Columbia agar containing 5 cattle blood

Isolation of Campylobacter spp. from meat and milk was conducted in compliance with the EN ISO 10272-1:2006 method with slight modification. Briefly, meat and cheese (25 g) were homogenized for 2 min in a stomacher with 225 mL buffered peptone water (Oxoid Limited, Basingstoke, United Kingdom) in the sterile plastic bags. Then, 10 mL of the homogenate solution and 10 ml of raw milk was added to 90 mL of Bolton broth containing the Bolton broth selective supplement and 5% laked horse blood (Oxoid Limited, Basingstoke, United Kingdom) and incubated at 42 °C for 48 h under microaerobic conditions (Generbox microaer-BioMerieux, Marcy l’Etoile, France). Next, the bacterial suspension was spread onto CCDA plates (Oxoid Limited, Basingstoke, United Kingdom) and then incubated at 42 °C for 48 h under microaerobic conditions. Characteristic growth from the CCDA plates was transferred to a blood plate (Columbia agar containing 5% cattle blood, Oxoid Limited, Basingstoke, United Kingdom) and incubated overnight at 42 °C. Colonies suspected as Campylobacter spp. were examined for cell morphology, motility, and oxidase reactions. Putative Campylobacter colonies were frozen at −80 in Microbanks (Pro-Lab Diagnostics, Birkenhead, United Kingdom) until species differentiation using polymerase chain reaction (PCR).

Isolation of Campylobacter spp. from water samples was performed according to International Organization for Standardisation (2019) . We filtered 100 ml of water sample through 0.45μm filters (Merc Millipore, Burlington, USA) and then each filter was transferred to 90 ml of Bolton broth (Oxoid Limited, Basingstoke, United Kingdom). The broth was incubated at 41°C in a microaerophilic atmosphere (Generbox microaer-BioMerieux, Marcy l’Etoile, France) for 48 hours and after preincubation 10μl of culture was performed on a solid CCDA medium (Oxoid Limited, Basingstoke, United Kingdom). Characteristic growth from the CCDA plates was transferred to a blood plate (Columbia agar containing 5% cattle blood, Oxoid Limited, Basingstoke, United Kingdom) and incubated overnight at 41°C. Colonies suspected as Campylobacter spp. were examined for cell morphology, motility, and oxidase reactions. Putative Campylobacter colonies were frozen at -80 in Microbanks (Pro-Lab Diagnostics, Birkenhead, United Kingdom) until species differentiation.