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.     

Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the budgerigar (Melopsittacus undulatus). IV. Ultrastructure of developing, mature and degenerating sarcocysts

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 ultrastructure 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 merozoites, 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.     

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.     

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.     

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 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.     

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.     

Identification of Sarcocystis cymruensis in wild Rattus flavipectus and Rattus norvegicus from Peoples Republic of China and its transmission to rats and cats

Sarcocystis cymruensis was initially identified in skeletal muscles of 22 (11.6%) of 189 wild rats (Rattus spp.) captured in 2008 in Anning and Kunming, Peoples Republic of China. Sarcocyst walls were thin (<1 μm) and smooth. Ultrastructurally, the parasitophorous vacuolar membrane had small, osmiophilic knob-like invaginations covered with numerous vesicle-like invaginations toward the interior of the cyst. Domestic cats (Felis catus) fed sarcocysts shed sporocysts measuring 10.3 (9.8-11.0) × 7.6 (7.2-9.5) μm with a prepatent period of 6 to 8 days. Sarcocysts were infective orally to Norway rats, and oocysts and sporocysts developed in the lamina propria of the small intestine of rats fed sarcocysts. Thus, rats were both intermediate and definitive hosts for S. cymruensis.     

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.     

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.     

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.     

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.     

Development of Ox-Coyote Cycle of Sarcocystis cruzi1

The ox-coyote cycle of Sarcocystis cruzi was studied by killing 38 calves between 4 and 153 days postinoculation (DPI) with 55 × 10³-5 × 10⁸ sporocysts from the intestines of coyotes. At 4 DPI, a zoite was found within the lumen of a mesenteric lymph node artery. At 7 DPI, zoites were found in mononuclear cells and in endothelial cells in mesenteric arteries. First generation meronts (41.0 × 17.5 μm in diameter) occurred 7–26 DPI in mesenteric lymph nodes. At 19–46 DPI, second generation meronts occurred in kidneys, muscles, and other tissues: renal meronts were 19.6 × 11.0 μm, and intramuscular meronts were 25.0 × 11.1 μm. Merozoites were found in the peripheral blood 17 DPI and later at 24–46 DPI. They divided by endodyogeny in mononuclear cells. Sarcocysts were seen first in the heart at 45 DPI and contained one or two metrocytes. At 55 DPI, sarcocysts containing only metrocytes were found in striated muscles, heart, and in smooth muscles of the urinary bladder, rumen, omasum, abomasum, and small intestine. At 67, 87, 112, and 153 DPI, sarcocysts were found only in striated muscles and in the heart. At 67 DPI, sarcocysts were up to 360 μm long. They contained only metrocytes and were not infective to the dog. At 86 DPI, sarcocysts contained mostly bradyzoites, a few metrocytes, and were infective to a coyote. The thin-walled sarcocysts grew to a maximum length of 800 μm and contained bradyzoites that were 10.9 × 3.0 μm. At 90 DPI, two mature sarcocysts were found in 2 of 73 sections of brain and spinal cord; hundreds of sarcocysts were present in sections of tongue and heart of this calf. Gametogony occurred in the small intestine of the coyote. Macro-and microgamonts were found in goblet cells of the small intestines of coyotes 6 h after the ingestion of infected meat. Microgamonts were few and contained 3–11 slender gametes. Oocysts were seen at 12 h and sporulation was completed 9 DPI. The prepatent period in the coyote was 8 days. The ox-coyote cycle is compared with ox-dog cycle.     

Muscular sarcocystosis in coyotes from Oklahoma

In a recent survey in Oklahoma (USA), 52 free-ranging coyotes were examined for the presence of sarcocysts. Two of these coyotes were found infected with sarcocysts in skeletal muscle. By light microscopy, the cyst wall was thin and smooth. Ultrastructurally, the cyst wall had minute villar protrusions. The sarcocysts were 14.4 to 50.4 microm wide and 46.8 to 99 microm long. This is the first report of Sarcocystis sp. sarcocysts in the skeletal muscle of coyotes.     

