Development and Ultrastructure of First-Generation Meronts of Sarcocystis cruzi in Calves Fed Sporocysts from Coyote Feces*

SYNOPSIS. The development of Sarcocystis cruzi Hasselmann (syn. S. fusiformis Railliet) meronts was studied in seven 7- to 10-day-old calves killed 4, 7, 11, 15, 22, 25 and 28 days postinoculation (DPI) with 5 × 10⁷ sporocysts from feces of coyotes. No meronts were found 4 and 7 DPI. Young and intermediate meronts with 1–16 nuclei were found in endothelial cells of arteries in mesenteric lymph nodes, but not in kidneys 11 DPI. Mature meronts were noted in endothelial cells of arteries, arterioles, or capillaries of many organs of calves killed 15 to 25 DPI. No first-generation meronts were found 28 DPI. By electron microscopy, all stages of the first-generation merogony were found free within the host cell cytoplasm and not within a parasitophorous vacuole. The appearance of intranuclear spindles preceded the formation of merozoites by endopolygeny. Mature meronts measured 41.0 × 17.5 (34–50 × 15–24) μm, contained ～ 100–350 merozoites, and had 2 to 4 relatively small residual bodies, 2.8 μm in diameter. Merozoites measured 6.3 × 1.5 (5.5–7 × 1 μm) and contained most of the organelles characteristically found in coccidian merozoites. Micropores were observed in merozoites, but not in young and intermediate meronts. Merozoites were seen free in the lumen of blood vessels, in intracellular areas, and free within the host cell cytoplasm.     

Fine Structure of Gametogony and Oocyst Formation in Sarcocystis sp. in Cell Culture

Fine structure of gametocytes and oocyst formation of Sarcocystis sp. from Quiscalus quiscula Linnaeus grown in cultured embryonic bovine kidney cells was studied. Microgametocytes measured up to ～5 μm diameter. During nuclear division of the microgametocyte, dense plaques were found adjacent to the nucleus just beneath the pellicle; occasionally microtubules were present within these plaques. These microtubules subsequently formed 2 basal bodies with a bundle of 4 microtubules between them. Microgametocytes also contained numerous mitochondria, micropores, granules, vacuoles, and free ribosomes. Each microgamete was covered by a single membrane and consisted of 2 basal bodies, 2 flagella, a bundle of 4 microtubules, a perforatorium, a mitochondrion, and a long dense nucleus which extended anteriorly and posteriorly beyond the mitochondrion. The bundle of 4 microtubules is thought to be the rudiment of a 3rd flagellum. Macrogametes were covered by a double membrane pellicle, and contained a large nucleus (～2.5 μm), vacuoles, and a dilated nuclear envelope connected with the rough endoplasmic reticulum (ER). In young macrogametes (～4 μm), the ER was arranged in concentric rows in the cortical region, and several sizes of dense granules were found in the cytoplasm. However, in later stages (～8 μm) the ER was irregularly arranged and was dilated with numerous cisternae; only large dark granules remained and a few scattered polysaccharide granules were found. No Golgi apparatus or micropores were observed. After the disappearance of dark granules 5 concentric membranes appeared. Four of these fused to form an oocyst wall composed of a dense outer layer (～66 nm thick) and a thin inner layer (～7 nm). The 5th or innermost membrane surrounded the cytoplasmic mass which was covered by a 2-layered pellicle and contained a nucleus, small amounts of ER, large vacuoles, and mitochondria. The sexual stages described greatly resemble those of Eimeria and Toxoplasma.     

On the Fine Structure of the Nucleus in Trypanosoma cruzi in Tissue Culture Forms. Spindle Fibers in the Dividing Nucleus*

The fine structure of the dividing nucleus in the intracellular amastigote forms of Trypanosoma cruzi from tissue cultures has been described. In the first phase of division the nucleus shows a homogenous structure owing to the dispersion of its chromatin and nucleolar material. Microtubules similar to those of a mitotic spindle in metozoan cells then appear, running from one pole to the other. They disappear when the division of the nucleus is complete and the chromatin and the nucleolar material reorganize into their former positions.     

Fine structure of immature cysts of Sarcocystis cruzi.

Sarcocystis cruzi forms cysts in striated muscle of the bovine host following schizogony. The fine structure of the immature cyst within muscle fibers of the ventricular myocardium was studied in relation to its development and to the multiplication of parasites within it. The young cyst is enclosed by a cyst wall containing numerous small protuberances. Metrocytes within the cyst are irregular in shape and are separated from each other and the cyst wall by a thin layer of ground substance. The parasite multiplies by endodyogeny within the metrocyte. As the cyst enlarges, the host muscle fiber is disrupted and large protrusions are present in the cyst wall.     

