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. 2009 Mar 13;4(3):e4813. doi: 10.1371/journal.pone.0004813

Pre-Columbian Origins for North American Anthrax

Leo J Kenefic 1,#, Talima Pearson 1,#, Richard T Okinaka 1, James M Schupp 1, David M Wagner 1, Jacques Ravel 2, Alex R Hoffmaster 3, Carla P Trim 1, Wai-Kwan Chung 1, Jodi A Beaudry 1, Jeffrey T Foster 1, James I Mead 1, Paul Keim 1,4,*
Editor: Igor Mokrousov5
PMCID: PMC2653229  PMID: 19283072

Abstract

Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ∼13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates. Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.

Introduction

The basic premises of disease tracking have changed little since John Snow first described the London cholera epidemic of 1854. The use of molecular genotyping technologies has allowed the epidemiological linkage of geographically disparate isolates, generating hypotheses about patterns and modes of disease dispersal. As might be expected, the distribution of human pathogens that cause persistent infections such as Helicobacter pylori, the Typhi serovar of Salmonella enterica, Mycobacterium tuberculosis and Polyomavirus JC reflect both recent and ancient human migratory patterns [1]–[7]. Conversely, pathogens that cause acute infections remain only briefly within a host and are therefore less likely to follow long term host distribution patterns [3]. The dispersal of Bacillus anthracis, Yersinia pestis, and human RNA viruses often reflect short term human movement frequently associated with trading contaminated animal products or inadvertently transporting primary vectors or hosts [1], [3], [8]–[12]. Such potentially frequent and long range dispersal of pathogens can obscure more ancient phylogeographic patterns. The history of B. anthracis in North America has certainly been affected by recent trade, and livestock movement [13], however here we present evidence that the introduction of this pathogen can be traced to much more ancient human migrations. We believe this to be an example of an opportunistic human pathogen reflecting ancient human dispersal patterns.

The recent and dramatic increase in the ability for extensive genomic sampling through whole genome sequencing coupled with extensive strain collections should enhance our ability to reconstruct even ancient epidemiological events. The strictly clonal reproductive patterns and low polymorphism frequency of evolutionarily stable molecular markers in B. anthracis makes this a model organism for tracking ancient epidemiological patterns. Whole genome sequencing of multiple B. anthracis strains has led to the construction of a highly accurate phylogenetic backbone [14] based upon an expansive world-wide strain collection [13]. Whereas it would be advantageous to sequence all available isolates within a lineage, this approach is still prohibitively expensive and requires SNP detection by whole genome comparisons. Targeting characters and taxa within specific lineages can further enhance the detection of evolutionary patterns which, when combined with sample spatial data, enables precise epidemiological tracking of disease, even for pre-historical events.

B. anthracis has dispersed globally via large and sequential radiations associated with human commerce and trade of animal products contaminated with B. anthracis spores [13]. Without human involvement, infected animals typically die within 7–10 days, seeding only the surrounding soil with spores thus keeping the spread of the disease relatively contained [15]. The potential for dispersion even among migratory herds is limited since infected animals typically die quickly before extensive dispersal can occur. Historically, an animal that died of anthrax was scavenged by people for its hair, hide, bones, and even consumed as food, facilitating the dispersal of spores away from a carcass. Indeed, imported spore-contaminated animal hides account for many of the recent US human anthrax cases [16], though such modern cases infrequently result in subsequent ecological establishment or further dispersal. Therefore, with the exception of the most recent human cases, the current distribution of B. anthracis can be traced to historical human dispersion, trade, and migratory patterns.

The most dramatic dispersal and clonal expansion of B. anthracis was the A-radiation [13], which is phylogenetically rooted in the Old World. Nested within the A-radiation is the highly successful trans-Eurasian subpopulation (TEA). Its prevalence in Europe and Asia is thought to be mediated by the east-west human trade routes, such as the “Silk Road”. One sublineage of this TEA population, western North America (WNA), was introduced into North America and has become highly successful within this geographic region. The WNA sublineage is dominant today in central Canada and much of the western United States.

In North America, two distinct types of anthrax cases are seen. Many have been observed along the East Coast and are associated with trade and industrial processing of contaminated animal products, often wool in textile mills [17]–[19]. These cases contribute to the overall genetic diversity of North American B. anthracis isolates, but generally represent small case clusters that do not become ecologically founded. This lack of establishment could be due to a requirement of suitable habitat for natural disease cycling [20]. In contrast, western North American grasslands are ideal for the ecological establishment of anthrax and may have persisted for much of the Holocene epoch, possibly over 10,000 years [21].

