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.

Cellular pathology in mouse embryonic brain cells following in vitro penetration by sporozoites of Eimeria papillata.

The pathology that occurs in mouse embryonic brain ( MEB ) cells that have been penetrated by sporozoites of Eimeria papillata was studied by light and electron microscopy. At the light microscopy level the greatest number of intracellular parasites was seen at 15 and 45 min postinoculation (PI). The monolayer of MEB cells had begun to round up by 45 min PI, and by 60 min PI most of the cells were stripped from the coverslip. Little ultrastructural damage was seen in MEB cells just penetrated by the parasites at 15 min PI, and no host cell membrane was seen around the sporozoites that had just entered the cells. Flexing and bending of the sporozoites within the MEB cell caused vacuolization of cell cytoplasm and in some cases rupture of host cell membrane. Sporozoites leaving the host cells at 15 min PI caused a rupture of the host cell membrane at the apical end of the parasite, and both host cell membrane and cytoplasm were attached to the surface of the parasite. MEB cells still attached to coverslips at 45 min PI demonstrated complete degeneration.

Light and electron microscope study of Sarcocystis sp. from the fallow deer (Cervus dama).

By means of light and electron microscopy a study was made of Sarcocystis sp. from 11 fallow deer (Cervus dama). Cysts of Sarcocystis sp. were found in the tongue and abdominal muscle of 3 of 11 deer from forests near Bonn (FRG). These measured 212-560 micron in length and 54-120 micron in width and contained metrocytes and merozoites. The cyst wall, which had narrow band-like protrusions, is compared with other Sarcocystis sp. from Cervidae.

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.

Electron microscope studies of cyst stages of Sarcocystis tenella: the origin of micronemes and rhoptries.

The origin and the development of the micronemes and rhoptries was investigated at the ultrastructural level in cyst stages of Sarcocystis tenella. During endodyogeny the micronemes appear in small vesicles distributed at the periphery of two large granular vacuoles in each daughter cell. Later, these vacuoles were divided into numerous vesicular spiral formation-centers, producing micronemes at the apical pole of young merozoites. Rhoptries were observed to originate from desnifications with in the same large vacuoles which gave rise to the micronemes.

Ultrastructure of macrogametogenesis of Eimeria mivati.

The development of the macrogamete of Eimeria mivati Edgar and Seibold 1964 was studied with the electron microscope. Development of the young gamont was characterized by a loss of organelles such as the apical complex, subpellicular microtubules, rhoptries and micronemes, followed by an increase in micropores, mitochondria, rough endoplasmic reticulum (rER), and Golgi complexes. Nuclear detachment bodies and canaliculi were present in maturing macrogamonts. Amylopectin was first observed as small electron-dense rod-like bodies that eventually became large electron-transparent bodies. Type II wall-forming bodies developed in the cisternae of the rER. Type I wall-forming bodies appeared shortly thereafter in close association with numerous Golgi complexes. Many small vesicles located between the cisternae of the rER and the Golgi complexes formed what appeared to be a secretory pathway whereby protein formed in the cisternae and, modified by the Golgi complex, may produce the type I wall body material. The outer wall of the oocyst developed between two distal membranes on the surface of the macrogamete. Although the actual mechanism of deposition of the wall material was not seen, it was probably by some secretory process. Wall-forming bodies did not fuse.

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.

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.