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

* Bruce Ivins experienced repeated irradiation failures with virulent Ames anthrax

Posted by DXer on June 13, 2015



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8 Responses to “* Bruce Ivins experienced repeated irradiation failures with virulent Ames anthrax”

  1. DXer said

    U.S. Army lab faulted over lax anthrax procedure

    Techniques used by a U.S. Army laboratory in Utah failed to neutralize live anthrax spores on many occasions over more than a decade and the lab should have realized the procedure was inadequate, a top health official told lawmakers on Tuesday.

    Despite warning signs, the lab, at the Dugway Proving Ground, persisted with the same process for inactivating anthrax samples for researchers, resulting in the unintentional shipment of live spores of the deadly bacteria to 192 labs in the United States and abroad, officials said.

    “This hearing is astounding, honestly,” Representative Larry Bucshon, a heart surgeon, told witnesses during testimony on an investigation into the shipments. “This is anthrax. We should have had policies for decades. It’s ridiculous.”

  2. DXer said

    If they developed INEFFECTIVE procedures in the past 10 years, what did they have before that?


    “Review Committee Report: Inadvertent Shipment of Live Bacillus anthraces spores by DoD,” July 13, 2015

    “The committee studied the irradiation and viability testing procedures used at DPG and noted that the amount of solution extracted from the inactivated sample, to be tested for viability, was the lowest amongst DoD laboratories (5% of the sample is used for viability testing). Additionally, within this production facility, DPG samples experienced a very short time period between the completed irradiation cycle and start of the viability testing. The confluence of large production quantities associated with the DPG, low sampling volume of the inactivated material for viability testing, and a very short time period between the completed irradiation cycle and start of the viability testing may have exacerbated the likelihood of not properly identifying live BA spores in inactivated samples. The development and implementation of ineffective irradiation and viability testing procedures took place over the last decade; this represents an institutional problem at DPG…. “

  3. DXer said

    Results of investigation into US anthrax testing to come within a week
    Posted on : Jul.16,2015 17:10 KSTModified on : Jul.16,2015 17:10 KST

    The US military is reportedly assessing the situation, paying heed to the possibility that an unexpected mutation may have occurred while the anthrax bacteria was being deactivated before shipment.

    “Generally, anthrax is deactivated before shipping by subjecting it to radiation. The US military is saying that some of the anthrax that was shipped in April was still live even after being radiated. The US suspects that the anthrax may have developed resistance to radiation,” the source added.

    • DXer said

      The issue of resistance came up, for example, in connection with the Amerithrax investigation in the context of the silicon signature and silicon encapsulation. One study involved acid resistance. The study concluded that the anthrax spores were harvested from culture on a silicate-containing medium.

      See, e.g., The Silicon Layer Supports Acid Resistance of Bacillus cereus Spores􏰀 Ryuichi Hirota,¶ Yumehiro Hata,¶ Takeshi Ikeda, Takenori Ishida, and Akio Kuroda*
      Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan
      Received 21 July 2009/Accepted 20 October 2009

      “When the anthrax powder sent to the U.S. Senate in 2001 was found to be coated with unusual silica, it was discussed whether the silica was related to spore dispersion. We concluded that Si encapsulation is not sufficient to make spores dispersible but does contribute to survival under acidic conditions. Our findings also strongly indicate that the anthrax spores were harvested from culture on a silicate-containing medium.”

    • DXer said

      Through the Looking Glass: Silicate in Bacterial Spores
      by Peter Setlow

      Amid the furor surrounding the anthrax attacks in the USA in 2001, significant attention was focused on whether the Bacillus anthracis spores used had been “weaponized” by the adding of a coating of fumed silica to aid in their dispersal. This information might have helped pinpoint where the spores had originated. However, a confusing factor was that for more than 20 years, significant levels of silicon had been reported in spores of at least some Bacillus species, including those of Bacillus cereus, a close relative of B. anthracis. Clearly, the natural presence of silicon in B. anthracis spores makes discrimination between weaponized and non-weaponized spores more difficult. However, in the older reports the silicon was not localized at the outermost surface of the spores, as would be expected if they had been artificially spiked with such compounds.

      Anyhow, what is silicon doing on a bacterial spore? A recent paper provides some clues. Starting with soil from a paddy field, these researchers identified a number of novel bacterial isolates that efficiently take up silicate present in media at concentrations comparable to levels found in the water in soils. Strikingly, all of these isolates were Bacillus species and most were members of the B. cereus group that includes the pathogens B. anthracis, B. cereus, and Bacillus thuringiensis. Silicate uptake by the novel isolates was associated only with spore formation (sporulation), and was a late event in this process. However, while sporulation was required for efficient silicate uptake, silicate uptake itself was not essential for sporulation.

