Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.
An IgG1 monoclonal antibody (mAb 35B9) developed against first-generation merozoites of Eimeria bovis was shown by immunoelectron microscopy to react selectively with antigens localized in amylopectin granules. Amylopectin does not contribute to the epitope, as enzymatic degradation of carbohydrates in the parasite did not alter the binding pattern of mAb 35B9. When tested by immunoblotting, despite its organelle specificity the mAb recognized a variety of E. bovis merozoite I components with predominant molecules of 135 and 200 kDa. The epitope was not affected by treatment with endoglycosidase H; thus, N-linked sugar residues should not be involved in it. Alkaline cleavage (beta-elimination), however, destroyed the epitope; thus, the involvement of O-linked carbohydrates cannot be excluded. Treatment of E. bovis merozoite extract with phospholipase C changed the binding pattern of mAb 35B9 in a way that suggests the presence of phosphorylcholine molecules on several antigens recognized by the mAb, albeit not belonging to the epitope but rather masking it. The epitope was not found in free sporozoites of E. bovis or young intracellular parasites up to day 4 after invasion of cells in vitro, whereas 5-day-old trophozoites were found to contain it. It seems to be species-specific, as it could not be shown in sporozoites or merozoites of E. tenella or in stages of several other Coccidia.
Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed after immunization of mice with homogenates of Eimeria bovis first-generation merozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targets. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recognized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 180 kDa after electrophoresis under reducing conditions and to a series of components when separated without reduction. The epitope of mAb 8-23F9 was destroyed by treatment of the antigen with endoglycosidase H and removal of phosphocholine (PC) by phospholipase C. Since mAb 8-23F9 does not recognize cytidine-linked PC, the data suggest that PC in combination with N-linked sugars and/or N-glycans is part of its epitope. In the case of mAb 9-21G9, endoglycosidase H did not alter the epitope. When E. bovis merozoite antigen was treated with phospholipase C the number of mAb 9-21G9-reactive constituents increased, suggesting that PC may otherwise mask the epitope. mAb 8-23F9 also bound to the apical area and the surface of E. bovis sporozoites and recognized a >200-kDa sporozoite component. When sporozoites invaded Vero cells in vitro, epitope-bearing components were released onto the host cell surface and became part of the early parasitophorous vacuole wall. At day 5 the binding of the mAb was again confined to the intracellular parasite. mAb 9-21G9 did not react with sporozoites but recognized the apical area of intra-cellular trophozoites on day 5 after invasion of host cells in vitro. When testing was done against a variety of other Apicomplexa in various assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gondii cystozoites.
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