Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma

Dogs across the globe are afflicted by diverse blood- and vector-borne bacteria (VBB), many of which cause severe disease and can be fatal. Diagnosis of VBB infections can be challenging due to the low concentration of bacteria in the blood, the frequent occurrence of coinfections, and the wide range of known, emerging, and potentially novel VBB species encounterable. Therefore, there is a need for diagnostics that address these challenges by being both sensitive and capable of detecting all VBB simultaneously. We detail the first employment of a nanopore-based sequencing methodology conducted on the Oxford Nanopore Technologies (ONT) MinION device to accurately elucidate the “hemobacteriome” from canine blood through sequencing of the full-length 16S rRNA gene. We detected a diverse range of important canine VBB, including Ehrlichia canis, Anaplasma platys, Mycoplasma haemocanis, Bartonella clarridgeiae, “Candidatus Mycoplasma haematoparvum”, a novel species of hemotropic mycoplasma, and Wolbachia endosymbionts of filarial worms, indicative of filariasis. Our nanopore-based protocol was equivalent in sensitivity to both quantitative PCR (qPCR) and Illumina sequencing when benchmarked against these methods, achieving high agreement as defined by the kappa statistics (k . 0.81) for three key VBB. Utilizing the ability of the ONT’ MinION device to sequence long read lengths provides an excellent alternative diagnostic method by which the hemobacteriome can be accurately characterized to the species level in a way previously unachievable using short reads. We envision our method to be translatable to multiple contexts, such as the detection of VBB in other vertebrate hosts, including humans, while the small size of the MinION device is highly amenable to field use. © 2022 Huggins et al.