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.     

Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis virginiana) faeces, and its differentiation from Sarcocystis falcatula

Sarcocystis neurona was isolated in nude mice and gamma-interferon knockout mice fed sporocysts from faeces of naturally infected opossums (Didelphis virginiana). Mice fed sporocysts became lethargic and developed encephalitis. Protozoa were first found in the brain starting 21 days post-inoculation. Sarcocystis neurona was recovered in cell culture from the homogenate of liver, spleen and brain of a nude mouse 11 days after feeding sporocysts. The protozoa in mouse brain and in cell culture multiplied by schizogony and mature schizonts often had a residual body. Sarcocystis falcatula, which has an avian-opossum cycle, was not infective to nude or knockout mice. Protozoa were not found in tissues of nude mice or knockout mice after subcutaneous injection with culture-derived S. falcatula merozoites and sporocysts from the faeces of opossums presumed to contain only S. falcatula. Results demonstrate that S. neurona is distinct from S. falcatula, and that opossums are hosts for both species.     

Comparative development and merozoite production of two isolates of Sarcocystis neurona and Sarcocystis falcatula in cultured cells

The development and merozoite production of Sarcocystis falcatula and 2 isolates (SN6 and SN2) of Sarcocystis neurona were studied in various cultured cell lines inoculated with culture-derived merozoites. All 3 parasites underwent multiple cycles of schizogony in VERO cells, bovine monocytes (M617 cells), and bovine pulmonary artery endothelial cells (CPA). Sarcocystis neurona strains SN6 and SN2 formed schizonts in rat myoblasts (L6) but not in quail myoblasts (QM7); S. falcatula formed schizonts in QM7 cells but not in L6 cells. Merozoites did not develop to sarcocysts in the myoblast cells lines. During a 47-day culture period in VERO cells, SN6 produced substantially more merozoites than did SN2 or S. falcatula. M617 cells produced substantially more merozoites of SN6 than did VERO or CPA cells. During a 17-day culture period of SN6, M617 cells produced mean totals of 4.7 x 10(8) merozoites, VERO cells produced 1.9 x 10(8) merozoites, and CPA cells produced 5.9 x 10(7) merozoites. At 4-12 days after inoculation of cultured cells with SN6, M617 cells cultured in the presence of 10% fetal bovine serum (FBS) produced a mean merozoite total of 5.1 x 10(8) compared to 3.6 x 10(8) for culture medium containing 1% FBS.     

Determination of the activity of diclazuril against Sarcocystis neurona and Sarcocystis falcatula in cell cultures

Diclazuril is a benzeneacetonitril anticoccidial that has excellent activity against the extraintestinal stages of Toxoplasma gondii and Neospora caninum. It also is highly active against intestinal coccidia of poultry. The present study examined the efficacy of diclazuril in inhibiting merozoite production of Sarcocystis neurona and Sarcocystis falcatula in bovine turbinate cell cultures. Diclazuril inhibited merozoite production by more than 80% in cultures of S. neurona or S. falcatula treated with 0.1 ng/ml diclazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 1.0 ng/ml diclazuril. Diclazuril may have promise as a therapeutic agent in the treatment of S. neurona-induced equine protozoal myeloencephalitis in horses and S. falcatula infections in birds.     

In vitro cultivation of schizonts of Sarcocystis speeri Dubey and Lindsay, 1999

Schizonts of Sarcocystis speeri Dubey and Lindsay, 1999 were cultured in vitro in bovine monocyte and equine kidney cell cultures inoculated with infected tissues of nude and gamma-interferon knockout mice fed sporocysts from opossums, Didelphis albiventris. At least 1 asexual cycle was completed in 3 days. In vitro-grown merozoites were structurally and antigenically distinct from those of Sarcocystis neurona and Sarcocystis falcatula. Culture-derived merozoites of S. speeri were not infective to budgerigars (Melopsittacus undulatus).     

