Fluorescent Eimeria bovis sporozoites and meront stages in vitro: a helpful tool to study parasite–host cell interactions

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 muM. 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.

Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

“…gondii rates from >15% to 70% have been described [29,30]. For Eimeria bovis infection rates >12% were reported [31]. Even the use of higher infection doses did not lead to more cells infected by I .…”

Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent

“…NET structures were visualized by staining extracellular DNA with 5 mM Sytox Orange dye (Invitrogen) for 10 min at RT according to Martinelli et al [27]. For the visualization of sporozoites within NET structures, sporozoites were stained with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE, 7.5 μM, 37 °C, 30 min; Invitrogen) according to Hermosilla et al [28] prior to PMN exposure. After fixation of the cells and three washings in sterile PBS, the samples were mounted in anti-fading buffer (Mowiol®, Sigma-Aldrich) and stored (4 °C, in the dark) until further use.…”

Neutrophil Extracellular Traps as Innate Immune Reaction against the Emerging Apicomplexan Parasite Besnoitia besnoiti

“…For the visualization of tachyzoites within NET structures, tachyzoites were stained with CFSE (7.5 μM, 37°C, 30 min; Invitrogen) according to Hermosilla et al [31] prior to PMN confrontation.…”