Initiation of a Sarcocystis neurona expressed sequence tag (EST) sequencing project: a preliminary report

To accelerate genetic and molecular characterization of Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), a sequencing project has been initiated that will generate approximately 7000-8000 expressed sequence tags (ESTs) from this apicomplexan parasite. Poly(A)(+) RNA was isolated from culture-derived S. neurona merozoites, and a cDNA library was constructed in a unidirectional lambda phage cloning vector. Sixty phage clones were randomly picked from the library, and the cDNA inserts were amplified from these clones using the T3 and T7 primers that flank the multi-cloning site of the lambda vector. This analysis demonstrated that 100% (60/60) of the clones selected from this library contained recombinant cDNA inserts ranging in size from 0.4 to 4.0 kilobases (kb) with an average size of 1.23kb. Single-pass sequencing from the 5' end of the 60 amplified cDNAs produced high-quality nucleotide sequence from 53 of the clones. Comparison of these ESTs to the current gene databases revealed significant matches for 10 of the ESTs, six of which are similar to sequences from other Apicomplexa (i.e., Toxoplasma gondii). Importantly, none of the ESTs were of obvious mammalian origin, thus indicating that the cDNAs in this library were derived primarily from parasite mRNA and not from mRNA of the bovine turbinate host cells. Collectively, these data indicate that the described cDNA library will provide an excellent substrate for generating a portion of the ESTs that are planned from S. neurona. This sequencing project will greatly hasten gene discovery for this protozoan pathogen thereby enhancing efforts towards the development of improved diagnostics, treatments, and preventatives for EPM. In addition, the S. neurona ESTs will represent a significant contribution to the extensive database of sequences from the Apicomplexa. Comparative analyses of these apicomplexan sequences will likely offer a multitude of important information about the biology and evolutionary history of this phylogenetic grouping of parasites.     

The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages

Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. In the present study, the expression of S. neurona merozoite surface antigens (SnSAGs) was evaluated in the sporozoite and bradyzoite stages. Western blot analysis was used to compare SnSAG expression in merozoites versus sporozoites, while immunocytochemistry was performed to examine expression of the SnSAGs in merozoites versus bradyzoites. These analyses revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed in sporozoites, while SnSAG5 was appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, it was found that SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. As shown for T. gondii, stage-specific expression of the SnSAGs may be important for the parasite to progress through its developmental stages and complete its life cycle successfully. Thus, it is possible that the SAG switching mechanism by these parasites could be exploited as a point of intervention. As well, the alterations in surface antigen expression during different life cycle stages may need to be considered when designing prospective approaches for protective vaccination.     

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.     

柔嫩艾美耳球虫表面抗原（EtSAG）基因的分离及鉴定

利用本实验室前期获得的柔嫩艾美耳球虫（Eimeria tenella）孢子化卵囊和未孢子化卵囊差异表达ESTs序列,选取编号为BW4-C03的孢子化卵囊,采用RACE技术,获得该基因全长序列。经BLAST分析,该序列与柔嫩艾美耳球虫表面抗原有72%以上的同源性,命名为EtSAG。利用荧光定量PCR（Real-time PCR）检测发现该基因在孢子化卵囊的转录拷贝数最高,且随着孢子化时间的延长,转录拷贝数逐渐增加。采用原核表达载体pET-28C表达该基因,得到的融合蛋白大小约为36 kDa,符合预期大小。经Western blot分析,该重组蛋白可被兔抗柔嫩艾美耳球虫的多克隆抗血清识别,表明该蛋白具有较好的反应原性。本研究结果为进一步研究该基因的生物学功能奠定了基础。     

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.     

First isolation of Sarcocystis neurona from the South American opossum, Didelphis albiventris, from Brazil

Sarcocystis neurona was isolated from sporocysts from two of eight South American opossums, Didelphis albiventris, from Brazil. Interferon gamma gene knock out (KO) mice fed sporocysts from two opossums developed neurologic sarcocystosis. S. neurona was demonstrated in the brains of infected KO mice by immunohistochemical staining with anti-S. neurona antibody. The parasite was cultivated in cell culture and S. neurona DNA was isolated from cultured merozoites. This is the first report of isolation of S. neurona from Brazil and the first report from its new host, D. albiventris.     

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.     

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.     

Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies

Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.     

Gene discovery and expression profiling in porcine Peyer's patch

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.     

