BACKGROUND
mycobacterium avium subspecies paratuberculosis  is a proven enteric pathogen with a wide host range that includes many domestic and wild animals
<cit> .
it is the causal agent of johne’s disease  in animals which is particularly common in countries with significant dairy industries leading to considerable economic losses
<cit> .
map can infect, disseminate and persist in humans and has been suggested as a contributory factor in the development of crohn’s disease
<cit> .
map vaccines are a major tool used in the control of jd in animals and can be highly profitable
<cit> .
they have advantages over herd management
<cit>  and culling strategies
<cit>  in being more cost efficient, easier to implement on a wide scale and less reliant on diagnostic testing.
it is clear however, that although able to prevent a majority of animals from reaching onset of clinical disease, their current formulations provide incomplete protection against infection and shedding
<cit> , thus failing to eradicate the organism
<cit> .
most current whole cell vaccine preparations rely on subcultures of classic strains that were generated over 70 years ago
<cit>  and some evidence suggests that, for killed preparations at least, more recently acquired local virulent strain types may be more effective
<cit> .
previous experience with bcg has shown that frequent in vitro passage of strains in different laboratories led to significant alterations in genomic profiles and diversities in attenuation and immunogenicity
<cit> .
it is of importance therefore to derive accurate definitions of map vaccine genotypes to better standardize vaccine manufacture and understand the critical mechanisms determining vaccine attenuations and protective efficacies.
the distribution and worldwide use of map vaccines has continued since live ‘attenuated’ strains were selected in france  and the uk  using a method of sequential passage similar to that applied for the generation of bcg
<cit> .
the degree and mechanism underlying their attenuation however is uncertain as virulence studies were not performed in any detail.
concerns in the 1980’s regarding the use of live vaccine strains because of low shelf life and spread to the environment promoted the use of killed vaccine formulations.
these were based on various combinations of three map strains comprising strain 2e from the uk, strain ii from canada and 316 f. a combination of these were used as live preparations for norwegian goats in the 1960’s and 1970’s
<cit>  and as a killed preparation delivered in mineral oil to icelandic sheep in the 1950’s
<cit> .
live vaccine formulations of 316 f alone were used in the 1960’s and 70’s in the uk
<cit>  and cyprus
<cit> , 1980’s in hungary
<cit> , 1990’s in germany
<cit>  and spain
<cit>  and up until  <dig> in new zealand
<cit> .
killed preparations of 316 f alone have been used extensively worldwide
<cit>  and are still available for commercial use.
these strains, due to the difficulty in retaining mycobacteria in frozen seed stocks, have been maintained through regular subculture on a variety of laboratory in-house media.
it is unsurprising therefore, that some reports suggest strain adaptation to growth in specialized media with loss of mycobactin j dependence
<cit>  and genome diversity
<cit>  has occurred amongst some lineages.
in this work we demonstrate attenuation and differential virulence of vaccine strains 2e, ii and 316 f in a mouse model and use a full map genome microarray, supported by pcr and sequencing to investigate the genomic shifts of vaccine strains from a variety of lineages, including one recently resuscitated 316 f strain, originally lyophilised in  <dig>  we describe large genomic regions with deletions and tandem duplications uniquely associated with each vaccine clade, demonstrate the functionality of some of these deleted genes and hypothesise as to their role in virulence attenuation.
RESULTS
comparative genomic hybridisation of vaccine strains
mapac hybridisations comparing each vaccine strain against a mapk <dig> reference control were made  and averaged values displayed as scatterplots .
significant loss of signals in contiguous genes representative of large variable genomic island  deletions were identified in a  <dig>  kbp region of 316fnor <dig>  and a  <dig>  kbp region in both iiuk <dig> and 2euk <dig> .
two fold increases in signals were also seen in contiguous genes within a  <dig>  kbp region of iiuk <dig> , a  <dig>  kbp region of 316 f-nld <dig>  and a  <dig>  kbp 316fuk <dig> .
* = map specific genes.
