The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.
The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.
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Eimeria tenella的生长：定量方法的表征和应用评估细胞培养中的孢子岩侵袭和内源性发育。
DOI：
10.3389/fcimb.2020.579833
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发表时间：
2020
影响因子：
5.7
通讯作者：
Tomley F
中科院分区：
文献类型：
--
作者：
Marugan-Hernandez V;Jeremiah G;Aguiar-Martins K;Burrell A;Vaughan S;Xia D;Randle N;Tomley F
在禽类上皮细胞原代培养中实现了艾美球虫完整生命周期的体外发育，但效率较低。永生化细胞系的使用简化了程序，但只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（MDBK）细胞的适用性，以支持对柔嫩艾美耳球虫孢子入侵和细胞内发育的定性和定量研究。通过透射电镜和连续块面扫描电镜对寄生虫的超微结构进行分析，证明该系统适合产生高质量的分裂体和第一代分裂子。通过质谱分析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不同类型的表面抗原，以及在入侵和内源性发育过程中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，而子孢子特异性基因和裂殖子特异性基因的反转录qPCR可以分别检测细胞分裂前和裂殖子形成后的早期变化。这些结果与使用ImageJ荧光寄生虫半自动图像分析的发育分析相关联，证明了MDBK培养系统的适用性和可重复性。该系统还允许评估当孢子子与抗球虫药物预先孵育时对入侵和发育的影响，显示出与之前报道的相似的效果。通过本研究，我们描述了一系列方法和实验，以进一步将该体外培养模型应用于更复杂的艾美耳球虫研究，包括寄生虫细胞生物学和宿主-寄生虫相互作用的基础研究以及抗球虫药物的筛选。
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.