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Studies of Sarcocystis in Malaysia. II. Comparative ultrastructure of the cyst wall and zoites of Sarcocystis levinei and Sarcocystis fusiformis from the water buffalo, Bubalus bubalis.

The two species of Sarcocystis--S. levinei and S. fusiformis from the water buffalo, Bubalus bubalis, show some ultrastructural similarities in their cyst wall and zoites. The zoites of both species are of about the same size, banana-shaped and have 22 subpellicular microtubules, numerous micronemes, eight rhoptries, a micropore in the region of the micronemes, an elongated mitochondrion, and a nucleus. S. levinei has 200--300 micronemes and S. fusiformis has about 400. The sarcocysts of both species are trabeculated and their cyst walls have cytophaneres containing annulated fibrils and coarse, electron dense granules. The cytophaneres of S. levinei are sloping, with irregular, wavy outlines, whereas S. fusiformis has the cauliflower-type of cytophaneres. This difference in the appearance of the cytophaneres, together with the difference in size of the sarcocysts and their definitive hosts, further confirms that S. levinei and S. fusiformis are two distinct species in the water buffalo.

Animals

Light and electron microstructure of a Sarcocystis sp. from the Malaysian long-tailed monkey, Macaca fascicularis.

The ultrastructure of the cyst wall and zoites of a species of Sarcocystis from the skeletal muscles of a naturally-infected Malaysian long-tailed monkey, Macaca fascicularis, is described in detail. The wavy, electron-dense primary cyst wall is thin (55 nm) and invaginated. Cytophaneres are absent. The ground substance contains electron-dense granules and bundles of parallel, fibrillar elements in some areas. Thin trabeculae are present. The zoites measure 1.2 X 4.7 microns and have an interior conoid, 22 subpellicular microtubules, 50-60 micronemes, 4-6 rhoptries, and a posteriorly situated nucleus. Some ultrastructural aspects of the cyst wall and the zoites of this parasite resemble those of Sarcocystis species of the moonrat, rhesus monkey, tamarin, and baboon. The light microscopic appearance of this species from M. fascicularis also bears some resemblance to that of parasites from the four cases of human Sarcocystis reported in Malaysia. The cyst in all these human cases were thin-walled, with no cytophaners. Although the final hosts of these species of Sarcocystis are not known, it is quite possible that man, monkeys, and perhaps the moonrat (an insectivore) may serve as common intermediate hosts for one or several species of Sarcocystis.

Animals

Sarcocystis booliati n.sp. and a parasite of undetermined taxonomic position, Octoplasma garnhami n. gen. n. sp., from the moonrat, Echinosorex gymnurus.

Sarcocystis booliati n.sp. is described from the moonrat Echinosorex gymnurus (Mammalia, Insectivora) from West Malaysia. The cysts are very thin-walled, not visible to the naked eye, and have no trabeculae or cytophaneres. They are found in skeletal but not heart muscle. The zoites are small, 5-8 by 2-3 mum with a mean of 6.5 by 2.2 mum, in dry fixed smears. Octoplasma garnhami n.gen. n.sp., a parasite of undetermined taxonomic status but belonging to the Coccidiasina, Apicomplexa, is also described from the same host. Only schizononts and pseudocysts with typically 8 zoites, have so far been seen in monocytes of the spleen and liver. The zoites are large, 15 by 3 mum and have a distinct nucleolus even in dry-fixed smears.

Animals

Ultrastructure of Sarcocystis sp. from the Malaysian house rat, Rattus rattus diardii.

The ultrastructure of Sarcocystis sp. from the Malaysian house rat, Rattus rattus diardii, was studied with the electron microscope. The thin, uniformly-dense primary cyst wall had a row of vesicular invaginations which were also seen along the wall of the villi-like projections or cytophaneres. Within the villi were spherical bodies and hollow, curled structures. The ground substance beneath the primary cyst wall extended into the cyst as thin septa or trabeculae separating the tightly-packed zoites into compartments. Merozoites had a double-layered membrane, a conoid, 2 conoidal rings, 22 subpellicular microtubules, 6 rhoptries, 80-100 micronemes, scattered lipid droplets, and sac-like mitochrondrion, beside which was a Golgi apparatus. A micropore was occasionally seen at the anterior third of the zoite whereas the nucleus occupied the posterior third. Metrocytes were few in number and peripheral in location.

Animals

[Ultrastructural localization of alkaline phosphatase and ATP-ase in cyst stages of Sarcocystis tenella (Sporozoa, Coccidia) parasitic in the esophagus of sheep (author's transl)].