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.     

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 dubeyella n. sp. and Sarcocystis phacochoeri n. sp. (Protozoa: Sarcocystidae) from the Warthog (Phacochoerus aethiopicus) in South Africa

ABSTRACT Sarcocystis dubeyella n. sp. and S. phacochoeri n. sp. from muscle fibers of the skeletal musculature of two warthogs in South Africa are described by light and and electron microscopy. Sarcocystis dubeyella sarcocysts are macroscopic (up to 12 mm long and 1 mm wide), with a parasite-induced encapsulation of the host muscle fiber in which the plasma membrane of the latter remained unaltered. The sarcocyst wall is characterized by evenly arranged, irregularly semicircular or rectangular villar protrusions (5.0 T. 2.8-11.0 μm) with indented margins and no specific content. Sarcocystis phacochoeri formed filiform microcysts (up to 4 mm long and 0.13 mm wide). Its cyst wall is provided with tightly packed, molarlike villar protrusions (1.6-3.3 T. 1.7-3.3 μm), with smooth margins, hollow on one side, and with longitudinal condensations of the fine granular matrix at various locations in the interior.     

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.     

Two new species of Sarcocystis (Apicomplexa: Sarcocystidae) infecting the wolverine (Gulo gulo) from Nunavut, Canada

Infection with Sarcocystis species is common in many species of animals, but it has not yet been reported in wolverines (Gulo gulo). Histological sections of tongues from 41 wolverines in the Kitikmeot Region, Nunavut, Canada, were examined for sarcocysts. Sarcocysts were found in 33 (80.4%) wolverines. Two structurally distinct types of sarcocysts were found. Type A sarcocysts were thin (<1 μm thick) walled. Ultrastructurally, the parasitophorous vacuolar membrane (Pvm) had minute undulations, but it lacked villar protrusions and was not invaginated into the granular layer. The bradyzoites were slender, about 5 × 1 μm in size. Structurally, these sarcocysts were distinct from known species of Sarcocystis and possessed a novel 18S and ITS-1 sequence, sharing 98% and 78% sequence similarity with Sarcocystis canis . A new species name, Sarcocystis kalvikus, is proposed for type A sarcocysts. In contrast, type B sarcocysts had relatively thicker (about 2 μm) cyst walls and larger bradyzoites, each about 10 × 2-3 μm. Ultrastructurally, the Pvm on the sarcocyst wall had villar protrusions that were either mushroom-like or sloping. Molecular analysis identified a unique 18S and ITS-1 sequence that placed them in a clade within the Sarcocystidae. Based on histology, TEM, and genetic data, the new name, Sarcocystis kitikmeotensis, is proposed. Sarcocystis kalvikus was found in 14 (34.1%), S. kitikmeotensis was found in 7 (17%), and both species were found in 12 (29.2%) of 41 wolverines.     

Completion of the life cycle of Sarcocystis zuoi , a parasite from the Norway rat, Rattus norvegicus

Transmission experiments were performed to elucidate the life cycle of Sarcocystis zuoi found in Norway rats ( Rattus norvegicus ) in China. Two king rat snakes ( Elaphe carinata ) fed sarcocysts from the muscles of 4 naturally infected Norway rats shed sporocysts measuring 10.8 ± 0.7 × 8.0 ± 0.7 μm, with a prepatent period of 8-9 days. Sporocysts from the intestine of 2 experimentally infected king rat snakes were given to the laboratory Sprague-Dawley (SD) rats ( R. norvegicus ) and Kunming (KM) mice ( Mus musculus ). Microscopic sarcocysts developed in the skeletal muscles of SD rats. No sarcocysts were observed in KM mice. Characters of ultrastructure and molecule of sarcocysts from SD rats were confirmed as S. zuoi . Our results indicate that king rat snake is the definitive host of S. zuoi .