INTRODUCTION

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Equine piroplasmosis, caused by Babesia equi, is an economically important tick-borne protozoan disease of horses in many regions of the world (6, 23). The disease is characterized by fever, anemia, and icterus (16). Complete clearance or prevention of B. equi infection by drug therapy or vaccination is not currently possible (5). The parasite is usually demonstrated during the acute phase of the infection by Giemsa-stained blood smears. However, horses that survive the primary infection are lifelong carriers of B. equi (11), and it is much more difficult to demonstrate the presence of parasites in these carrier animals, thus necessitating the use of a more sensitive test to detect carrier animals.

Equine piroplasmosis is becoming a disease of international importance and is a major constraint in the movement of horses for equestrian events such as the Olympic Games. A reliable, sensitive, and specific serological test for equine piroplasmosis is therefore very important not only for disease control but also in prevention of introduction of the parasites into countries regarded as areas free of the disease. The complement fixation test (CFT) and indirect fluorescent antibody test (IFAT) have been used for detecting B. equi and Babesia caballi antibodies in infected horses (7, 10, 18, 22, 25-27). Recently, a competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA) was established for detecting B. equi antibodies (13, 14, 24), and the sensitivity was compared with that of CFT, with sera from a number of different countries. The CFT and CI-ELISA agreed in 94% of the serum samples tested. However, discrepancies in five samples could not be definitely resolved, thus indicating the need for further improvement of the test.

A monoclonal antibody (MAb) with defined specificity and having functional inhibitory activity towards parasite development could be used to purify the antigens which might be promising vaccine candidates recognized by those antibodies. The aim of this study was to produce a MAb against B. equi and to characterize the location of the protein it detects and its effect on parasite growth in vitro. A further goal of the present study was to isolate the species-specific protein of B. equi bound to the MAb and to examine its potential application as an antigen in a serodiagnostic method.

	MATERIALS AND METHODS

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B. equi isolate. The USDA strain of B. equi adapted for in vitro culture was used as a source of antigenic material for production of MAbs. For preparation of free merozoites, B. equi cultures (parasitemia, >15%) from a six-well plate were transferred to 25-cm² culture flasks. Cultures in flasks were deprived of CO₂ for 4 to 6 h at room temperature, decanted into 15-ml centrifuge tubes, and sedimented at 400 × g for 20 min at 4°C to pellet erythrocytes. The top 70% of the supernatant was removed and centrifuged at 750 × g for 10 min at 4°C. The final supernatant was again centrifuged at high speed, 12,000 × g, for 20 min at 4°C, to collect free merozoites. After the last centrifugation, a smear was made from the pellet and no intact erythrocytes were observed. Viability of the free merozoites was checked by in vitro culture. These live, free merozoites of B. equi were used for the immunization of mice and for antigen for IFAT for the screening of hybridomas.

Production and purification of MAbs. Approximately 10⁷ free merozoites were inoculated intramuscularly into BALB/c mice with an equal volume of Freund complete adjuvant on day 0 and with Freund incomplete adjuvant on days 14 and 28. The mouse with the highest antibody titer was selected to be the spleen donor by IFAT with acetone-fixed B. equi parasites. Spleen cells (1.25 × 10⁸ cells) were fused with 10⁷ Sp2 myeloma cells, and hybridomas were cultured in S-Clone SF-B medium (Sanko Junyaku, Tokyo, Japan) supplemented with hypoxanthine, aminopterin, and thymidine (Boehringer Mannheim GmbH, Mannheim, Germany) in 96-well plates. Two to three weeks after fusion, screening for antibody-producing hybridomas was performed with undiluted supernatants by IFAT with acetone-fixed B. equi-infected erythrocytes. Hybridoma-producing MAb BEG3 was identified and cloned three times by limiting dilution. Ascitic fluid was produced by using Freund incomplete adjuvant-primed BALB/c mice (20). Purification of the MAb was performed by 50% ammonium sulfate precipitation and also with the Econo-Pac protein A kit (Bio-Rad Laboratories, Richmond, Calif.) as well as control normal mouse immunoglobulin G (IgG). Class and subclass of the purified MAb were determined with a mouse MAb isotyping kit (Amersham International plc, Little Chalfont, United Kingdom). To examine the specificity of the MAb, BEG3 was also screened against B. caballi, Babesia ovata, and Babesia microti acetone-fixed infected erythrocyte antigens in IFAT. B. caballi and B. ovata were prepared from in vitro culture (3, 12), and B. microti was prepared from infected mice.

