Exploring clonality of Mannheimia haemolytica in beef cattle.

Mannheimia haemolytica is a pathobiont bacterium of the upper respiratory tract in cattle and is associated with acute bovine respiratory disease (BRD). Despite research into bacterial pathogens and their associations with BRD, little is known about how bacterial variant dynamics interplay with disease progression. Therefore, our objective was to explore the clonal diversity of M. haemolytica in beef cattle utilizing whole genome sequencing. Ninety-nine M. haemolytica isolates from a commingled cohort of 33 cattle sampled in a prior study were selected for analysis. Long-read whole-genome sequencing was conducted to evaluate antimicrobial resistance genes, virulence factors, plasmids, and single nucleotide polymorphisms. Clonal lineages were derived using multi-locus sequence typing (MLST), a recently developed genomic sequence variant (GSV) method, and a core SNP-based clustering analysis with the average linkage method. We identified no differences in pathogenic profiles across isolates. We identified a single sequence type (ST1) and a single GSV (GSV 2) utilizing the MLST and GSV methods, respectively. The core SNP-based average linkage clustering analysis produced different clonal lineage estimations depending on the core SNP cutoff values (2 to 18 clusters). All estimations were greater than the single sequence type and GSV suggested by the MLST and the GSV methods. BRD status did not correlate with clonal interpretations. Our results suggest that method selection can influence the clonal interpretation of M. haemolytica within a population but may not reflect pathogenic capability. Alternate methods that increase discriminatory power or transcriptional profiles may provide new insights and should be evaluated.IMPORTANCEBovine respiratory disease remains a major challenge for cattle health and production, yet the population dynamics of key bacterial pathogens remain poorly understood. Using long-read whole-genome sequencing, this study shows that clonal diversity estimates of Mannheimia haemolytica are highly dependent on the analytical method employed, despite uniform virulence gene content. These findings highlight the importance of method selection in genomic epidemiology and caution against overinterpreting clonal structure as a proxy for pathogenic potential.