Effect of a multi-species synbiotic formulation on fecal bacterial microbiota of healthy cats and dogs as evaluated by pyrosequencing.

The effect of a multi-species synbiotic on the fecal microbiota of healthy cats (n = 12) and dogs (n = 12) was evaluated. The synbiotic (containing 5 × 10(9)  CFU of a mixture of seven probiotic strains, and a blend of fructooligosaccharides and arabinogalactans) was administered daily for 21 days. Fecal and serum samples were collected before, during, and up to 3 weeks after administration. Changes in the fecal microbiota were analyzed using denaturing gradient gel electrophoresis, 16S rRNA gene libraries, quantitative real-time PCR, and 16S rRNA gene 454-pyrosequencing. Probiotic species were detectable in 10/12 dogs and 11/12 cats during product administration. Abundances of Enterococcus and Streptococcus spp. were significantly increased in at least one time point during administration, and returned to baseline abundance after treatment was discontinued. No changes in the major bacterial phyla were identified on 454-pyrosequencing. No adverse gastrointestinal effects were recorded and no significant changes in gastrointestinal function or immune markers were observed during the study period. This study shows that while the ingestion of probiotics and prebiotics does not appear to alter the predominant bacterial phyla present in feces, supplementation with the investigated synbiotic leads to an increased abundance of probiotic bacteria in the feces of healthy cats and dogs.

Development and analytical validation of a radioimmunoassay for the measurement of alpha1-proteinase inhibitor concentrations in feces from healthy puppies and adult dogs.

Canine α(1)-proteinase inhibitor (cα(1)-PI), a proteolysis-resistant protein with a molecular weight similar to albumin, has been shown to be clinically useful as a marker for gastrointestinal protein loss in dogs. A competitive, liquid-phase radioimmunoassay was developed and analytically validated. Fecal samples were collected from 101 healthy pet dogs of various breeds and ages, and fecal cα(1)-PI (Fcα(1)-PI) concentrations were compared between dogs of different age groups. A reference interval for Fcα(1)-PI concentration was calculated using the central 95th percentile. Analytical sensitivity of the assay was 2.2 µg Fcα(1)-PI/g feces. Observed-to-expected ratios for the serial dilution and spiking recovery of 9 and 6 fecal extracts ranged from 90.4 to 152.0% and from 71.3 to 112.3%, respectively. Coefficients of variation for intra- and interassay variability for 6 fecal extracts were ≤10.8% and ≤12.5%, respectively. The reference intervals for the mean and maximum Fcα(1)-PI from fecal samples collected on 3 consecutive days were 2.2-13.9 µg/g and 2.2-21.0 µg/g, respectively. Fcα(1)-PI was significantly higher in dogs <1 year of age (P < 0.0001 for both mean and maximum Fcα(1)-PI for the 3 samples). The radioimmunoassay described is sensitive, linear, precise, reproducible, and accurate for clinical use, thus allowing reliable quantification of Fcα(1)-PI in clinical patients. Using this assay, a mean or a maximum Fcα(1)-PI for 3 sampling days of >13.9 µg/g or >21.0 µg/g, respectively, should be considered abnormal in dogs >1 year of age. Fecal cα(1)-PI concentrations in dogs <1 year of age were significantly higher and should be carefully interpreted in this age group.

Molecular analysis of the bacterial microbiota in duodenal biopsies from dogs with idiopathic inflammatory bowel disease.

An association between mucosa-adherent commensal bacteria and inflammatory bowel disease (IBD) has been proposed for humans. There are no reports characterizing the mucosa-adherent duodenal microbiota in dogs with idiopathic IBD using molecular methods. The aim of this study was to investigate differences in the mucosa-adherent duodenal microbiota between dogs with idiopathic IBD and healthy dogs. Duodenal biopsy samples were collected from seven dogs with IBD and seven healthy control dogs. DNA was extracted, 16S ribosomal RNA genes were amplified and 16S rRNA gene clone libraries were constructed and compared between groups. A total of 1035 clones were selected, and based on a 98% similarity criterion, 133 unique phylotypes were identified across all dogs. These phylotypes belonged to seven bacterial phyla: Proteobacteria (52.9%), Firmicutes (26.1%), Bacteroidetes (7.7%), Actinobacteria (8.6%), Fusobacteria (4.4%), Tenericutes (0.2%) and Verrucomicrobia (0.1%). Significant differences were identified in the relative abundance of several bacterial groups between dogs with IBD and healthy dogs (p<0.001). Healthy dogs and dogs with IBD clustered according to their disease status. Dogs with IBD had a significantly higher abundance of clones belonging to Alpha-, Beta-, and Gamma-proteobacteria (p<0.0001 for all classes), and a significantly lower abundance of Clostridia (p<0.0001). Bacteria of the genera Pseudomonas, Acinetobacter, Conchiformibious, Achromobacter, Brucella, and Brevundimonas, were significantly more abundant in dogs with IBD. In conclusion, significant differences of the mucosa-adherent duodenal microbiota were observed between dogs with idiopathic IBD and healthy dogs in this study. These results warrant further investigations into the role of the intestinal microbiota in the pathophysiology of canine IBD.