Ultrastructure of the invasion of Eimeria magna sporozoites into cultured cells.

Monolayers of bovine kidney cells were overlaid with Eimeria magna sporozoites and observed with phase-contrast optics until penetration of the cells by the parasites had begun. Cells and penetrating parasites were fixed with glutaraldehyde and OsO4-containing ruthenium red, dehydrated, and embedded in situ. Cells being penetrated were selected for study in the electron microscope. The lack of intracellular staining with ruthenium red and intact plasmalemmas of cells being penetrated, was accepted as evidence that the sporozoites did not disrupt the plasma membranes. The sporozoite caused invagination of the host cell plasmalemma until the parasite was entirely within the cell, after which the invagination was sealed off by short pseudopodia enclosing the sporozoite within a membrane=lined vacuole inside the cell. Often myelin-forms, apparently of host cell origin, were seen in the space between the sporozoite and the cell.     

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.     

Changes in the Cytoplasmic Elements of Cultured Cells Infected with Eimeria vermiformis Sporozoites

Epithelial-type (PK-15) and fibroblast-type (MDBK) mammalian cell cultures were inoculated with purified Eimeria vermiformis sporozoites. Matched samples from 0 to 93 h after inoculation (HAI) were processed for electron microscopy; half of the sample preparations were extracted with non-ionic detergent prior to fixation. Specimens were examined by both transmission and scanning electron microscopy. Numerous sporozoites were attached to the cultured cells from 2 to 93 HAI, usually near the cell periphery. Some host cell microvilli extended up and appeared attached to the sporozoites. Sporozoites fixed during the penetration process were markedly constricted at the site of entry; however, no noticeable changes occurred in the host cell membrane or surface microvilli during sporozoite invasion or in sporozoite-infected cells. In cells extracted with 1% Triton X-100, the host cytoskeleton was progressively reorganized about the parasites but changes were limited to the immediate area of the sporozoite. Around resident sporozoites, the cytoskeleton became less dense but also more ordered, which contrasted with adjacent cell areas. Cytoskeletal elements passed both over and under the parasites. The appearance of the cytoskeleton suggested that the host cell formed a loose, basket-like net of cytoskeletal elements about the parasite.     

Transmission electron microscopy of intracellular sporozoites of Eimeria vermiformis (Apicomplexa, Eucoccidiida) in the mouse

Sporozoites of Eimeria vermiformis from the mouse were first seen in the epithelial cells of villus tips and the crypts of Lieberkühn four hours after inoculation (HAI). They were always within a parasitophorous vacuole. By 12 HAI, most were in crypt epithelial cells between the basement membrane and host cell nucleus. The sporozoites in the villus tips had 26 subpellicular microtubules, two polar rings, two preconoidal rings, two refractile bodies surrounded by amylopectin-like granules, a lamellar Golgi apparatus, numerous micronemes, and rhoptries. The sporozoites in the crypt cells had fewer amylopectin-like granules, micronemes, and rhoptries. A nucleolus was visible, as were pieces broken off from the posterior refractile body. Later, the sporozoites folded over to become U-shaped; the infolded membranes fused; and then the inner membranes disappeared so that spherical meronts were formed. Folding sporozoites were first seen 16 HAI and persisted until 52 HAI.     

Effects of antiphagocytic agents on penetration of Eimeria magna sporozoites into cultured cells.

Madin-Darby Bovine Kidney cells were treated with sodium flouride, iodoacetate, and 2-deosyglucose, reagents that block glycolysis, and thus reduce phagocytosis. Sporozoites readily entered cells whose ATP stores were largely depleted. They also entered cells treated with colchicine, colcemid, and vinblastine. These latter agents did not inhibit sporozite motility after 6 hr incubation. Cytochalasin B prevented penetration of cells by inhibiting the motility of sporozoites. This effect was reversible. Warm sporozoites entered cold cells 4 times more radily than cold sporozoites into warm cells. The above findings suggest that phagocytosis is not the mechanism for entry of E. magna sporozoites into cultured cells, but that sporozoite motility is of primary importance.     

