Molecular Analysis of the Bacterial Microflora in Duodenal Biopsies From Dogs with Inflammatory Bowel Disease
An association between mucosa-adherent commensal bacteria and inflammatory bowel disease (IBD) has been proposed for humans. Inflammatory bowel disease is one of the most common causes of chronic diarrhea in dogs, but there are no reports characterizing the mucosa-adherent duodenal microflora in dogs using molecular methods. The aim of this study was to investigate differences in the mucosa-adherent duodenal microflora between dogs with IBD and healthy dogs.
Duodenal biopsy samples were collected from 7 dogs with a histopathological diagnosis of IBD during diagnostic gastroduodenoscopy. Duodenal biopsies were also collected from 7 healthy control dogs during endoscopy or immediately after euthanasia for unrelated studies. DNA was extracted and 16S ribosomal RNA genes (16S rDNA) were amplified using universal bacterial primers. 16S rDNA clone libraries were constructed and compared statistically between groups based on the UniFrac phylogenetic distance metric. The relative abundance of 16S rDNA clones at different phylogenetic ranks were compared using the Mann-Whitney test. Bacterial diversity indices were calculated and compared using t-tests.
A total of 1,035 16S rDNA clones were selected, and based on a 98% similarity criterion, 133 unique phylotypes were identified. These phylotypes belonged to 6 bacterial phyla: Proteobacteria (51.2%), Firmicutes (25.8%), Actinobacteria (9.0%), Bacteroidetes (8.3%), Fusobacteria (5.0%), and Verrucomicrobia (0.7%). There were no significant differences in bacterial diversity indices between groups. The UniFrac distance metric revealed significant differences in the relative abundance of several bacterial groups between dogs with IBD and healthy dogs (p<0.001). Healthy dogs and dogs with IBD clustered according to their disease status. Dogs with IBD had a significantly higher abundance of clones belonging to Alpha-, Beta-, and Gammaproteobacteria (p=0.002, p=0.02, and p=0.0058, respectively), and a significantly lower abundance of Clostridia (p<0.001). Members of Proteobacteria, especially Pseudomonadaceae (p=0.003), Neisseriaceae (p=0.009), and Brucellaceae (p=0.033), were significantly more likely to be present in dogs with IBD (p=0.006). Members of the Clostridiaceae family were significantly more likely to be present in healthy dogs (p=0.003).
In this study, significant differences of the mucosa-adherent duodenal microflora were observed between dogs with IBD and healthy dogs. These results warrant further investigations into the role of the intestinal microflora in the pathophysiology of canine IBD.