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.     

Sporogonic Development of the Gregarine Ascogregarina taiwanensis (Lien and Levine) (Apicomplexa: Lecudinidae) in Its Natural Host Aedes albopictus (Skuse) (Diptera: Culicidae)

ABSTRACT. Sexual reproduction of Ascogregarina taiwanensis occurred in pupal Malpighian tubules of its natural host Aedes albopictus , resulting in the formation of gametocysts within which oocysts developed. Sporogony proceeded in each newly formed unsporulated oocyst; eight sporozoites were formed after completion of nuclear divisions followed by the cytokinesis. Developing oocysts were separated by gradient centrifugation on percoll based on different buoyant densities. The slender sporozoite had a typical apical complex composed of a coiled conoid, polar rings, rhoptries with ductules, subpellicular microtubules and micronemes. An apical cavity was seen in the gland-like rhoptries. Mitochondria of gregarines were not seen in any stage during the sporogony. Howeever, amylopectin granules were frequently seen in the cytoplasm. These starch-related granules became scant when the sporozoite was formed. We assumed they were associated with the energy source. Since the apical complex was only present in the sporozoite stage, it was most likely related to the invasion of host epithelial cells of the midgut during the early phase of infection.     

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.     

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.     

Light and electron microscopy on the sporulation of the oocysts of Eimeria brunetti. II. Development into the sporocyst and formation of the sporozoite

The later stages of sporulation in oocysts of Eimeria brunetti were examined in samples which had been allowed to sporulate at 27 degrees C for 24, 36 and 48 hours. It was observed that the sporoblasts became ellipsoidal and the nucleus underwent the final division. A nucleus with associated Golgi bodies was not observed at either end of the organism. The cytoplasm was limited by two unit membranes and contained rough endoplasmic reticulum, dense bodies, electron translucent vacuoles and mitochondria. The first evidence of sporozoite formation was the appearance of a dense plaque at either end of the organism. This appeared in the vicinity of the nuclei, and adjacent to the limiting membrane of the soroblast. At this stage the sporocyst wall was still unformed. Then the two sporozoites were formed from opposite ends of the organism by growth of the dense plaques and invaginations of the plasmalemma which thus formed the pellicles of the developing sporozoites. A conoid and subpellicular microtubules were observed at this stage as development continued, a number of vacuoles were found between the nucleus and the conoid. These vacuoles constituted the precursors of the rhoptries and micronemes. At the same stage a large dense body had appeared within the forming sporozoite. As the sporozoite developed, this body, anterior refractile body, is followed by the nucleus and another dense body which formed the posterior refractile body. During this period, the thin sporocyst wall was formed and Stieda and sub-Stieda bodies were now present at one end of the sporocyst. Each mature sporocyst contained two sporozoites.     

Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites

Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120-180 kDa range.     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Ultrastructure of In Vivo-Produced Caryocysts Containing the Coccidian Caryospora bigenetica (Apicomplexa: Eimerüdae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35–1.00 μm thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Ultrastructure of in vivo-produced caryocysts containing the coccidian Caryospora bigenetica (Apicomplexa: Eimeriidae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35-1.00 microns thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Observations on First-Generation Schizogony of Leucocytozoon caulleryi in Chickens

ABSTRACT. First and second generation schizogony of Leucocytozoon caulleryi occurred in chickens infected with sporozoites. First generation schizogony was studied by light and electron microscopy. First-generation schizonts were first detected in capillary endothelial cells in the spleen, lung, liver, and bursa of Fabricius between 3 and 6 d post-sporozoite inoculation (DPI). The schizonts ranged from 15 to 65 μm in diameter and were surrounded by a thin pellicle. Early schizonts contained numerous round or oval nuclei, endoplasmic reticulum, and mitochondria. The schizonts reached maturity 5 DPI and produced first-generation merozoites which were released into the peripheral bloodstream. The merozoites. which were infective to chickens, measured 7.1 μm in length. They were slender and had a large nucleus, a mitochondrion, and an apical complex consisting of three polar rings, rhoptries, numerous micronemes. The morphology of first-generation merozoites was different from that of second-generation merozoites.     

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.     

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 micron in diameter); a small crystalloid body (0.5 micron 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 parasitophorous vacuole surrounding killed sporozoites but not live sporozoites.     

In vitro Development of First-Generation Schizonts of Eimeria canadensis (Bruce, 1921)*†

SYNOPSIS. In vitro development of Eimeria canadensis from cattle was studied in monolayer cultures of various bovine cell lines grown on coverslips in Leighton tubes. Excysted sporozoites were used for inoculation of the cell cultures. Sporozoites entered the host cells within a few minutes, but apart from a reduction in the number of refractile bodies, changed little in appearance during the first 9 days. Beginning at 91/2 days postinoculation, sporozoites developed into sporozoite-shaped schizonts or, less frequently, transformed into trophozoites. Sporozoite-shaped schizonts with as many as 8 nuclei were observed transforming into spheroid schizonts. At 111/2 days, intermediate schizonts had a characteristic single mass of refractile granules and 60–80 nuclei. Deep invaginations, which resulted in the formation of several blastophores, usually occurred when schizonts had about 100 nuclei. Merozoites were formed as a result of radial outgrowth from the surface of spheroid schizonts as well as of blastophores. Mature merozoites were seen 1st after 13 days.     

