Molecular Epidemiology of Anthrax Cases Associated with Recreational Use of Animal Hides and Yarn in the United States

To determine potential links between the clinical isolate to animal products and their geographic origin, we genotyped (MLVA-8, MVLA-15, and canSNP analysis) 80 environmental and 12 clinical isolates and 2 clinical specimens from five cases of anthrax (California in 1976 [n = 1], New York in 2006 [n = 1], Connecticut in 2007 [n = 2], and New Hampshire in 2009[n = 1]) resulting from recreational handling of animal products. For the California case, four clinical isolates were identified as MLVA-8 genotype (GT) 76 and in the canSNP A.Br.Vollum lineage, which is consistent with the Pakistani origin of the yarn. Twenty eight of the California isolates were in the A.Br.Vollum canSNP lineage and one isolate was in the A.Br. 003/004 canSNP sub-group. All 52 isolates and both clinical specimens related to the New York and Connecticut cases were MLVA-8 GT 1. The animal products associated with the NY and CT cases were believed to originate from West Africa, but no isolates from this region are available to be genotyped for comparison. All isolates associated with the New Hampshire case were identical and had a new genotype (GT 149). Isolates from the NY, CT and NH cases diverge from the established canSNP phylogeny near the base of the A.Br.011/009. This report illustrates the power of the current genotyping methods and the dramatically different epidemiological conditions that can lead to infections (i.e., contamination by a single genotype versus widespread contamination of numerous genotypes). These cases illustrate the need to acquire and genotype global isolates so that accurate assignments can be made about isolate origins

Comparison of two multiple-locus variable-number tandem-repeat analysis methods for molecular strain typing of human Brucella melitensis isolates from the Middle East

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-15IGM, was compared to an automated capillary electrophoresis-based method, MLVA-15NAU, with each MLVA scheme examining a unique set of variable-number tandem repeats. Both the MLVAIGM and MLVANAU methods were highly discriminatory, resolving 99 and 101 distinct genotypes, respectively, and were able to largely separate genotypes from Egypt and Qatar. The MLVA-15NAU scheme presented higher strain-to-strain diversity in our test population than that observed with the MLVA-15IGM 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

A new approach to in silico SNP detection and some new SNPs in the Bacillus anthracis genome

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis </it>is one of the most monomorphic pathogens known. Identification of polymorphisms in its genome is essential for taxonomic classification, for determination of recent evolutionary changes, and for evaluation of pathogenic potency.</p> <p>Findings</p> <p>In this work three strains of the <it>Bacillus anthracis </it>genome are compared and previously unpublished single nucleotide polymorphisms (SNPs) are revealed. Moreover, it is shown that, despite the highly monomorphic nature of <it>Bacillus anthracis</it>, the SNPs are (1) abundant in the genome and (2) distributed relatively uniformly across the sequence.</p> <p>Conclusions</p> <p>The findings support the proposition that SNPs, together with indels and variable number tandem repeats (VNTRs), can be used effectively not only for the differentiation of perfect strain data, but also for the comparison of moderately incomplete, noisy and, in some cases, unknown <it>Bacillus anthracis </it>strains. In the case when the data is of still lower quality, a new DNA sequence fingerprinting approach based on recently introduced markers, based on combinatorial-analytic concepts and called cyclic difference sets, can be used.</p

Genotyping of Bacillus cereus Strains by Microarray-Based Resequencing

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a “one reaction” genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing

Characterizing the gamma-ray long-term variability of PKS 2155-304 with H.E.S.S. and Fermi-LAT

Studying the temporal variability of BL Lac objects at the highest energies provides unique insights into the extreme physical processes occurring in relativistic jets and in the vicinity of super-massive black holes. To this end, the long-term variability of the BL Lac object PKS 2155-304 is analyzed in the high (HE, 100 MeV 200 GeV) gamma-ray domain. Over the course of ~9 yr of H.E.S.S observations the VHE light curve in the quiescent state is consistent with a log-normal behavior. The VHE variability in this state is well described by flicker noise (power-spectral-density index {\ss}_VHE = 1.10 +0.10 -0.13) on time scales larger than one day. An analysis of 5.5 yr of HE Fermi LAT data gives consistent results ({\ss}_HE = 1.20 +0.21 -0.23, on time scales larger than 10 days) compatible with the VHE findings. The HE and VHE power spectral densities show a scale invariance across the probed time ranges. A direct linear correlation between the VHE and HE fluxes could neither be excluded nor firmly established. These long-term-variability properties are discussed and compared to the red noise behavior ({\ss} ~ 2) seen on shorter time scales during VHE-flaring states. The difference in power spectral noise behavior at VHE energies during quiescent and flaring states provides evidence that these states are influenced by different physical processes, while the compatibility of the HE and VHE long-term results is suggestive of a common physical link as it might be introduced by an underlying jet-disk connection.Comment: 11 pages, 16 figure

Distribution and Molecular Evolution of Bacillus anthracis Genotypes in Namibia

The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP) and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983–2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA) and, in part, by twelve single nucleotide polymorphism (SNP) markers and four single nucleotide repeat (SNR) markers. A total of 37 genotypes (GT) were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate epidemiological relationships

Anthrax outbreak in a Swedish beef cattle herd - 1st case in 27 years: Case report

After 27 years with no detected cases, an outbreak of anthrax occurred in a beef cattle herd in the south of Sweden. The outbreak was unusual as it occurred in winter, in animals not exposed to meat-and-bone meal, in a non-endemic country

Yersinia pestis Lineages in Mongolia

BACKGROUND: Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia. METHODOLOGY/PRINCIPAL FINDINGS: Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches. CONCLUSIONS/SIGNIFICANCE: We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years

Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies

Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria

NAFTA Chapter 11 as Supraconstitution

More and more legal scholars are turning to constitutional law to make sense of the growth of transnational and international legal orders. They often employ constitutional terminology loosely, in a bewildering variety of ways, with little effort to clarify their analytical frameworks or acknowledge the normative presuppositions embedded in their analysis. The potential of constitutional analysis as an instrument of critique of transnational legal orders is frequently lost in methodological confusion and normative controversy. An effort at clarification is necessary. We propose a functional approach to supraconstitutional analysis that applies across issue areas, accommodates variation in kinds and degrees of supraconstitutionalization, recognizes its simultaneously domestic and transnational character, and reflects its uneven incidence and impacts. We apply this framework to NAFTA to consider whether and how it superimposes a supraconstitutional legal order on member states\u27 domestic constitutional orders. We show that the main thrust of this contemporary supraconstitutional project is to restructure state and international political forms to promote market efficiency and discipline, enable free capital movement, confer privileged rights of citizenship and representation on corporate capital, insulate key aspects of the economy from state interference, and constrain democratic decision-making