5% (vol/vol) glycerol, 2 mM asparagine,

10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment medium (Becton Dickinson, Oxford, Oxfordshire, United Kingdom), Selectatabs (code MS 24; MAST Laboratories Ltd., Merseyside, United Kingdom), and 2 μg ml-1 mycobactin J (Allied Monitor, Fayette, LGX818 cost Mo.); Herrold’s egg yolk medium with 2 μg ml-1 mycobactin J or Lowenstein-Jensen medium with 2 μg ml-1 mycobactin J. For the typing panel, three Map isolates were included to represent the three strain types described in Map [11, 12]. In addition, three isolates (one bovine, one ovine and one caprine) were duplicated in the panel as internal controls for the reproducibility of the typing methods and M. bovis BCG, M. phlei and IS901 positive M. avium (it is not known if this isolate is M. avium subsp. avium or M. avium subsp. silvaticum) were included as negative controls. The isolates were coded with an EU reference number (see supplementary dataset in Additional file 1) and genotyped in a blind study. IS900-RFLP method The typing laboratories were provided either with cultures or with DNA in agarose Tucidinostat mouse plugs that had been prepared for PFGE typing. DNA extraction from cultures and IS900-RFLP analysis was performed using the standardized procedure published by Pavlik et al. [50]. Where plugs were provided, the

restriction digests were carried out in the presence of agarose as described for PFGE [51]. Briefly, a 3-5 mm insert of agarose was cut from the plug, washed extensively in TE buffer and pre-incubated with the appropriate restriction buffer containing 0.1 mg ml-1 BSA. After one hr the buffer was discarded and replaced with fresh buffer containing the restriction endonuclease and incubated overnight at 37°C. The agarose containing the digested DNA was then

loaded into the wells of an Tangeritin agarose gel as described in the standardized procedure [51]. New profiles were designations assigned by the National Veterinary Institute, Brno using the standard nomenclature described. Profiles were analysed using Gel Compar (Biomathematics, Belgium). PFGE analysis PFGE analysis was carried out using SnaBI and SpeI according to the published standardized procedure of Stevenson et al. [11] with the following modifications. Plugs were prepared to give a density of 1.2 × 1010 cells ml-1 and the incubation time in lysis buffer was increased to 48 hr. The concentration of lysozyme was increased to 4 mg ml-1. Incubation with proteinase K was carried out for a total of seven days and the CA4P enzyme was refreshed after four days. Restriction endonuclease digestion of plug DNA by SpeI was performed with 10 U overnight in the appropriate restriction endonuclease buffer supplemented with 0.1 mg ml-1 BSA, after which the enzyme was refreshed and incubated for a further 6 hr.