BALB/c mice, 6-8 weeks old, purchased from SPF animal center (Koatech Company, Kyonggi-do, Republic of Korea) were used. Korean isolate-1 (KI-1) of T. gondii has been maintained by intraperitoneal injection of BALB/c mice [8]. Mice were starved for food for 1 day and then orally inoculated with 10⁵ or 10⁶ tachyzoites in 0.2 ml PBS using a narrow silicon tube inserted into the duodenum passing through the stomach. Control mice received 0.2 ml PBS without tachyzoites. For exact intraduodenal injection, it was confirmed that the whole length of silicon tube was properly intubated into the duodenum. Possibility for the tip of the tube remaining in the stomach could be ruled out by repeated preliminary practices. The introduction of this silicon tube was performed carefully to avoid any damage to the esophagus or stomach.

Each experimental group consisted of 6 mice. To confirm infection, 3 infected mice per group, were sacrificed under ethyl ether anesthesia, and the brain, heart, liver, lung, stomach, duodenum, jejunum, kidney, spleen, and mesenteric lymph nodes (MLN) were collected on days 2, 4, 6, and 8 after infection.

To examine the infection status of visceral organs after intraduodenal inoculation, the brain, heart, liver, lung, stomach, duodenum, jejunum, ileum, kidney, and spleen of mice were removed. These organs were fixed in 10% neutral formalin and embedded in paraffin. The embedded sections were cut into a thickness of 4 µm, stained with Giemsa, and examined for tachyzoites under a light microscope.

To genetically identify the presence of KI-1 tachyzoites in visceral organs, PCR analysis was performed to detect Toxoplasma B1 gene as a diagnostic gene [13]. DNA extraction was performed using the DNeasy®Tissue kit (Qiagen, Hilden, Germany). Forward- and reverse-primers were: 5'-AAA AAT GTG GGA ATG AAA GAG-3' and 5'-ACG AAT CAA CGG AAC TGT AAT-3', respectively. Amplification of the B1 gene was completed in the following conditions: 1 cycle of 5 min at 95℃ for initial denaturation followed by 30 cycles of 1 min at 95℃, 1 min at 60℃, and 3 min at 74℃. Amplification was performed using a DNA thermal cycler (GeneAmp™, PCR system 9600, PE Applied Biosystems, Foster City, California, USA). PCR amplification products were examined in 1.5% agarose gels and confirmed by staining with ethidium bromide and visualized under UV.

Six-week-old female BALB/c mice were inoculated with 10⁵ or 10⁶ T. gondii KI-1 tachyzoites into the duodenum using a silicon tube. The survival time of mice was recorded everyday post-infection (PI).

T. gondii KI-1 tachyzoites were maintained by serial passage in BALB/c mice. Tachyzoites were inoculated intraperitoneally and harvested by peritoneal lavage on day 5 or 6 PI. The peritoneal exudate was washed with sterile PBS and pelleted by centrifugation for 10 min at 3,000 rpm [14]. Tachyzoites were purified using 40% Percoll (Pharmacia Biotech, Uppsala, Sweden) in PBS. The purified tachyzoites were disrupted by 5 cycles of freezing at -70℃ and thawing at room temperature. The homogenates were centrifuged at 12,000 rpm at 4℃ for 30 min, and the supernatant was used as the antigen after filtration through a 0.45 µm membrane (Advantec MFS Inc., Pleasanton, California, USA).

The spleen and MLN were removed aseptically from the infected and uninfected control mice and kept in cold RPMI 1640 (Gibco BRL, Grand Island, New York, USA) containing 10% heat-inactivated FBS (Gibco BRL), 10 mM HEPES, 2 mM L-glutamine, 2 mM sodium bicarbonate, 5×10^-5 M 2-mercaptoethanol (2-ME), and antibiotics (100 IU/ml of penicillin G and 100 µg/ml of streptomycin) (Gibco BRL). Tissues were washed with RPMI 1640 containing 2% FBS and prepared to a single cell suspension [15]. After RBC lysis using a hypotonic buffer containing NH₄Cl, the single cell suspensions of MLN and spleen were washed 3 times by centrifugation. Finally, cells were resuspended in complete RPMI 1640 supplemented with 10% FBS, and viable cells were counted by trypan blue exclusion test. Concanavalin A (con A) (Sigma-Aldrich, St. Louis, Missouri, USA) was added at a predetermined final concentration of 5 µg/ml.

