Whole-transcriptome analysis and adenylate cyclase toxin expression in B. bronchiseptica strains RB50 and 253. Comparison of whole-transcriptome analysis (A) and cya-related genes (B) between strains RB50 and 253. The x axis indicates the order of genes along the B. bronchiseptica strain RB50 5.3-megabase (Mb) chromosome. The y axis indicates the fold change in expression (FCE) of each gene. Negative-fold-change values indicate decreased expression of strain 253 genes compared to strain RB50 genes, while positive-fold-change values indicate increased gene expression. Genes of interest are labeled with corresponding underscores. Φ, phage-related gene; wbm, O-antigen-related genes; ptx, pertussis toxin gene; fim, fimbria-related genes; bbuR, urease regulator gene; cyaA, adenylate cyclase toxin gene. Error bars represent the mean ± the standard error. (C) RT-PCR of cyaA and adk in RB50, RB50ΔcyaA, and 253 with samples either including RNase treatment or lacking reverse transcriptase. (D) Western blot analysis for the presence of CyaA in bacterial lysates. Recombinant CyaA, RB50, RB50ΔcyaA, or 253 lysates were probed for CyaA using the monoclonal antibody 3D1. (E) Western blot analysis for the presence of anti-CyaA antibodies in sera from mice immunized with strains RB50, 253, and RB50ΔcyaA or naïve sera. Using these sera, recombinant CyaA was probed. α, anti.