In this study, we examined genomic and phenotypic characteristics of a panel of MAP vaccine strains obtained from several laboratories around the world including both low and high passage examples of the 316 F lineage. Using a mouse model, we assessed the virulence ofrepresentative clades of three vaccine strains (2e, II, 316 F) with respect to a virulent MAP clinical isolate. The vaccine strains were clearly attenuated with regard to their ability to survive and persist in the mice as evidenced from the reduced numbers of MAP recovered and reduced numbers of leucocyte clusters containing AFB in the livers. This supports previous studies showing decreased persistence of the same 316 F and 2e strains in calves after 8 months
 and illustrates the utility of the C57BL/6 mouse model for virulence studies.
Using a pan-genomic MAP/MAH microarray we demonstrated that the genomes of all but one of the 316 F strains in the test panel contain the same full genome complement as the reference virulent bovine MAP type II strain MAPK10. One 316 F strain obtained from Norway (316FNOR1960) contained a single deleted region (vGI-19) spanning 21 ORF’s (including 10 MAP specific genes). Two strains not of the 316 F lineage (2eUK2000 and IIUK2000) contained a different deleted region (vGI-20), identical in both strains, spanning 34 ORF’s (including 10 MAP specific genes). Specific PCR for MAP1723 (Table
6), located within the vGI-20, on 2eUK2001 and IIUK2001 were negative confirming that the vGI-20 region was not present in these strains (Additional File
1). The inclusion of MAP-specific genes in these deleted regions is an important observation as these genes could provide the basis for differentiating infected from vaccinated animals (DIVA). Indeed, the deleted region vGI-19 contains part of the 38 kb pathogenicity island described by Stratmann et al. (2004)
 which contains genes encoding a number of antigens with diagnostic potential
. Both deleted regions vGI-19 and vGI-20 contain genes potentially involved in virulence and pathogenesis (Table
1) and their deletion could therefore have a profound effect on the virulence of these strains. In this study we demonstrated using a mouse model that both vaccine strains 2eUK2001 and IIUK2001 were attenuated with respect to a wild type MAP strain. In addition, vaccine strain IIUK2000 and IIUK2001 were found to contain a large 41 ORF tandem duplication (vGI-21) which includes copies of benzoate and lipid metabolic pathways. Vaccine strain 2eUK2000 comes from the same stock as 2eUK2001 and was maintained at the VLA, UK for over 50 years on a mineral deficient medium (Watson Reid ‘A’ block) whilst vaccine strain IIUK2000 was not. We suggest that the vGI-21 duplication in vaccine strain IIUK2000 was selected by these differences in media and fixed into the genome to compensate in vitro for the deletion of lipid biosynthesis and carbon usage repertoires, removed by the vGI-20 deletion.
The large deletion vGI-19 present in vaccine strain 316FNOR1960 was not present in any of the other 316 F strains including an early low passage lineage (316FCYP1966) and a more recent isolate (316 F2001) shown to be significantly attenuated in our virulence studies. Notably, part of vGI-19 is also present in the same gene order within the related MAH104 genome (GenBank reference CP000479). Together these suggest that any ancestral precursor and therefore the original 316 F strain would be unlikely to be missing vGI-19. We hypothesise that the vGI-19 deletion appeared in the 316FNOR1960 strain some point after its acquisition and transfer to Norway in 1960 from the VLA, UK. This strain is recorded as having been maintained, uniquely on Dubos medium with added pyruvate
 and we hypothesise that this medium was at some point selective. This is supported by the vGI-19 deletion in this strain including gene homologues of glyoxylate enzymes associated with pyruvate metabolism
. This strain previously has been used successfully as a live vaccine suggesting that it is attenuated. The knockout of the glyoxylate shunt could significantly affect the strain’s ability to control anaerobic respiration
 and intracellular persistence
, which may indicate that attenuation in this strain may be related to this loss. A 2002 isolate of this strain was shown to be highly immunogenic in goats
 and forms part of a 316 F/2e live commercial vaccine product (Paratuberkulose-vaksine, National Veterinaria Institute, Oslo, Norway) however there is no evidence that vGI-19 was deleted when the strain was used, albeit very effectively, on goats in the 1960-70’s
We tested the potential impact provided by deletion of the putative tellurite resistance gene (tehB) included in vGI-19 on 316FNOR1960 phenotype. Tellurite is highly toxic to bacteria due to its action on DNA synthesis. It is an important mechanism by which animals combat intracellular microorganisms
 and was used in early studies as a tuberculosis/leprosy therapeutic
. Bacterial resistance to tellurite is inducible, is associated with virulence
 and is linked to catalases which are required to process the superoxide anions generated as a result of bacterial metabolic mechanisms used to inactivate tellurite. We show a significantly increased sensitivity to tellurite in 316FNOR1960 whilst other 316 F strains either matched or exceeded the resistance of the two wildtype strains tested (K10:bovine, CAM87:caprine). Interestingly the strains most sensitive to tellurite were IIUK2000 and 2eUK2000 which lack the tehB gene. The metabolism of tellurite generates high reactive oxygen species which subsequently need to be de-toxified by catalase
. Significantly the vGI-20 deletion in these strains includes loss of the catalase gene homologue MAP1725c. Both vaccine deletion regions thus involve alterations in metabolic pathways associated with deactivation of high reactive oxygen species toxicity, which suggests this may be an important mechanism underlying attenuation in these strains.
