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

* NAS anthrax panel meetings – links for audio provided by NAS

Posted by Lew Weinstein on September 25, 2009

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******

NAS panel meetings 9/24/09 & 9/25/09

links for audio provided by NAS …

http://nationalacademies.org/newsroom/nalerts/20090925.html

The NAS is to be congratulated for the speed with which they provided these links

******

Agenda for September 24, 2009 Open Session

  • 2:00 Use of Preliminary Validated Assays in Exigent Circumstances, Steven Schutzer, University of Medicine and Dentistry of New Jersey
  • 2:30 Overview of the Scientific Investigation, Rita Colwell, University of Maryland College Park and Johns Hopkins University, Bloomberg School of Public Health
  • 3:00 Identification of Bacillus anthracis Ames Strain, Paul Keim, Northern Arizona University
  • 3:45 Identification of Morphological Variants, Patricia Worsham, USAMRIID
  • 4:15 Genetic Analysis of Bacillus anthracis Ames Strain, David Rasko, Institute for Genome Sciences, University of Maryland School of Medicine
  • 4:45 – 6:00 … Discussion

Agenda for September 25, 2009 Open Session

  • 9:00 Microscopy/Weaponization of Bacillus anthracis, Joseph Michael, Sandia National Laboratory
  • 9:40 Nano-scale secondary ion mass spectroscopy, Peter Weber, Lawrence Livermore National Laboratory



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75 Responses to “* NAS anthrax panel meetings – links for audio provided by NAS”

  1. DXer said

    Patricia Worsham, one of the leading speakers before the NAS, addresses the issue of making the dried powder in this deposition excerpt:

    Case 9:03-cv-81110-DTKH Document 154-13 Entered on FLSD Docket 07/15/2011

    IN THE UNITED STATES DISTRICT COURT SOUTHERN DISTRICT OF FLORIDA
    CASE NUMBER: 9:03-cv-81110-CIV-HURLEY/HOPKINS
    MAUREEN STEVENS, as Personal Representative of the Estate of ROBERT STEVENS, Deceased, and on behalf of MAUREEN STEVENS, Individually, NICHOLAS STEVENS, HEIDI HOGAN and CASEY STEVENS, Survivors
    Plaintiffs
    vs. UNITED STATES OF AMERICA
    Defendant ________________________________/
    The Videotaped deposition of PATRICIA L. WORSHAM, Ph.D. was held on Monday, February 7, 2011, commencing at 12:29 p.m., at the U.S. Army Research and Materiel Command, Office of the Staff Judge Advocate, 521 Fraim Street, Fort Detrick, Maryland 21702, before George W. Tudor, Notary Public.
    REPORTED BY: George W. Tudor
    All Florida Reporting, Inc. 800-898-7373

    *****

    Department of Justice, representing the defendant, United States.
    MS. WILKERSON: Kirsten Wilkerson with the Department of Justice, also for the United States.
    MR. WELLENS: Paul Wellens, with the FBI Office of General Counsel.
    MR. MILLER: Jeffrey Miller, Fort Detrick, United States Army.
    THE VIDEOGRAPHER: Would you administer the
    oath? Whereupon,
    PATRICIA L. WORSHAM, Ph.D., called as a witness, having been first duly sworn to tell the truth, the whole truth, and nothing but the
    truth, was examined and testified as follows:

    EXAMINATION BY MR. SCHULER:
    Q Would you state your name, please? A Patricia Lynne Worsham.
    Q And what is your professional address? A 1425 Porter Street, Bacteriology Division,
    USAMRIID, Fort Detrick.

    ******
    Q And what — do you have a Reader’s Digest
    4 version of — explanation of why you believe that?
    5
    A I think I summarized it before to a certain
    6 extent, in that I don’t believe that we had facilities
    7 at USAMRIID to make that kind of preparation. It would
    8 have taken a great deal of time; it would have taken a
    9 huge number of cultures; it would have taken a lot of
    10 resources that would have been obvious to other people
    11 within containment when they wanted to use those
    12 resources.
    13 We did not have anything in containment
    14 suitable for drying down anything, much less a quantity
    15 of spores. The lyophilizer that was part of our
    16 division was in noncontainment. If someone had used
    17 that to dry down that preparation, I would have
    18 expected that area to be very, very contaminated, and
    19 we had nonimmunized personnel in that the area, and I
    20 might have expected some of them to become ill.
    21 Q And maybe I misunderstood you, because I
    22 wanted to ask you those questions that you just
    23 answered, but what you just talked about, though, was
    24 the actual production capability, correct?
    25 A It’s part of the science.

    QJacques Ravel did the sequencing. Right. I think he’s a good sequencer. And have you had discussions with him about
    his work and what he did and how he arrived — he and, I think it’s Dr. Liggett, if I recall correctly?
    A There are a number of us on that paper.
    Q — how he arrived at the conclusions that RMR 1029 was the source?
    A He did not come to that conclusion. Jacques Ravel did the sequencing of the ancestral M strain and the variants that the FBI gave him to sequence. The actual interpretation of where they believe that material came from was done by the FBI, not by Jacques Ravel.

    ********

    Q So you’re saying that absolutely and without question, in your mind the equipment that’s at USAMRIID could not have been used to prepare the dried spore preparations used in the letters.
    evidence of anyone getting any training that would allow them to do that.
    To do what? Produce dried material of that quality. Is that something that is complicated,
    difficult to do?
    A I think it would be very difficult to do,
    Q of material correct?

    A Not any equipment that I have seen. And you mentioned that it was a huge amount that was involved in the attack letters,
    (Witness nods head.) Yes? Yes. Sorry. The — and is that based on visual or what is that based on?
    The sheer number of spores contained in those preparations.
    Q And the number of spores is determined,

    ********

    the years. I don’t recall specifically what we had in that year.
    Q And to get back to the expertise end of it, I know that’s not something that you all that work there customarily did, correct?
    A
    Correct.
    I mean, you developed spore preparations in media, right?
    Um-hmm.
    And then at some point, for aerosolyzing it had to turn into a vapor, right?
    There is what they call a nebulizer, and Collison nebulizer that sprays the liquid into very fine particles. So it retains its liquid state.
    Q So the aerosol tests are very small micron droplets, if you will, of the liquid that you prepare, right?
    A That’s generally true. They have the capability of doing polydispersed aerosols as well, which have multiple-sized particles, but generally we do small particles, which is at a micron.
    Q Did you ever discuss with Dr. Ivins — and again, outside the presence of any FBI folks — did you ever discuss with Dr. Ivins the process for accomplishing this type of an attack?

  2. DXer said

    http://biopreparat-mknaomi.blogspot.com/2009_12_01_archive.html

    Patrick Worsham explained:

    “So in my experience what causes the in vitro selection of oligosporogenic strains? In my hands, it was repeated passages of overgrown liquid cultures. So if you have a culture, and the organism is running out of nutrients, and the majority of the population begins to sporulate, they stop dividing. If you have a mutant in the population, that is not getting the signal to sporulate, and continues to replicate, it forms a larger percentage at the end of that culture, than it started. And if you continue to do this, it will eventually take over the culture. If you pick from the edge of colonies on a plate – particularly an old plate – that is, if you have a colony and that colony has reached a state where it would ordinarily start sporulating, sometimes what you’ll see are fingers coming out from the side of the colony, and these are mutant that have popped up, that are continuing to replicate under conditions where the parent has stopped growing and started to sporulate – and if you prepare stocks from old cultures, plate or broth, you tend to have this sort of an issue. …

    This reminds me that the Congo Red issue is how I initially found these variants many many years ago. And that is, I was interested in looking at virulence in surface proteins, and one of the ways – I’m from a gram-negative background – that we often look for changes in the surface properties of an organism, is by binding of dyes. And so I incorporated Congo Red into the growth media with Bacillus anthracis and I began to find that the wild-type formed pink colonies but then these sort of white outgrowths would appear – and those were actually oligosporogenic. They have decreased amounts of the surface array protein, EA1, there’s a brown pigment that they make which is a byproduct of catechol metabolism, and there’s diminished as I said spore production. I was interested in these from a virulence perspective, I tested the vegetative cells of some of these strains for virulence, and they really weren’t impaired compared to the wild-type, and that was pretty much the end of my love story with these strains for quite a while, because they didn’t seem to be important to virulence, and I had to move on.”

    Comment: Does this point to a supply associated with experiment with proteins and virulence?

    • DXer said

      William Broad, in reporting the research of Former Colleague #2, reports:

      “The pX02 plasmid carries genes that let the anthrax bacterium fashion an outer protein coat that acts as a defensive shield to thwart the immune system of hosts. ”

      Was Flask 1030, which contained a Silicon Signature, used in that research?

      Was the source of Flask 1030, #7738 as registered in Building 1412?

      Is it correct that there is no documentation as to whether the parent of 7738 was Flask 1029 or frozen stock?

      If the parent was 1029, then would it both be genetically identical and containing a silicon signature?

      Is it correct that the FBI does not know what happened to 7738?

      What does Martin Hugh-Jones about the FBI’s theory that Ivins is the processor and mailer? What does Kimothy Smith think?

      Does Dr. Hugh-Jones think that the Ivins Theory is hooey?

      Key to Strains of Anthrax Is Discovered
      By WILLIAM J. BROAD
      Published: March 27, 2003

      Scientists have discovered why different strains of the bacterium that causes anthrax differ so much in virulence, a finding that in theory could produce more effective vaccines and better tools for distinguishing and tracking the lethal germ.

      But the finding could also aid the creation of designer varieties of anthrax that are potentially deadlier to humans. Because of that potential danger, a debate occurred over whether the discovery should be kept secret, scientists said. In the end, it was decided that the benefits of publication outweighed the risks.

      The discovery was made by six scientists at Louisiana State University, the Lawrence Livermore National Laboratory and the United States Army Medical Research Institute of Infectious Diseases, the nation’s top center for studying germ defenses. It is published in the current Journal of Clinical Microbiology.

      The lead author, Dr. Pamala R. Coker, formerly at L.S.U. and now at the Livermore laboratory in California, spearheaded the research for her Ph.D. dissertation. The Livermore laboratory once pioneered nuclear arms but increasingly studies biology and germ defenses.