Immune sera. Serum samples (IFAT titer, 1:320) from a horse experimentally infected with B. equi (USDA strain) were obtained from the Equine Research Institute, The Japan Racing Association. Serum samples collected from eight Babesia-free horses in Japan and four negative serum samples (IFAT titer, <1:80) from Mongolian horses were used as negative controls in the ELISA. An additional 39 serum samples from Mongolian horses, which had previously been shown to be positive for anti-B. equi antibodies in IFAT (2), were also tested by ELISA. All serum samples were stored at 30°C until use.

IFAT. IFAT of acetone-fixed B. equi was used for screening of hybridomas as previously reported (2). The reactivity of the MAb was also examined by IFAT with viable free merozoites as described before (13). Bound murine antibodies were detected with fluorescein-conjugated goat anti-mouse IgG (Leinco Technologies, Inc.) diluted 1:100 in 0.01% Evans blue-phosphate-buffered saline (PBS; pH 7.4). The location of the antigen reacting with the MAb was further examined in thin films of B. equi-infected erythrocytes and observed with a confocal laser microscope (SARASTRO 2000; Molecular Dynamics Co., Sunnyvale, Calif.).

Immunoelectron microscopy. B. equi-infected erythrocytes were propagated in vitro as described above. B. equi-infected erythrocytes were collected when parasitemia was 15.5% and were washed three times in PBS. Infected erythrocytes were fixed in 4% paraformaldehyde in PBS for 2 h. After three washings in PBS, infected erythrocytes were mounted on glass slides coated with poly-L-lysine (Sigma, St. Louis, Mo.), incubated with MAb BEG3 for 24 h at 4°C, and then incubated with biotinylated goat anti-mouse IgG and then avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, Calif.) for 1 h at room temperature. The antigen-antibody reaction sites were visualized by incubating erythrocytes with diaminobenzidine tetrahydrochloride (DAB; Nakarai Chemicals, Kyoto, Japan)-0.01% hydrogen peroxide in 25 mM Tris-HCl buffer (pH 7.6). The immunostained erythrocytes were postfixed with 1% osmium tetroxide in PBS, briefly dehydrated with an alcohol series, and embedded in Epon 812. The ultrathin sections were counterstained only with uranyl acetate and examined with an electron microscope. The specificity of immunohistochemical staining was confirmed by replacing the MAb with normal mouse IgG.

SDS-PAGE and immunoblots. Crude antigen of B. equi was prepared from infected erythrocytes as described before (13), and the protein concentration of crude antigen was determined according to the method of Bradford (4). Extracted crude antigen was solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (final concentrations, 50 mM Tris [pH 6.8], 1% [wt/vol] SDS, 8% [vol/vol] glycerol, 4% 2-mercaptoethanol, and 0.001% bromophenol blue) and was electrophoresed in 15% polyacrylamide gels according to the method of Laemmli (17), and then the sample proteins were electrophoretically transferred from the gels onto polyvinylidene difluoride membranes for 60 min at 14 V with a semidry transfer cell (Bio-Rad Laboratories). The membrane strips were cut, and nonspecific sites of the membrane were blocked with 5% skim milk-PBS for 60 min at room temperature. Membranes were then washed three times with 0.5% skim milk-PBS containing 0.05% Tween 20 (SM-PBST) and incubated with the MAb or purified normal mouse IgG diluted in 1% SM-PBST for 90 min at room temperature. Bound antibody was detected by incubation with peroxidase-conjugated goat anti-mouse IgG (1:1,000) (Cappel Research Products, Durham, N.C.) in 0.5% SM-PBST for 60 min. After being washed, the strips were treated with freshly prepared substrate solution containing 5.3 mM DAB and 0.01% (vol/vol) hydrogen peroxide in 0.1 M Tris-HCl (pH 7.4).

