Studies of Sarcocystis in Malaysia. II. Comparative ultrastructure of the cyst wall and zoites of Sarcocystis levinei and Sarcocystis fusiformis from the water buffalo, Bubalus bubalis.

The two species of Sarcocystis--S. levinei and S. fusiformis from the water buffalo, Bubalus bubalis, show some ultrastructural similarities in their cyst wall and zoites. The zoites of both species are of about the same size, banana-shaped and have 22 subpellicular microtubules, numerous micronemes, eight rhoptries, a micropore in the region of the micronemes, an elongated mitochondrion, and a nucleus. S. levinei has 200--300 micronemes and S. fusiformis has about 400. The sarcocysts of both species are trabeculated and their cyst walls have cytophaneres containing annulated fibrils and coarse, electron dense granules. The cytophaneres of S. levinei are sloping, with irregular, wavy outlines, whereas S. fusiformis has the cauliflower-type of cytophaneres. This difference in the appearance of the cytophaneres, together with the difference in size of the sarcocysts and their definitive hosts, further confirms that S. levinei and S. fusiformis are two distinct species in the water buffalo.     

Sarcocystis cruzi: comparative studies confirm natural infections of buffaloes

Controversy exists concerning whether cattle and water buffalo sustain infections with cysts of distinct arrays of species in the genus Sarcocystis. In particular, morphologically similar parasites have been alternately ascribed to Sarcocystis cruzi or to Sarcocystis levinei, depending on their occurrence in cattle or water buffalo. We used light and transmission electron microscopy, genetic analysis, and experimental infections of definitive canine hosts to determine whether consistent differences could be identified from parasites derived from several natural infections of each host, examining several tissue types (esophagus, skeletal muscles, and heart). Cysts derived from cattle and water buffalo shared similar structure; variation among 18S rRNA sequences did not segregate consistently according to intermediate host type; parasites derived from cattle and water buffalo induced similar outcomes in the canine definitive host. One cattle specimen harbored unusually large (macroscopic) sarcocysts which nonetheless conformed to previously reported ultrastructural and genetic features of S. cruzi. Finding no consistent basis to differentiate between them, we conclude that the parasites infecting each host and tissue type correspond to S. cruzi. In our sample, no phylogenetically distinct taxon was sampled which might correspond to a distinct taxon previously described as S. levinei. Either that taxon was missed by our sampling effort, or it may represent a junior synonym to S. cruzi, which would then cycle between dogs and a broader range of intermediate bovine hosts than was previously considered.     

试验感染水牛体内枯氏住肉孢子虫内生发育的超微结构

通过人工感染试验在电镜下对一种住肉孢子虫在水牛体内的发育进行了研究。结果揭示在血管内皮细胞中的裂殖生殖和在肌细胞内的包囊发育过程与枯氏住肉孢子虫的一致,裂殖体、裂殖子、包囊壁和囊内母细胞、缓殖子的超微结构亦与枯氏住肉孢子虫的相同。首次通过试验感染的发育史研究证明,流行于我国湖南水牛的一种小包囊属于枯氏住肉孢子虫,水牛是黄牛枯氏住肉孢子虫的新宿主。     

Ultrastructural studies of Sarcocystis cruzi (Hasselmann, 1926) Wenyon, 1926 infection in cattle (Bos taurus): Philippine cases

This paper documents the first report of Sarcocysti s cruzi infection in domesticated cattle (Bos taurus) in the Philippines. Fusiform-shaped microscopic sarcocysts (183-578 microns long and 20-98 microns wide) with distinct septae were found in the skeletal, striated and heart muscle. The sarcocyst wall or parasitophorous vacuolar membrane, 1.37-2.75 microns thick consisted of closely-packed villar protrusions 80-400 nm in dm. Middle and distal segments of VP were bent approximately 90 degrees parallel to the cyst wall surface. The villar core lacked microtubules, and at some points, the distal ends of the VP collectively formed conical tufts. Primary cyst wall had numerous 70-100 nm bubble-like undulations, and the ground substance was 0.25-0.5 micron in thickness. The ultrastructure of S. cruzi cyst wall typifies the Type 7 sarcocyst wall, and bears close similarities with the Philippine and the Vietnam strain of bubaline Sarcocystis levinei.     

Studies on Sarcocystis in Malaysia. I. Sarcocystis levinei n. sp. from the water buffalo Bubalus bubalis.

Light and electron microscopic studies and feeding experiments have confirmed the presence of two species of Sarcocystis in the water buffalo Bubalus bubalis. One is the already known species with large macroscopic sarcocysts, Sarcocystis fusiformia (Railliet, 1897) Bernard and Bauche, 1912 and the other is S. levinei n. sp. which is being described in detail. The sarcocysts of S. levinei are 0.9 x 0.1 mm and the zoites in them 17.8 x 4.2 micrometer. Ultrastructurally, the primary cyst wall shows sloping villi with irregular wavy outlines. Within the villi are coarse granules and annulated fibrils. Trabeculae are present. The sexual stages of S. levinei occur in the subepithelial tissue of the small intestine of the dog and sporocysts shed by this definitive host are 15-16 by 10 micrometer.     

