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.     

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.     

Lack of Sarcocystis neurona antibody response in Virginia opossums (Didelphis virginiana) fed Sarcocystis neurona-infected muscle tissue

Serum was collected from laboratory-reared Virginia opossums (Didelphis virginiana) to determine whether experimentally infected opossums shedding Sarcocystis neurona sporocysts develop serum antibodies to S. neurona merozoite antigens. Three opossums were fed muscles from nine-banded armadillos (Dasypus novemcinctus), and 5 were fed muscles from striped skunks (Mephitis mephitis). Serum was also collected from 26 automobile-killed opossums to determine whether antibodies to S. neurona were present in these opossums. Serum was analyzed using the S. neurona direct agglutination test (SAT). The SAT was modified for use with a filter paper collection system. Antibodies to S. neurona were not detected in any of the serum samples from opossums, indicating that infection in the opossum is localized in the small intestine. Antibodies to S. neurona were detected in filter-paper-processed serum samples from 2 armadillos naturally infected with S. neurona.     

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.     

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.     

Biological characterisation of Sarcocystis neurona isolated from a Southern sea otter (Enhydra lutris nereis)

Sarcocystis neurona was isolated from the brain of a juvenile, male southern sea otter (Enhydra lutris nereis) suffering from CNS disease. Schizonts and merozoites in tissue sections of the otter's brain reacted with anti-S. neurona antiserum immunohistochemically. Development in cell culture was by endopolyogeny and mature schizonts were first observed at 3 days postinoculation. PCR of merozoite DNA using primer pairs JNB33/JNB54 and restriction enzyme digestion of the 1100 bp product with Dra I indicated the organism was S. neurona. Four of four interferon-gamma gene knockout mice inoculated with merozoites developed S. neurona-associated encephalitis. Antibodies to S. neurona but not Sarcocystis falcatula, Toxoplasma gondii, or Neospora caninum were present in the serum of inoculated mice. This is the first isolation of S. neurona from the brain of a non-equine host.     

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.     

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.     

Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona

A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.     

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.     

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.     

Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula

Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

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.     

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.     

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.     

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.     

The nine-banded armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona

Sarcocysts were dissected from the tongue of a nine-banded armadillo (Dasypus novemcinctus). DNA was extracted and characterised by PCR amplification followed by restriction fragment length polymorphism analysis and nucleotide sequencing. A total of 1879 nucleotides were compared; the sarcocyst DNA sequence was identical to that reported for Sarcocystis neurona. DNA was extracted from the sarcocysts of five more nine-banded armadillos. A 254-nucleotide sequence was determined for each and found to be identical to S. neurona. Western blot techniques for detection of anti-S. neurona antibody were developed for use with armadillo plasma and samples from 19 wild-caught and 17 captive-raised armadillos were examined. Whereas all of the 19 wild-caught armadillos had antibodies to S. neurona, only one of 17 captive-raised armadillos did. These results suggest that the nine-banded armadillo are naturally infected with S. neurona.     

Risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in California horses

The study objective was to assess the risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in foals from 4 California farms during 3 foaling seasons. Serum of presuckle foals and serum and colostrum of periparturient mares were tested using indirect fluorescent antibody tests for S. neurona and N. hughesi. Serum antibody titers were < or =10 in 366 presuckle foals tested. There was no serologic or histologic evidence of either parasite in aborted fetuses or placentas examined. Positivity for S. neurona and N. hughesi in mares increased with age. Mares < or =9 yr that originated from Kentucky were 3.8 and 1.4 times more likely to be positive for S. neurona and N. hughesi, respectively, than mares from California. The strength of association between positivity to either parasite and state of birth decreased as age increased. Mares positive for S. neurona and N. hughesi were 2.2 and 1.7 times more likely, respectively, to have a previous abortion than negative mares, adjusted for age and state of birth. The annual mortality rate for mares was 4%. The annual incidence rate of equine protozoal myeloencephalitis was 0.2%. In conclusion, there was no detectable risk of transplacental transmission of S. neurona and N. hughesi. Prevalence of antibodies against both parasites in mares increased with age.