Indeed, at least two B. anthracis clades are ecologically established in North America on a sub-continental scale [13]. The “Ames” clade (A.Br.Ames) has been associated with highly localized and sporadic outbreaks in south Texas since at least the early 1980s [22]. Only a short evolutionary time period, very few SNPs, separate Texas Ames isolates from Asian near relatives, suggesting a recent or perhaps colonial animal importation. In contrast, the WNA clade has been widely successful both in distribution and frequency across central and northerly North American regions and is clearly ecologically established in many geographic areas. WNA isolates have been recovered from near the Artic Circle in Canada to the U.S. Mexican border and even in insular Haiti, and account for 89% of non-human anthrax cases in North America. The WNA clade also exhibits greater genetic diversity than the Ames clade, and a longer evolutionary separation (106 SNPs) from its nearest Old World relatives (TEA), suggesting a more ancient introduction into North America. The ecological dominance and disease importance of the WNA clade led us to examine its evolutionary history in greater detail using whole genome sequence analysis and highly accurate phylogenetic reconstructions.

Results

SNP Analyses

Whole genome sequencing of seven diverse strains led to the discovery of 2,850 SNPs suitable for conversion to whole genome tiling microarrays. One of these seven strains was the WNA strain (A0193) [14]. These SNPs were screened among 128 diverse isolates (described by MLVA15 analyses) and identified WNA as a monophyletic group rooted in the Old World TEA group. These data also showed the WNA group to be separated by a long phylogenetic branch (106 SNPs) (Fig. 1) representing 53% of the total distance from the initiation of the A branch radiation to the sequenced WNA isolate. Ten of these 106 SNPs were developed into Real-time PCR assays and used to screen all 387 isolates in the study. These SNPs identified six sub-clades within the previously described WNA lineage.

Figure 1. Phylogeography of the WNA Bacillus anthracis clone.

Figure 1

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The evolutionary tree of the dominant North American B. anthracis lineage (WNA) and mapping of isolate locations supports southern dispersal of anthrax in North America after founding by a European or Asian ancestor (TEA). Circles indicate precise GIS coordinates, squares indicate state-level information, and colors indicate phylogenetic grouping.

Phylogenetic analyses

SNP characters are rare in the B. anthracis genome and almost never have character state reversals (∼0.1% homoplasy) [14]. Using a cladistic evolutionary model, there was one character-state inconsistency in these data (<1% homoplasy). Given such robust data, all approaches (e.g., MP, NJ) to construct phylogenetic trees generate the same evolutionary hypothesis (Fig. 1). Including any B. anthracis strain from outside this lineage allows us to accurately root this lineage using standard outgroup rooting methods.

Geographical mapping of WNA isolates

The six sub-clades identified by SNP analyses revealed a north-south phylogeographic pattern (Fig. 1), with short terminal branches indicating rapid radiation of the WNA group following the initial establishment in northern Canada. Additionally, there were correlations among the nodes identified with recovery from their respective hosts. For example, the yellow group was mostly associated with wildlife (n = 63) whereas the other groups were almost exclusively from livestock.

Discussion

SNPs are stable phylogenetic characters in B. anthracis and impart a highly accurate evolutionary hypothesis both in terms of branch lengths and branching order [14]. Our phylogenetic topologies are highly suggestive of an initial introduction of B. anthracis into the far north of North America and subsequent southerly dispersal (Fig. 1). The ancestral WNA phylogenetic nodes are northernmost, followed by progressively more southern locations for the more recently derived clades. More recent populations in southern states and a single human isolate from Haiti (not shown) are probably the result of recent commodity trading or infected livestock transport. In contrast, the northern-most and more ancient populations of WNA B. anthracis remain relatively localized, possibly due to association with bison (Bison bison) and restricted human commerce in this region. SNP-estimated evolutionary branch lengths provide additional support for a pre-Columbian introduction of WNA anthrax to North America. The accumulation of up to 106 SNPs since the split of the WNA lineage from the TEA lineage is a relatively large number for B. anthracis clades [14]. Interestingly, this indicates a long evolutionary separation followed by a genetic bottleneck and a single founding event in North America. In contrast, the Texas ecologically-established Ames lineage accumulated only ∼8 SNPs since introduction from Asia [23]. Molecular clock calibration is always problematic, but this >10-fold difference offers dramatic contrast between a recent and ancient New World introduction. Moreover, divergence times within branches of the A-radiation may be greatly affected by the number of generations in natural disease cycling. The model introduced in Van Ert et al (2007) was based upon 1 and 0.5 generations per year (approximately 3500 to 7000 ybp respectively for the major A-radiation). However, a recent overview of early outbreaks in Northern Canada provides greater insights into natural disease cycling in this region. Dragon and Elkin noted [24] that there were eight sporadic outbreaks between 1962 and 1991, and since 1993, there have been five more. Using these empirical data we can estimate 0.28 generations per year (13 outbreaks in 46 years). This empirical based estimation of this parameter significantly increases divergence times within the TEA-WNA lineage and suggests that divergence times provided in Van Ert et al. (2007) were underestimates.