Several of the other vaccine strains tested are also reported to have been maintained on markedly different media which may have similarly promoted or selected for genomic and phenotypic diversities. 316FNLD1978, available as a heat killed vaccination for dairy cattle since 1985
, was found to contain a large tandem duplication (vGI-22) unique to this strain. It is notable that this isolate was selectively subcultured on potato starch medium to enhance its growth (P. Willemsen personal communication) and now grows with difficulty on other media. It is tempting to speculate that the acquisition of extra copies of 14 ORFs including cell wall, fatty acid biosynthesis genes and two extra copies of IS900 are a direct result of the selective process performed on this strain.
We demonstrated in this study that vaccine strain 316FUK2001 was clearly attenuated with respect to wild type MAP strain JD87/107. The vGI-19 deletion found in 316FNOR1960 and the vGI-20 deletion found in 2eUK2000 and IIUK2000 were not detected by PCR in this strain suggesting that attenuation in this strain is due to different genetic polymorphisms. A duplicated region (vGI-1b) was detected in vaccine strain 316FUK2000, which may possibly have arisen as an adaptation to growth on liquid Watson Reid media.
Insertion sequences bordering specific genomic regions have previously been associated with variably tandem duplicated
 and possibly large genomic inversions
 and contribute highly to the degree of plasticity in the MAP genome. Variations in copy number of insertion elements including IS900, IS1311, IS256 and IS1652-like elements were seen between vaccine strains and virulent isolates. An IS1311 was found immediately bordering the vGI-1b region duplicated in 316 F-UK2000 but not other 316 F strains. Similar genomic variations including vGI-1b have been observed in virulent MAP strains
. IS900, a definitive element of MAP found in all clinical and vaccine strains, was also shown to be present in a variety of copy numbers. This work used comparative ratios of qPCR signals to estimate the average number of IS900 copies per cell per culture relative to two single copy MAP genes using an assumption determined from a MAP assembled genome sequence that MAPK10 would contain 17 copies. Our results confirm previous studies showing the vaccine strain 316v used in Australia for ELISA testing
 contains one less genomic copy of IS900 than most other 316 F strains
. Vaccine strain 316FNLD1978 exhibited higher gene signal ratios consistent with the two extra copies of IS900 copies inside the duplication of vGI-22. Vaccine strains IIUK2000 and 2eUK2000 contained lower signal ratios consistent with loss of an IS900 copy inside the deletion region vGI-20. Consistently however the calculated IS900 copy number in these strains was much lower than expected using the ratio method. Using site specific PCR we confirmed 16 IS900 filled insertion sites in the genomes of these strains whereas the ratio method, using MAPK10 as a standard, predicted only 13 copies. The reason could be technical, perhaps involving incomplete bacterial lysis of these unusual strains, however IS900 is known to replicate in episomal minicircles
 and when all consensus insertion sites are filled they may exist as extra genomic components awaiting transposition. If this is indeed the case, virulent MAP strains would have the capacity to contain more than the predicted 17 IS900 copies per cell. This could be an important factor in studies relying on qPCR to determine accurate estimates of MAP load
MIRU3 is a short tandem repeat sequence located within the sensX3-regX3 two component signalling system that controls carbon source usage and mechanisms reducing damaging reactive oxygen species generated by aerobic metabolism
. The attenuated BCG vaccine characteristically contains a low MIRU3 tandem repeat copy number which has been suggested to be involved in the control of sensX3-regX3 expression
. In this study 316 F strains (316FNLD1978, 316FUK2001, 316FNLD2008) had low MIRU3 copy numbers whilst others, mostly originating from older culture stocks, were larger. This may represent a potential for genomic drift within recent 316 F lineages and could be used as a marker for the most current 316 F VLA, UK strain but may also be suggestive that loss of intracellular persistence in full genome complement 316 F strains may be associated with reduced transcriptomic signalling capacity. It will be of interest therefore in future total genome sequencing studies to compare dysfunctional SNP variations within signalling features of 316 F strain genomes.