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

* a recent article by Greg Gordon raises the potentially critical importance of b. subtilis contaminant found in the Brokaw and New York Post anthrax letters … not connected to Dr. Ivins … and substantially ignored by the FBI

Posted by Lew Weinstein on April 21, 2011

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has the FBI told the whole truth about its case against Dr. Ivins?

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Greg Gordon writes for McClatchy Newspapers (4/20/11) …

  • Scouring the anthrax-laced mail that took five lives and terrorized the East Coast in 2001, laboratory scientists discovered a unique contaminant – a microscopic fingerprint that they hoped would help unmask the killer.
  • One senior FBI official wrote in March 2007, in a recently declassified memo, that the potential clue “may be the most resolving signature found in the evidence to date.”
  • Yet once FBI agents concluded that the likely culprit was Bruce Ivins – a mentally troubled but highly regarded Army microbiologist – they stopped looking for the contaminant, after testing only a few work spaces of the scores of researchers using the anthrax strain found in the letters.
  • They quit searching, despite finding no traces of the substance in hundreds of environmental samples from Ivins’ lab, office, car and home.
  • But the FBI’s decision not to fully test for the distinct bacterial contaminant, pieced together by McClatchy Newspapers in interviews with scientists, federal law enforcement officials and in a review of recently declassified bureau records, could reignite the debate over whether its agents found the real killer.
  • FBI agents locked on Ivins after 2007 tests showed a genetic match between the mailed anthrax and spores in a flask in his lab. He’d shared the contents with others. Testing all samples submitted by labs, the FBI found eight with mutations matching those in Ivins’ anthrax, and soon eliminated all suspects but Ivins.
  • Retired Army Lt. Col. Jeffrey Adamowicz, who supervised Ivins in 2003 and 2004, expressed dismay that the search for the contaminant was cut short.
  • Adamowicz said that anyone with access to spores from Ivins’ flask – or to anthrax he shipped to other labs – needed only “a teeny tiny microscopic drop of that culture to grow their own.”
  • In February, a National Academy of Sciences panel challenged the bureau’s finding that a genetic match meant that the wet anthrax in Ivins’ flask was the “parent” of the dry powder in the envelopes. The panel said that link wasn’t definitive.
  • Lab scientists didn’t identify the genetically unique strain of b. subtilis until December 2005. It was in letters sent to NBC News anchor Tom Brokaw and The New York Post, but wasn’t in the Senate letters.
  • B. subtilis is harmless, but looks and behaves so much like anthrax that researchers have used it to simulate how anthrax spores would act if made into an airborne spray.
  • Its presence in the letters, LSU’s Hugh-Jones said, suggests that somebody grew anthrax using equipment contaminated during earlier b. subtilis experiments.
  • But once the four mutations in the mailed anthrax were linked to Ivins’ flask, there seemed little value to testing the equipment, countertops and b. subtilis stocks in the labs of researchers whose anthrax didn’t match Ivins’ spores, another of the law enforcement officials said.
  • Jacques Ravel, a lab scientist who aided the FBI while with the Institute for Genomic Research in Rockville, Md., shrugged off the b. subtilis lead as “a long shot,” saying that the contaminant is found “everywhere” in the air and soil and wasn’t used much at the time by bio-weapons labs.
  • However, a 2004 paper in a science journal described a study of b. subtilis by researchers at Dugway, the Battelle Memorial Institute’s operations at Dugway, and the Army’s Aberdeen Proving Ground in Maryland. Unlike Ivins, researchers at Dugway and Battelle both worked with dry anthrax powder.
  • Nonetheless, the National Academy of Sciences’ panel accepted the FBI’s finding that the incomplete testing for b. subtilis lead “did not provide useful forensic information.” But, the panel said deep in its report, such clues “should be investigated to their fullest” in the future.

read the entire article at … http://www.miamiherald.com/2011/04/20/2177735/was-fbi-too-quick-to-judge-anthrax.html

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LMW COMMENT …

The FBI’s publicly presented case against Dr. Ivins is seriously flawed: no evidence, no witnesses, an impossible timeline, science that proves innocence instead of guilt. So what really happened? And why doesn’t the FBI offer America a credible story?