* = map specific genes.
signal ratios from genes in multiple copies in the genome such as insertion elements are more difficult to interpret as changes in signal are related to copy number.
nevertheless is <dig> signal ratios were significantly reduced in iiuk <dig>  2euk <dig>  316fnor <dig>  316v, 316fuk <dig> and elevated in 316fnld <dig> and 316fnld <dig>  increases in signals for is1652-like elements were also seen in iiuk <dig>  2euk <dig>  316fuk <dig>  316fnld <dig> and 316fnld <dig> and decreases for this element in 316fnor <dig>  is256-like elements were more abundant in 2euk <dig> and also raised in 316v and 316fuk <dig> but decreased in 316fnor <dig> relative to mapk <dig>  is1311-like elements were increased in 316v and decreased in 316fnor <dig>  one of these elements  borders the previously described vgi-1b region which is bounded by short inverted repeat sequences and duplicated in map type iii strains
<cit> .
the tandem duplication vgi- <dig> was confirmed using primer set map <dig> seq <dig> and map <dig> r  orientated to amplify only the end of one tandem repeat and the beginning of the other.
this produced an amplicon of 651 bp only with vaccine strain iiuk <dig> and iiuk <dig> .
sequencing of this amplicon showed the duplication event to occur relative to the reference strain k <dig> genome  at position  <dig> within the first copy of map2733c  followed by a unique region of 16 bp  and then a second copy of map <dig>  followed by the vgi- <dig> duplication thus comprising a tandem duplication of  <dig>  bp spanning  <dig> orfs .
pcr using primer pairs specific for this tandem duplication map <dig> seq <dig> and map <dig> r was negative for all other strains included in this study.
map gene annotation corresponds to genbank ae <dig>
vgi- <dig> was confirmed as a tandem duplication using primer set map <dig> f and map <dig> r  which produced an amplicon of 2967 bp with vaccine strain 316 f-nld <dig> only.
sequencing of this region showed the duplication event to comprise  <dig>  bp spanning  <dig> orfs .
the duplication site occurs at genbank accession ae <dig> position  <dig> within the first copy of map <dig>  followed by an overlap region of 3 bp  and then the second copy of map1748c  followed by the vgi- <dig> duplication.
pcr with primer pairs specific for this tandem duplication  was negative for all other strains included in this study.
several attempts to localise the vgi-1b duplication using outward facing primers around the region in a similar manner to vgi- <dig> and vgi- <dig> were unsuccessful and this region may not be present in 316uk <dig> as a tandem duplication.
qpcr was performed using both map <dig> and map <dig> specific primer pairs located inside vgi-1b and the relative copy number of each compared against a single copy genome target control represented by primer pairs to map2114c.
both map <dig> and map <dig> pairs showed a doubling of signal strengths relative to map2114c in strain 316fuk <dig> whilst all other strains included in this study showed signal consistent with single copies of both these genes .
nd = not done.
* vgi status for vgi-1b, vgi- <dig> - vgi- <dig> either duplicated  or deleted , else no entry designates present as a single copy region.
is <dig> insertion site analysis
to determine which is <dig> sites were absent relative to the k <dig> reference genome, pcr primers were designed to specifically amplify each of the known  <dig> is <dig> loci .
these were used to confirm the insertion of is <dig> into each locus in the reference strain k <dig> and were also all positive in all 316 f strains and a caprine isolate cam <dig>  both vaccine strains iiuk <dig> and 2euk <dig> were missing is <dig> whereas is <dig> was also missing from vaccine strain 2euk <dig> but present in all other strains including vaccine strain iiuk <dig>  comparative qpcr of is <dig> copy number relative to map2114c, demonstrated a range of is <dig> copies in vaccine strains that corresponded to the trend in hybridisation signals observed in mapac scatterplots .
the ratio of copy number however was surprisingly higher than predicted, with vaccine strains iiuk <dig> and 2euk <dig> having only  <dig> copies whilst mapk <dig> and 316 f strains gave signals correspondent with 16– <dig> relative is <dig> copy numbers .
functional analysis of tellurite resistance
one map specific gene predicted to be deleted in vgi- <dig> was map <dig> , a s-adenosylmethionine-dependent methyltransferase with homologues to tellurite resistance genes  involved in bacterial virulence and persistence
<cit> .
the functionality of this gene in mycobacteria has not previously been investigated.
using a solid culture plate assay we compared tellurite resistance  of map strains with and without the vgi- <dig> deletion .
this demonstrated a wide mic range  between strains, with significant reductions associated with vgi- <dig>  deletion over full genome complement strains.
of note however was the very low level of tellurite resistance  found in strains containing the vgi- <dig>  deletion.
assessment of virulence using a mouse model
the virulence of vaccine strains 316fuk <dig>  iiuk <dig> and 2euk <dig> was compared with wild type strain jd87/ <dig> in a mouse model.
ten mice from each of five groups  were killed at  <dig>   <dig> and 12 weeks post inoculation.