      The team’s finding centers on the anthrax genome, which consists of a single large chromosome and two small circles of DNA, known as plasmids, that carry extra genes. The scientists found that, contrary to common belief, each anthrax bacterium carries not just one set of plasmids but up to 243 copies of the first and up to 32 copies of the second, which is known as pX02. The more copies of this plasmid in a bacterial strain, the more it is capable of causing disease, the scientists said.

      The research was conducted in guinea pigs. The scientists found, for example, that an anthrax strain from Mozambique that possessed just one pX02 plasmid killed 25 percent of the test animals. But a strain from Australia with 32 copies of the plasmid left all the guinea pigs dead.

      The team of scientists also reported that added factors like subtle features of the bacterium’s DNA chromosome appeared to help determine virulence. Thus, the anthrax that killed five Americans in the germ attacks of 2001 — the so-called Ames strain — was found to possess just two copies of pX02. But it nonetheless killed 62 percent of the guinea pigs.

      The pX02 plasmid carries genes that let the anthrax bacterium fashion an outer protein coat that acts as a defensive shield to thwart the immune system of hosts. The scientists suspect that multiple copies of pX02 thicken that coating, letting the germ escape immune damage and multiply to do extensive harm.

      Scientists had previously identified 89 types of anthrax as genetically distinct but had failed to discover what determined their wide differences in virulence. The plasmid findings, they said, opened a new window on that question.

      • DXer said

        7738, Dr. Ivins explained, was a dilution of 7737 (which was Flask 1029). It consisted of leftover aerosols and yet had a silicon signature. Where did it come to have a silicon signature?

        What if it had not been diluted? Might the silicon signature be that much more pronounced?

        Did the aerosol experiments involve microencapsulation? If so, where were they done?

        • DXer said

          http://docs.google.com/viewer?a=v&q=cache:pz_3CSwt2UIJ:foia.fbi.gov/amerithrax/847423.PDF+&hl=en&gl=us&pid=bl&srcid=ADGEESh-7_2LsuVaGUYcU4-LQ1-QpOpP1hTLjNZbsAbLqr_Sb9MHNajojwZ8t3Ix46E-nrofJ9e22iCXOffovc18SLPRwxA-Wnnii_nfnr67fhIBv40yet2mQku0QS90h41lS7PjI2YC&sig=AHIEtbRnr0Cpmso_-09fwcBs9menYhyAJA

          _________ frequently grew Bacillus anthracis (B.a.) Ames spores during the time ______________________________________________________ ____ kept ___ laboratory notes in lab notebooks that were organized according to study. All of ___________ would be located in ________________________________________________________________________________________________________________ ___ did not photocopy ____ notes from the laboratory notebooks, nor did ___ ___ own side notes.

          ______ used to grow ____________________________________________ would have used IVINS’ spore stock was kept in a tube in the walk-in refrigerator and was not frozen. _______ would have gone back to the same stock to start each batch, rather than to plate from the previous batch. Although ____ was not certain what the sample name was for the seed stock that ____ used, ____ knew that it would have been the same as what IVINS used for his seed stock. Specific batch information can be found in IVINS’ laboratory notebooks. Approximately 100 (mL) of spores were being produced per week at a concentration of 8.5 x 10 or 10 and up to approximately 10 spores per mL

        • DXer said

          It seems that Flask 1030 (7738 as registered in Building 1412) could be aerosol leftovers but could not have Flask 1029 as its parent because 1029 dates to 1997 and Flask 1030 reportedly, according to this 302, dates to before that. Thus, is it 7739a, 7739b, 7739c that would be genetically identical and have a silicon signature? Were they also aerosol leftovers?

          10/2/2003:

          IVINS listed his Ames samples as follows. He explained that his lab technican submitted the samples in April 2002 to the Repository.

          1) Original 1981 slant from Texas, 255414B
          2) 7800a
          3) 7800b
          4) 7737
          5) 1030 Reference Material
          6) 7739a
          7( 7739b
          8 7739c

          The last four samples listed above are spore preparations which were produced by various individuals at USAMRIID. Reference Material 1030 is a multiple batch lot of spores produced by IVINS ______________ from 11/20/1995 to 11/18/1996. _________________ produced lot 7739a on 7/25/1997. ___________________ produced lots 7739b and 7739c on 12/08/1999 and 3/28/2001 respectively. IVINS will save the above four preparations for future submission to the Repository. IVINS also had two additional preparations of Ames BA spores, lots 7736 and 7738, but the spores were used and are no longer available.

          The first four samples of Ames listed above were submitted to the Repository by ________ during April, 2002. The 7800a and b samples are from IVINS ___________________ respectively. 7800a is a sample of IVINS’ Ames stock and was prepared from a single colony of the original Ames slant by IVINS in 1985. 7800b is a sample from the BA collection of PERRY MIKESELL, dated July, 1991, and labeled ___________. IVINS believes that the MIKESELL sample was derived from __________ stock culture around 1985. The sample labeled ______________ and submitted to the Repository in April, 2002 may be from MIKESELL’s collection and not directly from __________ stocks of BA. ____________ submitted IVINS’ samples to the Repository and would be able to provide information on the origin of the ___________ sample in the Repository. ______ is now employed by ____________________________________________________________

        • DXer said

          In 2003, isn’t he saying that Former Colleague #2 submitted the samples to the Repository and is that what the April 24, 2002 email shows?

        • DXer said

          https://caseclosedbylewweinstein.wordpress.com/2010/04/05/who-wrote-this-email-who-submitted-the-slants-to-the-fbi/

        • DXer said

          Is the woman who submitted the slants the aerobiology expert, Former Colleague #2 Pat Fellows, who was thanked for her technical assistance by the former Zawahiri associate Tarek Hamouda (who worked alongside Bruce Ivins). Pat then went to head the BL-3 lab at Southern Research Institute (“SRI”). Tom Voss who had led SRI refuses to say when they first acquired virulent Ames. Dave Franz, SRI VP at the time, too. SRI had a subcontract for the DARPA research requiring a BL-3 being done by the Ames researchers at the DARPA-funded GMU Center for Biodefense. There the top researchers, Ken Alibek and Charles Bailey, shared a suite with Ali Al-Timimi, the scientist coordinating with Anwar Awlaki.

          Meanwhile, leading the FBI science effort, we have the FBI scientist examining the powder and collecting the samples from Ivins, John Ezzell, who made a dried powdered aerosol for DARPA using Flask 1029 and we were never told. And we have the collection scientist for American Type Culture Collection, Jason Bannon, which co-sponsored Al-Timimi’s program. We have Doug Beecher who was with Ezzell in the Hazardous Materials Response Unit. The pair have participated in withholding of FBI documents now for two years in violation of the Federal Adivsory Committee Act. And as the genetics expert in 2002, we have Kimothy Smith, the fellow who provided the BL-3 to the former Zawahiri associate. All these scientists have acted in good faith, IMO. In fact, Dr. Ezzell has acted downright heroically coming forward to answer questions. Given that none of these FBI scientists or USAMRIID scientists were complicitous, and instead are all just motivated by the same career-oriented and personal motivations of us all, isn’t it time that they come forward and say that the emperor has no clothes? Do these scientists not realize that Leiter, a very bright guy, ranks Awlaki right up there with Bin Laden in terms of the threat posed. Napolitano too. Holder also. Don’t these people realize that this is not the type of matter where the country can afford to get the analysis wrong?

          Who is going to step forward and say “Enough is enough — the emperor has no clothes.”

          Isn’t the obfuscation due to CYA relating to the infiltration … even though it is only through hindsight that the infiltration becomes apparent.

          We’ll cover your back, guys if you come forward now. But someone needs to come forward and do a dead guy — who deserved better from his friends — some justice. Martin Hugh-Jones wept for Dr. Ivins. A lot of people who didn’t even know him did also. It just isn’t right.

        • DXer said

          Anthrax and Al Qaeda: The Infiltration of US Biodefense (440 pages fully viewable text)
          http://www.blurb.com/bookstore/detail/1443811

          100+ graphics of the documentary evidence from a guy who was taught like Leiter, by Charles Nesson what evidence suffices in seeking to do justice
          http://www.anthraxandalqaeda.com

        • DXer said

          America has a new Public Enemy No. 1

          U.S.-born cleric. Al-Awlaki replaces bin Laden as ‘most significant risk’

          Read more: http://www.montrealgazette.com/news/America+Public+Enemy/4255387/story.html#ixzz1DYkTgc00

          The greater danger, Leiter said, now comes from American-born cleric Anwar al-Awlaki and his Al-Qa’ida ties in the Arabian Peninsula. Not only has al-Awlaki demonstrated some capability to strike at the U.S. from outside the country, he also has emerged as the primary influence over homegrown terrorists plotting to strike within the United States, he said.

          “I actually consider Al-Qa’ida in the Arabian Peninsula, with al-Awlaki as a leader within that organization, probably the most significant risk to the U.S. homeland,” Leiter said.

          It was the most explicit statement yet from an Obama administration official that al-Awlaki, a 39-year-old U.S. citizen now hiding in Yemen, has surpassed bin Laden in his ability to carry out plots against the U.S. homeland.

          U.S. President Barack Obama’s chief counterterrorism adviser, John Brennan, last December had described Al-Qa’ida in the Arabian Peninsula as “most operationally active node” of the Al-Qa’ida network.

          http://www.montrealgazette.com/news/America+Public+Enemy/4255387/story.html#ixzz1DYkOuBHd

        • DXer said

          Pat says the parent of 7739a was from the freezer. What about 7739b and 7739b. There doesn’t seem to be any contemporaneous documentation. Was the parent Flask 1029?

          12/12/03 302 IVINS Interview:

          IVINS provided the four samples of Ba Ames strain (labeled Reference Material 1030, 7729 a, b, c) to the FBI repository in October 2003. 7739 a, b, c) to the FBI repository in October 2003. IVINS provided Agents with a typewritten description of the 4 samples (The description summary will be submitted to the 1A section of the subfile). IVINS provided the following labels and descriptions for each of the four samples:

          “2) 7739a was produced by ____________ – The spores porduced were made in Leighton and Doi media. IVINS revised ____________ notebook for a description of how 7739a was made. __________ wrote in the notebook that she obtained the inoculum used to grow 7739a from a freezer tube in the freezer. No other details were listed.