As regular readers of this blog well know, I can imagine only 3 possible “actual” scenarios …

  1. The FBI has more evidence against Dr. Ivins but is, for some undisclosed reason, withholding that evidence … POSSIBLE BUT NOT SO LIKELY
  2. The FBI, despite the most expensive and extensive investigation in its history, has not solved the case and has no idea who prepared and mailed the anthrax letters that killed 5 Americans in 2001 … EVEN LESS LIKELY
  3. The FBI knows who did it (not Dr. Ivins) but is covering up the actual perpetrators, for undisclosed reasons …THE MOST LIKELY SCENARIO
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21 Responses to “* a recent article by Greg Gordon raises the potentially critical importance of b. subtilis contaminant found in the Brokaw and New York Post anthrax letters … not connected to Dr. Ivins … and substantially ignored by the FBI”

  1. DXer said

    Who decided not to swab the suspect labs for the subtilis contaminant?

  2. DXer said

    “This was not an incidental finding,” leading anthrax expert Martin Hugh-Jones said. “The FBI had what I would call an institutional fingerprint. Whoever had that strain of (bacteria) has to answer to the investigators.”
    http://www.globalsecuritynewswire.org/gsn/nw_20110421_3330.php

  3. DXer said

    McClatchy Points Out Key FBI Failure in Amerithrax Investigation

    By: Jim White Thursday April 21, 2011 5:58 am
    http://my.firedoglake.com/jimwhite/2011/04/21/mcclatchy-points-out-key-fbi-failure-in-amerithrax-investigation/

  4. BugMaster said

    The bacillus is most likely an environmental contaminant, from the soil in the vicinity the material was made. Assuming it was a lab strain is the LEAST likely possiblity.

    Nevertheless, isolate a genetically identical bacillus from a soil sample (from say, Columbus, Ohio) and you now have identified the geographic location the material was produced.

    The contaminant is genetically unique. The analysis done by Novazymes describes it as a very efficient spore-former, and conclude that it was able to contaminate the material because of the high number of spores it can produces and therefore can contaminate a given location (so where is it?).

    The FBI should be required to deposit cultures of the contaminating subtilus with the ATCC. I believe they can be compelled to do so under FOIA.

    Then, independent investigators lacking the FBI’s bias can go bioprospecting, and find the critter!

    • DXer said

      “The bacillus is most likely an environmental contaminant, from the soil in the vicinity the material was made. Assuming it was a lab strain is the LEAST likely possibility.”

      Dr. Martin Hugh-Jones, oft-quoted expert on anthrax who has the largest repository in the world (collecting those samples from throughout the globe, says:

      “Its presence in the letters, LSU’s Hugh-Jones said, suggests that somebody grew anthrax using equipment contaminated during earlier b. subtilis experiments.”

      Note that FOIA applies to documents. Is there something you see in the wording of the statute that makes you think it would apply to a bacteria?

      • DXer said

        Martin is currently in touch with Kimothy, the FBI genetics expert, who had worked closely with him at LSU. Doesn’t Kimothy agree with Martin?

    • Old Atlantic said

      Suppose it was a contaminant in 2001 from the environment. So the person growing the anthrax picked it up on their hands outside the lab and then put their hands on the equipment (before putting in the anthrax) and that transferred it.

      Suppose this lab also keeps stocks of subtilis. Those are not likely kept in a BSL3. Nor do they worry very much about subtilis from the environment getting into their subtilis stock. They can take the subtilis in an ordinary lab, open the container and leave it open. And stick equipment into it, etc.

      If this subtilis is so prevalent in the environment it got into the anthrax one time out, then it is more likely in the last 10 years to have gotten into the subtilis stock.

      This is a different strain of subtilis according to the article. It isn’t just a tiny segment of DNA with some change that is hard to find. This is a different strain. So it will be easier to type than finding bits of DNA that are different in the same strain, which was the task with the morphs in the anthrax.

      So subtilis stocks are the best place to look, even if they did not exist in 2001 at that institution. This is because the environment is presumably the same in that location and so even in the last 5 years subtilis stocks at an institution would be likely to pick it up.

