Abstract

Besnoitia besnoiti is an apicomplexan parasite, causal agent of bovine besnoitiosis. This disease is characterized by cyst formation in skin and mucosa. During early infection, fast proliferating tachyzoites invade and replicate within host endothelial cells. In non-excitable cells, extracellular signals activate the inositol-triphosphate/calcium (InsP3/Ca2+) pathway which depends on phospholipase C (PLC) activation, inducing IP3 levels increasement, followed by intracellular Ca2+ release. Despite the understanding of Ca2+ signalling, this process in B. besnoiti is unclear. The aim of this work was to study Ca2+ dynamics during B. besnoiti in bovine umbilical vein endothelial cells (BUVEC) and the role of the InsP3/Ca2+ pathway during B. besnoiti infection. Ca2+ dynamics during tachyzoite replication were determined in B. besnoiti-infected BUVEC loaded with fluo-4-AM. The role of InsP3/Ca2+ signalling for parasite invasion was evaluated by treatments with Ca2+ chelators (BAPTA, EGTA) or PLC inhibitors (U73122, D609). PLC activation was studied in fluo-4-loaded free tachyzoites with using the PLC activator m-3M3FBS, in the presence or absence of PLC inhibitors. Current data show an infection-driven increase in total Ca2+ signals in B. besnoitiinfected BUVEC over time. BAPTA, but not EGTA treatments of tachyzoites affected their invasion, reducing infection rates by 85.4 ± 9.3 %, suggesting that intracellular Ca2+ sources are necessary for B. besnoiti invasion. In line, treatments with U73122 and D609 reduced infection rates by 79.3 ± 9.4 % and 49.7 ± 8.9 %, respectively, demonstrating that PLC participation for host cell infection. Finally, m-3M3FBS treatments induced a PLC-independent Ca2+ flux in B. besnoiti tachyzoites. © The Author(s), 2025.