          3). 7739b was produced by _____________ on 12/8/1999. The spores porduced were made in Leighton and Doi media. No details are known about the inoculum and methods used.

          4) 7739c was produced by _______________________ on 3/28/2001. The spores were made in Leighton and Doi media. No details are known about the inoculum and methods used.”

          ___________________________________ would be able to provide more information regarding inoculum and production method used to make _________________________.

        • DXer said

          When 7739b was produced by _____________ on 12/8/1999, no details are known about the inoculum and methods used, but note that there is no withdrawal known to have been made from Flask 1029 on that date.

          Similarly, when 7739c was produced by _______________________ was produced on 3/28/2001, no details are known about the inoculum and methods used, , but note that there is no withdrawal known to have been made from Flask 1029.

          What do we know about 7739b and 7739c? (The numbers are numbers assigned upon it being stored in Building 1412).

          https://caseclosedbylewweinstein.wordpress.com/2010/02/25/amerithrax-inventory-control-sheet-31798-to-10401/

  3. DXer said

    WEDNESDAY, September 30- Live Webcast Today on Security in Research Using Bio Agents and Toxins

    This briefing will discuss the findings of the new report ‘Responsible Research With Biological Select Agents and Toxins’ which assesses safeguards against the deliberate misuse of biological select agents and toxins (BSAT) used in research. Listen to the live audio webcast from 1:30 p.m. to 2:30 p.m. EDT.
    More Information

    Please refresh this page after 1:25 p.m. for a link to the webcast audio.
    http://www.nationalacademies.org/

    http://www.anthraxandalqaeda.com

  4. DXer said

    At the August 18, 2008 briefing to the science media, the FBI says they can’t speculate (don’t know) what equipment Dr. Ivins would have used to dry the spores.

    “DR. HASSELL: They were regrown. I mean, they weren’t just pulled out of that flask of 1029 and then dried and used. There were chemical differences. There were some chemical constituents in the flask that were not present on any of the samples. So we do know that there was regrowth there. Post-processing, we just really can’t speculate without treading too far on the other side of things. I mean, he did have access to some post-processing equipment, but that doesn’t necessarily say that was used.”

  5. DXer said

    Dr. Hassell on the subtilis contamination at Aug. 18 briefing to Science media (for which no match was found in Ivins’ lab)

    QUESTION: I’ve read a little bit of that there was some sort of contamination of Bacillus Subtilis in these samples. Is that true and is there any significance to that?

    DR. HASSELL: Like Dr. Majidi mentioned in the beginning, there were differences between the letters themselves. There were, essentially he showed that there were two batches that were made from this and one of those batches appeared to have been contaminated with Subtilis.

    QUESTION: Is it typical to see or is it unusual to see Subtilis contamination with Anthrax?

    BACKGROUND OFFICIAL: It is possible always to contaminate potentially a liquid culture or if you’re growing large quantities of an organism where you’re growing lawns of a bacteria on a Petri dish it could be difficult to see other members of the bacillus species because they would be occluded by and look very similar to the organism that you are culturing. So it appears at some point a low level of bacillus contamination occurred in the preparation of the material and that was detectable and was able to be isolated and characterized.

    QUESTION: Is there any sense of where that might have come from along the process?

    DR. HASSELL: Well, it doesn’t exactly answer — well, I’ll get to that but one other point is that the Subtilis contamination was also in the samples that also had which looked like a rougher preparation, so there was more darker color. They were rougher.

    QUESTION: That was the first one.

    DR. HASSELL: The particle sizes and other things wasn’t as refined a sample. So it’s consistent. If we found that in the other ones it might have raised more question but it just looked like one was just a rougher preparation than the other and that rougher one had the Subtilis.

    BACKGROUND OFFICIAL: The B Subtilis is a common environmental organism and so incomplete sterilization or perhaps some sort of settling in media would enable it to be co-cultivated.

    QUESTION: I understand that. If Ivins was such a good microbiologist I’m wondering how likely he would be to contaminate his sample to where it might come from.

    DR. HASSELL: I consider myself a good microbiologist and I’ve contaminated cultures in my life. [Laughter.]

    http://www.anthraxandalqaeda.com

  6. DXer said

    On the more general subject of the infilitration of US biodefense, and the Bush Administration’s gross negligence in permitting it to occur, the National Research Council (research arm of the NAS) has issued a report RESPONSIBLE RESEARCH WITH BIOLOGICAL SELECT AGENTS AND TOXINS. The report assesses the efficacy of regulations, procedures, and oversight that have been instituted to safeguard against the deliberate misuse of biological select agents and toxins (BSAT) used in research. The report looks at security programs designed to protect against external threats, as well as internal threats from laboratory personnel. The report also makes recommendations on refining security programs and procedures with the aim of informing policy discussions in the U.S. on balancing the security risks and benefits of BSAT research.

    As to whether this is closing the door after Dr. Red has left the building, see

    http://www.anthraxandalqaeda.com

  7. DXer said

    Dr. Michael at August 18, 2008 briefing to science media –

    DR. HASSELL: Well, as the work progressed on this and more and more sophisticated techniques were brought to bear on just the silicon question, just to take that as an example, we brought more people in to advise on this, to look at it in much more detail.

    So I’m going to ask Dr. Michael if he might comment on it and then we can bring it back and then talk about some others, if we need to.

    [DR. MICHAEL]: Yeah. It was — when we got the evidence the first thing we did was put them in assay and take a look at them and we indeed did see signature of silicon and oxygen. What we didn’t see was any sort of nanoparticles on the outside of the spores. They just looked like regular spores to the experts that were advising us.

    So at that point, we knew there was silica or silicon and oxygen within the spores, but we weren’t sure and so we used some more sophisticated techniques. I’ve been working at Sandia for quite awhile now with hyper-spectral imaging and particularly for x-ray imaging on the scan (inaudible).

    Whenever you hit a sample with electron-generated x-rays that are characteristic of (inaudible) and so we use hyper-spectral imaging so that every pixel and image we would get would have a full analytical signature of silica.

    We then take this data and put it through some statistical analysis that then shows us how the various signatures are related. In fact, we found that when we took the spores and put them in the transmission electron microscope, we would run sections of spores, just take them three to the power, we would get the silica signature, but now they were sort of located on the outside of the spores. It wasn’t until we actually had ultra microtome sections in the transmission electron microscope and the scan transmission electron microscope where we could localize the silicon and the oxygen signal to the spore code and not to the excess correlate. So it was on the inside of the spore and not on the outside of the spore.”

    QUESTION: Is that common? Does the anthrax usually have some kind of spore —

    DR. MAJIDI: You know, this is a — if you look, ultimately what we’re saying is that that is not a postproduction additive to make the anthrax more disbursable. That’s what the whole concept or methodology of weaponization comes from, is to weaponize. That’s really — that’s an ambiguous word, but what people mean by weaponize is that postproduction of the spores was silica added to it to make it more disbursable.”

  8. DXer said

    Still more from Background Official on mutants at August 18, 2008 science briefing to science media –

    QUESTION: I’m going to follow up further on that. Rachel Erenberg from Science News.

    And my understanding is both within the flask and within the Petri dish, you do get the stochastic mutations arising. How, in terms of doing sequencing, do you make sure — how do you prevent actual growth of a dish because those mutations may arise in the course of the dish sitting in the cabinet? How do you know that the samples that you’re sampling from, the time that you take the sample, are the same samples in terms of the genes, the genome snapshot that you’re taking as they were when they arrived? How do you make sure they’re the same?

    BACKGROUND OFFICIAL: For essentially of the genome work that we have done, we have tried, I think in every case we have followed this protocol that I outlined, that when we have either prepared material ourselves or in the majority of cases they have provided material from outside collaborators, we have gone through this process of going back to low passage freezer stocks.

    QUESTION: So what does that mean exactly?

    BACKGROUND OFFICIAL: Clonal selection.

    QUESTION: Sorry?

    BACKGROUND OFFICIAL: Clonal selection.

    BACKGROUND OFFICIAL: Clonal selection. It’s really —

    QUESTION: So you’ve got the addition of freezer that is highly —

    BACKGROUND OFFICIAL: No, it’s a slant or it’s a frozen stock in a freezer that is — it’s going back to a stock that is not grown. Then you plate that material. It’s plated out. A single colony is selected, grown as minimally as possible and for genome projects, you need a minimal amount of material. We’re talking about a very different order of magnitude of material here for genome sequencing projects as compared to vaccine development efforts and that material is used for preparation of DNA, as a template for genome sequencing projects.

    But you raise an important point, that there is the possibility that during that process, additional stochastic mutations might arise, but I think what’s important to bear in mind is that if they do, these are not random mutations that come and go. If mutations arise, certainly over the time frames that we’re talking about in terms of genetic analysis that we carried out, they should persist.

    We’re not talking about large numbers of passages where minority representations in a population would be overgrown and the fact that these morphotypes that have been described were seen through repeated passage on plates could be identified through various assays that were developed gives us great confidence that in fact they were — they represented stable components of these cultures. They were not — they may have represented random mutations, but they were not random members of the population, if you will, that came and went at random.

    DR. MAJIDI: Can I add one more item?

    BACKGROUND OFFICIAL: I’d like to make a point of clarification. Many bacteria have phenotypic variations when you grow them as well as overnight in some bacteria. Anthrax is not known for that same practice. In fact, it’s very homogeneous.

    When one passes anthrax from one culture to another through passage, it’s rare to experience any type of phenotypic variation or the mutations that we’re talking about. In vaccine work, the Code of Federal Regulation actually limits for most bacteria the number of passages from your seed stocks to production of the vaccine to avoid this problem of mutation. Often that’s as little as five to seven passages.

    So in anthrax, it is very homogeneous. You don’t see phenotypic variation and you rarely see mutations occurring with just a few passages. It was noteworthy that the anthrax powders that was in the letters had significant phenotypic variant numbers of phenotypic variants. This was unusual for anthrax and in particular unusual for AMES.

    If one passes the 1981 AMES strain, the original AMES wildtype strain, one does not normally see mutations arising at a very high frequency. So it was noted that this was unusual in the anthrax powder.

  9. DXer said

    Background Official at August 18, 2008 briefing to Science media on mutants –

    DR. MAJIDI: Let me see if I can ask you to just put a little more texture on that and talk about how stable these mutations are. Are these things just randomly appearing or are they stable over multiple generations, the particular four markers we saw?

