Ultrastructure of Sporozoites and Zoites of Hammondia heydorni

The ultrastructure of sporozoites and zoites of Hammondia heydorni was studied in cultured bovine cells. In addition to ultrastructural features typical of coccidian parasites, H. heydorni sporozoites and zoites contain rhoptries that are located posteriorly as well as anteriorly. Also, sporozoites contain a posteriorly located crystalloid body (1.2 μm in diameter); a small crystalloid body (0.5 μm in diameter) was occasionally seen in the anterior end. Zoites resulting from the 1st division of endodyogeny contain a posteriorly located crystalloid body, which is absent in zoites formed by subsequent divisions. Zoites contain posteriorly located amylopectin granules and a relatively large anterior vacuole which is not present in sporozoites. During penetration, the host cell plasmalemma ballooned laterally around the sporozoite creating a large cavity, which later disappeared. Sporozoites and zoites undergoing cell penetration usually exhibit partially empty anterior rhoptries; no changes occur in posterior rhoptries. Lysosomes fuse with the par-asitophorous vacuole surrounding killed sporozoites but not live sporozoites.     

Sarcocystis neurona (Protozoa: Apicomplexa): description of oocysts, sporocysts, sporozoites, excystation, and early development

Equine protozoal myeloencephalitis is a major cause of neurological disease in horses from the Americas. Horses are considered accidental intermediate hosts. The structure of sporocysts of the causative agent, Sarcocystis neurona, has never been described. Sporocysts of S. neurona were obtained from the intestines of a laboratory-raised opossum fed skeletal muscles from a raccoon that had been fed sporocysts. Sporocysts were 11.3 by 8.2 microm and contained 4 sporozoites. The appearance of the sporocyst residuum was variable. The residuum of some sporocysts was composed of many dispersed granules, whereas some had granules mixed with larger globules. Excystation was by collapse of the sporocyst along plates. The sporocysts wall was composed of 3 layers: a thin electron-dense outer layer, a thin electron-lucent middle layer, and a thick electron-dense inner layer. The sporocyst wall was thickened at the junctions of the plates. Sporozoites were weakly motile and contained a centrally or posteriorly located nucleus. No retractile or crystalloid body was present, but lipidlike globules about 1 microm in diameter were usually present in the conoidal end of sporozoites. Sporozoites contained 2-4 electron-dense rhoptries and other organelles typical of coccidian zoites. Sporozoites entered host cells in culture and underwent schizogony within 3 days.     

In vitro development from sporozoites to first-generation merozoites in Eimeria contorta Haberkorn, 1971. A fine structural study.

The asexual development of Eimeria contorta from sporozoites to first-generation merozoites in tissue culture was investigated with the electron microscope. Sporozoites with a three-layered pellicle, 26 subpellicular microtubules, a conoid, 4-7 rhoptries, and an abundance of micronemes actively entered host cells and showed direct contact to the host cell's cytoplasm. Shortly after penetration, small vacuoles surrounding the parasite merged into a parasitophorous vacuole. Inside this vacuole, sporozoites assumed a definite U-shape before transformation into schizonts took place. This process was characterised by the occurrence of subpellicular microtubules exclusively in the anterior half of the sporozoite, by a degeneration of the 2 inner pellicular membranes, by an outpocketing of the parasite's surface, and by the arrangement of microtubules in clusters. About 25 merozoites were formed at the surface of mature schizonts, to which they remained attached at their posterior pole. A polar ring was present at that area. Anterior and posterior refractile bodies were conspicuous in merozoites and showed close association with mitochondria. The significance of a fibrillar substructure in rhoptries and micronemes is discussed, and special attention is drawn to the pathway of nutrient transport from host cell mitochondria and dictyosomes through intravacuolar folds, parasitophorous vacuole and crescent body into the parasite's food vacuoles.     

Early Developmental Stages of Sarcocystis cruzi in Calf Fed Sporocysts from Coyote Feces1

ABSTRACT. The development of Sarcocystis cruzi was studied in an 11-day-old calf killed seven days postinoculation with 5 × 10⁸ sporocysts from feces of coyotes. Uninucleate zoites were found in arteries of mesenteric lymph nodes but not in other organs. Zoites measured 4.9 × 3.0 (3.5–7.0 × 2.1–3.5) μm. Of the 36 zoites studied, 31 were in endothelial cells, four were in macrophages in the lumen of arteries, and one was free in the lumen of an artery. Infected endothelial cells were two to three times larger than uninfected cells. Zoites appeared structurally similar to sporozoites. The occurrence of zoites in macrophages suggests that sporozoites of Sarcocystis might use such cells to reach the site of their first merogony.     

