Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the Budgerigar (Melopsittacus undulatus). IV. infrastructure of Developing, Mature and Degenerating Sarcocysts

ABSTRACT. Sarcocysts in cardiac and skeletal muscles of budgerigars (Melopsittacus undulatus) were examined transmission electron microscopically 5 to 168 days after experimental infection with Sarcocystis falcatula. The ultrastnicture of the primary cyst wall, amorphous substance, metrocytes and bradyzoites in developing, degenerating and mature sarcocysts is described and compared with precystic merozoites studied previously. Sufficient morphologic differences between precystic rnerozoites, metrocytes and bradyzoites (cystozoites) were found which seem to justify their semantic differentiation. Significant differences in immature and mature primary cyst wall morphology were encountered. If primary cyst wall morphology is to be used in determination and differentiation of species of Sarcocystis , then caution must be used to employ only mature sarcocysts.     

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.     

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.     

The cytochemical demonstration of glycoproteins and glycolipids in Sarcocystis muris sarcocysts]

An electron cytochemical study of glycoproteins and glycolipids was made for the mature sarcocysts of Sarcocystis muris. Glycoprotein structures as branched fibrilles were seen on the surface of the sarcocyst wall. The fibrillar and granular glycoprotein structures were found in the ground substance of sarcocysts near the cyst wall and in the septae. In the plasmalemma of two types of cyst stages (merozoites and intermediate cells), glycoprotein fibrillar structures were revealed connecting these two cell types with each other. The third type cyst stages, i.e. the metrocytes, are situated separately without any fibrillar connections between them and other cyst stages being observed. This question is discussed in terms of the problem of cytodifferentiation. The fibrillar and granular glycoprotein material is scattered over the cytoplasm of the cyst stages, being especially concentrated in micronemes, rhoptries and around amylopectin granules. The control ultrathin sections were treated with saliva or pronase for the aims of protein identification in the material under study. In addition to glycoprotein, some glycolipids material was detected in the sarcocysts in the form of drops surrounded with thin glycoproteinaceous layers. Glycolipids were found in the ground substance of sarcocysts near the cyst stages and in the parasite cell cytoplasm around the micronemes and rhoptries. The data obtained are discussed in connection with the functional role glycoproteins and glycolipids play in S. muris.     

Morphological studies on the muscle cysts of Sarcocystis dirumpens (Hoare 1933) H?fner and Matuschka 1984 in several host species revealing endopolygeny in metrocytes

Muscle cysts from rodents (Mastomys natalensis, Mus musculus, Rattus norvegicus, Meriones unguiculatus, Phodopus sungorus) experimentally infected with Sarcocystis dirumpens were examined by light and electron microscopy. The corrugated primary wall showed a pattern of densely arranged invaginations surrounded by minute bleb-like evaginations. True protrusions were absent. The length of the blebs and the thickness of the primary wall varied insignificantly but not remarkable host-dependent morphological differences could be noticed between the cyst wall structures 60-418 days p.i. In the mature parts of the cysts merozoites and metrocytes showed typical apicomplexan features of fine structure and mode of multiplication (endodyogeny). In the tips of the cysts large metrocytes simultaneously formed more than two daughter cells (at least up to 12) by endopolygeny.     

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.     

Ultrastructure of Sarcocystis sp. from the muscle of a white-tailed deer (Odocoileus virginianus)

Sarcocystis sp. from the muscle of naturally infected white-tailed deer (Odocoileus virginianus) was examined by transmission electron microscopy. The primary cyst wall forms regularly spaced protrusions filled with electron-lucent ground substance; no fibrils are present in the protrusions. The cysts are divided by septa into compartments containing typical coccidian metrocytes and merozoites. Taxonomy of the protozoon from the white-tailed deer-dog is discussed.     

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.     

Light and electron microscope study of Sarcocystis sp. from the fallow deer (Cervus dama)

By means of light and electron microscopy a study was made of Sarcocystis sp. from 11 fallow deer (Cervus dama). Cysts of Sarcocystis sp. were found in the tongue and abdominal muscle of 3 of 11 deer from forests near Bonn (FRG). These measured 212-560 micron in length and 54-120 micron in width and contained metrocytes and merozoites. The cyst wall, which had narrow band-like protrusions, is compared with other Sarcocystis sp. from Cervidae.     

