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Label-free enrichment of avian Leucocytozoon using flow cytometric sorting

Published online by Cambridge University Press:  19 July 2012

NAYDEN CHAKAROV ,
JOHANNES F. W. GREINER ,
STEFAN HAUSER ,
DANIEL SCHUETZMANN ,
OLIVER KRÜGER ,
MARTINA BOERNER  and
DARIUS WİDERA
NAYDEN CHAKAROV
Affiliation:
Department of Animal Behaviour, Bielefeld University, Bielefeld, Germany
JOHANNES F. W. GREINER
Affiliation:
Department of Cell Biology, Faculty of Biology, Bielefeld University, Bielefeld, Germany
STEFAN HAUSER
Affiliation:
Molecular Neurobiology, Department of Cell Biology, Faculty of Biology, Bielefeld University, Bielefeld, Germany
DANIEL SCHUETZMANN
Affiliation:
Molecular Neurobiology, Department of Cell Biology, Faculty of Biology, Bielefeld University, Bielefeld, Germany
OLIVER KRÜGER
Affiliation:
Department of Animal Behaviour, Bielefeld University, Bielefeld, Germany
MARTINA BOERNER
Affiliation:
Department of Animal Behaviour, Bielefeld University, Bielefeld, Germany
DARIUS WİDERA*
Affiliation:
Department of Cell Biology, Faculty of Biology, Bielefeld University, Bielefeld, Germany
*
*Correspondence: Department of Cell Biology, Faculty of Biology, Bielefeld University, Bielefeld, Germany. E-mail: darius.widera@uni-bielefeld.de
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Summary

The group of haemosporidian parasites is of general interest to basic and applied science, since several species infect mammals, leading to malaria and associated disease symptoms. Although the great majority of haemosporidian parasites appear in bird hosts, as in the case of Leucocytozoon buteonis, there is little genomic information about genetic aspects of their co-evolution with hosts. Consequently, there is a high need for parasite-enrichment strategies enabling further analyses of the genomes, namely without exposure to DNA-intercalating dyes. Here, we used flow cytometry without an additional labelling step to enrich L. buteonis from infected buzzard blood. A specific, defined area of two-dimensional scattergramms was sorted and the fraction was further analysed. The successful enrichment of L. buteonis in the sorted fraction was demonstrated by Giemsa-staining and qPCR revealing a clear increase of parasite-specific genes, while host-specific genes were significantly decreased. This is the first report describing a labelling-free enrichment approach of L. buteonis from infected buzzard blood. The enrichment of parasites presented here is free of nucleic acid-intercalating dyes which may interfere with fluorescence-based methods or subsequent sequencing approaches.

Keywords

Leucocytozoonflow cytometrylabel-free enrichment
Type
Research Article
Information
Parasitology , Volume 139 , Issue 12 , October 2012 , pp. 1547 - 1552
Copyright
Copyright © Cambridge University Press 2012

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