    BACKGROUND OFFICIAL: Well, the four markers that we saw have turned out to be stable over multiple generations and this was an assay that was — this was something that was looked at very specifically because these mutations were deemed to be so potentially critical.

    But if I might just make another more general comment to, I think, pick up on the discussion here, and it gets to the comments that Paul made. When I learned microbiology 30+ years now, it was clear that this notion of stochastic mutation was understood. We clearly didn’t have genomic approaches to track these mutations, to measure them, but it’s true not only in bacterial cultures, it’s true in mammalian cell culture, and this has been well described in many, many publications, that repeated passage of bacterial cultures or mammalian cell lines tends to be associated with phenotypic changes.

    This can be loss of virulence. It doesn’t have to be, but when I was learning microbiology, I remember very distinctly what I learned was that in liquid culture, a large batch liquid culture should be considered whenever possible to be an end state in what you were doing and whenever possible you went back to low passage stocks as a starting point and from there, the next step was to plate out bacteria on a Petri dish or an appropriate solid substrate so you could actually look at to see what you had and colonies that appeared different would be, in most cases, apparent to the naked eye. They could have a different morphology. They could be pigmented or not pigmented, depending upon what you were working with, and it would be selection of a single colony that you would use to inoculate a flask.

    It was considered bad technique to then take a liquid culture and use that for repeated inoculations because these stochastic mutations can accumulate and so if you think about it, if that’s a natural process, but for all of the reasons why you don’t in the process of vaccine development ideally want to be going back and generating multiple batches of material for development and testing, perhaps this puts all of this in a different context.

    This is a trade-off between having enough material and some of the risks, if you will, or risks is perhaps not the best word, but some of the caveats that come with having to acquire that through the preparations of multi-flask use.

  10. DXer said

    August 18, 2008 science media briefing on mutants –

    BACKGROUND OFFICIAL: So we published several papers on this and in fact, you know, the theory of how mutations arise go back to the classic Gloria Dellbrick experiments and so at least the idea is, is that, experiments occur by random chance and different regions of the genome will mutate at different rates and that’s been shown in this whole syntheses and so just by stochastic processes, you will end up with mutations occurring and depending upon the rate, the intrinsic rate of those, they’ll happen faster or slower and some regions mutate faster and some mutate slower, and understand that a population of cells that has, you know, 10 to the 9th cells has actually undergone almost 10 to the 9th generations of growth and so that’s a very large number and if you have 10 the 12th, almost 10 the 12th generations of growth and so almost any mutation that you can imagine will arise during the growth of that.

    The question is, is how soon does it arise and hence if it arises early, its frequency is going to be larger than if it arises late.

  11. DXer said

    It still seems surprising that the fact that it was a mixture of two strains would not have been sufficient initially to zero in on the flask. Dr. Ivins knew it was a mixture. Although the FBI focused on his email with Dr. Keim as to whether he would know the capability of genetic inquiry in this regard, certainly all of his email is relevant on the issue. He also knew it would have mutations in it. See 2002 patent. So why would he use a supply that is provably a mixture, and a unique mixture with mutants at that?

    From August 18 briefing of Science media –
    “QUESTION: I’m going to ask a clarification question on that. Christine Piggee with Local Chemistry, American Chemical Society.

    Are you saying that the mixture of different mutations of the same strain in the flask was an artifact of production?

    BACKGROUND OFFICIAL: Eventually could have been the case.

    QUESTION: So it was a mixture of substrains?

    BACKGROUND OFFICIAL: Correct. That’s right. So — but remember, the majority of the material is the wildtype AMES anthrax in that container and through various processes, you have the spore distinct mutation that appears in that flask.

    Now, we don’t know what the origin of the mutation is. We don’t know if it started all in one batch or if it started in three of the combined batches or in two of the combined batches. We don’t know. What we know is that RMR 1029 and its four genetic mutants within that container are uniquely RMR 1029. We will not find it in any of our others.”

  12. DXer said

    Dr. Worsham says the reason Ames was chosen for vaccine research was due to an article by Dr. Little in the early 1980s suggesting that it was good at overcoming/defeating vaccines.

  13. DXer said

    Patricia Worsham says that the Ames spores with the mutations she previously worked on were sent to repository and they would have been screened with all the others. What repository did she submit them to? Where was the repository kept? Dr. Keim’s lab? Where were they kept before being sent? Were they from flask 1029?

    With regard to Patricia Worsham’s talk about the use of red Congo agar and the resulting variants defective in spoliation upon repeated passages of overgrown cultures, Bruce Ivins seems to have known all about it.

    See Can. J. Microbiol. 45(1): 1–8 (1999) | doi:10.1139/cjm-45-1-1 | © 1999 NRC Canada
    Isolation of an asporogenic (spoOA) protective antigen-producing strain of Bacillus anthracis
    Patricia L. Worsham and Michele R. Sowers

    Bruce Ivins, in a patent filed with Dr. Worsham, wrote on March 2000:

    “This strain, ΔSterne-1(pPA102), was then subjected to Congo Red agar selection for mutants displaying an inability to bind the dye, a characteristic known to correlate with an asporogenic phenotype (Worsham, submitted).”

    Thus, while the process supports pointing to the Ivins’ flask, it seems to point not to Ivins but away from Ivins. He seems to have known about the mutants.

    Instead it seems to point to one of the 100-300 with access to the stream of isolates.

    The documentary evidence does not point to Ivins.

    http://www.anthraxandalqaeda.com

    Method of making a vaccine for anthrax
    United States Patent 6387665

    Abstract:
    A method of making a vaccine for anthracis that inolves a bacterial expression system and production and use of protective antigen (PA) against Bacillus anthracis. The PA immunogen is useful in a vaccine against human anthrax. The PA can be produced by an asporogenic organism which produces the desired antigen, which is then harvested from the supernatant.

    Inventors:
    Ivins, Bruce (Frederick, MD)
    Worsham, Patricia (Jefferson, MD)
    Friedlander, Arthur M. (Gaithersburg, MD)
    Farchaus, Joseph W. (Frederick, MD)
    Welkos, Susan L. (Frederick, MD)
    Application Number:
    09/520215
    Publication Date:
    05/14/2002

    1. A method of making a vaccine comprising: incorporating a protective antigen produced by recombinant asporogenic B. anthracis with a pharmaceutically acceptable carrier, wherein said recombinant asporogenic B. anthracis was isolated from a ΔSterne-1(pPA102) strain of bacteria and said recombinant asporogenic B. anthracis does not have the ability to bind a dye when grown on Congo Red Agar.

    2. The method of claim 1, wherein the recombinant asporogenic B. anthracis is B. Anthracis ΔSterne-1(pPA 102)CR4.

    3. The method of claim 1, wherein the vaccine is in the form of a suspension.

    4. The method of claim 1 wherein the vaccine is in the form of buffered suspension.

    5. The method of claim 1 wherein said carrier is an adjuvant.

    Description:
    FIELD OF THE INVENTION

    This invention relates to the bacterial expression system, production and use of protective antigen (PA) against Bacillus anthracis . The PA immunogen is useful in vaccine against human anthrax. The PA can be produced by an asporogenic organism which overproduces the desired antigen, which is then harvested from the supernatant.
    BACKGROUND OF THE INVENTION

    Bacillus anthracis is the etiologic agent responsible for anthrax, a disease often found in persons exposed to infected animals or their products. Persons particularly exposed to animals include veterinarians, laboratory technicians, ranchers and employees working with skin or hair of animals. The mode of entry into the body may be the skin or, when contaminated meat is eaten, the gastrointestinal tract. Inhaling of spores can cause inhalation anthrax, a disease that can be fatal. Vaccines against Bacillus anthracis have been available. Virulent strains of the organism produce two toxins and a poly-D-glutamic acid capsule which are coded for on two endogenous plasmids, pX01 and pX02, respectively. Loss of either of the plasmids results in an attenuated strain of reduced virulence, while loss of both results in an avirulent organism. The history of the USAMRIID Sterne strain of B. anthracis prior to 1981 is uncertain, though it is believed to be derived from the Sterne strain isolated at the Onderstpoort Research Laboratory in Pretoria, South Africa.

    In 1985 the Bacillus anthracis protective antigen (PA) gene was cloned into a plasmid (pUB110) resulting in the formation of a recombinant plasmid identified as pPA102, which was reported in the literature (Ivins and Welkos, Infection and Immunity , 54:537-542 (1986)). The production of vaccines lacking lethal factor was possible thereby. However, a primary problem remained, since the Bacillus anthracis formed spores. Once spores have formed, they persist in the environment for months and years. Once the laboratory environment contains such spores, it is very difficult to free the environment of the spores.
    ***
    DETAILED DESCRIPTION OF THE INVENTION

    ***
    This strain, ΔSterne-1(pPA102), was then subjected to Congo Red agar selection for mutants displaying an inability to bind the dye, a characteristic known to correlate with an asporogenic phenotype (Worsham, submitted). The selected isolate, now designated ΔSterne-1(pPA102)CR4 was further subcultured three times to insure that a single clone was isolated. This clone has served as the seed stock for all research and development of fermentation conditions, and purification of PA.

    Materials and Methods:
    Fermentation Conditions

    Media: FA medium was used for all plates and liquid cultures described here unless otherwise specified. FA medium consisted of 33 g/l tryptone (Difco), 20 g/l yeast extract (Difco), 2 g/l L-histidine, 8 g/l Na2HPO4, 7.4 g/l NaCl, 4 g/l KH2PO4 adjusted to pH 7.4 with NaOH.