Electron microscopic study of the cyst of the sarcosporidian, Sarcocystis ovifelis

Cysts of S. ovifelis, examined from the sheep oesophagus muscles have been shown to be covered by a cyst wall made of a primary and a secondary envelopes. Within the cyst, three morphologically different cell types are distinguished: metrocytes, merozoites, and interstitial cells. The latter have been first discovered for the genus Sarcocystis, in addition to earlier literature evidence of their availability in cysts of the genus Frenkelia.     

Light optical and electron microscopic study of the cystic stages in Sarcocystis fusiformis from the buffalo

Three different cell types are distinguished in the sarcocysts of Sarcocystis fusiformis from the water buffalo: metrocytes, intermediate cells, and merozoites. The former lines the cyst near the border, the two latter lie more inside forming groups of cells separated from each other by septae. The pattern of metrocyte division giving rise to another metrocyte population has not been observed, still remaining obscure. Merozoites do not divide asexually due to their gamont nature to be realized in the final host only. The intermediate cells divide asexually by endodyogeny giving rise, on the one hand, to another population of intermediate cells, and on the other--to merozoites which divide no longer. Cytophotometrical measurements (G. D. Gaibova, 1987) revealed the amounts of DNA per cell nucleus within 1 and 2 c. This quantity corresponds to the stable DNA content in the most numerous population of merozoites (gamonts), whereas the amount between those higher than 1 c and 2 c may be attributed to the nuclei of metrocytes and intermediate cells, respectively.     

Development of Sarcocystis suicanis Erber, 1977 in the pig

Twenty-eight weanling pigs inoculated with sporocysts from an isolate of Sarcocystis suicanis from Georgia were examined at intervals ranging from 2 to 90 days postinoculation (DPI). Merogony was first observed histologically within the heart muscle 12 DPI and within 23 of 35 tissues examined 13 DPI. Most infected cells were "floating" in extravascular spaces and were near intact endothelial cells. In some cases, the infected cell clearly was an endothelial cell comprising a portion of the capillary wall. Immature sarcocysts containing metrocytes were observed in striated muscle 27 DPI, and bradyzoites were detected by digestion techniques 52 DPI. Sarcocysts matured between 27 and 80 DPI, after which thickness of the cyst wall and morphology of bradyzoites changed little. Dissolution of sarcocysts was detected as early as 38 DPI and was accompanied by ingress of plasma cells, lymphocytes, and occasionally, eosinophils. Based on information presented herein, feeder pigs reared on pasture may become infected, and infections mature well within the 100-day period usually considered necessary for production of marketable swine.     

Sarcocystis sigmodontis n. sp. from the cotton rat (Sigmodon hispidus)

Sarcocystis sarcocysts were found in 3 of 4 cotton rats (Sigmodon hispidus) from Atlanta, Georgia. Sarcocysts were several centimetres long and were present only in skeletal muscles. The sarcocyst wall appeared thin (less than 1 micron), with minute projections in the light microscope. By transmission electron microscopy, the sarcocyst wall had 0.6-1.0 x 0.21-0.36-micron villar protrusions without microtubules. The metrocytes were 6.5 x 3.8 micron, and the bradyzoites were 8 x 2.7 micron. The sarcocysts were not infectious for dogs and cats. The parasite was named Sarcocystis sigmodontis because it differed from all sarcocysts in rodents.     

The cytochemical study of polysaccharides in coccidia of the genus Sarcocystis. I. Sulfated glycosaminoglycans in Sarcocystis muris sarcocysts]

An electron microscope study of sulfatized glycosaminoglycans (SG) was made for cyst stages of S. muris. The polysaccharides were detected in the submembranous and subwall layers of the sarcocysts, in addition to the ground substance and septae. Moreover SG were discovered in the cyst stages themselves--metrocytes, intermediate cells and merozoites (gamonts). SG were discernible as electron dark spots in vacuoles of the metrocytes. SG shaped as granules were scattered in the cytoplasm of both intermediate cells and merozoites. More granules of SG were seen in the cytoplasm of the merozoites compared to the intermediate cells. Thus, the quantity, localization and structure of SG are seen to follow the process of differentiation in muscle cysts of S. muris.     