Human migrations are the most likely source for the introduction and establishment of the WNA lineage of anthrax in North America through an ice-free corridor that connected Beringia to the southern areas east of the Rocky Mountains. Contiguous grassland habitat created by the partial retreat of the Laurentide and Cordilleran ice sheets would have been ideal for anthrax susceptible grazing herd animals. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem [25]. This grassland refugium allowed animals and humans to freely cross what would become a water barrier (Bering Sea) in the Holocene. Humans could have transported B. anthracis to this land bridge from Asia and then moved it further south as the ice-free corridor and developing grassland opened in central Canada ∼13,000 ybp. Bison and other potential herding hosts were already widespread in North America, yet the limited range of the most ancient northern WNA B. anthracis populations suggest that anthrax was not present at this time south of the bottleneck created by the coalescing glaciers. Furthermore, as the ice-free corridor expanded, there was a simultaneous northward movement of these grazers like bison, yet the phylogenetic directionality of anthrax spread is southerly. Humans, however, did move southward through this corridor and could have brought contaminated animal products with them, which eventually enabled the ecological introduction of WNA B. anthracis in North America.

This northern-origin hypothesis is highly consistent with phylogeographic patterns and rates of SNP accumulation observed in recent B. anthracis isolates. While isolates from many different lineages have been observed in North America, their presence can be attributed to post colonial trade. Continent-wide dispersal of WNA B. anthracis may have involved later European colonizers, but some of the first inhabitants of the continent likely seeded the initial North American populations.

Materials and Methods

Strains

The 387 strains (Table 1) used in this study were obtained from various sources and linked to both livestock and wildlife outbreaks in North America. Four of these isolates were associated with human infections. Of the 387 strains, 352 cluster with the major ecologically established Western North American clade (WNA: A.Br.WNA) whereas 17 isolates were included to represent the TEA population as an outgroup. Genotypic data for each strain is provided in Table 2.

Table 1. Strains used in this study (Epidemiologic data)1.

