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ABSTRACT. Exploring the antigenic and genetic diversities of Babesia ovata, we obtained several field isolates from grazing cattle in the Okushiri island, Japan. Parasite isolation was greatly facilitated by using bovine red blood cell-substituted SCID mice (Bo-RBC-SCID mice), into which the blood samples of the cattle were inoculated. Isolates from different individuals within a herd of cattle were compared in immunoblot analysis with an anti-B. ovata serum and also in Southern blot analysis with a probe for the small subunit ribosomal RNA gene. In both analyses, the isolates exhibited banding patterns that were significantly different from each other. We were also able to obtain a series of parasite isolates from a single cow in different seasons of a nine months period, including winter when active vector ticks were not in the field environment. Different seasonal isolates showed different banding patterns in both immunoblot and Southern blot analyses. By contrast, these analyses detected little difference among the parasites that had been passed various times in Bo-RBC-SCID mice, where no specific immune responses should be generated. These results indicate that individual animals within a herd of cattle were infected with antigenically and genetically diversified populations of B. ovata, and that the parasites could persistently infect a single animal with dynamic change in their predominant subpopulations. -KEY WORDS: Babesia ovata, diversity, persistent infection.

ABSTRACT. To investigate the surface antigens of the bovine red blood cells (RBCs) parasitized by Babesia ovata or Theileria sergenti, attempts were made to produce monoclonal antibodies (mAbs) with BALB/c mice. Comparable numbers of hybridomas producing antipiroplasm mAbs, as well as anti-bovine RBC mAbs, were obtained from the mice immunized with B. ovata-or T. sergenti-PRBCs. However, mAbs directed to the surface of parasitized RBCs (PRBCs) were obtained only from the mice immunized with B. ovata-PRBCs, but not from those immunized with T. sergenti-PRBCs. When serum samples from the immunized mice and the infected cattle were examined, antibodies recognizing B. ovata-PRBC surface were detected in the sera against B. ovata, but analogous antibodies were undetectable in the sera against T. sergenti, despite that the sera showed substantial antibody titers to T. sergenti piroplasms. The results suggest that significant antigenic modifications occur on the surface of B. ovata-PRBCs, but not on the surface of T. [12,19]. Blood samples containing 30-60% parasitemia were harvested from the Bo-RBC-SCID mice and immunized into BALB/c mice (SLC Inc.). For each parasite antigen, six mice were intravenously injected 3 times at 3 days intervals with 100 µl of 20% suspension of infected RBCs in 0.85% NaCl. Four weeks after the last injection, the mice were boosted intraperitoneally with an equal amount of infected RBCs. Three days later, they were euthanatized by CO 2 asphyxiation, and the spleens were removed to obtain lymphocytes. Serum samples were prepared from all the mice, and they were absorbed with an excess amount of uninfected bovine RBCs prior to being tested. The method to produce mAbs was described previously [12]. Briefly, the spleen cells from two immunized mice were pooled, and 1.5 × 10 8 of the cells were fused with 1.5 × 10 7 of mouse myeloma P3-X63-Ag8-U1 cells using polyethylene glycol 4000 (Merck). Cells were plated 3.5 × 10 6 /well in 96-well plates, and hybridomas were selected in Dulbecco's modified Eagle medium (D-MEM; Flow laboratories) containing 16 µM hypoxanthine, 0.4 µM aminopterin, 100 µM thymidine and 10% fetal bovine serum (FBS). Cultured supernatants were examined for antibody production by the two immunofluorescence assays (IFA) described below. Cells in the antibody-positive wells were cloned twice by limiting dilution.Fixed-cell IFA: Approximately 30% suspension of B. ovata-or T. sergenti-infected RBCs, which have 10-40% parasitemia, was prepared in phosphate buffered saline (PBS), pH 7.2, containing 1% bovine serum albumin (BSA). Roughly 0.3 µl aliquots were spread and dried out in each well of 24-well HT Coating Slide (MS342BL; Bokusui Brown). The slides were fixed in acetone for 5 min, and immediately transferred into PBS. Following removal of solution by blotting with a filter paper, 15 µl of cultured supernatant was added to each well. After 1 hr incubation