Effect of the MAb on the growth of B. equi. To evaluate the inhibitory effect of the MAb on the growth of B. equi parasites in vitro, infected erythrocytes from B. equi cultures (with 12% parasitemia at 48 h) were diluted to 0.5 to 1.0% with normal horse erythrocytes. The MAb was added to culture medium (M199 supplemented with 40% horse serum) to a final concentration of 100, 500, or 1,000 µg/ml. Purified mouse IgG was used as a control, each sample was tested in triplicate, and parasitemia was monitored daily with Giemsa-stained smears.

Partial purification of B. equi antigen. High-pressure liquid chromatography (HPLC) was carried out with a TSKgel DEAE-5PW column to partially purify B. equi antigen. One milliliter of crude antigen, prepared as described above, was solubilized in an equal volume of 25 mM Tris-HCl-1 mM EDTA, pH 8.0, and the total 2 ml of antigen solution was applied to a TSKgel DEAE-5PW column (Tosoh, Tokyo, Japan). Antigens were eluted stepwise with 1 M NaCl. Eluted proteins were separated by SDS-PAGE, evaluated by being stained with silver stain, and transferred to nitrocellulose membranes for immunoblotting with MAb BEG3.

ELISA for detection of antibody. For the detection of antibodies in horse serum by ELISA, 96-well microtiter plates (Nunc S/A, Roskilde, Denmark) were coated with 100 µl of partially purified antigen (40 µg/ml in 0.1 M carbonate-bicarbonate buffer, pH 9.6) overnight at 4°C, washed three times with PBS containing 0.05% Tween 20 (PBST), and blocked with 3% bovine serum albumin-PBS. The negative or positive controls and sample sera diluted 1:80 were added to each well in volumes of 0.1 ml. The plates were then incubated for 2 h at room temperature and washed as described above. Goat anti-horse IgG horseradish peroxidase conjugate (Cappel Research Products) diluted 1:1,000 with PBST was added to each well and again incubated for 1 h at room temperature. After washing, 0.1 ml of substrate (0.2 mM amino-di-[3-ethylbenthiazoline]sulfonic acid) was used for color development. The optical density (OD) was read at 415 nm on an MTP-120 (Corona Electric, Tokyo, Japan) ELISA plate reader. A serum sample was considered positive for antibody to B. equi if it showed an OD higher than the mean plus 3 standard deviations of negative serum samples.

	RESULTS

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Production of MAb binding to B. equi. Nine hybridomas producing antibodies which bound to parasites, but not to the erythrocytes or uninfected erythrocytes, were identified by screening with IFAT. The surface reactivity of one MAb (BEG3) was demonstrated by its binding to live merozoites. This MAb, BEG3, isotyped as IgG1(), was selected for further study. This MAb also did not react with acetone-fixed B. caballi-, B. ovata-, or B. microti-infected erythrocytes in IFAT (Table 1). The fluorescence of the MAb to B. equi-infected erythrocytes was observed not only on the single ring form (Fig. 1a) but also on the round form with cytoplasm (Fig. 1b), double ring-like forms (Fig. 1c), and the Maltese cross form (Fig. 1d). Intensity analysis with a confocal laser microscope indicated that MAb BEG3 reacted with the surface of the parasite (Fig. 2).

View larger version (94K): [in this window] [in a new window]   FIG. 1.   MAb BEG3 binding to different forms of B. equi as observed with a confocal laser microscope. (a) Single ring form; (b) round form with cytoplasm; (c) double ring-like form; (d) Maltese cross form. Bar, 5 µm.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   Intensity of fluorescence on round form (trophozoite) of B. equi after reaction with the MAb. (a) Intensity of fluorescence was scanned along the yellow line. Bar, 5 µm. (b) Two peaks were observed in the scanning pattern of intensity.

Immunoelectron microscopy of B. equi-infected erythrocytes. Figure 3 is an electron micrograph of a B. equi-infected erythrocyte incubated with MAb BEG3. The MAb binding was visualized by the presence of DAB immunoreaction deposits with goat anti-mouse IgG. Immunoreaction deposits were observed along the surface of the B. equi parasite in the infected erythrocyte (Fig. 3a). No immunoreaction deposits were seen in the cytoplasm of the parasite or in the membrane or cytoplasm of infected erythrocytes. Uninfected erythrocytes in the preparation did not have immunoreaction deposits (Fig. 3b), nor did infected erythrocytes incubated with control mouse IgG have any deposits bound to them (data not shown).