    Precultures: A working stock of ΔSterne-1(pPA102)CR4 was prepared from the seed culture by streaking cells on an FA medium plate containing 40 μg/ml of kanamycin. A sweep from the confluent growth zone on plate was cultured one time in liquid FA medium supplemented with kanamycin 40 μg/ml to a final O.D. 600nm of 4.0. This culture was checked for purity by streaking on SBA plates, and diluted into multiple vials containing sterile 100% glycerol to a final glycerol concentration of 50% (v/v). These stocks were stored at −70° C. A single vial was removed at the start of each fermentation cycle and discarded after use. The defrosted cells were streaked onto FA plates containing 40 μg/ml kanamycin and incubated at least 16 hrs at 37° C. After 16 hrs the plated cells were used to inoculate 50 mls of FA medium supplemented with 40 μg/ml kanamycin in a 250 ml baffled-Erlenmeyer flask (Bellco Laboratories). The culture was incubated at 37° C. at 200 rpm for 6 hrs or until an O.D. 600nm of 4-6 was obtained. The cells were then subcultured into 50 mls of FA medium in an identical flask under identical conditions. After 6 hrs, or a culture O.D. 600nm of 6.2-6.5, a 1.6% (v/v) inoculum was transferred to 300 mls of PA medium supplemented with 40 μg/ml kanamycin in a 2 liter baffled Erlenmeyer and incubated at 37° C. at 200 rpm for 7 hrs, or until a final O.D. 600nm of 3.5-3.7 was achieved.

    Fermentation conditions: The fermentations described here were carried out using a New Brunswick Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and hemispherical bottom cooling dish. Four liters of FA medium were added to the vessel, which had been previously completely disassembled, scrubbed in a dilute Envirochem solution and autoclaved for 15 min after the addition of 4 liters of H2O. The polarographic DO 2 probe (Ingold) and pH probes (either liquid or gel filled, Ingold) were also inserted and all addition and sampling ports were sealed or clamped and wrapped in aluminum foil. Addition lines consisted of surgical grade autoclavable Tygon tubing (Thomas Scientific) and all lines were sealed with the exception of the condenser, which was left open to permit pressure release, but covered with aluminum foil. The vessel was autoclaved using a 10 min exposure time at 121° C. and removed from the autoclave as soon as sufficient cooling had occurred to allow opening of the autoclave. The vessel was then immediately connected to the fermentor unit and the condenser line was connected to a sterile liquid trap and 0.2μ capsule filter to avoid the introduction of contaminants during the cooling process. The vessel was then cooled to 37° C. using the fermentor driven temperature control and positive pressure was provided using compressed sterile filtered air. Once the vessel had cooled to 37° C. sterile filtered kanamycin was added to a final concentration of 40 μg/ml. The agitation was activated at 150 rpm and aeration was adjusted to 1-1.2 volume/volume/min (vvm) and antifoam C (DOW), that had been diluted 10-fold into H 2 O and autoclaved, was added to a final concentration of 200 ppm.

    • BugMaster said

      Note the FA media. Really rich stuff! 33 g/l typtone, and 20 g/l yeast extract.

      Lots of iron in a media containing 20 g/l yeast extract.

    • BugMaster said

      “Media: FA medium was used for all plates and liquid cultures described here unless otherwise specified. FA medium consisted of 33 g/l tryptone (Difco), 20 g/l yeast extract (Difco), 2 g/l L-histidine, 8 g/l Na2HPO4, 7.4 g/l NaCl, 4 g/l KH2PO4 adjusted to pH 7.4 with NaOH.”

      DXer,

      Have you located any other documents or other examples of this patent that describes the FA medium?

      Seems to me that almost everything here is present about 10 times the normal concentration. 53 g/l total yeast extract and tryptone? 10 g/l total phosphates, anhydrous at that?

      Perhaps this description refers to a 10X concentration of the media.

      As formulated, if you were to grow the RMR-1029 material on plates of this media, I would suspect you would get lots of debris and vegetative cells, and low amounts of spores (the media as formulated is too rich).

      And no sporulation in liquid.

      • DXer said

        Vaccination against anthrax with attenuated recombinant strains of Bacillus anthracis that … – ►nih.gov
        DL Burns, JP Barnard, AM Friedlander – Infection and immunity, 1999 – Am Soc Microbiol

        … Five-milliliter aliquots of FA medium (3.3% tryptone, 2% yeast extract [dialyzed
        overnight against water], 0.2% L-histidine, 0.8% Na 2 HPO 4 , 0.4% KH 2 PO 4 …

        A Mohawk Valley Family Portrait

      • BugMaster said

        Wow. That is a very rich media. Probably why the yeast extract had to be dialyzed overnight to remove excessive amounts of iron and other low molecular weight compounts.

        This media must have been formulated to avoid sporulation in case the asporous RPA-102 bug underwent any revertance. Also note the presence of histidine, which would stimulate any spores produced to germinate, also limiting the possiblity of any spore production (the desired product here being the protective antigen, which is a toxin that the RPA-102 vaccine is based on).

  14. DXer said

    Lew, if you could upload pretty powerpoints explaining Peter Weber’s group’s work with NanoSIMS, it would drive home how important the work is to the resolution of Amerithrax.

    Decoding the Origin Of A Bioagent

    https://www.llnl.gov/str/Sep06/Velsko.html

    Livermore’s NanoSIMS provides a spatial resolution of 50 nanometers, 20 times better than that of conventional SIMS. Its sensitivity is 50 times better. Weber’s team is hoping to gain insight into possible links between the chemical composition of microorganisms and how, when, and where they were produced. These studies might also help resolve some of the uncertainty that exists with regard to conclusions drawn from research using surrogates of lethal pathogens. Weber says, “Biological weapon agents can be produced using a variety of methods. In addition to the microorganism, there may be a matrix of agents intended to enhance delivery. Plus samples collected from different environments may contain contaminants from external material.”

    The team has also used a focused-ion- beam instrument to extract slices from targeted areas of a sample before analyzing the composition with NanoSIMS. Bacterial spores are composed of approximately 50 percent carbon, 15 percent nitrogen, 3 percent calcium, 1 percent phosphorus, and trace amounts of about 20 other elements. Chemically related elements can substitute for one another. For example, strontium and barium are related to calcium and can substitute for it. Substitutions may be signatures of certain growth or processing methods.

    The high resolution achievable with NanoSIMS allows Weber’s team to analyze not only individual spores but also the elemental distribution inside the spores. These analyses can reveal microstructural production signatures.

    • DXer said

      Boxer, Kraft and Weber, “Advances in Imaging Secondary Io Mass Spectrometry For Biological Samples,” Annual Review of Biophysics, June 2009
      http://www.stanford.edu/group/boxer/papers/paper263.pdf

    • Anonymous scientist said

      The most interesting thing about Weber’s presentation are the silicon concentrations in the reverse engineered spores. Even using a solution saturated with silica they could not get anywhere near the concentrations of silica as were found in the mailed spores – it was orders of magnitude less. Clearly they were using the wrong silicon species as an additive.

      Also Michael appeared to contradict his colleague Paul Kotula. Kotula had previously said out of 200 samples they anlyzed for the FBI no spores matched the silicon signature of the mailed spores. And yet yesterday Michael said there were spores in RMR-1030 with an identical silica signature – Si and O on the spore coat.

      Incidentally, the “looking for a mother cell” doesn’t really prove anything. A siliconzing monomer added post sporulation would easily penetrate the cell wall of the mother cell – just as it penetrates the exosporium.

      • BugMaster said

        Perhaps Dr. Michael’s better off as an archeologist.

        I think he has a way with “artifacts”.

      • BugMaster said

        “The FBI used Inductively Coupled Plasma mass spectrometry (ICP) to determine the silicon content of the Leahy spores.
        They admitted that they found the record breaking level of 1.45% silicon.”

        There’s your artifact!

      • BugMaster said

        O.K., what did they find in the spores from the NYC mailings?

      • BugMaster said

        I suppose the readings for the NYC material (using a different technique) were equally ridiculous, determining the silica to be between 15% and 40% of the total.

        Wait a minute. Isn’t that in fact what they found!?

      • DXer said

        And assuming they did, it would seem that they are developing a total body of evidence that will be very powerful — the different puzzle pieces are coming together to form a context for the “evidence” they gave to Sandia’s lab in May 2008. But I would need a scientist familiar with such things to explain whether the AFIP EDX is able to target the agent and accurately separate out the exogenous silica. That is heralded as an advantage of NanoSIMS. If not, then the difference in percentage weight between the two mailings is explainable simply by repeated centrifugation — with AFIP’s figures for silica including exogenous silica that then was removed by the centrifugation.

        Although the webcast is the best guide to what he said, Dr. Weber, as I recall, explains that his group’s method can be useful in understanding whether the same culture was used or whether there were two different growths. If you then take the “massive” silica (Anonymous Scientists’ phrase based on rumored figures) and then the Sandia puzzle piece relating to the location of the Silicon,

        and add the LLNL piece,

        it seems that they have a body of evidence that is shaping up nicely.

        It is to be expected that data and evidence has not yet been shared with the public. It is a confidential investigation. And giving the FBI the benefit of the doubt, it presumably will be stronger than their “evidence” of Dr. Ivins editing Wikipedia. The agents said that they knew him better even than his family. I’ll credit that –which is why it was startling they thought the Wikipedia editing was evidence of anything other than Wikipedia editing. They knew him really well and still came up with no evidence he was guilty of the mailings. They cited a 2005 interview rather than a 2001 interview. Why?

      • DXer said

        BugMaster,

        Quick. What were you doing October 5, 2005 in the early evening?

        They failed to rely on the best evidence in their affidavit. They relied on Dr. Ivins’ recollection of events four years earlier in a then-sealed affidavit for a search.

        If the earlier 2001 interview supported them or if the Lab Notebook 4010 supported them, why didn’t they cite it?

      • DXer said

        The EDX spectrum of silica is well known and has been well characterized. It consists of silicon and oxygen peaks in certain speficic ratios. SEM images are always obtained simultaneously with EDX spectra – then EDX images are also obtained. SEM images are composed of electrons scattering from the surface of a sample. EDX images are composed of x-ray peaks from the surface of a sample.

        Any analytical lab who uses SEM/EDX to analyze for the presence of silica would do the following:

        (1) Obtain the SEM image of the sample – say an anthrax spore containing features of an unkown origin on the surface.

        (2) Place the electron beam on one of the features and measure it’s EDX spectrum.

        (3) Match the EDX spectrum obtained with a known reference spectrum (in this case silica). Make sure the EDX spectrum exactly matches the reference spectrum in peak ratio and peak shape.

        (4) Turn the SEM to EDX image mode then (a) Run a EDX image of silicon x-rays, (b) run a EDX image of oxygen x-rays.

        (5) Superimpose the original SEM image with EDX images 4a and 4b – check that all the Si and O signals originate from the same spots and coincide with the original unknown features.