Single cell suspensions of the spleen and MLN were prepared as described above. Cell suspensions were adjusted to 1×10⁷ cells/ml with complete RPMI, and 100 µl (1×10⁶ cells) of the cell suspension was then used for FACS staining. To avoid non-specific binding of fluorescein-conjugated antibody, Fc blocker (anti-CD16/32 [Fcγ III/II receptor]) (eBioscience, Boston, Massachusetts, USA) was used before labeling specific antibodies with anti-mouse CD3, CD4, CD8, DX5, or F4/80. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (eBioscience), phycoerythrin (PE)-conjugated anti-CD4 (eBioscience), PE-conjugated anti-DX5 (eBioscience), cyanin 5-phycoerythrin (Cy5-PE)-conjugated anti-CD8 (eBioscience), and Cy5-PE-conjugated anti-F4/80 (eBioscience). After staining, the cells were washed with 2% FBS/HBSS and then fixed with 2% paraformaldehyde. Phenotypic analysis of CD4⁺- or CD8⁺-T cells, NK cells, and macrophages was performed by FACScan flow cytometer (Becton-Dickinson, Rutherford, New Jersey, USA). Gates were set to exclude nonviable cells and adjusted to detect specifically stained cells. Data are presented by the percentage of positive cells.

The spleen and MLN were removed aseptically from infected mice at days 2, 4, and 6 PI. Single cell suspensions were prepared and adjusted to 1×10⁷ cells/ml with complete RPMI, and 40 µl (4×10⁵ cells) was used for the cell proliferation assay. The assays were carried out in a 96-well flat-bottomed plate (Nalge Nunc Int., New York, USA) in triplicate in 0.2 ml volumes with following conditions: 4×10⁵ cells per well in 0.2 ml of culture media alone or in media containing 100 µg/ml Toxoplasma KI-1 lysate antigen (TLA) or 5 µg/ml con A. Plates were incubated for 72 hr in 5% CO₂ at 37℃ and viable cells were converted to a colored product by XTT solution, i.e., 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, a mixture of 1 µl phenazine methosulphate (PMS; electron-coupling reagent) and 50 µl of the XTT labeling reagent were added to each well and incubated at 37℃ during the last 4 hr of culture. Absorbance was read at 450 nm with a reference wavelength of 650 nm (ELx808, BioTek Instruments, Winooski, Vermont, USA). Average absorbance values were obtained from the net absorbance values, i.e., absorbance of seeded wells minus absorbance of blank wells.

The spleen was removed at days 0, 6, and 9 PI to examine cytokine signals. The total RNA was extracted by homogenization of the spleen in TRIzol Reagent (Invitrogen, Calsbad, California, USA) according to the manufacturer's protocol. After adding chloroform (20% of the total volume), the mixture was shaken for 15 sec, incubated at 15-30℃ for 2-3 min, and then centrifuged at 12,000 g for 15 min at 4℃. An equal volume of isoprophyl alcohol was added, and the mixture was incubated at room temperature for 10 min. After centrifugation at 12,000 g for 10 min at 4℃, the pellet was washed with 75% ethanol in diethyl pyrocarbonate (DEPC) water, air-dried and dissolved in an appropriate volume of DEPC water for cDNA preparation.

Reverse transcription for the first strand DNA was performed using the Superscript™III First-Strand Synthesis system for RT-PCR (Invitrogen). Briefly, total RNA (2 µg) was added to reaction mixture containing 10 mM dNTPs and Oligo (dT), incubated at 65℃ for 5 min, and then placed on ice for 1 min. It was then added to the mixture containing SuperScript™III RT, and incubated at 50℃ for 50 min and 85℃ for 5 min. The cDNA was amplified with each cytokine primer by GeneAmp PCR system 9600 (PE Applied Biosystems, Foster City, California, USA). The primers for mouse cytokines (IL-10, IFN-γ, TNF-α, and 18s RNA) were prepared: IL-10; sense: 5'-CGG GAA GAC AAT AAC TG-3'; anti-sense: 5'-CAT TTC CGA TAA GGC TTG G-3'; IFN-γ; sense: 5'-AAC GCT ACA CAC TGC ATC TTG G-3'; anti-sense: 5'-GAC TTC AAA GAG TCT GAG G-3'; TNF-α; sense: 5'-GAT CTC AAA GAC AAC CAA CTA GTG-3'; anti-sense: 5'-CTC CAG CTG GAA GAC TCC TCC CAG-3'; 18s RNA sense: 5'-CGC GGT TCT ATT TTG TTG GT-3'; anti-sense: 5'-AGT CGG CAT CGT TTA TGG TC-3'. There were 35 PCR cycles for all cytokines, except for TNF-α which had 32 cycles. Annealing temperatures were set for IL-10 (48℃), IFN-γ (48℃), TNF-α (55℃), and 18s rRNA (60℃). The amplified PCR products were electrophoresed in 2% agarose gel and photographed under UV light after ethidium bromide stain. The density of each band was analyzed using the densitometrical analysis program, TINA (Raytest, Straubenhardt, Germany). The optical densities of the bands were normalized by the housekeeping gene, 18s rRNA, and the quantity of naive mRNA was calculated. When the value of IFN-γ/18s RNA of uninfected mice was "1" (standard value), the values of IFN-γ/18s RNA of infected mice were presented as a fold change.