body, spleen and liver weights were recorded.
samples of the liver were taken for bacteriological culture and histopathology.
mean bodyweights increased with age, but no statistically significant difference was observed in mean body weight between any of the vaccine strains and the control wild type strain at any of the time points .
there was strong evidence of a difference in pattern over time between the mean rank spleen weight expressed as a percentage of body weight of the mice inoculated with the different map strains relative to those receiving pbs, where those inoculated with map had increasingly higher mean rank percentage weights .
differences between the map strains were not formally statistically significant  although the control virulent strain jd87/ <dig> showed an increase in mean rank spleen weight percentage between weeks  <dig> and  <dig>  and 316fuk <dig> had an increase between weeks  <dig> and  <dig>  there was no statistical evidence for differences in the mean levels of liver weight expressed as a percentage of body weight either for different strains or over time for any of the map strains .
however, there was some evidence of a difference between the means for the map strains and the lower mean weights associated with pbs .
map was recovered from the liver tissue of mice four weeks post inoculation in all groups except the control group inoculated with pbs.
by 12 weeks post infection, map was recovered from the tissues of only one mouse inoculated with vaccine strain 2euk <dig> , from  <dig> mice inoculated with iiuk <dig>  and from all the mice inoculated with the virulent jd87/ <dig> strain  suggesting attenuation of each of the vaccine strains .
mean rank counts increase over time for the jd87/ <dig> strain, while dropping for all the other map strains, this being most rapid for the 2euk <dig> strain but ultimately most notable for strain 316fuk <dig>  statistical assessment of the effect of the strain by time interaction on the mean rank count indicate that differences exist in the abilities of the map vaccine strains to survive or persist in mice .bmc1010
infections of the liver result in multifocal hepatitis characterised by clusters of inflammatory cells.
to measure the effect of the experimental infection with map, the densities of mononuclear leucocyte clusters were counted.
small numbers of leucocyte clusters  were observed in liver samples from mice inoculated with pbs, though no acid fast bacilli  were detected in any of these samples.
large differences in the mean ranked density of leucocyte clusters between strains were identified  with the wild type strain jd87/ <dig> having the highest mean ranked densities of clusters .
strain 2euk <dig> showed evidence of higher mean rank densities than the 316fuk <dig> and iiuk <dig> strains .
the ranked density of leucocyte clusters with afb showed highly statistically significant differences between the means of map strains , with the jd87/ <dig> strain consistently showing higher mean densities, with this effect being more pronounced from 8 weeks post infection .
the vaccine strains all tended to exhibit increasingly lower mean ranked densities over the lifetime of the experiment , with consistent patterns of differences between strains : strain iiuk <dig> showed the largest mean rank densities, strain 316fuk <dig> the lowest, with 2euk <dig> intermediate.
the histopathology results show that all strains elicited a similar inflammation at 4 weeks.
only thereafter some differences between the inflammatory responses to the strains became apparent.
in addition, the analysis of mean bacterial counts and afb positive clusters showed the reduced ability of the vaccine strains to survive and persist within mice.
overall, these results provide proof of attenuation of the vaccine strains with respect to a wild type map strain.
discussion
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  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
<cit>  and illustrates the utility of the c57bl/ <dig> 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 mapk <dig>  one 316 f strain obtained from norway  contained a single deleted region  spanning  <dig> orf’s .
two strains not of the 316 f lineage  contained a different deleted region , identical in both strains, spanning  <dig> orf’s .
specific pcr for map <dig> , located within the vgi- <dig>  on 2euk <dig> and iiuk <dig> were negative confirming that the vgi- <dig> region was not present in these strains .
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 .
indeed, the deleted region vgi- <dig> contains part of the 38 kb pathogenicity island described by stratmann et al.
<cit>  which contains genes encoding a number of antigens with diagnostic potential
<cit> .
both deleted regions vgi- <dig> and vgi- <dig> contain genes potentially involved in virulence and pathogenesis  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 2euk <dig> and iiuk <dig> were attenuated with respect to a wild type map strain.
in addition, vaccine strain iiuk <dig> and iiuk <dig> were found to contain a large  <dig> orf tandem duplication  which includes copies of benzoate and lipid metabolic pathways.
vaccine strain 2euk <dig> comes from the same stock as 2euk <dig> and was maintained at the vla, uk for over 50 years on a mineral deficient medium  whilst vaccine strain iiuk <dig> was not.
