we obtained clinical samples and background information from 15 glanderous equids in punjab , pakistan from 1999 through 2007 ( table ; 
 appendix table ) . research on equine subjects was approved by the synopsis scrutiny committee and animal ethics committee , faculty of veterinary science , university of agriculture , faisalabad , pakistan . samples were plated on brain - heart infusion ( bhi ) agar with 4% glycerol and incubated for 2430 hours at 37c . individual colonies were inoculated into bhi broth containing 4% glycerol and were incubated with shaking for 36 h at 37c . an aliquot of broth ( 1.5 ml ) was centrifuged at 13,000 rpm for 15 min . genomic dna was extracted from the resulting pellets using standard digestion buffer and phenol - chloroform extraction protocols ( 6 ) . samples from equines with similar histories were assigned to the same epidemiologic group ( e.g. , group 1 , group 2 ) . subgroups were defined based on vntr data ; samples with identical vntr genotypes were assigned to the same subgroup ( e.g. , prl11 and prl13 in group 1b ) . data are comparing subgroups 3a and 3b to each other ( prl1 and prl41 ) and excluding subgroup 3c ( prl7 ) .  data are comparing subgroups 3a and 3b combined ( prl1 with prl41 ) to subgroup 3c ( prl7 ) . for genotyping , we screened 23 loci ( appendix table ) from a previously established 32-marker multiple locus variable number of tandem repeats ( vntr ) analysis system designed for b. pseudomallei and b. mallei ( 7 ) . in silico genotyping of the same loci was also performed for 10 whole genome sequences ( wgs ) of b. mallei ( 8 ; appendix table ) . vntr markers have higher mutation rates than other genetic markers which make them inappropriate for determining deep levels of evolutionary relatedness , however vntrs are appropriate for 1 ) discrimination between closely related isolates , 2 ) determination of the degree of relatedness among isolates , and 3 ) discernment of population structure on a spatial scale ( 7,9,10 ) . this utility is especially important for b. mallei because it is a recently emerged clone of b. pseudomallei and has been shown to be genetically monomorphic with typing methods such as multilocus sequence typing ( 11 ) . to compare the genetic diversity of our punjab isolates to that of sequenced strains , we performed a phylogenetic analysis on the 23 loci using the neighbor - joining algorithm in paup * 4.0b ( 9 ) . to determine the genetic relationships among the punjab population itself , we performed the same analysis using only the punjab isolates and polymorphic loci ( n = 15 loci ) . combined analysis of the punjab isolates and wgs showed that the punjab isolates are phylogenetically distinct from wgs ( figure 1 ) . this finding was also demonstrated in the values for average pairwise distance ( apd ) , where the apd among punjab isolates is 2 lower than the apd calculated for either the entire phylogeny or the wgs alone ( figure 1 ) . the punjab isolates represent only a small amount of the genetic diversity demonstrated in this pathogen . phylogenetic analysis of the punjab isolates alone placed 14 of the 15 samples into 3 distinct clades with 1 sample standing alone ( figure 2 ) . most samples ( 9/15 ) belong to clade a , whose isolates are both temporally and geographically diverse , suggesting that this lineage is ecologically established in punjab . unrooted neighbor - joining tree based on 23 variable number tandem repeat loci demonstrating that the punjab isolates ( black text and prl-20 ) are genetically distinct from and less diverse than available whole genome sequences ( wgs , red text ) ( 8) . statistical supports for branches based on 1,000 bootstrap iterations are shown . sample prl-20 is shown in red text because it is also available as a whole genome sequence ; therefore , it was used in all 3 situations where an average pairwise distance ( apd ) was calculated . among 10 wgs , the average pairwise distance was 0.607 ; between 10 wgs and punjab isolates , average pairwise distance was 0.627 ; and among 15 punjab isolates , average pairwise distance was 0.312 . these results indicate that the punjab isolates are more closely related to each other than to the sequenced strains because the apd among punjab isolates is 2 lower than the apd calculated in the other 2 situations . unrooted neighbor - joining tree showing phylogenetic relationships among 15 samples of burkholderia mallei from the punjab province , pakistan . sample names are color - coded to match their district of origin in reference to the inset map of the punjab province . approximate linear distances between districts are faisalabad to lahore 120 km , faisalabad to sargodha 84 km , lahore to sargodha 168 km . because of the limited sample size , many of the patterns observed from these data may result from sampling bias . however , even a limited amount of genotypic data can be useful in formulating hypotheses regarding the dispersal of b. mallei . for example , the presence of samples from faisalabad in each clade suggests that this district may be a center of diversity in the province ( figure 2 ) but this does not indicate a lesser degree of diversity in other districts where fewer samples were collected . the diversity seen in the district of faisalabad may result from either 1 ) the industrial nature of faisalabad or 2 ) from high endemism of b. mallei in the region . currently , the district has 10,000 horses and mules and > 44,000 donkeys , plus other transient equines ( 12 ) . many equines move through and work in the city , potentially introducing strains from surrounding regions . because horses and mules can be positive but asymptomatic for glanders ( 13 ) , many hosts are available to maintain strains throughout the region . communal stables and water troughs are common throughout the district and b. mallei has been isolated from these water troughs ( a. naureen , unpub . furthermore , b. mallei can remain viable in contaminated stables for up to 6 weeks ( 14 ) and in sterile tap water for up to 4 weeks ( 15 ) , which provides an environment for establishment and retention of b. mallei populations in faisalabad . combining phylogenetic with epidemiologic data for example , epidemiologic data suggests that 2 horses from a farm in the district of sargodha ( prl3 and prl4 ) contracted glanders while at a polo club in the lahore district . this is supported by vntr data , as these 2 isolates clustered phylogenetically with one of the samples obtained from an outbreak that occurred at the same polo club 3 months prior ( groups 5 and 6 , table ) . furthermore , at the time of the prl3 and prl4 infections , a co - resident mule with no previous travel history ( prl44 ) was negative for glanders , making it unlikely that these horses acquired glanders from their farm . this mule was positive for glanders 1.5 years later , and the isolate obtained from its infection clustered phylogenetically with the samples from the polo club and sargodha horses . therefore , we hypothesize that the infected horses either directly transferred the disease to the mule or they contaminated a source on the farm which subsequently led to the mules infection . environmental sampling would be required to identify the original infection source for the horses and subsequent transmission route to the mule . nevertheless , this case shows a strain that was transferred a distance of 168 km , demonstrating that human - mediated movement of equines can influence the distribution of b. mallei genotypes . other cases in the province demonstrate that infections either stem from similar strains or are caused by multiple strains . for example , samples that were placed in the same epidemiologic group cluster together phylogenetically ( groups 1 , 2 , and 4 ; table ) , indicating communal infections similar to the cases described above . in contrast , epidemiologic group 5 ( prl19 and prl20 ) was separated into 2 distinct clades ( figure 2 ) , indicating that this outbreak was caused by multiple strains . therefore , it should not be assumed that an outbreak of glanders is always caused by a single strain . our study suggests that numerous lineages of burkholderia mallei are present in punjab , pakistan , and that these lineages persist across geographic space and time . despite this the economics and use of equines likely contribute to the persistence of glanders in this region because modern methods for control of this disease ( monitoring and euthanasia ) are not viable options . we suggest that a focus on finding methods to improve the sanitary conditions of communal water troughs and stables may lead to a practical solution for disease reduction and containment . finally , our study demonstrates the utility of vntrs paired with extensive epidemiologic data for analyzing the distribution of b. mallei genotypes throughout endemic regions . epidemiologic and 23-locus vntr data for 15 isolates of burkholderia mallei from the punjab province , pakistan , and 9 burkholderia mallei whole genome sequences *
we collected epidemiologic and molecular data from burkholderia mallei isolates from equines in punjab , pakistan from 1999 through 2007 . 
 we show that recent outbreaks are genetically distinct from available whole genome sequences and that these genotypes are persistent and ubiquitous in punjab , probably due to human - mediated movement of equines .