Conclusions Pets are members
Erismodegib of the North American family, with 37% of American and 33% of Canadian households containing pet dogs [25, 26]. As our understanding of Campylobacter pathogenicity increases, so must our understanding of its reservoirs and ecology. Domestic dogs are recognized as a risk factor for campylobacteriosis and this report reinforces those findings. We found human pathogens like C. jejuni, C. coli, C. upsaliensis, C. gracilis, C. concisus and C. showae in dog feces, with significantly higher levels present in dogs with diarrhea. As well, we see that disturbances to the intestinal microbiota related to diarrhea have an effect on Campylobacter ecology. How and why this is the case, as well as how this change in Campylobacter distribution relates to the overall intestinal community, are areas of future
investigation. Methods Sample Collection Fecal samples from healthy dogs were submitted for analysis by pet owners from the Saskatoon, SK, Canada metropolitan area (population 250,000) (Additional file 1: Table S1). All dogs were considered healthy by their owners and had not received antibiotic therapy for at least six months prior to sample collection. Samples were collected in accordance with the University of Saskatchewan Animal Research Ethics Board (protocol #20090054). Fecal specimens from dogs selleck chemicals suffering from diarrhea (of any etiology) were obtained from samples submitted to Prairie Diagnostic Services PND-1186 in vitro Inc., Saskatoon, SK for routine bacteriology Ribonucleotide reductase and/or parasitology
testing (Additional file 1: Table S1). All samples were stored at -80°C until processed for PCR analysis. DNA Extraction Total bacterial DNA was extracted from fecal samples using the QIAamp DNA stool kit (Qiagen), as per manufacturer’s instructions. Final DNA samples were diluted 1:10 with sterile water before analysis. This was done to improve the overall sensitivity of the assays used, which are known to be affected by PCR inhibitors carried through fecal DNA extractions . Quantitative PCR (qPCR) The detection and quantification of the 14 species of Campylobacter reported was done using assays targeting the cpn60 gene using the primer sets and PCR conditions described in . The lower detection limit of these assays is 103 copies/g of feces . Total bacterial DNA levels were measured by quantification of the 16S rRNA gene, using the primer set SRV3-1/SRV3-2 (with an annealing temperature of 62°C) described in . All assay reaction mixtures consisted of 1× iQ SYBR green supermix (Bio-Rad), 400 nmol/L concentrations of each of the appropriate primers, and 2 μL of template DNA in a final volume of 25 μL.