Sarcocystis canis n. sp. (Apicomplexa: Sarcocystidae), the etiologic agent of generalized coccidiosis in dogs

Sarcocystis canis n. sp. is proposed for the protozoon associated with encephalitis, hepatitis, and generalized coccidiosis in dogs. Only asexual stages are known in macrophages, neurons, dermal, and other cells of the body. The parasite is located free in the host cell cytoplasm without a parasitophorous vacuole; schizonts divide by endopolygeny. Schizonts are 5-25 x 4-20 microns and contain 6-40 merozoites. Merozoites are approximately 5-7 microns x 1 micron and do not contain rhoptries. The parasite is PAS-negative and reacts with Sarcocystis cruzi antiserum but not with Toxoplasma gondii, Neospora caninum, or Caryospora bigenetica antisera in an immunohistochemical test.     

Development of Hammondia heydorni in cultured bovine and ovine cells

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4-10 DAI, considerably more zoites were harvested from M617 cultures (80.1 x 10(6) zoites) than from CPA (17.4 x 10(6], MDBK (47.3 x 10(6], and WOMO (53.5 x 10(6]. Little or no parasite multiplication occurred at 10-16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis

Sarcocystis neuronan n. sp. is proposed for the apicomplexan taxon associated with myeloencephalitis in horses. Only asexual stages of this parasite presently are known, and they are found within neuronal cells and leukocytes of the brain and spinal cord. The parasite is located in the host cell cytoplasm, does not have a parasitophorous vacuole, and divides by endopolygeny. Schizonts are 5-35 microns x 5-20 microns and contain 4-40 merozoites arranged in a rosette around a prominent residual body. Merozoites are approximately 4 x 1 micron, have a central nucleus, and lack rhoptries. Schizonts and merozoites react with Sarcocystis cruzi antiserum but not with Caryospora bigenetica. Toxoplasma gondii, Hammondia hammondi, or Neospora caninum antisera in an immunohistochemical test.     

Transmission electron microscopy of intracellular sporozoites of Eimeria vermiformis (Apicomplexa, Eucoccidiida) in the mouse

Sporozoites of Eimeria vermiformis from the mouse were first seen in the epithelial cells of villus tips and the crypts of Lieberkühn four hours after inoculation (HAI). They were always within a parasitophorous vacuole. By 12 HAI, most were in crypt epithelial cells between the basement membrane and host cell nucleus. The sporozoites in the villus tips had 26 subpellicular microtubules, two polar rings, two preconoidal rings, two refractile bodies surrounded by amylopectin-like granules, a lamellar Golgi apparatus, numerous micronemes, and rhoptries. The sporozoites in the crypt cells had fewer amylopectin-like granules, micronemes, and rhoptries. A nucleolus was visible, as were pieces broken off from the posterior refractile body. Later, the sporozoites folded over to become U-shaped; the infolded membranes fused; and then the inner membranes disappeared so that spherical meronts were formed. Folding sporozoites were first seen 16 HAI and persisted until 52 HAI.     

Transmission Electron Microscopy of Intracellular Sporozoites of Eimeria vermiformis (Apicomplexa,Eucoccidiida) in the Mouse1

ABSTRACT. Sporozoites of Eimeria vermiformis from the mouse were first seen in the epithelial cells of villus tips and the crypts of Lieberkühn four hours after inoculation (HAI). They were always within a parasitophorous vacuole. By 12 HAI, most were in crypt epithelial cells between the basement membrane and host cell nucleus. The sporozoites in the villus tips had 26 subpellicular microtubules, two polar rings, two preconoidal rings, two refractile bodies surrounded by amylopectin-like granules, a lamellar Golgi apparatus, numerous micronemes, and rhoptries. The sporozoites in the crypt cells had fewer amylopectin-like granules, micronemes, and rhoptries. A nucleolus was visible, as were pieces broken off from the posterior refractile body. Later, the sporozoites folded over to become U-shaped; the infolded membranes fused; and then the inner membranes disappeared so that spherical meronts were formed. Folding sporozoites were first seen 16 HAI and persisted until 52 HAI.     

