BACKGROUND
sika deer tuberculosis is caused by the mycobacterium tuberculosis complex ; most commonly, m. bovis.
this disease has led to significant economic losses for deer producers in china.
however, previous efforts in controlling mtbc have been hampered by a lack of data, as genotyping mtbc members is an important tool in epidemiological analysis for studying the spread of mtbc  <cit> .
in  <dig>  vntr genotyping method using miru units was developed  <cit>  and used to study the molecular epidemiology of mycobacterium tuberculosis  <cit> .
miru-vntr has high efficiency and reproducibility, because miru-vntr typing results are easier to compare between different laboratories than restriction fragment length polymorphisms , which were the previous “gold standard” for mtbc typing  <cit> .
another advantage of miru-vntr is that it does not require large quantities of template dna for typing  <cit> .
in addition to m. tuberculosis, this technique has also been proven in m. bovis in different countries and epidemiologic scenarios  <cit> .
earlier studies frequently used the loci designated as mycobacterial interspersed repeat units   <cit>  and exact tandem repeats   <cit> .
to obtain better resolution, other vtnr loci were investigated  <cit> .
recently, novel m. tuberculosis miru-vntr loci groups, such as the queen’s university belfast  and mtub groups  <cit> , have been identified.
various combinations of miru-vntr loci have been reported to give better differentiation of m. tuberculosis strains  <cit> .
however, no specific set of loci have been agreed upon as a standard, and the allelic diversity of loci can vary from country to country and between m. tuberculosis complex species, requiring localized selection of suitable loci.
this study was therefore developed to determine the miru-vntr loci most appropriate to use for typing mtbc organisms isolated from sika deer in china.
RESULTS
analysis of individual miru-vntr locus
miru-vntr typing results of m.bovis isolated in this study was described in additional file  <dig>  the copy numbers and allelic diversity  in miru-vntr loci were recorded .
the diversity analysis showed that the discriminatory power of individual loci differed greatly, with h ranging from  <dig>  to  <dig> .
the etra, miru <dig>  qub11b, and mtub <dig> loci had high discriminatory power .
the miru <dig>  miru <dig>  miru <dig>  miru <dig>  miru <dig>  miru <dig>  qub <dig>  and mtub <dig> loci were moderately discriminatory .
miru <dig>  mtub <dig>  and mtub <dig> were poorly discriminatory .
seven loci displayed no allelic diversity.table  <dig>
allelic diversity of individual miru-vntr locus
loci
no.
of copies
allelic diversity
1
2
3
4
5
6
7
8
9
10
evaluation of miru-vntr loci combinations
based on all  <dig> miru-vntr loci with non-zero allelic diversity,  <dig> different types  were identified; of these,  <dig> was unique and  <dig> were clustered.
clusters contained  <dig>  to  <dig>  identical strains , and the  <dig> clinical isolates were classified into two groups .
group  <dig>  which included  <dig> genotypes, was more complex.
this group included isolates from four different regions—shuangyang, liaoyuan, tonghua, songyuan and siping—strongly suggesting interregional transmission.table  <dig>
molecular differentiation of isolates by miru-vntr
miru-vntr type
miru-vntr allelic profile
isolates id
number of isolates
etra
miru4
qub11b
mtub4
miru2
miru23
miru31
miru39
miru40
qub26
mtub21
miru27
mtub30
mtub34
miru16
the discrimination ability was compared with different combinations of loci .
the four highly discriminatory loci and the eight moderately discriminatory loci, also identified  <dig> genotypes .
the miru loci with moderate discriminatory power  resolved the  <dig> isolates into  <dig> types .
if the miru <dig> and miru <dig> loci were also used,  <dig> types  were resolved.
with the mtub <dig>  mtub <dig>  mtub <dig>  and mtub <dig> loci,  <dig> types  were resolved.
based on these results, we consider etra, miru <dig>  qub11b, and mtub <dig> to be robust enough for initial molecular epidemiological studies when rapid results are of paramount importance.table  <dig>
evaluation of the discriminatory capacity of the combinations of miru-vntr locus
locus combination
no.
of clusters
no.
