Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development

Françoise I Bussière,Anne Silvestre,Evelyne Esnault,Alisson Niepceron,Alix Sausset,Pascale Quéré,Nicholas C Smith,Robert A Walker,Fabrice Laurent

doi:10.1186/s13071-018-2622-1

Françoise I Bussière, Anne Silvestre + Show 7 more

Open Access

https://doi.org/10.1186/s13071-018-2622-1

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Abstract

BackgroundEimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide. The life-cycle of E. tenella is monoxenous with the chicken as the exclusive host; infection occurs in caecal epithelial cells. However, in vitro, the complete life-cycle of the parasite has only been propagated successfully in primary chicken kidney cells, which comprise undefined mixed cell populations; no cell line model has been able to consistently support the development of the sexual stages of the parasite. We therefore sought to develop a new model to study E. tenella gametogony in vitro using a recently characterised chicken cell line (CLEC-213) exhibiting an epithelial cell phenotype.MethodsCLEC-213 were infected with sporozoites from a precocious strain or with second generation merozoites (merozoites II) from wild type strains. Sexual stages of the parasite were determined both at the gene and protein levels.ResultsTo our knowledge, we show for the first time in CLEC-213, that sporozoites from a precocious strain of E. tenella were able to develop to gametes, as verified by measuring gene expression and by using antibodies to a microgamete-specific protein (EtFOA1: flagellar outer arm protein 1) and a macrogamete-specific protein (EtGAM-56), but oocysts were not observed. However, both gametes and oocysts were observed when cells were infected with merozoites II from wild type strains, demonstrating that completion of the final steps of the parasite cycle is possible in CLEC-213 cells.ConclusionThe epithelial cell line CLEC-213 constitutes a useful avian tool for studying Eimeria epithelial cell interactions and the effect of drugs on E. tenella invasion, merogony and gametogony.

Highlights

Eimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide
We used specific markers described by Walker et al [15]: EtGAM-56, a macrogamete specific protein incorporated into the oocyst wall and EtFOA1, a flagellar outer arm protein, which is specific to microgametes
CLEC-213 were infected with merozoites II from the wild-type Wisconsin strain (Wis) strain of E. tenella, and Etfoa1 and Etgam56 gene expression levels were measured by RT-qPCR

Summary

Introduction

Eimeria tenella infection leads to acute intestinal disorders responsible for important economic losses in poultry farming worldwide. In vitro, the complete life-cycle of the parasite has only been propagated successfully in primary chicken kidney cells, which comprise undefined mixed cell populations; no cell line model has been able to consistently support the development of the sexual stages of the parasite. Coccidiosis has a high economic impact on poultry farming worldwide [1] This disease is caused by species of Eimeria, a genus of obligate intracellular parasites belonging to the phylum Apicomplexa; Eimeria spp. invade and multiply in intestinal epithelial cells [2]. Given that Eimeria spp. are highly host-specific [13], it may be possible to develop an in vitro assay based on chicken epithelial cell lines to study sexual stages of E. tenella life-cycle, host-pathogen interactions and largescale drug screening

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