View larger version (62K): [in this window] [in a new window]   FIG. 3.   Electron micrograph of MAb BEG3 binding to the B. equi parasite. (a) The immunoreaction deposits (arrows) were observed on the surface of the B. equi parasite. (b) No immunoreaction deposits were observed in the cytoplasm of uninfected erythrocytes. Bar, 0.2 µm.

Immunoblots of B. equi-infected erythrocytes. Immunoblotting of B. equi-infected erythrocyte lysates was performed to identify antigens recognized by MAb BEG3. The MAb recognized one major protein band of B. equi of approximately 19 kDa, but it did not react with antigens from erythrocytes infected with B. caballi, or uninfected erythrocyte antigens, in Western blot analysis (Fig. 4). The 19-kDa protein was not detected with normal mouse IgG.

View larger version (65K): [in this window] [in a new window]   FIG. 4.   Recognition of 19-kDa B. equi parasite antigen by MAb BEG3. Lanes: 1, B. caballi-infected erythrocyte lysate; 2, B. equi-infected erythrocyte lysate; 3, noninfected erythrocyte lysate. MW, molecular weight markers (in thousands).

Effect of the MAb on growth of B. equi. The MAb was added to culture medium at different concentrations to examine its effect on the growth of B. equi. No effect of the MAb on parasite growth could be observed for the first 2 days. However, significant inhibitory effects were observed at concentrations of 500 and 1,000 µg/ml on day 5 (Table 2). The inhibitory activity of the MAb was associated with morphological damage of the parasite (Fig. 5). About 38% of infected erythrocytes were visibly damaged by the presence of the MAb. The affected parasites were observed to be vacuolated and larger than normal. Many dot forms of the parasite in the cultures with the MAb were also demonstrated.

View larger version (82K): [in this window] [in a new window]   FIG. 5.   Morphological changes of B. equi parasites caused by MAb BEG3. (a) Parasites incubated with normal mouse IgG; (b) parasites incubated with MAb BEG3. Larger and vacuolated parasites in erythrocytes were observed. Bar, 5 µm.

Partial purification of B. equi antigen by means of HPLC. HPLC was performed to isolate the antigen which would react with MAb BEG3. Crude antigens were eluted into 25 fractions with 1 M NaCl. Although crude antigen contained several major proteins, only two proteins were detected in fraction 9 obtained by HPLC of immune serum (Fig. 6, lanes 1 and 2). The 19-kDa protein in this fraction was confirmed to react with the MAb (Fig. 6, lane 4). Therefore, fraction 9, which contained the 19-kDa protein, was collected as partially purified antigen and used for ELISA to detect antibody to B. equi.

View larger version (63K): [in this window] [in a new window]   FIG. 6.   Immunoblot analysis of crude and partially purified antigen (fraction 9) obtained by HPLC. Crude (lanes 1 and 3) and partially purified (lanes 2 and 4) antigens were probed with anti-B. equi immune serum (lanes 1 and 2) and MAb BEG3 (lanes 3 and 4). MW, molecular weight markers (in thousands).

ELISA with partially purified antigen. ELISA with partially purified antigen was conducted on 12 negative control serum samples and 39 horse serum samples from Mongolia which had previously been shown to be positive for antibody to B. equi in IFAT. All 12 control samples showed low ODs. Thirty-eight of these serum samples were positive, while one was negative in the ELISA (Fig. 7). The agreement of IFAT and ELISA with partially purified antigens was 97.4% for detection of antibody to B. equi.

View larger version (19K): [in this window] [in a new window]   FIG. 7.   Correlation of IFAT titers and ELISA values. Titer of <80 is negative in IFAT. Circles show values of individual horse sera; squares show mean values with standard deviations of horse sera at different IFA titers. Serum samples showing OD values more than those of the means plus 3 standard deviations of negative serum samples (dotted line) were considered positive by ELISA.