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.     

Fine Structure of the Endogenous Stages of Eimeria labbeana. 5. Schizonts and Merogony,with Special Reference to the Rhoptry-Microneme System*

SYNOPSIS The fine structure of the 3 generations of meronts, merogony, and merozoites of Eimeria labbeana Pinto from the ileal mucosa of experimentally infected pigeons, Columba livia Linnaeus, was described and compared to that of similar stages in other species of Eimeria. Sporozoite-trophozoite transition stages, trophozoites (5.8 × 4.2 μm), young meronts (10.1 × 8.4 μm), and mature meronts with free merozoites of the first generation, were observed at 20, 28, 36, and 48 hr post-infection, respectively. The 2nd and 3rd generation merogony were completed at 96 and 144 hr. Merogony was essentially of the ectomerogonous type without cytomere formation, as in most species. The average number of merozoites per meront in the 3 generations was 10 (5–15), 14 (8–19), and 7.5 (6–16); and the average size was 4.4 × 2.1 (4.1–5.9 × 1.8–2.2) μm, 4.2 × 1.8 (4.0–4.8 × 1.5–2.0) μm, and 5.4 × 1.8 (5.2–7.8 × 1.6–2.0) μm, respectively. Aggregation and subsequent degeneration of micronemes within membrane-bounded vesicles in the sporozoite-trophozoite stage, was observed as a possible mode of eliminating certain organelles present in the motile stages. Centrioles with (9 + 1) microtubular composition, and centrocones, were frequently seen in early meronts. Anlagen of micronemes, without any apparent association with the Golgi complex and the merozoite bud, were seen to develop in the cytoplasm of the meront. A single, median structure, probably representing the anlage of the rhoptry-microneme system was observed within the conoid of an early merozoite bud. Connections between the micronemes and the bulbous portion of the rhoptries, and a branched (interconnected ?) structure of the rhoptries observed in the present study, substantiate the present contention that the micronemes and rhoptries are functional forms of the same complex of organelles, the rhoptry-microneme system.     

Fine Structure of Schizonts and Merozoites of Eimeria nieschulzi*

SYNOPSIS. Schizonts of E. nieschulzi lie in a vacuole within the host cell. After nuclear division the cell membrane invaginates forming merozoites. Differentiation of the pellicle and other organelles occurs while merozoites are still attached to the schizont cytoplasm. Merozoites have a pellicle thickened at the anterior end to form a polar ring. Radiating posteriorly from the ring, directly beneath the pellicle, are about 25 microtubules. Within the polar ring is a dense conoid. Extending posteriorly from within the conoid is a paired organelle. The paired organelle varies in size and shape in each generation of merozoites. Numerous toxonemes occupy the anterior half of the merozoites. Two paranuclear bodies are present in 1st generation merozoites. One or 2 granular bodies were seen in the anterior end of 2nd generation merozoites. In 3rd generation merozoites 6 or more granular bodies were seen anterior to the nucleus. Each merozoite has a single nucleus containing diffuse chromatin material. Elongate mitochondria and glycogen granules are present. The vacuole surrounding mature merozoites contains residual cytoplasm of the schizont and some granular material. Microvilli project into the vacuole from the host cell membrane.     

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.     

Sarcocystis cruzi: comparative studies confirm natural infections of buffaloes

Controversy exists concerning whether cattle and water buffalo sustain infections with cysts of distinct arrays of species in the genus Sarcocystis. In particular, morphologically similar parasites have been alternately ascribed to Sarcocystis cruzi or to Sarcocystis levinei, depending on their occurrence in cattle or water buffalo. We used light and transmission electron microscopy, genetic analysis, and experimental infections of definitive canine hosts to determine whether consistent differences could be identified from parasites derived from several natural infections of each host, examining several tissue types (esophagus, skeletal muscles, and heart). Cysts derived from cattle and water buffalo shared similar structure; variation among 18S rRNA sequences did not segregate consistently according to intermediate host type; parasites derived from cattle and water buffalo induced similar outcomes in the canine definitive host. One cattle specimen harbored unusually large (macroscopic) sarcocysts which nonetheless conformed to previously reported ultrastructural and genetic features of S. cruzi. Finding no consistent basis to differentiate between them, we conclude that the parasites infecting each host and tissue type correspond to S. cruzi. In our sample, no phylogenetically distinct taxon was sampled which might correspond to a distinct taxon previously described as S. levinei. Either that taxon was missed by our sampling effort, or it may represent a junior synonym to S. cruzi, which would then cycle between dogs and a broader range of intermediate bovine hosts than was previously considered.     

