This protein was more variable in amino acid sequence among these strains (Figure 3). Two other genes encoding filamentous hemaggultinins, pfhB3 and pfhB4, were absent in strain Pm70, with pfhB3 present in strains P1059, X73, and 36950, and pfhB4 present in strains P1059, HN06, and 3480. Finally, lipoproteins plpP, plpB, and plpD BIBW2992 in vitro were present in all sequenced strains, and all were highly conserved

except plpP, whose product shared only 82-98% amino acid similarity between strains. Table 3 Similarity of proteins of interest in sequenced avian Pasteurella multocida genomes Protein name Pm70 P1059 X73 36950 HN06 3480 HgbA 100A 87 96 89 99 99 HgbB 100 – 95 – 84 – Omp16 100 100 100 99 100 100 OmpH1 100 84 83 83 84 99 OmpH2 100 98 98 99 98 97 OmpH3 100 97 – 98 97 98 TbpA 100 99 99 98 100 99 PtfA 100 100 100 100 100 99 ComE 100 99 100 99 99 99 PlpE 100 94 94 – - – PlpP 100 84 82 98 72 76 PlpB 100 99 100 99 100 100 PlpD 100 100 100 100 100 100 PfhB1 (PM0057) 100 99 98 – - 99 PfhB2 (PM0059) 100 90 90 97 – - PfhB3 – 100B 98 96 – - PfhB4 – 100 – - 93 93 APercent amino acid similarity to same protein from strain Pm70. BPercent amino acid similarity to same protein from strain P1059. Single nucleotide polymorphisms The three avian source P.

multocida genomes were also compared for SNPs within the conserved regions of their genomes using MAUVE [42], and the SNPs were

analyzed for their coding effects using SNPeff [44] (Table 4). A total of 31,021 SNPs were identified between strains Pm70 and P1059, and 26,705 SNPs were identified between Akt inhibitor strains Pm70 and X73. The density of SNPs varied considerably across the P. multocida genome, with some regions containing a much higher density of SNPs than the rest of the core genome (Figure 4). This suggests that some regions of the genome are under diversifying selection, while the majority of the genome is under neutral or purifying selection. The ratio between non-synonymous to synonymous substitutions (dN/dS) is commonly Arachidonate 15-lipoxygenase used as a measure of purifying versus diversifying selection [56]. The overall dN/dS ratios of all coding regions of strains P1059 and X73 compared to strain Pm70 were 0.40 and 0.38, respectively. Proteins were then divided into groups based upon predicted subcellular localization of each protein using PSORT-B version 3.0. Using this approach, the dN/dS ratios varied considerably, with higher ratios (0.76-0.93) found within proteins predicted as extracellular or outer membrane [57]. Amongst specific outer membrane proteins, the highest dN/dS ratios were observed within PfhB2, HgbA, HemR, pm0591 (a secreted effector protein), pm0803 (an iron-regulated outer membrane protein), TadD-F (pilus assembly proteins), RcpB-C (pilus assembly proteins), and PlpP.