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Use of flow cytometry to separate Leucocytozoon caulleryi gametocytes from avian blood

Published online by Cambridge University Press:  12 July 2010

SUMIE OMORI ,
YUKITA SATO ,
HIDEAKI TODA ,
KAZUE SASAKI ,
TAKASHI ISOBE ,
TERUYUKI NAKANISHI ,
KOICHI MURATA  and
MASAYOSHI YUKAWA
SUMIE OMORI
Affiliation:
Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa 252-0880, Japan
YUKITA SATO*
Affiliation:
Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa 252-0880, Japan
HIDEAKI TODA
Affiliation:
Laboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa 252-0880, Japan
KAZUE SASAKI
Affiliation:
Scientific Feed Laboratory Co. Ltd, Otawara 324-0045, Japan
TAKASHI ISOBE
Affiliation:
National Institute of Animal Health, Tsukuba 305-0856, Japan
TERUYUKI NAKANISHI
Affiliation:
Laboratory of Fish Pathology, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa 252-0880, Japan
KOICHI MURATA
Affiliation:
Laboratory of Wildlife Science, Department of Animal Resource Sciences, College of Bioresource Sciences, Nihon University, Fujisawa 252-0880, Japan
MASAYOSHI YUKAWA
Affiliation:
Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa 252-0880, Japan
*
*Corresponding author: Laboratory of Biomedical Science, Department of Veterinary Medicine, Nihon University, Fujisawa 252-0880, Japan. Tel: +81 466 84 3378. Fax: +81 466 84 3445. E-mail: sato.yukita@nihon-u.ac.jp
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Summary

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.

Keywords

Leucocytozoon caulleryigametocyteflow cytometryseparation
Type
Research Article
Information
Parasitology , Volume 137 , Issue 13 , November 2010 , pp. 1899 - 1903
Copyright
Copyright © Cambridge University Press 2010

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References

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