PFGE typing was undertaken at the Moredun Research Institute, Scotland, UK and VISAVET, Madrid, Spain. IS900-RFLP typing Everolimus research buy was carried out at the Veterinary Research Institute in Brno, Czech Republic and VISAVET. Published standardized typing procedures were used as described in Materials and Methods. The only difference

in procedures between laboratories was that at VISAVET the IS900-RFLP analysis was performed using the Enzalutamide manufacturer agarose plugs prepared for PFGE to avoid having to perform two separate DNA preparations for the different typing techniques. The correct profiles were reported by all laboratories for the duplicate isolates included to check reproducibility. All typing techniques correctly reported that the Mycobacterium phlei (M. phlei), Mycobacterium bovis BCG (M. bovis BCG) and IS901 positive M. avium were not Map. One field isolate, EU112 was found to be IS901 positive M. avium (it see more is not known if the isolate is M. avium subsp. avium or M. avium subsp. silvaticum) and not Map as was originally suspected. Another isolate, EU169 was

found to be a mixed culture. Isolates one to 50 were typed at Institut für Mikrobiologie Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany using the Type I/Type II PCR as described by Dohmann et al. [17]. EU25 and EU30 were identified as Type I and all other field isolates as Type II. These results correlated with the strain type as determined Phosphoglycerate kinase by PFGE. This PCR [17] cannot

discriminate between Type I and Type III and as strain types could be discerned from the PFGE profiles, it was not considered necessary to determine the strain type of the remaining isolates by PCR. It was not possible to type all of the isolates with all typing methods as some laboratories had difficulties in subculturing some isolates to prepare sufficient cells for analyses. A total of 123 Map isolates were typed by IS900-RFLP, PFGE and MIRU-VNTR. IS900-RFLP typing IS900-RFLP typing data were obtained for 147 Map isolates (Table 1 and see supplementary dataset in Additional file 1). It was not possible to obtain PstI profiles for 55 isolates or clear BstEII profiles for five isolates. There was a problem using agarose plug DNA for IS900-RFLP typing with PstI as the enzyme would not cleave in the presence of agarose. Extraction of the DNA from the agarose and repeat PstI digestion was not attempted. As expected, profiles were not obtained for the negative control strains M. bovis BCG, M. phlei and IS901 positive M. avium. A total of six PstI profiles were found among 93 isolates: B (n = 88); G (n = 1); I (n = 1); K (n = 1); R (n = 1); and U (n = 1). Seventeen BstEII profiles were detected among 142 isolates: C1 (n = 71); C17 (n = 49); C5 (n = 5); C9 (n = 3); C16 (n = 2) and single isolates with C10, C18, C22, C27, C29, C35, C36, C38, C39, S4, I4 and I5.