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

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.

In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR)

“…However, RT-qPCR is a more accurate and sensitive means to assess parasite reproduction. In fact, RT-qPCR is nowadays commonly used for quantitative evaluation and has been established successfully to assess reproduction of coccidia (e.g., Marugán-Hernández et al 2020 , Rentería-Solís et al 2020 , Thabet et al 2019 , Bussiere et al 2018 , Hiob et al 2017 , Thabet et al 2017 , Raj et al 2013 , Khalafalla et al 2011 ).…”

“…MDBK cells are a bovine-derived permanent line used as in vitro model for a broad variety of applications. MDBK cells are well established for in vitro studies on E. tenella (Marugán-Hernández et al 2020 , Rentería-Solís et al 2020 , Thabet et al 2019 , Bussiere et al 2018 , Thabet et al 2017 , Khalafalla et al 2011 ). Marugán-Hernández et al ( 2020 ), for example, conducted a comprehensive description of intracellular development of E. tenella in MDBK cells.…”

“…MDBK cells are well established for in vitro studies on E. tenella (Marugán-Hernández et al 2020 , Rentería-Solís et al 2020 , Thabet et al 2019 , Bussiere et al 2018 , Thabet et al 2017 , Khalafalla et al 2011 ). Marugán-Hernández et al ( 2020 ), for example, conducted a comprehensive description of intracellular development of E. tenella in MDBK cells. In this study, the authors tracked via RT-qPCR and reverse-transcriptase real-time PCR cellular division and stage development of transgenic strains of E. tenella .…”

“…Moreover, they can be a useful tool providing baseline data for future therapies (Thabet et al 2017;Khalafalla et al 2011). Due to its ability to grow in non-avian cell lines (Marugán-Hernández et al 2020;Thabet et al 2017), E. tenella has been widely used as model organism in in vitro research (Marugán-Hernández et al 2020;Thabet et al 2019Thabet et al , 2017Khalafalla et al 2011) and considerable efforts have been directed to E. tenella in vitro and in vivo research, including the use of molecular approaches such as real-time quantitative PCR (RT-qPCR) (Marugán-Hernández et al 2020;Thabet et al 2019Thabet et al , 2017Hiob et al 2017;Raj et al 2013). Less efforts have been reported for other Eimeria species, including E. acervulina (Naciri-Bontemps 1976;Itagaki et al 1974;Strout et al 1965;Hiob et al 2017).…”

“…Animal models are a valuable element in infection research. However, in vitro studies of coccidian parasites can contribute to the baseline understanding of the disease on a cellular level (Marugán-Hernández et al 2020 , Bussiere et al 2018 , Thabet et al 2017 ). Moreover, they can be a useful tool providing baseline data for future therapies (Thabet et al 2017 ; Khalafalla et al 2011 ).…”