Search

Abstract   Neospora caninum, Toxoplasma gondii and Eimeria bovis are coccidian parasites of veterinary importance. Tachyzoites of N. caninum and T. gondii and sporozoites of E. bovis are able to invade and replicate in endothelial cells in vivo and in vitro. As it holds true for all eukaryotic cells, the survival of parasitized host cells and the parasites themselves should be dependent on ion balances, especially on extra- and intracellular calcium concentrations. Addition of the calcium ionophore A23187 reliably did release merozoites from mature N. caninum and T. gondii meronts grown in cultured primary bovine umbilical vein endothelial cells (BUVEC). Extent and time course of merozoite release depended on both, maturity of the meronts and concentration of the calcium ionophore. Attempts, however, to achieve synchronous release of merozoites from E. bovis first generation meronts by ionophore treatment failed, suggesting a different biological behaviour of this parasite. According to microscopical observations, the quite variable time of E. bovis macromeront maturation and a hampered merozoite exit owing to dense parasite-induced cytoskeleton elements surrounding the meront may be a reason for the lack of inducible synchronous release. [ABSTRACT FROM AUTHOR]

Abstract  A fluorescence-based technique was established to trace intracellular sporozoites of Eimeria bovis for tests on gliding motility, invasion, replication and quantification of infection rates in cultured bovine umbilical vein endothelial cells (BUVEC) by laser scanning confocal microscopy and flow cytometry (FCM) analyses. Employing the fluorescent dye 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), we determined its effects on sporozoites at various concentrations and duration of staining. More than 98% of sporozoites were labelled with the dye at a concentration of 2.5 μM. Staining was predominantly found in refractile bodies and presumptive micronemes. Upon infection of BUVEC, CFSE-labelled sporozoites developed into fluorescent immature macromeronts, which were traceable inside the cells until 22 days postinfection (p. i.). Consistent with a peripheral localisation of the fluorescence signal in macromeronts merozoites released from these lacked detectable fluorescence. As example of use, a multicolour FCM approach for the simultaneous determination of E. bovis infection and host cell surface molecule expression was established. The approach proved suitable to quantify major histocompatibility complex (MHC-I) and MHC-II expression, thereby clearly distinguishing between infected and uninfected BUVEC up to day 14 p. i. In conclusion, CFSE labelling of E. bovis sporozoites facilitates monitoring of intracellular stages in vitro and will be a highly useful tool for studying host cell responses towards parasite invasion. [ABSTRACT FROM AUTHOR]

Abstract  The first schizogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macroschizonts within 2–3 weeks. In this study, we analyse early cellular immune responses to infected host cells on the basis of peripheral blood mononuclear cell (PBMC) adhesion on and transmigration through infected bovine umbilical vein endothelial cell (BUVEC) monolayers. Adhesion of PBMC was upregulated by an E. bovis infection. Most marked effects were observed 1 day p.i.; thereafter, PBMC adhesion declined reaching control levels from day 8 p.i. onward. CD8 T cells adhered more frequently to infected BUVEC (42%) than CD4 T cells (25%). About one third of attached PBMC were represented by γδ-TCR T cells. Adhesion of T cells was not restricted to parasitised host cells, but occurred almost equally on non-infected BUVEC within the same monolayer. Furthermore, we found moderately enhanced levels of PBMC transmigration through infected BUVEC monolayers, in particular on day 2 p.i. The data presented here suggest that E. bovis infection of BUVEC induces endothelial cell-derived proinflammatory reactions, which appear suitable for the initiation of both adaptive and innate immune responses. [ABSTRACT FROM AUTHOR]