In vitro development of the complete life cycle of Eimeria species has been achieved in primary cultures of avian epithelial cells with low efficiency. The use of immortalized cell lines simplifies procedures but only allows partial development through one round of parasite invasion and intracellular replication. We have assessed the suitability of Madin-Darby Bovine Kidney (MDBK) cells to support qualitative and quantitative studies on sporozoite invasion and intracellular development of Eimeria tenella. Analysis of parasite ultrastructure by transmission electron microscopy and serial block face—scanning electron microscopy proved the suitability of the system to generate good quality schizonts and first-generation merozoites. Parasite protein expression profiles elucidated by mass spectrometry corroborated previous findings occurring during the development of the parasite such as the presence of alternative types of surface antigen at different stages and increased abundance of proteins from secretory organelles during invasion and endogenous development. Quantitative PCR (qPCR) allowed the tracking of development by detecting DNA division, whereas reverse transcription qPCR of sporozoite- and merozoite-specific genes could detect early changes before cell division and after merozoite formation, respectively. These results correlated with the analysis of development using ImageJ semi-automated image analysis of fluorescent parasites, demonstrating the suitability and reproducibility of the MDBK culture system. This systems also allowed the evaluation of the effects on invasion and development when sporozoites were pre-incubated with anticoccidial drugs, showing similar effects to those reported before. We have described through this study a series of methods and assays for the further application of this in vitro culture model to more complex studies of Eimeria including basic research on parasite cell biology and host-parasite interactions and for screening anticoccidial drugs.

体外 开发完整的生命周期 艾美球虫在鸟类上皮细胞的原代培养中已经获得了低效率的物种。永生化细胞系的使用简化了程序，但仅允许通过一轮寄生虫入侵和细胞内复制进行部分发育。我们评估了Madin-Darby牛肾脏（MDBK）细胞是否适合支持定性和定量研究子孢子的侵袭和胞内发育。艾美球虫。用透射电子显微镜和串联块面扫描电子显微镜分析寄生虫的超微结构，证明该系统适用于生成高质量的裂殖子和第一代裂殖子。通过质谱法阐明的寄生虫蛋白表达谱证实了在寄生虫发育过程中发生的先前发现，例如在不同阶段存在表面抗原的替代类型，以及在入侵和内源发育过程中来自分泌细胞器的蛋白质丰度增加。定量PCR（qPCR）可以通过检测DNA分裂来跟踪发育，而反转录qPCR的子孢子和裂殖子特异性基因可以分别检测细胞分裂之前和裂殖子形成后的早期变化。这些结果与使用ImageJ半自动图像分析荧光寄生虫进行的发育分析相关，证明了MDBK培养系统的适用性和可重复性。当子孢子与抗球虫药预孵育时，该系统还可以评估对子孢子侵袭和发育的影响，显示出与以前报道的相似的作用。通过这项研究，我们描述了一系列方法和测定方法，以进一步应用该方法 表现出与以前报道的相似的效果。通过这项研究，我们描述了一系列方法和测定方法，以进一步应用该方法 表现出与以前报道的相似的效果。通过这项研究，我们描述了一系列方法和测定方法，以进一步应用该方法体外 文化模型以进行更复杂的研究 艾美球虫 包括有关寄生虫细胞生物学和宿主-寄生虫相互作用以及筛选抗球虫药的基础研究。