Fujiko Sunaga

Azabu University, Sagamihara, Kanagawa-ken, Japan

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Publications (12)19.63 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study is to determine the efficacy of exoantigens derived from Babesia gibsoni cultures to induce protective immunity against challenge exposure of virulent organisms. An attenuated B. gibsoni Oita strain was maintained in vitro by the microaerophilus stationary phase (MASP) method, and exoantigens-containing supernatant fluids were collected for preparation of the immunization. Two dogs received three subcutaneous immunizations with a 20-day interval of B. gibsoni exoantigens plus 0.5 mg saponin (Quil A). On day 68 after the prime immunization, the immunized dogs and control dogs were challenged intravenously with 2 × 10(8) virulent parasites of a homologous B. gibsoni strain. The results showed that exoantigens could induce a high degree of protection against virulent homologous challenge exposure. Two dogs immunized with exoantigens showed a lower parasitemia, accompanied by a slight decrease in the PCV that returned to normal values. Control dogs developed typical acute clinical signs, including severe anemia and hyperthermia. The immunization elicited humoral immune responses. In dogs immunized with exoantigens, the maximum antibody titer was 2,560 and 5,120 by indirect fluorescent antibody test (IFAT), respectively. Preliminary Western blot analysis of the immunogen revealed five dominant proteins of molecular weights of 18, 37, 43, 50, and 57 kDa. These results suggested that the culture-derived exoantigens were candidates for non-viable vaccine.
    Parasitology Research 02/2014; · 2.85 Impact Factor
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    ABSTRACT: The virulence of the Babesia gibsoni Oita isolate was attenuated by serial passages in vitro by using the microaerophilus stationary phase (MASP) technique. After 400 serial passages, the virulence of the isolate was found to be attenuated. This was evidenced by the response of two dogs inoculated intravenously with 10(9)B. gibsoni passaged isolate. Specific antibodies were produced at a titer of 1:20,480, as detected by the fluorescent antibody test (IFAT). These results suggested that the serial passages of B. gibsoni reduced its virulence while retaining its antigenicity. The dogs that were inoculated with the attenuated isolate (1 and 2) and two naïve dogs (3 and 4) were challenged by intravenous inoculation of 2×10(8) infected erythrocytes of the virulent Oita isolate. Protection afforded by exposure to the attenuated isolate was evidenced by a lower parasitemia in dogs 1 and 2 with a rapid decrease to nondetectable levels, accompanied by a slight decrease in the PCV that returned to normal values. Dogs 3 and 4 developed typical acute clinical signs, including severe anemia and hyperthermia. These results suggested that the attenuated isolate was a candidate for live vaccine.
    Veterinary Parasitology 05/2013; · 2.38 Impact Factor
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    ABSTRACT: We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results.
    Parasitology International 12/2011; 61(2):364-8. · 2.30 Impact Factor
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    ABSTRACT: The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260microM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6microM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10mg/kg of body weight on days 8-10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.
    Parasitology International 09/2010; 59(3):481-6. · 2.30 Impact Factor
  • Journal of Electrophoresis. 01/2010; 54(1):9-12.
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    ABSTRACT: It is not known whether precursors of methotrexate, such as 2, 4-diamino-6-hydroxymethyl-pteridine (DAP) and 2, 4-diamino-N10-methyl-pteroic acid (DAMPA), could target the dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme of Babesia and inhibit the parasite growth. Therefore, we have determined whether DAP and DAMPA as well as other chemically related compounds like pteroic acid (PA) and N10-Triflouropteroic acid (N10TFPA) could target the DHFR-TS enzyme of B. gibsoni and inhibit its growth. DAMPA was a more-potent inhibitor of the B. gibsoni growth in vitro (50% inhibition concentration [IC50] = 2.4 ± 0.20 μM) [mean ± standard error of the mean] than DAP (IC50 = 78 ± 15 μM). Moreover, DAMPA potently inhibited enzymatic activity of recombinant DHFR-TS of B. gibsoni (IC50 = 2.6 ± 0.15 μM) than DAP (IC50 > 100 μM). In contrast, PA and N10-TFPA did not inhibit the activity of the recombinant enzyme and growth of B. gibsoni. The inhibition of the recombinant enzyme activity by DAMPA mirrored with inhibition of the parasite growth indicating that the purified recombinant enzyme could be used for preliminary screening of some antifolate precursors. Therefore, both DAP and DAMPA inhibit growth of B. gibsoni by targeting the DHFR-TS enzyme of the parasite.
    J. Protozool. Res. 20, 70-81 (2010). 01/2010; 20:70-81.
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    ABSTRACT: Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus chromogenes are known to cause skin infections in human or animals by producing exfoliative toxins (ETs). Staphylococcus pseudintermedius can also cause canine pyoderma, but no exfoliative toxins or similar toxins have been reported. PCR with degenerate primers targeted to the conserved regions in ETA, ETB, and ETD from S. aureus and SHETB from S. hyicus, and subsequent chromosome walking identified a novel gene, designated as exi (exfoliative toxin of pseudintermedius) in S. pseudintermedius. EXI had significant homologies with the exfoliative toxins (43-68% identity), particularly with ETB (67.1%), ETD (67.9%), and SHETB (65.1%). Phylogenetic analysis showed close relation between EXI and ETB with a bootstrap value of 80%. Neonatal mice injected with the crude proteins from the culture supernatant or recombinant EXI showed gross blisters and/or characteristic skin exfoliation. The prevalence of exi assessed by dot-blot hybridization was 23.3% (10/43) in S. pseudintermedius isolates from canine pyoderma. The EXI reported herein is the first exfoliative toxin identified in S. pseudintermedius.
    FEMS Microbiology Letters 10/2009; 301(2):176-80. · 2.05 Impact Factor
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    ABSTRACT: A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity.
    Parasitology International 11/2008; 58(1):55-60. · 2.30 Impact Factor
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    ABSTRACT: Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed Km values of 4.70 ± 0.059 (mean ± standard error of the mean) and 9.75 ± 1.64 μM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC50] = 68.6 ± 5.20 nM) than pyrimethamine (IC50 = 55.0 ± 2.08 μM) and trimethoprim (IC50 = 50 ± 12.5 μM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target.
    Antimicrobial Agents and Chemotherapy 10/2008; · 4.57 Impact Factor
  • The Japanese Journal of Veterinary Dermatology 01/2006; 12(1):11-15.
  • The Japanese Journal of Veterinary Dermatology 01/2005; 11(4):205-206.
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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 degrees 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 large 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.
    Journal of Veterinary Medical Science 08/2002; 64(7):571-5. · 0.88 Impact Factor

Publication Stats

53 Citations
19.63 Total Impact Points

Institutions

  • 2002–2013
    • Azabu University
      • • Laboratory of Infectious Diseases
      • • Department of Veterinary Medicine
      • • School of Veterinary Medicine
      Sagamihara, Kanagawa-ken, Japan
  • 2008–2011
    • Obihiro University of Agriculture and Veterinary Medicine
      • National Research Center for Protozoan Diseases
      Obihiro, Hokkaido, Japan