BACKGROUND
mycobacterium avium subsp.
paratuberculosis  is the etiological agent of paratuberculosis in cattle.
this disease can affect many ruminant species and other wildlife animals  <cit> .
paratuberculosis is characterized in cows by severe gastroenteritis, diarrhoea, weight loss, reduced milk production and premature culling, leading to economic losses  <cit> .
moreover, although still debated, some data suggest a possible involvement of map in crohn's disease  <cit> .
the knowledge of the causative agent of paratuberculosis, both in terms of epidemiology and biodiversity within different strains, could be useful, especially in case of new outbreaks or in following map's diffusion pathway.
according to their genetic differences  <cit> , map strains have been classified into four groups.
however, this classification only partially reflects their host-specificity.
type s , also designed as type i, seems to be largely prevalent in sheep, while type c , also known as type ii, represents the type most widely recovered from various hosts and sources.
type b  is considered a subtype of type c. in addition, a sub-type of type i, designed as type iii, has been reported  <cit> .
beyond the previously described classification, many methods have been proposed for map sub-typing ; in particular, those based on the amplification of repetitive-element loci, at present, are considered the emerging map sub-typing techniques  <cit> .
the repetitive-element loci herein analysed, both mini-and microsatellite loci , have been previously described  <cit>  and their discriminatory power has been evaluated  <cit> .
the aim of this study was to explore the effectiveness of this technique in isolates from the same country and host species.
for this purpose, we evaluated a combination of mini-and microsatellite loci in a panel of  <dig> type c map isolates, each recovered from a different italian cattle herd.
in order to further investigate the effectiveness of this technique, the proposed method was also applied to  <dig> map isolates originated from  <dig> herds .
RESULTS
all the isolates were classified as type c, showing a pcr product of  <dig> bp  and the typical type c pattern in is <dig> pcr-rea  .
minisatellite loci
ten minisatellite loci were considered for the calculation of the simpson's index of diversity .
locus miru <dig> showed the highest allelic diversity  , followed by loci vntr <dig>  vntr <dig>  vntr <dig>  vntr <dig>  vntr <dig>  miru <dig> and vntr <dig>  finally, loci miru <dig> and vntr <dig> did not show any allelic diversity in our isolates.
taken together, minisatellite loci revealed  <dig> clusters , with a sid value of  <dig>  .
two main genotypes  clustered the 51% and 21% of the total isolates .
type mv  <dig> clustered the 8% and type mv  <dig> and type mv  <dig> the 6% and the 5%, respectively.
these five genotypes differed from each other for three loci: miru <dig>  vntr <dig> and vntr <dig>
a miru loci according to bull et al.
<cit> .
b vntr-miru according to overduin et al.
<cit>  for loci  <dig> and  <dig> and according to thibault et al.
<cit>  for loci  <dig>   <dig>   <dig>   <dig>   <dig>
microsatellite loci
among the  <dig> ssr loci proposed by amonsin  <cit> , three loci  were selected for the analysis.
the selection was made according to previous evidence indicating that these loci showed the highest allelic diversities  <cit> .
these observations have been preliminarily confirmed analysing a set of  <dig> italian isolates  <cit> .
the locus ssr <dig> showed five alleles , while the loci ssr <dig> and ssr <dig> showed four and three alleles respectively.
overall, the analysis of ssrs loci grouped the isolates into  <dig> clusters, with a sid of  <dig>  .
four main genotypes  clustered the 75% of the total, differentiating each other only for the locus ssr <dig>
a ssr according to amonsin et al.
<cit> .
combination of both mini-and microsatellite loci
a total of  <dig> different clusters originated from the combination of the  <dig> loci, both mini-and microsatellites .
the final sid was  <dig> , ranging from  <dig>  to  <dig>  .
three main genotypes resulted from the analysis of the combination between miru-vntr and ssr typing .
these three genotypes showed always the same miru-vntr type  but differed for the ssr type at ssr <dig> locus level.
the types mvs  <dig> and mvs  <dig> seem to be the most recovered genotypes in the northern italy, while the type mvs  <dig> was present even in the centre and south of the country .
a according to table 1
b according to table 2
c in bold and underlined are indicated herds in the centre and south of the country, respectively, while the rest of the herds are placed in the north of italy.
considering the three loci with the highest h , we obtained  <dig> clusters, with a sid value of  <dig>   .
adding more loci to the sid calculation, the slope of the graph increased slowly, finally reaching the plateau phase after the locus miru <dig>
the proposed method was then used to test the inside-herd variability.
even after this analysis , the genotypes most frequently found were mvs  <dig> and mvs  <dig>  while others, like types mvs  <dig> and mvs  <dig>  seem to be confined to only two herds placed in two adjacent regions .