        This is very standard SEM/EDX analysis – it can be done in a few minutes especially with the nifty software available on modern SEM/EDX tools.

        There is no reason to think the AFIP operator Frank, now deceased, was in error. He was highly expert in detecting silica and was interviewed by a family friend of mine. But are there limitations in this regard of the equipment available to him relative to NanoSIMS? I’ll wait to hear from Anonymous Scientist on that before researching the literature or asking Dr. Weber.

        Peter Weber says he was startled at the figures relating to the bulk analysis that he was hearing for the first time. (I would have to listen to the webcast again to recall the context).

        As I recall the webcast, Dr. JM from Sandia heard the AFIP figures for the first time last Winter at a meeting.

        Maybe the FBI just hasn’t played that card yet. And maybe when the high percentage silicon weight (Si and O signal co-located in a way pointing to silica) is revealed, the process used will be indicated. … a process by which excess silica is removed by repeated centrifugation. (See Microdroplet Cell Culture Technique that uses silica in the culture medium to concentrate anthrax and then in a second step the excess silica is removed through repeated centrifugation.)

        A Spring 2008 report said that the suspects had been narrowed to four — a leading anthrax scientist, a former deputy USAMRIID Commander, a microbiologist and perhaps one other. For all we know, under the FBI’s theory from Spring 2008, Dr. Ivins will turn out to be the “possibly one other.” I’m on record saying I have no basis to dispute the distinguished law professor/defense counsel who says that his client, who was coordinating with the 911 imam and was in the suite with the leading anthrax scientist and a former deputy USAMRIID commander, is an anthrax weapons suspect. The suite members co-invented the process that involved removing the silica used to concentrate the anthrax by repeated centrifugation. The international patent shows that it is also useful in aerosols, such as an inhaled vaccine.

        Although I am not a scientist, that is what I liked so much about Weber’s presentation. He did not seem to draw conclusions or implications that went beyond his data. He was just offering up the puzzle piece.

        The NAS last week should have had a witness address the AFIP data.

      • DXer said

        It’s now been about 6 years that Dr. Alibek told me in a telephone conversation that the FBI suspected Dr. Al-Timimi of being involved.

      • DXer said

        As I vaguely recall, the chemical identified by Anonymous Scientist as having been detected is used in making Aerogel which as I recall was first commercially available in 2001. Mostly air. As a layperson, my impression was that silica off-the-shelf had just gone nano in 2001. So Dr. Weber could be asked if he used the best silica nanopowder available in 2001.

      • Anonymous scientist said

        DXer wrote:

        “Dr. Weber found grew spores with up to 4% silicon.”

        This is not the case – if you go back and re-listen he said the highest percent silicon they got in any spore was 0.4%. They tried extensive reverse-engineering and the silicon content of the spores varied from 20ppm (0.00002%) to 4000ppm (0.4%).

        When asked if he analyzed samples containing as much silicon as the mailed spores he said “no, we never got anywhere close to that”.

      • DXer said

        Thanks! I’ll go re-listen.

      • DXer said

        Anonymous Scientist is correct. I had heard 4% but he said .4%.

        And so it appears that the clear result is that given that there a direct relationship between iron and silicon, you don’t get the levels observed with just G Media being the source of the iron. The implication seems to be that there needs to be added iron. I don’t see that this study tested that.

        I wouldn’t know sheep blood agar if it was put in my cereal and so let me just correct my mistaken reporting of the percentages. Lew could you correct it so that no one reading just that post is misled?

  15. DXer said

    [audio src="http://www.nationalacademies.org/podcast/20090925part2.MP3" /]

    Nano-scale secondary ion mass spectroscopy, Peter Weber, Lawrence Livermore National Laboratory

    In what I think is a very important talk, there was one scientist who spoke of his effort to manipulate the amount of silicon grown into the spores.

    It would take a scientist like Dr. Kiel or Dr. Popov to explain this to me and I’m sure only that there will be mistakes in my rough notes below.

    There is a very close relation between iron and silica. Previously, I’ve noted the articles as they came out at the time that it appeared they believed heme was in the culture medium. But the clear implication of this talk is that it points to silica in the culture medium. The scientist points out that there is a natural increased tendency for the silicon to be absorbed due to hydroferric oxide in particular.

    This talk seems to be very important. He explains that the silica appears to be following the iron.

    resulted in up to about 3% weight consistent with absorption of silica in the culture medium due to hydroferric oxide.

    They looked at 57 spore samples. They studied Ames, Sterne, bt, bg, cereus (from 1980 paper).

    Iron must be particulate, colloidal or chelated. Why the iron becomes absorbed into spores is unclear. But then silica follows the iron into the spore.

    His method can detect silica down to 10 ppm so they see it everywhere. Dissolved silica is ubiquitous. Even in deonized water at ppm level. 2-30 ppm per liter as silica. At high end, you can only get up to 70 ppm. So dynamic range is small.

    They studied about 10 spores per sample. There was significant variability between individual spores (about 25%).

    They were manipulating the medium… adding silicon, adding iron, …

    Considered three mechanisms.

    First, a passive model… sponge… left behind during drying… can only get up to 100 ppm in spore.

    Second, primary model is absorption model… to include metal oxide … specifically… hydroferric oxide
    up to 1800 ppm.

    Third, precipitation… not as conclusive.

    Two media.. G media and nutrient broth.

    Two levels of silicon… second was dissolved Cabosil.

    heat shocking, various washing, drying… no interesting effect…

    including washing with a detergent that contained silica.

    Post-production aqueous silica solution experiments.

    Adding silica to G media has an effect.

    If you look at the same treatment in nutrient broth, no relationship.

    Follows geomicrobiology (?) literature.

    Pretty direct relationship between iron and silica.

    So, putting it lay terms, I can try to understand — THE SILICA WAS IN THE CULTURE MEDIUM, LIKE THE WMD CHIEF SAID, AND WAS ABSORBED BECAUSE OF THE USE OF IRON IN EITHER A PARTICULATE, COLLOIDAL OR CHELATED FORM.

    I’ll pull up the literature and listen to it again and try to improve on my accuracy of his explanation.

    I think this talk really advances things (for those who were unwilling to take the plain statement of the WMD Chief at the August 18 roundtable and process it.

    The FBI rocks!

  16. DXer said

    Joseph Michael says that they found tin and iron in weaponized samples they tested (which lacked the “Silicon Signature”).

    The FBI has suggested that the iron and tin may have come from a water source.

    Okay, so what water supplies contain high levels of iron and tin?

    More Anthrax Revelations Emerge | GroundReport
    Mar 5, 2009 … Bannan suggests that the growth medium may have contained iron and tin may have come from a water source. The meeting offered scientists who …
    http://www.groundreport.com/…/More-Anthrax-Revelations-Emerge

    • DXer said

      Don’t they want to hear a tin expert?

      Was the stuff stored in a tin can?

      Agency finds high levels of tin in Heinz canned spaghetti in tomato…
      Free with registration – M2 Presswire – AccessMyLibrary.com – Feb 1, 2001
      High levels of tin have been found by the Food Standards Agency, during a survey of canned fruit and vegetable products. The survey identified a problem …

    • DXer said

      In “Immunomagnetic-Electrochemilumenscent Detection of Bacillus anthrax Spores in Soil Matrices,” Applied and Environmental Microbiology, Sept. 1996, p. 3474-3476, involved Ames supplied by John Ezzell. This study was published the same year John tells me he supplied aerosolized Ames to DARPA. One soil was a moist dark brown to black soil (soil 1) and the other was a dry light yellowish sandy soil (soil 2) take from two different locations in the vicinity of Aberdeen Proving Ground.

      It doesn’t seem that the NAS is yet placing sufficient emphasis on understanding the “Tin Signature.” Might irradiation have been unsuccessful as it was in repeated instances in 2001? TK in Texas had a $100,000 grant from the CIA studying the persistence of anthrax in soil and later published on the general subject. Was there something about genetic exchange in a soil suspension that researchers wanted to take advantage of in seeking to develop a more effective vaccine?

      Virginia Tech has a lab experienced at testing soil suspensions.
      http://pubs.ext.vt.edu/452/452-881/452-881.html

    • DXer said

      Homeland Security Department funded this study published by Joe and Paul in 2008.

      Guess where the sample with the tin was made?

      Guess the source of the tin/iron?

      Forensic analysis of bioagents by X-ray and TOF-SIMS hyperspectral imaging star, open ….. This work was supported by Department of Homeland Security. …
      linkinghub.elsevier.com/retrieve/pii/S0379073808001734

  17. DXer said

    As to the suggestion that antifoam was used, Anonymous Scientist should support his claim that antifoam was used given it is not mentioned in the 1980 article.

    On antifoam issue, I was struck by Dr. M’s sentence, as I best recall it, “I think we looked at a lot of samples with antifoam and sometimes they mentioned…”

    Although a layperson, it occurs to me that the relevant witness is the one who can explain controlled studies using antifoam or not etc. and the resulting Si and O signature. Not someone saying “sometimes they mentioned”…

  18. DXer said

    “We can argue what ‘natural’ means all day long.” — Joe Michael

    Instead, it would increase clarity if the term, when used, is defined.

  19. DXer said

    Tom Geisbert said that there mother cells in the process of forming the spore and sent JM on a hunt to find one.

    Even then we see the Si and O signature on that spore.

    This points to use of silica in the growth medium, right? like the WMD Chief said? See Microdroplet Cell Culture Technique.

    Sometimes it would be mentioned to Sandia whether antifoam was used in preparing a sample and they couldn’t correlate that sort of appearance with use or not of antifoam.

    “We were dependent on what the FBI gave us to look at.” (and apparently what they casually happened to mention or happened to volunteer)

    He said that there was sample of “evidence” that was close that he urged the panel to probe.

    As I’ve suggested, Dr. Ivins wrote Patricia Fellows that he heard from [I’ll redact the name but later will name names] that the closest product was made by FBI anthrax expert John Ezzell, who tells me he aerosolized Ames at the request of DARPA.

    There was one sample that he urges the NAS panel “to pull the string on.” I think he said it went from 18% in silica content down to 1% or no percent.

    He doesn’t know the source of the series but says it would be interesting to understand the growth conditions.