of isolates in individual clusters
hunter–gaston discriminatory index
discussion
m. bovis is the primary pathogen for tuberculosis in sika deer.
because it also infects humans, molecular epidemiologic studies on m. bovis are of major importance in tuberculosis prevention.
genotyping of mtbc isolates is a useful tool, not only for regional investigations, such as tracebacks, but to allow comparisons of tuberculosis isolates worldwide  <cit> .
different typing methods published for m. bovis include is <dig>  rflp, vntr, and spoligotyping  <cit> .
while is <dig> has been the most common, the discriminatory power of is <dig> is generally low for m. bovis typing  <cit> .
because the resolution provided by miru-vntr is adequate for most situations and this method is relatively cheap and rapid, it is quickly becoming the standard as the initial step in m. bovis epidemiology  <cit> .
the individual discriminatory power of  <dig> miru-vntr loci were analyzed.
the qub11b locus produced the highest individual discriminatory index , followed by mtub <dig>  etra, and miru <dig>  this correlates well with previous studies  <cit> .
mtub loci exhibit good application potential; however, the discriminatory power of mtub <dig>  mtub <dig>  mtub <dig>  and mtub <dig> differed greatly, with h ranging from  <dig>  to  <dig> .
these loci exhibited a high enough discrimination ability to be useful in early research  <cit> , and may prove useful in future m. bovis typing studies.
the etrc, miru <dig>  miru <dig>  miru <dig>  miru <dig>  qub <dig>  and qub <dig> loci showed no allelic diversity in this test.
miru <dig>  miru <dig>  miru <dig> and qub <dig> had similar results in studies on m. bovis isolated from xinjiang region  <cit> .
the published data on m. tuberculosis from ghana  <cit>  and other locations  <cit>  showed higher allelic diversities than our study, which suggests that not all miru-vntr loci are informative for m. bovis strains form sika deer in china.
the allelic diversity of miru-vntr loci are higher in our study than previous studies in china  <cit> , published data on m. tuberculosis from france  <cit> , south africa  <cit> , and the united states  <cit>  found that the discriminatory power of miru-vntr loci in sika deer is higher than in humans.
although these miru-vntr loci showed enough discrimination ability for genotyping current m. bovis isolates, additional studies with new miru-vntrs are needed to ensure accurate genotyping and epidemiological resolution for field studies.
the discriminatory power of miru-vntr loci may vary between countries, geographical regions, and epidemiological scenarios.
analysis of  <dig> vntr loci showed the expected results: when compared with previous studies the test had a relatively high hunter–gaston discriminatory index  and low clustering rates  <cit> .
the subset of  <dig> loci provides increased resolution compared to the original  <dig> miru loci.
the discriminatory power of miru-vntr loci also varies with high allelic diversity.
the etra, miru <dig>  qub11b, and mtub <dig> loci provide the greatest resolution  for individual loci, while the mirus alone  provided less resolution .
the moderately discriminatory mirus  had less resolution alone  than when combined with the other moderately loci , which had the greatest amount .
therefore, the discriminatory power of vntr loci appears greatly improved by applying novel combinations of vntr loci.
the  <dig> vntr loci methodology is a highly discriminatory method for first line typing of m. bovis isolated from sika deer and should be considered as a replacement for the original  <dig> miru loci method.
however, for in-depth studies, we recommend using etra, miru <dig>  qub11b, and mtub <dig> loci as a first set to obtain initial molecular epidemiology data quickly before proceeding on to the full set of loci.
CONCLUSIONS
the results of this study will be used in a molecular epidemiology study to identify risk factors for recent transmission of sika deer tuberculosis in china.
vntr studies will also help in tracing infection sources during sika deer tuberculosis outbreaks and help control transmission between sika deer and humans.
finally, this will improve chinese sika deer cultivation by reducing losses due to mtbc infections.

