Cloning and characterization of a 2-Cys peroxiredoxin from Babesia gibsoni.

Peroxiredoxins (Prxs) are a family of antioxidant enzymes. Here, we cloned a 2-Cys Prx, BgTPx-1, from the canine Babesia parasite B. gibsoni. Sequence identity between BgTPx-1 and 2-Cys Prx of B. bovis was 81% at the amino acid level. Enzyme activity assay by using recombinant BgTPx-1 (rBgTPx-1) indicated that BgTPx-1 has antioxidant activity. Antiserum from a mouse immunized with rBgTPx-1 reacted with parasite lysates and detect a protein with a monomeric size of 22 kDa and also a 44 kDa protein, which might be an inefficiently reduced dimer. BgTPx-1 was expressed in the cytoplasm of B. gibsoni merozoites. These results suggest that the BgTPx-1 may play a role to control redox balance in the cytoplasm of B. gibsoni.

Molecular and serological prevalence of Babesia bigemina and Babesia bovis in cattle and water buffalos under small-scale dairy farming in Beheira and Faiyum Provinces, Egypt.

In order to determine the molecular and serological prevalence of Babesia bigemina and Babesia bovis, a total of 247 blood samples were collected from cattle and water buffalos in Beheira and Faiyum Provinces in Egypt and examined by nested polymerase chain reaction (nPCR) and enzyme-linked immunosorbent assay (ELISA). In cattle, the prevalence of B. bigemina and B. bovis was 5.30% and 3.97% by nPCR and 10.60% and 9.27% by ELISA, respectively, whereas those of water buffalos were 10.42% and 4.17% by nPCR and 15.63% and 11.46% by ELISA, respectively. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and health status. Sequencing analysis revealed two genotypes for B. bovis spherical body protein-4. In conclusion, the current data provide valuable information regarding the epidemiology of B. bigemina and B. bovis infections in cattle and water buffalos from Egypt, which can be employed in developing future strategies for disease management and control.

Identification, cloning and characterization of BmP41, a common antigenic protein of Babesia microti.

Babesia microti is a rodent tick-borne blood parasite and the major causative agent of emerging human babesiosis. Here, we identified a candidate of common antigenic protein BmP41 of B. microti by serological screening of cDNA library of human-pathogenic Gray strain with antisera against rodent Munich strain. Immunofluorescent antibody test using mouse anti-recombinant BmP41 (rBmP41) serum revealed that native BmP41 was expressed in each of the developmental stages of B. microti merozoites. An enzyme-linked immunosorbent assay (ELISA) using rBmP41 detected specific antibodies in sera from hamsters infected with B. microti Gray strain and mice infected with B. microti Munich strain. Taken together, BmP41 could be a promising universal serological marker for diagnosis of human babesiosis.

Evaluation of in vitro and in vivo inhibitory effects of fusidic acid on Babesia and Theileria parasites.

Fusidic acid known to has antibacterial, antifungal, and antimalarial activities. Fusidic acid blocks translation elongation factor G gene in Plasmodium falciparum. In the present study, the inhibitory effects of fusidic acid on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of fusidic acid on the in vivo growth of Babesia microti was also assessed. The in vitro growth of four Babesia species that were tested was significantly inhibited (P<0.05) by micromolar concentrations of fusidic acid (IC(50) values=144.8, 17.3, 33.3, and 56.25 µM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Combinations of fusidic acid with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis and B. caballi. In B. microti-infected mice, fusidic acid caused significant (P<0.05) inhibition of the growth of B. microti at the dose of 500 mg/kg BW relative to control group. These results indicate that fusidic acid might be incorporated in treatment of babesiosis.

Molecular and immunological characterization of a novel 32-kDa secreted protein of Babesia microti.

A cDNA encoding the Babesia microti 32-kDa protein was identified by serological immunoscreening of a cDNA expression library and designated as BmP32. The full length of BmP32 contains an open reading frame of 918 base pairs consisting of 306 amino acids having a significant homology with B. microti secreted antigen 1. Antiserum raised against recombinant protein (rBmP32) specifically reacted with a 32-kDa native protein of the parasite lysate using western blot analysis. The indirect immunofluorescent antibody test showed a preferable localization of BmP32 in the cytoplasm of the intra- and extracellular parasites. Moreover, BmP32 was secreted in the cytosol of infected erythrocytes, especially during the peak parasitemia and the recovery phase of the infection. Next, the antigenicity of rBmP32 was examined by an enzyme-linked immunosorbent assay (ELISA) and sera from mice experimentally infected with either B. microti or closely related parasites. ELISA was highly specific and sensitive when used for the detection of B. microti antibody in a mouse model. Furthermore, mice immunized with rBmP32 emulsified with Freund's adjuvant were not significantly protected against challenge infection with B. microt i. However, high antibody titer was detected just before the challenge infection. Our data suggest that rBmP32 may be a specific diagnostic antigen but not a subunit vaccine.

Detection of Babesia gibsoni by reaction of phage display single chain antibodies with P50 proteins.

Babesia gibsoni (B. gibsoni) is a tick-borne hemoprotozoan parasite, which causes piroplasmosis in dogs. Diagnosis of canine babesiosis is commonly carried out using Giemsa-stained thin blood smears. However, at low levels of infection, it is difficult to detect Babesia organisms by observation of Giemsa-stained thin blood smears. We constructed a monoclonal phage display single chain antibody (scFv) against a B. gibsoni merozoite antigen, P50 protein. Intraerythrocytic B. gibsoni organisms are clearly stained using this antibody. The monoclonal scFv facilitated the detection of B. gibsoni organisms in canine blood samples.

Cloning, expression, and characterization of Babesia gibsoni dihydrofolate reductase-thymidylate synthase: inhibitory effect of antifolates on its catalytic activity and parasite proliferation.

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 K(m) values of 4.70 +/- 0.059 (mean +/- standard error of the mean) and 9.75 +/- 1.64 microM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC(50)] = 68.6 +/- 5.20 nM) than pyrimethamine (IC(50) = 55.0 +/- 2.08 microM) and trimethoprim (IC(50) = 50 +/- 12.5 microM). 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.