Isolates of Sarcocystis falcatula-like organisms from South American opossums Didelphis marsupialis and Didelphis albiventris from S?o Paulo, Brazil.

Isolates of Sarcocystis falcatula-like organisms from South American opossums were characterized based on biological and morphological criteria. Sporocysts from intestinal scrapings of 1 Didelphis marsupialis and 8 Didelphis albiventris from S?o Paulo, Brazil, were fed to captive budgerigars (Melopsittacus undulatus). Budgerigars fed sporocysts from all 9 isolates became ill and S. falcatula-like schizonts were identified in sections of their lungs by immunohistochemical staining. Sarcocystis falcatula-like organisms were cultured from lungs of budgerigars fed sporocysts from D. marsupialis and from lungs of budgerigars fed sporocysts from 3 of 8 D. albiventris. The 33/54 locus amplified by polymerase chain reaction from culture-derived merozoites contained both a HinfI endonuclease recognition site previously suggested to diagnose S. falcatula and a DraI site thought to diagnosed S. neurona. Development of the isolate from D. marsupialis was studied in cell culture; its schizonts divided by endopolygeny, leaving a residual body. Morphological and genetic variation differentiated this Sarcocystis isolate originating in D. marsupialis from the Cornell I isolate of S. falcatula. This is the first report of a S. falcatula infection in the South American opossum, D. marsupialis.     

Acute Sarcocystis falcatula-like infection in a carmine bee-eater (Merops nubicus) and immunohistochemical cross reactivity between Sarcocystis falcatula and Sarcocystis neurona

An unidentified Sarcocystis falcatula-like infection was diagnosed in a captive bee-eater (Merops nubicus) in a zoo in Florida. The bird died suddenly, probably due to protozoa-associated pneumonia. Protozoal schizonts were found in lungs and heart, and immature sarcocysts were seen in skeletal muscles. Ultrastructurally, schizonts were located in capillary endothelium and merozoites lacked rhoptries, consistent with the structure of Sarcocystis species. Sarcocysts were immature, microscopic, and contained only metrocytes. The sarcocyst wall had finger-like villar protrusions that were up to 0.7 microm long and up to 0.2 microm wide. The villar protrusions lacked microtubules, characteristically seen in sarcocysts of S. falcatula. Antigenically, parasites in lungs and muscles of the bee-eater reacted with a varying intensity with polyclonal rabbit antisera to S. falcatula and Sarcocystis neurona. Results indicated that sarcocysts in the bee-eater were morphologically different from the reported structure for sarcocysts of other S. falcatula 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.     

Neural sarcocystosis in a straw-necked ibis (Carphibis spinicollis) associated with a Sarcocystis neurona-like organism and description of muscular sarcocysts of an unidentified Sarcocystis species.

A Sarcocystis neurona-like parasite was associated with acute sarcocystosis in the brain of an ibis (Carphibis spinicollis). Numerous schizonts and merozoites were found extravascularly in encephalitic lesions. These schizonts reacted positively with anti-S. neurona and anti-S. falcatula polyclonal antibodies in an immunohistochemical test. Sarcocysts of an unidentified Sarcocystis species were present in the brain, heart, and skeletal muscles. Sarcocysts in skeletal muscles were microscopic, and the sarcocyst wall was up to 3 microm thick. The villar protrusions on the sarcocyst wall were up to 4.5 microm long, constricted at the base, and expanded laterally. Schizonts and sarcocysts distinct from those of S. falcatula.     

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.     

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.     

Characterization of Sarcocystis falcatula isolates from the Argentinian opossum, Didelphis albiventris

Two isolates of Sarcocystis falcatula were obtained from the lungs of budgerigars (Melopsittacus undulatus) fed sporocysts from two naturally-infected South American opossums (Didelphis albiventris). The two isolates were designated SF-1A and SF-2A. Both isolates induced fatal infections in budgerigars. Both isolates underwent schizogony in African green monkey kidney cells. The structure of schizonts in the lungs of budgerigars was more variable than that observed in cell culture. The two isolates were identified as S. falcatula by the two species-specific Hinf 1 restriction fragments dervied from digestion of a PCR amplification using primers JNB33/JNB54. Thus, the South American opossum, D. albiventris, is a definitive host for S. falcatula.     

