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Bacillus anthracis , the etiological agent of anthrax, is a well-established model organism. For B. anthracis and most other infectious diseases, knowledge regarding transmission and infection parameters in natural systems, in large part, comprises data gathered from closely controlled laboratory experiments. Fatal, natural anthrax infections transmit the bacterium through new host−pathogen contacts at carcass sites, which can occur years after death of the previous host. For the period between contact and death, all of our knowledge is based upon experimental data from domestic livestock and laboratory animals. Here we use a noninvasive method to explore the dynamics of anthrax infections, by evaluating the terminal diversity of B. anthracis in anthrax carcasses. We present an application of population genetics theory, specifically, coalescence modeling, to intrainfection populations of B. anthracis to derive estimates for the duration of the acute phase of the infection and effective population size converted to the number of colony-forming units establishing infection in wild plains zebra ( Equus quagga ). Founding populations are small, a few colony-forming units, and infections are rapid, lasting roughly between 1 d and 3 d in the wild. Our results closely reflect experimental data, showing that small founding populations progress acutely, killing the host within days. We believe this method is amendable to other bacterial diseases from wild, domestic, and human systems.

Bacillus anthracis, the causative pathogen of anthrax, is a spore-forming, environmentally maintained bacterium that continues to be a veterinary health problem with outbreaks occurring primarily in wildlife and livestock. Globally, the genetic populations of B. anthracis include multiple lineages, and each may have different ecological requirements and geographical distributions. It is, therefore, essential to identify environmental associations within lineages to predict geographical distributions and risk areas with improved accuracy. Here, we model the ecological niche and predict the geography of the most widespread sublineage of B. anthracis in the continental United States using updated MERRA-derived (Modern Era Retrospective analysis for Research and Applications; the NASA atmospheric data reanalysis of satellite information with multiple data products) bioclimate variables (i.e., MERRAclim data) and updated soil variables. We filter the occurrence data associated with the A1.a/Western North American sub-lineage of B. anthracis from historical anthrax outbreaks using the multiple-locus variable-number tandem repeat system. In addition, we also incorporate recent cases associated with B. anthracis A1.a sub-lineage from 2008 to 2012 in Montana, Colorado, and Texas. Our results provide the predicted distribution of the A1.a sub-lineage of B. anthracis for the United States with better predictive accuracy and higher spatial resolution than previous estimates. Our prediction serves as an improved disease risk map to better inform anthrax surveillance and control in the United States, particularly the Dakotas and Montana where this sub-lineage is persistent.

Significance This study applies coalescence modeling to a “slowly evolving” bacterial pathogen, Bacillus anthracis, to derive estimates of infection durations and founding population sizes from natural anthrax mortalities. Although coalescence modeling has been applied to highly mutable chronic pathogens (i.e., HIV), to date, methodological hurdles have prevented its wider application. Our findings show it is possible to obtain pathological data from infections, post hoc, which may be applicable to other pathogens and settings, including clinical. Given their higher resolution, microsatellites will remain useful in shorter evolutionary timeframe studies., Bacillus anthracis, the etiological agent of anthrax, is a well-established model organism. For B. anthracis and most other infectious diseases, knowledge regarding transmission and infection parameters in natural systems, in large part, comprises data gathered from closely controlled laboratory experiments. Fatal, natural anthrax infections transmit the bacterium through new host−pathogen contacts at carcass sites, which can occur years after death of the previous host. For the period between contact and death, all of our knowledge is based upon experimental data from domestic livestock and laboratory animals. Here we use a noninvasive method to explore the dynamics of anthrax infections, by evaluating the terminal diversity of B. anthracis in anthrax carcasses. We present an application of population genetics theory, specifically, coalescence modeling, to intrainfection populations of B. anthracis to derive estimates for the duration of the acute phase of the infection and effective population size converted to the number of colony-forming units establishing infection in wild plains zebra (Equus quagga). Founding populations are small, a few colony-forming units, and infections are rapid, lasting roughly between 1 d and 3 d in the wild. Our results closely reflect experimental data, showing that small founding populations progress acutely, killing the host within days. We believe this method is amendable to other bacterial diseases from wild, domestic, and human systems.

