A total of 24 Map S-type isolates were obtained from Scotland (n = 6), Spain (n = 11), Canada (n = 2), New Zealand (n = 2), Faroe Islands (n = 2) and Iceland (n = 1), isolated from sheep, goats and a pig (see Table 1 and Additional file 1: Table S1). Isolates were propagated on slopes of one of the following media depending on what was used routinely in the supply laboratories: modified Middlebrook 7H11 supplemented with 20% (vol/vol) heat-inactivated newborn calf serum, 2.5% (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, Mo.); Herrold’s egg yolk medium with 2 μg ml⁻¹ mycobactin J or Lowenstein-Jensen medium with 2 μg ml⁻¹ mycobactin J. In addition, genotyping information obtained previously 11 18 19 from 148 Map C-type strains, 31 Maa isolates, 4 Mas isolates and 82 Mah isolates were used for comparison and phylogenetic analyses and are described in Additional file 1: Table S1.

¹ Country and regions: ES, Spain; CA, Canada; UK, United Kingdom; FO, Faroe Island; IS, Iceland; NZ, New Zealand.

² nd, not determined; alphanumeric nomenclature as defined by Pavlik et al., 1999 17 , alphabetic nomenclature correspond to new profiles identified in this study.

³ Nomenclature as defined by Stevenson et al., 2002 8 .

⁴ Nomenclature as defined by Thibault et al., 2007 11 .

⁵ Number of repeats at locus 292-X3-25-47-3-7-10-32 defined by Thibault et al., 2007 11 .

⁶ +, presence; -, absence.

Map strains were typed by IS900-RFLP as described previously 11 . Profiles were designated according to nomenclature previously described 17 20 21 22 . Profiles were analysed using Bionumerics™ software version 6.5 (Applied Maths, Belgium).

PFGE analysis was carried out using SnaBI and SpeI according to the published standardized procedure of Stevenson et al. 8 with the following modifications. Plugs were prepared to yield 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 enzyme was refreshed after four days. Restriction of plug DNA by SpeI was performed with 10U overnight after which the enzyme was refreshed and incubated for a further 6 hr. The parameters for electrophoresis of SpeI restriction fragments were changed to separate fragments of between 20 and 250Kb as determined by the CHEF MAPPER and electrophoresis was performed for 40 hr. Gel images were captured using an Alphaimager 2200 (Alpha Innotech). Profiles were analysed using Bionumerics™ software version 6.5 (Applied Maths, Belgium).

Primers (Additional file 2: Table S2) were designed for both gyrA (GenBank accession no. 2720426 [Genome number: NC_002944.2]) and gyrB genes (GenBank accession no. 2717659 [Genome number: NC_002944.2]). The PCR mixture was composed as follows using the GoTaq Flexi DNA polymerase (Promega). Two microliters of DNA solution was added to a final volume of 50 μl containing 0.2 μl of GoTaq Flexi DNA polymerase (5 U/μl), 2 mM (each) dATP, dCTP, dGTP, and dTTP (Promega); 10 μl of 5x PCR buffer supplied by the manufacturer; 1 μM of each primers; and 1.5 mM of MgCl₂. The reactions were carried out using a TC-512 thermal cycler (Techne). PCR conditions were as follows: 1 cycle of 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C; and 1 cycle of 7 min at 72°C. PCR products were visualized by electrophoresis using 1.5% agarose gels (agarose electrophoresis grade; Invitrogen), purified using NucleoSpin® Extract II (Macherey-Nagel) and sequenced by GenomExpress (Grenoble, France). Sequence analysis and SNP detection were performed by using the Bionumerics™ software version 6.5 (Applied Maths, Belgium).

Primers were used according to Semret et al. 12 and described in Additional file 2: Table S2. The PCR mixture comprised 2 μl of DNA solution added to a final volume of 50 μl containing 0.2 μl of GoTaq Flexi DNA polymerase (5 U/μl Promega), 2 mM (each) dATP, dCTP, dGTP, and dTTP (Promega); 10 μl of 5x PCR buffer supplied by the manufacturer; 1 μM of each primers; 1 μL of dimethyl sulfoxide (Sigma) and 1.5 mM of MgCl₂. The reactions were carried out using a TC-512 thermal cycler (Techne). PCR conditions were as follows: 1 cycle of 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; and 1 cycle of 7 min at 72°C. To detect presence or absence of each LSP, PCR products were analyzed by electrophoresis using 1.5% agarose gels.

DNA in agarose plugs prepared for PFGE analysis was used for MIRU-VNTR analysis according to Stevenson et al. 23 . Small pieces of agarose plug, approximately 2 mm thick, were washed in TE buffer (pH 8) to remove residual EDTA in the storage buffer. One hundred microlitres of TE buffer were added to the agarose and the sample boiled for 10 min to melt the agarose. Five microlitres were used for PCR and MIRU-VNTR analysis interrogating eight polymorphic loci was performed as described by Thibault et al. 11 . The allelic diversity (h) at a locus was calculated by using Nei’s index (see Additional file 3: Table S4) h = 1 − ∑ x _i ² n/(n − 1)], where x _i is the frequency of the i ^th allele at the locus, and n the number of isolates 24 .

The Simpson Discrimination Index (DI) described by Hunter and Gaston 25 was used as a numerical index for the discriminatory power of each typing method PFGE, IS900-RFLP and MIRU-VNTR and combinations of the typing methods (see Table 2 and Additional file 3: Table S4). The DI was calculated using the following formula:

D I = 1 − 1 N N − ∑ j = 1 s n j n j − 1

¹: Panel of strains selected to represented the whole diversity of RFLP and MIRU-VNTR profiles of type C strains.

