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

Abstract

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 (BcSBP4) 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 BcSBP4. 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 BcSBP4 (rBcSBP4) 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 BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 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 rBcSBP4 based iELISA.

Conclusions: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 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.

Keywords: Babesia caballi; Equine piroplasmosis; SBP4; Serodiagnosis; iELISA.

Fig. 1

a Schematic representation for the synteny map framework of B. caballi spb4 gene in comparison with its orthologous genes in the genomes of B. bigemina, B. ovata and B. bovis. Babesia caballi genome: Ef G2, Elongation factor G2; B. bovis Ef G2, Elongation factor G2 BBOV_IV005400; B. bigemina Ef G2, Elongation factor BBBOND_0209390; B. ovata Ef G2, Elongation factor BOVATA_011720; B. ovata VW, Von Willebrand factor type ABOVATA_011730; B. bovis, BBOV_IV00539; B. bigemina, BBBOND_0209400; B. ovata, BOVATA_011740; HP, hypothetical protein; B. ovata Fib, Fibronectin type III domain containing 3C1-like BOVATA_011750; B. ovata FAD Ox, FAD-dependent oxidoreductase BOVATA_011760; B. ovata RPS 16, Ribosomal protein RPS16 BOVATA_011770; B. caballi Rib, Protein L13, Ribosomal protein L13; B. bovis Rib Prot L13, Ribosomal protein L13 BBOV_IV005370; B. bigemina Ric Prot, Ribosomal protein L13BBBOND_0209430; B. ovata Rib L13, Ribosomal protein L13 BOVATA_011780. Schematic rectangles represent gene location and not gene size. b Percentages of amino acid sequence identity and similarity and query coverage among the SBP4 proteins of B. caballi, B. bigemina, B. ovata and B. bovis

Fig. 2

Western blot analysis. a Specificity of the rabbit immune sera generated against BcSBP4 synthetic peptides. Lane 1: affinity purified rBc-SBP-4; Lane 2: native B. caballi antigens derived from sonicated in vitro cultured parasites; Lane M: pre-stained molecular weight protein ladder. b The pattern of reactivity of rBc-SBP4. Lane 1: pre-immune rabbit serum; Lane 2: rabbit immune sera generated against Bc-SBP4 synthetic peptides; Lane 3: sera from a B. caballi-infected horse; Lane 4: sera from a non-infected horse. Lane 5: sera from a T. equi-infected horse M: stained molecular weight protein ladder

Fig. 3

Immunofluorescence analysis of in vitro cultured B. caballi parasites with: rabbit anti-SBP4 cocktail peptides, rabbit pre-immune serum, monoclonal antibody 79/17.18.5 reactive with B. caballi RAP-1, and Tryp monoclonal irrelevant antibodies (the same isotype Ab as anti-RAP-1 79/17.18.5 mAb)

Fig. 4

In vitro neutralization of B. caballi in in vitro cultures using sera from B. caballi-infected horses and from anti-Sbp4 rabbits. a Representation of the decrease in PPE at day 3 of in vitro cultured B. caballi merozoites in the presence of an immune B. caballi horse (Ho A2034) and pre-immune (Ho A2034 Pre) sera. b Anti-Sbp4 rabbit (rab 7082) and pre-immune control (rab7082 Pre) sera. P < 0.05 indicates a significant difference

Fig. 5

Detection of anti-B. caballi SBP4 antibodies in B. caballi equid infected sera using ELISA. Optical density (OD) values obtained in an ELISA analysis based on SBP4 recombinant protein, with a sample of sera from 18 equids. LoD - limit of detection calculated according to the formula x + 3*SD [0.1614]. Samples results under the LoD indicate negative results vs above the line indicates the positive results. Samples 1–10 are donkeys and 11–18 are horses