Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).

Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni.

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 degrees C for 1 h, but not at 30 degrees 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-gamma 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.

C3 contributes to the cross-protective immunity induced by Babesia gibsoni phosphoriboprotein P0 against a lethal B. rodhaini infection.

We have studied the impact of complement component 3 (C3) deficiency on the progression of lethal Babesia rodhaini infection in immune mice. A B. gibsoni ribosomal phosphoprotein P0 (BgP0) previously reported to be a cross-protective antigen against Babesia infection was used to immunize C57BL/6 wild-type (WT) and C3-deficient (C3-/-) mice. Test mice were immunized intraperitoneally (i.p.) with recombinant BgP0 (rBgP0), while controls either were immunized with PBS or did not receive any immunization. Following the immunization regime, test WT mice induced a specifically strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2 associated with high production of IFN-gamma in the supernatant of splenocytes. While test C3-/- mice had significantly decreased total IgG, IgG1 and IgG2b responses, the secretions of IL-12 and IFN-gamma tended to be lower than those in WT mice. Furthermore, partial protection was only observed in rBgP0-immunized WT mice but not in C3-/- mice or controls. Indeed, rBgP0-immunized WT mice showed significant reductions in the initiation of parasitaemia correlated with delayed mortalities and considerable survival rates. Taken together, our results indicate that cross-protection was impaired in C3-/- mice in view of the decrease in the antibody responses and cytokine production and the high susceptibility to infection.

In-vitro uptake of vitamin A by Plasmodium falciparum.

The vitamin-A uptake of Plasmodium falciparum was investigated by culturing a standard isolate of the parasite (FCR-3) with (3)H-labelled vitamin A, at concentrations of the vitamin corresponding to those normally present in human serum. The (3)H-labelled vitamin A accumulated in the parasites from each culture in a parasitaemia-dependent manner. The radioactivity detected in the parasites increased with parasite maturation from the ring to the late-trophozoite stage. In addition, most of the radioactivity incorporated into the parasite cells was in the cytoplasm. The accumulation of vitamin A in the cytoplasm of late trophozoites indicates that P. falciparum may use vitamin A, from its human host, as an antioxidant, to protect itself from oxidative stress while intra-erythrocytic. The amount of the vitamin taken up by the parasite in vitro is small compared with the deficit that sometimes causes severe hypovitaminosis A in malaria cases. Consumption of vitamin A by the parasites together with the systemic decreases in non-enzymatic antioxidants that are seen in malaria may together cause this characteristic hypovitaminosis.

Leishmania mini-exon genes for molecular epidemiology of leishmaniasis in China and Ecuador.

The mini-exon gene is unique and is tandemly repeated in the Leishmania genome. The transcribed region is highly conserved, but the non-transcribed spacer region is distinct in length and in sequence among different Leishmania species. The usefulness of PCR amplification of the Leishmania mini-exon gene was examined for molecular epidemiology of visceral and cutaneous leishmaniasis. We previously described a PCR method for amplification of the mini-exon gene and obtained positive amplification in bone marrow aspirates of patients with visceral leishmaniasis in China. In this study, we have cloned and sequenced two PCR products from the patients. The sequences of two products revealed 100% identity and showed more similarity to the mini-exon gene of L. donovani Indian strain than those of L. donovani complex in Africa and South America. We also applied this PCR method to the diagnosis of cutaneous leishmaniasis. We obtained positive PCR amplification in skin biopsy materials taken from patients with cutaneous leishmaniasis in Ecuador. Since this PCR amplification is simple and requires only a pair of primers to detect all Leishmania species distributed in Ecuador, the method may be a useful tool for the detection of parasites, not only from patients, but also from sandflies and reservoir animals in this area of endemicity.