First-generation merozoites of caprine Eimeria christenseni are capable to invade and egress primary host endothelial cells in vitro

Liliana M. R. Silva^1,2*Antonio Ruiz³Emilio Barba^4*Sara López-Osorio⁵José Adrián Molina³José Manuel Molina⁴Anja Taubert⁶Carlos Hermosilla^6*

• ¹Egas Moniz Center for Interdisciplinary Research (CiiEM); Egas Moniz School of Health & Science, Caparica, Almada, Portugal
• ²Institute of Parasitology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany
• ³Universidad de Las Palmas de Gran Canaria Facultad de Veterinaria, Arucas, Las Palmas, Spain
• ⁴Department of Animal Pathology, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Arucas, Spain
• ⁵CIBAV Research Group, Veterinary Medicine School, Faculty of Agrarian Sciences, University of Antioquia, Medellín, Colombia
• ⁶Justus-Liebig-Universitat Giesen Institut fur Parasitologie, Giessen, Germany

The caprine Eimeria christenseni species belongs to the phylogenetic clade of pathogenic ruminant Eimeria replicating within 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 by using primary bovine umbilical vein endothelial cells (BUVEC) for development of first generation macromeronts. After 18-22 days post infection (p. i.), intracellular sporozoites developed into fully mature 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 are needed to assess whether primary endothelial cells of caprine origin might be suitable for completion of entire E. christenseni life cycle 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.