br Results br Discussion Understanding the IA outbreak has b

Results
Discussion Understanding the IA outbreak has been difficult because of the mostly sporadic nature of cases that are restricted to a subset of the population with hidden activities (Berger et al., 2014). Furthermore, efforts to directly monitor heroin for anthrax spore contamination by culture or PCR have failed (Booth et al., 2010; Team, 2011). Epidemiological analyses to identify IA trends using fewer strains and with lower resolution molecular subtyping (clade specific canSNPs and MLVA-31) have been informative, yet incomplete. In this report, we have increased our discriminatory power and phylogenetic resolution by increased sampling of IA cases, screening two large strain collections to identify other phylogenetically relevant strains, and employing WGS to all relevant isolates. The assumption that European heroin is produced in Afghanistan and then smuggled through intervening countries is well founded and has restricted hypotheses for the source of the IA anthrax spores to these regions. One use of what does the cytoskeleton do databases is for source attribution, and in Price et al. (Price et al., 2012), the affiliation with Turkish isolates fueled speculation that the spore contamination event occurred while the heroin was transiting through this country. The identification of other isolates in the IA subclade, possibly from other parts of the world, illustrates the strengths and weakness of this argument and the necessity for extensive sampling in the suspected geographic region. Detailed sampling of B. anthracis in Afghanistan and other potential regional smuggling routes within the Middle East is greatly needed for eliminating and pinpointing likely sources. For most recent molecular epidemiological investigations, the current databases have been sufficient to identify possible sources, but additional contemporary sampling is needed to differentiate alternative source hypotheses. In this report, the linkage to the Middle East and Turkey still exists, but new strains utilized in this study emphasize the uncertainty in attribution as discussed by Price et al. (Price et al., 2012). These strains are more closely related to the IA subclade than the Turkish ones, and while their exact origins are unknown, they emphasize the need to consider and sample neighboring geographical regions as possible sources. The phylogenetic and temporal patterns reported here indicate that at least two spore contamination events occurred. The core genome SNP analysis strongly supports the presence of two IA clusters, each of which is monophyletic and well supported by multiple synapomorphic SNPs. The phylogenetic presence of other isolates that are spatially and temporally unrelated to IA cases provides further evidence that these are distinct groups. Even though these non-IA strains probably originated from the same region as the IA strains, the dates of collection (1957 and 1988) are key to establishing that these isolates are not directly epidemiologically related to the IA outbreak and thus break apart the otherwise monophyletic clade of IA strains. The early isolation dates also demonstrate that strains representing these two clades could have been affecting human and animal health for decades before the IA was detected. Because of the lack of temporal calibration for B. anthracis evolution, we are limited to collection dates to make inferences about the duration of this outbreak. These two IA groups therefore must have arisen independently. There is also diversity within each group, suggesting diversity at the source and the possibility of even more contamination events. It is possible that variation is generated in a single host infection but some of the phylogenetic patterns shown here are best explained by diversity generated from several infection cycles and thus contamination of B. anthracis from several hosts. Although distinct, the two subclades are closely related with only 16 SNPs separating them. This level of variation is frequently seen within a single country (Khmaladze et al., 2014; Simonson et al., 2009; Kenefic et al., 2009) and could be indicative of a close geographic relationship for the two sources.