CIBAV Research Group, Faculty of Agrarian Sciences, University of Antioquia, Medellín, Colombia.
Institute of Parasitology, Biomedical Research Center Seltersberg, Justus Liebig University Giessen, Schuberstrasse 81, 35392, Giessen, Germany.
Parasitol Res. 2022 Apr;121(4):1179-1189. doi: 10.1007/s00436-021-07338-x. Epub 2021 Oct 11.
M onoxenous 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.
单卵球虫属物种是广泛存在的、具有高经济影响的肠道致病性顶复门原生动物。在牛中，感染 E. bovis 的动物持续排出坚韧的卵囊，无处不在，使犊牛感染几乎不可避免。为了成为感染性卵囊，外源性需氧 E. bovis 孢子发生必须发生，导致形成孢子化卵囊，每个卵囊含有四个孢子囊，每个孢子囊含有两个孢子虫。在文献中，由于常用的荧光染料无法穿透抗卵囊双层壁，因此仍然缺乏对反刍动物艾美球虫属物种孢子发生的活细胞成像技术研究。每天通过 3D Cell Explorer Nanolive 显微镜对孢子发生卵囊进行分析，以尽可能接近体内情况探索正在进行的需氧依赖性孢子发生。随后，使用 STEVE 软件（Nanolive）根据卵囊双层壁和周质的折射率（RI）对孢子化 E. bovis 卵囊进行数字染色，获得细胞形态计量参数。总的来说，记录了三个不同的 E. bovis 孢子发生阶段，每个阶段又分为两个亚阶段：（i）孢子母细胞/孢子体转化为孢子发生阶段，（ii）胞质分裂后核分裂，最后（iii）形成四个孢子囊，每个孢子囊含有两个完全发育的孢子虫。大约 60%的孢子化 E. bovis 卵囊以同步方式完成需氧孢子发生。与体内情况相比，E. bovis 孢子发生延迟（即 6 天），而在体内情况下，需要 2-3 天，但在最佳环境条件下。活细胞 3D 全断层摄影分析可能有助于评估新型消毒剂或抗球虫药物对反刍动物/禽类艾美球虫孢子发生的体外影响，因为基于紧凑度和干质量对孢子发生程度进行区分在这里得以成功实现。观察到卵囊面积、周长、紧凑度、范围和粒度的主要变化，表明这些参数是快速评估孢子化程度的有效工具。