      Visualizing silicate on the spores of these new isolates required some fancy techniques, namely scanning electron microscopy coupled with energy dispersive X-ray spectrometry (STEM-EDX). The silicate taken up was present, at least in part, as granules on the outer surface of the spore coat, with some perhaps in the coat itself. Note that the outermost layer in B. anthracis and B. cereus spores is not the coat but rather the exosporium, a loosely-fitting outermost structure found on some, but not all, bacterial endospores. Analysis of silicate uptake showed that laboratory B. cereus andBacillus thuringiensis strains, as well as a more distantly related Bacillus megaterium strain, also accumulated silicate on spores, although at lower levels than the novel Bacillus isolates. Interestingly, the well-characterized Bacillus subtilis 168 laboratory strain took up no detectable silicate. These new results make it clear that spores of at least some Bacillus species incorporate significant amounts of silicate and deposit it on the spore coat. It is notable that this silicate is not on the spore’s outer layer, and thus it should be possible to discriminate between weaponized and non-weaponized B. anthracis spores by STEM-EDX analysis.

      Bioterrorism aside, this work raises several interesting questions. The first concerns the mechanism of silicate accumulation on spores. It appears to be mediated by the mother cell in which the spore matures, but how the mother cell does this is not known. Finding this out may lead to an understanding of why some Bacillus strains/species accumulate silicate and some do not. The second obvious question is why would a spore accumulate silicate at levels up to 6% of the spore’s dry weight. One possibility is that this might facilitate dispersion of single spores, much as does an outer coating of fumed silica. However, according to this paper, high levels of silicate on the spore coat did not enhance the dispersal of dry spores. Thus, silicate accumulation must play another role.

      Indeed, whereas the spores’ silicate plays no role in spore resistance to heat, hydrogen peroxide, UV radiation or NaOH, it significantly increases spore resistance to killing by 0.1-0.4 N mineral acids. This increased acid resistance might be particularly important in spores of pathogens such asB. cereus and B. anthracis that may pass through an acidic mammalian digestive tract. On the other hand, this would not be important in the alkaline digestive tract of the insect forms for which B. thuringiensis is pathogenic. Therefore, it seems likely that the spores’ silicate layer may serve an additional function. Since silicate accumulation in other organisms can impart structural rigidity, perhaps silicate plays such a role for spores as well. This leaves us with yet more interesting questions to address to these spores.

      Peter Setlow is Professor of Molecular, Microbial and Structural Biology at the University of Connecticut Health Center in Farmington, CT.

    • DXer said

      Two experts advise me that this report seems like “hot air.” That would require selection that wasn’t occurring.

      I am further told that the to-be-released DoD report attributes DPG’s problems with radiation dosing viability testing, and aseptic procedures to “scientific uncertainty” concerning dose levels and time intervals between irradiation and viability testing

      (Which is suggested by one expert to be “worse than hot air” .)

  4. DXer said

    Note in this article by Alison Young about Ivins’ emails in the USA Today, that CDC signals which way it seems to be heading. The CDC seems focused on containment all right.

    But notice the blockbuster email that Ms. Young left yet for someone still to address:

    “Army lab lacked effective anthrax-killing procedures for 10 years”
    Alison Young, USA Today, June 17, 2015

    The emails from accused anthrax letter terrorist Bruce Ivins — a microbiologist at the Army’s elite infectious disease laboratory in Fort Detrick, Md. — offer possible clues about what may have happened at Dugway.

    Emails sent by Ivins during the normal course of his work with anthrax at the U.S. Army Medical Research Institute of Infectious Diseases, often called USAMRIID, provide a window into the difficulties scientists face killing the bacterium and its hardy spores. They also indicate that scientists did not follow a universal, standardized protocol for what percentage of anthrax specimens in an irradiated batch needed to undergo verification tests before the batch was considered sterile and safe for shipment and use without significant safety precautions.

    The emails are among a massive trove of Ivins’ correspondence released in 2010 and posted online in a Freedom of Information Act reading room of the U.S. Army Medical Research and Materiel Command after federal officials formally closed their investigation into the 2001 anthrax attacks. Ivins, 62, died from an intentional overdose of acetaminophen in July 2008 as prosecutors prepared to charge him with sending the anthrax-filled letters that killed five and sickened 17 others.

