Flow cytometry to evaluate Theileria sergenti parasitemia using the fluorescent nucleic acid stain, SYTO16.
BACKGROUND
Although the infection of Theileria sergenti is demonstrated by intraerythrocytic localization of this parasite, much time and labor are necessary in order to determine this. We applied flow cytometry to evaluate T. sergenti parasitemia using the fluorescent nucleic acid stain method.


METHODS
Peripheral blood samples from cattle infected with T. sergenti were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16, and hydroethidine (HE). Stained parasitized erythrocytes were measured by a flow cytometer equipped with a single argon laser operating at 488 nm.


RESULTS
SYTO16-stained intraerythrocytic parasites were detected on the FL1 (525 nm) and parasitized cells were separated completely from unparasitized cells. However, HE-stained erythrocytes could not be divided clearly into parasitized and unparasitized cells. SYTO16-stained parasites were reproducibly detected at a percentage above 0.1%. Contaminating leukocytes, which were indicated by CD18-positive cells, were eliminated from the analysis by narrowing the light scatter gate of the erythrocyte fraction. A correlation (r = 0.983) between the percentage of SYTO16-positive cells and parasitemia in grazing cattle was observed.


CONCLUSIONS
Flow cytometric detection using SYTO16 is a rapid and reliable method of monitoring parasitemia in T. sergenti-infected cattle.
Yagi
Y
Y
Hokkaido Research Station, National Institute of Animal Health, Hokkaido, Japan. yyagi@affrc.go.jp
Shiono
H
H
Kurabayashi
N
N
Yoshihara
K
K
Chikayama
Y
Y
D000818:Animals; D002417:Cattle; D016054:DNA, Protozoan / Q000032:analysis*; D004912:Erythrocytes / Q000469:parasitology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D018512:Parasitemia / Q000175:diagnosis*; D010617:Phenanthridines / Q000494:pharmacology; D016794:Theileria / Q000235:genetics / Q000302:isolation & purification*
D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D010617:Phenanthridines; C058428:hydroethidine