Tachyzoites of Neospora caninum (Kr2 strain) were propagated in Vero cells maintained in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and 20 µg/ml gentamicin (GIBCO-BRL, Grand Island, NY, USA). Parasites were harvested from their feeder cell cultures as described previously (Hempill and Gottstein, 1996). For purification of tachyzoites, the parasites and host cell debris were washed twice in cold phosphate-buffered saline (PBS, pH 7.2). The pellet obtained was resuspended in 2 ml of cold PBS and passed through a PD-10 column filled with Sephadex G25 M (Amersham Biosciences, Uppsala, Sweden) that had previously been equilibrated with PBS. The eluted, purified parasites were collected by centrifugation at 1,500 × g at 4℃ and resuspended in cold PBS. Purified tachyzoites were checked for viability using Trypan blue staining (0.25% in PBS). Whole lysates of tachyzoites were prepared as described previously (Andrianarivo et al., 1999)

Total RNA was isolated from N. caninum tachyzoites using Trizol reagent (GIBCO-BRL). mRNA was purified by using Oligotex® mRNA purification kit (Qiagen, Valencia, CA, USA) by following the manufacturer's instructions. Single-stranded cDNA was synthesized from isolated mRNA using a cDNA synthesis kit (Boehringer Mannheim GmbH, Mannheim, Germany) and oligo-dT primer. To amplify each gene, PCR was performed using total cDNA and primer sets for each of NcSAG1, NcSRS2, NcDG1, and NcDG2 (Table 1). Amplification reactions were performed using the following conditions; 94℃ for 10 min, 35 amplification cycles (94℃ for 1 min, 55℃ for 1 min, and 72℃ for 2 min), followed by a 72℃ extension for 10 min. Amplified PCR products were analyzed on 1.2% agarose gel, purified from gels with a QIAquick gel extraction kit (Qiagen), and ligated into pCR2.1 vector (Invitrogen, Carlsbad, CA, USA), respectively. Each plasmid was transformed into competent E. coli TOP10 cells (Invitrogen) and the cloned genes were sequenced. Plasmid DNA containing the entire coding region of each gene was digested with BamHI/EcoRI and XhoI, and ligated into pMal-c2x vector (New England BioLab, Beverly, CA, USA) predigested with the same enzymes. The resulting plasmids were transformed into competent E. coli TB1 cells (Amersham Biosciences) and spread on Luria-Bertani (LB) agar plates containing 100 µg of ampicillin per ml. The expressions of recombinant proteins were induced by adding isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 1 mM, and they were purified using an amylase affinity chromatography column (New England BioLab). The maltose-binding protein (MBP) was removed from the purified recombinant proteins using Factor Xa (New England BioLab) according to the manufacturer's instructions. The expressions and purities of the recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The polyclonal antisera directed against the whole lysates of N. caninum tachyzoites (NcWL) were produced by immunizing BALB/c mice with NcWL. Accordingly, mice were immunized intraperitoneally with NcWL (100 µg/mouse) emulsified with Freund's complete adjuvant. After primary immunization, animals were boosted 2 and 4 weeks later with the same antigen emulsified with Freund's incomplete adjuvant. Polyclonal antisera directed against the recombinant proteins, NcSAG1, NcSRS2, NcDG1, or NcDG2, were produced by immunizing 4 BALB/c mice with each purified protein. Mice were immunized intraperitoneally with purified protein (50 µg/mouse) emulsified with Freund's complete adjuvant, respectively. After primary immunization, animals were boosted 2 and 4 weeks later using the same protein emulsified with Freund's incomplete adjuvant. Sera were collected from immunized mice at 6 weeks after initial immunization, and immunoglobulin G was purified using a Protein G affinity column (Amersham Biosciences). Antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) using relevant recombinant proteins as antigens.

