Percorrer por autor "Hermosilla, C."
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- Comparative ex vivo responses of ovine and bovine polymorphonuclear neutrophils induced by Neospora caninum tachyzoitesPublication . Gutiérrez-Expósito, D.; Silva, L. M. R.; Wagner, H.; Gärtner, U.; Hermosilla, C.; Taubert, A.; Conejeros 1stNeospora caninum infection causes reproductive failure in ruminants with a traditionally higher incidence of bovine neosporosis compared to the ovine system. Differences in innate immune response could explain this observation. We here focused our study on the comparison of the N. caninum-induced response on bovine and ovine polymorphonuclear neutrophils (PMN) that might play a key role in the pathogenesis of neosporosis in both small and large ruminants. We here examined and compared in parallel PMN responses of healthy adult sheep (n = 6) and cows (n = 6) after exposure to N. caninum tachyzoites (ratio 1:4). PMN activation was evaluated by induction of NETosis, determined by immunofluorescence and scanning electron microscopy (SEM), extracellular reactive oxygen species (ROS) production via Amplex Red assays, and oxygen consumption rates (OCR) and proton efflux rates (PER) quantified by Seahorse XF technology. N. caninum tachyzoite-driven percentage of NETotic PMN was higher in the bovine system (24.4%) when compared to sheep (11.3%). For both, SEM analyses confirmed PMN activation and the formation of NET structures upon N. caninum tachyzoite exposure. The increase in tachyzoite-mediated ROS production proved higher in cattle than in sheep and these data were in line with significantly higher PER on bovine PMN indicating a differential glycolytic activity upon N. caninum exposure. Overall, this study documents early (minutes) and mid-late (hours) ovine and bovine PMN reactions after being exposed to N. caninum tachyzoites. The fundamental information here given contributes to the understanding of neosporosis in cows and sheep that should be complemented with in vivo studies.
- First-generation merozoites of caprine Eimeria christenseni are capable to invade and egress primary host endothelial cells in vitroPublication . Silva, L.M.R.; Ruiz, A.; Barba, E.; Lopéz-Osorio, S.; Molina, J. A.; Molina, J. M.; Taubert, A.; Hermosilla, C.The caprine Eimeria christenseni species belongs to the phylogenetic clade of pathogenic ruminant Eimeria, replicating within the endothelial cells of central lymph capillaries of the ileum villi in vivo. Investigations on E. christenseni-host endothelial cell interactions, including cell invasion, egress, apoptosis, senescence, cell cycle, cytoskeleton, cell metabolism and endothelium-derived innate immune reactions are possible to achieve through permissive in vitro culture systems. Therefore, we here established a suitable in vitro E. christenseni (strain GC) culture system using primary bovine umbilical vein endothelial cells (BUVEC) for the development of first-generation macromeronts. After 18–22 days post infection (p.i.), the intracellular sporozoites matured into fully developed E. christenseni-macromeronts, releasing viable merozoites I. Interestingly, two different types of E. christenseni-merozoites I were observed, i.e., thinner and thicker merozoites I. The thinner ones were more active, presented typical gliding motility, and were found intracellularly shortly after their release, while the thicker ones were less active and invasive to BUVEC. Thinner E. christenseni merozoites I actively invaded and egressed host cells by breaching the plasma membrane without host cell lysis, a phenomenon exclusively reported so far for apicomplexan sporozoites of Plasmodium yoelii and Eimeria bovis. Additionally, intracellular E. christenseni merozoites I were monitored over time (up to 30 days), thereby revealing no further development into meront II stages. Further research is needed to assess whether primary endothelial cells of caprine origin could support the complete life cycle of E. christenseni in vitro. This novel in vitro system will contribute not only for further studies on Eimeria-derived invasion- and egress strategies, endothelial cell-derived innate immune reactions, but also for merozoites I- and antigen production requested for vaccination strategies as already reported for other ruminant Eimeria species.