Distribution of Ca-ATPase, ATP-dependent Ca-transport, calmodulin and vitamin D-dependent Ca-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na⁺ + K⁺)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca²⁺-transport in fraction II (8.2 ± 0.3 nmol Ca²⁺/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca²⁺-transport activity (0.9 ± 0.1 nmol Ca²⁺/min per mg protein). The distribution of high-affinity Ca²⁺-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca²⁺-transport. The activity of Na⁺/Ca²⁺ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca²⁺-transport system, the Na⁺/Ca²⁺ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

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.     

Surface-membrane biogenesis in rat intestinal epithelial cells at different stages of maturation.

The biosynthesis of membrane proteins and glycoproteins has been studied in rat intestinal crypt and villus cells by measuring the incorporation of L-[5,6-3H] fucose, D-[2-3H] mannose and L-[3,4,5-3H] leucine, given intraperitoneally, into Golgi, lateral-basal and luminal membranes. Incorporation of leucine and mannose was approximately equal in crypt and villus cells, whereas fucose incorporation was markedly higher (3-4 times) in the differentiated villus cells. As previously reported [Quaroni, Kirsch & Weiser (1979) Biochem J. 182. 203-212] most of the fucosylated glyco-proteins synthesized in the villus cells and initially present in the Golgi and lateral-basal membranes were found re-distributed, within 3-4h of label administration, in the luminal membrane. A similar process appeared to occur in the crypt cells, where, however, only few fucose-labelled glycoproteins were identified. In contrast, most of the leucine-labelled and many mannose-labelled membrane components found in the lateral-basal membrane of both crypt and villus cells did not seen to undergo a similar re-distribution process. The fucosylated glycoproteins of the intestinal epithelial cells represent, therefore, a special class of membrane components, most of which appear with differentiation, that are selectively localized in the luminal portion of the plasmalemma. In contrast with the marked differences in protein and glycoprotein patterns between the luminal membrane of villus and crypt cells, only minor differences were found between their lateral-basal membrane components: their protein patterns on sodium dodecyl sulphate/polyacrylamide slab gels, and the patterns of fucose-, mannose- and leucine-labelled components (analysed 3-4h after label administration) were very similar. Although the minor differences detected may be of importance, it appears that most of the surface-membrane changes accompanying cell differentiation in the intestinal epithelial cells are localized in the luminal portion of their surface membrane.     

Cellular identity of a novel small subunit rDNA sequence clade of apicomplexans: description of the marine parasite Rhytidocystis polygordiae n. sp. (host: Polygordius sp., Polychaeta)

A new species of Rhytidocystis (Apicomplexa) is characterized from North American waters of the Atlantic Ocean using electron microscopy and phylogenetic analyses of small subunit (SSU) rDNA sequences. Rhytidocystis polygordiae n. sp. is a parasite of the polychaete Polygordius sp. and becomes the fourth described species within this genus. The trophozoites of R. polygordiae were relatively small oblong cells (L=35-55 microm; W=20-25 microm) and distinctive in possessing subterminal indentations at both ends of the cell. The surface of the trophozoites had six to eight longitudinal series of small transverse folds and several micropores arranged in short linear rows. The trophozoites of R. polygordiae were positioned beneath the brush border of the intestinal epithelium but appeared to reside between the epithelial cells within the extracellular matrix rather than within the cells. The trophozoites possessed a uniform distribution of paraglycogen granules, putative apicoplasts, mitochondria with tubular cristae, and a centrally positioned nucleus. The trophozoites were non-motile and lacked a mucron and an apical complex. Intracellular sporozoites of R. polygordiae had a conoid, a few rhoptries, micronemes, dense granules, and a posteriorly positioned nucleus. Phylogenies inferred from SSU rDNA sequences demonstrated a close relationship between R. polygordiae and the poorly known parasite reported from the hemolymph of the giant clam Tridacna crocea. The rhytidocystid clade diverged early in the apicomplexan radiation and showed a weak affinity to a clade consisting of cryptosporidian parasites, monocystids, and neogregarines.     

Ultrastructural development of first- to second-generation merozoites in Eimeria contorta Haberkorn, 1971.

The development of first-generation merozoites to second-generation schizonts and merozoites of Eimeria contorta in one of its natural hosts, the mouse, was investigated with the electron microscope. Merozoites inside a host cell show a marked U-shape and a degeneration of the inner-pellicular membrane complex prior to transformation into schizonts. These processes closely resemble those seen in transforming sporozoites. In young schizonts with about 3-5 nuclei, the Golgi-adjuncts (structures of unknown function) form a large interconnected network. Nuclear divisions in growing schizonts involve the formation of a centroc?ne, which develops in a pocket-like indentation of the nuclear envelope. At least one centriole is present immediately adjacent to this indentation. In a later stage, the centroc?ne forms a conical nuclear protrusion directed towards a merozoite-anlage. This developing merozoite contains anlagen of a conoid, of rhoptries, and of micronemes and a refractile body in addition to the nucleus, centrioles, and a Golgi-adjunct. The merozoite-anlage is limited by a triple unit membrane complex. Schizonts give rise to 8-15 second-generation merozoites. Interesting features of these merozoites are the high number of micronemes, the finding of one single large mitochondrion per merozoite, and the occurrence of 26 subpellicular microtubules, i.e. the same number as in sporozoites of E. contorta. At the end of their development, merozoites come into direct contact with the host cell cytoplasm as the parasitophorous vacuole breaks down.     

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.