Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4)

Background The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 ( Bcsbp4 ) gene and protein ( Bc SBP4) and analyzes its antigenicity in infected equids. Methods BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified Bc SBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant Bc SBP4 (r Bc SBP4) was tested with sera from field animals ( n = 18) using an indirect ELISA (iELISA). Results Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that Bc SBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of Bc SBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of Bc SBP4 predicted by in silico analysis. In vitro neutralization tests using anti- Bc SBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi -infected equids, but none out of ten negative equid control sera, gave a positive signal in an r Bc SBP4 based iELISA. Conclusions The Bcsbp4 gene is expressed in B. caballi blood stages. The Bc SBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.