²: No. Number of strains analyzed.

Where N is the total number of isolates in the typing scheme, s is the total number of distinct patterns discriminated by each typing method and strategy, and n _j is the number of isolates belonging to the jth pattern.

The existence of two major Map lineages was previously supported by the distribution of two LSPs specific for Map as described by Semret et al. 12 26 27 : the region corresponding to LSP^A20 is specifically absent from strains of the sheep lineage whereas that corresponding to LSP^A4 is specifically absent from strains of the cattle lineage. The distribution of these LSPs was thus investigated across our representative panel of Map S-type strains from various origins. As shown in Figure 1, analysis by PCR supports the association of the LSP^A20 region with C-type strains whereas the LSP^A4 region is present in all S-type strains. Presence of the LSP^A4 region was not related to PFGE subtype I versus III, of the country of origin and pigmentation status (Table 1).

Since SNPs found in gyrA and B genes have been reported to be subtype (I, II, III)-specific, the panel of Map S-type strains was subjected to SNP analysis and compared to C type K-10 strain. As shown in Table 3, consensus sequences obtained matched those previously published and distinguished types I, II and III of Map.

*K10 sequenced strain type II used as sequence reference.

PFGE analysis results were obtained for 15 S-type and 24 C-type strains (Figure 2A and 2B). The sequenced K10 type II strain was also included. SnaB1 or SpeI analyses segregated strains according to the two sheep and cattle lineages and at the subtype level I, II and III. With SnaBI and SpeI individually, 5 different profiles were obtained for the 5 type I strains and 9 different profiles for the 10 type III strains. The type II strains exhibited 15 different SnaBI profiles, with profile 2 being the most frequent (8 strains) and 14 different SpeI profiles with profile 1 being the most frequent (11 strains). The DI of the subtype I and subtype III were respectively 1 and 0.956 for SnaB1 and 1 and 0.978 for SpeI and that of C-type (Type II) was 0.895 for SnaBI and 0.801 for SpeI (see Table 2 and Additional file 3: Table S4). DI of 0.96 and 0.924 for SnaBI and SpeI respectively was achieved for the 39 Map strains presented in Figure 2A and 2B. The combination of both enzymes gave 39 unique multiplex profiles (see Table 1 and Additional file 1: Table S1).

IS900-RFLP typing clearly separated the strains into three groups that correlate with the PFGE subtypes I, II and III (Figure 3). Ten strains of S-type, subtype I cluster into two groups of profiles S1 (n = 2) and S2 (n = 8). The 14 strains of S-type, subtype III display more polymorphism with 9 profiles, including 6 new ones. Profiles previously described included I1 (n = 1), I2 (n = 1) and I10 (n = 2). The new profiles were called A (n = 3), B (n = 2), C (n = 2), D, E and F (n = 1 each) (indicated in the Additional file 4: Figure S1). The strains of C-type were well distinguished from S-type and were not highly polymorphic. In this panel of strains the most widely distributed profile R01 was found for 21 strains, then R09 (n = 2) and R34 (n = 2) and 10 profiles were identified in only one isolate, R04, R10, R11, R13, R20, R24, R27, R37, C18 and C20. With this Map panel of strains the discrimination index (DI) of RFLP was shown very variable depending on the type and the subtype of the strains. The DI of the subtype I was very low (0.356), for the subtype III high (0.934) and that of C-type (Type II) was low (0.644) (Table 2). A DI of 0.856 was achieved for the 59 Map strains presented in Figure 3.

The result of MIRU-VNTR typing of the S-type strains is shown in Table 1. MIRU-VNTR data from 148 C-type (type II) strains previously described 11 18 19 were included in the analysis (see Additional file 1: Table S1). MIRU-VNTR using the eight markers described previously 11 could differentiate between S- and C-type strains but not between the subtypes I and III. On this panel of strains, type III strains were the most polymorphic with a DI of 0.89 compared to 0.644 for type I strains and 0.876 for type II strains selected to represent the diversity of INMV profiles described. INMV profiles 21, 70 and 72 were shared by both type I and III strains. As described previously 11 IS900 RFLP and MIRU-VNTR typing may be used in combination to gain higher resolution. This was verified also on this panel of strains including S-type. In total, the combination of the two methods distinguished 32 distinct patterns comprising 59 isolates. Therefore, using carefully on the same set of strains, a DI of 0.977 was achieved for this panel by using IS900 RFLP and MIRU-VNTR typing in combination compared to 0.856 for IS900 RFLP typing alone and 0.925 for MIRU-VNTR typing (Table 2 and Additional file 3: Table S4). Because MIRU-VNTR is applicable to all members of the MAC, we wanted to know how the INMV profiles segregated within the MAC. None of the INMV profiles identified in the S-type strains matched those of other MAC members. The results presented by the minimum spanning tree in Figure 4, show that Map S-type strains are clearly separated from Map C-type strains, including 113 strains previously typed, and also from any strains belonging to the other subspecies hominissuis, avium or silvaticum. The allelic diversities of the various loci are shown in Additional file 5: Table S3. Five markers were monomorphic in Map S subtype III and 7 in Map S subtype I. In terms of the discriminatory hierarchy, locus 292 displayed the highest allelic diversity for both S- and C-type strains. This study shows that genotyping with MIRU-VNTR can distinguish MAC isolates to the species level and also distinguish with MAP subspecies to the strain type level.