Fine Structure of Penetration of Cultured Cells by Isospora canis Sporozoites

SYNOPSIS Monolayers of Embryonic Bovine Trachea (EBTr) cells were inoculated with Isospora canis Nemeséri spcrozoites. As penetration commenced, they were fixed, stained with OsO₄-ruthenium red, dehydrated, embedded and sectioned in situ. Examination by electron microscopy revealed that host cell membranes remained intact during penetration. The sporozoites caused an invagination of the cell's plasmalemma until the parasites were entirely within the cell, after which the invagination was sealed by short pseudopodia enclosing the parasite within a membrane-lined vacuole inside the cells. Rhoptries and micronemes, which appeared as branched elements of the same network, became less tortuous near the conoid and often became empty or partially empty during penetration. Concurrent with the appearance of these partially empty rhoptries, vesiculations were seen in the host cell cytoplasm opposite the apical tip of the sporczoite. Constrictions of the sporozoite during entry were probably due to bands of microfilaments beneath the plasmalemma and elsewhere in the cytoplasm of the host cell.     

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.     

Ultrastructural observations on the infection of rat liver by Plasmodium berghei sporozoites in vivo

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

Ultrastructural Observations on the Infection of Rat Liver by Plasmodium berghei Sporozoites In Vivo1

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

Caryospora uptoni n. sp. (Apicomplexa: Eimeriidae) from red-tailed hawks (Buteo jamaicensis borealis)

Oocysts of Caryospora uptoni n. sp. were described from the feces of red-tailed hawks, Buteo jamaicensis borealis. Sporulated oocysts were spherical or subspherical and measured 28.1 by 26.4 micron. The oocyst wall was composed of a yellowish outer layer and brownish inner layer and was about 1.5 micron thick. Neither micropyle, polar granules, nor oocyst residuum were present. A single, spherical sporocyst 18.2 by 17.9 micron was present; a Stieda body was absent. A spherical eccentrically located sporocyst residuum was present in many sporocysts, but it degenerated to form a dispersed granular residuum in other sporocysts. Eight randomly arranged sporozoites, 12.6 by 4.2 micron, were present in each sporocyst; they contained a centrally or slightly posteriorly located nucleus.     

Previously unknown apicomplexan species infecting Iceland scallop, Chlamys islandica (Müller, 1776), queen scallop, Aequipecten opercularis L., and king scallop, Pecten maximus L

Examination of three scallop species from three separate locations: Iceland scallop from Icelandic waters, king scallop from Scottish waters and queen scallop from Faroese and Scottish waters, revealed infections of a previously unknown apicomplexan parasite in all three scallop species. Developmental forms observed in the shells appeared to include both sexual and asexual stages of the parasite, i.e. merogony, gametogony and sporogony, which suggests a monoxenous life cycle. Meronts, gamonts, zygotes and mature oocysts were solely found in the muscular tissue. Zoites, which could be sporozoites and/or merozoites, were observed in great numbers, most frequently in muscles, both intracellular and free in the extracellular space. Zoites were also common inside haemocytes. Examination of the ultrastructure showed that the zoites contained all the major structures characterizing apicomplexans. This apicomplexan parasite is morphologically different from other apicomplexan species previously described from bivalves. Presently, its systematic position within the phylum Apicomplexa cannot be ascertained.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

Fine Structure of Oocyst Transformation and the Sporozoites of Leucocytozoon dubreuili*

SYNOPSIS. The sporogonic stages of Leucocytozoon dubreuili in the midgut and salivary glands of the simuliid vectors was studied by electron microscopy. Young uninucleate oocysts have a pellicle that initially resembles that of the ookinetc. Numerous electron-dense bodies and microtubules in the peripheral cytoplasm may be involved in the formation of the cyst wall. The dense bodies appear to give rise to the amorphous material of the wall. The tubules which run circumferentially beneath the oocyst's boundary probably serve as a skeletal support for the cell surface during deposition of the wall material. A subcapsular “space” which provides area for expansion of the developing sporozoites is formed in early multinucleate oocysts. The subcapsular “space” appears to be formed through a condensation of the peripheral cytoplasm, resulting in an osmotic gradient across the oocyst's limiting membrane. Consequently water diffuses out, creating a fluid-filled space. Sporozoite formation begins with localized thickenings on the oocyst's limiting membrane. Subsequent extension of the thickened regions into the subcapsular “space” marks the onset of sporozoite budding. The process is highly synchronized, and culminates with the production of up to 150 sporozoites about the sporoblastoid body. The structure of sporozoites from mature oocysts and of the salivary glands of the vector is basically similar, although salivary gland sporozoites are more elongate and have numerous electron-dense micronemes. The paired rhoptries in the latter sporozoites are more elongate and uniformly electron-dense than in oocyst sporozoites.     