A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa: Sarcocystidae) in Yunnan Province,PR China,reveals the water buffalo (Bubalus bubalis) as a natural intermediate host

A polymerase chain reaction-based restriction fragment length polymorphism (RFLP) approach is used to examine Sarcocystis cruzi-like taxa from the atypical intermediate host, water buffalo, in Yunnan, People's Republic of China. The loci examined lie within the 18S rRNA gene. A total of 15 water buffalo isolates are compared with those of 10 S. cruzi from cattle. RFLP patterns for the S. cruzi isolates from cattle and the S. cruzi-like taxon from water buffalo are found to be identical with all the 12 restriction enzymes used. Interpopulation variation between samples from Kunming and Gengma (Yunnan) is found to be undetectable at these loci for both S. cruzi and the S. cruzi-like taxon. But RFLPs are found between the S. cruzi taxa and S. suihominis from pigs at the same study sites. These findings support the hypothesis that S. cruzi is able to use the water buffalo as an intermediate host and is not restricted to cattle as was previously supposed.     

Experimental Sarcocystis hominis infection in a water buffalo (Bubalus bubalis)

A water buffalo (Bubalus bubalis) was fed 5.0 x 10(5) Sarcocystis hominis sporocysts from a human volunteer who had ingested S. hominis cysts from naturally infected cattle. A necropsy was performed on the buffalo 119 days after inoculation, and a large number of microscopic sarcocysts (approximately 5,000/g) were found in skeletal muscles. Ultrastructurally, the sarcocyst wall from buffalo muscles has upright villar protrusions measuring about 5.6 x 0.8 microm with numerous microtubules that run from the base to the apex. Sarcocysts from this buffalo were infective to 2 human volunteers, confirming their identity as S. hominis. Therefore, we believe that buffaloes can act experimentally as the intermediate host for S. hominis.     

Ultrastructure of the cyst wall of sarcocysts of the roe deer (Capreolus capreolus L.) (author's transl)]

The ultrastructure of the cyst wall of two types of sarcocysts from roe deer is described. In the thin-walled cyst (wall thickness 0.18-00.26 micrometer), the primary cyst wall forms long, finger-shaped protrusions distant from one another and running in parallel with the surface of the cyst (Figs. 1a--d, FP). No fibrils are observable in the protrusions. The primary cyst wall between them forms numerous bubble-like invaginations (Fig. 1c, arrows). In the thick-walled cysts (wall thickness 4.9--7.49 micrometer), the primary cyst wall forms massive, palisade-like protrusions lying close one to another (Figs. 2a, c). There are numerous fibrillar and tubular structures in these protrusions (Fig. 2d), and the primary cyst wall occasionally forms shallow invaginations at the base of some protrusions (Fig. 2b). The unit membrane onthe surface of some protrusions is slightly undulated and covered with a layer of short and thick bars on the outside. The sarcocysts found in roe deer are compared with those from cattle and sheep.     

Sarcocystis greineri n. sp. (Protozoa: Sarcocystidae) in the Virginia opossum (Didelphis virginiana).

Sarcocysts were found in the skeletal muscles of road-killed and live-trapped opossums collected in north central Florida. Sarcocysts were spindle-shaped and macroscopic and had an average measurement of 3.8 mm by 154.6 microm. Sarcocysts were only observed in skeletal muscle. Sarcocysts have invaginations throughout the sarcocyst wall, which is approximately 1 microm thick. Protrusions on the sarcocyst wall are stumpy and digitlike and contain fibrillar elements that extend from the interior portion of the cyst wall through the villi. A new name, Sarcocystis greineri, is proposed for this species.     

Ultrastructural development of the sarcocyst of Sarcocystis rauschorum (Apicomplexa: Sarcocystidae) in the varying lemming Dicrostonyx richardsoni

The development of the sarcocyst of Sarcocystis rauschorum in its intermediate host was studied. Lemmings were orally administered sporocysts of S. rauschorum obtained from snowy owls (Nyctea scandiaca). Beginning at 9 days postinoculation (DPI) and at various intervals to 84 DPI, skeletal muscle tissue taken from the infected lemmings was examined by electron microscopy. At 9 DPI the sarcocysts contained few metrocytes and the cyst wall was flat. The metrocytes underwent endodyogeny, and within a few days the cyst wall of the rapidly growing sarcocyst developed numerous tubulovesicular invaginations into the electron-dense layer, and the wall had a few irregular infoldings. By 21 DPI, banana-shaped bradyzoites appeared, and by 84 DPI the mature cysts were filled with bradyzoites in groups subdivided by septa and by deep infoldings of the cyst wall. The fine structure of the wall remained simple throughout maturation, with no conspicuous invagination or protrusion. The sarcocyst produced in response to S. rauschorum is unlike those from many species of Sarcocystis, which have complex walls that change markedly as the sarcocysts mature; however, its simple appearance is similar to other species that have rodents as intermediate hosts and raptorial birds as definitive hosts.     

