CASE CLOSED … what really happened in the 2001 anthrax attacks?

* Humana Press Has Published This 2012 “Chemical and Physical Signatures for Microbial Forensics”; such a treatise is all part of developing microbial forensics to the point where it can be more useful than it has been in the attribution of crimes using pathogens.

Posted by DXer on April 26, 2012




16 Responses to “* Humana Press Has Published This 2012 “Chemical and Physical Signatures for Microbial Forensics”; such a treatise is all part of developing microbial forensics to the point where it can be more useful than it has been in the attribution of crimes using pathogens.”

  1. DXer said

    Now the FBI never told the NAS the extent of their swabbing for the genetically distinctive subtilis — except to acknowledge that the issue was exculpatory of Dr. Ivins insofar as it was not found at USAMRIID. But there is no indiciation that they ever swabbed the workspaces of the subtilis expert, who worked with mutations, in touch with Ramzi Yousef’s telephone number up until the time the Blind Sheikh was arrested.

    If they had wanted to swab for subtilis — and it would have been negligent not to do so — there is learning on that too.

    Establishing the detection threshold for Bacillus subtilis in a complex matrix using an inorganic fingerprint approach.
    Lev, S.M.1
    Gasparich, G.2
    Choi, F.2
    King, L.1
    Moore, J.1
    Zimmerman, S.1
    Talanta; Sep2011, Vol. 85 Issue 4, p1734-1737, 4p

    Abstract: Methods for the detection and characterization of airborne biological warfare agents, such as bacteria, using their DNA or organic composition are fairly well developed. This approach is useful for identifying the type of bacterial strain once the organism has been isolated from the matrix sampled (e.g., dust particles) and can identify genetically related organisms, which might be helpful during a forensic investigation. However, this genetic signature will not reveal information related to the methods used to grow and weaponize the organism. Bacteria will take on an inorganic signature that is related to their growth and processing history. Therefore, the ability to characterize the inorganic fingerprint of a biological particle has the potential to detect the presence of a bio-agent and expand the forensic tools available to those investigating the origin of biological weapons. This investigation builds on previous work documenting the usefulness of the inorganic fingerprint and evaluates the limits of detection in the presence of background dust. Based on ICP-MS measurements and mixing models of digested mixtures of laboratory cultured Bacillus subtilis (anthrax stimulant) and NIST Standard Reference Material 2709 (dust stimulant), the inorganic fingerprint method is capable of detecting toxicologically relevant levels of a bio-warfare agent in the presence of a complex background matrix.

  2. DXer said

    For another example of DOJ withholding, one need look no further than the issue of “silicon signature”:

    Silicon Mystery Endures in Solved Anthrax Case.Detail Only Available
    By: BHATTACHARJEE, YUDHIJIT. Science, 3/19/2010, Vol. 327 Issue 5972, p1435-1435, 1p
    The article discusses the formal conclusion of U.S. investigations of the 2001 terrorist attacks involving anthrax-containing letters and the unresolved silicon content associated with many of th…

  3. DXer said

    On the subject of “elemental signatures,” see

    Developing an integrated proteo-genomic approach for the characterisation of biomarkers for the identification of Bacillus anthraces.

    Journal of Microbiological Methods; Feb2012, Vol. 88 Issue 2, p237-247, 11p
    Document Type:
    Subject Terms:
    *BACILLUS anthracis
    *BIOCHEMICAL markers
    *MASS spectrometry
    *LIQUID chromatography

  4. DXer said

    Dr. Wunschel and Dr. Fox had also published on the subject.

    Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture.
    Wunschel, David S.1
    Colburn, Heather A.1
    Fox, Alvin2
    Fox, Karen F.2
    Harley, William M.2
    Wahl, Jon H.1
    Wahl, Karen L.1
    Journal of Microbiological Methods; Aug2008, Vol. 74 Issue 2/3, p57-63, 7p

    Abstract: Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography–mass spectrometry (GC–MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC–MS–MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.

  5. DXer said

    Amber Dance, in “Ten years on from anthrax scare, analysis lags behind sequencing.” published in the October 2011 issue of Nature Medicine; Oct2011, Vol. 17 Issue 10, p1158-1159, 2p, explained that analysis techniques lag behind DNA sequencing.


    To start, if you don’t bother to interview the head of Al Qaeda’s anthrax for a full year after he’s captured, then it is unlikely that he submitted a voluntary sample of Ames to the FBI repository.

