5% (w/v). No viable bacteria could be obtained when plating the inactivated culture on TSA medium. The animal experiments were carried out according to the International Guiding Principles for Biomedical Research Involving Animals – 1985. Sixty 4-week-old female Selleckchem BTK inhibitor Balb/c mice (Hubei CDC, Wuhan, China) were divided into three groups of 20 each. A 50-μg aliquot of HP0245EC, dissolved in 200 μL

PBS and absorbed to a equal volume of aluminum hydroxide [Al(OH)3] gel adjuvant (Wuhan Chopper Biology Co. Ltd, Wuhan, China), was applied to immunize mice in group 1. Mice in group 2 were immunized with autogenous SS2 bacterin. The inactivated bacterial culture was concentrated fivefold, and 200 μL of the bacterin mixed with Al(OH)3 gel was injected to immunize mice. PBS/Al(OH)3 gel was used as the control for immunized mice in the third group. Mice were immunized twice at 2-week intervals by intraperitoneal injection. On the seventh day after the booster

immunization, sera were obtained from each group by tail vein bleeding. Ten mice of each group were intraperitoneally inoculated NSC 683864 mouse with 3 × 109 CFU of log-phase SC-19, and the remaining 10 mice were challenged with 7.5 × 109 CFU in 0.4 mL PBS. All mice were observed for a week for morbidity and mortality. The antibody titers were determined by ELISA as described before (Zhang et al., 2009b). Polyvinylchloride 96-well plates were coated at 4 °C overnight with 250 ng/100 μL of the purified recombinant protein HP0245EC diluted in sodium carbonate buffer (pH 9.6). After saturation of the plates with 5% skim milk solution for 2 h at 37 °C, serially diluted mice sera (initially in 1 : 100) were added and incubated Thymidylate synthase for 30 min at 37 °C. HRP-conjugated goat anti-mouse IgG was used as the secondary antibody and 3,3′,5,5′-tetramethylbenzidine (Tiangen, Beijing, China) was used as the HRP substrate to develop the reaction. Between each of the two steps, the plates were washed three times with PBS plus 0.05% Tween (v/v). The reaction

was stopped by adding 50 μL of 0.25% hydrofluoric acid to each well. The OD630 nm was read on a plate reader (Bio-Tek Instruments, Winooski, VT). End-point titers were calculated as the reciprocal of the last serum dilution that gave a value twofold higher than nonimmunized control with a minimum value of 0.05. The same method as described above was used to determine the titers of antibodies from the mice immunized with SS2 bacterin, except that the coated antigen was replaced by the killed bacteria, 5 μg per well. Isolation of porcine neutrophils was performed as previously described (Benga et al., 2008). Freshly collected heparinized blood from healthy piglets was mixed with an equal volume of 0.9% NaCl, then layered on Ficoll-Hypaque (Haoyang Biological Manufacture Co. Ltd, Tianjin, China) and subsequently centrifuged at 400 g, 20 °C for 30 min.