 sika deer tuberculosis is caused by the mycobacterium tuberculosis complex; most commonly, m. bovis. this disease has led to significant economic losses for deer producers in china. however, previous efforts in controlling mtbc have been hampered by a lack of data, as genotyping mtbc members is an important tool in epidemiological analysis for studying the spread of mtbc. in vntr genotyping method using miru units was developed and used to study the molecular epidemiology of mycobacterium tuberculosis. miru-vntr has high efficiency and reproducibility, because miru-vntr typing results are easier to compare between different laboratories than restriction fragment length polymorphisms, which were the previous “gold standard” for mtbc typing. another advantage of miru-vntr is that it does not require large quantities of template dna for typing. in addition to m. tuberculosis, this technique has also been proven in m. bovis in different countries and epidemiologic scenarios. earlier studies frequently used the loci designated as mycobacterial interspersed repeat units and exact tandem repeats. to obtain better resolution, other vtnr loci were investigated. recently, novel m. tuberculosis miru-vntr loci groups, such as the queen’s university belfast and mtub groups, have been identified. various combinations of miru-vntr loci have been reported to give better differentiation of m. tuberculosis strains. however, no specific set of loci have been agreed upon as a standard, and the allelic diversity of loci can vary from country to country and between m. tuberculosis complex species, requiring localized selection of suitable loci. this study was therefore developed to determine the miru-vntr loci most appropriate to use for typing mtbc organisms isolated from sika deer in china.  analysis of individual miru-vntr locus miru-vntr typing results of m.bovis isolated in this study was described in additional file the copy numbers and allelic diversity in miru-vntr loci were recorded. the diversity analysis showed that the discriminatory power of individual loci differed greatly, with h ranging from to. the etra, miru qub11b, and mtub loci had high discriminatory power. the miru miru miru miru miru miru qub and mtub loci were moderately discriminatory. miru mtub and mtub were poorly discriminatory. seven loci displayed no allelic diversity.table allelic diversity of individual miru-vntr locus loci no. of copies allelic diversity 1 2 3 4 5 6 7 8 9 10 evaluation of miru-vntr loci combinations based on all miru-vntr loci with non-zero allelic diversity, different types were identified; of these, was unique and were clustered. clusters contained to identical strains, and the clinical isolates were classified into two groups. group which included genotypes, was more complex. this group included isolates from four different regions—shuangyang, liaoyuan, tonghua, songyuan and siping—strongly suggesting interregional transmission.table molecular differentiation of isolates by miru-vntr miru-vntr type miru-vntr allelic profile isolates id number of isolates etra miru4 qub11b mtub4 miru2 miru23 miru31 miru39 miru40 qub26 mtub21 miru27 mtub30 mtub34 miru16 the discrimination ability was compared with different combinations of loci. the four highly discriminatory loci and the eight moderately discriminatory loci, also identified genotypes. the miru loci with moderate discriminatory power resolved the isolates into types. if the miru and miru loci were also used, types were resolved. with the mtub mtub mtub and mtub loci, types were resolved. based on these results, we consider etra, miru qub11b, and mtub to be robust enough for initial molecular epidemiological studies when rapid results are of paramount importance.table evaluation of the discriminatory capacity of the combinations of miru-vntr locus locus combination no. of clusters no. of isolates in individual clusters hunter–gaston discriminatory index discussion m. bovis is the primary pathogen for tuberculosis in sika deer. because it also infects humans, molecular epidemiologic studies on m. bovis are of major importance in tuberculosis prevention. genotyping of mtbc isolates is a useful tool, not only for regional investigations, such as tracebacks, but to allow comparisons of tuberculosis isolates worldwide. different typing methods published for m. bovis include is rflp, vntr, and spoligotyping. while is has been the most common, the discriminatory power of is is generally low for m. bovis typing. because the resolution provided by miru-vntr is adequate for most situations and this method is relatively cheap and rapid, it is quickly becoming the standard as the initial step in m. bovis epidemiology. the individual discriminatory power of miru-vntr loci were analyzed. the qub11b locus produced the highest individual discriminatory index, followed by mtub etra, and miru this correlates well with previous studies. mtub loci exhibit good application potential; however, the discriminatory power of mtub mtub mtub and mtub differed greatly, with h ranging from to. these loci exhibited a high enough discrimination ability to be useful in early research, and may prove