Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni.
OBJECTIVE
To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni.


METHODS
RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells).


METHODS
First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured.


RESULTS
B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer.


CONCLUSIONS
Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.
Yamasaki
M
M
Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Asano
H
H
Otsuka
Y
Y
Yamato
O
O
Tajima
M
M
Maede
Y
Y
D000596:Amino Acids / Q000097:blood*; D000818:Animals; D001403:Babesia / Q000254:growth & development*; D004285:Dogs; D004912:Erythrocytes / Q000469:parasitology* / Q000502:physiology*; D005978:Glutathione / Q000097:blood*; D066298:In Vitro Techniques; D007700:Kinetics; D011188:Potassium / Q000097:blood*; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood / Q000235:genetics; D013997:Time Factors
D000596:Amino Acids; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione; D011188:Potassium