Ultrastructure of developing Isospora suis in cultured cells

The ultrastructure of Isospora suis sporozoites, type-1 meronts, and type-1 merozoites was examined, using transmission electron microscopy of infected cultured cells. The ultrastructure of sporozoites and type-1 merozoites was similar. Each possessed trimembranous pellicles, subpellicular microtubules, a conoid, anterior and posterior polar rings, rhoptries, micronemes, a single vesicular nucleus, tubular mitochondria, Golgi complexes, ribosomes endoplasmic reticula, inactive micropores, amylopectin bodies, lipid bodies, dense bodies, and crystalloid bodies. Merozoites were produced by endodyogeny. Ultrastructural events associated with merozoite production by type-1 meronts are described.     

Ultrastructural study of globidian parasites infecting the abomasum of sheep in Germany.

Globidian parasites infecting the abomasum of sheep in Germany were investigated by means of electron microscopy. The frequency of infection was found to be 93 %. The globidian cyst-like bodies contained multinucleate schizonts, developing merozoites or fully developed merozoites. Among the latter there were two different types, namely short and long forms. The process of merozoite formation was described in detail. The giant schizonts were subdivided into multinucleate cell portions of irregular size and shape. Their nuclei were then arranged at the periphery of the cell portions and underwent their last division which was combined with the differentiation of merozoites. The long form merozoites were elongated cylindrical in shape with terminal nucleus. They measured 7.7 μm in length and 1.0 μm in width. The merozoites of the short type were spindle-shaped with a central nucleus. They were 5.0 μm long and 1.0 μm wide. The globidian parasites were located in a parasitophorous vacuole of an intact host cell.     

An electron microscopic study of intra-erythrocytic stages of Babesia bovis in the brain capillaries of infected splenectomized calves.

Splenectomized vaccine donor calves undergoing primary reactions to Babesia bovis infections may develop cerebral babesiosis which leads to death if not treated in time. A brain biopsy was performed on an artificially-infected animal showing nervous symptoms and the tissue was immediately processed for electron microscopic examination. Virtually every erythrocyte in the brain capillaries sectioned was infected with B. bovis. Intra-erythrocytic merozoites, trophozoites and dividing trophozoites were indentified. Important features of the piriform merozoites included a reduced apical complex consisting of the anterior polar ring, microtubules, rhoptries and micronemes. Unidentified membrane-bound bodies, mostly spherical in shape, were observed anterior to the nucleus. The trophozoites showed very little structural differentiation and no food vacuoles or micropores could be detected. Each trophozoite produced 2 identical merozoites and the parent cell became totally incorporated in the daughter merozoites in the multiplication process. Projections were seen radiating from the surface of infected erythrocytes which appeared to adhere to other surfaces on contact. This probably resulted in the sludging of infected erythrocytes in the capillaries. The latter observations coincide with those described for Babesia argentina.     

Life cycle of Eimeria krijgsmanni-like coccidium in the mouse (Mus musculus)

Life cycle of Eimeria krijgsmanni-like coccidium isolated from the feces of naturally infected mice purchased from commercial sources was examined. The parasite was purified by single oocyst isolation and maintained by passage in the mice before experiments. The sporulated oocysts were ovoid or ellipsoid, measuring 19.3 x 14.8 microm on average. One or two small polar granules were present. Micropyle and oocyst residuum were absent. Sporocysts were ellipsoid, measuring 11.6 x 7.2 microm on average with a small Stieda body and sporocyst residuum. Six groups of respective 5 mice (4-week-old) were inoculated with doses varying from 2.0 x 10(1) to 10(6) oocysts. All the mice examined began to shed oocysts from 7 day postinoculation (PI) and their maximum number of oocysts per gram of feces were 10(6) on day 8 PI. Patency was 6 or 7 days. This parasite had severe virulence to the mice that is, the mice given 10(6) oocysts showed anorexia, diarrhoea and rough hair from 1 day and all of them died on day 3 PI. The mice given 10(3) or more oocysts showed the clinical signs described above from day 5 and 4 of them received 10(5) died on day 9 or 10 PI. The parasites occurred within the epithelial cells of cecum, colon and rectum of infected mice. Sporozoites, 13.9 x 3.0 microm, with two large refractil bodies on side of the nucleus located subcentrally were observed on day 1 and 2 PI. Merozoites were first observed at 24 hr PI, and sexual stages were found from 4 day PI. No parasites were detected in the small intestine and mecenteric lymph nodes.     

