Abstract
Monoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2–3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.
Highlights
The genus Eimeria contains apicomplexan enteropathogenic protozoa with a high economic impact on livestock worldwide
In order to identify whether E. bovis–sporulated oocysts contained vital sporozoites, oocysts were submitted to an excystation protocol in vitro
Complete life cycle of E. bovis has been well documented in the past except for the exogenous sporogony generation (Hammond et al 1946; Fayer and Hammond 1967)
Summary
The genus Eimeria contains apicomplexan enteropathogenic protozoa with a high economic impact on livestock worldwide. All Eimeria species display a monoxenous life cycle composed of two phases, i.e., exogenous and endogenous phases. Freshly defecated non-sporulated oocysts are broadly spread into cattle environments; they are not still infective until undergoing aerobic sporogony to become infective. The sporulated oocysts within the genus Eimeria contain four sporocysts with two sporozoites in each. The endogenous phase of the life cycle includes numerous asexual merogonies, depending on Eimeria species (i.e., E. bovis developing two merogonies), in specific host cells, and some specific intestine sections, followed by a sexual gamogony leading after syngamy to the formation of non-sporulated oocysts, which are shed through feces into the environment (Hammond et al 1946; Fayer and Hammond 1967)
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