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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...

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genusBabesia gibsoni, the causative agent of canine babesiosis, is an important protozoan parasite that poses a major clinical health problem in dogs worldwide (19,29,43). Due to the emergence of resistance and failure of antibabesia agents to eliminate the parasite (5), novel chemotherapeutics with better potency and acceptable safety margins are required to treat the infection. To develop novel chemotherapeutics for apicomplexans related to Babesia, such as the Plasmodium parasites, many studies have focused on well-validated molecular targets, including the dihydrofolate reductase (DHFR) enzyme (10,22,30,39), and to a lesser extent the thymidylate synthase (TS) enzyme (20,25). The DHFR and TS domains are expressed as individual monofunctional enzymes in mammals and bacteria (21,36,41); however, in protozoans (and some plants), DHFR and TS exist as a bifunctional enzyme in which they are expressed on a single polypeptide chain (11,23,35). The DHFR domain is a validated target for chemotherapy of infectious diseases (42) and cancer (17) because it is important for the proliferation of cells due to its function in DNA biosynthesis and cell replication.The DHFR enzyme catalyzes the NADPH-dependent reduction of dihydrofolic acid (DHF) to tetrahydrofolate (4), an essential cofactor in the de novo biosynthesis of nucleotidic bases of DNA, while the TS domain catalyzes the reductive methylation of dUMP to dTMP with concomitant conversion of 5,10-methylenetetrahydrofolate to DHF (8). Therefore, the disruption of DHFR activity by antifolates depletes the reduced folate pool, thus blocking de novo dTMP biosynthesis by the TS enzyme and eventually inhibiting cell multiplication, leading to parasite death. Although antifolates inhibit the activities of bifunctional DHFR-TS enzymes of some pathogenic apicomplexan protozoans and disrupt folate metabolism (42), it has not been shown whether these drugs could also inhibit the activity of the corresponding enzyme in the genus Babesia, which includes the pathogen B. gibsoni.Furthermore, the results of earlier studies of the inhibition of the growth of Babesia bovis in vitro by various antifolates (33) and the inhibition of Babesia equi, as well as Babesia caballi, by pyrimethamine (31) suggested disruption of DHFR-TS activity only. Even in a case where the B. bovis dhfr-ts gene had been isolated and the resistance mechanism to pyrimethamine elucidated (16),

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Domesticated adult dogs with antibody titer classified as below 'high' to one or more of canine distemper virus (CDV), canine parvovirus type-2 (CPV-2) and canine adenovirus type-1 (CAdV-1) were then given an additional inoculation, and the effectiveness of this booster evaluated 2 months later. Consequently, CDV and CAdV-1 antibody titer experienced a significant increase, but the same effect was not observed in the antibody titer of CPV-2. These findings suggest that with additional inoculation, a booster effect may be expected in increasing antibody titers for CDV and CAdV-1, but it is unlikely to give an increase in CPV-2 antibody titer.