A freeze-fracture study of the host cell-parasite interface during and after invasion of cultured cells by cystozoites of Sarcocystis muris.

The parasite-host-cell interface of Sarcocystis muris in cell culture was studied by freeze-fracture electron microscopy. Cystozoites of S. muris have an intra membrane particle (IMP) density of 1347 ± 146 IMP/µm(2) on the P face and 638 ± 109 IMP/µm(2) on the E face. After exposure to trypsin during extraction from the cysts a reversal of IMP density to 820 ± 115 µm(2) on the P face and 1277 ± 140 µm(2) on the E face occurred. Invasion of S. muris cystozoites is followed by the enclosure of the parasite in a primary parasitophorous vacuole (PV 1), limited by a membrane which partly originates by invagination and inward expansion of the plasmalemma of the host cell. A decline of local densities of IMP on the P face of the infected host cell from 2557 ± 567 IMP/µm(2) to 417 ± 217 IMP/µm(2) in the membrane of PV 1 represents a significant decrease of protein elements. S. muris gamonts became translocated to a new vacuole (PV 2) 30 to 45 minutes after invasion. IMP reappeared on both fracture faces of the vacuole membrane. The rapid increase of IMP on the PV 2 membrane coincides with the release of large amounts of dense granule contents.

A scanning electron microscope study of the colon of the mouse (Mus musculus) infected with Eimeria ferrisi (Protozoa, Apicomplexa, Coccidia).

Micromorphological changes in the colon of mice infected with Eimeria ferrisi were studied by scanning electron microscopy. The damage observed consisted of swelling of epithelial cells, profound stretching of the host cell membranes, loss of microvilli and erosion of tissues. Mature meronts were visible at 3 days post-inoculation (PI) in localized areas of cellular rupturing. At 4-6 days PI, the cellular rupturing and epithelial disruption were more widespread, exposing intra- and extracellular meronts, merozoites, and sexual stages. Microgametes of E. ferrisi are equipped with 2 flagella. Even though large portions of the infected area become denuded of surface epithelium, recovery had begun by day 7 PI.

The endogenous stages of Eimeria utahensis (Protozoa: Eimeriidae) in the kangaroo rat, Dipodomys ordii.

The endogenous life cycle of Eimeria utahensis is described from experimentally infected kangaroo rats, Dipodomys ordii. The endogenous asexual cycle consisted of 4 generations of meronts. First-generation meronts were concentrated in the anterior third of the small intestine. The succeeding generations of meronts and the sexual stages were concentrated in the middle third of the small intestine. First-generation meronts had a mean diameter of 9.7 micrometer and contained 12 to 16 merozoites. Second-generation meronts had a mean diameter of 8.0 micrometer and contained 12 to 16 merozoites and a residual body. Third-generation meronts had a mean diameter of 12.4 micrometer and contained 4 to 8 merozoites. Fourth-generation meronts had a mean diameter of 8.6 micrometer and contained 16 to 24 merozoites. Young gamonts were located in epithelial cells of the crypts of the small intestine. Shortly after the parasites entered the epithelial cells, the infected cells became displaced into the lamina propria, and most of the mature gamonts were in this location. The nuclei of host cells containing young sexual stages became greatly elongated and flattened. A few young gamonts were seen in cells in which the host cell nuclei were dividing. During development, nuclei of microgamonts became arranged on the periphery of numerous compartments. Only one type of wall-forming body could be distinguished in the macrogamonts.

Cellular dynamics and cytokine responses in BALB/c mice infected with Eimeria papillata during primary and secondary infections.

BALB/c mice were infected with the intestinal intracellular parasite Eimeria papillata to characterize lymphocyte responses and cytokine profiles throughout primary and secondary infections. Lymphocytes from the mesenteric lymph node (MLN) and the gastrointestinal tract (GIT) of infected mice were phenotypically analyzed using flow cytometry and immunofluorescence microscopy, respectively. Lymphocytes isolated from the MLN during primary infections of BALB/c mice with E. papillata do not proliferate, compared to day 0 uninfected controls, when stimulated in vitro with conconavalin A and express TH2-type cytokines (interleukin [IL]-4 and IL-10) on day 3 PI followed by the release of TH1-type cytokines (IL-2 and interferon-gamma) during patency. In the small intestine, significantly more T cells and their subsets were observed during primary infection. During secondary infections, IL-2 was the only 1 of the 4 cytokines that was expressed earlier and at higher levels in the MLN when compared to primary infections. In the small intestine, significantly more alphabeta+ and CD8+ T lymphocytes were observed in mice during secondary infection. Oocyst antigens did not induce cellular proliferation at any time point during primary or secondary infections. We conclude that primary oral infection of BALB/c mice with E. papillata is associated with localized immunosuppression that may be mediated, in part, by early TH2-type cytokines. Immunity to secondary infection may be mediated by intestinal alphabeta+ CD8+ T lymphocytes through an IL-2-dependent mechanism.

Comparison of four murine Eimeria species in immunocompetent and immunodeficient mice.