  6. DXer said

    Book Description

    Publication Date: December 9, 2011 | ISBN-10: 1603272178 | ISBN-13: 978-1603272179 | Edition: 2012

    Combining the disciplines of biological, physical and chemical science, microbial forensics has a rapidly rising profile in a world increasingly troubled by the threat of ‘biocrime’ and ‘bioterrorism’. This valuable resource is a major addition to a body of literature reckoned to lack sufficient breadth. It presents a variety of phenotypic and trace signature methodologies associated with cultured microorganisms that, despite being genetically identical, may be characterized by differing cultural environments. One of the central challenges faced by those working in this field is the sheer diversity of potentially harmful agents, which in themselves total more than 1000 viruses, bacteria, fungi and protozoan parasites. Their numerous additional variants render the process of ‘fingerprinting’ biological agents notoriously difficult, especially when the limitations of genetic analysis are factored in. Attribution of crime is relatively easy through human DNA, but lacking the genetic individuation of humans and animals, microbial forensics has to complement phylogenetic techniques with chemical and physical ones. In the best case, genetic analysis in the ‘biocrime’ sector can exclude sources, narrow the population of possible sources and support associations with potential sources. To complement these genetic techniques, chemical and physical methods can be used to compare ‘signatures’ imparted to microbial samples by environments in which they are grown and processed. Collating a range of microbiological fingerprinting techniques in one volume, and covering everything from statistical analysis to laboratory protocols, this publication furthers the aim of forensic investigators who need robust and legally admissible forensic evidence to present in a courtroom.

    • DXer said

      K.H. Jarman explains:

      “It became clear early in the investigation of the anthrax letters that genetic identity alone was not necessarily sufficient to lead investigators to a perpetrator.”

      [Comment: Indeed, after a decade, genetics had only narrowed the field from 700-1000 to 200-300. Instead of millions spent on genetics, the FBI’s money might have been better spent asking those with Ames and access to a BL-3 to make copies of the documents about what they were doing those weeks and put them in an envelope and give the envelope to the polygrapher. The use of a polygraph is not validated science. But it at least provided an opportunity to sit down an individual and obtain self-collected alibi documents while they still existed. It would only have cost the ink used to copy the phone records, emails etc. If the FBI had done that, the Amerithrax investigators would have had the documents relating to Dr. Ivins’ work with rabbits at hand and would not have been led astray by Dr. Ivins’ inabiility to recreate what he had done on particular nights a half decade before. As it was, critical documents were lost such as the cards on the rabbit cages thrown out by 2004 by the animal caretaker, who had been tending to the rabbits in the B3 during the day during the first week of October 2001]. The AUSAs concocted the false narrative about his work in the B3 being unexplained by not disclosing the 25 documents eventually produced by USAMRIID after two years of wrangling over FOIA requests. Those documents were in the possession of the FBI but were withheld from production. GAO: See documents maintained by DOJ paralegal in database.

      Dr. Jarman continues:

      “Analytical methods were needed that could differentiate genetically identical organisms produced under different conditions and also provide information about how a particular batch of organisms was produced. In response, scientists have been exploring the use of a variety of analytical approaches to characterize changes in microbial signatures due to changes in culture conditions. Promising results have been presented; however, these analytical methods are still in their infancy. Significant sources of error have yet to be identified and characterized, and more needs to be learned about the way in which a microorganism interacts with its growth environment.”

      Here, the Silicon Signature has been most often discussed. A “silicon signature” existed only in the Flask 1030 that contained the aerosol leftovers — not Flask 1029 on which the FBI bases its Ivins Theory. SIlica was used, for example, as an antifoaming agent at the time of spraying — and for example is used in a spraydryer. An aerosol expert who makes anthrax simulant for the government explained to me years ago that a processor must know the right temperature to avoid killing the organism. I believe heat shocking may be thought by researchers to increase the permeability of the exosporium, which leads to absorption of the silica. But Dr. Bannan can produce to the GAO his research on heat shocking.

