Three farms were selected for parasite collection. Farm A and B were located in Chungnam Province and Farm C in Jeonnam Province. Blood samples were taken from 20 adult cattle of each farm; blood smears were prepared and then stained with Giemsa's stain. Five Holstein cows of Farm C only were found infected with Babesia, and 5 ml of blood were taken from each cow for later use.

An in vivo experimental system using SCID mice for Babesia species has been previously described (Tsuji et al., 1995). Briefly, C.B-17 scid/scid mice (Japan CLEA, Tokyo, Japan) of mixed sex and between 7 and 14 weeks old were used. The mice were housed in a vinyl-film isolator at around 23℃ and provided with λ-ray-irradiated pellets (FR-1; Funabashi Co., Japan) and autoclaved tap water ad libitum. All mice were splenectomized under anesthesia and were used for experiments after the surgical wounds had healed completely. They were treated according to Laboratory Animal Control Guidelines at Rakuno Gakuen University, which basically conform to the American Association of Laboratory Animal Control Guidelines issued by the National Institute of Health.

The SCID mice with circulating bovine red blood cells were prepared as described previously (Tsuji et al., 1995). To achieve the rapid substitution of circulating RBCs in SCID mice with Bo-RBCs, the authors developed a rat-mouse hybridoma, clone 2E11, producing anti-mouse RBC monoclonal antibody. The cultured supernatants of this hybridoma were concentrated into 1/20 of original volume by ammonium sulfate precipitation followed by dialysis against 0.85% saline. Splenectomized SCID mice were periodically administered subcutaneously with 200 ul of concentrated supernatants on Days -2, 1, and 5 in addition to intravenous transfusion of 0.5 ml packed cell volume of parasite-free Bo-RBCs.

The blood samples from two Holstein cows, which contained relatively high numbers of Babesia-infected RBCs, were chosen for parasite isolation. The RBC samples were washed three times with 0.85% saline, and 0.5 ml of packed cells was intravenously inoculated into a Bo-RBC-SCID mouse. Two mice were used for each sample. 10-20µl of tail blood samples of the mice were collected in heparinized tubes every day. The percentages of Bo-RBCs and parasitized Bo-RBCs in the peripheral blood cells was determined by Giemsa staining for blood smears and flow cytometry (Cyto ACE-150, JASCO Co., Japan) for RBC samples stained with FITC-labeled anti-Bo-RBC antibody, respectively (Tsuji et al., 1992). Babesia preparations that were free from Theileria were obtained by successive passages of the Babesia-infected RBCs in Bo-RBC-SCID mice as previously described (Tsuji et al., 1995; Terada et al., 1995). Babesia-infected Bo-RBCs obtained from the infected Bo-RBC-SCID mice were either passaged into new, uninfected Bo-RBC-SCID mice or suspended in a cell freezing solution (Cell Banker, Nippon Zenyaku Co. Ltd., Japan) followed by cryopreservation in liquid nitrogen. When needed, the frozen parasites were propagated by intraperitoneal inoculation into Bo-RBC-SCID mice. The paired, pyriform Babesia sp. in blood smears stained with Giemsa was measured, together with other B. ovata (strains Oshima and Miyake), B. bigemina (strain GJ29) and B. bovis (strain Mo7), in length and width with a real-time image analyzer (LUZEX F, Nikon, Japan).

Western blot analysis was performed as described (Arai et al., 1998). Frozen stocks of Babesia-infected RBC were thawed and washed five times with 10 mM Tris-HCl containing 10 mM EDTA (pH 7.5) at 4℃ and then centrifuged at 10,000g for 10 min. The resulting pellets were resuspended in 125 mM Tris-HCl (pH 6.5) containing 5% mercaptoethanol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.1% bromophenol blue, heated at 98℃ for 5 min, and vigorously vortexed.

The samples were diluted such that contained material from equivalent numbers of infected RBC and were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970) with 7.5% acrylamide gels, together with B. ovata (Oshima, Miyake and Okushiri strains), B. bigemina and B. bovis. Proteins were electrophoretically transferred to Fluorotrans membranes (Pall BioSupport, USA) for 1 h at 10 V. After blocking with PBS containing 0.5% casein, membranes were reacted with appropriately diluted immune sera, followed by reaction with secondary antibodies (alkaline phosphatase-conjugated Affinipure goat anti-mouse IgG (Organon Teknika, UK). Immunoreactive antigens were detected with a BCIP/NBT Alkaline Phosphatase Substrates Kit IV (Vector Laboratories, Inc., USA).

DNA samples were prepared from blood samples with a whole blood DNA extraction kit (GenTLE, TaKaRa Biochemical, Japan). Sequences encoding eukaryotic small-subunit rRNA (rDNA) were amplified from the DNA samples by PCR with primer set described by Medlin et al. (1988).

PCR mixtures contained 400 mM each deoxynucleoside triphosphates, 0.25 mM each primer (rRNA-3': 5'-GTCTTAGTATAAGCTTTTATACAGCG-3'; rRNA-5': 5'-GATAGGTCAGAAACTTGAATGATACATCG-3'), 10 to 100 ng of template DNA, and 2.5 U of La Taq DNA polymerase (TaKaRa Biochemical, Japan) in 50 ul of PCR buffer supplied together with enzyme. Thermal cycling was carried out in a Gene-Amp PCR system 9600 thermal cycler (Perkin-Elmer, USA), with 30 cycles of denaturation at 94℃ for 2 min, annealing at 55℃ for 2 min and extension at 72℃ for 90 sec.

The specific PCR products (1.7 kb) were purified by agarose gel electrophoresis, followed by cloning into EcoRV site of pT7blue T-vector (Novagen, Germany) according to the manufacturer's instruction. Nucleotide sequences were determined by the dideoxynucleotide chain termination method (Sanger et al., 1977) with double-stranded plasmid DNA as a template. Sequencing reactions were carried out with an AutoRead DNA sequencing kit (Pharmacia, Sweden) with fluorescein isothiocyanate-labeled primers. Samples were analyzed with an ALF DNA Sequencer II sequencer (Pharmacia, Sweden), and sequence data were processed with the associated software (ALF manager, version 3.02). The sequences of both strands of the rRNA gene (rDNA) were determined and were submitted to GenBank.

Analyses of DNA sequences and phylogenetic relationships were done by using the MacVector software package, version 7.0 (Genetic Computer Group Inc., USA). The rDNA sequences (accession numbers are given in parentheses) for phylogenetic analysis were from B. bigemina (X59604), B. divergens (U16370), B. canis (L19079), B. caballi (Z15104) B. equi (Z15105), B. rodhaini (AB04999), B. microti (U09833) and B. gibsoni (L13729). The sequences were aligned with Program Clustal W Alignment (Higgins et al., 1992), and a phylogenetic tree by neighbor-joining method (Saitou and Nei, 1987) was constructed from the aligned sequences by the programne Phylogenetic Analysis in the MacVector software. Support for tree nodes was calculated by 1,000 bootstrap replicates by the Bootstrap Tree algorithm.

The vector ticks of Korean Babesia were collected from 5 Holstein cows, which were infected with Babesia parasites. Twenty-one adult ticks were collected from the neck and ears of the cow; 15 young ticks were collected by flagging vegetation around pastureland. All ticks were sent to the Protozoology Section of the National Institute of Infectious Diseases in Japan for identification.