we suggest that the vgi- <dig> duplication in vaccine strain iiuk <dig> 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- <dig> deletion.
the large deletion vgi- <dig> present in vaccine strain 316fnor <dig> was not present in any of the other 316 f strains including an early low passage lineage  and a more recent isolate  shown to be significantly attenuated in our virulence studies.
notably, part of vgi- <dig> is also present in the same gene order within the related mah <dig> genome .
together these suggest that any ancestral precursor and therefore the original 316 f strain would be unlikely to be missing vgi- <dig>  we hypothesise that the vgi- <dig> deletion appeared in the 316fnor <dig> strain some point after its acquisition and transfer to norway in  <dig> from the vla, uk.
this strain is recorded as having been maintained, uniquely on dubos medium with added pyruvate
<cit>  and we hypothesise that this medium was at some point selective.
this is supported by the vgi- <dig> deletion in this strain including gene homologues of glyoxylate enzymes associated with pyruvate metabolism
<cit> .
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
<cit>  and intracellular persistence
<cit> , which may indicate that attenuation in this strain may be related to this loss.
a  <dig> isolate of this strain was shown to be highly immunogenic in goats
<cit>  and forms part of a 316 f/2e live commercial vaccine product  however there is no evidence that vgi- <dig> was deleted when the strain was used, albeit very effectively, on goats in the 1960-70’s
<cit> .
we tested the potential impact provided by deletion of the putative tellurite resistance gene  included in vgi- <dig> on 316fnor <dig> 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
<cit>  and was used in early studies as a tuberculosis/leprosy therapeutic
<cit> .
bacterial resistance to tellurite is inducible, is associated with virulence
<cit>  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 316fnor <dig> whilst other 316 f strains either matched or exceeded the resistance of the two wildtype strains tested .
interestingly the strains most sensitive to tellurite were iiuk <dig> and 2euk <dig> which lack the tehb gene.
the metabolism of tellurite generates high reactive oxygen species which subsequently need to be de-toxified by catalase
<cit> .
significantly the vgi- <dig> 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.
316fnld <dig>  available as a heat killed vaccination for dairy cattle since 1985
<cit> , was found to contain a large tandem duplication  unique to this strain.
it is notable that this isolate was selectively subcultured on potato starch medium to enhance its growth  and now grows with difficulty on other media.
it is tempting to speculate that the acquisition of extra copies of  <dig> orfs including cell wall, fatty acid biosynthesis genes and two extra copies of is <dig> are a direct result of the selective process performed on this strain.
we demonstrated in this study that vaccine strain 316fuk <dig> was clearly attenuated with respect to wild type map strain jd87/ <dig>  the vgi- <dig> deletion found in 316fnor <dig> and the vgi- <dig> deletion found in 2euk <dig> and iiuk <dig> were not detected by pcr in this strain suggesting that attenuation in this strain is due to different genetic polymorphisms.
a duplicated region  was detected in vaccine strain 316fuk <dig>  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
<cit>  and possibly large genomic inversions
<cit>  and contribute highly to the degree of plasticity in the map genome.
variations in copy number of insertion elements including is <dig>  is <dig>  is <dig> and is1652-like elements were seen between vaccine strains and virulent isolates.
an is <dig> was found immediately bordering the vgi-1b region duplicated in 316 f-uk <dig> but not other 316 f strains.
similar genomic variations including vgi-1b have been observed in virulent map strains
<cit> .
is <dig>  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 is <dig> copies per cell per culture relative to two single copy map genes using an assumption determined from a map assembled genome sequence that mapk <dig> would contain  <dig> copies.
our results confirm previous studies showing the vaccine strain 316v used in australia for elisa testing
<cit>  contains one less genomic copy of is <dig> <cit>  than most other 316 f strains
<cit> .
vaccine strain 316fnld <dig> exhibited higher gene signal ratios consistent with the two extra copies of is <dig> copies inside the duplication of vgi- <dig>  vaccine strains iiuk <dig> and 2euk <dig> contained lower signal ratios consistent with loss of an is <dig> copy inside the deletion region vgi- <dig>  consistently however the calculated is <dig> copy number in these strains was much lower than expected using the ratio method.
using site specific pcr we confirmed  <dig> is <dig> filled insertion sites in the genomes of these strains whereas the ratio method, using mapk <dig> as a standard, predicted only  <dig> copies.
the reason could be technical, perhaps involving incomplete bacterial lysis of these unusual strains, however is <dig> is known to replicate in episomal minicircles
<cit>  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  <dig> is <dig> copies per cell.
this could be an important factor in studies relying on qpcr to determine accurate estimates of map load
<cit> .
miru <dig> is a short tandem repeat sequence located within the sensx3-regx <dig> two component signalling system that controls carbon source usage and mechanisms reducing damaging reactive oxygen species generated by aerobic metabolism
<cit> .
the attenuated bcg vaccine characteristically contains a low miru <dig> tandem repeat copy number which has been suggested to be involved in the control of sensx3-regx <dig> expression
<cit> .
in this study 316 f strains  had low miru <dig> 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.
