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1.  Detection of Two Zoonotic Babesia microti Lineages, the Hobetsu and U.S. Lineages, in Two Sympatric Tick Species, Ixodes ovatus and Ixodes persulcatus, Respectively, in Japan 
The species Babesia microti, commonly found in rodents, demonstrates a high degree of genetic diversity. Three lineages, U.S., Kobe, and Hobetsu, are known to have zoonotic potential, but their tick vector(s) in Japan remains to be elucidated. We conducted a field investigation at Nemuro on Hokkaido Island and at Sumoto on Awaji Island, where up to two of the three lineages occur with similar frequencies in reservoirs. By flagging vegetation at these spots and surrounding areas, 4,010 ticks, comprising six species, were collected. A nested PCR that detects the 18S rRNA gene of Babesia species revealed that Ixodes ovatus and I. persulcatus alone were positive. Lineage-specific PCR for rRNA-positive samples demonstrated that I. ovatus and I. persulcatus carried, respectively, the Hobetsu and U.S. parasites. No Kobe-specific DNA was detected. Infected I. ovatus ticks were found at multiple sites, including Nemuro and Sumoto, with minimum infection rates (MIR) of ∼12.3%. However, all I. persulcatus ticks collected within the same regions, a total of 535, were negative for the Hobetsu lineage, indicating that I. ovatus, but not I. persulcatus, was the vector for the lineage. At Nemuro, U.S. lineage was detected in 2 of 139 adult I. persulcatus ticks (MIR, 1.4%), for the first time, while 48 of I. ovatus ticks were negative for that lineage. Laboratory experiments confirmed the transmission of Hobetsu and U.S. parasites to hamsters via I. ovatus and I. persulcatus, respectively. Differences in vector capacity shown by MIRs at Nemuro, where the two species were equally likely to acquire either lineage of parasite, may explain the difference in distribution of Hobetsu throughout Japan and U.S. taxa in Nemuro. These findings are of importance in the assessment of the regional risk for babesiosis in humans.
doi:10.1128/AEM.00142-12
PMCID: PMC3346458  PMID: 22389378
2.  Identification and Phylogenetic Analysis of Japanese Macaque Babesia-1 (JM-1) detected from a Japanese Macaque (Macaca fuscata fuscata) 
We demonstrate here the identification and phylogenetic characterization of Babesia microti (B. microti)-like parasite detected from a splenectomized Japanese macaque (Macaca fuscata fuscata) at a facility for laboratory animal science. On Day 133 after splenectomy, intra-erythrocytic parasites were found on light microscopic examination, and the level of parasitemia reached 0.3% on blood smear. Molecular characterization of the parasite using nested-polymerization chain reactions targeting the 18S rRNA, β-tubulin, and subunit 7 (eta) of the chaperonin-containing t-complex polypeptide 1 (CCT7) genes were identified as a B. microti-like parasite, designated the Japanese Macaque Babesia-1 (JM-1).
doi:10.4269/ajtmh.2011.11-0035
PMCID: PMC3183768  PMID: 21976563
3.  Cloning of a Novel Babesia equi Gene Encoding a 158-Kilodalton Protein Useful for Serological Diagnosis 
In this study, we characterized a Babesia equi Be158 gene obtained by immunoscreening a B. equi cDNA expression phage library with B. equi-infected horse serum. The Be158 gene consists of an open reading frame of 3,510 nucleotides. The recombinant Be158 gene product was produced in Escherichia coli and used for the immunization of mice. In Western blot analysis, mouse immune serum against the Be158 gene product recognized 75- and 158-kDa proteins from the lysate of B. equi-infected erythrocytes. In an indirect fluorescent-antibody test with the mouse immune serum, the Be158 antigen appeared in the cytoplasm of Maltese cross-forming parasites (which consist of four merozoites) and was located mainly in the extraerythrocytic merozoite body. When the recombinant Be158 gene product was used in an enzyme-linked immunosorbent assay as a serological antigen, it was found to react to B. equi-infected horse sera, indicating that the Be158 gene product is useful as a serologically diagnostic antigen for B. equi infection.
doi:10.1128/CDLI.12.2.334-338.2005
PMCID: PMC549306  PMID: 15699430
4.  Molecular Cloning of a Babesia caballi Gene Encoding the 134-Kilodalton Protein and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay 
A Babesia caballi gene encoding the 134-kDa (BC134) protein was immunoscreened with B. caballi-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate B. caballi-infected horse sera from Babesia equi-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of B. caballi infection.
doi:10.1128/CDLI.11.1.211-215.2004
PMCID: PMC321337  PMID: 14715570
5.  Rapid Immunochromatographic Test Using Recombinant SAG2 for Detection of Antibodies against Toxoplasma gondii in Cats 
Journal of Clinical Microbiology  2004;42(1):351-353.
