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Molecular Cloning of a Babesia caballi Gene Encoding the 134-Kilodalton Protein and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

ABSTRACT A Babesia caballi gene encoding the 134-kDa (BC134) protein was immunoscreened with B. caballi-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate B. caballi-infected horse sera from Babesia equi-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of B. caballi infection.

antigen could clearly distinguish the B. caballi-infected horse sera from noninfected or B. equi-infected horse sera (10). However, in order to cover all sera infected with various types of field strains, it is necessary to conduct further studies to search for other immunodominant antigens applicable to epidemiological surveillance. Such studies might lead to a more practical use of ELISA worldwide. In this study, we succeeded in obtaining a novel cDNA clone by immunoscreening a B. caballi cDNA expression library. On the basis of a nucleotide sequence that we determined, a recombinant antigen was designed, produced in an Escherichia coli expression system, and subjected to ELISA for the evaluation of its diagnostic utility for B. caballi infection.
A B. caballi (U.S. Department of Agriculture strain) cDNA expression phage library (2.5 ϫ 10 5 PFU/ml) was immunoscreened with experimentally B. caballi-infected horse serum according to the previously reported method (10,20). Nucleotide sequencing was performed as described previously (7). Open reading frame (ORF) and protein homology searches were performed using the MacVector program (Oxford Molecular, Ltd., Oxford, United Kingdom) and the National Center for Biotechnology Information database, respectively. Two oligonucleotide primers, 5Ј-caggatccGTCATGATGAGA GGGAAC-3Ј and 5Ј-gcgaattcGGCCTTAGTTGCTGGGG-3Ј (lowercase letters show BamHI and EcoRI restriction site linkers, respectively), were designed on the basis of the nucleotide sequence of the BC134 gene (accession number AB095267 in GenBank, EMBL, and DDBJ databases), and 2,876 bp of a DNA fragment containing the BC134 ORF was amplified by PCR (16). The amplified DNA product was digested with BamHI and EcoRI, purified with the QIAquick gel extraction kit (Qiagen, Inc., Hilden, Germany), and then ligated into the BamHI and EcoRI sites of a pGEX-4T E. coli expression plasmid vector (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom). The resulting plas-mid, designated pGEX/BC134, was used to produce a recombinant BC134 protein fused with glutathione S-transferase (GST) (designated the GST-BC134 protein) in the E. coli BL21 strain (Stratagene) according to standard techniques (18). Next, the protein was purified as described previously (10,22). The purified GST-BC134 protein was resuspended in phosphate-buffered saline to a final concentration of 0.1 mg/ml and used as an immune antigen for the production of an anti-BC134 protein mouse immune serum or as a diagnostic antigen for an ELISA system, as described below. Seven-weekold BALB/c mice (CLEA Japan, Tokyo, Japan) were intraperitoneally immunized with 20 g of the purified GST-BC134 protein emulsified with the same volume of complete Freund's adjuvant (Difco, Detroit, Mich.). At 2-week intervals, the mice were stimulated five additional times with the same amount of purified antigen emulsified with Freund's incomplete adjuvant (Difco). Sera were collected from the mice at 10 days after the last booster. The indirect fluorescent antibody test was performed as described previously (27). Purification of the B. caballi cells and B. equi (U.S. Department of Agriculture strain) cells was performed according to the previously reported method (13,14). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed as described previously (26). An ELISA was performed as described previously (10,25). Seven sera of noninfected control horses, eight sera of experimentally B. caballi-infected horses, and eight sera of experimentally B. equi-infected horses were obtained from the Equine Research Institute of the Japan Racing Association.
By immunoscreening with B. caballi-infected horse serum, the nucleotide sequence of one cDNA clone that showed the strongest immunoreactivity was determined. Computer-aided analysis demonstrated that the cDNA insert had a total of 3,227 bp (accession number AB095267 in GenBank, EMBL, and DDBJ) and contained a single ORF, which was designated the BC134 gene (Fig. 1). The ORF from the initial codon Of the deduced amino acids of the BC134 gene, 28.6% were charged. The predicted protein has a typical signal peptide sequence at the N-terminal end (Fig. 1, underlined). A recombinant BC134 protein (GST-BC134) with a molecular mass of approximately 160 kDa was expressed in the transformed E. coli with pGEX/BC134 and then purified using glutathione-Sepharose 4B beads, as shown in Fig. 2. The GST-BC134 protein was also recognized with the B. caballi-infected horse serum in Western blot analysis (data not shown). Since the GST-BC134 protein contains a 26-kDa GST tag, the molecular mass of the GST-BC134 protein was estimated at approximately 134 kDa after removal of the GST tag. The de-tected size of the GST-BC134 protein, however, was still larger than the size estimated by computer-aided analysis (about 104 kDa).
