Identification and expression of Babesia ovis secreted antigen 1 and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

Abstract

In order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5α cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis.

FIG 1

Nucleotide and predicted amino acid sequences for cDNA encoding BoSA1. The underlined region indicates the predicted signal peptide. The gray and boxed regions indicate 2 internal repeat domains.

FIG 2

SDS-PAGE analysis of the recombinant BoSA1 protein expressed in E. coli DH5α. Lane M, protein ladder (kDa). The predicted rBoSA1 protein band expressed in cells is indicated. The figure includes five randomly selected clones of E. coli DH5α cells transformed with recombinant plasmid pGEX-4T3/BoSA1. Lanes E1, E2, E3, E4, and E5, E. coli DH5α cells treated with IPTG. Lanes C1, C2, C3, C4, and C5, uninduced control E. coli DH5α cells.

FIG 3

Western blot analysis of recombinant and native BoSA1 proteins. (A) Western blots showing the reactivity of anti-B. ovis antibodies with rBoSA1. The anti-B. ovis antibodies reacted with several components of rBoSA1 between ∼70 and ∼40 kDa but not with the GST protein. (B) Western blots showing the reactivity of anti-rBoSA1 mouse antibodies with native BoSA1 proteins in the erythrocytes and plasma of B. ovis-infected blood. Lane M, protein ladder; lane iRBC, infected erythrocyte lysate extracted with saponin; lane iP, infected plasma proteins precipitated with saturated ammonium sulfate; lane niRBC, noninfected erythrocyte lysate extracted with saponin; lane niP, noninfected plasma proteins precipitated with saturated ammonium sulfate. Comparisons with molecular size markers indicated estimated molecular masses consistent with the monomeric, dimeric, and tetrameric forms of native BoSA1.

FIG 4

Localization of native BoSA1 proteins recognized by anti-rBoSA1 mouse antibodies in confocal laser micrographs. The images were derived from two sections. (A and E) Phase-contrast images of B. ovis merozoites. (B and F) Overlaid images of fluorescent reactivity and red PI staining on the phase-contrast images of the parasites. (C and G) Immunofluorescence images of parasite-specific antigens. (D and H) Propidium iodide staining of B. ovis merozoite nuclei. Bars = 5 µm.

FIG 5

Scatter diagrams of absorbance values measured in ELISAs with the recombinant BoSA1 protein. (A) Sera from 15 lambs experimentally infected with B. ovis, assayed on days 6, 7, 8, 11, 21, 30, 45, and 75 postinfection. The negative results obtained prior to infection and until day 6 of the experimental infection are not shown in the diagram. (B) Sera from 38 sheep naturally infected with B. ovis. Each dot above a horizontal line indicates an ELISA-positive sample. BT, before treatment; PT, 20 to 30 days posttreatment; C, negative-control samples.