Phylogenetic and epidemiologic relationships among Pasteurellaceae from Colorado bighorn sheep herds

We used 16S rRNA sequencing and leukotoxin gene (lktA) screening via PCR assay to clarify phylogenetic and epidemiologic relationships among Pasteurellaceae isolated from bighorn sheep (Ovis canadensis). Only six of 21 bighorn isolates identified as "Mannheimia haemolytica" in original laboratory reports appeared to be isolates of M. haemolytica sensu stricto based on 16S rRNA sequence comparisons; the remainder grouped with M. glucosida (n=8) or M. ruminalis (n=7). Similarly, 16S rRNA sequence comparisons grouped only 16 of 25 trehalose-fermenting bighorn isolates with reference strains of Bibersteinia trehalosi; nine other trehalose-fermenting bighorn isolates formed a clade divergent from B. trehalosi reference strains and may belong to another species. Of the 16 bighorn isolates identified as B. trehalosi by 16S rRNA sequences, only nine carried detectable lktA and thus seemed likely pathogens; none of the Bibersteinia clade isolates yielded detectable lktA despite reportedly showing β hemolysis in culture. Our findings suggest that traditional metabolism-based methods for identifying Pasteurellaceae isolates lack sufficient accuracy and resolution for reliably discerning bacterial causes of respiratory disease in bighorn sheep. Consequently, these traditional methods should minimally be augmented by molecular techniques to improve epidemiologic relevance. Streamlined surveillance approaches focused primarily on detecting pathogenic Pasteurellaceae (e.g., M. haemolytica sensu stricto and lktA-positive B. trehalosi) and other select pathogens may be most informative for investigating and managing bighorn respiratory disease.