within the bacillus genus , the most pathogenic species is bacillus ( b. ) anthracis , the causative agent of anthrax and a gram - positive spore - forming soil bacterium .
this zoonosis and infectious disease affects a wide range of species , including humans and herbivorous mammals .
the incidence of anthrax has been reduced by continuous research and disease control , but still occurs in undeveloped and developing countries . in 1905 ,
the first anthrax case occurred in animal was recorded in korea . during the next 5 years after the first official report in 1907 , the number of anthrax cases occurred in animal increased to 2,562 , causing serious damage to related industries that handle animal by - products like meat and animal skins .
more than 1,000 cases were subsequently reported annually , and until 1924 the rate of occurrence was approximately 500 cases per year .
the reduction of anthrax cases is due to development of live vaccines using non - virulent b. anthracis sterne and other methods of disease control .
however , there were two cases reported in 1994 ( kyungju and hongseong , respectively ) , one in 1995 ( hongseong ) , another two cases in 2000 ( changnyung ) , and one in 2008 ( yeongcheon ) .
the disease occurs annually in a seasonal manner , but the distribution of cases is most prominent in may and august . since the annual average rainfall is high in april and july to august , it can be inferred that contaminated soil is more exposed during the rainy season and pollutes pastures , thus transmitting the pathogen to cattle
discriminatory markers have long been used to identify and subdivide groups of these microbial pathogens .
tracking the epidemic and pandemic spread of particular strains may be possible using such markers , yielding a long - term overview of epidemiological patterns .
there are regions in the b. anthracis genome that vary greatly from strain to strain .
numerous studies have shown that genetic markers such as single - nucleotide polymorphisms ( snps ) , variable number tandem repeats ( vntrs ) , and single - nucleotide repeats ( snrs ) can be used to discriminate between strains in b. anthracis .
canonical snps ( cansnps ) , originally described by keim et al . , are a number of snps located at key phylogenetic junctions and are sufficient to create phylogenetic trees .
previously , a set of cansnps was able to distinguish three major lineages of b. anthracis isolates   and 12 clonal sub - lineages . in the present study
, we examined a genetic population of b. anthracis isolates from korea and compared it to a global genetic population using an eight - loci multiple - locus variable - number tandem repeat analysis ( mlva ) and 13-cansnp analysis .
the b. anthracis strains used in this study included four obtained from the korean centers for disease control and prevention ( kcdc ) , five reference strains , and 17 isolates from soil of four different regions in korea ( table 1 ) . atcc14185 and atcc14578 stain obtained from american type culture collection ( atcc ) , ( usa ) , and pasteur , delta sterne , and sterne strain obtained from national veterinary research and quarantine service ( nvrqs ) , ( korea ) .
the b. anthracis strains were cultured in brain heart infusion media ( becton dickinson , usa ) and grown at 37 with shaking at 200 rpm for 3 days .
total genomic dna was extracted was modified by hunter 's method . to isolate total bacterial dna ,
bacteria cells were harvested by centrifugation at 15,000 rpm for 10 min at 4 and resuspended in 1 ml te buffer ( ph 7.4 ) containing lysozyme ( 0.1 mg / ml ) , sds and proteinase k at 55 for 3 h. after the completion of this incubation , dna was extracted with phenol - chloroform , precipitated with isopropanol and dissolved in te buffer with rnase ( 20 g / ml ) . in order to determine whether b. anthracis isolates were full virulent ( pxo1 and/or pxo2 )
, we performed a polymerase chain reaction ( pcr ) assay with a primer set specific for b. anthracis .
each pcr mixture ( 20 l ) contained 10 ng of genomic dna , 10 pmole of each b. anthracis specific primer , and 2 ef - taq premix ii ( 10 l , solgent , korea ) .
the following thermal cycling ( bio - rad , usa ) conditions were used : an initial denaturation step ( 94 for 5 min ) followed by 30 cycles of denaturation at 94 for 1 min , annealing at 40 for 1 min , and primer extension at 72 for 1 min .
the pcr products were electrophoretically separated on 1% agarose gel and stained with ethidium bromide .
mlva was performed for the eight different vntr loci described by keim et al . .