Ultrastructure of Sarcocystis booliati Dissanaike and Poopalachelvam, 1975 from the moonrat, Echinosorex gymnurus, in Malaysia

The ultrastructure of the cyst wall and zoites of Sarcocystis booliati from the moonrat Echinosorex gymnurus, was studied with the electron microscope. The primary cyst wall was thin, smooth and filled with a finely-granular, electron-dense material. The surface of the cyst wall had a row of vesicular invaginations. The ground substance beneath the primary cyst wall did not extend into the cyst to form septae. The zoites were covered with a double-layered membrane or pellicle and had an anterior conoid, 2 conoidal rings, 22 subpellicular microtubules, about 8 rhoptries, 50–60 micronemes, scattered lipid droplets, a micropore and a posteriorly situated nucleus, in front of which was a sac-like mitochondrion with vesicular internal cristae. The distinctive features in the ultrastructure of S. booliati were the thinness of the cyst wall, the absence of cytophaneres or trabeculae and the comparatively small number of micronemes in the zoites.     

Structural and functional characterization of cyst cells in Sarcocystis sp. (Sporozoa, Apicomplexa)

By means of light and electron microscopy, the structural pattern of muscle cysts (sarcocysts) was examined for the four species of the genus Sarcocystis: S. muris (from murine skeletal muscles), Sarcocystis sp. and S. fusiformis (from, respectively, heart and skeletal muscles of buffalo), and S. ovifelis (from ovine tong muscles). The orderly fashion of the interior of the cyst is attained by partitition of its space into numerous compartments with the involvement of the intermediate filaments. These, in their turn, are bound to each other by thin filaments to make eventually a common filamentous net. The net limits separate groups of cells referred to as cyst zoites. The common net of filaments and microtubules (when present) may be regarded not only as the organizer of the cyst interior cytoskeleton, but also as the main mechanism of substance transportation in various directions: from the host cell to the sarcocyst, and within or outside the cyst. The role of dedifferentiation, proliferation and differentiation processes is suggested in the establishment of the fixed sequence of events throughout the unidirectional development of cyst cells and their interaction, from precystic meronts to cyst merozoites (gamonts). Special attention is paid to metrocyte morphogenesis and functioning. In the present work, metrocytes subjected to apoptosis were recognized. It is suggested that phenomenon of programmed cell death in metrocytes may be associated with the control of cell number in mature and ageing sarcocysts.     

Light and electron microscopical study on sarcocysts from muscles of the rhesus monkey (Macaca mulatta), Baboon (Papio cynocephalus) and Tamarin (Saguinus(=Oedipomidas) oedipus).

Sarcocystis cysts from muscles of monkeys (baboon, tamarin and rhesus monkey) were studied by means of light and electron microscopy. The differences in the morphology of the cyst wall and parasites clearly indicate that the three monkey species examined were each parasitized by at least a specific Sarcocystis species being not identical with S. nesbitti or S. kortei. The large numbers of cysts found within the muscle fibres point out the important role that have these monkeys as intermediate hosts in the life cycle of Sarcocystis species, where the final hosts are still unknown.     

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.     

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.     

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.     

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nuclotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of approximately 900 bp was used. A total of 26 electromorphs were detected. The electromorphs were sorted into seven different haplotypes that coincided with the six named species and an unidentified species from cattle. These findings support those of our morphological examinations, which suggested that the taxa resembling Sarcocystis hirsuta, S. hominis, both found in water buffalo, and S. sinensis found in cattle, are not new species but are in fact S. hirsuta and S. hominis as found in cattle, and S. sinensis as found in water buffalo; this finding supports the idea that these species can utilize both cattle and water buffalo as intermediate hosts and are not restricted to one or the other host group as previously thought. PCR-RFLP resolved by agarose gel electrophoresis is shown to be an easy and rapid method of discriminating between these species.     

Ultrastructure of the cyst and life cycle of Sarcocystis sp. from wild sheep (Ovis musimon)

Sarcocystis sp. (Eimeriina: Sarcocystidae) is described as a heteroxenous coccidian with domestic dogs as an experimental definitive host and wild sheep (Ovis musimon) as natural intermediate hosts. Mature sarcocysts of this Sarcocystis sp. were examined by transmission electron microscopy. Sarcocysts in various muscle tissues were microscopic, had a thin primary cyst wall and septa and measured 81.0 x 30.5 microns. The cysts were located within muscle cells and were limited by a primary cyst wall (PCW). The cyst surface was highly folded forming densely packed projections. Between the PCW projections the surface of the cyst was marked with pit-like invaginations. The ground substance of the cyst formed a layer at the periphery of the cyst, filled the projections and formed septa which divided the cyst into compartments. Sarcocysts contained numerous bradyzoites that were 15.2 x 3 microns and few metrocytes 11.5 x 3.5 microns. Twelve days after ingesting Sarcocystis sp.-infected wild sheep meat, four dogs began passing sporocysts in their feces: two domestic cats did not pass oocysts or sporocysts after ingesting meat from the same animals. Sporocysts measured 14.8 x 9.9 microns.