Sample ID 2 Country of Origin Host State/Province of Infection 3 County Nearest City/Town Latitude 4 Longitude
A1055 USA soil LA
A1065 USA bovine WY
A2051 USA Bovine CA Santa Clara San Jose
A2052 USA Bovine CA Santa Clara San Jose
A0394 USA goat kid TX Terrell
A1115 USA Bovine TX Leakey
A1117 USA Bovine TX
A2012 USA Human FL Palm Bay
A0169 Canada bovine
A0517 USA Vaccine
A0248 USA Human
A1057 USA bovine OK
A1056 USA Bovine
A0078 USA
A0156 USA sheep
A1058 USA bovine TX
A1062 USA MS
A1063 USA MD Ft. Detrick
A0032 China Fur
A0033 China Wool
A0149 Turkey human
A0241 Turkey goat
A0245 Turkey bovine
A0264 Turkey human
A0280 Italy sheep
A0303 Canada bovine
A0324 Slovakia
A0343 Hungary
A0362 Norway bovine
A0417 Hungary
A0463 Pakistan Sheep
A0480 Russia vaccine
A0596 China Marmot Xinjiang
A0604 China Soil Xinjiang
A0610 China Soil Xinjiang
A0669 China Soil Xinjiang
2000031001 Canada
2000031006 USA NM
2000031036 USA Moose WY
2000031764 Goat
2000031765
2000031766 USA Puritan Loom NC
2000031767
2000031770
2000031774
2000032818
2000032819
2000032820
2000032821
2000032822
2000032826
2000032828
2000032829
2000032831
2000032832
2000032833
2000032834
2000032888
2000032989
2000032990
2000032993
2000032994
2000032995
2000032996
2000032997
2002013003
2002013004
2002013007
2002013008
2002013009
2002013010
2002013011
2002013012
2002013013
2002013014
2002013015
2002013016
2002013017
2002013019
2002013020
2002013021
2002013022
2002013023
2002013024
2002013025
2002013026
2002013027
2002013028
2002013029
2002013030
2002013031
2002013032
2002013033
2002013034
2002013035
2002013053
2002013054
2002013055
2002013056
2002013057
2002013058
2002013059
2002013063
2002013064
2002013065
2002013068
2002013070 Canada Bison
2002013083 USA Mice WY
2002013086
2002013098
2002721542 USA Bovine UT
2002734033 USA UT
A0055 USA
A0056 USA
A0057 USA
A0071 USA
A0072 USA
A0073 USA
A0074 USA
A0075 USA
A0076 USA
A0117 USA Bovine NV
A0130 USA
A0142 Canada Moose
A0143 Canada bovine
A0144 Canada Bison
A0157 USA Pig
A0162 Canada Bison
A0163 Canada bovine
A0165 Canada Bison
A0166 Canada Bison
A0167 Canada bovine
A0168 Canada bovine
A0170 Canada bovine
A0171 Canada bovine
A0172 Canada bovine
A0173 Canada bovine
A0174 Canada bovine
A0175 Canada Bison
A0176 Canada Bison
A0177 Canada Bison
A0178 Canada Bison
A0179 Canada Bison
A0180 Canada bovine
A0181 Canada Ravens
A0182 Canada bovine
A0192 Canada bovine
A0193 USA bovine
A0194 Canada bovine
A0195 Canada bovine
A0197 Canada bovine
A0198 Canada bovine
A0246 USA bovine
A0268 USA Dog
A0295 Canada Bison
A0296 Canada Bison
A0297 Canada Bison
A0300 Canada bovine
A0301 Canada bovine
A0302 Canada bovine
A0304 Canada Bison
A0305 Canada Bison
A0306 Canada Bison
A0307 Canada bovine
A0308 Canada bovine
A0309 Canada bovine
A0310 Canada bovine
A0311 Canada bovine
A0312 Canada Bison
A0313 Canada Bison
A0369 Canada Bison
A0374 USA Bison
A0377 Haiti man
A0383 USA man CO
A0392 USA bovine
A0393 USA bovine
A0395 USA deer TX Uvalde
A0396 USA bovine TX Del Rio
A0397 USA deer TX Del Rio
A0398 USA deer TX Del Rio
A0407 USA bovine
A0408 USA bovine
A0409 USA bovine
A0410 USA bovine
A0418 Canada Bison
A0444 Canada bovine
A0445 Canada bovine
A0486 USA bovine
A0520 USA WT deer
A0526 USA Bovine MT Billings
A0767 Canada Red Fox Scat WBNP Parson's Lake 59 45′ 53.9″ N 112 16′ 55.4″W
A0768 Canada Red Fox Scat WBNP Parson's Lake 59 45′ 53.9″ N 112 16′ 55.4″W
