Different forms of <i>Babesia divergens</i> in human RBCs as seen on a Giemsa-stained smear from <i>in vitro</i> cultured parasites (ring, dividing figure eights, Maltese cross parasites, and multiply infected RBCs).

<p>Different forms of <i>Babesia divergens</i> in human RBCs as seen on a Giemsa-stained smear from <i>in vitro</i> cultured parasites (ring, dividing figure eights, Maltese cross parasites, and multiply infected RBCs).</p

Native BdRAP-1 localised to apical complex.

<p>A, Immunofluorescence staining with anti-rBdRAP-1 antibodies (FITC) on fixed cells infected with <i>B. divergens</i> parasites (DAPI) clearly shows that BdRAP-1 is localised to the apical ends of all 4 parasites in this Maltese Cross form of the intracellular parasite (see Merged panel). B, Electron microscopy of a singly-infected RBC (direct magnification of 30,000×, print magnification of ∼50,000×) shows the location of the rhoptry organelles and the presence of BdRAP-1. C, An enlarged section (direct magnification 49,000×, print magnification of ∼140,000×) clearly shows localisation of native BdRAP-1 is within the bulb of the rhoptry organelle.</p

Antigenicity of recombinant BdRAP-1 against sera from <i>B. divergens</i> –infected cows and hamsters.

<p>A, The reactivity of rBdRAP-1 in ELISA with pre-immune (PI) sera and experimentally <i>B. divergens</i>-infected sera from cows (n = 6). All infected bovine sera showed higher OD values than non-infected sera in a dilution-dependant manner at all dilutions. B, The reactivity of rBdRAP-1 in ELISA with pre-immune (PI) sera and experimentally <i>B. divergens</i>-infected sera from gerbils (n = 5). Four of the five gerbil sera showed s higher OD values than non-infected sera in a dilution-dependant manner at dilutions 1∶200 to 1∶6,400. One gerbil serum (G5) showed no reactivity. At dilutions 1∶12,800 and 1∶26,600, there is no difference in the reactivity of the gerbil sera and the pre-immune sera. C, Specificity for the animal serum against recombinant BdRAP-1 was confirmed independently by immunoblotting. Four of the six bovine sera and two of the three gerbil sera reacted strongly against rBdRAP-1. One gerbil sera (G3) reacted weakly. Sera which showed the greatest reactivity in ELISA (C3, G1 and G4) also showed greatest reactivity on the blots. Sera G2 and G3 showed moderate reactivity in ELISA but surprisingly very low reactivity in the Western analysis. Sera which did not show any significant reactivity in the ELISA (C5,C6 and G5) also did not react against rBdRAP-1 by immune-blotting.</p

Multiple sequence alignment of BdRAP-1 sequences shows homology to other babesial RAP-1 proteins is within the N-terminal portion.

<p>Clustal analysis of RAP-1 sequences of <i>B. divergens</i> (human host source) (accession number KJ699102) identified in this manuscript, <i>B. divergens</i> (bovine host origin) (accession number Z49818), <i>B. bovis</i> (accession number M38218) and <i>B. canis</i> (accession number M91168) shows that homology is restricted to the N-terminus region, which corresponds to the location of the RAP-1 superfamily structure (indication above the sequence by a sold line from BdRAP-1 Ala⁴¹ to Gly²⁹¹) as identified by BLAST analysis. The sites of non-synonymous differences between the BdRAP-1 proteins are shown in grey shading. Positions of identity are indicated by asterisk and similarity by dots, below the sequences. Four cysteine residues, at sites BdRAP-1 80, 89, 100, and 105 (bold underlined), are conserved across all species, and are also limited to the N-terminus region, further suggesting this portion of RAP-1 may contain the RBC binding site.</p

Anti-rBdRAP-1 antibodies identify a specific ∼46 kDa protein in parasite lysate.

<p>A, Immunoblotting of proteins from in vitro cultured <i>B. divergens</i> lysate showed anti-rBdRAP-1 antibodies (lane 1) are able to detect native BdRAP-1, as shown by the presence of a single band at ∼46 kDa. Pre-immune rabbit serum (lane 2) did not react with any native antigens. B, Immunoprecipitation with lysate from [³⁵S] methionine/cysteine-labelled <i>B. divergens</i> cultures showed native BdRAP-1 is present in the culture pellet (lane 1), as observed by the presence of a single band at ∼46 kDa. BdRAP-1 is secreted into the culture supernatant as a processed product, as shown by the presence of an abundant ∼17 kDa product, and a much less abundant product of ∼34 kDa, in the culture supernatant (lane 2; indicated by arrows).</p