Dysbiosis in a canine model of human fistulizing Crohn's disease

Abstract

Crohn's disease (CD) is a chronic immune-mediated inflammatory condition caused by the loss of mucosal tolerance toward the commensal microbiota. On average, 29.5% and 42.7% CD patients experience perianal complications at 10 and 20 y after diagnosis, respectively. Perianal CD (pCD) result in high disease burden, diminished quality of life, and elevated health-care costs. Overall pCD are predictors of poor long-term outcomes. Animal models of gut inflammation have failed to fully recapitulate the human manifestations of fistulizing CD. Here, we evaluated dogs with spontaneous canine anal furunculosis (CAF), a disease with clinical similarities to pCD, as a surrogate model for understanding the microbial contribution of human pCD pathophysiology. By comparing the gut microbiomes between dogs suffering from CAF (CAF dogs) and healthy dogs, we show CAF-dog microbiomes are either very dissimilar (dysbiotic) or similar (healthy-like), yet unique, to healthy dog's microbiomes. Compared to healthy or healthy-like CAF microbiomes, dysbiotic CAF microbiomes showed an increased abundance of Bacteroides vulgatus and Escherichia coli and a decreased abundance of Megamonas species and Prevotella copri. Our results mirror what have been reported in previous microbiome studies of patients with CD; particularly, CAF dogs exhibited two distinct microbiome composition: dysbiotic and healthy-like, with determinant bacterial taxa such as E. coli and P. copri that overlap what it has been found on their human counterpart. Thus, our results support the use of CAF dogs as a surrogate model to advance our understanding of microbial dynamics in pCD.

Keywords: canine furunculosis; dysbiosis; fistulizing Crohn’s disease; microbiome; perianal fistulas.

Figure 1.

Analysis of the microbiota composition in samples from CAF dogs and HC dogs. A) Shannon index of alpha diversity for HC dogs (green) and CAF dogs (orange). B) Relative abundance of the bacterial taxa found to discriminate between dogs with different disease status: HC dogs (green) and CAF dogs (orange); thick black lines indicate mean abundance. T-test, p values are expressed as: ****<0.0001, ***<0.001, and #0.1 (Corrected for multiple comparisons (5 bacterial taxa found significantly different by disease status) using the Bonferroni-Dunn method, with alpha = 0.05).

Figure 2.

Analysis of the microbiota clusters. A) NMDS visualization of samples from HC and CAF dogs. Bacterial taxa present were quantified by MetaPhlAn, distances were calculated using Bray Curtis dissimilarity index, and samples were plotted based on NMDS. Clusters were defined using PAMK and are colored green (HC dogs), orange (CAF dogs in the near cluster) and red (CAF dogs in the far cluster). Relative abundance of the bacterial taxa found to discriminate between dogs, separated by cluster assignment: B) Bacterial taxa overrepresented in HC dogs or CAF dogs in the near cluster compared to CAF dogs in the far cluster; C) Bacterial taxa overrepresented in CAF dogs in the far cluster compared to HC dogs or CAF dogs in the near cluster. Clusters are colored as described for panel A, and thick black lines indicate mean abundance. FDR t-test, p values are expressed as: ****<0.0001, ***<0.001, ** <0.005, * <0.05, and #0.1 (Corrected for multiple comparisons (8 bacterial taxa found significantly different among clusters and HC) using the Bonferroni-Dunn method, with alpha = 0.05).

Figure 3.

Clinical manifestation of CAF. A) The number of fistulas presented by CAF dogs according to cluster (orange, near cluster; red, far cluster). B) Percentage of dog DNA reads in metagenomic sequencing samples grouped by disease status. C) Shannon index of alpha diversity in samples from HC dogs (green, n = 8) or CAF dogs exhibiting fibrosis (orange pattern, n = 6). T-test, p values are expressed as: ****<0.0001, ***<0.001, ** <0.005, * <0.05, and #0.1.

Figure 4.

Microbial gene pathway analysis. A) Bray Curtis dissimilarity index beta diversity plot of dogs samples colored by clustering assignation: HC dogs (green) and CAF dogs belonging to the near (orange) or far (red) cluster. Bacterial pathways were quantified by HUMAnN2 and samples were plotted based on principal component analysis. B) The top 20 gene pathways that most strongly distinguish between CAF-dog samples and HC dog samples, identified using Random Forest.