon gene knockout (KO) mice fed S. neurona sporocysts. Mice were examined for S. neurona infection histologically and immunohistochemically and by bioassay in KO mice. For bioassay, blood and tissue homogenates were inoculated subcutaneously into KO mice. Parasitemia was demonstrated by bioassay in KO mice 1-8 days after feeding sporocysts (DAFS). Sporozoites were seen in histologic sections of all regions of the small intestine and in cells in Peyer's patches of a mouse killed 6 hr after feeding sporocysts. At 1 DAFS, organisms were present in all regions of the small intestine and were also seen in mesenteric lymph nodes. At 3 DAFS, organisms had begun to invade extraintestinal tissues. Sarcocystis neurona was demonstrated histologically in mouse brain as early as 4 DAFS.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Sarcocystis neurona infections in sea otter (Enhydra lutris): evidence for natural infections with sarcocysts and transmission of infection to opossums (Didelphis virginiana).

Although Sarcocystis neurona has been identified in an array of terrestrial vertebrates, recent recognition of its capacity to infect marine mammals was unexpected. Here, sarcocysts from 2 naturally infected sea otters (Enhydra lutris) were characterized biologically, ultrastructurally, and genetically. DNA was extracted from frozen muscle of the first of these sea otters and was characterized as S. neurona by polymerase chain reation (PCR) amplification followed by restriction fragment length polymorphism analysis and sequencing. Sarcocysts from sea otter no. 1 were up to 350 microm long, and the villar protrusions on the sarcocyst wall were up to 1.3 microm long and up to 0.25 microm wide. The villar protrusions were tapered towards the villar tip. Ultrastructurally, sarcocysts were similar to S. neurona sarcocysts from the muscles of cats experimentally infected with S. neurona sporocysts. Skeletal muscles from a second sea otter failed to support PCR amplification of markers considered diagnostic for S. neurona but did induce the shedding of sporocysts when fed to a laboratory-raised opossum (Didelphis virginiana). Such sporocysts were subsequently fed to knockout mice for the interferon-gamma gene, resulting in infections with an agent identified as S. neurona on the basis of immunohistochemistry, serum antibodies, and diagnostic sequence detection. Thus, sea otters exposed to S. neurona may support the development of mature sarcocysts that are infectious to competent definitive hosts.     

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.     

Prevalence of sarcocystis species sporocysts in wild-caught opossums (Didelphis virginiana)

Sarcocystis sporocysts were found in intestinal scrapings from 24 (54.5%) of 44 opossums (Didelphis virginiana). The number of sporocysts varied from a few (< 10,000) to 245 million. Sporocysts from 23 of 24 opossums were fed to captive budgerigars (Melopsittacus undulatas), and the inocula from 21 opossums were infective, indicating the presence of Sarcocystis falcatula. Sporocysts from 24 opossums were fed to gamma-interferon-knockout (KO) or nude mice; inocula from 14 opossums were infective to mice. Sarcocystis neurona was detected in tissues of KO mice by specific staining with anti-S. neurona antibodies, and the parasite was cultured in vitro from the brains of KO mice fed sporocysts from 8 opossums. Sarcocystis speeri was identified by specific staining with anti-S. speeri antibodies in tissues of KO mice fed inocula from 8 opossums; 3 opossums had mixed S. neurona and S. speeri infections. Thus, the prevalences of sporocysts of different species of Sarcocystis in opossums were: S. falcatula 21 of 44 (47.7%), S. neurona 8 of 44 (18.1%), and S. speeri 8 of 44 (18.1%) opossums. Sarcocystis neurona alone was found in 1 opossum, and S. speeri alone was found in 1 opossum. Mixed Sarcocystis infections were present in 21 opossums.