An immunochromatographic test using recombinant truncated surface antigen 2 for detection of antibodies against Toxoplasma gondii was developed. Evaluation of detection of the antibody in mice and cats suggests that this test is rapid, simple, accurate, relatively inexpensive, and suitable for use under field conditions.
doi:10.1128/JCM.42.1.351-353.2004
PMCID: PMC321666  PMID: 14715776
6.  Growth-Inhibitory Effects of Artesunate, Pyrimethamine, and Pamaquine against Babesia equi and Babesia caballi in In Vitro Cultures 
Three antimalarial drugs, artesunate, pyrimethamine, and pamaquine, were evaluated for their growth-inhibitory effects against Babesia equi and Babesia caballi in in vitro culture. B. equi was more resistant to pyrimethamine than B. caballi. B. equi was also found to be more sensitive to artesunate and pamaquine than B. caballi. Of the three compounds, pyrimethamine gave the most promise for in vivo effectiveness.
doi:10.1128/AAC.47.2.800-803.2003
PMCID: PMC151728  PMID: 12543697
7.  Identification of a Specific Antigenic Region of the P82 Protein of Babesia equi and Its Potential Use in Serodiagnosis 
Journal of Clinical Microbiology  2003;41(2):547-551.
The efficacy of the Be82 gene product fused with glutathione S-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Babesia equi infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST/Be82 antigen cross-reacted with Babesia caballi-infected horse sera, despite the high rate of detection of B. equi. These results suggested that GST/Be82 has an antigen in common with B. caballi or antigenicity similar to that of B. caballi. In the present study, we constructed a series of five clones with deletions in the Be82 gene product, each of which was fused with GST, and used them in ELISAs in order to overcome the cross-reactivity seen with B. caballi. One of the deletion clones, a clone with a deletion of the Be82 gene from positions 236 to 381 (Be82/236-381), specifically and sensitively detected B. equi-infected horse sera without cross-reactivity with B. caballi-infected horse sera. Assays with clones from which other gene products were deleted showed decreased sensitivities or remained nonspecific for the detection of B. equi-infected horse sera. These results suggest that the Be82/236-381 gene product is a novel antigen for the diagnosis of B. equi infection in horses.
doi:10.1128/JCM.41.2.547-551.2003
PMCID: PMC149686  PMID: 12574244
8.  Roles of the Maltese Cross Form in the Development of Parasitemia and Protection against Babesia microti Infection in Mice  
Infection and Immunity  2003;71(1):411-417.
Babesia microti, a hemoprotozoan parasite of rodents, is also important as a zoonotic agent of human babesiosis. The Maltese cross form, which consists of four masses in an erythrocyte, is characteristic of the developmental stage of B. microti. Monoclonal antibody (MAb) 2-1E, which specifically recognizes the Maltese cross form of B. microti, has been described previously. In the present study, we examined the roles of the Maltese cross form during the infectious course of B. microti in mice. The number of the Maltese cross form increased in the peripheral blood of infected mice prior to the peak of parasitemia. With confocal laser scanning microscopy, MAb 2-1E was found to be reactive with the ring form, with the parasites undergoing transformation to the Maltese cross form and subsequent division, and also with extracellular merozoites. Furthermore, the Maltese cross form-related antigen (MRA) gene was isolated from a B. microti cDNA library by immunoscreening with MAb 2-1E, and the nucleotide sequence was determined. Genomic analyses indicated that the MRA gene exists as a single-copy gene in B. microti. Immunization of mice with recombinant MRA induced significant protective immunity against B. microti infection. These findings indicate that the Maltese cross form plays important roles in both the development of parasitemia and the protective response against the infection.
doi:10.1128/IAI.71.1.411-417.2003
PMCID: PMC143159  PMID: 12496191
9.  Cellular Localization of Babesia bovis Merozoite Rhoptry-Associated Protein 1 and Its Erythrocyte-Binding Activity  
Infection and Immunity  2002;70(10):5822-5826.
The cellular localization of Babesia bovis rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca2+ accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in B. bovis merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.
doi:10.1128/IAI.70.10.5822-5826.2002
PMCID: PMC128354  PMID: 12228313
10.  Cloning of a Truncated Babesia equi Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay 
Journal of Clinical Microbiology  2002;40(4):1470-1474.
To isolate Babesia equi genes encoding immunodominant proteins, a cDNA expression library prepared from B. equi mRNA was immunoscreened with B. equi-infected horse serum. Eighteen positive cDNA clones were obtained, and the clone that showed the strongest immunoreactivity, designated Be82, was further characterized. The Be82 gene consisted of 1,953 bp and contained a partial open reading frame lacking the 5′-terminal sequence. As shown by Western blot analyses, immune sera from mice intraperitoneally injected with the Be82 gene product recognized the 82- and 52-kDa proteins of B. equi but not those of Babesia caballi. The glutathione S-transferase fusion protein expressed in Escherichia coli that was purified and used as the antigen in the enzyme-linked immunosorbent assay reacted specifically with B. equi-infected horse sera. These results suggest that the Be82 gene product is a potential diagnostic antigen candidate in the detection of B. equi infection in horses that will be useful both in the performance of epidemiological studies and in the granting of quarantine passes.
doi:10.1128/JCM.40.4.1470-1474.2002
PMCID: PMC140338  PMID: 11923375

Results 1-10 (10)