Defining a metagenomic threshold for detecting low abundances of in canine faecal samples.

Introduction: Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics.

Materials And Methods: To decrease the likelihood of false detection, we established a metagenomic threshold for by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for . Finally, the metagenomic threshold was used to determine the occurrence of in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project.

Results: The metagenomic assay and qPCR had a detection limit of 1.1x10 CFU per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics.

Discussion: Using our detection limit, the occurrence of in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.