Bolton broth

Upon arrival at the laboratory, samples were inoculated into Bolton Broth (Oxoid, Basingstoke, UK), containing the Bolton selective supplement for enrichment, and then incubated at 42 °C for 24 h in a microaerobic environment (5% O₂, 10% CO₂, and 85% N₂), with GENbox generators (BioMérieux, Craponne, France). After enrichment, putative Campylobacter-positive samples were streaked on Karmali agar (Oxoid, Basingstoke, UK) and incubated under the same conditions as described above for 48 h [24 (link)]. From each sample, suspected colonies were examined for the typical morphology and motility of Campylobacter, under a light microscope and using the oxidase/catalase tests. Thereafter, presumed Campylobacter colonies were subjected to PCR analysis for genus confirmation and species identification. Confirmed Campylobacter isolates were conserved at −80 °C in Mueller–Hinton broth containing 25% glycerol (v/v).

The material for the study consisted of field isolates of Campylobacter spp. obtained from the gut (cecum) of 140 broiler chickens directly after slaughter in slaughterhouses in southeastern Poland in September and October. The birds were from different indoor flocks. Presumptive identification of Campylobacter spp. isolates was based on colony morphology, Gram staining, and growth in microaerobic conditions. Initial isolation was carried out in Bolton Broth (Oxoid Ltd., UK). The cultures were incubated at 37°C for 48 hr in microaerophilic conditions (5% O₂, 10% CO₂, 85% N) in the CampyGen system (Oxoid Ltd.). On media that showed growth of gray, flat, and moist bacterial colonies with a tendency to expand, single colonies belonging morphologically to the Campylobacter spp. type were collected, directly transferred to selective mCCDA agar, and incubated at 41.5°C for 48 hr in microaerophilic conditions (Dudzic et al., 2016). The isolates were stored at −80°C in the Microbank system for storage of micro‐organisms (Biocorp, PL).

A total of 609 samples comprising of poultry ceca (n=116), poultry droppings (n=203), and feces of pigs (n=71), cattle (n=61), sheep (n=19), goat (n=17), human beings (n=88), and laboratory animals (n=34) (rats, rabbits, and guinea pigs) were collected from Uttarakhand state of India. Sterile 100 ml Whirl-Pak bags (Nasco, Fort Atkinson, WI) were used to collect the samples. The samples were collected aseptically and immediately brought to the laboratory for processing as per previously published protocols [9 ,10 ].
In brief, the poultry ceca and poultry fecal samples were streaked directly onto the modified charcoal-cefoperazone-deoxycholate agar (mCCDA, HiMedia, India) plates and incubated at 42°C with 5% CO₂ in a CO₂ incubator for 48 h [10 ]. However, human and other animal fecal samples (1 g) were initially enriched in 9 ml Bolton Broth (Oxoid, UK) supplemented with 5% sheep blood. Thereafter, a loopful of the enriched broth suspension was streaked onto mCCDA plates and was incubated at the same time-temperature combination. The characteristic Campylobacter colonies (1-2 mm size, circular, flat to slightly raised, sticky, spreading, and shiny gray) were selected from each plate and tested biochemically.

Enumeration was carried out in accordance with ISO 10272-2:2016. To 25g of each pooled faecal or caecal sample, 225ml of Bolton Broth (Oxoid, Basingstoke, United Kingdom) was added. Samples were stomached for 60s and serially diluted 10-fold in maximum recovery diluent (MRD, Oxoid, Basingstoke, United Kingdom). Each dilution was spread on modified charcoal cefoperazone deoxycholate agar (mCCDA, Oxoid, Basingstoke, United Kingdom) and incubated at 42°C microaerobically (10% CO₂, 5% O₂ and balancing N₂) in a controlled atmosphere incubator (MACS VA-500, Don Whitley) for 48h.

A 25 g meat sample was mixed with 225 mL Bolton broth (Oxoid, Hampshire, UK and homogenized using a stomacher for 2 min, and then incubated at 37 °C for 4–6 h under microaerobic conditions (CampyGen, Oxoid, Hampshire, UK) and at 41.5 °C for 48 h. A loopful of the suspension was inoculated on a modified charcoal cefoperazone deoxycholate (mCCD; Oxoid, Hampshire, UK) agar and Preston agar (Oxoid, Hampshire, UK) and incubated at 41.5 °C for 48 h. To identify Campylobacter, the typical flat and moist grayish colonies, frequently with a metallic sheen, were observed on mCCD agar and the moist, gray, flat spreading colonies were observed on Preston agar [40 (link)]. The suspected colonies were streaked on a Columbia blood agar (CBA; Oxoid, Hampshire, UK) supplemented with 5% (v/v) sterile defibrinated sheep blood (Clinag, Bangkok, Thailand) and incubated under microaerobic conditions as described at 41.5 °C for 48 h. The pure cultures were then confirmed by morphology, motility, and oxidase tests [41 ,42 ], and multiplex PCR for genus and species confirmation followed the primers and condition in Denis’ study [43 (link),44 (link)]. For Campylobacter enumeration, the homogenate of 25 g and 225 mL of the diluent underwent ten-fold dilution, and 50 μL of the sample was dropped onto mCCD agar and incubated at 41.5 °C for 48 h under microaerobic conditions [42 ].

After transferring carcasses into a rinse solution, massaging, and shaking, as previously described, 10 ml was added into 10 ml of Bolton broth (Oxoid) supplemented with a selective supplement (SR0155 E; Oxoid). Samples were incubated at 42°C for 48 hr under microaerophilic conditions generated by a gas‐generating pack (Campygen CN25; Oxoid). Following incubation, a loopful of Bolton's broth was streaked onto modified cefoperazone charcoal deoxycholate agar plate (mCCDA; Oxoid) supplemented with a selective supplement (SR0155 E; Oxoid) and incubated at 42°C for 48 hr under microaerophilic conditions generated by a gas‐generating pack (Campygen CN25; Oxoid).