Effects of cationized ferritin and neuraminidase on invasion of cultured cells by Eimeria meleagrimitis sporozoites

Primary turkey kidney cells and Eimeria meleagrimitis sporozoites were treated with cationized ferritin (CF) or neuraminidase ( NANase ), and the effects on the invasion of the cells by the sporozoites were measured. Cultures of host cells pretreated with either compound contained significantly fewer intracellular sporozoites than did control cultures. There was little additive effect if cultures were first treated with NANase and then with CF. In contrast, pretreatment of sporozoites with CF or low concentrations of NANase had no effect on invasion. The inhibition of invasion was apparently due to an interaction between treatment substances and host cell surface rather than to direct effect on the sporozoites. The CF bound to the randomly distributed anionic sites on the surfaces of both host cells and sporozoites and then rapidly aggregated. Sporozoites, probably in the process of invading cells, were invariably found with the conoid in close association with aggregates of CF on the host cell membrane. The CF on the sporozoites was apparently shed before or during invasion because all intracellular sporozoites were completely devoid of the label.     

Pathological Changes and Immunity Associated with Experimental Eimeria vermiformis Infections in Mus musculus1

ABSTRACT. Pathological changes and immunity induced by Eimeria vermiformis (Ernst, Chobotar & Hammond, 1971) were studied in outbred Swiss mice inoculated with 5000, 10,000, 20,000, or 40,000 oocysts. Cross immunity to E. ferrisi was also studied. In the case of E. vermiformis, mortality was dose dependent; most deaths were observed in the intermediate-dose groups. Most deaths also correlated with peak oocyst output. Histopathologic changes consisted of an early neutrophil and mononuclear cell infiltration in the small intestine. Later, villus atrophy and crypt hyperplasia caused a decrease in the villus-crypt ratio. During the acute phase (8-10 days after inoculation), villus tips were eroded and parasites with necrotic debris filled the cryptal and intestinal lumina. Vacuolar changes were observed in epithelial cells of the small intestine. Neither parasites nor significant pathological changes were observed in extra-intestinal organs. Mice were totally immune to reinfection with E. vermiformis 30 and 105 days after inoculation. Cross immunity was not observed between E. vermiformis and E. ferrisi.     

Eimeria tenella: stimulation of DNA synthesis in infected cultured animal cells.

An autoradiographic study was conducted to determine the influence of the intracellular coccidian parasite of chickens, Eimeria tenella, on the incorporation of tritium-labeled thymidine in kidney cell cultures. Evidence was obtained for a parasite-induced increase in thymidine incorporation in host cell cultures which is too great to be attributed to unscheduled DNA synthesis. The stimulatory effect became significant (P < 0.05) at 24 hr after inoculation and further increased at 48 and 72 hr postinoculation. Both parasitized and unparasitized cells in the infected cultures showed similar increases in thymidine incorporation. Furthermore, the increased incorporation of thymidine in the infected cultures was found to be independent of both the percentage of parasitized cells and development of the parasite.     

Transfer of extraintestinal stages of Eimeria vermiformis in the mouse

The oral administration of whole blood and liver or lung homogenates from mice donors inoculated 1-3 days previously with Eimeria vermiformis oocysts produced infection in coccidia-free recipient mice.     

Eimeria vermiformis: host strains and the developmental cycle

After confirmatory studies of the endogenous development of Eimeria vermiformis in hybrid mice, a comparison, both qualitative and quantitative, was made of the developmental cycle of the parasite in two strains of host of contrasting susceptibility. No differences were apparent between the resistant, BALB/c, and susceptible, C57BL/6, during the first 5 days of the cycle. Thereafter, more parasites were seen in the C57BL/6, they were more widely distributed along the intestine, and they persisted in the tissues for a considerably greater time. Schizogony continued for longer in the C57BL/6 mice, concurrently with gametogony, and there were probably up to four additional generations of schizonts, which resembled those of the accepted third generation. The kinetics of oocyst production, measured in the feces, reflected events in the intestinal epithelium. The findings are discussed in relation to eimerian life cycles and the immune response.