An Unusual Structure in the Primary Cyst Wall of Sarcocystis hemionilatrantis1

ABSTRACT. Disc-shaped plaques were found in the primary cyst wall of sarcocysts of Sarcocystis hemionilatrantis in mule deer in Montana. Ultrastructurally, the plaques were 190.5 nm in diameter, 161.6 nm thick and consisted of six distinct layers with microfilaments arising from the innermost layer. These unusual plaques have not been reported previously for any species of Sarcocystis.     

Ultrastructure of the cysts of Sarcocystis grueneri from cardiac muscle of reindeer (Rangifer tarandus tarandus)

Cysts of Sarcocystis grueneri from cardiac muscle of reindeer (Rangifer tarandus) in Norway were examined by transmission electron microscopy. The limiting unit membrane of the cyst proper formed regularly spaced invaginations into the cyst at numerous sites coinciding with interruptions in the underlying osmiophilic layer. The primary cyst wall formed numerous strip-like, sinuous protrusions, which were 30-40 nm thick, 150-300 nm wide and up to 4.5 microns long, and were running in parallel with the surface of the cyst. Generally the protrusions were arranged in several closely spaced layers compressed against the cyst. The nature and arrangement of the protrusions render them undetectable by light microscopy. Cyst ground substance divided the interior of the cyst into compartments containing typical sarcosporidian metrocytes and cystozoites. The cysts of S. grueneri from reindeer were ultrastructurally similar to cysts reported from red deer, roe deer and moose by other workers. The possibility that these cervids are hosts for a common Sarcocystis species is discussed.     

Sarcocystis stenodactylicolubris n. sp., a new sarcosporidian coccidium with a snake-gecko heteroxenous life cycle

Oocysts/sporocysts of Sarcocystis sp. measuring 9.7 (9-10) x 7.6 (7-8) microns were found in the intestinal contents of the Dahl's whip snake Coluber najadum. Of wide spectrum of experimentally inoculated hosts, only species of the family Gekkonidae--Ptyodactylus guttatus and Stenodactylus grandiceps--were found to be susceptible intermediate hosts. Transparent, barely visible sarcocysts found in tail, limbs and tongue striated muscles of the geckoes were 175-200 microns x 35-50 microns in size at 78 DPI. Ultrastructurally, the primary cyst wall was characteristic by spine-like villar protrusions up to 800 nm long, 200-250 nm in diameter at their base, tapering to thinner apex. Protrusions appear typically lobular or irregular in the cross-sections. Back-transmission from P. guttatus to Coluber rogersi leaded to oocysts/sporocysts excretion since 38 days post infection. Based on sarcocyst morphology and experimental data, Sarcocystis stenodactylicolubris is apparently a new species. Based on obtained and already published results, Sarcosporidia parasitising colubrid snakes as definitive hosts are suggested to be family specific on the level of their intermediate host.     

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

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

Diaplacental passage of Sarcocystis antibodies in man and rats (author's transl)]

Sera of babies up to the age of 3 months were tested for Sarcocystis antibodies using the indirect immunofluorescence test (IIFT). In addition titre of the Sarcocystis antibody level of litters born to serologic Sarcocystis positive mother rats was compared to those of their mothers. The results are as follows: 1. 45 (= 14.6%) out of 308 sera from babies up to the age of 3 months reacted positively in the Sarcocystis antibody test. 2. 28 (= 62.2%) out of the 45 positive sera were from babies up to 2 weeks old, 13 (= 28.9%) were from babies older than 2 weeks but not more than 4 weeks old, and 2 (=4.4%) each were from babies whose ages ranged from 4 to 8, and 8 to 12 weeks respectively. 3. These babies acquired their Sarcocystis antibodies which decreased in the first 3 months of life from their mothers. 4. Litters born to serologic Sarcocystis positive mother rats also demonstrated Sarcocystis antibodies. The titres of these antibodies were at the same level as their mothers' at birth but reduced gradually so that most of these young rats were negative at the age of 3 months. 5. Suckling rats born to Sarcocystis negative mothers but positive fathers remained negative. 6. Serologic positive mother and father rats still demonstrated Sarcocystis antibodies in the sera at a time when their litters have become negative. 7. Sarcocystis antibodies could be passed onto the newborn from their positive mothers in both man and rats. 8. These antibodies were probably passed onto the newborn through the placenta but their passage through the colostrum and the mother's milk cannot be ruled out.     