Early studies confirmed Bacillus anthracis in emesis and feces of flies under laboratory conditions, but there is little empirical field evidence supporting the roles of flies in anthrax transmission. We collected samples during outbreaks of anthrax affecting livestock and native and exotic wildlife on two ranches in West Texas (2009-2010). Sampling included animal carcasses, maggots, adult flies feeding on or within several meters of carcasses, and leaves from surrounding vegetation. Microbiology and PCR were used to detect B. anthracis in the samples. Viable B. anthracis and/or PCR-positive results were obtained from all represented sample types. Genetic analysis of B. anthracis samples using multilocus variable number tandem repeat analysis (MLVA) confirmed that each ranch represented a distinct genetic lineage. Within each ranch, we detected the same genotype of B. anthracis from carcasses, maggots, and adult flies. The results of this study provide evidence supporting a transmission cycle in which blowflies contaminate vegetation near carcasses that may then infect additional browsing animals during anthrax outbreaks in the shrubland environment of West Texas.

To mitigate the effects of zoonotic diseases on human and animal populations, it is critical to understand what factors alter transmission dynamics. Here we assess the risk of exposure to lethal concentrations of the anthrax bacterium, Bacillus anthracis, for grazing animals in a natural system over time through different transmission mechanisms. We follow pathogen concentrations at anthrax carcass sites and waterholes for five years and estimate infection risk as a function of grass, soil or water intake, age of carcass sites, and the exposure required for a lethal infection. Grazing, not drinking, seems the dominant transmission route, and transmission is more probable from grazing at carcass sites 1–2 years of age. Unlike most studies of virulent pathogens that are conducted under controlled conditions for extrapolation to real situations, we evaluate exposure risk under field conditions to estimate the probability of a lethal dose, showing that not all reservoirs with detectable pathogens are significant transmission pathways.

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters’ Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 of 12 subgroups. Reconstruction of the pagA gene also showed a unique SNP that defines a new lineage for B. anthracis.

Zoonoses, diseases affecting both humans and animals, can exert tremendous pressures on human and veterinary health systems, particularly in resource limited countries. Anthrax is one such zoonosis of concern and is a disease requiring greater public health attention in Nigeria. Here we describe the genetic diversity of Bacillus anthracis in Nigeria and compare it to Chad, Cameroon and a broader global dataset based on the multiple locus variable number tandem repeat (MLVA-25) genetic typing system. Nigerian B. anthracis isolates had identical MLVA genotypes and could only be resolved by measuring highly mutable single nucleotide repeats (SNRs). The Nigerian MLVA genotype was identical or highly genetically similar to those in the neighboring countries, confirming the strains belong to this unique West African lineage. Interestingly, sequence data from a Nigerian isolate shares the anthrose deficient genotypes previously described for strains in this region, which may be associated with vaccine evasion. Strains in this study were isolated over six decades, indicating a high level of temporal strain stability regionally. Ecological niche models were used to predict the geographic distribution of the pathogen for all three countries. We describe a west-east habitat corridor through northern Nigeria extending into Chad and Cameroon. Ecological niche models and genetic results show B. anthracis to be ecologically established in Nigeria. These findings expand our understanding of the global B. anthracis population structure and can guide regional anthrax surveillance and control planning., Author Summary Anthrax, caused by the soil-borne bacterium Bacillus anthracis, is a disease with important public health and national security implications globally. Understanding the global genetic diversity of the pathogen is important for epidemiological and forensic investigations of anthrax events. Toward this, we describe B. anthracis genetic diversity in Nigeria and confirm it belongs to a unique West African genetic group not yet reported beyond neighboring Cameroon and Chad and Mali. This refines the global phylogeny of B. anthracis, allowing the development of more accurate diagnostics. We coupled these efforts with ecological niche modeling to map the geographic distribution of this strain group across the region. Suitable habitat for the pathogen is predicted across central Nigeria from west to east into Cameroon and Chad. Understanding the geography of B. anthracis plays an important role in informing public health by targeting disease control to high risk regions. This is particularly important in resource limited areas where intervention strategies are constrained and zoonotic disease risk is high.

Background Anthrax, a soil-borne zoonosis caused by the bacterium Bacillus anthracis, is enzootic in areas of North America with frequent outbreaks in west Texas. Despite a long history of study, pathogen transmission during natural outbreaks remains poorly understood. Here we combined case-level spatio-temporal analysis and high resolution genotyping to investigate anthrax transmission dynamics. Carcass locations from a single white-tailed deer, Odocoileus virginanus, outbreak were analyzed for spatial clustering using K-function analysis and directionality with trend surface analysis and the direction test. Results The directionalities were compared to results of high resolution genotyping. The results of the spatial clustering analyses, combined with deer movement data, suggest anthrax transmission events occur within limited spatial areas, with carcass locations occurring within the activity space of adjacent cases. The directionality of the outbreak paralleled adjacent dry river beds. Isolates from the outbreak were represented by a single genotype based on multiple locus variable number tandem repeat analysis (MLVA); four sub-genotypes were identified using single nucleotide repeat (SNR) analysis. Conclusions Areas of high transmission agreed spatially with areas of higher SNR genetic diversity; however, SNRs did not provide clear evidence of linear transmission. Overlap of case home ranges provides spatial and temporal support for localized transmission, which may include the role of necrophagous or hematophagous flies in outbreaks in this region. These results emphasize the need for active surveillance and prompt cleanup of anthrax carcasses to control anthrax both during outbreaks and between seasons. Electronic supplementary material The online version of this article (doi:10.1186/s12898-015-0054-8) contains supplementary material, which is available to authorized users.