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.     

Characterization of Sarcocystis from four species of hawks from Georgia, USA

During 2001 to 2004, 4 species of hawks (Buteo and Accipiter spp.) from Georgia were surveyed for Sarcocystis spp. infections by examining intestinal sections. In total, 159 of 238 (66.8%) hawks examined were infected with Sarcocystis spp. Samples from 10 birds were characterized by sequence analysis of a portion of the 18S rRNA gene (783 base pairs). Only 3 of the 10 sequences from the hawks were identical; the remainder differed by at least 1 nucleotide. Phylogenetic analysis failed to resolve the position of the hawk Sarcocystis species, but they were closely related several Sarcocystis species from raptors, rodents, and Sarcocystis neurona. The high genetic diversity of Sarcocystis suggests that more than 1 species infects these 4 hawk species; however, additional molecular or experimental work will be required to determine the speciation and diversity of parasites infecting these avian hosts. In addition to assisting with determining species richness of Sarcocystis in raptors, molecular analysis should be useful in the identification of potential intermediate hosts.     

Experimental transmission of Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum (Didelphis albiventris) to the North American opossum (Didelphis virginiana)

Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum Didelphis albiventris was successfully transmitted to the North American opossum Didelphis virginiana. Sporocysts from a naturally infected D. albiventris from Argentina were fed to 2 gamma-interferon knockout (KO) mice. The mice were killed 64 and 71 days after sporocyst feeding (DAF). Muscles containing sarcocysts from the KO mouse killed 71 DAF were fed to a captive D. virginiana; this opossum shed sporocysts 11 days after ingesting sarcocysts. Sporocysts from D. virginiana were fed to 9 KO mice and 4 budgerigars (Melopsittacus undulatus). Schizonts, sarcocysts, or both of S. speeri were found in tissues of all 7 KO mice killed 29-85 DAF; 2 mice died 39 and 48 DAF were not necropsied. Sarcocystis stages were not found in tissues of the 4 budgerigars fed S. speeri sporocysts and killed 35 DAE These results indicate that S. speeri is distinct from Sarcocystis falcatula and Sarcocystis neurona, and that S. speeri is present in both D. albiventris and D. virginiana.     

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.     

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.     

Sarcocystis lindsayi n. sp. (Protozoa: Sarcocystidae) from the South American opossum, Didelphis albiventris from Brazil

A new species, Sarcocystis lindsayi n. sp., is proposed for a parasite resembling Sarcocystis falcatula. It was obtained from the lungs and muscles of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris, from Jaboticabal, Brazil. Sarcocysts of S. lindsayi n. sp. in budgerigars are microscopic, up to 600 microm long and up to 50 microm wide. The cyst wall is up to 2 microm thick. Ultrastructurally, the sarcocyst wall consists of numerous slender villar protrusions (up to 2.0 microm long and up to 0.3 microm wide), each with a stylet at its tip. Schizonts in cell culture divide by endopolygeny leaving a residual body. Sporocysts are approximately 12 x 7 microm. The parasite is genetically distinct from other organisms that also cycle between opossums and avian species and resemble S. falcatula. Diagnostic genetic variation has been observed in the nuclear large subunit ribosomal RNA gene, the internal transcribed spacer (ITS-1), and each of two other genetic loci. Although the structure of the sarcocyst wall may not provide sufficient grounds for differential diagnosis, several other attributes including schizont morphology and genetic variation at each of these genetic loci permit identification of S. lindsayi n. sp.. Natural intermediate hosts for S. lindsayi n. sp. are not known, and fuller characterization of these and other Sarcocystis species would benefit from experimental avian hosts that are more permissive to the maturation of sarcocysts.     

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.     

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.