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.     

Identification of Synapomorphic Characteris in the Genus Sarcocystis Based on 18S rDNA Sequence Comparison

ABSTRACT. In order to further investigate synapomorphic characters in the genus Sarcocystis , the small subunit ribosomal RNA gene sequences of Sarcocystis capracanis and Sarcocystis moulei were determined and used to infer the phylogenetic position of these two organisms within the cyst-forming coccidia. Phylogenies derived using distance, maximum parsimony and maximum likelihood methods demonstrated that S. capracanis groups with Sarcoystis tenella and Sarcocystis arieticanis as a clade that shares the characteristic of using canids as their definitive host. S. moulei was shown to group with Sarcocystis gigantea and Sarcocystis fusiformis as a clade that shares the characteristic of using felids as their definitive host.     

Multiple origin of the dihomoxenous life cycle in sarcosporidia

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.     

An undescribed species of Sarcocystis associated with necrotizing meningoencephalitis in naturally infected backyard chickens in the Midwest of Brazil

Sarcocistys -associated menigoencephalitis is virtually an unrecognized cause of neurological disease in chickens. An undescribed species of Sarcocystis cause fatal infection in two backyard chickens in the Midwest of Brazil. Infected chickens presented anorexia, weight loss, incoordination, ataxia and opisthotonos. Yellow necrotic foci in the gray and white matter of the telencephalon were the main gross lesion. Microscopically, necrotizing granulomatous and heterophilic meningoencephalitis with intralesional Sarcocystis -like schizonts and mezoites were observed in the central nervous system. Molecular analysis of frozen brain samples of the two chickens was identical and the protozoan was named Sarcocystis sp. Chicken-2016-DF-BR. Complete nested PCR- sequence of Sarcocystis sp. Chicken-2016-DF-BR was equally similar to Sarcocystis anasi (EU553477) and Sarcocystis albifronsi (EU502868). This is the first report of Sarcocistys –associated meningoencephalitis with molecular characterization in backyard chickens.     

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.     

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.     

Sarcocystis speeri N. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana)

The North American opossum (Didelphis virginiana) is host to at least 3 species of Sarcocystis: Sarcocystisfalcatula, Sarcocystis neurona, and a recently recognized Sarcocystis sp. A new name, Sarcocystis speeri, is proposed for the third unnamed Sarcocystis. Immunodeficient mice are an experimental intermediate host for S. speeri. Sarcocystis speeri sporocysts are 12-15 x 8-10 microm in size, and its schizonts are found in many organs of mice. Sarcocysts of S. speeri are found in skeletal muscles and they are up to 5 mm long and filiform. By light microscopy, the sarcocyst wall is thin (<1 microm thick); ultrastructurally, the cyst wall is up to 1.8 microm thick and has characteristic steeple-shaped villar protrusions surmounted by a spire. Sarcocystis speeri schizonts are morphologically and antigenically distinct from schizonts of S. neurona, and S. speeri sporocysts were not infective to budgerigars (Melopsittacus undulatus).     

Identification of Sarcocystis hominis-like (Protozoa: Sarcocystidae) cyst in water buffalo (Bubalus bubalis) based on 18S rRNA gene sequences

DNA templates were extracted from isolates of Sarcocystis hominis-like cysts collected from cattle and water buffalo, as well as from Sarcocystis fusiformis cysts and Sarcocystis suihominis cysts. The 18S rRNA genes were amplified using DNA from a single cyst as the templates. Approximately 1,367-1,440 bp sequences were obtained. The sequence difference in isolates of Sarcocystis hominis-like cysts from water buffaloes, and isolates of S. hominis cysts from cattle were very low, only about 0.1%, much lower than the lowest value (1.7%) among different species. Combined with their morphological structure, these sequence data indicate that the 4 isolates from cattle and water buffalo might be the same species, i.e., S. hominis, suggesting that both cattle and water buffalo may serve as the intermediate hosts for this parasite. Apparently, this is the first report using a single cyst to do such work and is a useful way to distinguish the Sarcocystis cyst in an intermediate host that may be simultaneously infected by several different Sarcocystis species.     

