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.     

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.     

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.     

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.     

Naturally occurring Sarcocystis infection in domestic cats (Felis catus)

Equine protozoal myeloencephalitis is an important neurological disease of horses in the United States. Consequently, there is an active research effort to identify hosts associated with the primary causative agent, Sarcocystis neurona. The purpose of this study was to determine whether the domestic cat (Felis catus) is a natural host for S. neurona. Muscle sections from 50 primarily free-roaming domestic cats were examined for the presence of sarcocysts. Serum from cats in this group and another group of 50 free-roaming cats were evaluated for the presence of S. neurona antibody. Sarcocysts were found in five of 50 (10%) cats, and S. neurona antibody in five of 100 (5%) cats. Morphological, molecular (including ribosomal RNA genes), and biological characterisation of these sarcocysts showed that they were not S. neurona or S. neurona-like. Sarcocysts found in the cats were identified morphologically as Sarcocystis felis, a common parasite of wild felids. The life cycle of S. felis is not known, and prior to this study, no molecular marker for S. felis existed. Although cats were found to be infected with S. felis sarcocysts, serological data provided evidence of possible infection with S. neurona as well. Further work is needed to determine the role of the domestic cat in the life cycle of S. neurona.     

Detection of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii in horses from Costa Rica

Serum samples from 315 horses from Costa Rica, Central America, were examined for the presence of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii by using the surface antigen (SAG) SnSAG2 enzyme-linked immunosorbent assay (ELISA), the NhSAG1 ELISA, and the modified agglutination test, respectively. Anti- S. neurona antibodies were found in 42.2% of the horses by using the SnSAG2 ELISA. Anti- Neospora spp. antibodies were found in only 3.5% of the horses by using the NhSAG1 ELISA, and only 1 of these horses was confirmed seropositive by Western blot. Antibodies to T. gondii were found in 34.0% of the horses tested, which is higher than in previous reports from North and South America. The finding of anti- S. neurona antibodies in horses from geographical areas where Didelphis marsupialis has wide distribution suggests that D. marsupialis is a potential definitive host for this parasite and a source of infection for these horses.     

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.     

Early migration of Sarcocystis neurona in ponies fed sporocysts

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM), a neurologic disease of the horse. In the present work, the kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 10(6) S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 postinoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI; the liver at 2, 5, and 7 days PI; and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of naive ponies.     

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.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.     

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.     

Life cycle of Sarcocystis neurona in its natural intermediate host,the raccoon,Procyon lotor

Sarcocystis neurona causes encephalomyelitis in many species of mammals and is the most important cause of neurologic disease in the horse. Its complete life cycle is unknown, particularly its development and localization in the intermediate host. Recently, the raccoon (Procyon lotor) was recognized as a natural intermediate host of S. neurona. In the present study, migration and development of S. neurona was studied in 10 raccoons that were fed S. neurona sporocysts from experimentally infected opossums; 4 raccoons served as controls. Raccoons were examined at necropsy 1, 3, 5, 7, 10, 14, 15, 22, 37, and 77 days after feeding on sporocysts (DAFS). Tissue sections of most of the organs were studied histologically and reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical test. Parasitemia was demonstrated in peripheral blood of raccoons 3 and 5 DAFS. Individual zoites were seen in histologic sections of intestines of raccoons euthanized 1, 3, and 5 DAFS. Schizonts and merozoites were seen in many tissues 7 to 22 DAFS, particularly in the brain. Sarcocysts were seen in raccoons killed 22 DAFS. Sarcocysts at 22 DAFS were immature and seen only in skeletal muscle. Mature sarcocysts were seen in all skeletal samples, particularly in the tongue of the raccoon 77 DAFS; these sarcocysts were infective to laboratory-raised opossums. This is the first report of the complete development of S. neurona schizonts and sarcocysts in a natural intermediate host.     

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.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in raccoons (Procyon lotor) from an urban area of Virginia

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural and experimentally induced cases of encephalitis caused by S. neurona have been reported in raccoons (Procyon lotor) and raccoons are an intermediate host for this parasite. A 3-yr-long serological survey was conducted to determine the prevalence of agglutinating antibodies to S. neurona in raccoons collected from Fairfax County, Virginia, a suburban-urban area outside Washington, D.C. Samples from 469 raccoons were examined, and agglutinating antibodies (> or = 1:50 dilution) were found in 433 (92.3%) of the raccoons. This study indicates that exposure to S. neurona is high in this metropolitan area.     

Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses

The objectives of this study were to evaluate the accuracy of the indirect fluorescent antibody test (IFAT) using serum and cerebrospinal fluid (CSF) of horses naturally and experimentally infected with Sarcocystis neurona, to assess the correlation between serum and CSF titers, and to determine the effect of S. neurona vaccination on the diagnosis of infection. Using receiver-operating characteristic analysis, the areas under the curve for the IFAT were 0.97 (serum) and 0.99 (CSF). Sensitivity and specificity were 83.3 and 96.9% (serum, cutoff 80) and 100 and 99% (CSF, cutoff 5), respectively. Titer-specific likelihood ratios (LRs) ranged from 0.03 to 187.8 for titers between <10 and 640. Median time to conversion was 22-26 days postinfection (DPI) (serum) and 30 DPI (CSF). The correlation between serum and CSF titers was moderately strong (r = 0.6) at 30 DPI. Percentage of vaccinated antibody-positive horses ranged from 0 to 95% between 0 and 112 days after the second vaccination. Thus, the IFAT was reliable and accurate using serum and CSF. Use of LRs potentially improves clinical decision making. Correlation between serum and CSF titers affects the joint accuracy of the IFAT; therefore, the ratio of serum to CSF titers has potential diagnostic value. The S. neurona vaccine could possibly interfere with equine protozoal myeloencephalitis diagnosis.     

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.     

Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells

Immunodeficient CD8 knockout mice were infected with Sarcocystis neurona merozoites, in order to determine the role of CD8 cells in protective immunity. Using a direct agglutination test, all infected mice seroconverted by selected time points. Infected mice developed splenomegaly and bilateral lymphadenopathy. Histological changes included marked follicular development in the spleen, endothelitis and moderate perivascular inflammation in the liver, and meningoencephalitis in the brain. Infected brains were positive for S. neurona by polymerase chain reaction. Corresponding to histopathological changes, there were decreased numbers of B-cells in the spleen. The mice did not have significant memory (CD44hi/CD4) or effector (CD45RBhi/CD4) populations present at the time of euthanasia. Flow cytometry confirmed the lack of CD8 cells. Taken together, these data support previous studies suggesting a critical role for CD8 cells in the prevention of menigoencephalitis in S. neurona-infected mice.     

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.