A0770 Canada Sand WBNP Parson's Lake 59 45′ 14.4″N 112 16′ 24.2″ W
A0771 Canada Sand WBNP Parson's Lake 59 45′ 14.4″N 112 16′ 42.5″ W
A0772 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0773 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0774 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0775 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0776 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0777 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0778 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0779 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0780 Canada Soil WBNP Falaise Lake 61 28′ 52.0″ N 116 15′ 54.0″ W
A0782 Canada Soil WBNP Falaise Lake 61 29′ 7.0″ N 116 13′ 29.0″ W
A0783 Canada Soil WBNP Falaise Lake 61 29′ 7.0″ N 116 13′ 29.0″ W
A0784 Canada Soil WBNP Falaise Lake 61 29′ 7.0″ N 116 13′ 29.0″ W
A0785 Canada Soil WBNP Falaise Lake 61 29′ 7.0″ N 116 13′ 29.0″ W
A0786 Canada Soil WBNP Falaise Lake 61 29′ 7.0″ N 116 13′ 29.0″ W
A0788 Canada Soil WBNP Falaise Lake 61 27′ 36.0″ N 116 18′ 23.0″ W
A0791 Canada
A0792 Canada Bovine Rocky Mountain House
A0793 Canada Bovine Rocky Mountain House
A0794 Canada Bovine Rocky Mountain House
A0795 Canada Bovine Rocky Mountain House
A0796 Canada Bovine Rocky Mountain House
A0797 Canada Horse Rocky Mountain House
A0798 Canada Bovine Eckville
A0799 Canada Bovine Eckville
A0800 Canada Bovine Eckville
A0801 Canada Bovine Eckville
A0802 Canada Bovine Eckville
A0803 Canada Bovine Rocky Mountain House
A0804 Canada Bovine Caroline
A0805 Canada Bovine Rocky Mountain House
A0806 Canada Bovine Rocky Mountain House
A0807 Canada Bovine Alhambra
A0808 Canada Bovine Alhambra
A0809 Canada Bovine Alhambra
A0810 Canada Bovine Alhambra
A0812 Canada Bovine Rocky Mountain House
A0913 USA Bovine NV
A0917 USA
A0948 USA Bovine ND Walsh Park River
A0949 USA Equine ND Steele Sharon
A0950 USA Bison ND Grand Forks Grand Forks
A0951 USA Equine ND Traill Portland
A0952 USA Bovine MN Clay Hawley
A0953 USA Bovine ND Stutsman Parkhurst
A0954 USA Bovine ND Foster Grace City
A0955 USA Bovine ND Griggs Cooperstown
A0956 USA Bovine ND Steele Sharon
A0957 USA
A0958 USA Bovine ND Nelson Aneta
A0959 USA Bovine ND Grand Forks Northwood
A0960 USA Bovine ND Traill Portland
A0961 USA Equine ND Steele Sharon
A0962 USA Bovine ND Grand Forks Emerado
A0963 USA Bovine MN Becker Lake Park
A0964 USA Bovine ND Grand Forks Grand Forks
A0965 USA Bovine ND Pembina Walhalla
A0966 USA Bovine ND Grand Forks Emerado
A0967 USA Bovine ND Pembina Hensel
A0968 USA Bovine MN Roseau Greenbush
A0969 USA Bovine ND Grand Forks Larimore
A0970 USA Bovine ND Adams Reeder
A0971 USA Bovine ND Nelson Aneta
A0972 USA Bovine ND Nelson Pekin
A0973 USA Bovine ND Eddy Hamar
A0974 USA Bovine ND Nelson Tolna
A0975 USA Equine ND Nelson Petersburg
A0976 USA
A0977 USA Bovine ND Grand Forks Larimore
A0978 USA Bovine MN Roseau Badger
A0979 USA Bovine MN Mower Lyle
A0980 USA Bovine MN Pennington Thief River Falls
A0981 USA Bovine ND Grand Forks Larimore
A0982 USA Bovine ND Pembina Hensel
A0983 USA
A0984 USA
A0985 USA
A0988 Canada Bison WBNP
A0989 Canada
A0990 Canada
A0991 Canada Bear
A0993 Canada Bison
A0994 Canada Bison
A0995 Canada Wolf
A0996 Canada Bovine
A0997 Canada Bovine
A0998 Canada Moose
A0999 Canada Bovine
A1000 Canada Bovine
A1001 Canada Bovine Manitoba Vita
A1002 Canada Bovine Manitoba Vita
A1003 Canada Bovine Manitoba Vita
