Campylobacter blood

For bacterial culture, methodology described by Hariharan et al., [38 (link)] was followed. The cloacal-swab samples were plated on Campylobacter blood-free selective agar (CBF) containing charcoal, cefoperazone and amphotericin B supplement (Oxoid Ltd. Basingstoke, Hampshire, England). The plates were incubated at 42°C for 48 hours under microaerophilic conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen) using Campy-gas generating pack (BBL, Becton Dickson and Co. Maryland, USA). Colonies were stained with Gram’s stain and examined under microscope. Positive colonies were sub-cultured on another CBF plate for isolation of pure cultures. To speciate the isolates, the cultures were subjected to biochemical tests including catalase and oxidase (BBL, Becton, Dickinson and Co., Sparks, MD, USA), and hippurate tests (Remel, Lennexa, KS, USA). Pure isolates were then transferred and stored in 10% skim milk in cryovials at -80°C [44 (link)] until the DNA extraction was performed. The above described culture methods were also performed on the reference strain Campylobacter jejuni subsp. jejuni ATCC® 33291^™ (ATCC, Manassas, VA, USA) and used as a positive control for subsequent experiments.

Rectal swabs isolated from 19 dogs with diarrhea were examined for Campylobacter species. The samples were plated on Campylobacter blood-free selective agar CCDA (Oxoid, UK) and cultured for 48 h at 42 ± 1 °C under microaerophilic conditions (Genbox microaer, bioMerieux, France). Species identification of the grown colonies was performed using a commercial, standardized system for the identification of Campylobacter—API Campy (bioMerieux, France) and PCR technique, performed according to protocols described previously (Table 5).