title: Use of flow cytometry to separate Leucocytozoon caulleryi gametocytes from avian blood.
abstract: 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.
authors.affiliation: Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Japan.
authors.forename: Sumie
authors.initials: S
authors.lastname: Omori
authors.forename: Yukita
authors.initials: Y
authors.lastname: Sato
authors.forename: Hideaki
authors.initials: H
authors.lastname: Toda
authors.forename: Kazue
authors.initials: K
authors.lastname: Sasaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Isobe
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Nakanishi
authors.forename: Koichi
authors.initials: K
authors.lastname: Murata
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Yukawa
mesh_terms: D000818:Animals; D001769:Blood / Q000469:parasitology*; D002645:Chickens / Q000469:parasitology*; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification; D005434:Flow Cytometry / Q000379:methods*; D016787:Haemosporida / Q000254:growth & development / Q000302:isolation & purification*; D018512:Parasitemia / Q000469:parasitology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods; D011201:Poultry Diseases / Q000469:parasitology*; D011529:Protozoan Infections, Animal / Q000469:parasitology*; D016053:RNA, Protozoan / Q000032:analysis / Q000302:isolation & purification
chemical_list: D016054:DNA, Protozoan; D016053:RNA, Protozoan