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

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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 β-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 β-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.

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Notes

  1. Kindly provided by Julie Clark and Fiona Tomley (submitted)

  2. Kindly provided by Julie Clark and Fiona Tomley (submitted)

References

  • Black M, Seeber F, Soldati D, Kim K, Boothroyd JC (1995) Restriction enzyme-mediated integration elevates transformation frequency and enables co transfection of Toxoplasma gondii. Mol Biochem Parasitol 74(1):55–63

    Article  PubMed  CAS  Google Scholar 

  • Coudert P, Licois D, Drouet-Viard F (1995) In: Eckert J, Brown R, Shirley MW (eds) Cost 89/820 Biotechnology: guidelines on techniques in coccidiosis research. European Comission, Luxembourg, p 61

  • del Cacho E, Pages M, Gallego M, Monteagudo L, Sanchez-Acedo C (2005) Synaptonemal complex karyotype of Eimeria tenella. Int J Parasitol 35(13):1445–1451

    Article  PubMed  CAS  Google Scholar 

  • Donald RG, Roos DS (1993) Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria. Proc Natl Acad Sci U S A 90(24):11703–11707

    Article  PubMed  CAS  Google Scholar 

  • Gubbels MJ, Li C, Striepen B (2003) High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein. Antimicrob Agents Chemother 47(1):309–316

    Article  PubMed  CAS  Google Scholar 

  • Hao L, Liu X, Zhou X, Li J, Suo X (2007) Transient transfection of Eimeria tenella using yellow or red fluorescent protein as a marker. Mol Biochem Parasitol. 153(2):213–215

    Article  PubMed  CAS  Google Scholar 

  • Hinterberg K, Mattei D, Wellems TE, Scherf A (1994) Interchromosomal exchange of a large subtelomeric segment in a Plasmodium falciparum cross. EMBO J 13(17):4174–4180

    PubMed  CAS  Google Scholar 

  • Hnida JA, Duszynski DW (1999) Taxonomy and phylogeny of some Eimeria (Apicomplexa:Eimeriidae) species of rodents as determined by polymerase chain reaction/restriction-fragment-length polymorphism analysis of 18S rDNA. Parasitol Res 85(11):887–894

    Article  PubMed  CAS  Google Scholar 

  • Hofmann J, Raether W (1990) Improved techniques for the in vitro cultivation of Eimeria tenella in primary chick kidney cells. Parasitol 76(6):479–486

    Article  CAS  Google Scholar 

  • Jeffers TK (1976) Genetic recombination of precociousness and anticoccidial drug resistance in Eimeria tenella. Z Parasitenkd 50(3):251–255

    Article  PubMed  CAS  Google Scholar 

  • Kelleher M, Tomley FM (1998) Transient expression of beta-galactosidase in differentiating sporozoites of Eimeria tenella. Mol Biochem Parasitol 97(1–2):21–31

    Article  PubMed  CAS  Google Scholar 

  • Kurth M, Entzeroth R (2008) Improved excystation protocol for Eimeria nieschulzi (Apikomplexa, Coccidia). Parasitol Res 102(4):819–822

    Article  PubMed  Google Scholar 

  • Matrajt M, Nishi M, Fraunholz MJ, Peter O, Roos DS (2002) Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii. Mol Biochem Parasitol 120(2):285–289

    Article  PubMed  CAS  Google Scholar 

  • Pfefferkorn LC, Pfefferkorn ER (1980) Toxoplasma gondii: genetic recombination between drug resistant mutants. Exp Parasitol 50(3):305–316

    Article  PubMed  CAS  Google Scholar 

  • Relman DA, Schmidt TM, Gajadhar A, Sogin M, Cross J, Yoder K, Sethabutr O, Echeverria P (1996) Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species. J Infect Dis 173(2):440–445

    PubMed  CAS  Google Scholar 

  • Rick B, Dubremetz JF, Entzeroth R (1998) A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa, Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion. Parasitol Res 84:291–296

    Article  PubMed  CAS  Google Scholar 

  • Schmatz DM, Crane MS, Murray PK (1984) Purification of Eimeria sporozoites by DE-52 anion exchange chromatography. J Protozool 31(1):181–183

    PubMed  CAS  Google Scholar 

  • Sheather AL (1923) The detection of intestinal protozoa and mange parasites by a flotation technique. J Comp Pathol 36:266–275

    Google Scholar 

  • Shirley MW, Ivens A, Gruber A et al (2004) The Eimeria genome projects: a sequence of events. Trends Parasitol 20:199–201

    Article  PubMed  CAS  Google Scholar 

  • Su X, Ferdig MT, Huang Y, Huynh CQ, Liu A, You J, Wootton JC, Wellems TE (1999) A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum.

  • van den Hoff MJ, Moorman AF, Lamers WH (1992) Electroporation in ‘intracellular’ buffer increases cell survival. Nucleic Acids Res 20(11):2902

    Article  PubMed  Google Scholar 

  • Wagner WH, Foerster O (1967) The first stages of the development of E. acervulina (Coccidia) in experimentally infected chicks. Z Parasitenkd 28(3):232–239

    Article  PubMed  CAS  Google Scholar 

  • Zhao X, Duszynski DW, Loker ES (2001) A simple method of DNA extraction for Eimeria species. J Microbiol Methods 44(2):131–137

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We appreciate and thank for financial support from the German Research Foundation (DFG). Grateful thanks go to Fiona Tomley and Julie Clark (Institute for Animal Health, Compton, UK) providing us with the plasmids and for the great support and successful collaboration. In this combination we thank the European Union for financing a research visit during the COST857 program. Many thanks go to Boris Striepen (University of Georgia, Athens, GA, USA) and Marc-Jan Gubbels (now Boston College, USA) for providing lab capacities and mentoring during a DFG founded research period. We also thank Bill Chobotar (Andrews University, USA) for reading and correcting the manuscript.

Declaration

We declare that the experiments comply with the current laws of Germany where they were performed.

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Authors and Affiliations

  1. Section Molecular Biotechnology, Technische Universität Dresden, 01062, Dresden, Germany

    Michael Kurth

  2. Institute of Zoology, Technische Universität Dresden, 01062, Dresden, Germany

    Rolf Entzeroth

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  1. Michael Kurth
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  2. Rolf Entzeroth
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Correspondence to Michael Kurth.

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Kurth, M., Entzeroth, R. Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa). Parasitol Res 104, 303–310 (2009). https://doi.org/10.1007/s00436-008-1192-0

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  • DOI: https://doi.org/10.1007/s00436-008-1192-0

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