      • DXer said

        Dr. Jarman first addressed these issues, along with her co-authors, in

        Bayesian-Integrated Microbial Forensics.
        Jarman, Kristin H.1
        Kreuzer-Martin, Helen W.1
        Wunschel, David S.1
        Valentine, Nancy B.1
        Cliff, John B.1
        Petersen, Catherine E.1
        Colburn, Heather A.1
        Wahl, Karen L.1
        Applied & Environmental Microbiology; Jun2008, Vol. 74 Issue 11, p3573-3582, 10p, 6 Diagrams, 3 Charts, 4 Graphs

        In the aftermath of the 2001 anthrax letters, researchers have been exploring ways to predict the production environment of unknown-source microorganisms. Culture medium, presence of agar, culturing temperature, and drying method are just some of the broad spectrum of characteristics an investigator might like to infer. The effects of many of these factors on microorganisms are not well understood, but the complex way in which microbes interact with their environments suggests that numerous analytical techniques measuring different properties will eventually be needed for complete characterization. In this work, we present a Bayesian statistical framework for integrating disparate analytical measurements. We illustrate its application to the problem of characterizing the culture medium of Bacillus spores using three different mass spectral techniques. The results of our study suggest that integrating data in this way significantly improves the accuracy and robustness of the analyses.

    • DXer said

      As quality assurance standards described in the chapter by Mark Wilson, note that the FBI scientist threw out the first sample Dr. Ivins submitted. (It contained all 4 morphs). Two samples were submitted. The scientist who threw out the sample should be identified and the quality protocols relating to the preservation of evidence should be disclosed. Some of the scientists working for the FBI had made a dried powder out of Flask 1029. If they are the ones who threw out the sample it is not something that should be kept secret. The need for transparency is keenly felt when the scientist collecting the sample had made a dried powder out of Flask 1029 and kept virulent Ames in her unlocked refrigerator. At one point, she told the FBI that she did not recall how she came to have the virulent Ames which was genetically matching. The fact that she and the FBI’s lead anthrax expert had made a dried powder out of virulent Ames from Flask 1029 was kept secret throughout the decade and was never voluntarily disclosed by the FBI. In July 2009 I had to call the FBI expert at home and ask him point-blank.

      For the FBI to jump up and down when its multi-million dollar genetics effort winnowed the field from 700-1000 to 200-300 was, well, er, stupid given that the same people at USAMRIID had access to both the genetically matching Ames and the Ames that wasn’t genetically matching. And having just a smidgen was enough to grow all you needed. In particular, it pointed to a massive conflict of interest of its leading scientists who had made a dried powder out of that Ames.

      • DXer said

        Of course, when the US Attorney Taylor said that the Ames was in Building 1425 rather than Building 1412 and 1425, he was wildly mistaken.
        Indeed, where exactly was the quality assurance in stating the findings?

      • DXer said

        Notice that the title is

        Quality Sample Collection, Handling, and PRESERVATION for an Effective Microbial Forensics Program.Detail Only Available
        By: Budowle, Bruce; Schutzer, Steven E.; Burans, James P.; Beecher, Douglas J.; Cebula, Thomas A.; Chakraborty, Ranajit; Cobb, William T.; Fletcher, Jacqueline; Hale, Martha L.; Harris, Robert B.; Michael A. Heitkamp; Keller, Frederick Paul; Kuske, Cheryl; LeClerc, Joseph E.; Marrone, Babetta L.; McKenna, Thomas S.; Morse, Stephen A.; Rodriguez, Luis L.; Valentine, Nancy B.; Yadev, Jagjit. Applied & Environmental Microbiology, Oct2006, Vol. 72 Issue 10, p6431-6438, 8p; DOI: 10.1128/AEM.01165-06
        The article presents an overview of the October 2005 Department of Homeland Security (DHS) Banbury meeting. The meeting focused on identifying gaps and make recommendations on sample collection, …

        It is not quality sample collection [by someone with a massive conflict of interest], handling, and THROWING OUT of samples that then are deemed central to the FBI’s case by reason of narrowing things from 700-1000 to 200-300.

    • DXer said

      Spatially Resolved Characterization of Water and Ion Incorporation in Bacillus Spores.
      Ghosal, Sutapa1
      Leighton, Terrance J.2
      Wheeler, Katherine E.2
      Hutcheon, Ian D.1
      Weber, Peter K.1
      Applied & Environmental Microbiology; May2010, Vol. 76 Issue 10, p3275-3282, 8p

      We present the first direct visualization and quantification of water and ion uptake into the core of individual dormant Bacillus thuringiensis subsp. israelensis (B. thuringiensis subsp. israelensis) endospores. Isotopic and elemental gradients in the B. thuringiensis subsp. israelensis spores show the permeation and incorporation of deuterium in deuterated water (D20) and solvated ions throughout individual spores, including the spore core. Under hydrated conditions, incorporation into a spore occurs on a time scale of minutes, with subsequent uptake of the permeating species continuing over a period of days. The distribution of available adsorption sites is shown to vary with the permeating species. Adsorption sites for Li+, Cs+, and C1- are more abundant within the spore outer structures (exosporium, coat, and cortex) relative to the core, while F- adsorption sites are more abundant in the core. The results presented here demonstrate that elemental abundance and distribution in dormant spores are influenced by the ambient environment. As such, this study highlights the importance of understanding how microbial elemental and isotopic signatures can be altered postproduction, including during sample preparation for analysis, and therefore, this study is immediately relevant to the use of elemental and isotopic markers in environmental microbiology and microbial forensics.

