Abstract
Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.
Highlights
Neospora caninum is a heteroxenic apicomplexan parasite which belongs to the coccidian cyst-forming Sarcocystidae family (Lindsay and Dubey 2020)
The use of microscopy-based approaches of host cell-parasite interaction studies allows to characterize the role of Ca++ fluxes in coccidian biology (Lovett and Sibley 2003)
Despite that, using live cell fluorescence microscopy, the characterization of host cell-parasite interactions is usually limited to overlay approaches using light microscopic images
Summary
Neospora caninum is a heteroxenic apicomplexan parasite which belongs to the coccidian cyst-forming Sarcocystidae family (Lindsay and Dubey 2020). The successful replication cycle of tachyzoites starts with an active cell invasion, continues with intracellular parasite replication after parasitophorous vacuole (PV) formation and ends with active tachyzoite release which occurs after achieving full maturation (Behrendt et al 2008). Despite the well-described role of Ca++ in coccidian biology at functional level, the precise mechanisms underlying parasite egress are not fully understood (Caldas and de Souza 2018) In this context, egress-related studies on T. gondii have consistently demonstrated that treatments with calcium ionophores, such A23187 or ionomycin, induce an early egress of tachyzoites in a Ca++-dependent manner (Endo et al 1982; Black et al 2000; Caldas et al 2007; Behrendt et al 2008). In case of the coccidian parasite Eimeria bovis, treatments with A23187 failed to induce egress of merozoite I stages from macromeronts, but promoted a fast exit of sporozoites from recently invaded endothelial cells (Behrendt et al 2008), suggesting parasite species and stage-specific egress mechanisms in apicomplexan parasites
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