Factors associated with immune-mediated protection against coccidial parasites were examined in a series of experiments utilizing immunocompromised scid/scid(SCID) and scid/scid.beige/beige(SCID/Bg) mice, as well as immunocompetent BALB/c mice. Number of oocysts produced per g feces each day and prepatent and patent periods were assessed for 4 eimerian parasites (Eimeria papillata, Eimeria vermiformis, Eimeria falciformis, and Eimeria ferrisi) using the 3 murine strains. The number of infections required to elicit a protective immune response was also determined for each coccidial species in BALB/c mice. We report the first description of patent infections in inbred immunocompetent and immunodeficient mice infected with E. papillata. Results indicate that during primary infections, parasite replication is under partial immunological control for all Eimeria species. However, the control is mechanistically different for E. papillata because the adaptive immune response does not contribute to the control of primary infections. Both coccidial species infecting intestinal villar epithelial cells (E. papillata and E. ferrisi) were affected by the beige mutation using parasite output as an indicator, whereas E. falciformis, which infects intestinal crypt cells, is not. BALB/c mice were more resistant to challenge infections with upper intestinal parasites (E. papillata and E. vermiformis) in comparison to challenge infections with lower intestinal and cecal parasites (E. falciformis and E. ferrisi).

Ponazuril inhibits the development of Eimeria vermiformis in experimentally infected outbred Swiss mice.

We evaluated a 15% paste formulation of ponazuril in outbred Swiss mice that were experimentally infected with Eimeria vermiformis. Thirty, 8-week-old female mice (approximately 20 g) were placed in one group of 10 mice and one group of 20 mice. Mice in both groups were gavaged with approximately 5,000 sporulated oocysts of E. vermiformis on day 0. Mice in group 2 (n=10) were treated orally on days 3 and 4 with ponazuril (suspended in 30% propylene glycol) at the rate of 20 mg/kg. Mice in group 1 (n=20) were gavaged with a similar volume of 30% propylene glycol. Rates of oocyst passage (oocysts/g feces) were determined on day 10 (peak patency) for treated and nontreated mice using a fecal aliquot oocyst counting technique. Oocysts were not observed in the feces of treated mice using the aliquot technique. Control mice passaged oocysts at a geometric mean rate of >104,000 oocysts/g feces. Control mice also produced significantly less feces on day 10. These results indicate that ponazuril is effective against E. vermiformis under the conditions utilized in this study, and that the E. vermiformis mouse model could be useful in predicting the efficacy of new anticoccidial drugs.

Ultrastructure of Sarcocystis sp. from the muscle of a white-tailed deer (Odocoileus virginianus).

Sarcocystis sp. from the muscle of naturally infected white-tailed deer (Odocoileus virginianus) was examined by transmission electron microscopy. The primary cyst wall forms regularly spaced protrusions filled with electron-lucent ground substance; no fibrils are present in the protrusions. The cysts are divided by septa into compartments containing typical coccidian metrocytes and merozoites. Taxonomy of the protozoon from the white-tailed deer-dog is discussed.

Ultrastructural observations of host-cell invasion by sporozoites of Eimeria papillata in vivo.

Scanning and transmission electron microscopy were used to study the invasion of mouse small-intestinal epithelium by sporozoites of Eimeria papillata. Some mice received oocysts by gavage and others received either sporocysts or sporozoites by direct injection into the small intestine. The highest concentration of invaded cells were found in ligated intestinal tissues studied at 5-45 min after the inoculation of sporozoites. Sporozoites actively invaded anterior end first, which resulted in extensive damage to the host cell. Such cells showed disrupted microvilli; protuberances of cytoplasm into the lumen, apparently the result of a disrupted plasma membrane; vacuolization of the cytoplasm; and damage to the mitochondria. These damaged cells were rapidly vacated as the sporozoite moved laterally into one or more adjacent intact host cells without entering the lumen. It is suggested that the host cell initially entered from the lumen becomes so severely traumatized that the parasite of necessity enters an adjacent cell as a prelude to further development. Various aspects of host-cell invasion by coccidia and malarial parasites are reviewed.

Scanning and transmission electron microscopy of host cell pathology associated with penetration by Eimeria papillata sporozoites.

Scanning and electron microscopy was used to study the pathogenesis that occurred in mouse epithelial cells that had been penetrated by Eimeria papillata sporozoites. Optimal penetration of parasites injected into nonligated and ligated mouse intestine was found to occur at 4-15 min post-inoculation. During initial penetration, the parasite caused disruption of the microvilli of the intestinal cells, which led to detachment of the microvilli from the plasma membrane of the penetrated cell. Host cells penetrated by the parasite showed extensive destruction of the internal cellular organization together with blebbing of host-cell cytoplasm and release of internal organelles such as mitochondria. Ultimately, the penetrated cells completely broke down, leaving vacuolated areas next to ultrastructurally normal epithelial cells.

Ultastructural studies of Sarcocystis sp. from the camel (Camelus dromedarius) in Egypt.

