Continuous in vitro Culture of Erythrocytic Stages of Babesia gibsoni and Virulence of the Cultivated Parasite.

Abstract

ABSTRACT. Babesia gibsoni infected erythrocytes were collected from the blood of an experimentally infected dog. The parasite isolated could be continuously cultivated in vitro, with an average parasitemia of 18.2 ± 2.4% on day 3 of culture, in RPMI-1640 medium supplemented with 7.5% normal dog serum in a humidified atmosphere containing 5% CO 2 at 37°C. The parasites in the original culture were morphologically similar to those found in the peripheral blood of dogs, however, on the 4th generation of subculture, the l arge oval parasites, erythrocytes including many parasites and extracellular parasites were frequently observed. The B. gibsoni isolate was injected to the dog to test its infectivity after maintained in vitro for 738 days at the 214th subculture. The cultivated parasite did not cause a severe clinical sign in the dog. KEY WORDS: Babesia gibsoni, canine, in vitro culture, infectivity, morphology.J. Vet. Med. Sci. 64 (7): [571][572][573][574][575] 2002 Babesia gibsoni is an erythrocytic parasite, which is transmitted to dogs by ticks and causes hemolytic anemia, hemoglobinuria and marked splenomegaly in dogs, and the infected dogs occasionally show severe symptoms or die [3,6,16]. Long term in vitro cultivation of B. gibsoni is needed for the production of antigens for serologic tests and immunogens for the prevention of disease. The cultivation of B. gibsoni was reported by Murase et al. [8]. They cultivated the parasite obtained from a B. gibsoni-infected dog with a parasitemia of 1.0%-8.2%, using α-medium with 40% dog serum at 37°C in a humidified atmosphere containing 5% CO 2 , however, they could not maintain the culture for more than 15 days. Onishi et al. [13] examined the effect of serum concentration of, 5 culture media such as RPMI-1640, α-MEM, M-119, F-12 and L-15, gaseous environment and interval of medium exchange on the in vitro cultivation of B. gibsoni, and reported that higher parasitemia was obtained by using RPMI-1640 supplemented with 20% dog serum in a humidified atmosphere containing 5% CO 2 at 37°C and exchanging the medium at 24-hr interval. The parasites were maintained at least for 28 days by exchanging one third of the erythrocytes at every seven days after cultivation. However, long term in vitro cultivation of B. gibsoni with a high level of parasitemia has not been successfully achieved until now.We cultivated a B. gibsoni Oita isolate for 814 days with a high level of parasitemia and observed some changes in the morphology, infectivity and virulence of the isolate by in vitro culture. MATERIALS AND METHODSThree beagles, 1 to 2 years old were used in the experiments. The dogs were confirmed free of natural B. gibsoni infection by the indirect fluorescent antibody test and microscopic examination of Giemsa-stained blood smear before use. They received a standard amount of food daily and drinking water ad libitum. Two of the dogs were used as the donors of normal erythrocytes and serum and the other one was used for the examination of infectivity and virulence of cultu...

Increasing the in vitro proliferation rate of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis

“…Tissue lysate likely provided factors that are lacking or insufficient in our culture medium to support robust growth of P. mediterraneus. The proliferation of various protistan parasites has also been routinely stimulated with the addition of host components such as cell lines, cell lysates, tissue explants, blood, serum, organ infusions, bile, and mucus (Steinhagen et al 2000;Schuster and Sullivan 2002;Sunaga et al 2002). For example, Giardia duodenalis, an intestinal protistan of vertebrates is cultured in medium supplemented with bile (Binz et al 1992), and Trypanoplasma borreli, a pathogenic flagellate from the blood of cyprinid fishes, only grows in media containing carp serum (Steinhagen et al 2000).…”

Cloning, Expression, and Characterization of Babesia gibsoni Dihydrofolate Reductase-Thymidylate Synthase: Inhibitory Effect of Antifolates on Its Catalytic Activity and Parasite Proliferation

“…B. gibsoni strain NRCPD parasites (15) were maintained in splenectomized beagles, and B. gibsoni-infected dog erythrocytes were collected at peak parasitemia (14%) and stored at Ϫ80°C. In vitro stock cultures of B. gibsoni were grown in dog erythrocytes by using a previously established method (40).…”

“…For pyrimethamine and trimethoprim, 20 and 80 M of each drug was used for the inhibition. A 250-l mixture of the parasite culture per well was grown in a 48-well tissue culture plate using a previously described protocol (40). One-half volume (125 l) of the culture medium per well was replaced daily with an equivalent amount of fresh medium containing appropriate concentrations of the respective drugs for another two days.…”

Evaluation of doxycycline sensitivity in vitro against Babesia gibsoni by Real Time-PCR

“…To evaluate the effect of potential drugs against this pathogen, an in vitro susceptibility test would be important by Real Time-PCR (RT-PCR) but limited information is available. Because in vitro culturing of this parasite is difficult and it is successfully conducted only in a limited number of research institutes (Sunaga et al, 2002;Murase et al, 1991). B. gibsoni infected dogs either clinical patient or experimental are required for this method.…”