Comparison of the Fecal Microflora Between Healthy Cats and Cats with Inflammatory Bowel Disease Using Molecular Methods
In human beings, inflammatory bowel disease (IBD) is associated with alterations of the intestinal bacterial flora. In fact, some studies suggest that an altered bacterial flora might be an underlying cause for IBD. In cats, IBD is associated with clinical signs such as vomiting, diarrhea, and weight loss, but little is known about the pathogenesis of this disease. Also, the fecal microflora of cats with IBD has previously not been extensively characterized. Thus, the aim of this study was to characterize and compare the fecal microflora between healthy cats and cats with IBD based on direct sequence analysis of 16S ribosomal DNA (16S rDNA).
Four healthy cats and four cats with clinical signs and a histopathological diagnosis of IBD were evaluated in this study. One fecal sample was collected from each cat and stored at -80°C until further analysis. Purification of the bacterial DNA was performed by phenol:chloroform:iso-amylalcohol extraction, and an amplicon of approximately 450 bp of the hypervariable region of the 16S rDNA was amplified at low PCR cycle numbers, using universal bacterial primers. For identification of bacterial 16S rDNA sequences, a clone library was constructed. Each sequence was tested for possible chimeric structures and any identified chimeras were excluded from further analysis. Obtained sequences were compared to sequences listed in the Ribosomal Database Project (RDP) and subjected to phylogenetic analysis. Differences in the relative abundance of 16S rDNA clones obtained in healthy and IBD cats were analyzed using a Fisher's exact test and a Mann-Whitney U test. Statistical significance was set at p<0.05.
A total of 521 clones were analyzed. Based on a 98% similarity criterion, 47 non-redundant bacterial 16S rDNA sequences were identified. Four bacterial phyla were identified: 94.9% of identified phylotypes belonged to Firmicutes, 4.3% were Actinobacteria, and less than 1% each were Proteobacteria and Bacteriodetes. Clostridiales was the most abundant bacterial order in both healthy and IBD cats, representing 69.4% and 84.5% of the identified clones within each group, respectively. The proportion of clones belonging to the order Bacillales was significantly higher in healthy cats compared to cats with IBD (p<0.001). In contrast, IBD cats had a significantly higher proportion of clones classified as Clostridium perfringens-like organisms (27.7% vs. 5.1%; p<0.001).
These results indicate that the fecal bacterial flora of cats is comprised mainly of bacteria belonging to the phylum Firmicutes. Based on the results of this study, the fecal microflora of cats with IBD may be altered when compared to healthy cats. Thus, studies characterizing the fecal microflora in a larger population of cats with IBD are warranted.