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.     

In vitro quantitative analysis of (3)H-uracil incorporation by Sarcocytis neurona to determine efficacy of anti-protozoal agents

Parasite-specific incorporation of (3)H-uracil was used to assess the replication of Sarcocystis neurona, a protozoal parasite associated with equine protozoal myeloencephalitis (EPM). Anti-protozoal drugs, pyrimethamine (0.01, 0.1 and 1.0microg/ml PYR), sulfadiazine (5microg/ml; SDZ), sulfamethoxazole (5microg/ml; SMZ), diclazuril (100ng/ml; DCZ), atovaquone (0.04ng/ml; ATQ), tetracycline (5microg/ml; TET) and the herbicide glyphosate (1.5 and 4.5mM; GLY) were studied with varying S. neurona parasite densities (2x10(1)-1.2x10(6)merozoites/well). A microtiter plate format was used to test these compounds, and incorporation of (3)H-uracil was determined using a semi-automated plate harvester and liquid scintillation counter. When PYR, DCZ, ATQ, SMZ, SDZ, and TET were tested, the assay was most reliable when parasite densities were greater than 9.0x10(4) individual merozoites per well. When the herbicide GLY was tested, as few as 900 individual merozoites were sufficient to demonstrate reduction in parasite proliferation. Of the anti-protozoal drugs commonly used to treat EPM, PYR was the most potent anti-S. neurona agent tested. The herbicide GLY appears to be more potent than all of the other compounds tested in vitro; however information regarding in vivo use of GLY is not available, and central nervous system penetration by this compound is unlikely. Incorporation of (3)H-uracil by replicating S. neurona is quantitative and can be used in a semi-automated assay. This in vitro assay is capable of high throughput screening of candidate drugs that may have applications in a clinical setting. Further studies using a wider range of drug concentrations with optimal numbers of merozoites are necessary to determine true potency of these agents.     

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.     

鸡下丘脑组织表达序列标签初步分析

为了鉴定所构建的鸡下丘脑组织 c DNA文库能否用于下丘脑表达谱的建立 ,从 c DNA文库中随机挑取 12个克隆 ,对其表达序列标签 (expressed sequence tags,ESTs)进行了测定和初步分析。经 BL ASTn和 FASTA3.1分析后发现 ,1个 ESTs在 Gen Bank中可以找到鸡的同源序列 ,4个在其他物种中也可以找到同源序列 ,1个在 ESTs database中有同源 ESTs序列 ,还有 6个为未知功能基因。 12个 ESTs序列均已录入 Gen Bank。     

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

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

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.     

Generally applicable methods to purify intracellular coccidia from cell cultures and to quantify purification efficacy using quantitative PCR

The objective of this study was to evaluate the utility of a simple, efficient, and rapid method for the isolation of Sarcocystis neurona merozoites and Besnoitia darlingi tachyzoites from cultured cells. The efficacy of this purification method was assessed by microscopy, SDS-PAGE, Western blotting, immuno-fluorescence, and three novel quantitative PCR assays. Culture medium containing host cell debris and parasites was eluted through PD-10 desalting columns. This purification method was compared to alternatives employing filtration through a cellulose filter pad or filter paper. The estimated recovery of S. neurona merozoites purified by the column method was 82% (+/-3.7) of the original merozoites with 97.5% purity. In contrast, estimated recovery of S. neurona merozoites purified by filter pad and filter paper was 40% and 30% with 76% and 83% purity, respectively. The same procedures were applied to purify B. darlingi tachyzoites from cultured cells. Of the original cultured B. darlingi tachyzoites, 94% (+/-2.5) were recovered from the PD-10 column with 96.5%, purity whereas percentage recovery of B. darlingi tachyzoites purified by filter pad and filter paper were 51% and 35% with 84% and 88% purity, respectively. All described methods maintained sterility so that purified parasites could be subsequently cultured in vitro. However, purification using a PD-10 column minimized parasite loss and the loss of viability as determined by the trypan blue dye exclusion assay, the rate of parasite production, and plaque forming efficiency in cell culture. Moreover, column-purified parasites improved the sensitivity of an immuno-fluorescent (IFA) analysis and real-time quantitative PCR assays targeted to parasite 18S ribosomal DNA and hsp70 genes. This technique appears generally applicable for purifying coccidia grown in cell cultures.     

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.     

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.     

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.