Structure of Sarcocystis neurona sarcocysts.

The ultrastructure of Sarcocystis neurona sarcocysts was studied from muscle of an experimentally infected cat. The cat was killed 144 days after being fed sporocysts from a naturally infected opossum. Sarcocysts were microscopic, up to 700 microm long, and up to 50 microm wide. By light microscopy, the sarcocyst wall was 1-2 microm thick. Ultrastructurally, the sarcocyst wall consisted of numerous villar protrusions. The villar protrusions were up to 2.8 microm long and 0.4 microm wide, with a tapered end. Microtubules extended from the tip of the villus to the base and occasionally extended deep into the granular layer. The granular layer was approximately 0.5 microm thick. Longitudinally cut bradyzoites were 5.2 by 1.2 (4.8-6.5 by 1.0-1.3) microm in size. Micronemes in bradyzoites were numerous and located in the anterior 1/3 of the conoidal end.     

Biological control of rodents using Sarcocystis singaporensis

Parasites have been identified as an important factor in regulating vertebrate populations. In replicated field experiments (plots up to 4 ha) performed in Thailand we tested whether commensal and field rodents could be artificially infected and controlled with the host-restricted apicomplexan protozoon Sarcocystis singaporensis which is endemic in Southeast Asia. When bait-pellets containing high numbers of these parasites were consumed by rodents of three species (Rattus norvegicus, Rattus tiomanicus, Bandicota indica) in different agricultural habitats (chicken farm, oil palm plantation, ricefield), we observed a parasite-induced mortality ranging from 58% to 92%. Detection of merozoites of S. singaporensis in lung tissue samples of rats collected dead at the experimental sites using a species-specific monoclonal antibody confirmed that S. singaporensis was the causative agent of mortality. As observed with brown rats, the parasite's effect on the host was not related to sex. These experiments demonstrate for the first time that artificial infection of rodents with an endemic protozoon has the potential for effective population control.     

In Vitro Cultivation of the Vascular Phase of Sarcocystis singaporensis

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (± 3.8) μm in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (± 10.6) μm and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm² flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.     

Fine structure of the development of Sarcocystis singaporensis in Python reticulatus from macrogamont to sporulated oocyst stage

Three, 4-month old reticulated pythons (Python reticulatus) hatched from eggs laid by a newly caught female from Singapore Island, were fed on muscles of Sarcocystis singaporensis-infected Rattus rattus caught in Singapore. Snakes were sacrificed five, six and eight days later. The infected tissues were studied by transmission electron microscope. The present communication summarizes findings on macrogamont and oocyst stages. In the premature stages, rough endoplasmic reticulum consolidate into a large rectangular array; the electron-dense wall-forming-like bodies reveal a laminar structure. Macrogamont parasitophorous vacuoles became filled with granular matrix and electron-dense strands, which later on consolidate into a coat around the fertilized zygote. The oocyst wall is constructed from several formed membranes combined with deposited substance. All development to the sporulated oocyst stage occurs in the mucosal epithelium.     

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.     

Immunoglobulin subclass responses of wild brown rats to Sarcocystis singaporensis

Immunoglobulin subclass responses of wild brown rats (Rattus norvegicus) from southeastern Asia to the endemic cyst-forming coccidian Sarcocystis singaporensis were characterised. The antibody response of brown rats to wild-type parasites (high reproductive capacity) showed a Th1 profile during acute infection, namely elevated concentrations of parasite-specific IgG2b and IgG2c and absence of IgG1. Chronic infection (bradyzoite development) resulted in a mixed Th1/Th2 pattern whereby significant concentrations of IgG1 appeared. A primary infection with 1000 sporocysts eight days before challenge induced protection, accompanied by significant concentrations of IgA and IgG2, particularly IgG2a. Western blot analysis of rat sera, using sporozoite and bradyzoite-extracts as antigen, revealed that IgG1, IgG2a, and IgG2b predominantly recognised molecules between 70-80 kDa in one or the other stage. Some of the antibodies were possibly directed against a 79 kDa heat shock protein of sporozoites. An apparent unresponsiveness to molecules in the low molecular weight range, particularly of bradyzoite antigens, was observed. This was abrogated by infection of rats with an avirulent strain of S. singaporensis (low reproductive capacity) indicating that a parasite that was less adapted to its host provoked a stronger immune response. These results suggest the existence of an immune evasion strategy used by Sarcocystis and show that wild rodents may be suitable as immunological research objects, reflecting the natural situation.     