Observations on the ultrastructure of Haemogregarina simondi in the marine flatfish Solea solea (L.).

Developing stages of Haemogregarina simondi from the marine fish Solea solea (L.) were examined by electron microscopy. Merozoites lay in a parasitophorous vacuole and were bound by a pellicle of three unit membranes beneath which lay a ring of 45--61 microtubules. The cytoplasm contained 4--6 rhoptries, more than 169 micronemes, several mitochondria, and amylopectin granules. A conoid and one polar ring were observed at the anterior end. Intraleucocytic and intraerythrocytic schizonts with up to eight merozoites were described also. Intraerythrocytic and free gametocytes were characterized by distinct refractile bodies and a pellicle consisting of only two unit membranes. The number of micronemes was in excess of 194. The results were discussed in comparison with other members of the Haemogregarinidae.     

Salivary gland of the tick vector of East Coast fever. III. The ultrastructure of sporogony in Theileria parva

Sporogony of the sporozoan Theileria parva in the salivary gland of the tick vector of East Coast fever was studied in electron micrographs. The findings differ in several respects from previous interpretations based upon light microscopy. Cytokinesis of the primary sporoblast to form secondary and tertiary sporoblasts is not substantiated. Instead it is suggested that the parasite develops as a ramifying, multinucleate syncytium rapidly increasing in size and complexity until it gives rise to myriad sporozoites in a terminal episode of cytoplasmic fission. The proliferating nuclei initially occupy peripheral lobules that are continuous with a central labyrinth of branching and anastomosing processes which present a very large surface area for interchange of metabolites with the host cell cytoplasm. The membrane of the labyrinth is rich in cytostomes, but no evidence if found to bulk uptake of host cytoplasmic matrix or organelles into food vacuoles. Rhoptries are the first of the polar organelles of the parasite to develop and are associated with dense plaques irregularly distributed on the inner aspect of the parasite membrane. Micronemes form independently of the rhoptries at a later stage. After 3-4 days of tick feeding, sporogeny is complete and the infected salivary gland cell contains up to 50, 000 spherical or ovoid sporozoites about 1 micrometer in diameter. These are limited by a simple plasma membrane. The inner layer of the 'pellicle', the polar ring, and the conoid described for zoites of other Apicomplexa are lacking. Maturational changes are noted in sporozoites after sporogony is completed. Micronemes appear to increase in size, and possibly in number, from days 3-5 and the majority take up positions immediately subjacent to the plasmalemma.     

Zoosporulation of a new Perkinsus species isolated from the gills of the softshell clam Mya arenaria

A gill-associated Perkinsus sp. isolated from the softshell clam (Mya arenaria) is described as a new species, P. chesapeaki sp. nov. Examination of the parasite in seawater cultures revealed life cycle stages and zoosporulation processes similar to those described for other species of the genus Perkinsus. Prezoosporangia developed thickened cell walls upon contraction of the cytoplasm and development of a distinctive clear area between the cell wall and the protoplast. Successive bipartition of the protoplast led to the formation of hundred's of zoospores within mature sporangia. Zoospores were released into seawater through one or more discharge tubes. Ultrastructural studies revealed an oblong zoospore possessing two flagella that arose from a concave side located in the upper third of the zoospore body. The anterior flagellum possessed a unilateral array of hair-like structures. A large anterior vacuole and basolateral nucleus dominated the cytoplasm of the zoospore body. The presence of a rudimentary apical complex including an open-sided conoid, rhoptries, micronemes, and subpellicular microtubules were also discerned. Differences in zoospore morphology, and sequence analyses of two genes previously reported, support the designation of the gill-associated Perkinsus from the softshell clam as a new species.