An inoculum of C. jejuni ATCC 33291 (~104 colony-forming units (cfu/mL)) was used. Two types of media were used to activate the strain, Campylobacter blood-free selective medium (modified CCDA-Preston) (Oxoid CM 0739, Basingstoke, UK) supplemented with CCDA Selective Supplement (SR 0155) and Bolton Selective Enrichment Broth (Oxoid CM0983, Basingstoke, UK), and then cooled to 45 to 50 °C, and 25 mL of laked horse blood (SR0048) and one vial of Bolton Broth Selective Supplement (Oxoid SR0183 Basingstoke, UK), were aseptically added. The plates were incubated at 42 °C for 24 to 72 h.
Bolton selective enrichment broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Bolton Selective Enrichment Broth is a microbiological culture medium used for the selective enrichment of Campylobacter species from food, environmental, and clinical samples. The broth contains specific nutrients and growth supplements that support the growth of Campylobacter while inhibiting the growth of competing microorganisms.
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2 protocols using bolton selective enrichment broth
1
Campylobacter jejuni ATCC 33291 Activation
2
Campylobacter Isolation and Identification
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On fecal and liver samples, Campylobacter isolation and preliminary identification at the genus level were carried out after enrichment in Bolton Selective Enrichment Broth (Oxoid, Basingstoke, UK) following the ISO 10272-1:2017 method with a supplemental filtration phase of the incubated enrichment broth, as described in Pedonese et al. (2017) .
The isolates were cultured on Columbia blood agar plates at 42 °C for 48 h in microaerobic atmosphere and DNA was extracted using Maxwell instrument (Promega Corporation, Madison, WI, USA) and quantified using a Nanodrop Spectrophotometer (Nanodrop Technologies, Celbio Srl., Milan, Italy). Isolates were identified at the species level using a multiplex PCR (Wang et al. 2002) for the five most important Campylobacter species from a clinical point of view and also a simplex PCR (Di Giannatale et al. 2016) for C. jejuni.
Among all the sampled animals (193), 60 animals were chosen for the analysis of possible genetic resistance to Campylobacter and divided in two groups: Campylobacter negative (32 animals) and Campylobacter positive (28 animals). Of the 28 Campylobacter-positive wild boars, 22 were positive for C. coli and 6 for C. jejuni). In the positive animals, Campylobacter was isolated from the feces (78.57%), the liver (14.29%) and from both feces and liver (7.14%).
The isolates were cultured on Columbia blood agar plates at 42 °C for 48 h in microaerobic atmosphere and DNA was extracted using Maxwell instrument (Promega Corporation, Madison, WI, USA) and quantified using a Nanodrop Spectrophotometer (Nanodrop Technologies, Celbio Srl., Milan, Italy). Isolates were identified at the species level using a multiplex PCR (Wang et al. 2002) for the five most important Campylobacter species from a clinical point of view and also a simplex PCR (Di Giannatale et al. 2016) for C. jejuni.
Among all the sampled animals (193), 60 animals were chosen for the analysis of possible genetic resistance to Campylobacter and divided in two groups: Campylobacter negative (32 animals) and Campylobacter positive (28 animals). Of the 28 Campylobacter-positive wild boars, 22 were positive for C. coli and 6 for C. jejuni). In the positive animals, Campylobacter was isolated from the feces (78.57%), the liver (14.29%) and from both feces and liver (7.14%).
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