    In a March 2008 email about the planned shipment of some irradiated anthrax spores, Ivins references a recent division meeting and wrote: “As discussed at the meeting, 50% (5 ml) of the material was checked for sterility on SBA, and there was no growth at all. You may have seen [redacted] insistence that we need only check 10% of the material for sterility, but I think that a 50% check for B. anthracis samples is a good idea. We’ve had in the past some samples that failed sterility checks, so 50% leaves us ‘better safe than sorry.’ ”

    The email indicates that to verify that the irradiated spores were killed, samples were being put on a sheep blood agar plate to see if the bacteria would grow.

    It is unclear whom Ivins was writing to in the email or where the specimens were going to be sent. The Army has redacted all other names, including that of the person or organization Ivins said was advocating only testing 10% of the irradiated samples. Over the years, Ivins’ emails show he did work with anthrax researchers at Dugway Proving Ground and that USAMRIID had an anthrax spore production contract with Dugway.

    In 2006 and early 2007, Ivins’ emails indicate that he did verification tests only on 10% of irradiated anthrax specimens. “The spores were irradiated on October 30. On November 2, [redacted] plated out 10% of each of the preps onto SBA plates. All were negative for growth 24 hours later. They will be incubated over the weekend,” Ivins wrote in a Nov. 3, 2006, email.

    By the summer of 2007, however, Ivins and his colleagues ran into a concerning series of irradiation failures, the emails show.

    “The 27 spore samples that were irradiated on 30 MAY 07 (see enclosed file) came back still ‘hot,’ ” Ivins wrote in a June 7, 2007, email sent to eight other people whose names are redacted. The specimens, he wrote, would be sent back for another irradiation dose of 1 million rads “which should kill the remaining viable spores.” Emails in the string show the May irradiation dose was 5 megarads.

    But Ivins had bad news to report on June 13, 2007: “Irradiation sterilization failure … again,” said the subject line of his email, which said that even with the additional radiation dose, the spores could still grow. His email said that verification tests were done on 18 of the 27 samples — and 12 of the 18 were still “hot.” And so they were again going to be sent back to receive an additional 2 megarads of radiation. “Hopefully this will work. Stay tuned … – bruce,” wrote Ivins

    An unidentified recipient of the email replied: “What is going on with the irradiator? Is it not working properly? These things are going to be fried to a crisp!”

    Ivins was puzzled, too. “In the past, 4 megarads would do the job. This is 2 million rads over that, and still no sterility,” he replied.

    The next morning, June 14, 2007, Ivins emailed with more bad news: “After recheck of the 6 samples that appeared to pass sterility check after a total of 6 million rads, only two samples remained negative.” The other four, it turned out, weren’t dead and needed more radiation, he wrote.

    It’s unclear what may have caused the repeated irradiation failures. Issues relating to irradiation are again mentioned in the publicly available emails in August 2007, when an unidentified person wrote to Ivins asking for the paperwork on the 27 spore samples. In his reply, Ivins wrote that “several rounds of irradiation had to be done to finally get all of the spores sterile. It seems as though the new irradiator isn’t as reliable as the old one for some reason.”

    Military officials didn’t answer USA TODAY’s questions about the radiation doses used at the Dugway Proving Ground lab, which is the focus of the current international investigation of live anthrax shipments. They also didn’t answer questions about what percentage of anthrax specimens in each irradiated batch were undergoing verification tests for sterility at Dugway. They did, however, email a statement on Wednesday about Ivins’ emails detailing the 2007 irradiation failures:

    “It is clear from these emails that the investigators did their due diligence to determine what was causing the failure and that no live material was used in the lab or sent to other labs,” said the statement emailed by Maj. Eric Badger, a Defense Department spokesman. As part of the department’s comprehensive review of Dugway’s mishaps, investigators are “examining, among other things, the failure rates of gamma irradiation for killing anthrax.”

  5. DXer said

    Before 9/11, they were killing virulent Ames spores with 5 million rads.

    Then they came to sometimes use only 4 million rads.

    But then in 2007, to Ivins’ alarm, they found that 6 million rads was not working.

    But they also sometimes killed by formalin.

    Click to access 20061030_batch71(redacted).pdf

    From: Ivins, Bruce E Dr USAMRIID
    Subject: FW: Given to for Irradiation Date: Friday, November 03, 2006 5:18:53 PM

    Who do I give the material to, and when? Also, I have the Delta Sterne spores below which have NOT been irradiated. They can be given out. Also, (b) (6) has said that she’d be willing to learn (b) (6) procedure for formalin killing, so we could give them the various isolates killed by formalin.

    Bruce Ivins

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