A group of diseases have been shown to correlate with a phenomenon called microbiome dysbiosis, where the bacterial species composition of the gut becomes abnormal. The gut microbiome of an animal is influenced by many factors including diet, exposures to bacteria during post-gestational growth, lifestyle, and disease status. Studies also show that host genetics can affect microbiome composition. We sought to test whether host genetic background is associated with gut microbiome composition in the Norwegian Lundehund dog, a highly inbred breed with an effective population size of 13 individuals. The Lundehund has a high rate of a protein-losing enteropathy in the small intestine that is often reported as Lundehund syndrome, which negatively affects longevity and life-quality. An outcrossing project with the Buhund, Norrbottenspets and Icelandic sheepdog was recently established to reintroduce genetic diversity to the Lundehund and improve its health. To assess whether there was an association between host genetic diversity and the microbiome composition, we sampled the fecal microbiomes of 75 dogs of the parental (Lundehund), F1 (Lundehund x Buhund), and F2 (F1 x Lundehund) generations. We found significant variation in microbiome composition from the parental Lundehund generation compared to the outcross progeny. The variation observed in purebred Lundehunds corresponded to dysbiosis as seen by a highly variable microbiome composition with an elevated Firmicutes to Bacteroidetes ratio and an increase in the prevalence of Streptococcus bovis/Streptococcus equinus complex, a known pathobiont that can cause several diseases. We tracked several other environmental factors including diet, the presence of a cat in the household, living on a farm and the use of probiotics, but we did not find evidence of an effect of these on microbiome composition and alpha diversity. In conclusion, we found an association between host genetics and gut microbiome composition, which in turn may be associated with the high incidence of Lundehund syndrome in the purebred parental dogs.

In April to August 2021, we collected stool samples from Lundehund (P, n = 50), Lundehund x Buhund crosses (F1, n = 8), and F1 x Lundehund crosses (F2, n = 22). The lower number of individuals in the F1 cohort is due to the challenge in finding owners of Buhund females that were willing to let their dog being paired with a Lundehund. Owners were instructed in how to collect and handle fresh naturally deposed samples, avoiding contamination. The stool samples were stored at room temperature in Stool Nucleic Acid Collection and Preservation Tubes (Norgen BioTek Corp, Cat. 45660). In September 2021, the samples were analyzed with the ZymoBIOMICS® Targeted Sequencing Service (Zymo Research, Irvine, CA). The ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used to extract DNA using an automated platform. Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). The bacterial 16S rRNA primers amplified the V3-V4 region of the 16S rRNA gene. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, WA). The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e., blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process. The final library was sequenced on an Illumina® MiSeq™ with a v3 reagent kit (600 cycles). The sequencing was performed with a 10% PhiX spike-in. Unique amplicon sequences variants (ASVs) were inferred from raw reads using the DADA2 pipeline. Potential sequencing errors and chimeric sequences were also removed with the DADA2 pipeline. ASVs that were present in only one sample, i.e., singletons, were examined for each cohort and removed from the dataset for clustering analysis. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database. Any taxa that were not represented at over 1% relative abundance in at least one sample were removed. Five purebred Lundehund dogs older than 9 years and two F2 dogs that were under medication at sampling were also removed from further analyses, in order to obtain a sample more homogenous in age and without the influence of antibiotics. Normalization of the data was performed by calculating the relative abundance for each sample by library scaling.

The data can be analysed with different softwares, we used R version 4.1.3 with RStudio version 2022.07.2 and with the R packages Phyloseq and Microbiome. More details about scripts are in the README.md file.