The activity of adenosine triphosphatase and alkaline phosphatase was investigated at the fine structural level in the cyst stages of Sarcocystis tenella parasitic in the esophagus of sheep. Alkaline phosphatase reaction was observed along the outer membrane of the parasite's pellicle. The enzymatic activity was much higher on the surface of metrocytes than that of zoites, which proved to be infectious. No reaction was noted in the interior of the parasites. However, a significant amount of alkaline phosphatase activity occurred along the inner surface of the 25 nm thick primary layer of the cyst wall. No evidence of the reaction of this enzyme was seen in the secondary cyst wall, which consisted of degenerated host cells. ATP-ase activity was found in a considerable degree along the primary cyst wall (=directly limiting the cyst's interior), whereas the ground-substance of the cyst, surrounding the parasites, is free of deposits. In the parasites ATP-ase was localized in the endoplasmic reticulum, in the perinuclear space, and between the two inner membranes of the three-layered pellicle. Only rarely a slight reaction was seen in the mitochondria of the metrocytes, which are the reproductive cells. The other organelles typical for S. tenella were free of ATP-ase. The results indicate that the enzymes studied participate in the growing process of the cysts, in which finally the infectious zoites remain in a more or less inactive state. The localizations of the enzymes corresponded with the results known from metazoa.

Adenosine Triphosphatases

An epizootic among knots (Calidris canutus) in Florida. I. Disease syndrome, histology and transmission studies.

About 150 knots found sick or dead had distention of serosal blood vessels, and small, raised hemorrhagic foci on the serosal surface of the jejunum and ileum. The principal finding was endaortitis with large intimal multinucleated cysts that contained a protozoan parasite within a cytoplasmic vacuole. There was endarteritis of mesenteric arteries and muscular arteries of the external layers of the intestinal tract. Single or multiple schizont-like stages containing zoites were in the wall of the small intestine and close to affected muscular arteries. The more usual intestinal lesion was an aneurysm of the inflamed muscular artery. It was concluded that endarteritis led to colonic infarction. Schizonts in medullary renal tubular cells were smaller than stages in the intestine. There was necrosis of epithelial cells of themedullary collecting ducts and inflammatory and regenerative changes. Schizonts and zoites were free in collecting ducts and ureters.

Animals

Propagation of Toxoplasma gondii in suspension cultures of HeLa cells.

A novel method of Toxoplasma gondii cultivation in suspension cultures has been introduced using silicone-coated glass vessels (working volume 100 ml). The cells were kept in suspension by a magnetic impeller at 75 rpm at a temperature of 37 degrees C. HeLa cells grown on MEM with calf serum were used as host substrate. The HeLa cells were infected with zoites of Toxoplasma gondii virulent P strain. After seven days the host cells were destroyed by the toxoplasmas and the number of zoites was up to 24 x 10(6)/ml.

HeLa Cells

Light and electron microscope studies on the Sarcocystis of Rattus fuscipes, an Australian rat.

Light and electron microscope studies on the Sarcocystis of Rattus fuscipes showed that sarcocysts of two types occurred in this rat. These types could be distinguished from each other on the morphology of their cyst walls, on the size and micromorphology of their zoites, as well as by the changes they induced in the host cell. On the basis of these differences, it was concluded that the two sarcocyst types belonged to distinct Sarcocystis species. The possible life histories of the infections occurring in the rats were considered.

Animals

Suspected Sarcocystis infections of the bovine placenta and foetus.

Protozoan parasites resembling Sarcocystis schizonts were found in the brains of 2 bovine foetuses and the placentas of another 4. As typical of Sarcocystis, the organisms were located mainly within vascular endothelial cells, and frequently the zoites within the schizonts were arranged in a rosette or palisade fashion. Pathological changes noted were placentitis, myocarditis, pulmonary vasculitis and encephalitis. Attempts to demonstrate Toxoplasma by animal inoculation and serology were unsuccessful.

Abortion, Veterinary

Studies on Sarcocystis in Malaysia. I. Sarcocystis levinei n. sp. from the water buffalo Bubalus bubalis.

Light and electron microscopic studies and feeding experiments have confirmed the presence of two species of Sarcocystis in the water buffalo Bubalus bubalis. One is the already known species with large macroscopic sarcocysts, Sarcocystis fusiformia (Railliet, 1897) Bernard and Bauche, 1912 and the other is S. levinei n. sp. which is being described in detail. The sarcocysts of S. levinei are 0.9 x 0.1 mm and the zoites in them 17.8 x 4.2 micrometer. Ultrastructurally, the primary cyst wall shows sloping villi with irregular wavy outlines. Within the villi are coarse granules and annulated fibrils. Trabeculae are present. The sexual stages of S. levinei occur in the subepithelial tissue of the small intestine of the dog and sporocysts shed by this definitive host are 15-16 by 10 micrometer.

Animals

Light and electron microscopic observations of the life cycle of Sarcocystis orientalis sp. n. in the rat (Rattus norvegicus) and the Malaysian reticulated python (Python reticulatus).

A light and electron microscopic study of Sarcocystis orientalis sp. n. was made. The life cycle of this parasite is in two hosts. Gametogony is in the intestinal epithelial cells of a predator, Python reticulatus. Isospora-like oocysts developed. Sporocysts average 9.1 by 7.7 mum. Rats (Rattus norvegicus) were infected with sporocysts and asexual stages developed. Ten days after infection large zoites (average 7.85 by 2.48 mum) were observed free in peripheral blood and within white blood cells. Small schizonts producing merozoites 2-3 mum long were seen in lung tissue. Tissue cysts developed in skeletal muscle and produced numerous cystozoites (average 5.53 by 1.38 mum). Fine structure was similar to previously described Sarcocystis spp.