Hepatozoonosis in two species of Japanese wild cat

Hepatozoon sp. infections were detected in two species of Japanese wild cat, Iriomote wild cat (Felis iriomotensis) and Tsushima leopard cat (Felis bengalensis euptilura), between April 1993 and October 2005. The prevalence was 56.7% (17/30) and 14.3% (6/42), respectively. The most affected organ was the heart; all infected animals had organisms in their hearts. The parasitizing form was schizont and various developmental stages were observed. The size of schizont and merozoite was 22.3 +/- 3.1 x 15.3 +/- 2.2 mum and 6.1 +/- 0.6 x 2.3 +/- 0.2 mum, respectively. Few inflammatory reactions against the parasites were observed. Electron microscopically, organisms were located in parasitophorous vacuoles of unidentified host cells, and mature schizonts consisted of numerous merozoites. This is the first report of hepatozoonosis in Japanese felids.     

Influence of betaine and salinomycin on intestinal absorption of methionine and glucose and on the ultrastructure of intestinal cells and parasite developmental stages in chicks infected with Eimeria acervulina

The effect of betaine and salinomycin on absorption of methionine and glucose in tissue from the duodenal loops of Eimeria acervulina-infected chicks was determined. Differences in the ultrastructure of the intestinal cells and parasite developmental stages were also examined. With a drug-resistant isolate of E. acervulina, methionine absorption was significantly higher in chicks fed a basal diet supplemented with 0.15% betaine as compared with absorption in chicks fed the unsupplemented basal diet. Addition of 66 ppm salinomycin to the diet containing betaine did not further enhance absorption. Conversely, with a drug-sensitive isolate, methionine absorption was significantly higher in chicks fed a diet supplemented with both betaine and salinomycin than in chicks fed the unsupplemented basal diet. Tissue from chicks fed any of the supplemented diets was usually significantly heavier than that from chicks fed the unsupplemented diet, even when weight gains of the birds were similar. Glucose absorption was similar in all diet groups. Epithelial cells in coccidia-infected and uninfected chicks fed diets supplemented with betaine or betaine plus salinomycin were less electron dense than cells from chicks fed diets that were not supplemented with betaine. Merozoites of E. acervulina in chicks fed diets supplemented with salinomycin had extensive membrane disruption and vacuolization, but the damage was prevented when betaine was added to the diet. Numerous merozoites and intact schizonts were seen in the intestinal lumen of chicks fed the diet containing betaine plus salinomycin.     

An epizootic among knots (Calidris canutus) in Florida. II. Ultrastructure of the causative agent, a Besnoitia-like organism.

Multinucleated cysts near the luminal surface of the thoracic aortas of diseased knots (Calidris canutus) were similar to besnoitia cysts. Ultrastructurally, the cyst had four distinct layers. The central area included a vacuole that contained a sporozoan with a conoid, polar ring, micronemes, rhoptries, nucleus, mitochondria, dense bodies, a lipid-like vacuole and endoplasmic reticulum. External to the vacuole was a layer with organelles typical of vertebrate cells. The wall of the cyst was irregular in thickness and was bound by a strongly osmiophilic membrane. There was a loose, acellular area of intertwined strands between the cysts wall and layer of organelles.     

In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells

The growth of Sarcocystis neurona, isolate UCD1, in continuous culture was examined in 10 cell lines to identify growth conditions and methods for the preparation of parasites free of gross host cell contamination for molecular studies. The unpredictable, slow release of merozoites in most cell lines prompted development of a method to synchronously release the parasites from infected host cells. The calcium ionophore A23187 at a concentration of 1 microM was found to release intracellular merozoites with a 40 min treatment at 37 degrees C. The release of merozoites en masse from attached host cells allowed for the rapid collection of relatively pure parasites from the culture supernatant. This release of merozoites occurred in five different host cell lines. The ionophore-released parasites were highly infectious for host cells and appeared to be morphologically identical to naturally released merozoites, except that the treated merozoites had an increased number of micronemes when examined by electron microscopy. The ionophore did not enhance the release of sporozoites from sporocysts, but freezing in the presence of 5% DMSO released sporozoites that were infectious to bovine monocytes in in vitro culture.     

Ultrastructure of Babesai bovis (Babes, 1888)

An electron microscope study of thin sections of bovine erythrocytes parasitized with Babesia bovis (Texas isolate) revealed that the body of the parasite is covered with a pellicular complex consisting of three membranes, two located on the interior and one on the outer part of the pellicle. Parasites observed possessed one nucleus and one nucleolus containing aggregated nuclear material. Each of these aggregations was bounded by two nuclear membranes. Organelles such as polar rings, rhoptries, micronemes, vesicular structures, cisternae of the nuclear membrane, spherical bodies, mitochondria-like structures and Maurer's clefts were indentified and described. The basic similarities and differences in the intra-erythrocytic stages of Babesia bovis, in comparison with other Babesia spp. parasites, are discussed.     