after amplification , the pcr products were diluted 1 : 10 ( volume : volume ) in te buffer and 1 l of the dilution was added to a mixture containing 8 l of hi - ditm formamide ( applied biosystems , usa ) and rox - labeled mapmaker 1000 size standard [ bioventures , usa ; 20 : 1 ( volume : volume ) ] .
the dna was denatured at 95 for 5 min and then immediately placed on ice .
fragments were detected using an abi 3100 prism genetic analyzer ( applied biosystems , usa ) with a 36-cm capillary at a running voltage of 15 kv at 60. to determine the size of the detected fragments , peak height and peak area was measured using genescan analysis software ( ver .
an mlva bank developed by the university of orsay ( university paris xi , france ) was used . for cansnp analysis
, we used the primers described by van ert et al .   and purchased from genotech ( korea ) in which each primer was added a t7 primer sequence as a 5 ' tail on the primers for sequencing purposes .
after amplification , the pcr products were cloned using a t - a cloning vector ( rbc bioscience , taiwan ) .
each pcr mixtures contained 2 ef - taq premix ii ( solgent , korea ) , forward and reverse primers ( 0.5 pmol each ) , and genomic dna ( 400 ng/l ) .
the thermal cycling conditions were incubation at 95 for 5 min that was followed by 30 cycles of 95 for 1 min , 60 for 1 min , and 72 for 1 min .
the plasmids were then purified with hi - yield plasmid mini kit ( rbc bioscience , taiwan ) and sequenced using the t7 primer .
mlva and cansnp analysis data obtained for b. anthracis isolates from korea were used to calculate simple matching coefficients using mega ( ver .
to investigate the genetic diversity of b. anthracis strains isolated in korea , we used mlva and cansnp analysis to characterize 21 strains , including four that were obtained from the kcdc ( table 1 ) .
many researchers reported the pasteur strain to be pxo1 and pxo2 , but the one used in our study was pxo1 and pxo2 since pxo2 was cured during the culturing process .
b. anthracis strains obtained from the kcdc were pxo1 and pxo2 while the isolates obtained in korea ( hs , bch , kj , and ch ) contained both pathogenic plasmids ( pxo1 and pxo2 ; table 1 ) .
genotype analysis was performed using eight - locus mlva data acquired from an mlva bank .
b. anthracis strains obtained from the kcdc were placed into the a3b cluster while the ch isolates were in a b1 cluster ( fig . 1 and table 2 ) .
on the other hand , hs , bch , and kj strain were classified as a3a cluster and were not a part of the a3b or b1 clusters .
b. anthracis strains obtained from the kcdc ( s0303 , s0304 , s0307 , and s0308 ) had the same mlva type with # 07 strain of ryu 's study .
interestingly , all of the local isolates ( ch ) were pathogenic and contained identical sequences at eight hypervariable mlva loci .
sequence repeats in eight of the vntr loci ( vrra , vrrb1 , vrrb2 , vrrc1 , vrrc2 , cg3 , pxo1 , and pxo2 ) were identical to those of the other korean b. anthracis strains from changnyung ( # 02 strain of ryu ' study ) . in our analysis ,
sequences of the b. anthracis strains obtained from the kcdc ( pxo1 , pxo2 ) were identical in the vntr loci ( vrra , vrrb1 , vrrb2 , vrrc1 , vrrc2 , and cg3 ) and varied at one vntr locus ( pxo2 ) with respect to the sterne strain .
furthermore , the genotypes of the b. anthracis strains obtained from the kcdc and the sterne strain differed and were varied at three vntr loci ( vrra , vrrb1 , and vrrb2 ) with respect to the atcc14185 and atcc14578 strains ( table 2 ) .
the hs strain had the same mlva type as # 50 strain of keim 's study except vrrc1 .
the mlva type of the bch strain was identical to # 58 strain of keim 's study except pxo2 . additionally , the mlva type of the kj strain was same as # 32 strain of keim 's study except vrrc1 .
cansnps were used to calculate simple matching coefficients and subdivided the b. anthracis isolates according to major sub - lineages ( a and b ) .
these were further divided into six of 12 different sub - lineages ( table 3 and fig .