Ultrastructural Study of Schizogony of Eimeria bovis in Cell Cultures*

SYNOPSIS. First-generation schizogony of Eimeria bovis in bovine cell culture was studied by electron microscopy. The intracellular sporozoite retained its structure for at least 6 days at which time it rounded up and lost its apical complex. Although the refractile body underwent certain morphologic changes, it was retained throughout the parasite's growth. The beginning of mitosis was marked by the formation of a cytoplasmic funnel which traversed the nucleus opening on each side toward a pair of centrioles. Subsequently, there developed an intranuclear spindle. Separation of the daughter nuclei was preceded by the formation of typical centrocones. Differentiation of merozoites was accomplished by exogenesis during the last mitotic division. A dense fiber, interpreted as a link connecting the merozoite anlage with its nucleus, extended from the developing apical complex to the nearest division pole. In the anlage, the inner membrane complex was at first composed of patches associated with pairs of subpellicular microtubules. Rhoptries appeared early in merogenesis, whereas micronemes formed at the time the merozoites detached from the residuum. The level of amylopectin, low in schizonts, rose at the beginning of merozoite formation.     

The Fine Structure of Haemoproteus columbae Sporozoites*

SYNOPSIS. The fine structure of Haemoproteus columbae sporozoites has been studied and compared to sporozoite structure as revealed by the light microscope. The sporozoites are ultrastructurally similar to those of other Haemosporidia in that they possess a 3-layered pellicle, subpellicular microtubules, polar ring, micropore, free ribosome-like particles, micronemes, a structure resembling a Golgi complex, an irregular mitochondrion, and a large nucleus. In the anterior region of the sporozoite there are 21–22 regularly arranged longitudinal subpellicular microtubules located peripherally around the cell. In the apical region the microtubules appear thickened on 1 side. The sporozoite of H. columbae has a microneme system in which 1–3 micronemes are associated with the outer pellicular membrane at the anterior end. Micronemes are found throughout the cytoplasm, but occur in greater concentration in the anterior region of the sporozoite. A clear pellicular cavity, located between the polar ring and the termination of the inner pellicular layer, is present at the anterior end of the sporozoite. Vesicular invaginations of the inner pellicular layer have been observed in the anterior region; their function is unknown. Spherical osmophilic bodies are found throughout the cytoplasm.     

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co‐opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host–parasite interaction.     

Fine Structure of Scorpion Pectines for Odor Capture

The paper revealed the fine structure of the scorpion (Mesobuthus martensii) pectines and showed how the fine structure of the pecten influences odor flow.The first step of our investigation was to prove that scorpion pectines work as olfactory and this was done via experiments utilizing paraffin coverage.Subsequently,the location,morphology,section structure,and arrangement of the pectines were studied via stereomicroscopy and Scanning Electron Microscopy (SEM).The fine structure ofpecten comprises a comb-like structure with 24-30 knife-like teeth and thousands of micron bowl-like pecten sensilla in staggered arrangement on the surface of the tooth.Computational Fluid Dynamics (CFD) was applied to predict odor flow around the pecten via the relevant Reynolds numbers.The comb-like structure amplified the odor flow velocity similar to an amplifier,transporting the odor flow of increased velocity to the micron pecten sensilla,improving transport efficiency of the odor flow.The staggered arrangement of the pecten sensilla generated a vortex,improving contact duration between pecten sensilla and odorant molecules.Thus,the pecten finc structure was likely acting as an effective comb with non-smooth teeth for the transport and capture of odorant molecules.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.     

Observations on the Fine Structure of the Schizonts of Haemoproteus columbae Kruse*

SYNOPSIS. The schizonts of Haemoproteus columbae resemble the exoerythrocytic schizonts of avian Plasmodium in their fine structure. Haemoproteus infects endothelial cells and grows several hundredfold in volume, destroying the cytoplasm and nucleus of the host cell. The schizont's plasma membrane is trilamellar with a dense outer lamella. Some schizonts have micropores in their plasma membranes, but there is no evidence for ingestion thru them. Instead, numerous vesicles and channels fill the host cell cytoplasm and give its plasma membrane and periparasitic vacuolar membrane the appearance of active pinocytosis. The parasite's membrane shows no sign of pinocytosis, indicating that it probably feeds by diffusion. The growing schizont has numerous mitochondria, nuclei, and ribosome-rich cytoplasm which contains electron-lucent vacuoles and clefts. The latter appear to be artifacts of fixation.     