      So test any subtilis stocks, whether they existed in 2001 or not. Those are the most likely place to have accumulated the strain if in fact it is easily picked up and transmitted from the environment just by human hands touching equipment or similar transfers.

  5. Old Atlantic said

    They can search at labs and universities for their stocks and perhaps elsewhere. A single person can’t eliminate the stocks of subtilis at Battelle or another institution. So in some scenarios, they could still find the subtilis.

    One scenario is that an institution’s stock of subtilis was used by an individual at some stage of testing or preparation. That individual may then have passed what was grown to others outside the institution.

    The individual may not have anticipated genetic testing of the institution stocks of subtilis to find which institution’s stock corresponding to the subtilis contaminant.

    So in this scenario, routine testing of the stock of subtilis of each major/minor institution that had the anthrax or even just had subtilis stocks could find the institution.

    They already did that for USAMRIID it appears. So USAMRIID is eliminated. So try the others.

  6. DXer said

    The FBI only provided one peer-reviewed article in its 3500 pages uploaded in February 2010.

    It involved DARPA-funded research involving virulent Ames and subtilis.

    In the article, Dr. Ivins was thanked for supplying virulent Ames. Former Colleague #1 (Mara Linscott) and #2 (Patricia Fellows) were thanked for providing technical assistance.

    Where was the subtilis research done? USAMRIID? Johns-Hopkins? Edgewood? LSU? Or Dugway? University of Michigan?

    What strain of subtilis was used.
    In vitro sporicidal activity

    To assess the sporicidal activity of BCTP, spores from four species of Bacillus genus (B. cereus, B. circulans, B. megaterium, and B. subtilis) were tested.

    We thank Shaun B. Jones, Jane Alexander, and Lawrence DuBoise (Defense Science Office, Defense Advanced Research Project Agency) for their support; Bruce Ivins, Patricia Fellows, Mara Linscott, Arthur Friedlander, and the staff of USAMRIID for their technical support and helpful suggestions in the performance of the initial anthrax studies; Martin Hugh-Jones, Kimothy Smith, and Pamala Coker for supplying the characterized B. anthracis strains and the space at Louisiana State University (Baton Rouge); Robin Kunkel (Department of Pathology, University of Michigan) for her help with electron microscopy preparations; and G. Morris and A. Shih for their technical assistance with manuscript preparation.

    A Novel Surfactant Nanoemulsion with Broad-Spectrum Sporicidal Activity against Bacillus Species

    Tarek Hamouda1,
    Michael M. Hayes1,a,
    Zhengyi Cao1,
    Richard Tonda1,
    Kent Johnson2,
    D. Craig Wright3,
    Joan Brisker3 and
    James R. Baker Jr.1

    • DXer said

      I called researcher Michael Hayes, who worked alongside Bruce Ivins in the BL-3 in connection with the research involving the virulent Ames supplied by Bruce Ivins, and he said “You don’t want to know.”

      What did Michael mean?

      • DXer said

        DARPA, you’ll recall, is the agency that asked the FBI anthrax expert JE to make a dried powder out of the Ames supplied by Bruce Ivins from Flask 1029.

        The 302 interview previously interviewed explains that special facilities were built. What were those facilities?

        • DXer said

          Spore preparation

          For induction of spore formation, B. cereus (ATCC 14579), B. circulans (ATCC 4513), B. megaterium (ATCC 14581), and B. subtilis (ATCC 11774) were grown for 1 week at 37°C on nutrient agar with 0.1% yeast extract and 5 mg/L MnSO4. The plates were scraped, and the bacteria and spores were suspended in sterile 50% ethanol and incubated at 22°C for 2 h with agitation to lyse the remaining vegetative bacteria. The suspension was centrifuged at 2500 g for 20 min, and the pellet was washed twice in cold distilled water. The spore pellet was resuspended in trypticase soy broth (TSB) and used immediately for experiments. B. anthracis spores, Ames and Vollum 1B strains, were supplied by Bruce Ivins (US Army Medical Research Institute of Infectious Diseases [USAMRIID], Fort Detrick, Frederick, MD) and were prepared as described elsewhere [5]. Four other strains of B. anthracis were provided by Martin Hugh-Jones (Louisiana State University, Baton Rouge). These strains (from South Africa; Mozambique; Bison, Canada; and Del Rio, TX) represent isolates with high allelic dissimilarity.