Certain cytologic features of the porcine adrenal medulla

Adrenal glands were collected from pigs of various ages under general anesthesia. Glutaraldehyde-fixed medullary tissue was postfixed with OsO4 for electron microscopy and with potassium dichromate or potassium iodate for light microscopy. Columnar epinephrine (E) cells formed cords between wide sinusoidal capillaries at the corticomedullary junction and were arranged in palisade fashion along the central vein and its major tributaries. The E cells usually were polarized, with the nuclei located away from the sinusoidal capillaries. Clusters of polygonal norepinephrine (NE) cells formed large central aggregates surrounded by E cells. Granulated vesicles were the predominant cytoplasmic feature of both E and NE cells. Round or oval E granules were bounded by a crenated membrane separated from the granule by a clear halo. The more electron-dense, elongate NE granules were bounded by a closely apposed, smooth membrane. The average longest granule axis was 270 nm for E granules and 305 nm for NE granules. Many cytoplasmic organelles were congregated in a granule-free paranuclear zone, which contained a prominent Golgi complex. Thin nonmyelinated nerve fibers (singly or in small groups) were interposed between the E and NE cells. Nerve fibers often were located close to the nucleus in a depression of the cell surface and often were wrapped by thin E or NE cell processes. The medulla of newborn pigs was composed predominantly or exclusively of NE cells. In both adults and pigs, E or NE cell cords radiated through the cortex toward the capsule, and isolated clusters of E or NE cells frequently were found in the capsule or zona glomerulosa.     

A light and electron microscopic study on abomasal globidiosis in Somali goats

Abomasum from apparently healthy Somali goats with globidiosis showed pin-head sized nodules embedded in the mucosa. The nodules consisted of encapsulated cysts, containing mature or immature schizonts. Glandular atrophy and lymphohistiocytic cell reaction were often found in the vicinity of these cysts. The fine structure of immature and mature cysts is described in details. The mature cysts contained elongated, spindle shaped merozoites (type I) or shorter, ovoidal merozoites (type II). Some mature cysts also had basophilic granular bodies among the merozoites. Type I and type II merozoites were morphologically different from those earlier described in goats.     

Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria bovis.

Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to first-generation E. bovis merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites of Isospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. bovis sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.     

Studies on Globidial Schizonts in the Abomasum of Norwegian Sheep: The Fine Structure of One of the Four Merozoite forms Investigated

Norwegian sheep were investigated for globidial schizont infection in the abomasum. The frequency of infection was found to be 78.2 %. Light microscope studies of the various mature schizonts revealed the existence of four morphologically different merozoites, small A, small B, intermediate and long forms. Each globidial schizont was found to contain only one form of these merozoites. However, these four schizont types occurred in the same abomasum.The intermediate form of globidial schizont merozoites was investigated by the aid of an electron microscope, with the aim of comparing its internal morphology with that previously published for Eimeria species. A striking resemblance was observed between the fine structures of the intermediate merozoite and that of Eimeria species, particularly the first generation merozoites described in giant schizonts of Eimeria bovis.The present status of globidial schizonts infecting the abomasum of sheep was discussed. It was concluded that these four forms of merozoites could represent different generations of one Eimeria species or different E. species producing giant schizonts in the abomasum.Due to the practical difficulties in studying the life histories of the different Eimeria species infecting sheep, it was proposed that the in vitro propagation of the individual species in cultured cells may shed some light on the corresponding asexual, as well as the sexual, stages. This would offer a new approach to the study of the ultra-structure of the developing parasite.     

<Emphasis Type="Italic">Eimeria bovis</Emphasis> meront I-carrying host cells express parasite-specific antigens on their surface membrane

Host immune responses conducted against antigens of Eimeria bovis are key factors for the development of protective immunity against this protozoan disease. In this study we investigated the expression of E. bovis-derived antigens on the host cell surface membrane during E. bovis first merogony in vitro. Host cells carrying E. bovis-meront I stages expressed E. bovis host cell surface antigens (EbHCSAg) on their surface membrane which were recognised by hyperimmune sera of calves and by sera from rats immunized with E. bovis merozoites I, when tested by indirect immune fluorescent antibody test (IIFAT), laser scanning confocal microscopy (LSCM) and immune electron microscopy. Expression of EbHCSAg on permissive host cells was earliest detected 7 days p. i., thus coinciding with the onset of the parasite replication. Membrane-associated EbHCSAg were removed from infected host cells by proteinase K, partially by Triton X-100, Triton X-114 and Triton X-405, but not by 1 M NaCl, CHAPS or phospholipase C treatment. Antibodies, affinity-purified on paraformaldehyde/glutardialdehyde (PAGA)-fixed E. bovis meront I-infected bovine host cells bound to the surface meront I-carrying cells and to merozoites I (IIFAT, LSCM) but, in contrast to untreated sera, not to sporozoites. When tested on methanol-fixed merozoites I and sporozoites by IIFAT, affinity-purified antibodies bound to structures in the apical complex area of merozoites I, but not to sporozoites, whilst untreated sera caused diffuse labelling of internal structures of both parasite stages. Immune electron microscopy demonstrated binding of affinity-purified antibodies to micronemes and dense granules of merozoites I. Although the function of EbHCSAg is still unknown, results of this study might suggest an involvement in the development of protective immunity against E. bovis infections.     

