Genbox microaer system

Rectal swabs were plated onto Campylobacter blood-free selective agar with cefoperazone and amphotericin B supplements (Modified CCDA-Preston plates, Oxoid Ltd., Basingstoke, UK). The plates were incubated under microaerophilic conditions at 42°C for 48 h using a microair atmosphere generating system (GENbox microaer system, 96126 BioMerieux UK Ltd, Basingstoke UK). Presumptive Campylobacter colonies were identified by their typical grey-white, mucoid flat appearance. Campylobacter morphology was confirmed by Gram staining. Growths of pure cultures were transferred into 2% sterile skim milk in cryovials and stored at −85°C. To speciate the isolates, the hippurate test was used as previously described [18 ]. A multiplex PCR that differentiates between C. jejuni, C. coli, C. lari, and C. upsaliensis on the basis of amplicon size of the IpxA gene was also used [19 (link)].

Bacteria were cultured at 37 °C or 42 °C. LB-medium (BioShop, Burlington, ON, Canada) was used to cultivate Gram-negative bacteria. For S. aureus, E. faecalis, L. lactis, and L. acidophilus, a BHI medium (Graso Biotech, Starogard Gdański, Poland) was used. Bacteriological agar (BioShop, Burlington, ON, Canada) at a final concentration of 1% was used in solidified media (LB-agar or BHI-agar). L. lactis and L. acidophilus were cultivated under microaerophilic conditions using the GenBox microaer system (BioMérieux, Marcy l’Etoile, France).

The faecal and caecal contents samples were stirred immediately before inoculating the tip of a swab onto mCCDA and spreading for single colonies. Both left and right caecal for a bird were cultured individually. The inoculated agar plates were incubated at 42 °C for 48 hours in a microaerobic atmosphere using the GenBox Microaer system (BioMerieux Ltd, UK). Presumptive Campylobacter colonies were identified by characteristic appearance, Gram, catalase and oxidase reactions. Where colonies formed distinct, rather than spreading colonies, up to five samples per flock were selected for subculture of between 22 and 50 colonies onto separate Columbia Blood agar plates (Oxoid, UK). These plates were then incubated for a further 48 hours at 42 °C in a microaerobic environment and examined for purity. The identity of each of the colonies was confirmed to be Campylobacter using porA short variable region (SVR) Sanger sequencing^{22 (link)}. In addition, all bacterial growth from the mCCDA plate, referred to as a ‘plate sweep’, was harvested in 400 µl TE buffer for each of the samples and immediately stored at −80 °C.