useful in future m. bovis typing studies. the etrc, miru miru miru miru qub and qub loci showed no allelic diversity in this test. miru miru miru and qub had similar results in studies on m. bovis isolated from xinjiang region. the published data on m. tuberculosis from ghana and other locations showed higher allelic diversities than our study, which suggests that not all miru-vntr loci are informative for m. bovis strains form sika deer in china. the allelic diversity of miru-vntr loci are higher in our study than previous studies in china, published data on m. tuberculosis from france, south africa, and the united states found that the discriminatory power of miru-vntr loci in sika deer is higher than in humans. although these miru-vntr loci showed enough discrimination ability for genotyping current m. bovis isolates, additional studies with new miru-vntrs are needed to ensure accurate genotyping and epidemiological resolution for field studies. the discriminatory power of miru-vntr loci may vary between countries, geographical regions, and epidemiological scenarios. analysis of vntr loci showed the expected results: when compared with previous studies the test had a relatively high hunter–gaston discriminatory index and low clustering rates. the subset of loci provides increased resolution compared to the original miru loci. the discriminatory power of miru-vntr loci also varies with high allelic diversity. the etra, miru qub11b, and mtub loci provide the greatest resolution for individual loci, while the mirus alone provided less resolution. the moderately discriminatory mirus had less resolution alone than when combined with the other moderately loci, which had the greatest amount. therefore, the discriminatory power of vntr loci appears greatly improved by applying novel combinations of vntr loci. the vntr loci methodology is a highly discriminatory method for first line typing of m. bovis isolated from sika deer and should be considered as a replacement for the original miru loci method. however, for in-depth studies, we recommend using etra, miru qub11b, and mtub loci as a first set to obtain initial molecular epidemiology data quickly before proceeding on to the full set of loci.  the results of this study will be used in a molecular epidemiology study to identify risk factors for recent transmission of sika deer tuberculosis in china. vntr studies will also help in tracing infection sources during sika deer tuberculosis outbreaks and help control transmission between sika deer and humans. finally, this will improve chinese sika deer cultivation by reducing losses due to mtbc infections. 
this study was planned to assess the discriminatory power of each mycobacterial interspersed repetitive unit -variable number tandem repeats locus and evaluate the most appropriate combination of miru-vntr loci for molecular epidemiological studies on sika deer in china. of the loci tested, the four highly discriminative loci and eight moderately discriminative loci proved to be most appropriate for first line typing of m. bovis from sika deer, with the same resolving ability as all loci. the discriminatory power of miru-vntr typing based on known loci were assessed in 96 mycobacterium bovis strains collected sequentially from sika deer at a slaughterhouse in northeastern china. bovine tuberculosis has led to serious economic losses for sika deer producers in china. miru-vntr typing is quick and effective for typing bovine tuberculosis isolates from sika deer in china. mycobacterium bovismiru-vntr typingsika deerissue-copyright-statement© the author 2015
 bovine tuberculosis has led to serious economic losses for sika deer producers in china. strategies for controlling the spread of mycobacterium bovis are often hampered by a lack of epidemiological data. specifically, tracing infections requires the ability to trace back infections, which, in turn, requires the ability to determine isolates with a common source. this study was planned to assess the discriminatory power of each mycobacterial interspersed repetitive unit -variable number tandem repeats locus and evaluate the most appropriate combination of miru-vntr loci for molecular epidemiological studies on sika deer in china.  the discriminatory power of miru-vntr typing based on known loci were assessed in 96 mycobacterium bovis strains collected sequentially from sika deer at a slaughterhouse in northeastern china. we defined four loci as highly discriminative, eight loci as moderately discriminative, and three loci as poorly discriminative. the final locus showed no polymorphism between strains. miru-vntr typing as a whole was highly discriminative, with an overall allelic diversity of. of the loci tested, the four highly discriminative loci and eight moderately discriminative loci proved to be most appropriate for first line typing of m. bovis from sika deer, with the same resolving ability as all loci.  miru-vntr typing is quick and effective for typing bovine tuberculosis isolates from sika deer in china. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mycobacterium bovismiru-vntr typingsika deerissue-copyright-statement© the author 2015 