  7. DXer said

    Where was subtilis used prior to 9/11 in aerosol biodefense testing?

    The strain that was the contaminant was genetically distinctive.

    What strains were used in the biodefense testing?

    Once identified: The question is: Were they tested? (And if not, why not?) How much would it have cost to swab for subtilis?

    • DXer said

      A broader search might be “anthracis” and subtilis

      Journal of Bacteriology, February 2003, p. 1443-1454, Vol. 185, No. 4
      0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.4.1443-1454.2003
      Copyright © 2003, American Society for Microbiology. All Rights Reserved.
      Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

      Received 9 August 2002/ Accepted 21 November 2002

      The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

      • DXer said

        The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis

        Article first published online: 5 JAN 2002

        • DXer said

          Applied and Environmental Microbiology, January 2002, p. 227-234, Vol. 68, No. 1
          0099-2240/02/$04.00+0 DOI: 10.1128/AEM.68.1.227-234.2002
          Copyright © 2002, American Society for Microbiology. All Rights Reserved.
          Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant Bacillus anthracis Protective Antigen from B. subtilis

        • DXer said

          Journal of Bacteriology, April 2003, p. 2418-2431, Vol. 185, No. 8
          0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.8.2418-2431.2003
          Copyright © 2003, American Society for Microbiology. All Rights Reserved.
          Methionine Regeneration and Aminotransferases in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis

          Defence R&D Canada-Suffield, Medicine Hat,1 Canada West Biosciences, Calgary, Alberta, Canada2

          Received 8 November 2002/ Accepted 27 January 2003

        • DXer said

          Infection and Immunity, February 2003, p. 801-813, Vol. 71, No. 2
          0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.2.801-813.2003
          Copyright © 2003, American Society for Microbiology. All Rights Reserved.

          Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines

        • DXer said

          Bacillus Spore Identification via Proteolytic Peptide Mapping with a Miniaturized MALDI TOF Mass Spectrometer

          Anal. Chem., 2003, 75 (24), pp 6886–6893
          DOI: 10.1021/ac034624+
          Publication Date (Web): November 6, 2003

        • DXer said

          The articles I cite above show Porton Down (the UK biodefense lab) and Suffield (the UK biodefense lab) both using subtilis. Here is another example – this one from PNNL.

          Appl Spectrosc. 2003 Aug;57(8):893-9.
          Identification of bacterial spores using statistical analysis of Fourier transform infrared photoacoustic spectroscopy data.

          Thompson SE, Foster NS, Johnson TJ, Valentine NB, Amonette JE.
          Source

          Pacific Northwest National Laboratory, P.O. Box 999, MS K8-96, Richland, Washington 99352, USA.

          Abstract

          Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores. A total of forty specimens representing five strains of Bacillus spores (Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis subsp. kurstaki, and Bacillus globigii Dugway) were analyzed. Spores were deposited, with minimal preparation, into the photoacoustic sample cup and their spectra recorded. Principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations were used on this spectral library to develop algorithms for step-wise classification at three levels: (1) bacterial/nonbacterial, (2) membership within the spore library, and (3) bacterial strain. Internal cross-validation studies on library spectra yielded classification success rates of 87% or better at each of these three levels. Analysis of fifteen blind samples, which included five samples of spores already in the spectral library, two samples of closely related Bacillus globigii 01 spores not in the library, and eight samples of nonbacterial materials, yielded 100% accuracy in distinguishing among bacterial/nonbacterial samples, membership in the library, and bacterial strains within the library.

    • DXer said

      For additional example,s Adrian Ponce used subtilis in his 2001 experiments in Southern California relating to detecting aerosols.

      Experiments at ISU used subtilis.

      Even the most cursory googling shows that it subtilis was widely used as a simulant in 2001.

      The dried aerosol experiments at USAMRIID used simullants. Was one simulant used subtilis? Or was only BG used.

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