2 , diamond ) or sub - groups ( circle ) . in order to make comparisons with global genetic populations , data
obtained for b. anthracis korean isolates from our study along with the results of an eight - loci mlva and 13-cansnp analysis for 1,033 reported b. anthracis isolates from 42 countries were used to calculate simple matching coefficients ( figs . 2 and 3 ) .
b. anthracis vntr sequences found in many different chromosomal and plasmid locations exhibit a few or even dozens of different allelic states , showing that this bacterium has a degree of diversity frequently lacking among pathogenic microbes .
mlva is currently utilized to identify different sub - populations and particular strains of interest . for b. anthracis , six chromosomal loci and one locus on each of pxo1 and pxo2
, a global b. anthracis ( over 400 strains ) analyzed and subdivided two major clonal lineages ( a and b ) and 86 unique mlva 8 genotype using eight - loci mlva .
this eight - loci mlva has also been used to examine the genotypes of b. anthracis in france , poland , italy , and countries in southern   and northern africa . in 2007 , van ert et al .
, used a 15 marker - loci , mlva15 , to examine a collection of 1,033 b. anthracis isolates from 42 countries .
this group described three major lineages ( a , b , and c ) that were further subdivided into 12 clonal sub - lineages or sub - groups , and finally 211 unique mlva15 genotypes .
le flche et al .   augmented the mlva8 assay by proposing 14 additional markers . additionally , lista et al .   expanded the mlva21 assay with the addition of four markers ( 25-loci mlva ) and described two new branches , d and e. the eight - locus mlva was used in our study of vrrb1 , vrrb2 , vrrc1 , vrrc2 , and cg3 identified by sequencing b. anthracis amplified fragment length polymorphism markers .
vrra was identified by andersen et al . , and the remaining two loci were identified by analysis of the pxo1 and pxo2 plasmid sequences .
vrrc is located in a gene encoding a ftsk - spooiiie dna translocase family homologue .
vrra , vrrb , and vrrc markers are found in protein coding regions of the b. anthracis genome . as the mlva marker expands or contracts ,
this genotyping method had been previously used for consistent subspecies typing worldwide for discrimination of the b. anthracis genome with the highly monomorphic nature . among the isolates analyzed in our study , eight vntr markers had different levels of variability in four loci ( vrra , vrrb1 , vrrb2 , and pxo2 ) and
based on the mlva profiles , korean b. anthracis isolates were assigned to both the a and b branches .
similar genetic dissimilarity has been found in b. anthracis isolates originating from several geographic regions in africa , europe , north america , and south america . in a previous analysis ,
b. anthracis isolates originating from asia , turkey , and china were assigned to branch a .
it is interesting that our korean isolates belong to both the a and b branches although our data agree with the results of a previous study .
variations in the number of repeat sequences at a given locus , or in - sequence heterogeneity among individual isolates , allows discrimination among strains .
these may be due to slipped - strand mispairing and can occur in combination with inadequate dna mismatch repair during replication .
however , the discriminative power of vntr markers becomes limited when analyzing strains of close geographical origins .
in such situations , diversity can be demonstrated by either analyzing a greater number of vntr markers   or using more sensitive markers .
snps have been used in several studies as important markers that linked sequence variations to phenotypic changes , thus elucidating the molecular basis of diseases .
these are also considered extremely valuable to perform phylogenetic analysis as snps have very low mutation rates and are less prone to homoplasy .
comparative full - genome sequencing between eight strains of b. anthracis which led to the discovery of about 3,500 snps was reported in the study of read et al .   along with 990 snps in 26 diverse isolates .
these results demonstrate the usefulness of a few strategically placed snps that may replace a large number of snps for typing b. anthracis strains and generating an snp - based tree .
cansnp have also been previously used to successfully differentiate b. anthracis isolates and identify worldwide patterns of distribution in which the major clonal lineage a had been shown to be widespread globally . in the study of van ert et al .