      • DXer said

        Dr. Weber’s June 2010 article on NanoSIMS imaging of Bacillus spores is here:

        NanoSIMS imaging of Bacillus spores sectioned by focused ion beam.

        WEBER, P. K.1
        GRAHAM, G. A.1
        TESLICH, N. E.1
        CHAN, W. MOBERLY1
        GHOSAL, S.1
        LEIGHTON, T. J.2
        WHEELER, K. E.2
        Journal of Microscopy; Jun2010, Vol. 238 Issue 3, p189-199, 11p, 3 Color Photographs, 3 Black and White Photographs, 1 Chart, 1 Graph

        Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top-sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift-out. The lift-out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top-cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is ∼300 nm for top-cut sections versus ∼100 nm for electron-transparent sections.

    • DXer said

      Dr. Kreuzer-Martin and Dr. Ehleringer, consulting scientists for the FBI, were co-authors.

      Stable Isotope Ratios as a Tool in Microbial Forensics–Part 2. Isotopic Variation Among Different Growth Media as a Tool for Sourcing Origins of Bacterial Cells or Spores.
      Kreuzer-Martin, Helen W.1
      Chesson, Leley A.1
      Lott, Michael J.1,2
      Dorigan, Janet V.3
      Ehleringer, James R.1,2
      Journal of Forensic Sciences (Blackwell Publishing Limited); Sep2004, Vol. 49 Issue 5, p961-967, 7p

      Evaluates the carbon, nitrogen, and hydrogen stable isotope ratios in samples of bacteriological culture media. Consistency of the observed variation in the ratios with the expected isotopic variation in the plant and animal products; Observation of substantial isotope variation in cultures grown on different batches of media; Potentials of seized media to produce seized bioweapons agents.
      Author Affiliations:
      1Stable Isotope Ratio for Environmental Research, Department of Biology, University of Utah, Salt Lake City, Utah
      2IsoForensics, Inc., Salt Lake City, Utah
      3Central Intelligence Agency, Washington, DC

      • DXer said

        Dr. Kreuzer-Martin has also co-authored on the subject with Dr. Jarman:

        Stable Isotope Ratios and Forensic Analysis of Microorganisms.
        Kreuzer-Martin, Helen W.1
        Jarman, Kristin H.1
        Applied & Environmental Microbiology; Jun2007, Vol. 73 Issue 12, p3896-3908, 13p

        In the aftermath of the anthrax letters of 2001, researchers have been exploring various analytical signatures for the purpose of characterizing the production environment of microorganisms. One such signature is stable isotope ratios, which in heterotrophs, are a function of nutrient and water sources. Here we discuss the use of stable isotope ratios in microbial forensics, using as a database the carbon, nitrogen, oxygen, and hydrogen stable isotope ratios of 247 separate cultures of Bacillus subtilis 6051 spores produced on a total of 32 different culture media. In the context of using stable isotope ratios as a signature for sample matching, we present an analysis of variations between individual samples, between cultures produced in tandem, and between cultures produced in the same medium but at different times. Additionally, we correlate the stable isotope ratios of carbon, nitrogen, oxygen, and hydrogen for growth medium nutrients or water with those of spores and show examples of how these relationships can be used to exclude nutrient or water samples as possible growth substrates for specific cultures.

  7. DXer said

    Silicon signature:

    Elemental Signatures for Microbial Organisms, by John Cliff –

    “Although their experiments [Weber] failed to replicate the signature of the attack samples, they showed that high amounts of Si could be assimilated by the organism from the growth medium and their development of quantitative methods for single microbial cells marks a milestone in the field. Unfortunately, despite the great developments in this technique following the 2001 attacks, data are yet to be published in peer-reviewed literature rigorously linking elemental content of spores with growth conditions using NanoSIMS.”

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