Animals

[Instability of the DNA content in Entamoeba histolytica].

DNA content was repeatedly determined during a 10-month period on axenically cultured tropho-zoites from three strains (HK-9:NIH, HM-2:IMSS and HM-3:IMSS) and a clone of HK-9:NIH strain of Entamoeba histolytica, by means of the diphenylamine reaction. Average DNA content was more or less variable in all of them. According to their extent, variations were: 1) large (almost four times in strain HM-2:IMSS), or 2) small (1.5 times in the clonal isolate). With this relatively small sample, three statistically different groups were formed with nuclear DNA averages (1.35, 2.05 and 7.78 picograms respectively). Changes of ploidy and/or genomic heterogeneity in trophozoites could be the main causes of the striking instability in their DNA content.

Animals

Serologic test for antibody to Sarcocystis in cattle.

Soluble antigen was prepared from Sarcocystis zoites obtained from heart muscle of a bovine inoculated with sporocysts from canine feces and killed 120 days after infection. The antigen was used in an indirect hemagglutination (IHA) test and an agar gel diffusion test to detect antibody to Sarcocystis in experimentally infected cattle. IHA serum titers began to rise 30 to 45 days after infection and reached levels up to 1:39,000 90 days after infection. Sera collected under field conditions from 21 dairy cows had IHA titers ranging from 1:54 to 1:486. Since all cows appeared in good health, titers of 1:486 or less can probably be considered nonsignificant with regard to diagnosis of clinical disease. No positive Sarcocystis IHA titers were obtained with human sera previously found to be IHA positive for toxoplasma, indicating a lack of cross reactivity between antigens. Precipitins in the agar gel diffusion test appeared 30 days postinoculation and became very pronounced at 65 to 90 days.

Animals

Experimental transmission of Sarcocystis from icterid birds to sparrows and canaries by sporocysts from the opossum.

Cowbirds (Molothrus ater) and grackles (Cassidix mexicanus) infected with muscle cysts of Sarcocystis were fed to opposums (Didelphis virginiana) and fecal sporocysts from the latter were given to sparrows (Passer domesticus, Family Ploceidae), canaries (Serinus canarius, Family Fringillidae) and ducks (Anas platyrhynchos, Family Anatidae). Asexual parasites were found in the endothelium of sparrows and canaries but not in ducks. When birds were kept 10 weeks or more after infection, muscle cysts were found grossly and microscopically in the majority of sparrows, and in 1 canary, but not in ducks. Muscle zoites were found in digests of all sparrows and canaries but not in that of ducks. Metrocytes and forms dividing by endodyogeny also were found in the digest. Thus, avian Sarcocystis was transmitted experimentally from 2 genera of 1 family (Icteridae) to 2 different families of passerine intermediate hosts by sporocysts from the definitive host. This is the broadest intermediate host spectrum known for a species of Sarcocystis.

Animals

Canine cholecystitis in the presence of Hammondia spp. infection.

Biliary protozoal infections are becoming increasingly recognized in dogs. Sarcocystidae protozoal infections (i.e Toxoplasma, Neospora, Hammondia, Besnoitia, and Sarcocystis spp.) are uncommon and host-specific. Diagnosis is traditionally dependent on tissue or serologic evaluation; sensitivity and specificity for these techniques can vary. PCR amplification and genetic sequencing provide a technique to expedite diagnosis and species identification in clinical biliary protozoal infections. Bile samples were collected percutaneously from two dogs presented to a referral hospital with mixed hepatopathies and hyperbilirubinemia. Diagnostic imaging revealed changes consistent with hepatobiliary inflammation, and tachyzoites were identified via bile cytology. Sera were positive for anti-Toxoplasma sp. (n = 1/2) and anti-Neospora sp. (n = 2/2) antibodies. Genomic isolation from bile and pan-Sarcocystidae (18S rRNA) PCR amplification were consistent with Hammondia spp. Additional 28S rRNA (LSU), alpha-tubulin (aTUB) and cytochrome b (CytB) gene-specific fragments were PCR amplified and sequenced. Phylogenetic analysis of LSU DNA fragment (584 bp) suggested both patients were infected with Hammondia spp. closely related to H. heydorni. Sequencing of aTUB (234 bp) and CytB (344 bp) revealed Patient 1 Hammondia spp. was more closely related to H. triffittae, while Patient 2 was more similar to H. heydorni. Treatment with clindamycin, and in one case additional enrofloxacin, resolved clinical and clinicopathologic changes in both dogs. This report highlights the importance of multilocus sequencing in protozoal identification in atypical infections, clinical manifestations of canine hepatobiliary hammondiasis, and need for regional Sarcocystidae prevalence studies.

Animals