Coletsos medium

In a restricted set of samples (18 ONL, 13 BAL and 12 fecal samples from 5 wild boar and 7 red deer) bacteriological culture for M. bovis detection was performed. Briefly, 15 mL of lavages or 15 g of feces were decontaminated for 2 h with 30 mL of 0.75% hexa-decyl-pyridinium chloride solution, after which they were centrifuged at 2566g for 30 min; most supernatant was discarded and 0.25 mL aliquots of the sediment-supernatant inoculated in Coletsos medium (2 tubes for each sample) (BioMerieux, Marcy l’Étoile, France). The inoculated tubes were incubated at 37 °C for 15 weeks, checked weekly for any growth suspected to be MTC, which was then re-inoculated again in Coletsos medium. Isolates were identified by PCR for a panel of selected genes: 16S RNA, IS1081, Rv3120 and Rv1510 following the protocol by Huard et al. [32 (link)]. Briefly, 250 ng DNA were added to a solution of 6.5 μL of NZYTech Green Master Mix (NZYTech, Lisbon, Portugal), containing 1.3 U Taq polymerase, 1.5 mM MgCl₂, 1 μL of each primer at 20 mM and 5% DMSO, in a final volume of 25 μL. This mix was submitted to the following PCR protocol: denaturation at 94 °C for 5 min, 35 cycles at 94 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 1 min, with a final extension step of 72 °C for 10 min.