By means of electron microscopy a study has been made on Sarcocystis from 29 camels (Camelus dromedarius) in Egypt. In oesophagus and diaphragm muscles sarcocysts have been observed. The micromorphology of metrocytes, merozoites as well as of the cyst wall has been described.

Fine structure of microgametogenesis of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus.

The microgamogony of Eimeria ferrisi from experimentally infected mice was investigated with the electron microscope. Microgamonts were recognizable by the presence of peripherally arranged nuclei and the presence of single or paired centrioles between each nucleus and the limiting membrane of the parasite. Often an intranuclear centrocone directed toward the centriole was present. Differentiation of the microgamete began when elevations of the limiting membrane, which indicated the commencement of flagellar development, appeared above the centrioles. This event was accompanied by the segregation of nuclear content into a dense osmiophilic portion and an electron-pale portion. Then followed a gradual protrusion of the dense portion of the nucleus and developing flagella into the parasitophorous vacuole. A dense ring developed at the base of the differentiating microgamete, resulting in the formation of a stalk which was occupied by the residual portion of the nucleus. Fully developed microgametes became detached and occupied the parasitophorous vacuole along with residual cytoplasm. Microgametes had an anterior perforatorium, a dense elongate nucleus, with an anteriorly positioned mitochondrion in a small groove of the nucleus. Usually two flagella were present but one microgamete appeared to have three. Polysaccharide first appeared when differentiation was in progress and increased until large numbers of granules were present in the microgamont cytoplasm.

Clinical and histologic observations of actively induced resistance to Eimeria ferrisi Levine and Ivens, 1965 (Protozoa: Eimeriidae) in the mouse (Mus musculus).

Actively induced resistance to Eimeria ferrisi was studied clinically and histologically in Mus musculus. Results indicated that a partial resistance to challenge was obtained. Initially infected animals had severe symptoms of coccidiosis but the symptoms diminished as the infections progressed. Previously infected mice had only slight symptoms of infection when challenged. Challenge doses produced severe coccidiosis in the non-immune controls although none died. Six mice died at peak patency during the immunizing inoculations. Histologic examination indicated that parasite numbers were reduced in resistant animals. The reduction appeared greatest during late sexual development. Tissues from resistant animals showed little evidence of damage and appeared to contain an increased amount of lymphoid tissue. Tissues from non-immunized mice killed after challenge were heavily infected and contained extruded blood, mucous, and cellular debris in the gut lumen. Considerable destruction of host epithelial cells occurred. The observations are discussed and compared to other coccidial immunity studies.

The ultrastructure of macrogametes of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus.

Macrogametes of Eimeria ferrisi occurred in epithelial cells of the cecum and colon of Mus musculus and were studied by electron microscopy. Young stages were identified as macrogamonts by the presence of wall-forming bodies. At first an outerlimiting membrane and remnants of the inner membrane complex of the former merozoite pellicle were present; the latter was later lost but in mature macrogametes 3 limiting membranes were observed. Type II wall-forming bodies appeared before type I; the former developed in expanded cisternae of the endoplasmic reticulum whereas the latter were smaller in size and appeared in the ground substance of the cytoplasm. After formation of the oocyst wall the bodies of the 2 types were no longer visible. The presenceodies of the 2 types were no longer visible. The persistence of micronemes in mature macrogametes and the presence of numerous layers of rough endoplasmic reticulum during wall formation have not been previously reported.

A fine structural study of asexual stages of the murine coccidium Eimeria ferrisi Levine and Ivens 1965.

The schizogony of Eimeria ferrisi was studied in experimentally infected Mus musculus. Developmental stages occurred in epithelial cells of the cecum and colon. During transformation of invasive stages into schizonts the inner membrane complex of the pellicle, the conoid, subpellicular microtubules and micronemes gradually disappeared. The micropore, however, seemed to persist. Dividing nuclei had eccentric intranuclear spindles consisting of microtubules which extended between 2 centrocones, in close relationship with centrioles. During the last nuclear division anlagen of merozoites appeared as extensions on the surface of schizonts. The outer single membrane of the schizont became the outer membrane of the merozoite pellicle. Cytoplasmic organelles, typical of eimerian merozoites were incorporated into the developing merozoites. Finally the merozoite became detached leaving behind a residual cytoplasm. Fully developed merozoites had a 3-layered pellicle, the outer single unit membrane was continuous around the merozoite with the inner complex having interruptions at the anterior and posterior poles and at the micropores. Thirty-two subpellicular microtubules, originating at the anterior polar ring extended to the posterior region of each merozoite. The apical complex consisted of a conoid, preceded by 2 rings and surrounded by a polar ring. Two rhoptries were present having club-shaped terminal ends and slender ductules in the conoid region. Some merozoites had enlarged rhoptries, with the distal vesical appearing dense and osmiophilic. The Golgi complex, endoplasmic reticulum, mitochondria, polysaccharide granules were similar to those seen in other eimerian merozoites.