	DISCUSSION

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The primary aims of the present study were to produce a specific MAb against B. equi and to characterize the MAb with regard to the location of the antigen and effect on parasite growth. The MAb (BEG3) was produced against B. equi parasites, was species specific against B. equi, and did not react to B. caballi. The production of MAbs against B. equi was also described by Knowles et al. (13, 14), Ali et al. (1), and Brüning et al. (6). The surface location of antigen associated with the epitope recognized by MAb BEG3 was demonstrated by confocal laser microscopy and binding of the MAb to live merozoites. Moreover, immunoelectron microscopy confirmed that the MAb reacted directly with the surface of the B. equi parasites and did not react with other organelles of parasites or infected erythrocytes. These results suggested that the antigen recognized by the MAb was species specific to B. equi and was located on the parasite surface membrane. It is also of interest to note that the MAb bound to all stages of the parasite in infected erythrocytes, because the antigen recognized by the MAb was always expressed on the parasite at the stage at which it infected erythrocytes and to which the immune system of the host was exposed. Therefore, this antigen might be a good target for detecting antigen or antibody to it for the diagnosis of B. equi infection.

Immunoblot analysis demonstrated that the MAb recognized a 19-kDa antigen in lysate of erythrocytes infected with B. equi but did not react with any B. caballi antigen. The 19-kDa protein recognized by the MAb in this study was also recognized by immune serum from a horse experimentally infected with B. equi. Ali et al. (1) also reported a surface membrane protein with similar molecular weight in B. equi. Analysis of the genes encoding these antigens (the 19-kDa protein of the present study and the 18-kDa protein described by Ali et al.) may be of interest regarding their functions and differences.

The addition of the MAb to in vitro cultures of B. equi significantly inhibited parasite growth. Perrin et al. (21), Winger et al. (28), and Figueroa and Buening (9) reported the production of MAbs which have growth-inhibitory activity in vitro against protozoan parasites such as Plasmodium falciparum, Babesia divergens, and Babesia bigemina. The inhibitory effect of MAb BEG3 against parasite growth was associated with morphological change in the parasite. However, the mechanism of the inhibitory effect remains unknown. Perrin et al. (21) hypothesized that (i) the MAb blocks the process of parasite invasion of the erythrocytes by interfering with the parasite ligand-erythrocyte receptor, (ii) the MAb binds to the parasite antigen at the surface of infected erythrocytes and somehow modifies the parasite's metabolism, and (iii) the MAb would reach the intracellular parasites at the last stage of development in infected erythrocytes, due to increased permeability. The first and third possibilities cannot be excluded, because the protein recognized by the MAb was observed in all stages of the parasite in erythrocytes. The second possibility may be less likely in the present study, because the MAb did not bind the surface of infected erythrocytes. Further studies are necessary to examine the role of the protein recognized by the MAb in the immune responses of the host, since antigens on the merozoite membranes or on erythrocyte membranes seem to be good targets for the protective immune response in Babesia infections (19).

The secondary aims of the present study were to isolate the antigen with the MAb and to examine whether the antigen recognized by the MAb can be used as a suitable antigen for serological tests. Although several serological tests have been developed for the detection of antibodies against Babesia infection (8, 25, 27), the contamination of the erythrocyte component has hampered the development of a specific and sensitive test. Knowles et al. (13-15) developed a CI-ELISA for horse babesiosis, with a MAb raised against an epitope found on several proteins, including a 34-kDa surface protein of B. equi. They reported that CI-ELISA proved to be more sensitive than CFT. However, Brüning et al. (6) also produced a MAb which recognized the 34-kDa antigen of B. equi and found that CI-ELISA with this MAb was less sensitive than CFT. In the present study, a 19-kDa B. equi antigen recognized by the MAb was partially purified by means of HPLC, and a clear difference between negative and positive serum samples was observed when ELISA for B. equi was tested on serum samples from field-infected horses in Mongolia with the partially purified antigen. These results suggested its potential usefulness for more sensitive and specific identification of B. equi infection. Since another protein was detected in partially purified antigen from immune serum, further refinement of the test with a recombinant 19-kDa antigen instead of partially purified antigen would make the ELISA a favorable replacement for the CFT, as suggested by Knowles et al. (15) and Schelp et al. (24). For this purpose, cloning of the gene encoding this protein and DNA sequencing are under way.