Assessment of the canine intestinal microflora using molecular methods and serum markers
Previous studies examining the canine intestinal microflora have focused on cultivation of bacteria from intestinal content. Recently, it has been recognized that the majority of bacteria cannot be identified using standard culture techniques. The aim of this study was to describe the composition and dynamics of the canine intestinal microflora using molecular methods based on identification of the 16S ribosomal DNA (16S rDNA) and to evaluate the clinical use of a 13C-glycocholic acid blood test (13CGCBT) as a serum marker for small intestinal bacterial biomass. Intestinal content was obtained from healthy dogs and the microflora was characterized in different compartments of each dog by denaturing gradient gel electrophoresis (DGGE) and comparative 16S rDNA analysis. A 13C-glycocholic acid blood test (13C-GCBT) was developed as a marker for small intestinal bacterial biomass and the influence of tylosin administration on the 13C-GCBT, serum concentrations of cobalamin, folate, and unconjugated cholic acid (SUCA) was evaluated. There was marked variation in DGGE profiles between individual dogs and also between different intestinal compartments within dogs. DGGE profiles from duodenal juice samples collected endoscopically at different time-points varied within individuals, possibly due to variations over time or a slight variation in sampling location. Direct sequencing revealed 106 individual 16S rDNA sequences. Forty-two sequences showed less than 98% similarity to described sequences in public databases and may constitute previously uncharacterized bacterial species. Serum folate concentrations, SUCA, and the cumulative percent dose/min of 13C administered as 13C-glycocholic acid (CUMPCD) increased significantly following tylosin administration (p<0.01). The results indicate that dogs have a complex intestinal microflora with marked differences between individual dogs. Different intestinal compartments appear to host a unique microflora and the assessment of a fecal sample does not yield accurate information about the composition of the microflora in proximal compartments of the gut. The intestine harbors many previously uncharacterized bacterial species. The clinical significance of these uncharacterized intestinal bacterial species needs to be further investigated in dogs with gastrointestinal disease. Increased serum folate, SUCA, and CUMPCD in the 13C-GCBT suggest that, in the dogs described here, tylosin administration increased the biomass of organisms carrying out these metabolic functions.