A1004 Canada Bovine Manitoba Vita
A1005 Canada Bovine Manitoba Vita
A1006 Canada Bovine Manitoba Vita
A1007 Canada Bovine Manitoba Vita
A1008 Canada Bovine Manitoba Vita
A1009 Canada Bovine Manitoba Vita
A1010 Canada Bovine Manitoba Vita
A1011 Canada Bovine Manitoba Vita
A1012 Canada Bovine Manitoba Vita
A1013 Canada Bovine Manitoba Vita
A1014 Canada Bovine Manitoba Vita
A1015 Canada Bovine Manitoba Vita
A1016 Canada Bovine Manitoba Vita
A1017 Canada Bovine Manitoba Vita
A1018 Canada Bovine Manitoba Vita
A1019 Canada Manitoba Vita
A1020 Canada Manitoba Vita
A1021 Canada Manitoba Vita
A1022 Canada Bison Manitoba Vita
A1023 Canada Bovine Manitoba Vita
A1024 Canada Bovine Manitoba Vita
A1025 Canada Bovine Manitoba Vita
A1026 Canada Bovine Manitoba Vita
A1027 Canada Bovine Manitoba Vita
A1028 Canada Bovine Manitoba Vita
A1029 USA Bovine NV Vita
A1030 USA Bovine NV Washoe Reno
A1031 USA Bovine NV Washoe Reno
A1040 USA bovine SD
A1041 USA bovine SD
A1042 USA bovine SD
A1047 USA bovine IA
A1118 USA Bison MN Greenbush
A1119 USA Bovine MN Greenbush
A1120 USA Bovine MN Greenbush
A1121 USA Bovine MN Greenbush
A1122 USA Bovine MN Greenbush
A1123 USA Bovine MN Greenbush
A1124 USA Bovine MN Greenbush
A1125 USA Bovine MN Greenbush
A1126 USA Bovine MN Greenbush
A1127 USA Bovine MN Greenbush
A1128 USA Bovine MN Greenbush
A1129 USA Bovine MN Greenbush
A1130 USA Bovine MN Greenbush
A1131 USA Bovine MN Greenbush
A1132 USA Bovine MN Greenbush
A1133 USA Bovine MN Greenbush
A1134 USA Bovine MN Greenbush
A1135 Canada Manitoba Sprague
A1137 USA SD
A1138 USA SD
A1139 USA SD
A2014 Mexico Bovine Acuna
A2015 Mexico Bovine Acuna
A3455 USA Bovine SD 44.83663 100.33589
A3456 USA Bovine SD 45.66154 98.41541
A3457 USA Bovine SD 46.90731 98.10566
A3458 USA Bovine SD 44.77967 100.42004
A3459 USA Bovine SD 44.83672 100.33584
A3460 USA Bovine SD 44.70675 100.19478
A3461 USA Bovine SD North
A3462 USA Bovine SD North
A3463 USA Bovine SD 44.98544 100.36005
A3464 USA Bovine SD 44.69881 100.46175
A3465 USA Bovine SD 44.98094 100.30310
A3466 USA Bovine SD 44.68186 98.05983
A3467 USA Bovine SD Central
A3468 USA Bovine SD 44.38436 99.61700
A3469 USA Bovine SD 44.41030 99.80588
A3470 USA Bovine SD 44.72853 100.45644
A3471 USA Bovine SD 44.91887 99.79668
A3472 USA Bovine SD 44.72853 100.45644
A3473 USA Bovine SD Central
A3474 USA Bovine SD 44.67489 98.05903
A3475 USA Bovine SD 44.90751 100.48686
A3476 USA Bovine SD North
A3477 USA Bovine SD Central
A3478 USA Bovine SD 44.95312 100.49400
A3479 USA Bovine SD 45.70536 98.39379
A3480 USA Bovine SD 45.25863 100.23270
A3481 USA Bovine SD 44.58392 99.87045
A3482 USA Bovine SD 45.92200 98.05015
A3483 USA Bovine SD North
A3484 USA Bovine SD 44.94745 100.51275
A3485 USA Bovine SD 45.03621 100.30700
A3486 USA Bovine SD 45.16650 100.11835
A3487 USA Bovine SD 44.79834 100.55977
A3488 USA Bovine SD 44.95903 99.90616
A3489 USA Bovine SD 45.13020 100.33318
A3490 USA Bovine SD 44.84012 98.87670
A3491 USA Bovine SD 45.52951 97.72276
A3492 USA Bovine SD 45.35902 97.49963
A3493 USA Bovine SD 44.53633 103.51009
A3494 USA Bovine SD 44.91887 99.79668
A3495 USA Bovine SD 44.10121 99.86757
A3496 USA Bovine SD Central
A3497 USA Bovine SD 45.51513 100.48982
A3498 USA Bovine SD 43.78920 99.26104
A3499 USA Bovine SD 44.17555 99.50194
A3500 USA Bovine SD 44.10859 99.45870
A3501 USA Bovine SD 45.06766 100.39238
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1