Redescription of the sarcocysts of Sarcocystis rileyi (Apicomplexa: Sarcocystidae)

The intermediate hosts for Sarcocystis rileyi (Stiles 1893) Minchin 1913 are ducks (Anas spp.), and the striped skunk (Mephitis mephitis) is its definitive host. The structure of sarcocysts from an experimentally infected shoveler duck (Anas cylpeata) fed sporocysts from an experimentally-infected M. mephitis was studied and compared with type specimens from a naturally infected duck. The experimentally infected duck was killed 154 d after feeding sporocysts. By light microscopy the sarcocyst wall was 3-5 microm thick with indistinct villar protrusions. Ultrastructurally, the sarcocyst wall was a type-23 cyst wall with anastomosing villar protrusions that were up to 7.5 microm long. The villar projections contained filamentous structures. The bradyzoites were 12-14 microm long. Structurally, the sarcocyst from the naturally infected and experimentally infected ducks appeared similar.     

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.     

Experimental Sarcocystis hominis infection in cattle: lesions and ultrastructure of sarcocysts

Five calves inoculated orally with 10(5)-10(6) sporocysts of Sarcocystis hominis from human feces were necropsied 10, 18, 24, 111, and 222 days postinoculation (DPI). Calves became febrile (greater than 40-41 C) between 10 and 24 DPI and developed mild anemia (packed cell volumes were reduced by 40% of initial values) between 29 and 57 DPI but otherwise remained clinically normal. Focal hepatitis, mesenteric lymphadenitis, and myocarditis were seen in calves at 10, 18, and 24 DPI. No stages of the parasite were found at any of these times except for a few merozoites in macrophages associated with myocardial lesions in the calf necropsied 24 DPI. Mature sarcocysts at 111 and 222 DPI were up to 950 microm long and their walls were up to 6 microm thick. They were found only in skeletal muscles. One immature sarcocyst was seen in the myocardium of the calf at 222 DPI.     

Reduction of transmission stages concomitant with increased host immune responses to hypervirulent Sarcocystis singaporensis, and natural selection for intermediate virulence.

Parasite virulence (pathogenicity depending on inoculum size) and host immune reactions were examined for the apicomplexan protozoan Sarcocystis singaporensis. This parasite is endemic in southeastern Asia and multiplies as a proliferation (merozoite) and transmission stage (bradyzoite) in rats. Virulence in wild brown rats of parasites freshly isolated in the wild (wild-type) was surprisingly constant within the endemic area and showed an intermediate level. In contrast, serially passaged parasites either became avirulent or virulence increased markedly (hypervirulence). Production of transmission stages was maximal for the wild-type whereas numbers were significantly reduced for hypervirulent and avirulent (shown in a previous study) parasites. Analyses of B and T cell immunity revealed that immune responses of WKY rats to the transmission stage were significantly higher for hypervirulent than for wild-type parasites. These results suggest that it is the immune system of the host that is not only responsible for reduction of transmission stages in individual rats, but also could act as a selective force that maintains intermediate virulence at the population level because reduction of muscle stages challenges transmission of S. singaporensis to the definitive host. Collectively, the presented data support evolutionary theory, which predicts intermediate rates of parasite growth in nature and an ‘arms race’ between host immunity and parasite proliferation.     

Development of the cyst-forming coccidian Sarcocystis muris in the body of its intermediate host

On examining the cyst merogony of S. muris, two patterns of development were established. The one occurs in young cysts where metrocytes underwent endopolygeny, giving rise to the intermediate cells. No dividing metrocytes were seen in the older cysts. Unlike, metrocyte differentiation was observed in these from 1, 2 and 3 step metrocytes to the intermediate cell state, which is the other pattern of development. An original scheme of development within the cysts of S. muris is proposed.     

