Pellets containing cell membrane materials were collected by centrifugation at 200 000 g for 45 min at 4 °C and solubilized in 10 mM HEPES buffer (pH 7.4). Finally, OMPs were separated on SDS-PAGE and visualized by Coomassie blue staining. Normal rabbit serum was obtained from the Laboratory Animal PARP inhibitor Center of South China in Guangzhou, China. Porcine serum consisted of a pool of sera collected from five healthy piglets (3–4 weeks old) from a farm free of Glässer’s disease.
Both sera were filter-sterilized (0.22 μM) and aliquots were stored at −80 °C. Some aliquots of the sera were treated at 56 °C for 30 min to inactivate the complement. The serum bactericidal assay was performed with porcine and rabbit sera as previously described (Cerda-Cuellar & Aragon, 2008) with some modifications. Briefly, 100 μL of each aliquot of fresh serum or heat-treated www.selleckchem.com/products/3-methyladenine.html serum was mixed with 100 μL of bacterial suspension (approximately 1 × 108 CFU mL−1) to achieve a final concentration of 50% serum. Then, 180 μL of each aliquot of fresh serum or heat-treated serum was mixed with 20 μL of bacterial suspension (approximately 1 × 107 CFU mL−1) to achieve a final concentration of 90% serum. The mixtures were incubated at 37 °C for 1 h with gentle shaking. After incubation, 10-fold serial dilutions of the samples were made and placed on TSA plates containing inactive bovine serum and NAD. The plates were incubated at 37 °C with 5% CO2 for
36 h, Dapagliflozin at which point the colonies were counted. The percent survival was calculated by the ratio of colonies in fresh serum to those in heat-treated serum. Each H. parasuis strain was tested in three independent experiments. Comparison of several test series was evaluated by analysis of variance (anova). The significance of differences was determined using Student’s t-test. A P value of < 0.05 was considered statistically significant. Using the method of Bigas et al. (2005), no transformants were obtained when the seven different clinical isolates and four reference strains
listed in Table 1 were transformed with the pZB2 plasmid carrying the ompP2::GmR cassettes. This result suggested that the strains might not share the reported USS (5′-ACCGAACTC) or might be non-transformable strains. Therefore, we searched the H. parasuis SH0165 strain genome (GenBank accession no. NC_011852) to determine the prevalence of the alternative motif, 5′-ACCGCTTGT. In total, 523 occurrences of this motif were found, a much higher number than of the reported USS (13 occurrences, including its complement). Recently, Xu et al. (2011) also reported the 5′-ACCGCTTGT motif as a DNA USS in the SH0165 strain genome. To confirm that the 5′-ACCGCTTGT motif was required for H. parasuis transformation, the hepII gene, containing this motif 842 bp from its translational start point was selected for test transformations. Of the seven isolates and four reference strains, only the SC096 strain was transformable with plasmid pZB3 under the conditions tested.