moreover, this investigation allowed finding out eight further genotypes not detected with the previous analysis.
three or more distinct genotypes, differing from each other for both mini-and microsatellite loci, were isolated from three herds placed in the same area , while in four herds, two  or three different genotypes , diverging each other only for the ssr <dig> locus, were recovered .
discussion
this study was mainly conducted to evaluate the effectiveness of a panel of selected mini-and microsatellite loci for sub-typing closely related isolates.
the  <dig> isolates included in this study have not been previously sub-typed with any other method and were identified as type c. most of the isolates analysed were from two northern italian regions , where about the 60% of the national dairy farms is located.
few isolates derived from other northern regions , while the rest of the isolates came from herds located in the centre and south of italy.
as a result, the sampling was not representative of the whole country, so epidemiological conclusions about the distribution of the different genotypes could not be drawn.
moreover, it is very plausible the existence of links related to cattle trading which can further complicate the epidemiological analysis.
considering the  <dig> isolates from different herds, despite the above mentioned limits, type mvs  <dig> profile was recovered in the whole country, while those containing types mvs  <dig> and mvs  <dig> seem to be confined in the northern italy.
these genotypes showed the most frequent miru-vnr pattern profile and differed for the locus ssr <dig>  in a recent paper, douarre et al.
<cit>  reported similar results for  <dig> type c isolates from ireland.
these authors concluded that the analysis of minisatellite loci could be useful for producing broad epidemiological data, especially in terms of establishing the interrelatedness of the isolates, while microsatellite loci seem to be more informative producing unique genetic fingerprints.
even the intra-herd analysis confirmed that the types most frequently found were mvs  <dig> and mvs  <dig>  while others types seem to be limited to some herds .
as recently reported  <cit> , we noticed that herds characterised by an elevated turnover of cows showed many sub-types of map , which differed each other for both mini-and microsatellite loci .
interestingly, the herds  <dig> is the only herd in the south of the country where type mvs  <dig> was recovered, suggesting, as a possible route of infection, the introduction of infected animals from the northern italy.
more in general, the presence of different types of map within the same herd might suggest i) multiple infection due to the introduction of infected animals or ii) divergence of the isolates caused by locus instability.
regarding this last hypothesis, as inferred from previous reports  <cit> , locus ssr <dig> showed a very high variability.
the stability of the ssrs loci has been demonstrated "in vitro" by testing three strains after ten subcultures  <cit> , but no data are available about the "in vivo" stability.
nevertheless, in m. avium  <dig> genome, the locus ssr <dig> is localised in an intergenic region between "rph" and "rdgb" genes, while the same region on map k <dig> does not seem to code for any protein, suggesting the hypothesis that this locus could be a potential hot spot for mutation occurrence.
loci ssr <dig> and ssr <dig> showed lower variability in comparison with locus ssr <dig>  the two most frequently found alleles for the locus ssr <dig> had seven and more than  <dig> repeats .
similar results were achieved for locus ssr <dig> with the alleles containing four and five repeats .
for this latter locus we found one isolate containing the allele with three repeats, which has been observed only in map type s strains  <cit> .
the dna region target of miru <dig> locus seems to be related to some components of the regulatory system involved in oxidative response in other mycobacteria  <cit> .
in our isolates the predominant allele carried five repetitions, a variant previously associated with a higher degree of virulence compared to those with three repeats .
however, we found two isolates with this last allele  in two farms placed in adjacent provinces of the northern italy, confirming that, although rare, it is circulating even in field strains  <cit> .
guidelines for validation and application of typing methods for epidemiology  <cit>  proposed that the discriminatory index value for the evaluation of epidemiological data should be upper than  <dig> .
in our study, the analysis of  <dig> loci enabled a sid of  <dig> .
however, some authors considered even  <dig>  as an acceptable cut-off value, because map is an organism characterised by limited genetic diversity  <cit> .
in this regard, an exceeding result  can be reached considering only the three loci with the highest allelic diversity  .
accordingly, these loci could be considered the first choice for sub-typing field isolates.
finally, our data indicated that the use of minisatellite loci alone did not seem appropriate to reach good discriminatory indexes in homogeneous group of isolates .
these findings suggest its application should be coupled with other techniques  or eventually used to sub-type among different map types  <cit> .
CONCLUSIONS
our data confirm the combined use of mini-and microsatellite loci as a suitable tool to sub-type map, even in isolates recovered from the same host species , originating from the same country  and belonging to the same type .
miru-vntr loci analysis is a robust and simple technique, but, in order to reach appropriate discriminatory indexes among homogeneous isolates, it should be associated with the analysis of ssr loci or other techniques.
finally, these data suggest that the combination of loci here proposed, coupled with other epidemiological information , could be a useful molecular tool for both national and regional epidemiological studies.

 mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis in cattle. this disease can affect many ruminant species and other wildlife animals. paratuberculosis is characterized in cows by severe gastroenteritis, diarrhoea, weight loss, reduced milk production and premature culling, leading to economic losses. moreover, although still debated, some data suggest a possible involvement of map in crohn's disease. the knowledge of the causative agent of paratuberculosis, both in terms of epidemiology and biodiversity within different strains, could be useful, especially in case of new outbreaks or in following map's diffusion pathway. according to their genetic differences, map strains have been classified into four groups. however, this classification only partially reflects their host-specificity. type s, also designed as type i, seems to be largely prevalent in sheep, while type c, also known as type ii, represents the type most widely recovered from various hosts and sources. type b is considered a subtype of type c. in addition, a sub-type of type i, designed as type iii, has been reported. beyond the previously described classification, many methods have been proposed for map sub-typing; in particular, those based on the amplification of repetitive-element loci, at present, are considered the emerging map sub-typing techniques. the repetitive-element loci herein analysed, both mini-and microsatellite loci, have been previously described and their discriminatory power has been evaluated. the aim of this study was to explore the effectiveness of this technique in isolates from the same country and host species. for this purpose, we evaluated a combination of mini-and microsatellite loci in a panel of type c map isolates, each recovered from a different italian cattle herd. in order to further investigate the effectiveness of this technique, the proposed method was also applied to map isolates originated from herds.  all the isolates were classified as type c, showing a pcr product of bp and the typical type c pattern in is pcr-rea. minisatellite loci ten minisatellite loci were considered for the calculation of the simpson's index of diversity. locus miru showed the highest allelic diversity, followed by loci vntr vntr vntr vntr vntr miru and vntr finally, loci miru and vntr did not show any allelic diversity in our isolates. taken together, minisatellite loci revealed clusters, with a sid value of. two main genotypes clustered the 51% and 21% of the total isolates. type mv clustered the 8% and type mv and type mv the 6% and the 5%, respectively. these five genotypes differed from each other for three loci: miru vntr and vntr a miru loci according to bull et al.. b vntr-miru according to overduin et al. for loci and and according to thibault et al. for loci microsatellite loci among the ssr loci proposed by amonsin, three loci were selected for the analysis. the selection was made according to previous evidence indicating that these loci showed the highest allelic diversities. these observations have been preliminarily confirmed analysing a set of italian isolates. the locus ssr showed five alleles, while the loci ssr and ssr showed four and three alleles respectively. overall, the analysis of ssrs loci grouped the isolates into clusters, with a sid of. four main genotypes clustered the 75% of the total, differentiating each other only for the locus ssr a ssr according to amonsin et al.. combination of both mini-and microsatellite loci a total of different clusters originated from the combination of the loci, both mini-and microsatellites. the final sid was, ranging from to. three main genotypes resulted from the analysis of the combination between miru-vntr and ssr typing. these three genotypes showed always the same miru-vntr type but differed for the ssr type at ssr locus level. the types mvs and mvs seem to be the most recovered genotypes in the northern italy, while the type mvs was present even in the centre and south of the country. a according to table 1 b according to table 2 c in bold and underlined are indicated herds in the centre and south of the country, respectively, while the rest of the herds are placed in the north of italy. considering the three loci with the highest h, we obtained clusters, with a sid value of. adding more loci to the sid calculation, the slope of the graph increased slowly, finally reaching the plateau phase after the locus miru the proposed method was then used to test the inside-herd variability. even after this analysis, the genotypes most frequently found were mvs and mvs while others, like types mvs and mvs seem to be confined to only two herds placed in two adjacent regions. moreover, this investigation allowed finding out eight further genotypes not detected with the previous analysis. three or more distinct genotypes, differing from each other for both mini-and microsatellite loci, were isolated from three herds placed in the same area, while in four herds, two or three different genotypes, diverging each other only for the ssr locus, were recovered. discussion this study was mainly conducted to evaluate the effectiveness of a panel of selected mini-and microsatellite loci for sub-typing closely related isolates. the isolates included in this study have not been previously sub-typed with any other method and were identified as type c. most of the isolates analysed were from two northern italian regions, where about the 60% of the national dairy farms is located. few isolates derived from other northern regions, while the rest of the isolates came from herds located in the centre and south of italy. as a result, the sampling was not representative of the whole country, so epidemiological conclusions about the distribution of the different genotypes could not be drawn. moreover, it is very plausible the existence of links related to cattle trading which can further complicate the epidemiological analysis. considering the isolates from different herds, despite the above mentioned limits, type mvs profile was recovered in the whole country, while those containing types mvs and mvs seem to be confined