To verify N. caninum immunostaining, IFAT was performed with mouse sera collected at 45 d.a.i. as previously described [29]. Slides Dasatinib containing formolized tachyzoites were incubated with serum samples diluted 1:50, and then with FITC-labeled goat anti-mouse IgG (1:50; Sigma). Slides were overlaid with buffered glycerol and examined in fluorescence microscope (EVOS, Advanced Microscopy Group, Inc., Mill Creek, WA). Two weeks after the last immunization (45 d.a.i.), three mice from each group were euthanized and
their spleens were aseptically removed for cell culture and cytokine production assay. Mouse spleens were dissociated in RPMI medium and cell suspensions were washed in medium, treated with lysis buffer (0.16 M NH4Cl and 0.17 M Tris–HCl, pH 7.5), washed again and resuspended in complete RPMI medium containing 10% CFS. Viable cells (2 × 105 cells/200 μl/well) were cultured in triplicate in
96-well plates in the presence of antigen (NLA, 10 μg/ml), mitogen (Concanavalin A – ConA, 2.5 μg/ml) or medium alone and incubated at 37 °C in 5% CO2. After 48 h, cell-free supernatants were collected and stored at −70 °C for cytokine quantification. IL-10 and IFN-γ measurements were carried out by sandwich ELISAs according to manufacturer’s selleck inhibitor instructions (R&D Systems, Minneapolis, MN). The limit of detection for each assay was 31 pg/ml and intra-assay variation coefficients were below 15%. After 30 days of the last immunization (60 d.a.i.), the remaining animals of each group (10 per group) were challenged intraperitoneally (200 μl/mouse) with 2 × 107 low-passage Nc-1 tachyzoites. Animals were observed daily for clinical signs through morbidity scores, body weight changes
and mortality during 30 days post-infection (d.p.i.). Morbidity scores were calculated as described elsewhere [32], with minor modifications as follows: sleek/glossy coat, bright and active (score 0); ruffled coat (score 1); hunched, tottering gait, starry stiff coat (score 2), reluctance to move (score 3). Results were expressed as the mean of the scores given daily to each animal for each group. After 30 days of challenge, surviving animals were euthanized and blood PAK6 samples and brain tissues were collected. Serum samples were tested for N. caninum serology and brain tissues were sliced longitudinally, being half of them stored at −70 °C for polymerase chain reaction (PCR) assay. The remaining tissue was fixed in 10% buffered formalin, embedded in paraffin and routinely processed for immunohistochemical and histological assays. Brain parasite load was determined by quantitative real-time PCR as previously described [29], using primer pairs (sense 3′ GCTGAACACCGTATGTCGTAAA-5′; antisense 3′-AGAGGAATGCCACATAGAAGC-5′) to detect the N. caninum Nc-5 sequence through SYBR green detection system (Invitrogen, San Francisco, CA). DNA extraction was performed from 20 mg of murine brain tissues (Genomic DNA kit, Promega Co.