    • Anonymous Scientist said

      DXer wrote:
      “Tom Geisbert said that there mother cells in the process of forming the spore and sent JM on a hunt to find one.

      Even then we see the Si and O signature on that spore.

      This points to use of silica in the growth medium, right? like the WMD Chief said? See Microdroplet Cell Culture Technique.”

      This is not necesssarily true at all. Post sporulation treatment with a polymerized glass monomer could also create a coating of polymerized SiOx on spores inside the mother cell. The polymerized glass monomer is easily capable of penetrating the cell wall as well as the exosporium. This has been demonstrated in peer reviewed scientific papers on treatment of cellular material with siliconizing agents.

      The bottom line is – PROPER reverse engineering needs to be demonstrated. These people have had eight years to reverse engineer the spores. They failed. And their pitiful answer to that failure is “oh well, reverse engineering wasn’t that important after all”. The real situation is that they DO NOT UNDERSTAND HOW THE POWDER WAS MADE.

      It needs to be rigourously demonstrated exactly what spores treated with polymerized glass look like. If that has not been done, it needs to be done and it all needs to be openly discussed.

    • DXer said

      He says if you understand the growth conditions you could understand the reason for the silica in the spore coat. Rather than just say si and o signature, he stops and expressly says silica. He is talking about silica here, not merely silicon.

      John Ezzell provided Edgewood Ames in a soil suspension. Would a soil suspension explain the “Silicon Signature?”

    • DXer said

      He was given the “evidence” in May 2008.

    • BugMaster said

      “Tom Geisbert said that there mother cells in the process of forming the spore and sent JM on a hunt to find one.”

      Michael reported that he spent three days searching for this elusive “evidence” before he found what he was looking for.

      And we are all to accept the conclusions based on such an astonishing finding (by a non-microbiologist).

      This is really strange! If such a finding is so significant, how come it took three days to come up with this “forgone conclusion”?

      • DXer said

        Toward the end, did I hear JM say that aerosolized virulent Ames was not used in experiments? If so, to the contrary, as the Dugway scientist told NPR in 2001, it was sometimes used, for example, to test the kill rate of decontamination agents.

        In the 2008 study funded by Homeland Security Department by Dr. Kotula, Michael and others, they explain hyperspectral imaging. The presence of elements in the silicates is most likely due to overlapping interaction volume between the particulates and the spore matrix. The samples used were selected from archives of simulated agents produced by a variety of processing agents. In one picture, the green represented the silica particles and the blue compoent was composed of Mg-like signature. My vague, lay understanding of things is that in the 2008 Homeland Security funded article, the authors are suggesting that a Fe/Sn spike could have resulted from a stainless steel particle. I believe the authors further are urging that hyperspectral imaging is more sensitive and might be able to distinguish between different sources of stainless steel. In other words, Dr. Popov’s hypothetical possible explanation of the Tin Signature is strengthened. But I am still interested in whether a soil suspension of Ames might result in such a spike. There was considerable use of soil suspensions and one of the leading scientists said that the mutations may have resulted precisely because of treatment it underwent. (I don’t have his phrasing; I think it might have been Dr. Budowie). Lab Notebook 4010 likely would be huge in putting all this in context.

        As a layperson, I still don’t understand how someone who has never aerosolized anthrax can do more than get the runner on first base and identify the location of the silicon.

  20. DXer said

    He refers to silica being on the outside as the “traditional” method of weaponization.

    Is the thought the FBI should only protect us against attacks done in the conventional manner? See 9/11.

    • DXer said

      Somlyo’s widow, his co-author, reportedly says the AFIP results disclosed to her were completely different than what that Somlyo paper showed.

      Stewart M, Somlyo AP, Somlyo AV, Shuman H, Lindsay JA, Murrel WG. “Distribution of calcium and other elements in cryosectioned Bacillus cereus T-spores, determined by high-resolution scanning electron-probe X-Ray microanalysis.” J Bacteriol, 1980 vol. 143(l):481-491.

      Stewart M, Somlyo AP, Somlyo AV, Shuman H, Lindsay JA, Murrel WG. “Scanning electron probe microanalysis óf elemental distributions in freeze-dried cryosections of Bacíllus coagulans spores.” J Bacteriol, 1981 vol. l47(2):670-674.

  21. DXer said

    Here is the link for Joe’s presentation on weaponization.

    [audio src="http://www.nationalacademies.org/podcast/20090925part1.MP3" /]

    Query: Why wouldn’t the panel ask to hear from an expert on weaponizaton of anthrax — someone who has aerosolized anthrax simulants using different methods, in testing in a controlled way the methods that result in a comparable silicon spike. I recommend Dr. Kiel whose lab has done such research and is independent of the FBI.

    • DXer said

      He says in a different study they did they have seen a spike for tin. There the “tin” peak was not associated with the Si and O peaks.

      But Anonymous Scientist, we should find the other study he mentions that they did for separate purposes to see the reason for the tin (or at least the labs).

      The journal Nature summarized the background of the tin issue:

      “At a biodefence meeting on 24 February, Joseph Michael, a materials scientist at Sandia National Laboratories in Albuquerque, New Mexico, presented analyses of three letters sent to the New York Post and to the offices of Senators Tom Daschle and Patrick Leahy. Spores from two of those show a distinct chemical signature that includes silicon, oxygen, iron, and tin; the third letter had silicon, oxygen, iron and possibly also tin, says Michael.”

      Ivins flask did not contain tin. There was no iron or oxygen or silicon or tin detected in the spore coat of those spores.Dr. Michael speculated it might have been in the water. But Ft. Detrick water did not have high levels of tin.

      Former Russian bioweapons expert Sergeui Popov comments:

      “Although the tin and iron may have come from the water used for cultivation, their amount, in my opinion, far exceeds the levels commonly present in the water used in a laboratory. Another possibility to consider is that the suspect used a primitive but a sturdy and a widely-available container to dry the spores, namely a tin can. It would explain a simultaneous presence of both elements. This suggestion is easy to test in experiments.”

      Dr. Popov reports: “I don’t remember the exact levels from the presentation, but it spikes out like hell.”

      Dr. Popov walks me through the scenario:

      “Let’s do a hypothetical spore prep in the simplest way and try to suggest something to prepare a dry powder. You cultivate the bacteria and end up with a wet paste (doesn’t matter if you used a fermenter, flasks, or plates). Now, you have to dry it and make it dispersable. The lyophilization is out of question (slow, unproductive, visible, requires equipment, generates powder in the flasks with narrow necks, difficult to dispose or decontaminate the flasks). Plastic is out of question either (disposable, but not heat resistant, comes mainly as awkwardly capped tubes or jars with poorly sealed lids). A tin is perfect, unbreakable, easy to seal and heat up (if necessary). Many of them are lying around. You first try a slow evaporation with a low heat. In order to agitate a dry residue you can use a spatula, then put the powder in the envelope. It doesn’t work good, the brown powder is too coarse (Florida anthrax). A desiccation is quiet, can be done in the same container, simple, does not produce any contamination, and produces better powder. Just open the lid, put a can into a bigger jar and forget about it for a few days. Then put a few glass beads into the container, close the lid. Shake it and the beads will make a powder. No contamination, no aerosols, everything in the same container, and no traces.”

      Even those “tin cans” nowadays made of aluminum or steel are commonly plated with a thin layer of tin. For example, some Heinz products in 2001 involved contamination of a spaghetti product in tomato sauce with high levels of tin.

      Sergei first described use of such a tin container in September 2008 in discussing the possible drying method used and Professor Rosenberg’s opinion addressing the drying method.

      “Prof. Rosenberg

      One of those methods, azeotropic drying, was used recently at Dugway-the only US laboratory that has admitted to making dry anthrax—for drying pelleted anthrax spores that were intended to simulate closely the spores used in the attack. The azeotropic method used is “proprietary.” Bill Patrick said in 1996 that he had taught Dugway to use an azeotropic drying method developed at Fort Detrick in the 1950’s. […] Knowledge of appropriate azeotropic drying methods is esoteric, and methods developed for anthrax spores are classified. Expertise and experience of this sort is another discriminator that could be used to screen the list of 100+ potential suspects.

      Comment

      It is nice that the method have been classified, but the knowledge cannot be a discriminator of guilt. Scientists can do things right without a direct knowledge but with a sufficient background. My internet search turned out several articles on ambient drying without heating, based on the properties of the water-organic emulsions. Drying is a key to the successful preparation of the spore powder, and a poor quality of Florida anthrax demonstrates that the person made several small-batch attempts to discover an appropriate procedure. Therefore, we may conclude that the person experimented without a preliminary knowledge of a previously established protocol. In my opinion, the requirement to conceal the experiments was extremely important for the perpetrator in the choice of cultivation approaches as well as the drying procedure. The perpetrator had to use minimal amount of medium and equipment; produce minimal amount of suspicious waste, including the organic solvents. Ideally, the cultivation, sporulation, as well as drying would be performed in the containers routinely used in the lab, which could be unsuspiciously dumped into trash for autoclaving.

      Here is the most interesting part, but you don’t have to take it seriously. During my internet research, I also came across a procedure for the preparation of fungal spores. The author described how to buy all necessary equipment at Wal-Mart. He used a desiccant in a small jar to dry the spores. Here is a hypothetical scenario based on my experience (please, don’t consider it as an indication of my guilt). Take a small amount of spores and inoculate several agar plates (this is a part of a microbiological routine). Nobody will notice the “missing” amount of bacteria. Forget about the plates for several days in order to let the culture grow and sporulate. If somebody finds the plates and asks questions, say “sorry, I forgot to dispose of them” (it happens all the time). If not, scrape the spores, which are now almost “theoretically” pure, and put a paste into an unsuspicious container. It may be an empty vial left after a used reagent. Plenty are lying around. Attach a cap loosely and put a vial into a bigger container with some desiccant in it. These tin or plastic containers come with many reagents and protect the content from moisture. Close the lid and put the container aside, and it will look like somebody forgot to dispose of it. Again, if somebody asks, say it is trash. Two days later, stay late in the lab, take the vial out, use a spatula to disperse the dry stuff, and put it into an envelope. Next time, you may try to add a drop of silicate to the spore paste before drying. All these procedures will take minutes to accomplish. Everything is disposable, and there will be no traces left behind. Now, try to criticize this scenario, and maybe we will get a little bit closer to what really had taken place.”

