Comparative genomics of Ornithobacterium spp. isolated from free range layer chickens with respiratory infections unveils marked genetic diversity and putative new species.

The bacterium Ornithobacterium rhinotracheale causes upper respiratory tract infections (URTI) in commercial poultry worldwide. Efficient diagnostic and control of this emerging pathogen require accurate understanding of its classification, prevalence and distribution. The present study explores the genetic diversity of sixty-seven organisms presumptively identified as Ornithobacterium and recovered from chickens with URTIs in Australian free-range layer farms. Rep-PCR fingerprinting revealed wide diversity of isolates between and within farms and sites of infection. Forty representative isolates were sequenced entirely and compared to published genomes. Sequence alignments of the rpoB gene supported their classification into the genus Ornithobacterium, and 16S rRNA analysis revealed 98.08 % to 100 % identity with O. rhinotracheale type-strain DMS15997. However, most isolates gave non-interpretable profiles with the current Multi Locus Sequence Typing (MLST) scheme. Average Nucleotide Identity (ANI) analysis separated the dataset into four genetically divergent clusters. Most of the published O. rhinotracheale genomes, including DMS15997, belonged to the largest group, whereas the other clusters contained isolates with ANI values ranging from 84 % to 92 % against DMS15997, suggesting the presence of new species or sub-species. Pan-genome analysis was consistent with these observations, identifying only a small set of core genes (n = 254) in the dataset, while delineating distinct subsets of accessory proteins for each ANI cluster. Core single nucleotide polymorphism phylogeny confirmed further the substantial genetic diversity of the isolates. This study underlines the complex epidemiology and taxonomy of Ornithobacterium-associated URTIs in poultry farms, and is expected to improve diagnostic and control programs for this pathogen.