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 due to human-mediated movement of equines.

temporally and geographically diverse, suggesting that this lineage is ecologically established in Punjab.
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. data). 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 reveals how B. mallei disseminates throughout a region. 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 mule's 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 infl uence the distribution of B. mallei genotypes. This case also suggests that a strain can persist for ≈1.5 years.
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.

Conclusions
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, these isolates appear to be genetically distinct from other studied strains. 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.  †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). ‡See Figure 2. §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).
Therefore, other solutions to curbing the spread of glanders need to be identifi ed. We suggest that a focus on fi nding 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.