To investigate the inhibitory effect of immunized sera on the invasion of N. caninum tachyzoites into host cells, in vitro inhibition assays were performed. Purified N. caninum tachyzoites (10⁵) in PBS were mixed with two-fold stepwise dilutions (from 1:10 to 1:10,000) of each polyclonal antibody. Tachyzoites in PBS containing diluted normal mouse antibodies were used as negative controls. The mixtures were preincubated for 30 min at 37℃ and then added to Vero cells (2 × 10⁵ cells) cultured in 8 well chamber slides. After 1 h incubation, the medium in each chamber was changed with fresh complete medium and incubated for 1 day at 37℃. Subsequently, infected parasites were differentiated by Giemsa staining and counted. Inhibition percent was expressed as [(% of infected cells in the negative control - % of infected cells after antibody treatment)/% of infected cells in the negative control] × 100. Neutralization titer was defined as the reciprocal of the antibody dilution that reduced parasite infection by 50% versus the negative control. All experiments were repeated at least three times.

Gerbils (Meriones unguiculatus, 5-6 weeks of age) were used for the challenge experiments. Uninfected control group animals (n = 23) were injected intraperitoneally with only PBS. The other experimental groups (n = 24 to 40/group) were injected intraperitoneally with NcWL or recombinant proteins. Animals in the first experimental group were injected with NcSAG1, NcSRS2, NcDG1, or NcDG2 at 100 µg/gerbil intraperitoneally, whereas animals in the second group were injected intraperitoneally the recombinant proteins in combinations. Gerbils were boosted with the same inoculums 2 weeks after the first injection. For parasite challenge, gerbils were inoculated intraperitoneally with live N. caninum tachyzoites (10⁵) suspended in PBS, 1 week after boosting (3 weeks after the first injection). The protective efficacies of the vaccinations against infection were determined by comparing survival rates after challenge infection of the control group and each of the experimental groups using a Kruskal-Wallis non-parametric test and a distribution-free multiple comparisons method (Mutapi and Roddam, 2002). Significance was accepted at the p < 0.05 level.

Four major antigens of N. caninum, NcSAG1, NcSRS2, NcDG1, or NcDG2, were expressed in E. coli and purified (Fig. 1A). Each purified recombinant antigen was immunized into mice to produce polyclonal antibodies against the proteins. Antibody reactivities were evaluated by Western blotting using each antibody and recombinant protein. Antibodies reacted specifically with their counterpart proteins and no cross reactivity was found (Fig. 1B). Antibody titers against the relevant antigens were < 1:10,000 by ELISA (data not shown).

In vitro inhibition assays were performed to determine the inhibitory effect of each polyclonal antibody on the invasion of N. caninum tachyzoites into host cells (Fig. 2A). All antibodies had inhibitory activities ranging from 61.2 to 87.8%, as determined by parasite invasion into host cells at a 1:400 dilution. Neutralization titer, the reciprocal of antibody dilution giving 50% reduction of invasion of the parasite into host cells, of the antibodies were; 1:4,000, 1:900, 1:1,700, 1:2,500, and 1:600 for NcWL, NcSAG1, NcSRS2, NcDG1, and NcDG2, respectively (Fig. 2B). These neutralization titers indicate that NcSAG1, NcSRS2, NcDG1, or NcDG2 evoke antibody production that can effectively inhibit the invasion of N. caninum tachyzoites into host cells.

It was difficult to evaluate the protective efficacies of recombinant antigens in mice, because most mice, e.g. BALB/c and C57BL/6, are resistant to N. caninum infection (Long et al., 1998). According to our preliminary study, gerbils are highly sensitive to N. caninum infection and all animals inoculated with 4 × 10⁵ of N. caninum tachyzoites intraperitoneally died within 11 days with an LD₅₀ value of 4 × 10³ (data not shown). Therefore, in this study, we used gerbils as experimental animals to evaluate vaccination efficacy. Vaccination efficacy was evaluated by determining the survival durations and the survival rates of vaccinated gerbil groups after challenge infections. The protective efficacies of vaccinations of single- or multiple-recombinant antigens followed by challenge with N. caninum tachyzoites are summarized in Table 2. When negative controls were treated with N. caninum tachyzoites, 4 of the 23 gerbils (17.4%) survived. In animals vaccinated with NcWL, 17 of the 24 gerbils (70.8%) survived. All groups of gerbils vaccinated with single- or multiple-recombinant antigens also demonstrated meaningful levels of protection against the challenge infections. The survival rates of animals vaccinated with these recombinant antigens varied from 42.5 to 67.5%. The highest survival rate (67.5%) was obtained after vaccination with the recombinant antigens, NcSRS2 and NcDG1 in combination.