Molecular Epidemiology of Glanders, Pakistan

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 because of humanmediated movement of equines. G landers is an equine disease that was recognized byHippocrates and Aristotle (1).It is caused by the bacterium Burkholderia mallei, an obligate pathogen of horses, donkeys, and mules (Equidae), with occasional infections in felines, canines, and humans (2,3).Strict regulation of equines has reduced the range of this once globally distributed disease to a few endemic foci in South and Central America, the Middle East, and parts of Africa and Asia (2,4).This emerging disease has only recently regained attention following the listing of B. mallei as a Category B agent by the US Centers for Disease Control and Prevention (2).Although outbreaks are common in regions of disease endemicity, much of what is known about the ecology and natural population dynamics of B. mallei relies on indirect evidence and expert opinion, with little to no knowledge concerning its genetic diversity (2,3).We genetically characterized 15 samples of B. mallei from recent outbreaks in Pakistan to provide additional knowledge of how this disease of antiquity is transmitted throughout endemic regions today. The StudyWe obtained clinical samples and background information from 15 glanderous equids in Punjab, Pakistan from 1999 through 2007 (Table; online Appendix Table, available from www.cdc.gov/EID/content/15/12/2036-appT.htm).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 brainheart infusion (BHI) agar with 4% glycerol and incubated for 24-30 hours at 37°C.Individual colonies were inoculated into BHI broth containing 4% glycerol and were incubated with shaking for 36 h at 37°C.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).For genotyping, we screened 23 loci (online 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; online Appendix Table).VNTR markers have higher mutation rates than other genetic markers, which make them inappropriate for determining deep levels of evolutionary relatedness.VNTRs, however, 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 fi nding 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).Therefore, 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