, b. anthracis isolates are subdivided into two previously recognized distinct major sub - lineages ( a and b ) according to the cansnp data , and further divided into 6 of 12 different sub - lineages . in this study , data from an eight - locus mlva also clearly classified these strains into two major lineages .
the more common genotype among the b. anthracis strains previously analyzed and globally distributed strains is the a genotype .
strains obtained from the kcdc ( s0303 , s0304 , s0307 , and s0308 ) and pasteur strain belong to the a.br.ames sub - group , which is found in central and eastern china .
later , other isolates were shown to be closely related to the ames strain , indicating the rarity of this strain in nature . despite its scarcity
, the ames strain is widely used as a reference strain in many laboratories for genotyping analysis .
two similar strains of b. anthracis ( 14578 ) from the kcdc and from the laboratory stock used in our study were found to belong to the a.br.vollum sub - lineage .
this sub - lineage genotype is dominant in southern africa and is also present in europe .
three strains ( sterne , hs , and delta sterne ) were found to belong to the a.br.001/002 sub - group , and a similar genotype has been identified in central and eastern china .
cansnp analysis of one laboratory strain ( 14185 ) showed that it belongs to the a.br.003/004 sub - group in which is common in south america .
one of the b. anthracis isolates ( kj ) collected recently from korea belonged to the a.br.005/006 sub - group , which is common in southern africa .
the korean b. anthracis isolates from ch analyzed in our study belonged to the b.br.001/002 cansnp sub - group .
the major clonal lineages found in asia are usually assigned to lineage a , which is more common than the other lineages and distributed globally .
b lineage isolates have been found in southern africa ( b.br.kruger sub - lineage and b.br.001/002 cansnp sub - group genotypes ) , some parts of europe ( b.cneva-9006 sub - lineage ) , and a small region in california , usa . additionally , a major clonal lineage b genotype was identified in korean isolates from ch in this study , and these isolates were closely related to the b1 cluster of mlva reported by ryu et al . .
anthrax is rare among humans but very common among grazing animals due to the natural spore transmission cycle .
spores that are formed in a place where an animal died from anthrax are the main source of infection for grazing animals .
it is also plausible that livestock grazing over dry , dusty , and contaminated soil inhale spores , leading to infection .
humans can be infected through contact with diseased animals or animal products depending on the route of spore entry : cutaneous ( the most frequent form of natural infection ) , inhalation , and gastrointestinal . throughout history
, anthrax had been widely spread around the world through the trade of infected live animals or meat , hides , hair , wool , or bones from such animals .
infected animals and products are often transported in long distances for industrial , food , or handicraft purposes , and the anthrax spores may find their way to livestock in the local community , thus spreading the disease in locations distant from the original infection source . restricting
the geographical distribution of b. anthracis is primarily a result of niche specialization associated with adaptive differences among the different lineages .
however , genetic evidence indicates that human activities have dramatically influenced the global population structure of b. anthracis .
given that natural outbreaks of anthrax are extremely rare , the introduction of new lineages in korea might be the result of agricultural development and increased international trade .
results of the present study have added further insight into b. anthracis phylogeny and the distribution of korean isolates .
 bacillus ( b. ) anthracis is the pathogen that causes fatal anthrax . 
 strain - specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics . in the present study , we employed molecular approaches including multiple - locus variable - number tandem repeat analysis ( mlva ) and canonical single - nucleotide polymorphism ( cansnp ) analysis to perform molecular typing of b. anthracis strains isolated in korea . according to the mlva 
 , 17 b. anthracis isolates were classified into a3a , a3b , and b1 clusters . 
 the cansnp analyses subdivided the b. anthracis isolates into two of the three previously recognized major lineages ( a and b ) . 
 b. anthracis isolates from korea were found to belong to four cansnp sub - groups ( b.br.001/2 , a.br.005/006 , a.br.001/002 , and a.br.ames ) . 
 the a.br.001/002 and a.br.ames sub - lineages are closely related genotypes frequently found in central asia and most isolates were . on the other hand , 
 b. anthracis ch isolates were analyzed that belonged to the b.br.001/002 sub - group which found in southern africa , europe and california ( usa ) . 
 b.br.001/002 genotype is new lineage of b. anthracis in korea that was not found before . 
 this discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in korea . 
Introduction
Materials and Methods
Results
Discussion