We genotyped 15 Bacillus anthracis isolates from Chad, Africa, using multiple-locus variable-number tandem repeat analysis and three additional direct-repeat markers. We identified two unique genotypes that represent a novel genetic lineage in the A cluster. Chadian isolates were susceptible to 11 antibiotics and free of 94 antibiotic resistance genes.

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation ofBacillus anthracis. The use ofSNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan® mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene ofB. anthracis. The assay permits specific, low-level detection (25fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena ofbiodefense and microbialforensics.

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Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICRMS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.

Precise identification of Bacillus anthracis isolates has aided forensic and epidemiological analyses of natural anthrax cases, bioterrorism acts and industrial scale accidents by state-sponsored bioweapons programs. Because there is little molecular variation among B. anthracis isolates, identifying and using rare variation is crucial for precise strain identification. We think that mutation is the primary diversifying force in a clonal, recently emerged pathogen, such as B. anthracis, since mutation rate is correlated with diversity on a per locus basis. While single nucleotide polymorphisms (SNPs) are rare, their detection is facilitated by whole genome discovery approaches. As highly stable phylogenetic markers, SNPs are useful for identifying long branches or key phylogenetic positions. Selection of single, diagnostic "Canonical SNPs" (canSNPs) for these phylogenetic positions allows for efficient and defining assays. We have taken a nested hierarchal strategy for subtyping B. anthracis, which is consistent with traditional diagnostics and applicable to a wide range of pathogens. Progressive hierarchical resolving assays using nucleic acids (PHRANA) uses a progression of diagnostic genomic loci that are initially highly stable but with low resolution and, ultimately, very unstable but with high resolution. This approach mitigates the need for data weighting and provides both a deeply rooted phylogenetic hypothesis and high resolution discrimination among closely related isolates.

The cyclins are tightly regulated elements governing eukaryotic cell cycle progression by means of sequential activation-inactivation of cyclin-dependent kinases. In one manifestation of this regulation, the mRNA levels of several cyclin genes oscillate during the cycle in mammalian cells. Such cycle-dependent fluctuations in transcript levels could result from changes not only in rates of transcription, but also in mRNA stability. Here we used a new, minimally-disturbing method for producing multi-cycle synchronous growth of human MOLT-4 cells, in combination with quantitative real-time RT-PCR, to compare cell cycle-dependent transcript levels and half-lives of cyclin A2, B1, D3, E and PCNA mRNAs. While all mRNA levels except cyclin D3 varied in the cycle, there were no apparent variations in message half-lives. This differs from several previous reports of dramatic fluctuations in the stabilities of cyclin mRNAs, and infers that fluctuations in cyclin mRNA transcript levels during the MOLT-4 cell cycle are not due to variations in half-lives. The discrepancy in mRNA stability determinations could be due to differences in cell types or synchronization methods, but our findings may be representative of mRNA processing in the cycle of cells in unstressed steady-state growth.