in the northern italy. these genotypes showed the most frequent miru-vnr pattern profile and differed for the locus ssr in a recent paper, douarre et al. reported similar results for type c isolates from ireland. these authors concluded that the analysis of minisatellite loci could be useful for producing broad epidemiological data, especially in terms of establishing the interrelatedness of the isolates, while microsatellite loci seem to be more informative producing unique genetic fingerprints. even the intra-herd analysis confirmed that the types most frequently found were mvs and mvs while others types seem to be limited to some herds. as recently reported, we noticed that herds characterised by an elevated turnover of cows showed many sub-types of map, which differed each other for both mini-and microsatellite loci. interestingly, the herds is the only herd in the south of the country where type mvs was recovered, suggesting, as a possible route of infection, the introduction of infected animals from the northern italy. more in general, the presence of different types of map within the same herd might suggest i) multiple infection due to the introduction of infected animals or ii) divergence of the isolates caused by locus instability. regarding this last hypothesis, as inferred from previous reports, locus ssr showed a very high variability. the stability of the ssrs loci has been demonstrated "in vitro" by testing three strains after ten subcultures, but no data are available about the "in vivo" stability. nevertheless, in m. avium genome, the locus ssr is localised in an intergenic region between "rph" and "rdgb" genes, while the same region on map k does not seem to code for any protein, suggesting the hypothesis that this locus could be a potential hot spot for mutation occurrence. loci ssr and ssr showed lower variability in comparison with locus ssr the two most frequently found alleles for the locus ssr had seven and more than repeats. similar results were achieved for locus ssr with the alleles containing four and five repeats. for this latter locus we found one isolate containing the allele with three repeats, which has been observed only in map type s strains. the dna region target of miru locus seems to be related to some components of the regulatory system involved in oxidative response in other mycobacteria. in our isolates the predominant allele carried five repetitions, a variant previously associated with a higher degree of virulence compared to those with three repeats. however, we found two isolates with this last allele in two farms placed in adjacent provinces of the northern italy, confirming that, although rare, it is circulating even in field strains. guidelines for validation and application of typing methods for epidemiology proposed that the discriminatory index value for the evaluation of epidemiological data should be upper than. in our study, the analysis of loci enabled a sid of. however, some authors considered even as an acceptable cut-off value, because map is an organism characterised by limited genetic diversity. in this regard, an exceeding result can be reached considering only the three loci with the highest allelic diversity. accordingly, these loci could be considered the first choice for sub-typing field isolates. finally, our data indicated that the use of minisatellite loci alone did not seem appropriate to reach good discriminatory indexes in homogeneous group of isolates. these findings suggest its application should be coupled with other techniques or eventually used to sub-type among different map types.  our data confirm the combined use of mini-and microsatellite loci as a suitable tool to sub-type map, even in isolates recovered from the same host species, originating from the same country and belonging to the same type. miru-vntr loci analysis is a robust and simple technique, but, in order to reach appropriate discriminatory indexes among homogeneous isolates, it should be associated with the analysis of ssr loci or other techniques. finally, these data suggest that the combination of loci here proposed, coupled with other epidemiological information, could be a useful molecular tool for both national and regional epidemiological studies. 
these data suggest that the combination of loci here proposed could be a useful molecular tool for regional epidemiological studies. our results revealed the advantage of using both mini-and microsatellite approaches for successfully discriminating among map type c isolates from the same geographic area, host species and herd. the intra-herd analysis revealed more than one genotype in most herds with a similar distribution of clusters. paratuberculosis is the etiological agent of paratuberculosis.
 mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis. the aim of our study was to combine mini-and microsatellite loci analysis in order to explore the effectiveness of this sub-typing method in a group of map isolates. for this purpose, italian type c map isolates, each from a different cattle herd, were submitted to miru-variable-number tandem-repeats typing and short sequence repeats sequencing. moreover, the method was used to analyse the variability inside herds.  the molecular sub-typing, carried out using three ssr and miru-vntr loci, differentiated the isolates into clusters, reaching a simpson's discriminatory index value of. among all considered loci, six showed relevant allelic variability. thirty-eight% of the isolates were clustered into four genotypes, differing from each other for the ssr locus. the other isolates, characterised by differences in two or more loci, were spread among the rest of the clusters. the intra-herd analysis revealed more than one genotype in most herds with a similar distribution of clusters.  our results revealed the advantage of using both mini-and microsatellite approaches for successfully discriminating among map type c isolates from the same geographic area, host species and herd. these data suggest that the combination of loci here proposed could be a useful molecular tool for regional epidemiological studies. 