The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture.

Virginia Marugan-Hernandez,Kelsilandia Aguiar-Martins,Georgia Jeremiah,Dong Xia,Alana Burrell,Sue Vaughan,Fiona Tomley,Nadine Randle

doi:10.3389/fcimb.2020.579833

Abstract

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.

Highlights

Eimeria tenella is a host- and tissue-specific parasite, replicating in vivo only in the epithelial cells that line the caeca of the domestic chicken
In vitro Madin-Darby Bovine Kidney (MDBK) Cell Culture Supported the Endogenous Development of E. tenella Leading to Fully Formed First-Generation Merozoites
Cell culture conditions were optimized for an optimal rate of invasion and development of E. tenella sporozoites without damaging the MDBK monolayers

Summary

Introduction

Eimeria tenella is a host- and tissue-specific parasite, replicating in vivo only in the epithelial cells that line the caeca of the domestic chicken. The most reliable cells used to date for the study of E. tenella sporozoite invasion and intracellular growth are Madin-Darby Bovine Kidney (MDBK) epithelial cells (Brown et al, 2000; Bumstead and Tomley, 2000; Tierney and Mulcahy, 2003) These have been used to examine phenotypes of transgenic E. tenella parasites generated by transfection (Clark et al, 2008; Marugan-Hernandez et al, 2017b) and to test the impacts of anticoccidial drugs (Zhu et al, 1994; Jenkins et al, 2014; Thabet et al, 2015, 2017), antibodies (Whitmire et al, 1988), cytokines (Kogut and Lange, 1989), natural compounds (Allen, 2007; Alnassan et al, 2015), and competition with other organisms (Tierney et al, 2004)

Methods

Results

Conclusion