  22. BugMaster said

    From Ed’s website today:

    “A couple comments are worth noting. The first confirms that some of the evidence pointing to Dr. Bruce Ivins’ is still considered confidential, and will be considered so until the case is offically closed :

    [Patricia] Worsham, who works in USAMRIID’s bacteriology division, said she was prevented by a federal gag order from talking about certain aspects of the case involving Ivins. Instead, she presented how the lab identified morphological variants to identify the strain used in the attack.”

    Ed:

    Worsham reported that she was prevented from discussing certain ASPECTS of the case.

    So where do you come up with “evidence pointing to Dr. Bruce Ivins’ is still considered confidential”?

    “Certain aspects of the case” does not neccessarily mean “confidential evidence”.

    • DXer said

      One aspect of the case is that Dr. Ivins mapquested a route to a female co-workers home. The concern was that he was angry at her. That would be an example of information that is confidential but not probative. One’s anger would be greatest when the claim is thought to be false or malicious. The identity of the female co-worker has not been disclosed.

      Henry Heine is under a gag order and he will come forward to say that an Ivins Theory is a provable and total crock.

      Does it violate the First Amendment of people to come forward (under Section 1983) to gag them from coming forward to defend a co-worker who they think is being falsely accused by the Government?

    • DXer said

      What other “aspects of the case involving Ivins” are there which are under a gag order?

      There are several:

      1) One is that the anthrax deemed closest to the anthrax was made in the late 1990s by FBI anthrax expert John Ezzell who tells he made aerosolized Ames at the request for DARPA.

      2) Bruce Ivins supplied virulent Ames to former Zawahiri associate Tarek Hamouda.

      http://www.anthraxandalqaeda.com

      3) Dr. Ivins’ colleague Patricia Fellows worked on a project involving making Ames more lethal by copying of the virulence plasmids. It was published by Pamala Coker, who along with FBI genetics expert and evidence custodian Kimothy Smith, was thanked by former Zawahiri associate Tarek Hamouda.

      4) Dr. Ivins’ Lab Notebook and polygraph would be the best evidence of how he spent his time in the lab and the reason for the Silicon Signature in flask 1030. Not his best recollection many years later in an interview.

      All these tend to be exculpatory which is why Henry Heine, who is also under a gag order, says he will be in a position to explain that the FBI’s Ivins Theory is a total crock once freed by the gag order.

    • BugMaster said

      Ed:

      The gag order could pertain to confidential evidence against Ivins. It could also pertain to other aspects of the case that have nothing to do with the “evidence” against Ivins. Exculpatory evidence, perhaps? Information that does NOT back up the government’s case against Ivins?

      I can think of at least two aspects of the case the government would’t want discussed. (And I’m not going to discuss them).

      The fact is, the gag order is not “confirmation” of anything here, except for the fact that there was a gag order.

  23. DXer said

    Here is a link to the audio from yesterday. I would say that’s pretty prompt service. Kudos to the NAS staff for working so hard on such a wide variety of important matters involving dozens of scientists.

    http://nationalacademies.org/newsroom/nalerts/20090925.html

    • DXer said

      Paul Keim (part 4) says the culture from Stevens’ spinal fluid came about 8 p.m. on a Thursday night.

      By the next morning they were able to call the CDC and FBI and be able to identify that it was the Ames strain.

      They were aware that what they did would have to be admissible in Court under Daubert and were constantly in contact with the FBI.

      He says at the time they thought the Ames strain was from Ames, Iowa.

      Question: What date was this Thursday night?

      What date was the Ames inventory destroyed?

      Who did the FBI contact? (Dr. PK elsewhere has said he was not consulted).

      My intelligence source says that the FBI did not provide the NAS regarding the destruction of the inventory at Ames.

      US Postal Inspector Richter, if I am remembering the name correctly, has said that Ames in fact did have Ames.

      What was the date of the destruction of the inventory relative to the prompt identification of the strain as Ames?

      The Coen/Nadler book reports/claims without citation that the USDA inventory at the strip mall was also destroyed at the same time as the ISU inventory. Upon emailed inquiry, no support for the claim was provided by the authors.

    • DXer said

      Dr. Keim’s lab became one of the repositories of 2,000 pieces of evidence in their BL-3 lab — all under a chain of custody.

      So the first question relating to the sample alleged to be false is: Who was in the chain of custody?

      The question relating to the destruction of the Ivins sample sent in the wrong format is: Who was in the chain of custody?

    • DXer said

      Dr. Keim says he has given 120 talks on these issues and scientists have had the question him –important for purposes of Daubert.

      Yet, no answer has been provided as to who the FBI consulted when asked if it was okay to destroy the inventory in Ames. It’s a simple question with a simple answer and now is the time to answer it. (It wasn’t PK; was ANY genetics consultant asked if it was okay. If not, why not? They were on board from the start).

      PK says his big problem with the FBI was funding. “Patriotism only goes so far when you need to do things like that” (put gas in your car).

      (The criticism related to the funding issue, not being a repository for evidence). Funding came from the DOE.

    • DXer said

      He says researchers did not send them their collections — they destroyed them. He says “I’ll name names later.”

      We know who the researchers involved in the destruction are at Ames. What we don’t know is who, if anyone, the FBI consulted about it being okay to destroy the inventory.

  24. DXer said

    Pat Worsham said “Ames had a good history.” “It had not been passed through the laboratory a great deal.”

    Dr. Keim said he had criticism for the FBI, which used his lab as a repository for evidentiary samples.

    That means Dr. Keim’s colleague, Kimothy Smith, who made available the space at LSU for the former Zawahiri associate, Tarek Hamouda, to do research with virulent Ames, had custody of the evidentiary samples. (Dr. Hamouda thanked Bruce Ivins for supplying virulent Ames, Dr. K. Smith for supplying the space, and Dr. Ivins’ colleague Patricia Fellows for technical assistance. (See literature and numerous patents.) Bruce emailed Patricia about how the aerosol created by FBI anthrax expert JE most closely matched the mailed anthrax). See Fox News 2008 report.

    http://www.anthraxandalqaeda.com

    Even presuming everyone’s good faith in all things (as I do), any case against the crazy dead guy was a mess from the start. There is no just evidentiary basis disclosed yet warranting not presuming Dr. Ivins’ innocence and good faith. If I were the FBI, I wouldn’t let the NAS release documents or webcast live either.

    The documentary evidence points to Zawahiri’s plan to recruit scientists at universities to aerosolize anthrax for use against US targets — not any plan by Dr. Ivins. The documentary evidence against Dr. Ivins relates to his accurate editing of a Wikipedia entry involving sororities.

    Now the science is being guided by the scientist, Dr. Bannan, formerly a collections scientist at the bacteriology division of the American Type Culture Collection. ATCC was the sponsor of the program of Ali Al-Timimi, of the Ann Arbor charity IANA, who shared a suite with the leading anthrax scientist Alibek and former deputy USAMRIID Commander Bailey, who co-invented the process to concentrate anthrax using silica in the culture medium.

    The ongoing briefing involving Dr. Al-Timimi is highly classified — the federal district judge is not allowed assistance by his clerk and the defense counsel was not privy to the US DOJ’s briefing. His counsel says his client is an “anthrax weapons suspect.”

    I simply have no reason to doubt distinguished defense counsel.

    • DXer said

      As I recall the case against Dr. Ivins, he allegedly submitted the first sample in the wrong format. Now what did the FBI do? They destroyed the sample. Who made that decision? Who destroyed the sample?

      Then the FBI claimed that what he submitted was a false sample. He denied it. Who determined it was a false sample? Who was in the chain of custody?

      It was found later that a what he had submitted originally to Dr. Keim for the genetics study was an identical match.

      This is the sort of issue that the NAS panel needs to focus on. It relates more broadly to the issue of contamination which is part of the assigned task.

      • DXer said

        Turning back to the GAO Report issued on Monday about Dr. Ivins, here is a passage that describes additional background on the contamination issue insofar as it related to the lab at the USAMRIID Bacteriology Division and Dr. Ivins assistance with the investigation:

        “Immediately following the anthrax mailings in 2001, FBI took contaminated evidence to USAMRIID for analysis. Dr. Ivins was tasked by USAMRIID management to analyze the samples of spores sent through the mail and was also a technical consultant to the FBI in the early months of investigation. In March 2003, Dr. Ivins and two of his colleagues at USAMRIID received the Decoration for Exceptional Civilian Service–the highest award given to DOD civilian employees–for helping solve technical problems in the manufacturing of licensed anthrax vaccine.

        In December 2001, one of Dr. Ivins` coworkers told Dr. Ivins that she observed on several occasions unsafe handling procedures by Diagnostic System Division personnel. She also told him that she might have been exposed to anthrax spores when handling an anthrax-contaminated letter. Dr. Ivins began sampling areas in the laboratory space that might have been contaminated with anthrax. He took samples from the shared office areas and later decontaminated her desk, computer, keypad, and monitor. However, he neither documented this incident in the Army record log book nor notified his superiors. He later acknowledged to Army officials that this was a violation of protocol. Dr. Ivins` behavior was detailed in an Army investigation[Footnote 40] conducted in response to a second round of sampling he conducted in April, but his name did not surface at that time as a suspect in the anthrax attacks.

        After a spill incident inside of suite B-3 in building 1425 in April 2002, Dr. Ivins conducted a second round of unauthorized sampling of his shared office space and cold side areas outside of suite B-3. These findings were reported and sparked a buildingwide sampling inspection. An inspection conducted by the Army 7 months after the anthrax mailing found that suite B-3 in building 1425 at USAMRIID was contaminated with anthrax in four rooms of suite B-3 (306, 304, cold room, and 313 (Dr. Ivins`s laboratory)) and that the bacteria had escaped from secure to unprotected areas in the building. All the areas outside of suite B-3 that tested positive were associated with Dr. Ivins and members of the Bacteriology Division. The inspection report stated that “safety procedures at the facility and in individual laboratories were lax and inadequately documented; that safety supervision sometimes was carried out by junior personnel with inadequate training; and that exposures of dangerous bacteria at the laboratory, including anthrax, had not been adequately reported.“ (See appendix V for additional information on the U.S. Army`s requirements for high-containment laboratories at the time of the 2001 anthrax incidents.)”

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