Comparative genomic analysis identifies potential adaptive variation in Mycoplasma ovipneumoniae

Mycoplasma ovipneumoniae is associated with respiratory disease in wild and domestic Caprinae globally, with wide variation in disease outcomes within and between host species. To gain insight into phylogenetic structure and mechanisms of pathogenicity for this bacterial species, we compared M. ovipneumoniae genomes from six countries (Australia, Bosnia and Herzegovina, Brazil, China, France, USA) and four host species (domestic sheep, domestic goats, bighorn sheep, caribou).

Methods
Deep nasal swab samples were collected from domestic sheep, domestic goats, bighorn sheep, and caribou. Swab samples were selectively enriched for Mycoplasma ovipneumoniae using a two-step procedure involving incubation in mycoplasma broth, followed by incubation in SP4 broth with glucose. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen). Whole-genome shotgun libraries were prepared using Illumina Nextera DNA kits and sequenced on an Illumina MiSeq using the v3 600 cycle sequencing kit. Oxford Nanopore Technologies (ONT) shotgun sequencing was also performed for samples with sufficient genomic DNA remaining after Illumina sequencing. Barcoded ONT libraries were created using the SQK-LSK109 library prep kit, and sequencing was performed for 48 hours on FLO-MIN106D flowcells. 

Raw Illumina sequence reads were cleaned using HTStream to filter PhiX reads, PCR duplicates, adapter sequences, and low-quality reads. Raw ONT sequence reads were demultiplexed by barcode and base-called using Guppy v.4.2.2. For samples with ONT sequence data, genome assembly was performed using Trycycler v0.4.1, and assemblies were polished using cleaned Illumina sequence reads with Pilon v.1.23. For samples with only Illumina sequence data and no ONT sequence data, cleaned Illumina reads were assembled de novo using SPAdes v3.13.1.

Comparative genomic analyses were performed using the assemblies generated in this study, along with additional Mycoplasma ovipneumoniae genome assemblies from prior studies that were publicly available on NCBI at the time of the study.