The ultrastructural changes of Sarcocystis ovifelis infested sheep tongue myofibrils

Structural changes were observed in filaments of Sarcocystis ovifelis infected sheep tongue myofibrils. In sarcocysts containing myofibrils, actin filaments and Z-disks, myosin filaments and M-line were seen destroyed. Protein bridges, uniting actin and myosin filaments into a joint complex (net), eventually become not visible, and as a result separate Z-disks and free filaments appear. Fibrils, referred to as leptomeric, have been first revealed between protrusions of the sarcocyst surface apparatus. These are striated filaments with periodic 100 nm striation of dark and light bands, made of thin and short 120-200 nm long filaments 5 nm in diameter. The genesis of leptomeric fibrils still remains obscure. In sarcocysts infected myofibrils these may be involved in metabolite transportation to the intercellular space and back.     

Prevalence of Sarcocystis spp. cysts in Japanese and imported beef (Loin: Musculus longissimus)

A survey was carried out to investigate the occurrence of Sarcocystis infection in the loin (Musculus longissimus) of Japanese and imported beef. In all, the muscle tissue of 482 samples were examined by histological method. The prevalence of Sarcocystis unspecified species cysts was lower in Japanese beef (total 6.31%: 0% in Holstein castrated, 12.96% in Holstein milk cow, 3.33% in Japanese shorthorn and 11.58% in Japanese black cattle) than in beef imported from America (36.78%) or Australia (29.49%). The infection density of imported beef, especially in American, was higher than in Japanese beef. All detected cysts except one were identified as Sarcocystis cruzi. One thick walled cyst was found in Australian beef but it could not be distinguished as either Sarcocystis hirsuta or Sarcocystis hominis.     

Sarcocystis dubeyi (Huong and Uggla, 1999) infection in water buffaloes (Bubalus bubalis) from Egypt

Water buffaloes (Bubalus bubalis) are intermediate hosts for 4 species of Sarcocystis , i.e., Sarcocystis fusiformis and Sarcocystis buffalonis with cats as definitive hosts; Sarcocystis levinei with dogs as definitive hosts; and Sarcocystis dubeyi with an unknown definitive host but thought to be zoonotic. Currently, the latter species has been identified with certainty only from Vietnam. In the present study, sarcocysts of S. dubeyi are reported in 11 (30%) of 35 Egyptian water buffaloes from which the esophageal muscles were examined histologically. Sarcocysts were microscopic, measuring 180-250 × 70-110 μm in size. Ultrastructurally, the sarcocyst wall was 3.5-6.5 μm thick and had palisade-like villar protrusions which give it a striated appearance. The villar protrusions contained microtubules that were distributed along the whole villus. This is the first report of S. dubeyi from water buffaloes in Egypt.     

Effects of frozen storage on the structure of sarcocysts in pig muscle and implications in taxonomic studies

The morphology of the cyst wall of Sarcocystis has unique characteristics that can be used in species identification. To find a suitable way to preserve Sarcocystis cyst samples for species identification, by light microscopy and electron microscopy, we recorded the morphological changes in the cysts of Sarcocystis suihominis and Sarcocystis miescheriana from pig muscle, induced by storage at -20 degrees C. Comparisons were made between fresh cysts and those subjected to frozen storage for periods of 3 days, 20 days and 30 days. Results: cyst wall of the two Sarcocystis species appeared unaffected by storage. There was no obvious change in the length, nor in the width of the protrusions after storage (P>0.05), but the structure of the bradyzoite in the sarcocyst was in many cases disintegrated at -20 degrees C in 20 days for S. miescheriana and 30 days for S. suihominis. To our knowledge this is the first report that Sarcocystis cyst in muscle can be stored at -20 degrees C before and remain suitable for ultrastructural morphological study. Consequently, this paper proposes freezing as a convenient storage method for samples used in taxonomic studies of Sarcocystis.     

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.