A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by Babesia bovis merozoites.

OBJECTIVE: The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA. METHODS: Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay. RESULTS: RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival. CONCLUSION: B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.

Water Buffalos as carriers of Babesia bovis in Argentina.

The tick-transmitted hemoprotozoan Babesia bovis is a major causative agent of bovine babesiosis, an often fatal disease of cattle. The disease is widespread in the northeastern region of Argentina, where an increasing part of the livestock is composed of water buffalos. Although clinical cases of buffalo babesiosis have not been reported so far, the pathogen-transmitting tick vector has been occasionally observed by us to be feeding on water buffalos. We therefore set out to examine whether buffalos may constitute a reservoir of the parasite. Competitive enzyme-linked immunosorbent assay (cELISA) detected B. bovis-specific antibodies in 20% of investigated buffalos (21/103), while direct detection of the pathogen by nested PCR was demonstrated in 34% of the animals (35/103). In one field, more than 60% of investigated animals (22/36) tested positive by nested PCR. These results are discussed in the context of buffalo babesiosis reported in other countries and in view of the currently effected control measures against bovine babesiosis in the region.

Use of a monoclonal antibody against Babesia bovis merozoite surface antigen-2c for the development of a competitive ELISA test.

Bovine babesiosis caused by Babesia bovis is a disease that hampers the production of beef and dairy cattle in tropical and subtropical regions of the world. New diagnostic methods based on recombinant antigens constitute valuable biotechnological tools for the strategic control of this disease. We have developed a competitive enzyme-linked immunosorbent assay that includes a recombinant form of the merozoite surface antigen-2c and a novel monoclonal antibody against it. Preliminary results showed that this test is able to identify specific antibodies against B. bovis from experimentally and naturally infected cattle.