Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa)

Abstract

The rat parasite Eimeria nieschulzi is a suitable model for transfection studies and was used as an additional model organism for the genus Eimeria. We describe the transfection of this apicomplexan parasites and the cultivation of transformed stages in cell culture and in vivo. The beta-galactosidase or yellow fluorescent protein was expressed in all parasitic stages up to the second merozoite generation in vitro under control of the heterologous promoter region of Eimeria tenella mic1 gene previously described for E. tenella transfection. Pyrimethamine resistant E. nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr/ts-m2m3 gene. Co-transfection experiments with an YFP-plasmid resulted in pyrimethamine resistant and fluorescent parasitic stages. Infection of rats with transfected E. nieschulzi sporozoites directed to expression of beta-galactosidase or YFP in oocysts. Co-transfection with YFP/DHFR-TS allowed selection of resistant parasites in vivo. Excreted transgenic oocysts showed arrangement of YFP expression which lead to questions about meiotic recombination frequency and mechanisms.

Development ofEimeria nieschulzi(Coccidia, Apicomplexa) Gamonts and Oocysts in Primary Fetal Rat Cells

“…As affirmed by various cell lines in this study and in previous studies, the in vitro development of Eimeria nieschulzi is limited to the second-generation merozoite [5–7]. Tilley and Upton [10] reported a development until the fourth generation in vitro under reducing conditions.…”

“…In that manner, the genus Eimeria could even acts as a model for Toxoplasma gondii because such experiments are more difficult to accomplish in cats than in rats or chicken. The transfection technology is already established for Eimeria species infecting these host animals [7, 11, 26, 27] and can give the opportunity to deepen the knowledge of sexual processes in oocyst-forming Apicomplexa.…”

“…In previous studies with the rat parasite Eimeria nieschulzi, the stage development in vitro did not exceed the second merozoite stage [5–7]. Whereas, up to four schizont generations are found in the host before an in vivo gamont development occurs [8, 9].…”

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

“…Additionally, the development of protocols supporting genetic manipulation including transient and stable transfection of Eimeria tenella is beginning to provide complementary tools for functional genomic and transcriptomic analyses [6], [7], [8], [9], [10]. Nonetheless, despite these efforts functional genomic studies remain limited for Eimeria because of the lack of powerful and user-friendly molecular tools.…”

Laboratory Growth and Genetic Manipulation of Eimeria tenella

“…A number of 5ʹ and 3ʹ endogenous regulatory regions have been successfully identified from Eimeria genomes and used to drive expression of different exogenous genes (normally fluorescent reporters), most commonly in E. tenella . These include promoters from constitutively expressed genes—such as actin, beta tubulin, and histone H4—but also other regulatory regions from genes expressed during specific stages of the parasite life cycle such as those encoding microneme proteins (MIC) 1, 2, 3, and 5; the surface antigen (SAG) 13 from the zoite stages; and the Gam56 protein from the sexual stages (Clark et al., ; Hanig, Entzeroth, & Kurth, ; Hao, Liu, Zhou, Li, & Suo, ; Kelleher & Tomley, ; Kurth & Entzeroth, ; Marugán‐Hernandez et al., , ; Tang et al., ). Thus, the choice of promoter can determine whether the transgene is expressed throughout the entire parasite life cycle or if it is restricted to specific life cycle stages.…”