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

Alignment of partial gene sequences of glutathione <i>S</i>-transferases (GSTs) from <i>O. volvulus</i> (<i>OvGST1a</i>, <i>OvGST1b</i>) and <i>O. ochengi (OoGST1)</i> (A) and primer sets targeting <i>OvGST1a</i> (B).

<p>Primers are indicated by solid black arrows and dash arrows represent the binding regions of the loop forward (LFP) and loop back (LBP) primers respectively.</p

Diagrammatic view of the similarity of <i>Onchocerca</i> sigma-class GST gene models for <i>O. volvulus</i> GSTs 1a and 1b and the homologous <i>O. ochengi</i> sigma-class GST t09064.

<p>Gene models were aligned over the full-length sequence (total distance, 3,870 bp). Numbers associated with gene model exons (<i>I–VII shaded blocks</i>) and introns (<i>1–8 non-shaded blocks</i>) display the number of base-pairs within those sections over which the alignment is spaced. The three major differences between the genes (all insertions in <i>O. volvulus GST1a</i> intron 3) are highlighted in the diagram.</p

Phylogenetic neighbour-joining tree showing the relationship of the sigma-class GSTs of <i>Onchocerca ochengi</i> to similar enzymes of nematodes, mammals and insects.

<p>Numbers shown alongside branches are bootstrap values of 1,000 replications. The key for protein sequence accession numbers and organisms displayed in the tree is as follows: <u>Nematodes</u>: Oo_GST_t09064, Oo_GST_t03844 and Oo_GST_t06414 glutathione transferase [<i>Onchocerca ochengi</i>]; Ov_GST_1b AAG44696.1 glutathione <i>S</i>-transferase Ia [<i>Onchocerca volvulus</i>]; Ov _GST_1a AAG44695.1 glutathione <i>S</i>-transferase Ia [<i>Onchocerca volvulus</i>]; Ll_GST XP_003139665.1 hypothetical protein LOAG_04080 [<i>Loa loa</i>]; Bm_GST_4 XP_001901855.1 glutathione <i>S</i>-transferase 4 [<i>Brugia malayi</i>]; As_GST_1 ERG83753.1 glutathione <i>S</i>-transferase 1 [<i>Ascaris suum</i>]; As_GST_4 ERG81431.1 glutathione s-transferase 4 [<i>Ascaris suum</i>]; Ce_GST-11 NP_508625.1 protein GST-11 [<i>Caenorhabditis elegans</i>]; Ce_GST-36 NP_509652.2 protein GST-36 [<i>Caenorhabditis elegans</i>]. <u>Mammals:</u> Hs_PGD NP_055300.1 hematopoietic prostaglandin D synthase [<i>Homo sapiens</i>]; Bt_PGD_x1 XP_002688181.1 PREDICTED: hematopoietic prostaglandin D synthase isoform X1 [<i>Bos taurus</i>]; Rt_PGD NP_113832.1 hematopoietic prostaglandin D synthase [<i>Rattus norvegicus</i>]; Mm_PGD NP_062328.3 hematopoietic prostaglandin D synthase [<i>Mus musculus</i>]. <u>Insects:</u> Md_GST_ NP_001273827.1 glutathione <i>S</i>-transferase [<i>Musca domestica</i>]; Dm_GST_s1 NP_725653.1 glutathione <i>S</i>-transferase S1, isoform A [<i>Drosophila melanogaster</i>]; Ph_GST XP_002426887.1 glutathione <i>S</i>-transferase, putative [<i>Pediculus humanus corporis</i>]; Tc_GST XP_970714.1 PREDICTED: glutathione <i>S</i>-transferase [<i>Tribolium castaneum</i>]. The GSTs from <i>O. volvulus</i> and their closest relative in <i>O. ochengi</i> are shown in bold.</p

Sensitivity of LAMP and PCR methods for the detection of <i>O. volvulus</i> using ten-fold serial dilutions of <i>O. volvulus</i> genomic DNA ranging from 0.001–1.0 ng.

<p>Detection of LAMP product using turbidity (<b>A</b>) or hydroxy napthol blue (<b>B</b>). PCR amplification of a ∼200 bp product using LAMP primers F3 and B3 was obtained when <i>O. volvulus</i> genomic DNA was used (<b>C</b>). Molecular weight marker (MW) is indicated.</p

A comparison of LAMP and PCR methods to detect varying amounts of genomic DNA isolated from pools of black flies using different methods.

<p>A comparison of LAMP and PCR methods to detect varying amounts of genomic DNA isolated from pools of black flies using different methods.</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

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

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

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