To the Editor: Forensic and epidemiologic investigation of the 2001 bioterrorism-associated anthrax attacks used multiple-locus variable-number tandem-repeat analysis (MLVA) to identify the attack strain as Ames (1). Strain identity was essential for subsequent molecular epidemiologic and forensic investigations of this biocrime. To more easily identify this particular strain, comparative whole-genome sequencing (2) and phylogenetic analyses were used to identify single-nucleotide polymorphisms (SNPs) that seem highly specific for Ames strain identification (3). Because Bacillus anthracis is a recently emerged clonal pathogen, these SNPs represent highly evolutionarily stable markers (4) that are amenable to many rapid and cost-effective analytical techniques. MLVA and the Ames-specific SNP assay indicate that the Ames strain has been isolated from nature only 1 time, in southern Texas, USA. Several lineages of B. anthracis (5) have been ecologically established in North America. The A.Br.009 clade is the most successful and widely dispersed in North America, but it is not closely related to the Ames strain (5), which is a member of the A.Br.001 clade. Although A.Br.001 is not as successful as A.Br.009, it appears to be ecologically well established in southern Texas. Analyses of outbreaks in this region from 1974 to 2000 found 190 culture-confirmed cases clustered mainly in 5 counties (6). A major epizootic in Texas in 2001 paralleled this trend. This outbreak (6) affected many deer species, horses, and bovids (total 1,637), which suggests that this clade is well established and not limited to cultivated areas and domesticated livestock. Previous molecular and epidemiologic analyses (3) of isolates from this region identified close, but not identical, matches to the Ames strain, which suggests that more intense surveillance in this region would likely yield more Ames and Ames-like isolates. Two recent (2006 and 2007) outbreaks in Texas confirmed this suggestion. Isolates from the 2006 and 2007 outbreaks were initially screened by using an 8-marker MLVA system (MLVA8) as described by Keim et al. (7). The MLVA8 genotypes were identical to the B. anthracis Ames strain (GT62). Additional analysis by a 15-marker (MLVA15) variable-number tandem repeats (VNTR) system (5) again generated an MLVA15 genotype that was identical to the original Ames strain (A0462) and to the 2001 bioterrorism-associated attack strain (A2012) (Figure). Given the identical MLVA genotypes, could these natural strains be differentiated from the laboratory or biocrime Ames strain by using higher resolution genotyping? Figure Geographic and phylogenetic relationships among strains closely related to Bacillus anthracis Ames strain. A) Spatial relationships among Ames-like isolates from southern Texas. 1, location of the original Ames strain, isolated from Jim Hogg County, Texas, ... We developed 6 Ames strain–specific SNPs to address the potential that the Ames strain might reappear naturally and hinder epidemiologic and forensic investigations (3). We found that 5 of 6 SNP loci could be used to distinguish between all known natural isolates and laboratory or biocrime isolates (Figure). Results were consistent with our previous identification of a B. anthracis isolate from a goat kid in Texas in 1997 (A0394) as being closely related to the Ames strain (3). However, the 2006 and 2007 isolates from Texas were even more closely related to the Ames strain because they also shared the MLVA15 genotype with Ames. In contrast, the 1997 goat isolate differed by a single mutational step at the BaVNTR16 locus (Figure). Hence, 5 of 6 SNP markers enabled differentiation among Ames and Ames near relatives even when VNTR profiles were identical. Resolution of nearly identical genotypes might also be accomplished by using additional VNTRs (8) or hypermutable loci (9). However, we doubt that this approach would be better than whole-genome sequencing with interrogation of resultant SNPs because these markers would most likely result in topologic conflicts due to homoplasy (10). The available epidemiologic data from other isolates included in this clade show that although the Ames clade is well established in southern Texas, no subsequently recovered natural isolates completely match the original Ames isolate. The precision of subtyping assays is a matter of importance and debate for epidemiologic and, recently, forensic investigations. Strain identity is commonly used to infer a common source even when spatial and temporal data are not congruent. Moreover, the definition of a strain is somewhat unclear and relies on analytical methods that vary widely. Therefore, isolates may be erroneously excluded or included into a strain definition and disease outbreak as illustrated with the Ames strain and 2 contrasting approaches to identification. MLVA15 ties naturally occurring isolates to bioterrorism-associated attacks, while specific SNP assays can distinguish among them. MLVA is an unbiased approach and can be used on any set of B. anthracis strains, although, as in the 2006 and 2007 Texas outbreaks, it can be limited in resolving power. In contrast, our SNP assays have great resolving power but are useful only for differentiating the Ames strain, thus limiting their value to categorical inclusion or exclusion in outbreaks. Future rational use of a battery of different molecular signatures will yield far greater insights into strain identity than the application of 1 specific signature.

We modeled the ecological niche of a globally successful Bacillus anthracis sublineage in the United States, Italy and Kazakhstan to better understand the geographic distribution of anthrax and potential associations between regional populations and ecology. Country-specific ecological-niche models were developed and reciprocally transferred to the other countries to determine if pathogen presence could be accurately predicted on novel landscapes. Native models accurately predicted endemic areas within each country, but transferred models failed to predict known occurrences in the outside countries. While the effects of variable selection and limitations of the genetic data should be considered, results suggest differing ecological associations for the B. anthracis populations within each country and may reflect niche specialization within the sublineage. Our findings provide guidance for developing accurate ecological niche models for this pathogen; models should be developed regionally, on the native landscape, and with consideration to population genetics. Further genomic analysis will improve our understanding of the genetic-ecological dynamics of B. anthracis across these countries and may lead to more refined predictive models for surveillance and proactive vaccination programs. Further studies should evaluate the impact of variable selection of native and transferred models.