Scanning and transmission electron microscopic observations on the host-parasite relationship in intestinal cryptosporidiosis of neonatal calves

The association of cryptosporidia with the intestinal epithelium of three neonatal calves was studied by scanning (SEM) and transmission (TEM) electron microscopy. Trophozoites and schizonts were observed embedded in the microvillous brush border of epithelial cells. Merozoites, released from schizonts, were seen free in the lumen and penetrating epithelial cells by SEM and TEM. Incorporation of microvilli into the parasitophorous envelope of trophozoites was seen by TEM. These findings indicate that cryptosporidia develop at an intracellular position in the apex of the epithelial cells following merozoite penetration.     

Development and use of hybridoma antibodies directed against Eimeria acervulina merozoites for cross-reactive and ferritin-labeling studies

Hybridoma antibodies (Hab) were produced against Eimeria acervulina merozoites that had been separated from extraneous intestinal material by a fiber column technique before injection into mice. The Hab demonstrated three different immunofluorescent-antibody (IFA) patterns of tip, surface, or surface-internal fluorescence in or on the merozoites. Some Hab reacted with round immature schizonts, which were also present in the fiber-cleaned merozoite material. Variations in cross-reactivity were seen with a number of Hab tested by IFA with merozoites, sporozoites, and immature schizonts of different coccidial species. Certain Hab were species- and stage-specific, whereas others cross-reacted with some or all stages or species tested. One Hab apparently reacted with only a small percentage of the E. acervulina merozoites in the fiber-cleaned material. The ferritin (Fe)-labeling technique showed that with one Hab, which gave a surface-internal IFA pattern, there was an irregular clumping of the Fe label along the surface of the immature schizont. A heavier deposit of Fe label was seen on the area of the schizont where the merozoite was beginning to form. A heavy uniform labeling of Fe was seen on the surface of the pellicle of the mature merozoites. These results demonstrate that stage-specific and cross-reactive antigens are present in or on the merozoites of E. acervulina, and as shown with one Hab, surface antigens present on the immature schizont are incorporated onto the mature merozoite.     

Buffalo (Bubalus bubalis) Pre-antral Follicle Population and Ultrastructural Characterization of Antral Follicle Oocyte

The main objectives of the present study were to determine the ultrastructural modifications occurring in the oocyte during late folliculogenesis and to estimate pre-antral follicle population in buffalo. Half the collected ovaries were fixed and prepared for optic microscopy; the antral follicles from the other ovaries were measured and individually punctured. The cumulus–oocyte complexes (COCs) were processed for transmission electron microscopy. The number of pre-antral follicles in buffalo ovaries was estimated at 19 819 structures. Cumulus–oocyte complexes derived from 1-mm antral follicle had an eccentrical nucleus and compact corona radiata , ooplasm vilosities were fully embedded in zona pellucida (ZP) and a well-defined junction could be observed. Mitochondria were predominantly round and well distributed in ooplasm, as were small lipid vacuoles. In COCs derived from 2-mm antral follicles, the initial formation of perivitelline space was observed. The nucleus was peripherally located and the number of pleomorphic mitochondria increased. Cortical granules were clustered at oocyte periphery and lipid vacuoles increased in number and size. In COCs derived from 6-mm antral follicles, the organelles were located mainly in the perinuclear region. Golgi complexes and smooth endoplasmic reticulum (SER) were more developed. Mitochondria migrated to the cortical region and lipid vacuoles migrated to the medullar region. In COCs derived from 10-mm antral follicles, the lipid vacuoles coalesced and occupied the medullar region of the oocyte, together with a well-developed SER. Mitochondria were pleomorphic and located at the oocyte periphery. In conclusion, the morphological differences described in this paper could be responsible for some functional differences observed in in vitro embryo production and follicular dynamics for buffalo, when compared with cattle.     

Merogony in in vitro cultures of Theileria parva

In vitro studies were focussed on the duration and cessation of merogony in Theileria parva infected blood lymphocyte cell cultures. The cultures were infected using purified tick stabilates as an alternative to in vitro infections, using sporozoites obtained by labour intensive dissections of salivary glands from infected ticks. After establishment of infection in peripheral blood lymphocytes (PBL), merozoites were temporarily produced for about 2 months after which lymphoblasts only contained schizonts.