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.