Missing or Unknown data has been intentionally left blank.

2

Samples having an “A” preceeding a four digit code are samples for which we possess live culture material. Samples having a 10-digit code were received as DNA from part of a historic collection maintained at the Centers for Disease Control and Prevention.

3

Two letter state codes are used when referencing states within the United States, otherwise the state is written out. WBNP is Wood Buffalo National Park.

4

Centroids were used for locations where state of origin information was available but Global Positioning System data was unavailable.

Table 2. Strains used in this study (Genotyping data)1.

Sample ID2 canSNP group3 A.Br.WNA4 wna237471 wna1141774 wna2994131 wna3368524 wna3631093 wna3682247 wna3732539 wna3774186 wna4461234 wna4718500
A1055 C.Br.A1055 A T C C A G G T T G G
A1065 C.Br.A1055 A T C C A G G T T G G
A2051 B.Br.001/002 A T C C A G G T T G G
A2052 B.Br.001/002 A T C C A G G T T G G
A0394 A.Br.001/002 A T C C A G G T T G G
A1115 A.Br.001/002 A T C C A G G T T G G
A1117 A.Br.001/002 A T C C A G G T T G G
A2012 A.Br.Ames A T C C A G G T T G G
A0169 A.Br.001/002 A T C C A G G T T G G
A0517 A.Br.001/002 A T C C A G G T T G G
A0248 A.Br.Aust94 A T C C A G G T T G G
A1057 A.Br.Aust94 A T C C A G G T T G G
A1056 A.Br.003/004 A T C C A G G T T G G
A0078 A.Br.Vollum A T C C A G G T T G G
A0156 A.Br.Vollum A T C C A G G T T G G
A1058 A.Br.Vollum A T C C A G G T T G G
A1062 A.Br.Vollum A T C C A G G T T G G
A1063 A.Br.Vollum A T C C A G G T T G G
A0032 A.Br.008/009 A T C C A G G T T G G
A0033 A.Br.008/009 A T C C A G G T T G G
A0149 A.Br.008/009 A T C C A G G T T G G
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Genotyping data are presented as the SNP state at the particular locus. Each locus is presented with reference to the genome position in the Ancestral Ames strain (GenBank ID: AE017334).

2

Sample IDs with “A” numbers indicate isolates for which we have live culture. Sample IDs bearing a 10-digit number are from an historical strain collection maintained by the Center for Disease Control and Prevention.

3

Canonical SNP groups as defined in Van Ert et al. (2007).

4

The A.Br.WNA canonical SNP was also definied in Van Ert et al. (2007).

5

Rules of parsimony place this isolate (A0303) in the A.Br.WNA group. This single data point is homoplastic.

DNA Extraction

DNA for Affymetrix whole genome tiling microarrays was extracted on 21 geographically dispersed and genetically diverse (MLVA15) strains within the A.Br.009 clade by a modification of the chloroform∶isoamyl alcohol method described in Keim et al. (1997). DNA for sub-clade specific TaqMan® MGB dual-probe real-time PCR SNP assays was extracted by a simplified heat lysis protocol described in Keim et al. (2000).

SNP Discovery

Seven diverse B. anthracis strains, including one from the WNA clade, which had previously been characterized by Multi-Locus Variable Number of Tandem Repeats Analyses (MLVA), were further characterized by shotgun cloning and whole genome sequencing by the Sanger method. These efforts lead to the discovery of ∼3,000 SNPs within the B. anthracis genome.

Construction of a whole genome tiling microarray

A custom Affymetrix genotyping microarray was constructed using 2850 SNPs. We genotyped 128 diverse strains using this format; relevant to this study, 10 strains were from the trans-Eurasian population (TEA: A.Br.008/009) and 21 were from the Western North American clade (WNA: A.Br.WNA). Of the 2,850 SNPs, 78 separated TEA from WNA, while 28 split the WNA clade into 6 genotypes (Fig. 1).

Real-Time PCR analyses

We selected 10 SNPs for conversion into Taqman® MGB dual-probe real-time PCR SNP assays (Table 3). Assays were designed using Primer Express (Applied Biosystems, Foster City, CA). This is a highly sensitive technology that is fast and cost-effective when analyzing hundreds of samples. Table 4 describes the associated phylogenetic groups for each SNP: Branch separating TEA from WNA (4 SNPs) and WNA subtypes (6 SNPs). These rapid PCR assays were used to genotype a total of 352 isolates from the WNA clade (Table 2). Most of the WNA members had been previously identified using the canonical SNP assay for A.Br.009 [13].

Table 3. Primers and Probes used to detect WNA clade specific SNPs.