Ultrastructural study of the development of Sarcocystis singaporensis sarcocysts in the muscles of its rat host

Laboratory rats fed sporocysts of Sarcocystis singaporensis (Zaman & Colley, 1975) Zaman & Colley, 1976 originating from Singapore were euthanized 22, 23, 33 and 80 days later. Sporocysts were extracted from feces of either naturally or laboratory-infected Python reticulatus. Electron microscopically examined longue and esophageal muscles yielded images of successive developing stages of sarcocysts. The primary wall evolved from a continuous thin layer into folds and later, into villar protrusions. At all stages the wall was interrupted by pinocytotic-like indentations. Young sarcocysts contained only metrocytes, they divided by endodyogeny into daughter metrocytes. The first bradyzoites appeared only 33 d.p.i. Sarcocysts by 80 d.p.i. were enclosed in a fully differentiated primary wall and contained almost entirely bradyzoites.     

Current conception of the Sarcosporidia (Sarcocystis, Eimeriidae, Sporozoa, Apicomplexa): their morphofunctional organization, life cycle and practical importance

Advances in sarcosporidian research within the latest decade are critically reviewed and analysed in respect to some recent cytological findings of the author and her colleagues on the subject. Three rather than two morpho-functional cell types (metrocytes and merozoites) are distinguished within the sarcocyst, the third one being the intermediate cell. Division by endodyogeny in the cyst of Sarcocystis is very likely confined to the latter cell type. The pattern of nuclear chromatin and the constancy in DNA value per nucleus in the cystic merozoites, revealed by flow cytometry, is rather indicative of their incapability of dividing within the cyst, i.e. in the intermediate host. This enabled us to consider these merozoites as homologs of coccidian gamonts. The obvious differences between cysts and cystic stages in Sarcocystis and Toxoplasma may account in part for the rare, if any, reported cases of congenital sarcocystosis in the intermediate host.     

Light optical and electron microscopy research on the cyst-forming coccidium Sarcocystis muris

Part of the complicated life cycle of Sarcocystis muris, confined to the muscle cyst (sarcocyst), has been studied by light and electron microscopy. The early development of the sarcocyst proceeds strictly intracellularly, whereas the older and larger cysts tend to destroy the harbouring muscle cell, and since then their development seems to be intercellular rather than intracellular. Three different cell types are distinguished within the growing sarcocyst of S. muris differing from each other both structurally and functionally: metrocytes, intermediate cells and merozoites. These differ as well in the structure of their nuclei. The metrocyte nuclear chromatin is mainly in decondensed state with some minute granules taking the central part of the nucleus. The condensed chromatin of the intermediate cell is accumulated into some relatively large peripheral granules, whereas numerous RNP-granules appear in the karyolymph. The nuclear chromatin of merozoites is condensed to be seen as separate chromocenters scattered over the nucleus; the karyolymph is packed with RNP-granules. Metrocytes are seen to divide in young sarcocysts, although the mode of their division is still obscure. In sarcocysts of advanced age (2.5 months or more), only intermediate cells are seen to divide, their mode of division being endodyogeny.     

The initial phase of cyst formation in Sarcocystis dispersa sarcosporidians

Six white mice were inoculated orally with Sarcocystis dispersa sporocysts and killed on days 10, 12 and 14 post inoculation (p. i.). Precyst merozoites were found in leukocytes and muscular cells on days 10 and 12 p. i. Young cysts containing only metrocytes were found on days 12 and 14 p. i. Precyst merozoites in muscular cells were situated freely, without any parasitophorous vacuole. The process of merozoite transformation into a primary metrocyte runs parallel with the process of sarcocyst wall formation.     

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.