Comparison of microbiological, histological, and immunomodulatory parameters in response to treatment with either combination therapy with prednisone and metronidazole or probiotic VSL#3 strains in dogs with idiopathic inflammatory bowel disease

Abstract

Background: Idiopathic inflammatory bowel disease (IBD) is a common chronic enteropathy in dogs. There are no published studies regarding the use of probiotics in the treatment of canine IBD. The objectives were to compare responses to treatment with either combination therapy (prednisone and metronidazole) or probiotic strains (VSL#3) in dogs with IBD.

Methodology and principal findings: Twenty pet dogs with a diagnosis of IBD, ten healthy pet dogs, and archived control intestinal tissues from three euthanized dogs were used in this open label study. Dogs with IBD were randomized to receive either probiotic (D-VSL#3, n = 10) or combination drug therapy (D-CT, n = 10). Dogs were monitored for 60 days (during treatment) and re-evaluated 30 days after completing treatment. The CIBDAI (P<0.001), duodenal histology scores (P<0.001), and CD3+ cells decreased post-treatment in both treatment groups. FoxP3+ cells (p<0.002) increased in the D-VSL#3 group after treatment but not in the D-CT group. TGF-β+ cells increased in both groups after treatment (P = 0.0043) with the magnitude of this increase being significantly greater for dogs in the D-VSL#3 group compared to the D-CT group. Changes in apical junction complex molecules occludin and claudin-2 differed depending on treatment. Faecalibacterium and Turicibacter were significantly decreased in dogs with IBD at T0, with a significant increase in Faecalibacterium abundance observed in the animals treated with VSL#3 strains.

Conclusions: A protective effect of VSL#3 strains was observed in dogs with IBD, with a significant decrease in clinical and histological scores and a decrease in CD3+ T-cell infiltration. Protection was associated with an enhancement of regulatory T-cell markers (FoxP3+ and TGF-β+), specifically observed in the probiotic-treated group and not in animals receiving combination therapy. A normalization of dysbiosis after long-term therapy was observed in the probiotic group. Larger scale studies are warranted to evaluate the clinical efficacy of VSL#3 in canine IBD.

Figure 1. Results for histology scores, CIBDAI, CD3+ cells, FoxP3+ cells, TGF-β cells, and plasma citrulline concentrations.

Significant differences between baseline (T0) and 30 days after the end of therapy (T1) were observed for all parameters in both treatment groups except the expression of FoxP3+ T-cells in the CT group (P = 0.3296). While TGF-β increased significantly in both treatment groups, the magnitude of the increase was significantly higher in dogs treated with VSL#3 (P = 0.0008). Data for CD3+ cells, FoxP3+ cells, TGF-β cells expressed as cells per 62,500 μm².

Figure 2. Histology of intestinal mucosa of dogs with IBD after treatment with VSL#3 (A, C, E) and CT (B, D, F).

A residual inflammatory infiltrate with lymphocytic-plasmacytic cells (arrows) is evident after the therapy in both samples (H&E, 40X). In both treatment groups ssimilar patterns of mucosal infiltrations with CD3+ T-lymphocytes are evident (C and D). Infiltration with Fox-P3+ cells are proportionally increased in a sample belonging to a VSL#3 treated dog (E) compared to the sample from CT treated dog (F). Note the particular Fox-P3+ T-cells concentrations at the apical portion of villi in the VSL#3 treated dog (E) (arrow-heads) (IHC, ABC method, Harris haematoxylin nuclear counterstain, 40X).

Figure 3. Expression of AJC proteins in the intestinal mucosa of control dogs (ED group) (A, D, G) and dogs treated with VSL#3 (B, E, H) or CT (C, F, I).

No discernible differences in the distribution or staining intensity of E-cadherin are observed between normal mucosa (A) and IBD samples (B and C); the overall intensity of E-cadherin staining decreased from the luminal epithelium to the distal crypts. Occludin-specific labelling is most intense at the epithelial cell AJC (arrows) of the luminal epithelium covering the apical portion of villi in ED (D) and VSL#3 (E); a weak to absent expression is observed in the luminal epithelium and in some intestinal glands of the small intestine of the CT sample (F). In colonic samples belonging to ED (G) and VSL#3 (H) groups, claudin-2 is readily detectable only in the colonic crypt epithelium, decreasing in intensity from the distal to the proximal crypt and becoming barely detectable at the luminal surface of the colon. In contrast, claudin-2 expression is increased in the proximal crypt and luminal epithelium of all samples from CT dogs (I).

Figure 4. Expression of AJC proteins.

Mucosal biopsies were evaluated after the end of treatment (T1) either with the probiotic (VSL) or combination drug therapy (CT), and compared to archived mucosal samples from dogs euthanized for non-gastrointestinal disorders (ED). (*significantly different to the other 2 groups; line denotes median).

Figure 5. Results of quantitative PCR assays for selected bacterial groups.

Dogs with IBD (in both treatment groups) had significantly decreased abundance of Faecalibacterium spp. (p = 0.008) and Turicibacter spp. (p = 0.0078) compared to the healthy dogs. Faecalibacterium spp. increased significantly in the VSL#3 treated dogs at T1 but not in the CT group. (*significantly different compared to healthy dogs; **significantly different after treatment compared to pre-treatment).