CONCLUSIONS
this study has shown that significant genomic diversity exists between map vaccine strains and within the 316 f lineage.
these include large deletions, duplications and changes in insertion sequence copies.
these mutations were probably derived in a classical manner by selective subculture on laboratory media and in some cases have led to significant alterations of phenotype and attenuation.
there were  <dig> map specific gene deletions identified of which at least one could be linked to phenotypic change that would disadvantage its persistence in the host and thus associates it with virulence.
furthermore, these map-specific gene deletions could provide the basis for a diva diagnostic for use with these vaccines.
overall, this work illustrates that map genome plasticity can be influenced by in vitro culture over long periods and a robust definition of vaccine strain genome lineage will be necessary in the future to guarantee consistency between studies.

 mycobacterium avium subspecies paratuberculosis is a proven enteric pathogen with a wide host range that includes many domestic and wild animals. it is the causal agent of johne’s disease in animals which is particularly common in countries with significant dairy industries leading to considerable economic losses. map can infect, disseminate and persist in humans and has been suggested as a contributory factor in the development of crohn’s disease. map vaccines are a major tool used in the control of jd in animals and can be highly profitable. they have advantages over herd management and culling strategies in being more cost efficient, easier to implement on a wide scale and less reliant on diagnostic testing. it is clear however, that although able to prevent a majority of animals from reaching onset of clinical disease, their current formulations provide incomplete protection against infection and shedding, thus failing to eradicate the organism. most current whole cell vaccine preparations rely on subcultures of classic strains that were generated over 70 years ago and some evidence suggests that, for killed preparations at least, more recently acquired local virulent strain types may be more effective. previous experience with bcg has shown that frequent in vitro passage of strains in different laboratories led to significant alterations in genomic profiles and diversities in attenuation and immunogenicity. it is of importance therefore to derive accurate definitions of map vaccine genotypes to better standardize vaccine manufacture and understand the critical mechanisms determining vaccine attenuations and protective efficacies. the distribution and worldwide use of map vaccines has continued since live ‘attenuated’ strains were selected in france and the uk using a method of sequential passage similar to that applied for the generation of bcg. the degree and mechanism underlying their attenuation however is uncertain as virulence studies were not performed in any detail. concerns in the 1980’s regarding the use of live vaccine strains because of low shelf life and spread to the environment promoted the use of killed vaccine formulations. these were based on various combinations of three map strains comprising strain 2e from the uk, strain ii from canada and 316 f. a combination of these were used as live preparations for norwegian goats in the 1960’s and 1970’s and as a killed preparation delivered in mineral oil to icelandic sheep in the 1950’s. live vaccine formulations of 316 f alone were used in the 1960’s and 70’s in the uk and cyprus, 1980’s in hungary, 1990’s in germany and spain and up until in new zealand. killed preparations of 316 f alone have been used extensively worldwide and are still available for commercial use. these strains, due to the difficulty in retaining mycobacteria in frozen seed stocks, have been maintained through regular subculture on a variety of laboratory in-house media. it is unsurprising therefore, that some reports suggest strain adaptation to growth in specialized media with loss of mycobactin j dependence and genome diversity has occurred amongst some lineages. in this work we demonstrate attenuation and differential virulence of vaccine strains 2e, ii and 316 f in a mouse model and use a full map genome microarray, supported by pcr and sequencing to investigate the genomic shifts of vaccine strains from a variety of lineages, including one recently resuscitated 316 f strain, originally lyophilised in we describe large genomic regions with deletions and tandem duplications uniquely associated with each vaccine clade, demonstrate the functionality of some of these deleted genes and hypothesise as to their role in virulence attenuation.  comparative genomic hybridisation of vaccine strains mapac hybridisations comparing each vaccine strain against a mapk reference control were made and averaged values displayed as scatterplots. significant loss of signals in contiguous genes representative of large variable genomic island deletions were identified in a kbp region of 316fnor and a kbp region in both iiuk and 2euk. two fold increases in signals were also seen in contiguous genes within a kbp region of iiuk, a kbp region of 316 f-nld and a kbp 316fuk. * = map specific genes. * = map specific genes. signal ratios from genes in multiple copies in the genome such as insertion elements are more difficult to interpret as changes in signal are related to copy number. nevertheless is signal ratios were significantly reduced in iiuk 2euk 316fnor 316v, 316fuk and elevated in 316fnld and 316fnld increases in signals for is1652-like elements were also seen in iiuk 