Characterization of the Bacterial Microflora and Innate Immunity Response in German Shepherd Dogs with Antibiotic-Responsive Diarrhea
The interaction of receptors mediating innate immunity and the intestinal bacterial microflora is believed to play an important role in the etiology of antibiotic-responsive diarrhea (ARD) in German Shepherd Dogs (GSDs). The aim of this study was to compare Toll-like receptor (TLR) 2 and TLR4 mRNA expression in duodenal biopsies from GSDs with ARD and healthy controls and to simultaneously assess the mucosa-adherent bacterial microflora in the same dogs.
Seven GSDs with ARD and 11 healthy retired racing Greyhounds were evaluated for this study. TLR2 and TLR4 mRNA expression was assessed using quantitative real-time PCR (qRT-PCR). The microflora was evaluated by obtaining cytology brush samples during gastroduodenoscopy followed by DNA extraction and PCR amplification using universal 16S rDNA primers. Constructed clone libraries were compared between groups using the UniFrac distance metric and the RDP Classifier. Indices for bacterial diversity and species richness were calculated and compared using t-tests.
TLR2 and TLR4 mRNA expression was significantly higher in the GSDs compared to healthy controls (TLR2 in GSDs vs controls: p=0.03, TLR4 in GSDs vs controls: p=0.01). In addition, dogs with ARD showed a 2-4 times higher expression of TLR2 mRNA compared to TLR4 (p=0.02). Principal component analysis of the bacterial intestinal clone libraries revealed clustering of individual dogs within each group, indicating that the small intestinal microflora of GSDs and control dogs are composed of distinct microbial communities. The abundance of several bacterial groups was altered in GSDs compared to control dogs. There was a trend for GSDs to be enriched in sequences belonging to Proteobacteria (p=0.08). Healthy dogs were more likely to harbor members of Bacteroidetes (including Bacteroides spp. and Prevotella spp.) and Clostridiaceae (p<0.001).
In conclusion, TLR2 and TLR4 mRNA expression is upregulated in the duodenum of GSDs with ARD, which is accompanied by an imbalance of the intestinal microflora compared to healthy control dogs. The increased TLR mRNA expression could contribute to an abnormal immune response, resulting in the development of ARD in GSDs.