All data are expressed as the mean±standard deviation. The statistical significance of differences between experimental groups was analyzed by the Mann Whitney U test; P<0.05 was considered to be statistically significant.

The virulence of T. gondii KI-1 tachyzoites was estimated by the survival of intraduodenally infected mice (Fig. 1). At the inoculum dose of 10⁵ tachyzoites, 4 (66.7%) of 6 infected mice lived until day 10 PI, and all mice died by day 11 PI. At the dose of 10⁶ tachyzoites, 4 (66.7%) of 6 infected mice lived until day 8 PI, and all mice died by day 9 PI (Fig. 1).

The presence of KI-1 tachyzoites in organs and tissues of infected mice was determined by light microscopy of histopathological slides (Table 1; Fig. 2) and by PCR using the B1 gene primer (Fig. 3). At the inoculum dose of 10⁵ tachyzoites, tissue samples stained with Giemsa revealed T. gondii tachyzoites in the lungs and spleen at day 6 PI (Table 1; Fig. 2). In mice inoculated with 10⁶ tachyzoites, tachyzoites were seen in sections of the liver and spleen at day 8 PI (Table 1). However, PCR appeared to be more sensitive than histopathology in detecting tachyzoites. In mice inoculated with 10⁵ tachyzoites, the B1 gene was detected in the liver, lungs, stomach, kidney, MLN, and spleen on day 6 or 8 PI (Table 1). In mice inoculated with 10⁶ tachyzoites, the B1 gene was detected in the duodenum and ileum as early as day 2 PI, and they continued to be detected in the duodenum on days 4, 6, and 8 PI (Table 1). The B1 gene was also expressed in the liver, jejunum, kidney, MLN, and spleen at day 8 PI. In both inoculum doses, there was no evidence of tachyzoite invasion into the brain in this experimental setting.

When splenocytes and MLN cells were harvested and stimulated with mitogen (con A) or Toxoplasma lysate antigen (TLA), the absorbance of con A-treated splenocytes was high at days 0 and 3 PI, and then gradually decreased until day 9 PI (Fig. 4). The absorbance of TLA-treated splenocytes was low at day 0 PI, increased in the early stage of infection (days 3 and 6 PI), and then decreased in the late stage of infection, with mortality of infected mice (Fig. 4). The proliferative responses of MLN cells were high by stimulation with con A at day 0 PI but became low during days 3-9 PI, suggesting immunosuppression (Fig. 4).

Among the splenocytes and MLN cells from the infected mice, the proportions of NK cells and macrophages did not change significantly throughout the experimental period (Fig. 5). However, in the spleen, the proportions of CD4⁺ T-cells and CD8⁺ T-cells decreased through the experimental period. In the MLN cells, similar decreases of CD4⁺ T-cells were seen. However, CD8⁺ T-cells almost maintained their proportions throughout the experimental period (Fig. 5).

The level of IFN-γ in the spleen of T. gondii KI-1 tachyzoite-infected mice decreased 0.6 fold compared to normal uninfected mice at day 6 PI and increased 1.45 fold at day 9 PI although the difference was not statistically different (P>0.05). The level of TNF-α was similar between the infected and uninfected mice (data not shown). IL-10 decreased 0.57 fold at day 3 PI and returned to the normal value by day 9 PI (Fig. 6).