2euk 316fuk 316fnld and 316fnld and decreases for this element in 316fnor is256-like elements were more abundant in 2euk and also raised in 316v and 316fuk but decreased in 316fnor relative to mapk is1311-like elements were increased in 316v and decreased in 316fnor one of these elements borders the previously described vgi-1b region which is bounded by short inverted repeat sequences and duplicated in map type iii strains. the tandem duplication vgi- was confirmed using primer set map seq and map r orientated to amplify only the end of one tandem repeat and the beginning of the other. this produced an amplicon of 651 bp only with vaccine strain iiuk and iiuk. sequencing of this amplicon showed the duplication event to occur relative to the reference strain k genome at position within the first copy of map2733c followed by a unique region of 16 bp and then a second copy of map followed by the vgi- duplication thus comprising a tandem duplication of  bp spanning orfs. pcr using primer pairs specific for this tandem duplication map seq and map r was negative for all other strains included in this study. map gene annotation corresponds to genbank ae vgi- was confirmed as a tandem duplication using primer set map f and map r which produced an amplicon of 2967 bp with vaccine strain 316 f-nld only. sequencing of this region showed the duplication event to comprise  bp spanning orfs. the duplication site occurs at genbank accession ae position within the first copy of map followed by an overlap region of 3 bp and then the second copy of map1748c followed by the vgi- duplication. pcr with primer pairs specific for this tandem duplication was negative for all other strains included in this study. several attempts to localise the vgi-1b duplication using outward facing primers around the region in a similar manner to vgi- and vgi- were unsuccessful and this region may not be present in 316uk as a tandem duplication. qpcr was performed using both map and map specific primer pairs located inside vgi-1b and the relative copy number of each compared against a single copy genome target control represented by primer pairs to map2114c. both map and map pairs showed a doubling of signal strengths relative to map2114c in strain 316fuk whilst all other strains included in this study showed signal consistent with single copies of both these genes. nd = not done. * vgi status for vgi-1b, vgi- - vgi- either duplicated or deleted, else no entry designates present as a single copy region. is insertion site analysis to determine which is sites were absent relative to the k reference genome, pcr primers were designed to specifically amplify each of the known is loci. these were used to confirm the insertion of is into each locus in the reference strain k and were also all positive in all 316 f strains and a caprine isolate cam both vaccine strains iiuk and 2euk were missing is whereas is was also missing from vaccine strain 2euk but present in all other strains including vaccine strain iiuk comparative qpcr of is copy number relative to map2114c, demonstrated a range of is copies in vaccine strains that corresponded to the trend in hybridisation signals observed in mapac scatterplots. the ratio of copy number however was surprisingly higher than predicted, with vaccine strains iiuk and 2euk having only copies whilst mapk and 316 f strains gave signals correspondent with 16– relative is copy numbers. functional analysis of tellurite resistance one map specific gene predicted to be deleted in vgi- was map, a s-adenosylmethionine-dependent methyltransferase with homologues to tellurite resistance genes involved in bacterial virulence and persistence. the functionality of this gene in mycobacteria has not previously been investigated. using a solid culture plate assay we compared tellurite resistance of map strains with and without the vgi- deletion. this demonstrated a wide mic range between strains, with significant reductions associated with vgi- deletion over full genome complement strains. of note however was the very low level of tellurite resistance found in strains containing the vgi- deletion. assessment of virulence using a mouse model the virulence of vaccine strains 316fuk iiuk and 2euk was compared with wild type strain jd87/ in a mouse model. ten mice from each of five groups were killed at and 12 weeks post inoculation. body, spleen and liver weights were recorded. samples of the liver were taken for bacteriological culture and histopathology. mean bodyweights increased with age, but no statistically significant difference was observed in mean body weight between any of the vaccine strains and the control wild type strain at any of the time points. there was strong evidence of a difference in pattern over time between the mean rank spleen weight expressed as a percentage of body weight of the mice inoculated with the different map strains relative to those receiving pbs, where those inoculated with map had increasingly higher mean rank percentage weights. differences between the map strains were not formally statistically significant although the control virulent strain jd87/ showed an increase in mean rank spleen weight percentage between weeks and and 316fuk had an increase between weeks and there was no statistical evidence for differences in the mean levels of liver weight expressed as a percentage of body weight either for different strains or over time for any of the map strains. however, there was some evidence of a difference between the means for the map strains and the lower mean weights associated with pbs. map was recovered from the liver tissue of mice four