A targeted PCR approach for the detection of IOLA in canine infectious respiratory disease samples during an atypical...

During winter 2023, an atypical canine infectious respiratory disease (aCIRD) outbreak was associated with high case-fatality rates and poor antibiotic response. Preliminary metagenomics investigations claimed partial sequences resembling Infectious Organism Lurking in Human Airways (IOLA), a poorly characterized bacterium first described in humans, in canine respiratory samples. However, its detectability remained uncertain and required systematic molecular investigation.

We screened 777 veterinarian-submitted canine respiratory samples from the United States using 16S targeted sequencing for samples positive for Rickettsiales, the lowest taxonomic classification for IOLA. Samples containing sequencing reads classified as for Rickettsiales were tested by PCR assay targeting two IOLA genes (16S rRNA and PrfA). Assays were optimized at 58 °C with 500 nM primers, and products visualized by agarose gel and capillary electrophoresis. Analytical sensitivity was 10⁴ copies/μl and 10⁴ copies/μl for 16S and PrfA assay, respectively. Candidate amplicons were verified by Sanger sequencing and BLAST analysis.

Of the 777 samples screened, 55 contained sequencing reads classified as Rickettsiales. Forty-five of the 55 samples were negative by 16S rRNA PCR, while 10 samples produced amplicons near the expected size. The PrfA PCR assay was negative across all samples. Sequencing representative samples from those that produced 16S amplicons confirmed nonspecific amplification. Therefore, all 55 samples were negative for IOLA.

Dual-target PCR identified no evidence of IOLA in respiratory samples from the 2023 aCIRD outbreak. Non-specific amplification in the 16S PCR assay highlights the need for multi-target validation in novel pathogen detection and supports prioritization of established CIRD pathogens over unverified organisms.