Fine Structure of Trichomitus batrachorum (Perty)*

SYNOPSIS. Trichomitus batrachorum (Perty) shares with Trichomonadinae most of its fine-structural characteristics, especially those pertaining to the undulating membrane that consists of a high body fold, enclosing the loop-shaped, periodic marginal lamella, and of the recurrent flagellum applied to the fold. This flagellate has also certain structures, i.e. the costal base, comb-like organelle, and well developed marginal lamella, in common with Monocercomonas. It shares with Hypotrichomonas the costal base; large pelta; very fine fibers perpendicular to, and connecting the axostylar microtubules; structural details of the most proximal segment of the marginal lamella; and general relationships between dorsal body fold (poorly developed in Hypotrichomonas) and the recurrent flagellum. All these electronmicroscopic findings support the crucial position of Trichomitus in the evolutionary sequence Monocercomonas→Hypotrichomonas→Trichomitus→ Trichomonadinae suggested previously by lightmicroscopic observations. Further, Trichomitus shares with all Tritrichomonadinae the comb-like structure, not found either in Hypotrichomonas or Trichomonadinae; and it has in common with Tritrichomonas also the costa with Type A periodicity, being the only member of Trichomonadinae with this type of supporting organelle. It appears, therefore, that Trichomitus-type organisms represent an important link in the evolution of all Trichomonadidae from Monocercomonadidae.     

On the Fine Structure of Trypanosoma cruzi in Tissue Cultures of Pigment Epithelium from the Chick Embryo. Uptake of Melanin Granules by the Parasite*

Trypanosoma cruzi has been cultured in pigment epithelial cells of the iris from the chick embryo. Melanin granules, identical with those of the host cells were found in the intracellular, amastigote (leishmania) forms. In many of the intracellular forms cytostome-like structures were seen, often in intimate contact with the pigment granules of the host cells, which suggests the uptake of melanin granules through the cytostomes by the process of intracellular phagotrophy.     

Fine Structure of the Sporogonic Stages of Nosema parkeri

SYNOPSIS The fine structure of sporogonic stages of Nosema parkeri Krinsky, a microsporidan from the argasid tick, Ornithodoros parkeri Cooley, is described. Developmental changes in the spore coat and cytoplasmic organelles are discussed. As a sporoblast transforms into a spore, the organelles become more compact and the membranes surrounding them appear to become more taut. It is suggested that the polaroplast complex is involved in fluid transport during development of the spore. Organelles in the mature spore include 2 contiguous nuclei enveloped in a lattice containing ribosome-like particles, a polaroplast complex composed of laminar and saccular regions, and a coiled tubular polar filament attached to a polar sac. Sporogonic stages do not appear to have mitochondria, Golgi apparatus, or a posterior vacuole. The fine structure of the spore of N. parkeri is very similar to that of species of Nosema found in insects, crustaceans, and trematodes.     

Structure and expression of three gp82 gene subfamilies of Trypanosoma cruzi

The glycoprotein gp82 is a GPI-anchored cell surface protein of Trypanosoma cruzi and is involved in cell invasion. Gp82 is encoded by multiple genes. To investigate the genetic basis of its biological function, we analyzed structure and expression of gp82 multigene family members in the Peruvian and Guatemalan strains. Three major groups of gp82 genes (A, B and C) were categorized by analyzing multiple DNA clones from the genomic PCR products. Within each group, 95–97% homology was observed, whereas between the groups, homology was 67–79%. The copy numbers of groups A, B and C as determined by real-time PCR were 18, 8 and 7 copies, respectively, in the Peru-2 strain. Significant elevation of the mRNA expression levels (5–10 times more) of all the subfamily genes was observed in the metacyclic stage compared with the epimastigote stage. When we focused on the binding motif sequence reported previously, we found substantial difference between that of A and C. However, the peptide inhibition invasion assay showed no functional difference. Taken together, we demonstrated that three subfamilies of gp82 were in the genome of T. cruzi and maintained their functional structure, and that the mRNA expressions of those genes were equally controlled in a stage-specific manner.