A mouse immune serum was prepared against the purified GST-BC134 protein. In Western blot analysis (Fig. 3), the anti-recombinant BC134 protein mouse serum recognized only 134 kDa of the native protein in the lysate of B. caballi-infected erythrocytes (lane a) and not in the lysates of B. equi-infected and noninfected erythrocytes (lanes b and c). Next, in the indirect fluorescent antibody test with mouse antiserum to the recombinant BC134 protein, positive reactions were observed in all of the developmental stages of B. caballi in erythrocytes as well as in extracellular parasites (Fig. 4). In the intraerythrocytic stage of merozoites (the ring and subsequent pearshaped forms), the BC134 protein appeared as a dense accumulation in the parasite cytoplasm (Fig. 4a, b, and c), which later blanketed the membrane of ruptured erythrocytes (Fig.  4c), and retained a denser mass or accumulation in the cytoplasm of extracellular merozoites (Fig. 4b and c).
To evaluate the efficacy of the recombinant BC134 protein as a diagnostic antigen, the purified GST-BC134 antigen was subjected to diagnostic ELISA (Fig. 5). All of the sera used were confirmed to be nonreactive to the GST control antigen (data not shown). Positive reactions to the GST-BC134 antigen were detectable in six out of eight B. caballi-infected horse sera at optical densities at 415 nm (OD 415 ) higher than 0.25 (lane 1). However, the antigen showed a cross-reaction or a nonspecific reaction to B. equi-infected horse or healthy horse sera with OD 415 below 0.25 in the ELISA. These results demonstrate that the ELISA with GST-BC134 antigen is able to distinguish between the sera of B. caballi-infected horses and those of B. equi-or noninfected horses at OD 415 of 0.25 and higher.
In the present study, a B. caballi-cDNA expression phage library was screened with serum from a horse experimentally infected with B. caballi, and a novel recombinant BC134 protein of B. caballi was identified as the most immunoreactive antigen. The deduced amino acid sequence of the BC134 gene has a signal peptide sequence at the N-terminal end, suggesting that it might function as a membrane-associated or -secreted protein (21). The complete nucleotide sequence of the BC134 gene was identified, but the native BC134 protein, as well as the synthesized GST-BC134 protein, was found to be about 30 kDa larger than expected. This discrepancy in molecular masses may be attributed to the presence of highly charged amino acid residues and to the low pI of the predicted protein, which may have disrupted the binding of sodium dodecyl sulfate to the protein (6). Similar findings have been reported for Ag322 of Plasmodium falciparum (15), the 200-kDa protein of Babesia bigemina (23), and the Be82 protein of B. equi (6). Despite the discrepant molecular sizes, the mouse immune serum against the recombinant BC134 protein specifically recognized the native protein in the lysate of B. caballi-infected erythrocytes, and the recombinant BC134 protein was reactive with the B. caballi-infected horse serum. These findings indicate that the BC134 protein is a specific antigen of B. caballi and also an immunoreactive component applicable for the development of diagnostic ELISA against the infection.
In the indirect fluorescent antibody test, the native BC134 protein was observed in the cytoplasm and/or membrane of the infected erythrocytes as well as in the cytoplasm at all developmental stages of the intracellular parasites (the ring and subsequent pear-shaped forms) and free merozoites. The BC134 protein also seemed to have interacted with the cytoskeleton and/or the membrane of erythrocytes at the later phase of parasite development, which is associated with merozoite maturation and release and host cell rupture, similarly to the B. bovis rhoptry-associated protein 1 (27).
Although we already developed a diagnostic ELISA for B. caballi infection using a recombinant BC48 antigen (9, 10), we consider that further study is necessary to search for other immunodominant antigens applicable to diagnostic ELISA in order to cover all of the sera infected with various types of field strains. In the present study, an ELISA using a newly identified recombinant BC134 protein was developed and proved to be highly specific for the detection of B. caballi-specific equine antibodies at OD 415 higher than 0.25. Although the diagnostic ELISA that we have developed detected B. caballi infection clearly, in the ELISA using the recombinant BC134 protein two B. caballi-infected serum samples tested negative. Although the number of serum samples was not enough to examine whether the cross-reactivity or nonspecific reactivity of the GST-BC134 protein with B. equi-infected sera and healthy horse sera was authentic, there is a possibility that B. equi parasites may share a region bearing antigenicity that is similar to the recombinant BC134 protein. Furthermore, there is nonspecific reactivity with uninfected sera. Therefore, further studies of ELISA with the recombinant BC134 antigen are thus necessary and should use a large number of horse sera in order to ascertain which region of the BC134 antigen is specific to the serological diagnosis of B. caballi infection in the field.
Nucleotide sequence accession number. Nucleotide sequence data reported in this paper are available in the Gen-Bank, EMBL, and DDBJ databases under accession number AB095267.