title: Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16.
abstract: In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vive were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vive and in vitro.
authors: Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. masayama@vetmed.hokudai.ac.jp
authors: Masahiro
authors: M
authors: Yamasaki
authors: Shiang-Jyi
authors: SJ
authors: Hwang
authors: Hiroshi
authors: H
authors: Ohta
authors: Osamu
authors: O
authors: Yamato
authors: Yoshimitsu
authors: Y
authors: Maede
authors: Mitsuyoshi
authors: M
authors: Takiguchi
mesh_terms: D000818:Animals; D001403:Babesia / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D002478:Cells, Cultured; D016054:DNA, Protozoan / Q000032:analysis*; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D004912:Erythrocytes / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D005456:Fluorescent Dyes; D018512:Parasitemia / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*
chemical_list: D016054:DNA, Protozoan; D005456:Fluorescent Dyes