Prevalence and site specificity of Sarcocystis greineri sarcocysts in Virginia opossum (Didelphis virginiana) in Florida

Sarcocysts of Sarcocystis greineri in the Virginia opossum (Didelphis virginiana) were observed for documenting sarcocyst prevalence, seasonal prevalence, and muscle specificity. Characteristics of sarcocysts found in striated muscle were recorded, as were light microscopy measurements. Overall prevalence of sarcocysts in striated muscle was 10.0% (24/240). No statistical difference (P = 0.156) in prevalence was detected between summer (13.1%; 16/122) and fall (6.7%; 8/118). Sarcocysts were found in muscles of the diaphragm, leg, breast, tongue, back, and esophagus. Diaphragm had the highest specificity of 72.7% (8/11), which was significantly different (P = 0.05) when compared with tongue and esophagus at 16.6% (1/6). Breast and leg muscle had a specificity of sarcocysts of 54.5% (6/11), whereas 27.2% (3/11) of back muscles contained sarcocysts.     

Ultrastructural study of microsporidian development

Summary Vegetative growth of Nosema sp. occurs within the gut submucosal cells of Callinectes sapidus. Vegetative cell morphology is dominated by profiles of endoplasmic reticulum, numerous free ribosomes and aggregates of vesicles enclosed by a membranous sac. The dikaryotic vegetative cell is the earliest stage found in the target area for sporogenesis, the sarcoplasm of the striated muscle cell. The next obvious stage is the sporoblast mother cell; it undergoes karyokinesis without breakdown of the nuclear envelope. Intranuclear mitotic microtubules extend from the chromosomes to the intact nuclear envelope. After repeated nuclear divisions, the sporoblast mother cell undergoes delayed cytokinesis and a series of sporoblast progeny develops.The polar filament is the first visually apparent system to develop during sporogenesis. It appears to be of dual origin: (1) the central core component is condensed in Golgi-like saccules, and (2) the envelopes around the core originate from the endoplasmic reticulum.The polaroplast, which forms after early polar filament development, appears to originate as an elaboration of the endoplasmic reticulum.Supported in part by a training grant from the National Institutes of Health (GM-669-05) and research grants from the National Science Foundation (GB-3036, GB-5235, and GB-7938) to Prof. F. Sogandares-Bernal. The skillful guidance of Prof. F. Sogandares-Bernal is acknowledged. Special thanks are extended to Prof. D. E. Copeland for the use of a Siemens Elmiskop IA electron microscope. I also wish to thank Mr. Julian King, professional fisherman of Irish Bayou, Louisiana, for providing hundreds of blue crabs used in the course of this study.     

Life cycle of Sarcocystis neurona in its natural intermediate host,the raccoon,Procyon lotor

Sarcocystis neurona causes encephalomyelitis in many species of mammals and is the most important cause of neurologic disease in the horse. Its complete life cycle is unknown, particularly its development and localization in the intermediate host. Recently, the raccoon (Procyon lotor) was recognized as a natural intermediate host of S. neurona. In the present study, migration and development of S. neurona was studied in 10 raccoons that were fed S. neurona sporocysts from experimentally infected opossums; 4 raccoons served as controls. Raccoons were examined at necropsy 1, 3, 5, 7, 10, 14, 15, 22, 37, and 77 days after feeding on sporocysts (DAFS). Tissue sections of most of the organs were studied histologically and reacted with anti-S. neurona-specific polyclonal rabbit serum in an immunohistochemical test. Parasitemia was demonstrated in peripheral blood of raccoons 3 and 5 DAFS. Individual zoites were seen in histologic sections of intestines of raccoons euthanized 1, 3, and 5 DAFS. Schizonts and merozoites were seen in many tissues 7 to 22 DAFS, particularly in the brain. Sarcocysts were seen in raccoons killed 22 DAFS. Sarcocysts at 22 DAFS were immature and seen only in skeletal muscle. Mature sarcocysts were seen in all skeletal samples, particularly in the tongue of the raccoon 77 DAFS; these sarcocysts were infective to laboratory-raised opossums. This is the first report of the complete development of S. neurona schizonts and sarcocysts in a natural intermediate host.     

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.