This study provides useful baseline data for guiding future disease control programs., To map the distribution of anthrax outbreaks and strain subtypes in Kazakhstan during 1937–2005, we combined geographic information system technology and genetic analysis by using archived cultures and data. Biochemical and genetic tests confirmed the identity of 93 archived cultures in the Kazakhstan National Culture Collection as Bacillus anthracis. Multilocus variable number tandem repeat analysis genotyping identified 12 genotypes. Cluster analysis comparing these genotypes with previously published genotypes indicated that most (n = 78) isolates belonged to the previously described A1.a genetic cluster, 6 isolates belonged to the A3.b cluster, and 2 belonged to the A4 cluster. Two genotypes in the collection appeared to represent novel genetic sublineages; 1 of these isolates was from Krygystan. Our data provide a description of the historical, geographic, and genetic diversity of B. anthracis in this Central Asian region.

Brucella species are highly monomorphic, with minimal genetic variation among species, hindering the development of reliable subtyping tools for epidemiologic and phylogenetic analyses. Our objective was to compare two distinct multiple-locus variable-number tandem-repeat analysis (MLVA) subtyping methods on a collection of 101 Brucella melitensis isolates from sporadic human cases of brucellosis in Egypt ( n = 83), Qatar ( n = 17), and Libya ( n = 1). A gel-based MLVA technique, MLVA-15 IGM , was compared to an automated capillary electrophoresis-based method, MLVA-15 NAU , with each MLVA scheme examining a unique set of variable-number tandem repeats. Both the MLVA IGM and MLVA NAU methods were highly discriminatory, resolving 99 and 101 distinct genotypes, respectively, and were able to largely separate genotypes from Egypt and Qatar. The MLVA-15 NAU scheme presented higher strain-to-strain diversity in our test population than that observed with the MLVA-15 IGM assay. Both schemes were able to genetically correlate some strains originating from the same hospital or region within a country. In addition to comparing the genotyping abilities of these two schemes, we also compared the usability, limitations, and advantages of the two MLVA systems and their applications in the epidemiological genotyping of human B. melitensis strains.

Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.

Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis , whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (B. anthracis .

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia .

We used multiple-locus variable-number tandem repeat analysis (MLVA) to type 64 Bacillus anthracis isolates from outbreaks that have occurred during the past 40 years in Italy. MLVA of the 64 isolates revealed 10 unique genotypes; 9 of these genotypes and the majority of isolates (63/64) belonged to the previously described genetic cluster A1.a. Within the A1.a isolates, two previously described genotypes (G1 and G3), which differ by a single mutation in the pX01 locus, account for the majority of isolates in the country (53/63). The low diversity of B. anthracis genotypes in Italy suggests a single, dominant historical introduction, followed by limited localized differentiation.

A TaqMan-minor groove binding assay designed around a nonsense mutation in the plcR gene was used to genotype Bacillus anthracis , B. cereus , and B. thuringiensis isolates. The assay differentiated B. anthracis from these genetic near-neighbors and determined that the nonsense mutation is ubiquitous across 89 globally and genetically diverse B. anthracis strains.

We used multiple-locus variable-number tandem repeat analysis (MLVA) and pagA sequencing to genotype a Bacillus anthracis isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.

Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B . anthracis , showing that it lies closer to a newly described “C” branch than to either of two previously described “A” or “B” branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.

The cyclins are tightly regulated elements governing eukaryotic cell cycle progression by means of sequential activation-inactivation of cyclin-dependent kinases. In one manifestation of this regulation, the mRNA levels of several cyclin genes oscillate during the cycle in mammalian cells. Such cycle-dependent fluctuations in transcript levels could result from changes not only in rates of transcription, but also in mRNA stability. Here we used a new, minimally-disturbing method for producing multi-cycle synchronous growth of human MOLT-4 cells, in combination with quantitative real-time RT-PCR, to compare cell cycle-dependent transcript levels and half-lives of cyclin A2, B1, D3, E and PCNA mRNAs. While all mRNA levels except cyclin D3 varied in the cycle, there were no apparent variations in message half-lives. This differs from several previous reports of dramatic fluctuations in the stabilities of cyclin mRNAs, and infers that fluctuations in cyclin mRNA transcript levels during the MOLT-4 cell cycle are not due to variations in half-lives. The discrepancy in mRNA stability determinations could be due to differences in cell types or synchronization methods, but our findings may be representative of mRNA processing in the cycle of cells in unstressed steady-state growth.