Locus Name 1 (GenBank ID: AE017334) Primers 5′→3′ 2 Probes 5′→3′ 3
wna2994131 F-GCACGGTCTTTCTAAATTCATTGTT VIC-AAAGAACATAGGAGTTTAC
R-TGCGATTGGAGTTGCAAATAAT FAM-AAAGAACATACGAGTTTAC
wna3631093 F-CAGAACCTACAGAACCATCATTAAAGAA VIC-ATTGCTTACACTTCAGA
R-GTAAACCCATTACCACCACACTATGT FAM-ATTGCTTACACTTCGGAC
wna3682247 F-ACATGTTCACTTCACACATTTTCTCA VIC-ACTCTTGAACAAACCA
R-GCAATTGCAACAAGTCATCCA FAM-ACTCTTGAACAAGCCAA
wna3732539 F-CTAAAAGCTCCAAATGCAATAGCA VIC-CTGTTCCTGATAACAA
R-TGGTGGATCAAATGCAGTTAACTT FAM-CTGTTCCTGATAATAA
wna3774186 F-CTTTGGTTTTCCTTTGGTATAATCTCTT VIC-ATGGCACCTTTACATCT
R-TGACGTTGAAGGATGGAATATTTTTA FAM-CAGATGGCATCTTT
wna4461234 F-TTTTGATGGAGAGATTTTGCTTTCT VIC-TACGTTCTACAAATGGTACGT
R-GCGAAATCGAGCAAGGATTC FAM-TACGTTCTACAGATGGT
wna4718500 F-GCATCACCATTTAGATCATAAACCA VIC-AGCCATGTGTATAGAA
R-TGTCGTCGTACAGAAAGAAACGTT FAM-AGCCATGTGTATGGAA
wna237471 F-TCGATGGTGCGAGCTTTTATATT VIC-AATGAGCTCGGCACCAT
R-TGGTCATTGGTGGTATTTGCA FAM-AATGAGCTCTGCACCA
wna1141774 F-CGGCTTTTTTTCATTACGCATTA VIC-CATTTACCGTATTGTTTTG
R-AAAGAAAACAGAACATGCATTGATG FAM-ATTTACCGCATTGTTTTG
wna3368524 F-CACGCTTATCGCCATCGAT VIC-TCTACTGGCATTTCA
R-TGACGGAAGTGTAACGGAAGGT FAM-TCTACTGGAATTTC
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1

The locus name correlates to the position on the whole genome sequence of the Ancestral Ames strain (AE017334).

2

F refers to the Forward primer whereas R refers to the Reverse primer.

3

Allele states are designated in red. VIC is the fluor conjugated to the 5′ end of the probe for one allele whereas FAM is the fluor conjugated to the 5′end of the alternate allele.

Table 4. SNPs used in this study to define clades in WNA.

Branch Name 1 Locus Name Base Change (Ancestral→Derived)
A.Br.0092 canSNP2589947* A→G
A.Br.0182 wna0237471 T→G
A.Br.0182 wna1141774 C→T
A.Br.0182 wna3368524 A→C
A.Br.0193 wna3631093 A→G
A.Br.0193 wna4718500 G→A
A.Br.0204 wna3774186 T→C
A.Br.0215 wna2994131 C→G
A.Br.0226 wna3682247 A→G
A.Br.0226 wna4461234 G→A
A.Br.0237 wna3732539 T→C
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1

SNPs are arranged in this table from basal to derived nodes.

*

The A.Br.009 canonical SNP was previously reported5 and was used to identify most of the WNA clade samples used in this study.

2

These four SNPs are located on the Fig. 1 basal node separating TEA from the “yellow” clade.

3

These two SNPs are located on the Fig. 1 node separating the “yellow” from “red” clades.

4

This SNP is located on the Fig. 1 node separating the “red” from “green” clades.

5

This SNP is located on the Fig. 1 node separating the “green” from “blue” clades.

6

These two SNPs are located on the Fig. 1 node separating the “blue” from “black” clades.

7

This SNP is located on the Fig. 1 node separating the “black” and the terminal clades.

Spatial Analysis

Spatial data were then linked to genotypic analysis for each isolate and plotted with a Geographic Information System (ArcView 3.3). Some isolates were retrieved from archival collections and were not associated with geographic information, hence only 285 isolates were spatially mapped.

Phylogenetic analysis

Phylogenetic analysis using a cladistic approach was accomplished with PAUP 4.0 [26].

Acknowledgments

We are grateful to Paul Jackson and Martin Hugh-Jones for strain acquisition efforts.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This work was supported by NIH NIGMS (1R01GM060795), DHS (NBCH2070001) and NIH NIAID (N01-AI15447). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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