weeks post inoculation in all groups except the control group inoculated with pbs. by 12 weeks post infection, map was recovered from the tissues of only one mouse inoculated with vaccine strain 2euk, from mice inoculated with iiuk and from all the mice inoculated with the virulent jd87/ strain suggesting attenuation of each of the vaccine strains. mean rank counts increase over time for the jd87/ strain, while dropping for all the other map strains, this being most rapid for the 2euk strain but ultimately most notable for strain 316fuk statistical assessment of the effect of the strain by time interaction on the mean rank count indicate that differences exist in the abilities of the map vaccine strains to survive or persist in mice.bmc1010 infections of the liver result in multifocal hepatitis characterised by clusters of inflammatory cells. to measure the effect of the experimental infection with map, the densities of mononuclear leucocyte clusters were counted. small numbers of leucocyte clusters were observed in liver samples from mice inoculated with pbs, though no acid fast bacilli were detected in any of these samples. large differences in the mean ranked density of leucocyte clusters between strains were identified with the wild type strain jd87/ having the highest mean ranked densities of clusters. strain 2euk showed evidence of higher mean rank densities than the 316fuk and iiuk strains. the ranked density of leucocyte clusters with afb showed highly statistically significant differences between the means of map strains, with the jd87/ strain consistently showing higher mean densities, with this effect being more pronounced from 8 weeks post infection. the vaccine strains all tended to exhibit increasingly lower mean ranked densities over the lifetime of the experiment, with consistent patterns of differences between strains: strain iiuk showed the largest mean rank densities, strain 316fuk the lowest, with 2euk intermediate. the histopathology results show that all strains elicited a similar inflammation at 4 weeks. only thereafter some differences between the inflammatory responses to the strains became apparent. in addition, the analysis of mean bacterial counts and afb positive clusters showed the reduced ability of the vaccine strains to survive and persist within mice. overall, these results provide proof of attenuation of the vaccine strains with respect to a wild type map strain. discussion 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 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/ 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 mapk one 316 f strain obtained from norway contained a single deleted region spanning orf’s. two strains not of the 316 f lineage contained a different deleted region, identical in both strains, spanning orf’s. specific pcr for map, located within the vgi- on 2euk and iiuk were negative confirming that the vgi- region was not present in these strains. 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. indeed, the deleted region vgi- contains part of the 38 kb pathogenicity island described by stratmann et al. which contains genes encoding a number of antigens with diagnostic potential. both deleted regions vgi- and vgi- contain genes potentially involved in virulence and pathogenesis 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 2euk and iiuk were attenuated with respect to a wild type map strain. in addition, vaccine strain iiuk and iiuk were found to contain a large orf tandem duplication which includes copies of benzoate and lipid metabolic pathways. vaccine strain 2euk comes from the same stock as 2euk and was maintained at the vla, uk for over 50 years on a mineral deficient medium whilst vaccine strain iiuk was not. we suggest that the vgi- duplication in vaccine strain iiuk 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- deletion. the large deletion vgi- present in vaccine strain 316fnor was not present in any of the other 316 f strains including an early low passage lineage and a more recent isolate shown to be significantly attenuated in our virulence studies. notably, part of vgi- is also present in the same gene order within the related mah genome. together these suggest that any ancestral precursor and therefore the original 316 f strain would be unlikely to be missing vgi- we hypothesise that the vgi- deletion appeared in the 316fnor strain some point after its acquisition and transfer to norway in 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- 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 isolate of this strain was shown to be highly immunogenic in goats and forms part of a 316 f/2e live commercial vaccine product however there is no evidence that vgi- 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 included in vgi- on 316fnor 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 316fnor whilst other 316 f strains either matched or exceeded the resistance of the two wildtype strains tested. interestingly the strains most sensitive to tellurite were iiuk and 2euk 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- 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. 