Phylogenetic relationships and diversity within the Pasteurella haemolytica complex based on 16S rRNA sequence comparison and outer membrane protein and lipopolysaccharide analysis.

The outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 30 untypeable isolates of Pasteurella haemolytica were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with the profiles of typeable isolates. The phylogenetic relationships of 28 isolates representing each of the serotypes of P. haemolytica and Pasteurella trehalosi, as well as untypeable isolates of P. haemolytica, were determined by comparing 16S rRNA sequences. The analysis of the OMP and LPS profiles of the untypeable isolates revealed five groups, which were designated untypeable groups 1 (UG1) through UG5. The UG1 and UG2 isolates had OMP and LPS profiles identical to the profiles of certain serotype A1 and A2 isolates, respectively. Furthermore, UG1 isolates originating from cattle and sheep could be clearly differentiated on the basis of their OMP profiles. The OMP and LPS profiles of UG3 isolates were similar appearance to the profiles of serotype A11 isolates, suggesting that these two groups are closely related. The OMP profiles of UG4 and UG5 isolates were unique and different from the OMP profiles of the UG1 through UG3 isolates. A comparison of 16S rRNA sequences revealed that typeable isolates of P. haemolytica could be divided into the following three groups: (i) serotype A1, A5 through A9, A12 through A14, and A16 isolates, (ii) serotype A2 isolates, and (iii) serotype A11 isolates. the isolates belonging to the first group all had identical sequences, whereas the sequences of isolates belonging to the second and third groups differed from the sequences of the isolates belonging to the first group at two and four base positions, respectively. The sequence data for the untypeable isolates confirmed the conclusions derived from the OMP and LPS analysis. Isolates belonging to UG1 and UG2 were identical to serotype A1 and A2 isolates, respectively; isolates belonging to UG3 were related to serotype A11 isolates, although there was some sequence heterogeneity within this group; and isolates belonging to UG4 and UG5 were more distantly related to P. haemolytica than were isolates belonging to UG1 through UG3 and were clearly members of two different species. As expected, isolates of P. trehalosi were event more distantly related to P. haemolytica than were the untypeable isolates, but there was significantly more sequence variation among the four serotypes of this species than there was among the serotypes of P. haemolytica. The correlation of the OMP and LPS data with the 16S rRNA sequence data suggested that OMP and LPS analyses might be useful for preliminary screening and comparing large numbers of isolates in taxonomic and epidemiological studies of the Pasteurellaceae.