M. Alaa Terkawi

Artesunate, a potential drug for treatment of Babesia infection.

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.

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.

Molecular and immunological characterization of Babesia gibsoni and Babesia microti heat shock protein‐70

Serological immunoscreening was used to identify a gene encoding heat shock protein‐70 from Babesia gibsoni (BgHSP‐70) that showed high homology with HSP‐70s from other apicomplexan parasites. This gene corresponded to a full‐length cDNA containing an open reading frame of 1968 bp predicted to result in a 70‐kDa mature protein consisting of 656 amino acids. Analysis of the expression levels of BgHSP‐70 indicated elevated transcription from cultured parasites incubated at 40°C for 1 h, but not at 30°C. Interestingly, antiserum raised against recombinant BgHSP‐70 protein reacted specifically not only with a 70‐kDa protein of B. gibsoni but also with a corresponding native protein of B. microti (BmHSP‐70), indicating the high degree of conservation of this protein. The BmHSP‐70 gene was then isolated and characterized and the immunoprotective properties of recombinant BgHSP‐70 (rBgHSP‐70) and rBmHSP‐70 were compared in vitro and in vivo. Both proteins had potent mitogenic effects on murine and canine mononuclear cells as evidenced by high proliferative responses and IFN‐γ production after stimulation. Immunization regimes in BALB/c and C57BL/6 mice using rBgHSP‐70 and rBmHSP‐70 elicited high antibody levels, with concurrent significant reductions in peripheral parasitaemias. Taken together, these results emphasize the potential of HSP‐70s as a molecular adjuvant vaccine.

Genetic diversity of two selected antigen loci in Babesia gibsoni Asian genotype obtained from Japan and Jeju island of South Korea

Previous reports have shown that the secreted antigen 1 of Babesia gibsoni (BgSA1) and the thrombospondin-related adhesive protein of B. gibsoni (BgTRAP) are promising diagnostic reagents and vaccine candidates. Therefore, we determined the extent of nucleotide sequence variation in the BgSA1 and BgTRAP genes, obtained from eight isolates of B. gibsoni got from clinically infected dogs in geographically distinct areas of Japan and one isolate from Jeju island of South Korea. Sequence analyses have revealed that nucleotide diversity is lower in BgSA1 than that in BgTRAP. The mean number of non-synonymous (dn) nucleotide substitutions was significantly greater than that of synonymous (ds) ones per site in region II of BgTRAP. Overall, the results predict more allele-specific immunity to BgTRAP than that to BgSA1, which could be useful in designing and testing efficacy of diagnostic reagents as well as vaccine candidates for the B. gibsoni isolates from Japan and Jeju island of South Korea.

Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis.

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.

Identification of Secreted Antigen 3 from Babesia gibsoni

ABSTRACT A cDNA expression library of Babesia gibsoni was screened with the serum collected from a dog experimentally infected with B. gibsoni. A novel antigen sharing homology with secreted antigen 1 of B. gibsoni was isolated. The genomic analysis indicated that the BgSA3 gene exists as multicopies in the genome of B. gibsoni. The putative peptide encoded by the BgSA3 gene showed some characteristics of secreted proteins. The serum raised in mice immunized with the recombinant BgSA3 expressed in Escherichia coli could recognize a native parasite protein with a molecular mass of 70 kDa. Moreover, a sandwich enzyme-linked immunosorbent assay with anti-BgSA3 antibodies could detect the circulating BgSA3 in the blood plasma of dogs experimentally infected with B. gibsoni. The identification of BgSA3 provided a useful target for the development of a diagnostic test for detecting specific antibodies and circulating antigens.

Four promising antigens, BgP32, BgP45, BgP47, and BgP50, for serodiagnosis of Babesia gibsoni infection were classified as B. gibsoni merozoite surface protein family

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.