316fnld available as a heat killed vaccination for dairy cattle since 1985, was found to contain a large tandem duplication unique to this strain. it is notable that this isolate was selectively subcultured on potato starch medium to enhance its growth and now grows with difficulty on other media. it is tempting to speculate that the acquisition of extra copies of orfs including cell wall, fatty acid biosynthesis genes and two extra copies of is are a direct result of the selective process performed on this strain. we demonstrated in this study that vaccine strain 316fuk was clearly attenuated with respect to wild type map strain jd87/ the vgi- deletion found in 316fnor and the vgi- deletion found in 2euk and iiuk were not detected by pcr in this strain suggesting that attenuation in this strain is due to different genetic polymorphisms. a duplicated region was detected in vaccine strain 316fuk 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 is is is and is1652-like elements were seen between vaccine strains and virulent isolates. an is was found immediately bordering the vgi-1b region duplicated in 316 f-uk but not other 316 f strains. similar genomic variations including vgi-1b have been observed in virulent map strains. is 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 is copies per cell per culture relative to two single copy map genes using an assumption determined from a map assembled genome sequence that mapk would contain copies. our results confirm previous studies showing the vaccine strain 316v used in australia for elisa testing contains one less genomic copy of is than most other 316 f strains. vaccine strain 316fnld exhibited higher gene signal ratios consistent with the two extra copies of is copies inside the duplication of vgi- vaccine strains iiuk and 2euk contained lower signal ratios consistent with loss of an is copy inside the deletion region vgi- consistently however the calculated is copy number in these strains was much lower than expected using the ratio method. using site specific pcr we confirmed is filled insertion sites in the genomes of these strains whereas the ratio method, using mapk as a standard, predicted only copies. the reason could be technical, perhaps involving incomplete bacterial lysis of these unusual strains, however is 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 is copies per cell. this could be an important factor in studies relying on qpcr to determine accurate estimates of map load. miru is a short tandem repeat sequence located within the sensx3-regx 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 miru tandem repeat copy number which has been suggested to be involved in the control of sensx3-regx expression. in this study 316 f strains had low miru 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.  this study has shown that significant genomic diversity exists between map vaccine strains and within the 316 f lineage. these include large deletions, duplications and changes in insertion sequence copies. these mutations were probably derived in a classical manner by selective subculture on laboratory media and in some cases have led to significant alterations of phenotype and attenuation. there were map specific gene deletions identified of which at least one could be linked to phenotypic change that would disadvantage its persistence in the host and thus associates it with virulence. furthermore, these map-specific gene deletions could provide the basis for a diva diagnostic for use with these vaccines. overall, this work illustrates that map genome plasticity can be influenced by in vitro culture over long periods and a robust definition of vaccine strain genome lineage will be necessary in the future to guarantee consistency between studies. 
current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown. using mouse virulence studies we confirm that map vaccine strains 316 f, ii and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. mycobacterium avium subspecies paratuberculosis whole cell vaccines have been widely used tools in the control of johne’s disease in animals despite being unable to provide complete protection. mycobacterium avium subspecies paratuberculosisvaccinecomparative genomicsvariable genomic islandattenuationmicroarray we show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted map-specific genes and their possible relation to attenuation.
 mycobacterium avium subspecies paratuberculosis whole cell vaccines have been widely used tools in the control of johne’s disease in animals despite being unable to provide complete protection. current vaccine strains derive from stocks created many decades ago; however their genotypes, underlying mechanisms and relative degree of their attenuation are largely unknown.  using mouse virulence studies we confirm that map vaccine strains 316 f, ii and 2e have diverse but clearly attenuated survival and persistence characteristics compared with wild type strains. using a pan genomic microarray we characterise the genomic variations in a panel of vaccine strains sourced from stocks spanning over 40 years of maintenance. we describe multiple genomic variations specific for individual vaccine stocks in both deletion and tandem duplicated large variable genomic islands and insertion sequence copy numbers. we show individual differences suitable for diagnostic differentiation between vaccine and wild type genotypes and provide evidence for functionality of some of the deleted map-specific genes and their possible relation to attenuation.  this study shows how culture environments have influenced map genome diversity resulting in large tandem genomic duplications, deletions and transposable element activity. in combination with classical selective systematic subculture this has led to fixation of specific map genomic alterations in some vaccine strain lineages which link the resulting attenuated phenotypes with deficiencies in high reactive oxygen species handling. mycobacterium avium subspecies paratuberculosisvaccinecomparative genomicsvariable genomic islandattenuationmicroarray 